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© The Author(s), under exclusive license to Springer Nature
Switzerland AG 2022
J. Zhang, V. Serpooshan (eds.), Advanced Technologies in Cardiovascular
Bioengineering, https://doi.org/10.1007/978-3-030-86140-7_1
From Simple Cylinder to Four-Chambered
Organ: A Brief Overview of Cardiac
Morphogenesis
Carissa Lee, Sharon L. Paige, Francisco X. Galdos, Nicholas Wei,
and Sean M. Wu
1 Introduction
CHDs are the most common type of birth defects, accounting for approximately
13% of deaths in the US in 2017, or 365,914 deaths [1]. In considering the origins
of CHDs and related malformations, a fundamental understanding of cardiac growth
and morphogenesis is requisite. This article reviews the salient morphogenic phases
of the developing heart, beginning with the incipience of the two embryonic axes
and concluding with the completion of complex septation and trabeculation pro-
cesses that characterize the mature embryonic heart. We also discuss the origins of
four major cardiac lineages, namely derivatives of the FHF, SHF, PEO, and cNCC
progenitors. In this article, we have chosen to focus on the murine model of cardiac
C. Lee (*) · F. X. Galdos · N. Wei
Stanford Cardiovascular Institute, Stanford University School of Medicine,
Stanford, CA, USA
e-mail: smwu@stanford.edu
S. L. Paige
Stanford Cardiovascular Institute, Stanford University School of Medicine,
Stanford, CA, USA
Department of Pediatrics, Division of Pediatric Cardiology, Stanford University School of
Medicine, Stanford, CA, USA
S. M. Wu
Stanford Cardiovascular Institute, Stanford University School of Medicine,
Stanford, CA, USA
Department of Medicine, Division of Cardiovascular Medicine, and Stanford University
School of Medicine, Stanford, CA, USA
Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of
Medicine, Stanford, CA, USA](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-15-320.jpg)
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development due to its similarity to human models and popularity in recent and
ongoing embryology research.
1.1
Early Gastrulation and Formation of the Cardiac Crescent
(E5.0 – E7.5)
Prior to gastrulation at approximately embryonic day E5.0, the mouse embryo
resembles an elongating cylinder consisting of a single proximal-distal axis. The
TGF-beta signaling protein nodal growth differentiation factor (NODAL), expressed
along a concentration gradient with its antagonists (Cer1 and Lefty1), in both
embryonic and extraembryonic tissues induces patterning along a second anterior-
posterior axis by E5.5 as shown in Fig. 1 [2].
Gastrulation is an essential process for embryogenesis, beginning at approxi-
mately E6.0 in mice [3]. During this period, a pluripotent group of embryonic cells
called the epiblast ingresses through a strip-like structure known as the primitive
streak (PS) to generate the three germ layers of the early embryo: endoderm, meso-
derm, and ectoderm. While cells from these germ layers collectively give rise to the
body and all its organs, the heart is specifically established by a group of myocardial
Fig. 1 At E5.5, NODAL expression originates from the Node, a structure located at the posterior
side of the mouse embryo. NODAL antagonists Lefty1 and Cer1 are expressed anteriorly, allowing
for formation of the primitive streak on the posterior side. Upon induction of the primitive streak
around E6.5–7.5, NODAL, Bmp, and canonical Wnt signaling gradients direct commitment of
migrating epiblast cells to various endoderm and mesodermal lineages, including cardiac meso-
derm that gives rise to the heart. (Adapted from [2])
C. Lee et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-16-320.jpg)
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progenitor cells which derive from the mesoderm. Notably, the heart is the first
functioning organ of the embryo, as it pumps blood carrying oxygen and nutrients
necessary for embryonic development [4].
By E6.5, these precardiac mesodermal progenitors on the posterior end of the
embryonic “cylinder” will migrate anteriorly and laterally to a region named the
anterior lateral plate mesoderm (ALPM). Along the way, these cells acquire cardiac
fates due to the patterned expression of bone morphogenic protein (BMP), wingless-
related integration site (Wnt), fibroblast growth factor (FGF), and other signaling
molecules that guide their organization at the ALPM into the two main cardiac
progenitor cell populations: the first and second heart fields. These two cell popula-
tions contribute to distinct structures of the developing heart. Fate-mapping studies
have shown that the FHF lineage primarily establishes the myocardium of the left
ventricle (LV), while the SHF lineage gives rise to the right ventricle (RV) and out-
flow tract (OFT) [5]. Initially, FHF precursors differentiate rapidly, becoming beat-
ing cardiomyocytes (CMs) which form the early cardiac crescent [6]. As SHF
precursors settle medially to the FHF, they together form the completed cardiac
crescent, which is typically visible by E7.5 [2]. The temporal delay between the
formation of FHF and SHF progenitors from the precardiac mesoderm, FHF pre-
ceding SHF, is the primary driver of organization into each heart field.
Recent findings have shown that coordinated flow of calcium ions between CMs
triggers the first heartbeat [7]. This explains evidence of primitive pacemaker activ-
ity originating near the inflow tract and sinus venosus of the linear heart tube at this
stage of development [8]. Furthermore, this portion of the heart tube includes the
primordium of the sinus node, which later becomes the chief pacemaker of the
mature cardiac conduction system [9].
1.2
The Linear Heart Tube (E8.0)
As development proceeds, the next few processes—heart tube formation, heart tube
elongation, and early chamber establishment —all overlap in time across the embry-
onic heart. Additionally, these morphogenic stages from E8.0–11.0 contribute sig-
nificantly to growth, facilitating a 100-fold increase in CM number and cardiac
volume [10].
Following formation of the cardiac crescent, a process characterized by rapid
differentiation of FHF progenitors into CMs, the embryonic heart enters a period of
more extensive morphogenesis. Splanchnic mesoderm slides over endoderm, tem-
porarily pausing CM differentiation as the heart tube (HT) is assembled. When dif-
ferentiation resumes upon completion of the primitive HT, newly formed CMs
instead contribute to SHF-derived regions of the HT and initiate closure of the dor-
sal side. The influx of SHF cells also strengthen the heartbeat such that by E8.25,
the embryonic “cylinder” is transformed into a beating linear HT [6].
The linear HT is made up of three distinct layers, with the inner endocardial layer
and outer myocardial layer separated by a thick band of extracellular matrix (ECM)
From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-17-320.jpg)
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referred to as the cardiac jelly [8]. The ECM plays a role in the maturation of cardiac
cells into highly vascularized, densely compacted myocardium.
Immunohistochemistry techniques have designated four essential ECM proteins—
collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN)—that
are found within the LV of the mouse heart [11]. The first beating CMs are found in
the outer heart tube, while the cells of the inner heart tube retain an endothelial cell
identity [2].
Subsequent migration of SHF progenitors to the arterial and venous poles facili-
tates gradual elongation of the heart tube as these cells undergo proliferation. The
proepicardial organ (PEO), a transitory mesenchymal structure responsible for gen-
erating the embryonic epicardial cell lineage, also appears near the venous pole by
E8.5 [12]. Importantly, this cluster of coelomic cells is highly conserved among
vertebrates [13].
1.3
Cardiac Looping (E8.5)
As elongation slows, the linear heart tube undergoes a characteristic process of
rightwards looping in which the posterior regions begin to move anteriorly as shown
in Fig. 2. This establishes the structural basis of the heart’s four distinct chambers:
right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV) [3].
Inversion of the “S” loop during this phase is a common malformation observed
in patients with heterotaxy syndrome (HS), a disorder characterized by develop-
mental abnormalities in the left-right axis [14]. Early defects in the looping process
underscore its importance to proper heart formation as patients with HS may pres-
ent with dextrocardia, a condition in which the apex of the heart points towards the
right instead of the left.
Cardiac valve formation also initiates around E8.5 with the formation of the
dorsal and ventral endocardial cushions following heart looping [15]. These “swol-
len” protrusions of the ECM are lined with endocardial cells, a portion of which will
migrate into the cushion ECM and adopt mesenchymal identities through a process
known as the endothelial-to-mesenchymal transition (EndMT). This thickening of
the endocardial cushions provides the foundation for later remodeling into valve
leaflets.
Finally, epicardium development proceeds simultaneously as vesicles from the
PEO either directly adhere to the surface of the beating heart or gradually drift
towards the myocardium following their release into the pericardial cavity [13].
Upon making contact, the attached cells will collapse and proliferate, generating a
primitive layer of epicardium that covers the heart. These epicardially derived cells
(EPDCs), a subset of which will undergo an epithelial-to-mesenchymal transition
(EMT) and migrate into the myocardium, have the potential to differentiate into
coronary smooth muscle cells and interstitial fibroblasts while reports of their dif-
ferentiation into coronary endothelium and cardiomyocytes require further investi-
gation [16].
C. Lee et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-18-320.jpg)
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1.4
The Four-Chambered Heart (E9.5)
The looped heart tube consists of four main structural elements: the atrium, atrio-
ventricular canal (AVC), ventricle, and outflow tract [5]. Partitioning the left from
right and the atrial from ventricular regions of the heart constitutes the most com-
plex stage of heart morphogenesis, beginning around E9.5 and lasting approxi-
mately until E14.5 as depicted in Fig. 3. Proper chamber formation is crucial to the
Fig. 2 Diagram illustrating the process of cardiac looping beginning at E8.5, whereby the linear
heart tube is first transformed into an S-shaped curve before organization into primitive chambers.
(a) The linear heart tube. (b) Looping. (c) The primitive 4-chambered heart. (Key: T truncus arte-
riosus, BC bulbus cordis, SV sinus venosus, PV primitive ventricle, PA primitive atrium, pRV prim-
itive right ventricle, pLV primitive left ventricle, pRA primitive right atrium, pLA primitive left
atrium, OFT outflow tract). (Adapted from [17])
From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-19-320.jpg)
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developmental pathway, as failure to establish structures capable of sustaining sys-
temic circulation results in defects such as atrial septal defects (ASDs), ventricular
septal defects (VSDs), and atrioventricular septal defects (AVSDs) that can progress
to end-stage heart failure.
At E9.5, active proliferation and migration of the SHF leads to the formation and
elongation of the outflow tract (OFT), a transient structure which, in its most primi-
tive state, connects the developing right ventricle to the aortic sac [18].
Subsequent infiltration of cNCCs into the looped heart initiates septation of the
elongated OFT into the aorta (Ao) and pulmonary trunk (PT) [19]. From their start-
ing point in the cardiac neural crest, these cells migrate through the aortic arches
and cluster near the distal OFT, forming truncal cushions. Interactions between
these truncal cushions and the proximally-located conal cushions of the OFT cre-
ates a spiral septum that divides the OFT into the Ao and PT, allowing for separate
systemic and pulmonary circulations [5]. Congenital malformations of the OFT
may result in conotruncal defects, which include conditions such as Tetralogy of
Fallot (TOF) and Transposition of the Great Arteries (TGA).
Fig. 3 Illustrations depicting ventral surface cuts of embryonic mouse hearts between E9.5 and
E17.5. (Key: AVC atrioventricular canal). (Adapted from [10])
C. Lee et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-20-320.jpg)
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By E10.5, “well-defined chambers” are visible in the heart despite persistence of
the primitive tubular structure [3]. Histological samples indicate the presence of a
functioning sinoatrial node, which is responsible for initiating heart beats [8]. The
epicardium is fully formed, creating a protective envelope around the heart. At this
point, the atrial and ventricular chambers septate, a process that begins with the
expansion of the mesenchymal cushions, leading to the formation of the right and
left atria and ventricles.
The development of the two major atrioventricular (AV) cushions, the inferior
and superior cushions, in the centralAVC is facilitated by EndMT [20]. Endocardium
derived cells (ENDCs) populate the cushions, displacing the existing ECM within
the AV canal. As such, lineage tracing studies have shown that the majority of mes-
enchymal cells infiltrating the cushions are derived from endocardium [21].
As shown in Fig. 4, atrial septation occurs from E10.5–E13.5, beginning when
the major AV cushions fuse at the AVC along with two mesenchymal structures: the
vestibular spine and mesenchymal cap. Muscularization of the mesenchymal tissue
results in two muscular tissue structures, the septum primum and septum secundum,
which together septate the atrial chamber into right and left [22]. At E11.5, within
the ventricular chamber, an outgrowth called the interventricular muscular septum
fuses with the AV cushions to create distinct right and left ventricles [5]. The
“minor” left and right lateral AV cushions, which form after the inferior and supe-
rior AV cushions, are also formed from ENDCs. These minor cushions become the
Fig. 4 Depiction of the atrial septation process, occurring approximately from E10.5–E13.5. (a)
Formation of the first of two muscular septa, the septum primum, begins at the roof of the primitive
atrial chamber. (b) As the septum primum elongates, the foramen primum and foramen secundum
allow for continued communication between the right and left sides of the atrium. (c) The septum
secundum grows to the right of the septum primum, forming an oval-shaped hole called the fora-
men ovale. (d) Both septa begin to fuse. (e) The foramen ovale remains open, allowing for blood
flow from the right to left atrium. (Key: RA right atrium, LA left atrium). (Adapted from [25])
From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-21-320.jpg)
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septal leaflets of the mitral and tricuspid valves, which are necessary to prevent
retrograde flow of blood from the ventricles to the atria [23]. Failure of the tricuspid
valve tissue to delaminate from the ventricular myocardium at this stage results in
right ventricular myopathy and a apically-displaced tricuspid valve, which are char-
acteristic of a rare congenital defect called Ebstein’s anomaly [24].
Myocardial trabeculation, which also involves the endocardium, is another
essential developmental step that begins at this timepoint, extending through the end
of the embryonic stage. Within the heart wall, the myocardial layer projects into the
cardiac jelly, the ECM layer between the endocardium and myocardium, as endo-
cardial cells invaginate, the resulting finger-like structures are called trabeculae.
This process aids in the gradual dissipation of the cardiac jelly as the trabeculae
mature and eventually collapse to join with the compact myocardium, completing
the inner wall of the heart.
1.5
The Mature Embryonic Heart (E15.0)
The conclusion of chamber and OFT septation around E15.0 prepares the heart for
postnatal separation of the pulmonary and systemic circulatory pathways of the
blood [3]. In the fetal heart, oxygenated blood flows from the placenta to the umbili-
cal vein, entering the ductus venosus and passing through the inferior vena cava
(IVC) before entering the RA. It then travels across the foramen ovale to the LA,
down to the LV, and out the Ao to the brain and upper body. Deoxygenated blood
from the superior vena cava (SVC) drains to the RA, down to the RV, through the
pulmonary artery, and across the ductus arteriosus to the rest of the developing
embryo. The fetal circulation pathway ensures that the most oxygenated blood in
the fetus goes to the brain, with limited blood entering the lungs as oxygenation of
this area occurs postnatally. In the final fetal morphogenic phase, the heart tissue
undergoes “fine tuning” modifications that improve cardiac conduction, coronary
circulation, and control of blood flow.
Cardiac Conduction System The cellular origins of the cardiac conduction system
have yet to be detailed in full. Currently, it is known that signaling from arterial
endothelial cells induces the differentiation of Purkinje conduction cells from myo-
cardium [26]. Fast-conducting chamber myocardium makes up the contractile fibers
of the bundle of His, while slow-conducting myocardium from the inflow tract and
AV canal creates the SA and AV nodes [4]. The sinus venosus region’s important
role as a primitive pacemaker provides evidence of SAN progenitors, but the mech-
anisms behind the formation of the AV node remain poorly understood.
Coronary Vessels The appearance of coronary endothelial cells has been recorded
as early as E12.5, with endocardium giving rise to coronary arterial endothelium
and the sinus venosus generating coronary venous endothelium [27]. SV-derived
“sprouts” of venous cells cover the fetal heart before proliferating to form the
C. Lee et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-22-320.jpg)
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immature coronary plexus [28]. Recent single-cell RNA sequencing experiments
found that a subset of these venous endothelial cells dedifferentiate and undergo
pre-
arterial specification via transcriptional changes that take place prior to the
establishment of blood flow [29]. The connecting of the plexus to the Ao initiates
blood flow and is crucial to arterial morphogenesis. Epicardial cells from the PEO
also play a role in coronary vessel formation, assembling the smooth muscle wall of
the coronary vasculature through EMT [30].
Valves The mitral and tricuspid valves are derived from the AV endocardial cush-
ions. As the superior and inferior AV cushions fuse and divide the AVC, they give
rise to the anterior mitral and septal tricuspid leaflets. Further remodeling of these
primitive leaflets results in formation of the mature mitral and tricuspid valves that
ensure unidirectional atrial to ventricular blood flow.
Atrioventricular EPDCs (AV-EPDCs) give rise to the AV sulcus, a transient mes-
enchymal structure that separates the atrial and ventricular myocardial walls. A por-
tion of the AV-EPDCs within the sulcus infiltrate the AV myocardial junction where
they begin to form the annulus fibrosus, a divider made up of fibrous tissue respon-
sible for physically and conductively isolating the working atrial myocardium from
its ventricular counterpart. Yet another group of AV-EPDCs will continue on from
the annulus fibrosus to merge with the parietal AV valve leaflets and eventually
become valve interstitial cells [23].
Trabeculation By E19.0, trabeculation concludes with the complete degradation
of the cardiac jelly layer and resultant compaction of the ventricular wall [8]. This
compaction is associated with greater strength of contraction, allowing the blood to
penetrate deeper layers of the myocardium before the coronary vasculature fully
develops in the post-embryonic stage. With the completion of trabeculation, the
prenatal mouse heart is ready for postnatal modification following gestation, which
typically occurs 20 days post-fertilization.
2 Challenges and Opportunities
While much of early heart development has been documented through fate-mapping
and histological examination of mice, chick, and zebrafish embryos, a number of
challenges still remain in documenting human heart morphogenesis, including lack
of data, inconclusive literature, and poor imaging capabilities.
Due to a combination of technical, legal, and ethical complications, human fetal
heart cells are exceedingly difficult to obtain for data collection. Use of human
induced-pluripotent stem cells (hiPSCs), while generally considered more ethically
sound, suffer from difficulties with chamber and cell type identification. Ongoing
research focuses on single-cell RNA sequencing or lineage tracing-based solutions
that enable determination of distinct genetic markers within the embryonic heart.
From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-23-320.jpg)
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Recent studies have used sequencing data to identify candidate genes that may con-
tribute to CHDs such as HS [14].
An emerging topic of research is the transcriptomic correlation between murine
and human embryonic heart cells. A recent single-cell RNA sequencing (scRNA-
seq) study of cardiac cells from 18 human embryos showed several differences
between developing mouse and human cardiac cells in terms of certain gene expres-
sion levels, targets, and corresponding developmental timepoints, suggesting that
correlating mouse transcriptomics to human may not be practical. However, pre-
liminary research into using mouse scRNA-seq data in human cardiac cell classifi-
cation models have yielded promising results. Ongoing research is looking into
whether machine learning algorithms can be trained on mouse scRNA-seq data and
fine-tuned on preliminary human data to identify human embryonic heart cells, as
well as heart cells derived from induced pluripotent stem cells. The impact of this
research would be two-fold. First, a mouse-to-human classification model can lever-
age the vast amount of mouse transcriptomic data that exists (as well as future epig-
enomic data) to create a highly useful tool providing insights into the mechanisms
that control the signaling pathways of human cardiac development and regenera-
tion. Second, a successful classification model could be used to identify cardiac
cells derived from human induced pluripotent stem cells (hiPSCs), enabling scien-
tists to use these findings in order to better study cardiac cells in vitro.
Another area of interest is improving existing functional and molecular defini-
tions of certain cell types and processes. For instance, EndMT remains poorly
understood in comparison to EMT [31] as cell culture conditions severely impact
the process. Furthermore, endocardial precursors originate from a variety of regions,
making it difficult to establish precise molecular criteria. Standardizing markers for
both the presence of and definitive stages of ongoing EndMTs may improve under-
standing of endothelial dysfunctions that cause both CHDs and adult cardiovascular
diseases.
The “fine tuning” steps that occur during the septation phase, most notably the
fusion of the OFT and AVC, are complex and difficult to record. Additionally, while
heart formation is thus far understood to unfold continuously, viewing morphoge-
netic events in real time may reveal new insight into the kinetics of development.
Although limited by embryo survivability, whole-embryo live-imaging methods,
such as the two-photon microscopy approach used by Ivanovitch and colleagues [6],
may harbor potential to capture these nuances at cellular resolution.
In summary, cardiac morphogenesis involves an extensive process of looping,
septation, and remodeling that transforms the early heart fields into a matured, four-
chambered organ capable of systemic and pulmonary circulation. As the intricacies
of the embryonic heart render it susceptible to disruption, the understanding of
human fetal heart development will remain a popular topic that continues to gener-
ate novel research directions in hopes of finding solutions to prevalent the develop-
ment of CHDs.
C. Lee et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-24-320.jpg)
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© The Author(s), under exclusive license to Springer Nature
Switzerland AG 2022
J. Zhang, V. Serpooshan (eds.), Advanced Technologies in Cardiovascular
Bioengineering, https://doi.org/10.1007/978-3-030-86140-7_2
Lineage Tracing Models to Study
Cardiomyocyte Generation During
Cardiac Development and Injury
Kamal Kolluri, Bin Zhou, and Reza Ardehali
1 Introduction
Lineage tracing is a powerful method to mark a finite number of progenitors at a
specific developmental stage and interrogate the progeny of the founder cell at later
time points [1]. Lineage tracing has been particularly important in studying cardio-
vascular development by identifying cardiac progenitors that contribute to specific
myocardial lineages and their clonal activities. Fundamental to understanding car-
diac development is the ability to determine the identity of stem/progenitor cells,
their ancestry and when and how their progeny move to reside in their final location.
Lineage tracing, especially the clonal analysis of a single progenitor, is the main
approach used to address these issues. Lineage tracing of cardiomyocytes is
achieved by using cardiomyocyte-type specific marker genes that permanently label
K. Kolluri
Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine,
University of California, Los Angeles, California, USA
B. Zhou
State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology,
Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences,
Shanghai, China
R. Ardehali (*)
Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine,
University of California, Los Angeles, California, USA
Eli and Edythe Broad Stem Cell Research Center, University of California,
Los Angeles, California, USA
Molecular, Cellular and Integrative Physiology Graduate Program, University of California,
Los Angeles, California, USA
Molecular Biology Institute, University of California, Los Angeles, CA, USA
e-mail: RArdehali@mednet.ucla.edu](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-27-320.jpg)
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any cell expressing those genes as well as all subsequent progeny. By using markers
expressed by cardiomyocytes or cardiac progenitors, researchers can gain insight
into how these cells progress over the course of development and growth, or in
response to injury by analyzing their proliferative capacity as well as the expression
profile of the generated clones. In this chapter we will review two powerful lineage
tracing tools (Mosaic Analysis with Double Markers [MADM] and Rainbow) that
have been successfully used for clonal analysis of cardiomyocytes during normal
development and after injury. Both models allow for precise fate tracking and lin-
eage tracing, making them applicable to many different types of studies.
2
Mosaic Analysis with Double Markers (MADM)
The extent of cardiomyocyte proliferation during development and in the postnatal
heart remains an area of controversy. Recent studies suggest that there is a low turn-
over of cardiomyocytes that declines with age in mice and humans [2–6]. It has
come to consensus in the field that newly generated cardiomyocytes originate from
pre-existing cardiomyocytes through proliferation rather than differentiation from
stem cells [5]. Studies of cardiomyocyte proliferation have been limited by reliance
on indirect assays of markers for cell proliferation and on surrogates for cell divi-
sion [7, 8]. These studies are particularly challenging to interpret, due to the con-
founding issues of cardiomyocyte polyploidy, multi-nucleation, and DNA repair
after injury in adult hearts [9, 10]. Therefore, it is important to develop a system that
differentiates between karyokinesis and cytokinesis in adult cardiomyocytes.
Mosaic Analysis with Double Markers (MADM) uses Cre-Recombinase technol-
ogy that induces genetic recombination upon cell division, which indelibly labels
clones of proliferating cells with a single fluorescent marker. Analysis of clonal
expansion using MADM has numerous applications in studying cellular prolifera-
tion in development, stem cell biology, and regenerative medicine.
2.1 Methodology
The MADM strategy uses two reciprocally chimeric genes that are knocked into the
same location on homologous chromosomes, with each containing the N-terminus
of one reporter and the C-terminus of the other reporter, interrupted by a lox-P site.
After DNA replication, genetic recombination induced by Cre-LoxP creates func-
tional reporter genes (green fluorescent protein [GFP] or red fluorescent protein
[RFP]). Upon G2-X-segregation during cell division, the divided cells express
either GFP or RFP, and this feature allows MADM to be used for genetically record-
ing cytokinesis events upon G2-X segregation. Considering that inter-chromosomal
Cre-LoxP recombination after S phase is a rare event, labeling a fraction of cells of
interest allows for clonal analysis of marked cells and their progeny. Since G2-X
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-28-320.jpg)
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Fig. 1 Mosaic Analysis with Double Markers (MADM). (a) Schematic detailing how MADM
works. Upon DNA replication and MADM recombination, the cell can result in three different
situations. Upon G2-X segregation, two single-labeled daughter cells will arise, with one being
GFP+
, the other RFP+
. G2-Z segregation results in one double-labeled (GFP+
RFP+
) cell and one
unlabeled (GFP−
RFP−
) cell. G0 recombination with no division results in one double-labeled cell
(GFP+
RFP+
). (b) Two distinctly, single-labeled, sibling cardiomyocytes exhibiting intimate end-on
contact (Scale bar, 10 μm) [13]. (c) Myocardial infarction (MI) was induced by Left Anterior
Descending Artery (LAD) ligation. The number of cardiomyocytes in Sham versus MI is similar,
suggesting that MI does not result in any appreciable increase of cardiomyocyte proliferation in the
left ventricle (LV). White arrowheads point to single-labeled cells (Scale bars, 120 μm) [13]. (d)
The utility of MADM in post-mitotic and mitotic tissues. Hprt-Cre was used to generate MADM
labeled cells in post-mitotic tissues (liver, kidney, heart, spleen). Actin-Cre was used to label cells
in the epidermis while keratin5-Cre was used to label keratinocytes in the epidermis (Scale bars:
(Aa)-(Da) and (Ab)–(Db), 2 mm; (Ac)–(Dc), 50 μm; (Ea) and (Fa), 100 μm; (Eb) and (Fb), 10 μm)
[12]. (e) Two single-labeled daughter cardiomyocytes generated by a MADM recombination/divi-
sion event in the Myh6/MADM model (Scale bar, 15 μm) [13]. (f) Section from a P12 Myh6/
MADM heart revealing the presence of sparse single-labeled and double-labeled cells (Scale bar,
100 μm). (i) GFP+
single-labeled cardiomyocyte with its sibling RFP+
single-labeled cell, and
double-labeled cells. (ii) GFP+
single-labeled cell with a double-labeled cell. The GFP+
cell con-
tains visible sarcomeric elements, demonstrating the fidelity of this model in marking cardiomyo-
cytes. (iii) RFP+
single-labeled cardiomyocyte (Scale bars (i-iii), 10 μm) [13]
Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-29-320.jpg)
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mitosis is required to generate single-labeled cells, the MADM system unambigu-
ously labels divided cells [11, 12]. Figure 1a details a schematic for how
MADM works.
In addition, MADM allows for asymmetric labeling of daughter cells so that a
relationship between precursor-progeny lineages can be established [14]. This is
particularly important in studies involving stem cell differentiation, organ develop-
ment and tissue regeneration in response to injury. Since MADM couples mitosis
with labeling, MADM can be used to identify the progenitor cell or single cells that
can be tracked for clonal expansion. The two differentially labeled MADM daugh-
ter cells can be retrospectively analyzed to investigate clonal analysis and patterns
of cell division. MADM has been successfully used to track stem cell division [12,
15, 16]. In the case of an asymmetric stem cell division where a daughter stem cell
and a differentiated cell are generated, each daughter cell is differentially labeled. If
the differentiated cell continues to proliferate, then a clone of single-labeled cells
can be identified. This is particularly useful for in vivo stem cell tracking, since
MADM events are rare, hence labeling a small number of cells and their progeny.
This allows for retrospective tracing of cellular expansion through easily identifi-
able sparse clones. In addition, multilineage cell potential can be studied by track-
ing whether certain clones generate multiple different cell types [14].
The labeling efficiency of MADM is partly dependent on the Cre line used.
Obviously, ubiquitous Cre lines result in more doublelabeled cells, which is due to
the increase in post-mitotic cells, which undergo G0 recombination [12]. Tissue-
specific inducible Cre lines are routinely used for two main reasons: 1) to control
the extent of recombination events, and 2) to control the timing of recombination
events according to the study design. When experimental planning requires clonal
analysis at specific time points (i.e. at precise stages of development or after injury),
temporal control of MADM labeling can be achieved using transiently expressed
Cre lines [12]. While the amount of tamoxifen induces a desired recombination
event, the timing of tamoxifen administration allows for temporal control of recom-
bination and fluorescent labeling of cells.
2.2
Labeling Rare Populations and Lineage Tracing
The first study to use MADM for clonal analysis in the heart was reported by Ali
et al. [13]. First, they used an HprtCre+/−
/MADM-11GT/TG
model, which can label
any cell, and observed distinct clusters of RFP+
and GFP+
cells in the heart. In addi-
tion, Tasic et al. generated another transgenic mouse model where they inserted the
reconstituted GFP gene into one Hipp11 locus and the reconstituted RFP gene into
the other Hipp11 locus of each cell [17]. In this model, MADM-11GG/TT
, they
observed only double-labeled cells and found no evidence of single or unlabeled
cells. This further validated the use of MADM in the heart because there was no
observed silencing of the MADM transgene, thereby ensuring no false negative
results. After confirming the validity of MADM in the heart, Ali and colleagues
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-30-320.jpg)
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created an inducible Myh6CreERT2;MADM-11GT/TG
mouse model in which upon
tamoxifen induction, Myh6 dividing cells may undergo homologous recombina-
tion, resulting in two distinctly labeled daughter cells (Fig. 1b). Newborn pups were
given tamoxifen and their hearts were analyzed at P12. They observed that approxi-
mately 11% of labeled cells were single-labeled, which were progeny of Myh6-
expressing cardiomyocytes. These cells all expressed alpha-actinin and contained
sarcomeric elements, demonstrating that dividing cardiomyocytes gave rise to new
cardiomyocytes. In addition, they observed equivalent frequencies of GFP+
and
RFP+
labeled cells, demonstrating that cardiomyocytes divide to generate further
cardiomyocytes in a symmetric fashion. In many cases, the cardiomyocyte clones
were noncontiguous, separating from each other after division.
They next induced MADM recombination at E13.5 and analyzed the labeling of
Myh6 expressing cells during development. They found that a majority of the cells
were single-labeled, confirming that a majority of these cells are mitotically active.
In contrast, they observed a significant number of double-labeled cardiomyocytes
after birth, likely arising from G0 inter-chromosomal recombination, indicating that
after birth, cardiomyocytes are not mitotically active. While their work showed that
cardiomyocytes were the source of proliferating cells during development, Ali et al.
demonstrated that only a very small portion of cardiomyocytes divide in the adult
heart. Their lineage tracing experiments confirmed that pre-existing cardiomyo-
cytes generate cardiomyocytes in adults at a low rate after birth.
Next, they used a β-ActinCreER/MADM-11GT/TG
model, which permits MADM
recombination in any cell-type. The use of the β-ActinCreER/MADM model
allowed the investigators to determine if there is a stem/progenitor cell source for
cardiomyocytes, and their findings clearly and unambiguously argued against the
existence of a multipotent progenitor cell in the adult heart akin to canonical stem
cells in other tissues. To investigate the proliferative behavior of cardiomyocytes
after injury, MI was induced by ligation of the left anterior descending artery (LAD)
at 8 weeks of age, with tamoxifen administration for 2 weeks. They analyzed the
hearts 4 weeks after MI and found similar frequencies of single-labeled cells in both
sham and MI, suggesting that injury in mice does not necessarily induce cardiomyo-
cyte proliferation above the basal level (Fig. 1c). An alternative explanation could
be the inefficient inter-chromosomal recombination in the setting of induced Cre
recombinase in the adult cardiomyocytes. Further iteration of MADM for more
efficient recombination would improve our understanding of adult cardiomyocyte
proliferation in homeostasis and after injuries. The study by Ali et al. demonstrated
for the first time how cardiomyocytes can be labeled through MADM and how their
fate can be tracked both in response to injury and through several stages of
development.
MADM has also been used to study cardiomyocyte division stimulated by cell-
cycle gene induction. Mohamed and colleagues used a combination of four cell
cycle regulators (CDK1:CCNB and CDK4:CCND complexes) to induce cardio-
myocyte proliferation and growth in vitro and in vivo [18]. Particularly, they used
MADM lineage tracing and demonstrated that adult cardiomyocyte division could
be induced in vivo at an efficiency of at least 15%.
Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-31-320.jpg)
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2.3
Utility of MADM in Other Organ Systems
Zong et al. first reported the development of the MADM system [12]. In their
groundbreaking report, they demonstrated that Cre-dependent inter-chromosomal
recombination can be induced efficiently in vivo in mitotic and post-mitotic cells
(Fig. 1d). They used this system for conditional gene knockout, lineage analysis,
and neural connection tracing. To illustrate the utility of MADM, they studied the
fate of granule cell progenitors in the cerebellar cortex. Using the MADM system,
they identified 26 distinct subclusters of granule cells in the cerebellar cortex. They
showed that these granule cell clusters exhibit limited dispersion, that there was a
low frequency of generating these clusters, and that there was a small chance that
clusters were generated from two separate clonal lineages. They reasoned that each
cluster was likely the progeny of a single-labeled clone. Thus, Zong et al. success-
fully used the MADM system for the first time to demonstrate that granule cell
progenitors are fated to give rise to adult granule cells which distinctly localize and
project axons to specific sublayers of the cerebellar cortex.
MADM has been used extensively for developmental studies in the field of neu-
roscience. Mihalas and Hevner used MADM to study the differentiation of early
intermediate progenitors (IP) and their role in the developing cerebral cortex [16].
IP cells are derived from radial glial progenitor cells and give rise to pyramidal
projection neurons in the cerebral cortex. Their data suggested three main models
for IP cell differentiation. Their analysis revealed that IP cells can have asymmetric
fates and generate multilayered clones, or undergo rapid or delayed terminal dif-
ferentiation to produce either upper or lower-layer cortical neurons, respectively. In
all of the suggested processes, the authors observed asymmetric cell death.
MADM has since been used in other systems, particularly cancer. Liu et al. used
a MADM-based model for glioma to lineage trace neural stem cells (NSC) and
distinguish between cell-of-mutation and cell-of-origin [15]. The lineage tracing
feature of MADM allowed this group to track clones throughout the process of
tumorigenesis. The mutant cells were labeled GFP+
, whereas the wild-type cells,
which served as internal controls, were labeled RFP+
. Importantly, the MADM sys-
tem was utilized to trace the cells at pre-malignant stages. They induced p53 and
NF1 mutations in NSCs and observed that oligodendrocyte progenitor cells exhib-
ited higher proliferative capacity, thus pointing to these as the cell-of-origin in their
glioma model. By using MADM to differentiate between mutant and wild type
cells, Liu et al. were able to conduct a detailed analysis of the precise physiological
changes that occur during tumor formation and identify a cell-of-origin in their
glioma model.
The MADM system has also been used in a variety of genetic imprinting studies.
A particular advantage is that MADM can be used to study uniparental disomy
(UPD) by indelibly and unambiguously labeling either unimaternal or unipaternal
disomic cells. Hippenmeyer et al. studied the effects of genomic imprinting in chro-
mosome 7 and 12 and explored chromosome and cell-type specific imprinting [19].
In addition, Laukoter et al. found that UPD in the neocortex results in highly
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-32-320.jpg)
![21
cell-type specific genome-wide changes [20]. They also used MADM to reveal dif-
ferences in paternal dominant and maternal dominant UPD within cortical astrocytes.
2.4 Limitations/Future Directions
The major limitation of traditional systems for conditional gene knockout has been
the difficulty to achieve strict coupling of knockout and labeling. Since there is a
single chromosomal exchange event in MADM, generation of homozygous cells
and labeling is coupled, hence leaving little chance for ambiguity. However, there
are several limitations with MADM. First, the efficiency of interchromosomal
recombination is much less than intrachromosomal recombination used in tradi-
tional knockout systems. Although this could be a desirable feature for analyzing
single-cell autonomous gene function, it may become a problem where high fre-
quency of gene knockout is desired. Also, since MADM is based on the availability
of a pair of MADM knock-ins between the gene of interest and the centromere,
there is a need to generate knock-in cassettes for other chromosomes, an effort that
has been realized in recent publications [12, 15, 19, 20].
For lineage tracing and clonal analysis studies, a potential limitation of the
MADM system is performing event quantification. A binucleated daughter cell
resulting from G2-Z segregation without cytokinesis would be double-labeled, an
observation that is frequently encountered when analyzing heart tissue during
development (Fig. 1e). However, if cytokinesis occurs, G2-Z segregation could lead
to one double-labeled daughter cell while its sibling would be unlabeled (Fig. 1f).
This ultimately could cause underestimation of the number of proliferated cells.
Another limitation is the fact that MADM cannot be used as effectively in post-
mitotic cells as it depends on mitosis in order to label cells [12]. Terminally differ-
ential cells in G0 can also undergo recombination without cytokinesis, leading to
the presence of a double-labeled cell.
3 Rainbow Reporter
The rainbow reporter system is a novel stochastic four-color Cre-dependent reporter
system that has been used for clonal analysis studies. The advantage of this system
lies in its ability to randomly assign different fluorescent labels to cells of interest,
allowing for retrospective tracing of their progeny with easily distinguishable clones
in vivo. When used in combination with an inducible tissue-specific Cre mouse line,
recombination events can be controlled in a spatiotemporal manner. Random recom-
bination events in proliferating cells will result in clones of cells that retain the same
fluorescent label as the parental cell. When rare recombination events occur (i.e. as
a result of limited amount of tamoxifen administration), sparse clones are direct
Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-33-320.jpg)
![22
evidence of cell proliferation, whereas the size of clones is suggestive of their pro-
liferative capacity through a specific time window.
3.1 Methodology
The Rainbow system relies on Cre-dependent recombination to induce indelible
labeling of one of three random fluorescent markers. Rainbow mice carry a cassette
of four fluorescent genes (GFP, mCerulean, mOrange, and mCherry) inserted in the
Rosa26 (R26) locus. Without Cre-mediated recombination, all tissues express the
default GFP reporter label. When the Rainbow mouse is crossed with a tissue-
specific inducible Cre line, tamoxifen administration leads to random excision of a
pair of the mutated LoxP sites, resulting in permanent and exclusive expression of
one of the three fluorescent proteins. Rainbow can be used to retrospectively trace
cell lineages by identifying and counting same-colored clones that arise from a
common progenitor. When crossed with a mouse line that expresses Cre/CreER
under the promoter of a certain marker gene, this model can be used to label and
track the fate of a population of cells of interest. The permanent labeling feature also
allows for analysis over a long period of time in processes such as cellular dynamics
and symmetry of cell divisions in a single cell lineage [21]. Figure 2a illustrates a
schematic of how the Rainbow reporter works.
We previously reported the utility of the Rainbow system to retrospectively iden-
tify the source of new cardiomyocytes during fetal and neonatal development, as
well as in adult hearts after injury [23]. Through 3D clonal analysis of cardiovascu-
lar progenitors and cardiomyocytes, we demonstrated that cardiac progenitors are
the main source of cardiomyocytes during murine cardiac development. The lineage
tracing experiments revealed that immature cardiomyocytes maintain their prolif-
erative potential throughout embryonic development, however, there is a decline in
their proliferation as they progress to more mature cardiomyocytes. In this study,
several inducible mouse models were used in combination with the Rainbow system
which allowed for distinguishable reporter expression in the heart as opposed to a
mosaic pattern generated by a non-inducible Cre model. Clones of cells were per-
manently labeled with one of the three fluorescent proteins and further staining for
α-sarcomeric actinin confirmed their cardiomyocyte identity. In order to determine
the size of the generated clonal clusters, the cell counter tool on ImageJ software
was used to quantify the number of cells per clone (Fig. 2b). Additionally, for a
more detailed three-dimensional clone volume analysis and anatomical localization,
a modified CLARITY technique was used in which the heart was transformed into
an optically translucent but structurally preserved organ. The cleared hearts were
subsequently imaged by confocal and light-sheet fluorescence microscopy. This
advanced imaging modality facilitated an accurate measurement of clone volumes
at different time points during heart development.
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-34-320.jpg)
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3.2
Labeling of Rare Cell Populations and Lineage Tracing
Sereti et al. were the first to use Rainbow mice to study the regenerative capacity of
the heart [22]. In order to first demonstrate the utility of Rainbow in the heart, they
utilized 3 different transgenic Cre mouse models, under the control of either the
cardiovascular progenitor genes Mesp1 and Nkx2.5 or the more mature cardiomyo-
cyte marker αMHC. Analysis of hearts at embryonic day 14.5 (E14.5), postnatal
day 1 (P1) and P21 revealed that the hearts marked by progenitor markers formed
clonal clusters while those marked by αMHC showed a mosaic pattern of singletons
(Fig. 2c). The use of constitutively active Cre lines, although very informative, nor-
mally produces high levels of recombination, and one cannot exclude the possibility
that the observed single-color cell clusters are the results of random recombination
events. On the other hand, a tamoxifen-inducible Cre line permits spatiotemporal
control of recombination events. By administering a limiting amount of tamoxifen,
one can achieve a handful of labeled cells in an organ and follow their fate
retrospectively.
The authors next crossed Rainbow mice with mice harboring an inducible Cre
under the control of a βactin, Nkx2.5, or αMHC promoter. This approach allowed
for distinction between non-cardiomyocyte-derived clonal expansion (βactinCreER
;
R26VT2/GK
), cardiac progenitor-derived clonal expansion (Nkx2.5CreER
; R26VT2/GK
), or
mature cardiomyocyte (αMHCCreER
; R26VT2/GK
) clonal expansion. When a limiting
amount of tamoxifen was administered at E9.5 or E12.5 to βactinCreER
; R26VT2/GK
mice, postnatal heart analysis revealed clear clones of cardiomyocytes, fibroblasts,
endothelial and vascular smooth muscle cells (Fig. 2d). Clonal analysis of
Nkx2.5CreER
; R26VT2/GK
mice labeled at E9.5 or E12.5, and analyzed postnatally, also
revealed a similar pattern of clonal expansion with comparable clone size and vol-
ume to those observed in βactinCreER
; R26VT2/GK
mice. These findings supported the
Fig. 2 (continued) Marking a cardiomyocyte clone for counting purposes. Cells are pseudo-col-
ored red and WGA staining is used to mark cell boundaries (Scale bar, 100 μm) [22]. (c)
Longitudinal sections from E14.5, P1 and P21 transgenic mice under the control of the progenitor
markers Mesp1 (i-iii) and Nkx2.5 (iv-vi) or the adult CM marker αMHC (vii-ix). Fluorescent
microscope images from hearts under the control of progenitor markers (ii, v) revealed the pres-
ence of clear clonal clusters. Images from hearts under the control of αMHC (viii) revealed a
mosaic pattern of singletons, with no definite clonal clusters (Scale bar (iii, vi, ix), 500 μm; all
others 50 μm) [22]. (d) The utility of the βactinCreER
; R26VT2/GK
model in marking other cell types
in the heart. (i) shows a close up confocal image of a clone (blue) containing vascular smooth
muscle cells in a P7 heart stained for smooth muscle Myosin Heavy Chain (smMHC). (ii) shows a
confocal image of cardiomyocyte clones in a P15 Nkx2.5CreER
; R26VT2/GK
heart stained
for α-sarcomeric actinin (Scale bar (i), 50 μm; (ii), 100 μm) [22]. (e) Limited tamoxifen adminis-
tration allows for rare recombination events. Section of a βactinCreER
; R26VT2/GK
adult heart, in
which recombination was induced at E9.5, shows the presence of sparse single-colored clones
labeled with mOrange and mCherry (Scale bar, 500 μm) [22]. (f) Representative confocal images
of sections from βactinCreER
; R26VT2/GK
(i), Nkx2.5CreER
; R26VT2/GK
(ii) and αMHCCreER
; R26VT2/GK
(iii) neonatal mice that received LAD ligation at P0. Clonal analysis performed 21 days after MI
reveals the presence of sparse single-labeled clones, suggesting that neonatal mice undergo cardio-
myocyte regeneration in response to injury [22]
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-36-320.jpg)
![25
proliferative capacity of progenitor cells to generate cardiomyocytes during early
fetal development (Fig. 2e).
In order to explore the proliferative capacity of cardiomyocytes during fetal
development, recombination was induced in cardiomyocytes at different embryonic
time points using αMHCCreER
; R26VT2/GK
mice. When tamoxifen was administered at
E12.5, postnatal analysis revealed mostly singleton cardiomyocytes with few small
size clones. However, interrogation of αMHCCreER
; R26VT2/GK
mice labeled at E9.5
revealed similar size cardiomyocyte clones compared to βactinCreER
; R26VT2/GK
or
Nkx2.5CreER
; R26VT2/GK
mice. These data suggest that αMHC-expressing cardiomyo-
cytes at E9.5 retain the ability to proliferate and that this capacity is significantly
diminished by E12.5.
They went on to perform single cell transcriptional analysis of αMHC-expressing
cardiomyocytes at E9.5, E12.5 and P1. Their investigation demonstrated the exis-
tence of a heterogeneous population of cardiomyocytes within the early stages of
cardiac development and their transition into a mature, less proliferative, and
homogenous population by the early postnatal period. Overall, the use of the
Rainbow model revealed that clonal dominance of differentiating progenitors medi-
ates cardiac development, while a distinct subpopulation of cardiomyocytes may
have the potential for limited proliferation during late fetal and early postnatal life.
Such precise analyses at a single cell resolution would be challenging and prone to
inaccurate interpretations if traditional lineage tracing experiments were utilized. It
would be important to incorporate new technology in future, eg. DNA barcoding,
for high-throughput analysis of a large number of individual cardiomyocytes and
their progenies in developing hearts.
Sereti et al. also used Rainbow to study clonal expansion in neonatal and adult
cardiomyocytes in response to cardiac injury. Newborn (P0) αMHCCreER
; R26VT2/GK
,
βactinCreER
; R26VT2/GK
and Nkx2.5CreER
; R26VT2/GK
mice received tamoxifen followed
by LAD ligation or Sham operation at P1. At 21 days after MI, hearts were analyzed
and frequent clones of cardiomyocytes were observed in the infarct and border zone
areas of αMHCCreER
; R26VT2/GK
mice (Fig. 2f). Similar observations were made with
βactinCreER
; R26VT2/GK
and Nkx2.5CreER
; R26VT2/GK
mice after injury. This suggested
that regeneration of the heart after injury in neonates was largely due to cardiomyo-
cyte proliferation, which was confirmed by recent fate mapping studies of cardio-
myocytes and non-cardiomyocytes in neonates [24]. In adults, LAD ligation was
performed at 8 weeks of age and analysis at 21 days post-MI revealed the presence
of sparse single-labeled clones in the infarct and border zone areas. These observa-
tions suggested that injury induces cardiomyocyte proliferation in the neonatal heart
but not in the adult heart.
Wang et al. used the Rainbow system to study the clonal expansion of smooth
muscle cells (SMC) in atherosclerosis [25]. Myh11CreERT2
; R26VT2/GK
; ApoE−/−
mice
were fed a high-fat Western diet to induce atherogenesis. The authors observed that
during early atherogenesis, there was a distinct subpopulation of SMCs that de-
differentiated and upregulated Sca1 (a stem cell marker). Sca1 staining was most
intense within the core of the dominant clone and Sca1+
cells appeared to colocalize
near the necrotic core of the atherosclerotic plaque. Single cell RNA sequencing
Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-37-320.jpg)
![26
analysis of this Sca1+
population revealed that these cells down-regulated SMC
marker genes and upregulated genes relating to inflammation and the complement
cascade (complement C3 was one of the most significantly up-regulated factors).
These findings translated to human specimens as well. Histological analysis of post-
mortem carotid artery specimens revealed localization of C3 expression to the
necrotic core of human atherosclerotic plaque, similar to mice. Furthermore, this
Sca1+
SMC signature was found in humans and was associated with coronary artery
disease, with an enriched expression of inflammatory genes (including C3).
Production of C3 by the Sca1+
SMC population may play a role in triggering further
SMC proliferation and vascular inflammation, which could explain the rapid expan-
sion of SMCs during early atherogenesis. Overall, Wang et. al’s study reveals the
potential of the Rainbow system to identify rapidly proliferating and differentiating
populations of cells to gain insight into the pathophysiology of complex diseases
such as atherosclerosis.
3.3
Utility of Rainbow in Other Organ Systems
Rinkevich et al. used an inducible lineage tracing mouse model (βactinCreER
; R26VT2/
GK
) to perform lineage tracing and clonal analysis of individual cells of mouse hind
limb tissue during regeneration of the digit tip, cutaneous wound healing, and nor-
mal maintenance [26]. They removed nerve supply and observed clonal expansion,
revealing that cellular regeneration remains largely intact in the absence of nerve
supply. In a study of the kidney, Rinkevich et al. used the Rainbow mouse model for
clonal analysis and lineage tracing of cells that contribute to the development, main-
tenance and regeneration of the kidney [27]. In all three processes, they found that
cells generating distinct parts of the nephron (i.e. Proximal, Distal tubules or col-
lecting duct) were fate-restricted and stayed within their lineage. Furthermore, they
used an Axin2CreER
; R26VT2/GK3
mouse line to track Wnt pathway responsive cells
(WRC). They showed that WRCs increased their proliferative capacity and that
their clones were restricted to either a proximal tubule or collecting duct fate.
Recent studies using dual recombinases and Confetti reporter (also random
labeling by one of the three fluorescences) demonstrated that bronchioalveolar stem
cells residing in the bronchioalveolar duct junction could clonally expand to form
bronchial epithelial cells and/or alveolar type I and II cells during lung repair and
regeneration [28, 29]. This demonstrates the utility of clonal analysis by rainbow/
confetti reporters for resolving the uni- or bi-differentiation potential of stem cells
in tissue regeneration.
Interestingly, Rainbow has also been used in cancer models. For example, Corey
et al. lineage traced endothelial cells within the tumor microenvironment and con-
cluded that clonal expansion within the microvasculature is crucial to an invasive
melanoma phenotype [30]. Particularly, their studies showed that there is a dimin-
ishing of founder clones to produce subclones, with tumor blood vessels upregulat-
ing genes associated with angiogenesis and a downregulation of lymphocyte
K. Kolluri et al.](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-38-320.jpg)
![27
adhesion molecules. Thus, the Rainbow mouse model allowed lineage tracing of
single cells and their progeny to conclude that clonal evolution within melanoma
can induce changes within the microvasculature to confer cancer cells an advantage.
3.4 Limitations/Future Directions
The ability of Rainbow to precisely track the fate of certain cells across multiple
organ systems makes it a strong candidate for clonal analysis and lineage tracing
within the heart. This model allows researchers to differentiate between proliferat-
ing and quiescent cardiomyocytes, a phenomenon that remains controversial in the
field of cardiac development and regeneration. However, it is not without limita-
tions. A major consideration when using the Rainbow mouse model is the efficiency
of recombination events that partially depends on the Cre line used. While in many
mouse lines, Rainbow faithfully induces equal expression of fluorescent proteins in
labeled cells, some Cre lines have been reported to show uneven expression of the
markers. Additionally, the model’s dependence on tamoxifen could leave the pos-
sibility that a smaller dose does not induce enough recombination to mark all pos-
sible clones, thus underestimating the number of clones [22]. Furthermore, since in
clonal expansion studies, only a small number of cells are labeled and their fate is
monitored retrospectively, it is possible that rare populations of cells cannot be
labeled by this strategy. The Rainbow system can be used for clonal analysis studies
and the presence of clusters of single-colored cells confidently supports the exis-
tence of proliferating cells. However, the absence of an observation does not con-
firm its lack of existence.
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Evidence for cardiomyocyte renewal in humans. Science. 324(5923), 98–102 (2009). https://
doi.org/10.1126/science.1164680
3. Bergmann, O., Zdunek, S., Felker, A., Salehpour, M., Alkass, K., Bernard, S., et al.: Dynamics
of cell generation and turnover in the human heart. Cell. 161(7), 1566–1575 (2015). https://
doi.org/10.1016/j.cell.2015.05.026
4. Cai, C.L., Molkentin, J.D.: The elusive progenitor cell in cardiac regeneration: slip Slidin’
away. Circ. Res. 120(2), 400–406 (2017). https://doi.org/10.1161/CIRCRESAHA.116.309710
5. Eschenhagen, T., Bolli, R., Braun, T., Field, L.J., Fleischmann, B.K., Frisén, J., et al.:
Cardiomyocyte regeneration: a consensus statement. Circulation. 136(7), 680–686 (2017).
https://doi.org/10.1161/CIRCULATIONAHA.117.029343
6. Vagnozzi, R.J., Molkentin, J.D., Houser, S.R.: New myocyte formation in the adult heart:
endogenous sources and therapeutic implications. Circ. Res. 123(2), 159–176 (2018). https://
doi.org/10.1161/CIRCRESAHA.118.311208
Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-39-320.jpg)
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HSC Hematopoietic stem cell
Mlc2v Myosin light chain 2v
Myf5 Myogenic factor 5
MyHC Myosin heavy chain
Myod Myoblast determination protein 1
Nkx2-5 Homeobox Protein Nkx2–5
OE Overexpression
Pdx1 Pancreatic and duodenal homeobox 1
qPCR Quantitative polymerase chain reaction
scRNA-seq Single cell RNA sequencing
Tie2 Endothelial cell specific receptor tyrosine kinase 2
Ve-Cad Vascular endothelial cadherin
1
Cardiovascular Diseases Are Common and Have
Considerable Morbidity and Mortality
Peripheral artery disease affects more than 10 M Americans resulting in more than
150,000 limb amputations each year in the U.S. In addition, more than 300,000
patients have coronary artery bypass grafting (surgical revascularization) [1]. These
diseases collectively are amplified by the rising incidence of diabetes, obesity and
cardiovascular disease. These complications result in considerable morbidity and
mortality [1, 2]. Current medical therapies for vascular diseases include limb ampu-
tation and vascular bypass grafting. However, these therapeutic interventions have
significant limitations. These diseases are chronic, debilitating, lethal and they war-
rant new and novel therapies. The definition of the molecular mechanisms that gov-
ern the endothelial lineage and vascular development will provide a platform to
modulate these pathways and promote vasculogenesis as a therapeutic initiative.
The overall goal for this chapter is to highlight the key regulators that govern endo-
thelial and vascular development.
2
Master Regulators Govern Fate Decisions
and Lineage Development
Loss of function and gain of function genetic studies have defined essential factors
that govern cell fate and lineage development (Fig. 1a). These factors also known as
master regulators occupy or sit at the top of a regulatory hierarchy [3, 4]. Perhaps a
prototypic example are members of the MYOD family of transcription factors. The
MYOD family consists of bHLH transcription factors that have distinct and over-
lapping functional roles for the regulation of the myogenic lineage [5]. These mas-
ter regulators also have the capacity to convert another differentiated cell type
(usually a fibroblast) to a specific lineage (i.e. skeletal muscle) using a
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-43-320.jpg)
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promoter-
reporter construct that demonstrates expression with lineage specific dif-
ferentiation (Fig. 1) [6–9]. These assays were initially referred to as conversion
assays. Using conversion assays in combination with gene disruption and transgenic
technologies, hundreds of master regulators have been described [3]. In addition to
the MYOD family, PDX1 (pancreas), OCT4/SOX2/NANOG (pluripotency), SCL/
TAL1 (blood), HIF1 (hypoxia), and others have been identified (Fig. 1b) [6–8, 10–
12]. ETV2 is a recently identified master regulator that has been shown to be essen-
tial for the specification and development of the endothelial lineage (Fig. 1b)
[13, 14].
Fig. 1 Master regulators specify lineage-specific development. (a) Adaptation of Waddington’s
landscape that outlines the role of master regulators to govern fate decisions during embryogene-
sis. (b) Schematic outlining examples of master regulators for specific lineages. PDX1 is a master
regulator for pancreatogenesis and ETV2 is master regulator for hematoendothelial lineages
Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-44-320.jpg)
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3
Developmental Milestones for Endothelial Development
The initial developmental stage for vasculogenesis occurs as specified progeni-
tors (i.e. angioblasts) that migrate from the primitive streak in the developing mouse
embryo to the yolk sac [15]. Later, these angioblasts migrate from the extraembry-
onic yolk sac to the embryo proper to form cord like structures, lumens (a process
known as tubulogenesis) and ultimately form a vascular plexus (Fig. 2) [15]. This
process continues and is associated with the onset of cardiac contractility in the
E8.25 heart tube, the appearance of primitive blood cells associated with the primi-
tive circulation (E8.5) and the establishment of a complete circulation with the
propagation of blood throughout the E10-E10.25 mouse embryo [16]. This circula-
tion is impacted by the growth of the embryo and the transition from diffusion to the
circulation of blood in response to hypoxic signals (HIF1 and HIF2) and signaling
pathways (VEGF1-FLK1/FLT1/FLT2, SHH-GLI1/2/3, SRC-CDH5, ANG1/2-
TIE2, etc.) [16, 17]. This latter process is termed angiogenesis, which is character-
ized by the formation of new vessels that originate or sprout from pre-established or
Fig. 2 Overview of hematoendothelial development. (a) Schematic highlighting the role of angio-
blasts being recruited to form a vascular plexus, tubulogenesis and sprouts to generate vascular
networks and remodeling. (b) Schematic highlighting the role of hemogenic endothelium during
primitive (yolk sac) and definitive (AGM) hematopoiesis
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-45-320.jpg)
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pre-existing vessels [17]. The vasculature continues to architecturally evolve and
mature with the determination of venous and arterial endothelial fates. This matura-
tion phase is marked by the expression of integrins, the balance of apoptosis and cell
proliferation and the impact of the extracellular matrix [15]. Overall, the endothelial
and vascular lineages are coordinated and responsive to microenvironmental cues
and signals.
The endothelial-endocardial relationship is established early during embryogen-
esis. The endothelial lineage is characterized by a single cell thickness of develop-
ing vascular networks that form a tight syncytium separating the luminal space with
the underlying vascular wall. This endothelial lining is contiguous with the endocar-
dium, which lines the four-chambered heart. The ontogeny of the endocardium is
distinct from the endothelial lineage and is reflected in the expression of a lineage
specific molecular program [18].
4
The Common Origin of Endothelial
and Hematopoietic Lineages
The endothelial and hematopoietic lineages are both derivatives of the mesodermal
germ layer [19]. As both lineages develop in close proximity (i.e., blood islands of
the yolk sac and the blood containing vessels) and express overlapping molecular
programs, studies support a common origin for the lineages [19]. The extraembry-
onic yolk sac is the source for primitive hematopoiesis (Fig. 2b) [20]. Indeed, stud-
ies have demonstrated that multilineage hematopoietic stem cells are derivatives of
hemogenic endothelium. Hemogenic endothelium, despite its endothelial gene
expression profile, loses its endothelial potential early in development and only
gives rise to blood [21]. Hemogenic endothelium are flat shaped cells that undergo
endothelial-to-hematopoietic transition (EHT and marked by CD44 expression) and
acquire a spherical shape characteristic of blood cells [20, 22]. These hemogenic
endothelial cells are found within the allantois, the yolk sac, the endocardium and
the aorta-gonad-mesonephros (AGM) of the developing embryo [20, 23]. The AGM
is closely associated with the ventral wall of the dorsal aorta (~E9.5-E11.5) and has
been shown to produce hematopoietic stem cells that are capable of engrafting and
reconstituting the irradiated bone marrow. Therefore, the AGM is recognized for its
critical role in definitive hematopoiesis (Fig. 2b) [23]. Single cell RNA-seq of the
AGM in the developing mouse have identified multiple cell types and defined the
expression of GATA2, RUNX1, LYL1, ERG, FLI1, LMO2 and TAL1 transcription
factors, which result in the loss of endothelial gene expression and acquisition of
hematopoietic gene expression [22].
Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-46-320.jpg)
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5
ETV2 Is Necessary and Sufficient for Endothelial
Lineage Development
ETV2 (Ets Variant Transcription Factor 2) was initially sequenced from the testis by
Steve McKnight’s laboratory [24]. Later, ETV2 was co-discovered as an essential
factor for hematoendothelial (HE) lineage development by the Choi laboratory and
the Garry laboratory [25, 26]. These independent efforts resulted in studies by the
Choi laboratory, which defined a BMP/NOTCH/WNT-ETV2 axis during mouse
embryogenesis and demonstrated that Etv2 null embryos were lethal and lacked
hematopoietic and vascular lineages [25]. Similarly, the Garry laboratory used a
Nkx2-5-reporter transgenic strategy and identified Etv2 as a putative downstream
target. Further, they demonstrated that Etv2 null embryos were nonviable at E9-E9.5
and had an absence of blood and endothelial lineages and defined that ETV2 was a
direct upstream regulator of the Tie2 gene [26]. Studies further demonstrated the
essential role for ETV2 in zebrafish, xenopus, pig and human [13, 14, 27–35]. These
latter results support the evolutionary conserved role for ETV2 as an essential factor
for hematoendothelial development. Furthermore, forced overexpression (OE) of
ETV2 using mouse embryonic stem cells/embryoid bodies (ES/EB) differentiation
assays demonstrated that it was sufficient for HE development (Fig. 3) [25, 35–37].
Collectively, these studies provided a foundation for ETV2 as a master regulator for
the HE lineages.
6
ETV2 Expression during Mouse Embryogenesis
and the Postnatal Period
Using in situ hybridization, PCR, immunohistochemistry and the 3.9Kb Etv2-EYFP
reporter transgenic expression, ETV2 expression was restricted to the angioblasts at
E6.5, hematoendothelial cells and endocardium until ~E10-E10.5 after which it was
rapidly extinguished with the exception of a small subpopulation of cells associated
with the dorsal aorta (Fig. 4) [25, 26, 36–41]. Similarly, using the ES/EB differen-
tiation assay, ETV2 was robustly expressed on Days 3–4 following differentiation
after which it was extinguished (Fig. 3) [25, 42]. These expression studies support
the notion that ETV2 functions as a “rheostat” to tightly regulate HE lineage devel-
opment similar to other master regulators for the specification of other lineages.
While the mechanisms that regulate extinguished expression of ETV2 are incom-
pletely defined, the feedback mechanisms involving FLT1 contribute, in part, to the
negative regulation of Etv2 gene expression [42]. Future studies will need to focus
on the definition of these mechanisms to enhance our understanding of ETV2
expression.
ETV2 is expressed postnatally in the testis and in the HSC population (Lin-
Sca1+
cKit+
cells) in adult mouse bone marrow [44, 45]. ETV2 is induced and upreg-
ulated following tissue and vascular injury without sustained long-term or persistent
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-47-320.jpg)
![37
expression [46]. Furthermore, recent studies support the notion that ETV2 is
expressed in tumorigenic tissues and may be associated with the angiogenic
response observed with various solid tumors (as outlined below) [47–49]. These
expression patterns and its role as a master regulator suggest that ETV2 may be an
important target to promote or repress angiogenesis depending on the physiological
context.
7
ETV2 Is Dynamically and Transcriptionally Regulated by
Upstream Factors
Previous studies by our laboratory demonstrated that the 3.9 kb upstream fragment
of the Etv2 gene harbored all the modules and motifs necessary for the spatial and
temporal expression pattern of endogenous ETV2 (Fig. 5) [26, 31, 36, 41, 42, 50,
51]. These studies established that the EYFP reporter expression pattern recapitu-
lated endogenous ETV2 activity demonstrating the onset of expression and
Fig. 3 Embryoid body (EBs) differentiation assays recapitulate developmental mechanisms. (a)
Schematic highlighting ES cells differentiating to EBs and forming mesodermal derivatives
including: hematoendothelial, cardiac and skeletal muscle lineages. (b) FACS profile of dissoci-
ated EBs stained for FLK1 and PDGFR-a demonstrate the HE (FLK1+
/ PDGFR-a−
), cardiac
(FLK1+
/PDGFR-a+
) and skeletal muscle (FLK1−
/PDGFR-a+
) lineages. (c) Schematic of the
expression profile of Etv2 during mesodermal EB differentiation showing its transient pattern of
expression
Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-48-320.jpg)
![38
extinguished activity (Fig. 4) [41]. Bioinformatics analysis revealed evolutionary
conservation of two modules (CRI and CRII) within the 3.9 kb fragment and the
deletion of this fragment phenocopied the global deletion of Etv2 (Fig. 5b) [42].
Furthermore, the characterization of the Etv2 cis-regulatory modules in vitro
revealed the importance of the CRI and CRII modules using luciferase assays [26,
42, 52]. Collectively, these studies confirmed that the upstream regulation of this
gene utilized the binding motifs contained within the 3.9 kb fragment. Using gene
disruption models, transcriptional assays, EMSAs and mutagenesis, transcriptional
regulators of Etv2 gene expression included: ETV2, GATA2, VEFGF/FLK1-
Calcineurin-
NFAT, CREB1, MESP1, NKX2-5 and other signaling pathways (BMP,
Notch and Wnt signaling pathways) (Fig. 5c) [25, 26, 39, 42, 52, 53]. While all
Fig. 4 ETV2 fate mapping identifies ETV2 expressing lineages and its descendants in the heart.
Using the 3.9 kb Etv2-Cre transgenic mouse model, the ETV2 contribution to embryogenesis was
mapped during cardiogenesis in the developing mouse. E12.5 heart co-stained with NKX2–5 (red),
DAPI (blue), and ETV2 cells/descendants (green) [43] demonstrate that every endothelial and
endocardial cell is labeled with GFP including the developing valves (arrowhead) and aorta (open
arrowhead) (a, atria and v, ventricle)
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-49-320.jpg)
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these factors were important regulators, it appeared that MESP1-CREB1 may initi-
ate Etv2 gene expression in the mesodermal lineage. Gene activation may also be
context dependent, as in zebrafish, the overexpression of Nkx2-5 functions to
repress Etv2 gene activity whereas in the mouse it appears to function as a direct
upstream regulator of Etv2 in the endocardium [54].
8
Definition of Transcriptional Targets for ETV2 Mediate
Distinct Developmental Events
Master regulators have a number of functions that are mediated by their respective
downstream targets. ETV2 binds a canonical GGAA/T Ets motif and transactivates
the hematoendothelial molecular program including: Scl/Tal1, Lmo2, Tie2/Tek,
VE-Cadherin/Cdh5, Pecam1/CD31, Gata1, Gata2, Flk1, Elk3, Fli1, Sox7, Cepd,
and others (Fig. 6) [14, 26, 34, 37, 42, 43, 53, 55–61]. These targets were identified
and confirmed using CHiP-seq, transcriptional assays, EMSA, Standard ChIP and
other molecular techniques [14, 55, 62]. More recent studies have demonstrated that
ETV2 promotes cell proliferation by regulating Yes1 gene expression, which inter-
acts with the Hippo signaling pathway (Fig. 6) [63]. ETV2 also transcriptionally
regulates Rhoj gene expression to modulate endothelial progenitor cell migration
during embryogenesis (Fig. 6) [64]. Finally, ETV2 regulates microRNAs (i.e.
miR130a), which govern fate determination by promoting endothelial differentia-
tion but not hematopoietic differentiation (Fig. 6) [59, 60]. Context specific gene
regulation is observed in the testis where ETV2 is a direct upstream regulator of
Sox9 gene expression. In a positive feed-back loop, SOX9 binds to the Etv2 pro-
moter and serves to transactivate its regulator thereby maintaining the sertoli cell
phenotype [65]. ETV2-Chip-seq datasets are publicly available and are continuing
to be mined to further define and explore additional targets and functional roles for
this master regulator.
9
Protein-Protein Interacting Factors for ETV2 Are
Important Coregulators
The Etv2 gene harbors seven exons and encodes a protein that has carboxy terminal
domain, a DNA binding domain (amino acids 316–336 which overlaps with the
ETS domain that spans from 231 to 315 aa) and an amino terminal domain (amino
acids 1–157) (Fig. 5d) [44]. Using an array of biochemical and molecular tech-
niques (i.e. mass spectrometry, yeast two hybrid screening assay, FRET, etc.) inter-
acting factors have been identified for ETV2. Early studies identified FOXC2 as an
interacting factor for ETV2 and suggested that adjacent Ets-Fox binding motifs
(Ets-Fox enhancer motif) in more than 20% of endothelial specific genes were
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-51-320.jpg)
![41
potent coactivators of gene expression [66]. Furthermore, GATA2 has been shown
using multiple assays to interact with ETV2 and coactivate hematoendothelial gene
expression [53]. Other factors (OVol11311) have also been shown to cooperate with
ETV2 [67]. For example, forced overexpression of ETV2 and GATA2 in hiPSCs
promoted a hematopoietic fate [68]. These interacting factors are important context
dependent cofactors that function to amplify and modify the functional role
of ETV2.
10
ETV2 Is a Master Regulator for Hematoendothelial
Lineages Using Conversion Assays
Previous studies have demonstrated that forced overexpression of ETV2 promotes a
hematoendothelial cell fate (Fig. 7) [9, 36, 37]. Forced overexpression of ETV2
alone or in combination with other factors (i.e. GATA2, SCL, etc.) during murine
EB differentiation promotes a hematoendothelial fate (FLK1+
cells) (Fig. 7) [14, 36,
37, 55, 68]. Furthermore, the delivery of ETV2 converted cell populations into
Fig. 6 ETV2 functions as an upstream regulator of gene expression. Schematic which demon-
strates direct downstream targets of ETV2 and their functional role during vasculogenesis and
angiogenesis
Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-52-320.jpg)
![42
ischemic hindlimbs demonstrated that these converted cells were endothelial cells
as they participated in the repair of ischemic hindlimb mouse models [9].
Additionally, the delivery of lentivirus overexpressing ETV2 promoted repair in
response to ischemic injury and reduced the fibroproliferative response in mouse
hearts [69]. Collectively, these preliminary studies provide an important platform
for therapeutic initiatives focused on the overexpression of ETV2.
11
ETV2 Overexpression in Tumor Angiogenesis
Previous studies have demonstrated that master regulators not only promote fate
determination during embryogenesis but also are overexpressed in the context of
cancer [70]. Solid tumors require an enhanced vascular supply for growth, cell pro-
liferation and metastasis. Therefore, if ETV2 is at the top of the transcriptional
hierarchy for vasculogenesis/angiogenesis, then ETV2 should be expressed at dis-
tinct time-periods during tumorigenesis. Expression analysis demonstrated coex-
pression of ETV2 and the histone dymethylase, Junonji domain containing 2A in
Fig. 7 The Etv2 network regulates hematoendothelial lineage development. Upstream and down-
stream regulators and ETV2 effectors specify HE lineage development. ETV2 also represses non-
hematoendothelial lineages
D. J. Garry and J. Sierra-Pagan](https://image.slidesharecdn.com/19155330-250518214745-e907db30/85/Advanced-Technologies-In-Cardiovascular-Bioengineering-Jianyi-Zhang-53-320.jpg)
![43
neuroendocrine prostate tumors [49]. In a separate study, the knockdown of Etv2
using siRNA nanoparticles resulted in the inhibition of tumor angiogenesis and the
inhibition of tumor growth [48]. Furthermore, using a zebrafish xenotransplantation
model, ETV2 and FLI1b were shown to have redundant roles in promoting tumor
angiogenesis [71–73]. Collectively, these initial studies support the conclusion that
strategies focused on the inhibition of ETV2 may be effective therapies that target
tumor growth and angiogenesis.
12
ETV2 Functions to Repress Nonhematoendothelial
Fate Decisions
Master regulators function to reprogram the fate of differentiated cells [3, 4]. They
also have the capacity to repress other lineages and direct progenitor cell popula-
tions down a specified pathway as outlined in the Waddington’s landscape (Fig. 1)
[4, 74]. In the Etv2 global knockout mouse model, mesodermal progenitors that
typically are destined for the hematoendothelial lineage are redirected to the cardio-
myocyte lineage using genetic fate mapping techniques (Fig. 7) [26, 41, 75].
Similarly, the Etv2 null zebrafish endothelial progenitors are redirected to the skel-
etal muscle lineage (Fig. 7) [29, 30]. These studies using a gene disruption strategy
emphasize that ETV2 represses nonhematopoietic lineage formation during early
stages of embryogenesis.
13 Summary
The endothelial and vascular lineages require a complex network of gene expres-
sion that governs fate determination. ETV2 is an important master regulator for the
endothelial and vascular lineages. Definition of the ETV2 mediated molecular net-
works provide a platform for future therapeutic interventions.
Acknowledgements The authors acknowledge Cynthia Faraday for her assistance with the prep-
aration of the figures.
References
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CIR.0b013e3182456d46
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- 6. Advanced Technologies in Cardiovascular Bioengineering
- 7. Jianyi Zhang • Vahid Serpooshan Editors Advanced Technologies in Cardiovascular Bioengineering
- 8. ISBN 978-3-030-86139-1 ISBN 978-3-030-86140-7 (eBook) https://doi.org/10.1007/978-3-030-86140-7 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Editors Jianyi Zhang Biomedical Engineering University of Alabama at Birmingham Birmingham, AL, USA Vahid Serpooshan Biomedical Engineering Georgia Institute of Technology Atlanta, GA, USA
- 9. v Preface In recent decades, the convergence of discoveries in biological sciences and engi- neering have resulted in the development of new industries that offer the promise of revolutionary changes in society, as part of the Convergence Revolution (the Fourth Industrial Revolution). These advancements have offered the potential new man- agement options for some of humanity’s most intractable and deadly diseases. Within the cardiovascular sciences, many of the most provocative discoveries have emerged from studies of pluripotent stem cells, whose roles in the medical sciences and physiological and injury response are becoming increasingly acknowledged. In 2016, the National Institutes of Health (NIH) established the Progenitor Cell Translational Consortium (PCTC) to support research into the use of stem and pro- genitor cells for both biology and therapeutic applications. This book was inspired by the thought-provoking ideas and observations presented at the 2019 PCTC Cardiovascular Bioengineering (CVBE) Symposium and was written by leading scientists and physicians whose work in the CVBE field spans decades and was conducted on four different continents. Cardiomyocytes in the hearts of humans and other mammals are largely incapa- ble of self-replicating; thus, although advancements in the clinical management of cardiovascular conditions have led to substantial improvements in patient longevity and quality of life, the scarring caused by cardiac disease or injury is essentially permanent. Myocardial integrity can be fully restored via whole-heart transplanta- tion surgery, but the supply of donated hearts is far smaller than the number of patients who require treatment, so alternative strategies for replacing the myocardial scar with functional contractile tissue are urgently needed. The lack of cell-cycle activity in adult mammalian cardiomyocytes also severely restricts their availability for investigational work, so early studies of myocardial cell therapy were frequently conducted with stem cells which, though obtained from a variety of sources (e.g., the bone marrow, adipose tissue), were expected to differentiate into cardiomyo- cytes after transplantation. However, the benefits observed in subsequent clinical trials were only marginal and likely evolved from the cells’ paracrine activity, rather than through the production of new cardiomyocytes.
- 10. vi The scarcity of cardiomyocytes for therapeutic investigations was alleviated by the isolation of human embryonic stem cells (ESCs) and, especially, by the develop- ment of techniques for reprogramming somatic cells into induced-pluripotent stem cells (iPSCs). Both cell types can proliferate indefinitely and are capable of differ- entiating into diverse cellular lineage; however, direct stem cell transplantation can lead to tumor formation; so, ESCs and iPSCs must be differentiated into more spe- cialized cell types before administration to patients, and only in recent years the differentiation protocols achieved the adequate efficiency to meet such demands. In general, the most effective protocols are modeled after the mechanisms that regulate cell specification during embryogenesis, when the four major lineages of cardiac cells evolve from progenitor cells of the first and second heart fields, the proepicar- dial organ, and the cardiac neural crest. These protocols may become even more efficient as researchers continue to refine and develop novel methods for determin- ing the identity, ancestry, and progeny of progenitor cells during development and as the heart recovers from injury. Only a small fraction of transplanted cells are engrafted within the native tissue and survives for more than a few days after administration, which is perhaps not surprising, since the cytotoxic conditions responsible for the loss of endogenous cells are likely to endure longer than the initial injury. One of the chief requirements of a more salubrious environment for transplanted cells is adequate perfusion. Both the size and thickness of engineered tissues are typically limited by the access of nutrients and signaling molecules to the cells within the tissue. Thus, the success of cell-based regenerative therapies for treatment of cardiac disease, as well as periph- eral artery disease, critical limb ischemia, and other predominantly vascular condi- tions, will depend on understanding the mechanisms by which the vascular cell differentiation and proliferation can be manipulated to promote vessel growth. Tissues constructed from human ESC- or iPSC-derived cells can also provide researchers with an entirely human-specific platform for studying the pathogenesis of disease and for testing new pharmaceutical products. Notably, iPSC-derived cell and tissue models are powerful tools for personalized therapies, because the iPSCs can be reprogrammed from the patient’s own somatic cells and, consequently, reca- pitulate all of the genetic factors that regulate disease pathology and progression, as well as the patient’s response to treatment. Autologous iPSC-derived cells are also expected to be minimally immunogenic when re-administered to the same patient for treatment of chronic conditions such as heart failure; however, the reprogram- ming and differentiation procedures take several weeks, so cell-based treatments for emergency situations, such as acute myocardial infarction, will require the use of allogeneic cells, which have rarely been studied. Furthermore, one of the primary concerns associated with cardiac cell therapy is the potential for arrhythmogenic complications caused by inadequate electromechanical coupling between the endogenous and transplanted cells. Thus, researchers continue to develop increas- ingly sophisticated tools for assessing the integration and electrophysiological func- tion of engrafted cells and tissues, such as epicardial electrode arrays, genetically encoded fluorescent reporters, and catheter-based electroanatomic mapping. Preface
- 11. vii Although the regenerative capacity of adult mammalian hearts is extremely lim- ited, the hearts of at least some neonatal mammals (e.g., mice and pigs) can fully repair the damage caused by myocardial injury, provided that the injury occurs within the first few days after birth. Existing evidence suggests that this recovery is driven primarily by the proliferation of pre-existing cardiomyocytes, rather than the activity of stem or progenitor cells, which suggests that the cardiomyocytes of adult hearts may retain some latent proliferative capacity that could be therapeutically re-activated to improve cardiac performance in patients with heart disease. The mechanisms responsible for inducing proliferation in cardiomyocytes are just beginning to be explored. These works will be facilitated by advancements in single- cell genomics, which can characterize the gene expression profiles of thou- sands of individual cells; however, the resulting datasets are typically so enormous that they require the use of modern data science techniques, such as dimensionality reduction and clustering analysis, to identify the genes and pathways that are dif- ferentially activated in proliferating and non-proliferating cardiomyocytes. Machine-learning algorithms can even be applied to the text mined from the Medline database and other unstructured sources to identify relationships among specific genes, diseases, and disease symptoms, including those that may explain why out- comes of COVID-19 treatment are worse for patients with cardiovascular comorbidities. In summary, many of the greatest advancements in science, and in civilization as a whole, have occurred when previously disparate lines of inquiry come together in unanticipated ways. The fields of personal and public health will soon reap the ben- efits of the unprecedented degree of synergy that has recently developed among the life and physical sciences, computing, and engineering. The authors of this book hope to foster these advancements by sharing their knowledge and expertise with the broader community of scientists, engineers, and clinicians. Birmingham, AL, USA Jianyi Zhang Atlanta, GA, USA Vahid Serpooshan Preface
- 12. ix Contents Part I Cardiac Development and Morphogenesis From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac Morphogenesis������������������������������������������������ 3 Carissa Lee, Sharon L. Paige, Francisco X. Galdos, Nicholas Wei, and Sean M. Wu Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac Development and Injury �������������������������������� 15 Kamal Kolluri, Bin Zhou, and Reza Ardehali Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis������������������������������������������������������������������������������������������ 31 Daniel J. Garry and Javier E. Sierra-Pagan Part II Cellular Approaches to Cardiac Repair and Regeneration Remuscularization of Ventricular Infarcts Using the Existing Cardiac Cells�������������������������������������������������������������������� 51 Yang Zhou and Jianyi Zhang Allogeneic Immunity Following Transplantation of Pluripotent Stem Cell- Derived Cardiomyocytes�������������������������������������� 79 Yuji Shiba Vascular Regeneration with Induced Pluripotent Stem Cell-Derived Endothelial Cells and Reprogrammed Endothelial Cells���������������������������� 87 Sangho Lee and Young-sup Yoon The Guinea Pig Model in Cardiac Regeneration Research; Current Tissue Engineering Approaches and Future Directions���������������� 103 Tim Stüdemann and Florian Weinberger
- 13. x Part III Genetic Approaches to Study Cardiac Differentiation and Repair Analysing Genetic Programs of Cell Differentiation to Study Cardiac Cell Diversification������������������������������������������������������������ 125 Zhixuan Wu, Sophie Shen, Yuliangzi Sun, Tessa Werner, Stephen T. Bradford, and Nathan J. Palpant Recombinant Adeno-Associated Virus for Cardiac Gene Therapy������������ 169 Cindy Kok, Dhanya Ranvindran, and Eddy Kizana Part IV Bioengineering Approaches to Cardiovascular Tissue Modeling and Repair Microfabricated Systems for Cardiovascular Tissue Modeling������������������ 193 Ericka Jayne Knee-Walden, Karl Wagner, Qinghua Wu, Naimeh Rafatian, and Milica Radisic Bioengineering of Pediatric Cardiovascular Constructs: In Vitro Modeling of Congenital Heart Disease�������������������������������������������� 233 Holly Bauser-Heaton, Carmen J. Gil, and Vahid Serpooshan Biomaterial Interface in Cardiac Cell and Tissue Engineering������������������ 249 Chenyan Wang and Zhen Ma Stem Cell-Based 3D Bioprinting for Cardiovascular Tissue Regeneration���������������������������������������������������������������������������������������� 281 Clara Liu Chung Ming, Eitan Ben-Sefer, and Carmine Gentile Creating and Validating New Tools to Evaluate the Electrical Integration and Function of hPSC-Derived Cardiac Grafts In Vivo������������������������������������������������������������������������������������ 313 Wahiba Dhahri, Fanny Wulkan, and Michael A. Laflamme Part V Clinical Perspectives Understanding the Molecular Interface of Cardiovascular Diseases and COVID- 19: A Data Science Approach������������������������������������ 335 Dibakar Sigdel, Dylan Steinecke, Ding Wang, David Liem, Maya Gupta, Alex Zhang, Wei Wang, and Peipei Ping Clinical Application of iPSC-Derived Cardiomyocytes in Patients with Advanced Heart Failure������������������������������������������������������ 361 Jun Fujita, Shugo Tohyama, Hideaki Kanazawa, Yoshikazu Kishino, Marina Okada, Sho Tanosaki, Shota Someya, and Keiichi Fukuda Cell Therapy with Human ESC-Derived Cardiac Cells: Clinical Perspectives���������������������������������������������������������������������������������������� 375 Philippe Menasché Index������������������������������������������������������������������������������������������������������������������ 399 Contents
- 14. Part I Cardiac Development and Morphogenesis
- 15. 3 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. Zhang, V. Serpooshan (eds.), Advanced Technologies in Cardiovascular Bioengineering, https://doi.org/10.1007/978-3-030-86140-7_1 From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac Morphogenesis Carissa Lee, Sharon L. Paige, Francisco X. Galdos, Nicholas Wei, and Sean M. Wu 1 Introduction CHDs are the most common type of birth defects, accounting for approximately 13% of deaths in the US in 2017, or 365,914 deaths [1]. In considering the origins of CHDs and related malformations, a fundamental understanding of cardiac growth and morphogenesis is requisite. This article reviews the salient morphogenic phases of the developing heart, beginning with the incipience of the two embryonic axes and concluding with the completion of complex septation and trabeculation pro- cesses that characterize the mature embryonic heart. We also discuss the origins of four major cardiac lineages, namely derivatives of the FHF, SHF, PEO, and cNCC progenitors. In this article, we have chosen to focus on the murine model of cardiac C. Lee (*) · F. X. Galdos · N. Wei Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA e-mail: smwu@stanford.edu S. L. Paige Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA Department of Pediatrics, Division of Pediatric Cardiology, Stanford University School of Medicine, Stanford, CA, USA S. M. Wu Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA Department of Medicine, Division of Cardiovascular Medicine, and Stanford University School of Medicine, Stanford, CA, USA Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- 16. 4 development due to its similarity to human models and popularity in recent and ongoing embryology research. 1.1 Early Gastrulation and Formation of the Cardiac Crescent (E5.0 – E7.5) Prior to gastrulation at approximately embryonic day E5.0, the mouse embryo resembles an elongating cylinder consisting of a single proximal-distal axis. The TGF-beta signaling protein nodal growth differentiation factor (NODAL), expressed along a concentration gradient with its antagonists (Cer1 and Lefty1), in both embryonic and extraembryonic tissues induces patterning along a second anterior- posterior axis by E5.5 as shown in Fig. 1 [2]. Gastrulation is an essential process for embryogenesis, beginning at approxi- mately E6.0 in mice [3]. During this period, a pluripotent group of embryonic cells called the epiblast ingresses through a strip-like structure known as the primitive streak (PS) to generate the three germ layers of the early embryo: endoderm, meso- derm, and ectoderm. While cells from these germ layers collectively give rise to the body and all its organs, the heart is specifically established by a group of myocardial Fig. 1 At E5.5, NODAL expression originates from the Node, a structure located at the posterior side of the mouse embryo. NODAL antagonists Lefty1 and Cer1 are expressed anteriorly, allowing for formation of the primitive streak on the posterior side. Upon induction of the primitive streak around E6.5–7.5, NODAL, Bmp, and canonical Wnt signaling gradients direct commitment of migrating epiblast cells to various endoderm and mesodermal lineages, including cardiac meso- derm that gives rise to the heart. (Adapted from [2]) C. Lee et al.
- 17. 5 progenitor cells which derive from the mesoderm. Notably, the heart is the first functioning organ of the embryo, as it pumps blood carrying oxygen and nutrients necessary for embryonic development [4]. By E6.5, these precardiac mesodermal progenitors on the posterior end of the embryonic “cylinder” will migrate anteriorly and laterally to a region named the anterior lateral plate mesoderm (ALPM). Along the way, these cells acquire cardiac fates due to the patterned expression of bone morphogenic protein (BMP), wingless- related integration site (Wnt), fibroblast growth factor (FGF), and other signaling molecules that guide their organization at the ALPM into the two main cardiac progenitor cell populations: the first and second heart fields. These two cell popula- tions contribute to distinct structures of the developing heart. Fate-mapping studies have shown that the FHF lineage primarily establishes the myocardium of the left ventricle (LV), while the SHF lineage gives rise to the right ventricle (RV) and out- flow tract (OFT) [5]. Initially, FHF precursors differentiate rapidly, becoming beat- ing cardiomyocytes (CMs) which form the early cardiac crescent [6]. As SHF precursors settle medially to the FHF, they together form the completed cardiac crescent, which is typically visible by E7.5 [2]. The temporal delay between the formation of FHF and SHF progenitors from the precardiac mesoderm, FHF pre- ceding SHF, is the primary driver of organization into each heart field. Recent findings have shown that coordinated flow of calcium ions between CMs triggers the first heartbeat [7]. This explains evidence of primitive pacemaker activ- ity originating near the inflow tract and sinus venosus of the linear heart tube at this stage of development [8]. Furthermore, this portion of the heart tube includes the primordium of the sinus node, which later becomes the chief pacemaker of the mature cardiac conduction system [9]. 1.2 The Linear Heart Tube (E8.0) As development proceeds, the next few processes—heart tube formation, heart tube elongation, and early chamber establishment —all overlap in time across the embry- onic heart. Additionally, these morphogenic stages from E8.0–11.0 contribute sig- nificantly to growth, facilitating a 100-fold increase in CM number and cardiac volume [10]. Following formation of the cardiac crescent, a process characterized by rapid differentiation of FHF progenitors into CMs, the embryonic heart enters a period of more extensive morphogenesis. Splanchnic mesoderm slides over endoderm, tem- porarily pausing CM differentiation as the heart tube (HT) is assembled. When dif- ferentiation resumes upon completion of the primitive HT, newly formed CMs instead contribute to SHF-derived regions of the HT and initiate closure of the dor- sal side. The influx of SHF cells also strengthen the heartbeat such that by E8.25, the embryonic “cylinder” is transformed into a beating linear HT [6]. The linear HT is made up of three distinct layers, with the inner endocardial layer and outer myocardial layer separated by a thick band of extracellular matrix (ECM) From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…
- 18. 6 referred to as the cardiac jelly [8]. The ECM plays a role in the maturation of cardiac cells into highly vascularized, densely compacted myocardium. Immunohistochemistry techniques have designated four essential ECM proteins— collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN)—that are found within the LV of the mouse heart [11]. The first beating CMs are found in the outer heart tube, while the cells of the inner heart tube retain an endothelial cell identity [2]. Subsequent migration of SHF progenitors to the arterial and venous poles facili- tates gradual elongation of the heart tube as these cells undergo proliferation. The proepicardial organ (PEO), a transitory mesenchymal structure responsible for gen- erating the embryonic epicardial cell lineage, also appears near the venous pole by E8.5 [12]. Importantly, this cluster of coelomic cells is highly conserved among vertebrates [13]. 1.3 Cardiac Looping (E8.5) As elongation slows, the linear heart tube undergoes a characteristic process of rightwards looping in which the posterior regions begin to move anteriorly as shown in Fig. 2. This establishes the structural basis of the heart’s four distinct chambers: right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV) [3]. Inversion of the “S” loop during this phase is a common malformation observed in patients with heterotaxy syndrome (HS), a disorder characterized by develop- mental abnormalities in the left-right axis [14]. Early defects in the looping process underscore its importance to proper heart formation as patients with HS may pres- ent with dextrocardia, a condition in which the apex of the heart points towards the right instead of the left. Cardiac valve formation also initiates around E8.5 with the formation of the dorsal and ventral endocardial cushions following heart looping [15]. These “swol- len” protrusions of the ECM are lined with endocardial cells, a portion of which will migrate into the cushion ECM and adopt mesenchymal identities through a process known as the endothelial-to-mesenchymal transition (EndMT). This thickening of the endocardial cushions provides the foundation for later remodeling into valve leaflets. Finally, epicardium development proceeds simultaneously as vesicles from the PEO either directly adhere to the surface of the beating heart or gradually drift towards the myocardium following their release into the pericardial cavity [13]. Upon making contact, the attached cells will collapse and proliferate, generating a primitive layer of epicardium that covers the heart. These epicardially derived cells (EPDCs), a subset of which will undergo an epithelial-to-mesenchymal transition (EMT) and migrate into the myocardium, have the potential to differentiate into coronary smooth muscle cells and interstitial fibroblasts while reports of their dif- ferentiation into coronary endothelium and cardiomyocytes require further investi- gation [16]. C. Lee et al.
- 19. 7 1.4 The Four-Chambered Heart (E9.5) The looped heart tube consists of four main structural elements: the atrium, atrio- ventricular canal (AVC), ventricle, and outflow tract [5]. Partitioning the left from right and the atrial from ventricular regions of the heart constitutes the most com- plex stage of heart morphogenesis, beginning around E9.5 and lasting approxi- mately until E14.5 as depicted in Fig. 3. Proper chamber formation is crucial to the Fig. 2 Diagram illustrating the process of cardiac looping beginning at E8.5, whereby the linear heart tube is first transformed into an S-shaped curve before organization into primitive chambers. (a) The linear heart tube. (b) Looping. (c) The primitive 4-chambered heart. (Key: T truncus arte- riosus, BC bulbus cordis, SV sinus venosus, PV primitive ventricle, PA primitive atrium, pRV prim- itive right ventricle, pLV primitive left ventricle, pRA primitive right atrium, pLA primitive left atrium, OFT outflow tract). (Adapted from [17]) From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…
- 20. 8 developmental pathway, as failure to establish structures capable of sustaining sys- temic circulation results in defects such as atrial septal defects (ASDs), ventricular septal defects (VSDs), and atrioventricular septal defects (AVSDs) that can progress to end-stage heart failure. At E9.5, active proliferation and migration of the SHF leads to the formation and elongation of the outflow tract (OFT), a transient structure which, in its most primi- tive state, connects the developing right ventricle to the aortic sac [18]. Subsequent infiltration of cNCCs into the looped heart initiates septation of the elongated OFT into the aorta (Ao) and pulmonary trunk (PT) [19]. From their start- ing point in the cardiac neural crest, these cells migrate through the aortic arches and cluster near the distal OFT, forming truncal cushions. Interactions between these truncal cushions and the proximally-located conal cushions of the OFT cre- ates a spiral septum that divides the OFT into the Ao and PT, allowing for separate systemic and pulmonary circulations [5]. Congenital malformations of the OFT may result in conotruncal defects, which include conditions such as Tetralogy of Fallot (TOF) and Transposition of the Great Arteries (TGA). Fig. 3 Illustrations depicting ventral surface cuts of embryonic mouse hearts between E9.5 and E17.5. (Key: AVC atrioventricular canal). (Adapted from [10]) C. Lee et al.
- 21. 9 By E10.5, “well-defined chambers” are visible in the heart despite persistence of the primitive tubular structure [3]. Histological samples indicate the presence of a functioning sinoatrial node, which is responsible for initiating heart beats [8]. The epicardium is fully formed, creating a protective envelope around the heart. At this point, the atrial and ventricular chambers septate, a process that begins with the expansion of the mesenchymal cushions, leading to the formation of the right and left atria and ventricles. The development of the two major atrioventricular (AV) cushions, the inferior and superior cushions, in the centralAVC is facilitated by EndMT [20]. Endocardium derived cells (ENDCs) populate the cushions, displacing the existing ECM within the AV canal. As such, lineage tracing studies have shown that the majority of mes- enchymal cells infiltrating the cushions are derived from endocardium [21]. As shown in Fig. 4, atrial septation occurs from E10.5–E13.5, beginning when the major AV cushions fuse at the AVC along with two mesenchymal structures: the vestibular spine and mesenchymal cap. Muscularization of the mesenchymal tissue results in two muscular tissue structures, the septum primum and septum secundum, which together septate the atrial chamber into right and left [22]. At E11.5, within the ventricular chamber, an outgrowth called the interventricular muscular septum fuses with the AV cushions to create distinct right and left ventricles [5]. The “minor” left and right lateral AV cushions, which form after the inferior and supe- rior AV cushions, are also formed from ENDCs. These minor cushions become the Fig. 4 Depiction of the atrial septation process, occurring approximately from E10.5–E13.5. (a) Formation of the first of two muscular septa, the septum primum, begins at the roof of the primitive atrial chamber. (b) As the septum primum elongates, the foramen primum and foramen secundum allow for continued communication between the right and left sides of the atrium. (c) The septum secundum grows to the right of the septum primum, forming an oval-shaped hole called the fora- men ovale. (d) Both septa begin to fuse. (e) The foramen ovale remains open, allowing for blood flow from the right to left atrium. (Key: RA right atrium, LA left atrium). (Adapted from [25]) From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…
- 22. 10 septal leaflets of the mitral and tricuspid valves, which are necessary to prevent retrograde flow of blood from the ventricles to the atria [23]. Failure of the tricuspid valve tissue to delaminate from the ventricular myocardium at this stage results in right ventricular myopathy and a apically-displaced tricuspid valve, which are char- acteristic of a rare congenital defect called Ebstein’s anomaly [24]. Myocardial trabeculation, which also involves the endocardium, is another essential developmental step that begins at this timepoint, extending through the end of the embryonic stage. Within the heart wall, the myocardial layer projects into the cardiac jelly, the ECM layer between the endocardium and myocardium, as endo- cardial cells invaginate, the resulting finger-like structures are called trabeculae. This process aids in the gradual dissipation of the cardiac jelly as the trabeculae mature and eventually collapse to join with the compact myocardium, completing the inner wall of the heart. 1.5 The Mature Embryonic Heart (E15.0) The conclusion of chamber and OFT septation around E15.0 prepares the heart for postnatal separation of the pulmonary and systemic circulatory pathways of the blood [3]. In the fetal heart, oxygenated blood flows from the placenta to the umbili- cal vein, entering the ductus venosus and passing through the inferior vena cava (IVC) before entering the RA. It then travels across the foramen ovale to the LA, down to the LV, and out the Ao to the brain and upper body. Deoxygenated blood from the superior vena cava (SVC) drains to the RA, down to the RV, through the pulmonary artery, and across the ductus arteriosus to the rest of the developing embryo. The fetal circulation pathway ensures that the most oxygenated blood in the fetus goes to the brain, with limited blood entering the lungs as oxygenation of this area occurs postnatally. In the final fetal morphogenic phase, the heart tissue undergoes “fine tuning” modifications that improve cardiac conduction, coronary circulation, and control of blood flow. Cardiac Conduction System The cellular origins of the cardiac conduction system have yet to be detailed in full. Currently, it is known that signaling from arterial endothelial cells induces the differentiation of Purkinje conduction cells from myo- cardium [26]. Fast-conducting chamber myocardium makes up the contractile fibers of the bundle of His, while slow-conducting myocardium from the inflow tract and AV canal creates the SA and AV nodes [4]. The sinus venosus region’s important role as a primitive pacemaker provides evidence of SAN progenitors, but the mech- anisms behind the formation of the AV node remain poorly understood. Coronary Vessels The appearance of coronary endothelial cells has been recorded as early as E12.5, with endocardium giving rise to coronary arterial endothelium and the sinus venosus generating coronary venous endothelium [27]. SV-derived “sprouts” of venous cells cover the fetal heart before proliferating to form the C. Lee et al.
- 23. 11 immature coronary plexus [28]. Recent single-cell RNA sequencing experiments found that a subset of these venous endothelial cells dedifferentiate and undergo pre- arterial specification via transcriptional changes that take place prior to the establishment of blood flow [29]. The connecting of the plexus to the Ao initiates blood flow and is crucial to arterial morphogenesis. Epicardial cells from the PEO also play a role in coronary vessel formation, assembling the smooth muscle wall of the coronary vasculature through EMT [30]. Valves The mitral and tricuspid valves are derived from the AV endocardial cush- ions. As the superior and inferior AV cushions fuse and divide the AVC, they give rise to the anterior mitral and septal tricuspid leaflets. Further remodeling of these primitive leaflets results in formation of the mature mitral and tricuspid valves that ensure unidirectional atrial to ventricular blood flow. Atrioventricular EPDCs (AV-EPDCs) give rise to the AV sulcus, a transient mes- enchymal structure that separates the atrial and ventricular myocardial walls. A por- tion of the AV-EPDCs within the sulcus infiltrate the AV myocardial junction where they begin to form the annulus fibrosus, a divider made up of fibrous tissue respon- sible for physically and conductively isolating the working atrial myocardium from its ventricular counterpart. Yet another group of AV-EPDCs will continue on from the annulus fibrosus to merge with the parietal AV valve leaflets and eventually become valve interstitial cells [23]. Trabeculation By E19.0, trabeculation concludes with the complete degradation of the cardiac jelly layer and resultant compaction of the ventricular wall [8]. This compaction is associated with greater strength of contraction, allowing the blood to penetrate deeper layers of the myocardium before the coronary vasculature fully develops in the post-embryonic stage. With the completion of trabeculation, the prenatal mouse heart is ready for postnatal modification following gestation, which typically occurs 20 days post-fertilization. 2 Challenges and Opportunities While much of early heart development has been documented through fate-mapping and histological examination of mice, chick, and zebrafish embryos, a number of challenges still remain in documenting human heart morphogenesis, including lack of data, inconclusive literature, and poor imaging capabilities. Due to a combination of technical, legal, and ethical complications, human fetal heart cells are exceedingly difficult to obtain for data collection. Use of human induced-pluripotent stem cells (hiPSCs), while generally considered more ethically sound, suffer from difficulties with chamber and cell type identification. Ongoing research focuses on single-cell RNA sequencing or lineage tracing-based solutions that enable determination of distinct genetic markers within the embryonic heart. From Simple Cylinder to Four-Chambered Organ: A Brief Overview of Cardiac…
- 24. 12 Recent studies have used sequencing data to identify candidate genes that may con- tribute to CHDs such as HS [14]. An emerging topic of research is the transcriptomic correlation between murine and human embryonic heart cells. A recent single-cell RNA sequencing (scRNA- seq) study of cardiac cells from 18 human embryos showed several differences between developing mouse and human cardiac cells in terms of certain gene expres- sion levels, targets, and corresponding developmental timepoints, suggesting that correlating mouse transcriptomics to human may not be practical. However, pre- liminary research into using mouse scRNA-seq data in human cardiac cell classifi- cation models have yielded promising results. Ongoing research is looking into whether machine learning algorithms can be trained on mouse scRNA-seq data and fine-tuned on preliminary human data to identify human embryonic heart cells, as well as heart cells derived from induced pluripotent stem cells. The impact of this research would be two-fold. First, a mouse-to-human classification model can lever- age the vast amount of mouse transcriptomic data that exists (as well as future epig- enomic data) to create a highly useful tool providing insights into the mechanisms that control the signaling pathways of human cardiac development and regenera- tion. Second, a successful classification model could be used to identify cardiac cells derived from human induced pluripotent stem cells (hiPSCs), enabling scien- tists to use these findings in order to better study cardiac cells in vitro. Another area of interest is improving existing functional and molecular defini- tions of certain cell types and processes. For instance, EndMT remains poorly understood in comparison to EMT [31] as cell culture conditions severely impact the process. Furthermore, endocardial precursors originate from a variety of regions, making it difficult to establish precise molecular criteria. Standardizing markers for both the presence of and definitive stages of ongoing EndMTs may improve under- standing of endothelial dysfunctions that cause both CHDs and adult cardiovascular diseases. The “fine tuning” steps that occur during the septation phase, most notably the fusion of the OFT and AVC, are complex and difficult to record. Additionally, while heart formation is thus far understood to unfold continuously, viewing morphoge- netic events in real time may reveal new insight into the kinetics of development. Although limited by embryo survivability, whole-embryo live-imaging methods, such as the two-photon microscopy approach used by Ivanovitch and colleagues [6], may harbor potential to capture these nuances at cellular resolution. In summary, cardiac morphogenesis involves an extensive process of looping, septation, and remodeling that transforms the early heart fields into a matured, four- chambered organ capable of systemic and pulmonary circulation. As the intricacies of the embryonic heart render it susceptible to disruption, the understanding of human fetal heart development will remain a popular topic that continues to gener- ate novel research directions in hopes of finding solutions to prevalent the develop- ment of CHDs. C. Lee et al.
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- 27. 15 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. Zhang, V. Serpooshan (eds.), Advanced Technologies in Cardiovascular Bioengineering, https://doi.org/10.1007/978-3-030-86140-7_2 Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac Development and Injury Kamal Kolluri, Bin Zhou, and Reza Ardehali 1 Introduction Lineage tracing is a powerful method to mark a finite number of progenitors at a specific developmental stage and interrogate the progeny of the founder cell at later time points [1]. Lineage tracing has been particularly important in studying cardio- vascular development by identifying cardiac progenitors that contribute to specific myocardial lineages and their clonal activities. Fundamental to understanding car- diac development is the ability to determine the identity of stem/progenitor cells, their ancestry and when and how their progeny move to reside in their final location. Lineage tracing, especially the clonal analysis of a single progenitor, is the main approach used to address these issues. Lineage tracing of cardiomyocytes is achieved by using cardiomyocyte-type specific marker genes that permanently label K. Kolluri Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, USA B. Zhou State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China R. Ardehali (*) Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, USA Eli and Edythe Broad Stem Cell Research Center, University of California, Los Angeles, California, USA Molecular, Cellular and Integrative Physiology Graduate Program, University of California, Los Angeles, California, USA Molecular Biology Institute, University of California, Los Angeles, CA, USA e-mail: RArdehali@mednet.ucla.edu
- 28. 16 any cell expressing those genes as well as all subsequent progeny. By using markers expressed by cardiomyocytes or cardiac progenitors, researchers can gain insight into how these cells progress over the course of development and growth, or in response to injury by analyzing their proliferative capacity as well as the expression profile of the generated clones. In this chapter we will review two powerful lineage tracing tools (Mosaic Analysis with Double Markers [MADM] and Rainbow) that have been successfully used for clonal analysis of cardiomyocytes during normal development and after injury. Both models allow for precise fate tracking and lin- eage tracing, making them applicable to many different types of studies. 2 Mosaic Analysis with Double Markers (MADM) The extent of cardiomyocyte proliferation during development and in the postnatal heart remains an area of controversy. Recent studies suggest that there is a low turn- over of cardiomyocytes that declines with age in mice and humans [2–6]. It has come to consensus in the field that newly generated cardiomyocytes originate from pre-existing cardiomyocytes through proliferation rather than differentiation from stem cells [5]. Studies of cardiomyocyte proliferation have been limited by reliance on indirect assays of markers for cell proliferation and on surrogates for cell divi- sion [7, 8]. These studies are particularly challenging to interpret, due to the con- founding issues of cardiomyocyte polyploidy, multi-nucleation, and DNA repair after injury in adult hearts [9, 10]. Therefore, it is important to develop a system that differentiates between karyokinesis and cytokinesis in adult cardiomyocytes. Mosaic Analysis with Double Markers (MADM) uses Cre-Recombinase technol- ogy that induces genetic recombination upon cell division, which indelibly labels clones of proliferating cells with a single fluorescent marker. Analysis of clonal expansion using MADM has numerous applications in studying cellular prolifera- tion in development, stem cell biology, and regenerative medicine. 2.1 Methodology The MADM strategy uses two reciprocally chimeric genes that are knocked into the same location on homologous chromosomes, with each containing the N-terminus of one reporter and the C-terminus of the other reporter, interrupted by a lox-P site. After DNA replication, genetic recombination induced by Cre-LoxP creates func- tional reporter genes (green fluorescent protein [GFP] or red fluorescent protein [RFP]). Upon G2-X-segregation during cell division, the divided cells express either GFP or RFP, and this feature allows MADM to be used for genetically record- ing cytokinesis events upon G2-X segregation. Considering that inter-chromosomal Cre-LoxP recombination after S phase is a rare event, labeling a fraction of cells of interest allows for clonal analysis of marked cells and their progeny. Since G2-X K. Kolluri et al.
- 29. 17 Fig. 1 Mosaic Analysis with Double Markers (MADM). (a) Schematic detailing how MADM works. Upon DNA replication and MADM recombination, the cell can result in three different situations. Upon G2-X segregation, two single-labeled daughter cells will arise, with one being GFP+ , the other RFP+ . G2-Z segregation results in one double-labeled (GFP+ RFP+ ) cell and one unlabeled (GFP− RFP− ) cell. G0 recombination with no division results in one double-labeled cell (GFP+ RFP+ ). (b) Two distinctly, single-labeled, sibling cardiomyocytes exhibiting intimate end-on contact (Scale bar, 10 μm) [13]. (c) Myocardial infarction (MI) was induced by Left Anterior Descending Artery (LAD) ligation. The number of cardiomyocytes in Sham versus MI is similar, suggesting that MI does not result in any appreciable increase of cardiomyocyte proliferation in the left ventricle (LV). White arrowheads point to single-labeled cells (Scale bars, 120 μm) [13]. (d) The utility of MADM in post-mitotic and mitotic tissues. Hprt-Cre was used to generate MADM labeled cells in post-mitotic tissues (liver, kidney, heart, spleen). Actin-Cre was used to label cells in the epidermis while keratin5-Cre was used to label keratinocytes in the epidermis (Scale bars: (Aa)-(Da) and (Ab)–(Db), 2 mm; (Ac)–(Dc), 50 μm; (Ea) and (Fa), 100 μm; (Eb) and (Fb), 10 μm) [12]. (e) Two single-labeled daughter cardiomyocytes generated by a MADM recombination/divi- sion event in the Myh6/MADM model (Scale bar, 15 μm) [13]. (f) Section from a P12 Myh6/ MADM heart revealing the presence of sparse single-labeled and double-labeled cells (Scale bar, 100 μm). (i) GFP+ single-labeled cardiomyocyte with its sibling RFP+ single-labeled cell, and double-labeled cells. (ii) GFP+ single-labeled cell with a double-labeled cell. The GFP+ cell con- tains visible sarcomeric elements, demonstrating the fidelity of this model in marking cardiomyo- cytes. (iii) RFP+ single-labeled cardiomyocyte (Scale bars (i-iii), 10 μm) [13] Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
- 30. 18 mitosis is required to generate single-labeled cells, the MADM system unambigu- ously labels divided cells [11, 12]. Figure 1a details a schematic for how MADM works. In addition, MADM allows for asymmetric labeling of daughter cells so that a relationship between precursor-progeny lineages can be established [14]. This is particularly important in studies involving stem cell differentiation, organ develop- ment and tissue regeneration in response to injury. Since MADM couples mitosis with labeling, MADM can be used to identify the progenitor cell or single cells that can be tracked for clonal expansion. The two differentially labeled MADM daugh- ter cells can be retrospectively analyzed to investigate clonal analysis and patterns of cell division. MADM has been successfully used to track stem cell division [12, 15, 16]. In the case of an asymmetric stem cell division where a daughter stem cell and a differentiated cell are generated, each daughter cell is differentially labeled. If the differentiated cell continues to proliferate, then a clone of single-labeled cells can be identified. This is particularly useful for in vivo stem cell tracking, since MADM events are rare, hence labeling a small number of cells and their progeny. This allows for retrospective tracing of cellular expansion through easily identifi- able sparse clones. In addition, multilineage cell potential can be studied by track- ing whether certain clones generate multiple different cell types [14]. The labeling efficiency of MADM is partly dependent on the Cre line used. Obviously, ubiquitous Cre lines result in more doublelabeled cells, which is due to the increase in post-mitotic cells, which undergo G0 recombination [12]. Tissue- specific inducible Cre lines are routinely used for two main reasons: 1) to control the extent of recombination events, and 2) to control the timing of recombination events according to the study design. When experimental planning requires clonal analysis at specific time points (i.e. at precise stages of development or after injury), temporal control of MADM labeling can be achieved using transiently expressed Cre lines [12]. While the amount of tamoxifen induces a desired recombination event, the timing of tamoxifen administration allows for temporal control of recom- bination and fluorescent labeling of cells. 2.2 Labeling Rare Populations and Lineage Tracing The first study to use MADM for clonal analysis in the heart was reported by Ali et al. [13]. First, they used an HprtCre+/− /MADM-11GT/TG model, which can label any cell, and observed distinct clusters of RFP+ and GFP+ cells in the heart. In addi- tion, Tasic et al. generated another transgenic mouse model where they inserted the reconstituted GFP gene into one Hipp11 locus and the reconstituted RFP gene into the other Hipp11 locus of each cell [17]. In this model, MADM-11GG/TT , they observed only double-labeled cells and found no evidence of single or unlabeled cells. This further validated the use of MADM in the heart because there was no observed silencing of the MADM transgene, thereby ensuring no false negative results. After confirming the validity of MADM in the heart, Ali and colleagues K. Kolluri et al.
- 31. 19 created an inducible Myh6CreERT2;MADM-11GT/TG mouse model in which upon tamoxifen induction, Myh6 dividing cells may undergo homologous recombina- tion, resulting in two distinctly labeled daughter cells (Fig. 1b). Newborn pups were given tamoxifen and their hearts were analyzed at P12. They observed that approxi- mately 11% of labeled cells were single-labeled, which were progeny of Myh6- expressing cardiomyocytes. These cells all expressed alpha-actinin and contained sarcomeric elements, demonstrating that dividing cardiomyocytes gave rise to new cardiomyocytes. In addition, they observed equivalent frequencies of GFP+ and RFP+ labeled cells, demonstrating that cardiomyocytes divide to generate further cardiomyocytes in a symmetric fashion. In many cases, the cardiomyocyte clones were noncontiguous, separating from each other after division. They next induced MADM recombination at E13.5 and analyzed the labeling of Myh6 expressing cells during development. They found that a majority of the cells were single-labeled, confirming that a majority of these cells are mitotically active. In contrast, they observed a significant number of double-labeled cardiomyocytes after birth, likely arising from G0 inter-chromosomal recombination, indicating that after birth, cardiomyocytes are not mitotically active. While their work showed that cardiomyocytes were the source of proliferating cells during development, Ali et al. demonstrated that only a very small portion of cardiomyocytes divide in the adult heart. Their lineage tracing experiments confirmed that pre-existing cardiomyo- cytes generate cardiomyocytes in adults at a low rate after birth. Next, they used a β-ActinCreER/MADM-11GT/TG model, which permits MADM recombination in any cell-type. The use of the β-ActinCreER/MADM model allowed the investigators to determine if there is a stem/progenitor cell source for cardiomyocytes, and their findings clearly and unambiguously argued against the existence of a multipotent progenitor cell in the adult heart akin to canonical stem cells in other tissues. To investigate the proliferative behavior of cardiomyocytes after injury, MI was induced by ligation of the left anterior descending artery (LAD) at 8 weeks of age, with tamoxifen administration for 2 weeks. They analyzed the hearts 4 weeks after MI and found similar frequencies of single-labeled cells in both sham and MI, suggesting that injury in mice does not necessarily induce cardiomyo- cyte proliferation above the basal level (Fig. 1c). An alternative explanation could be the inefficient inter-chromosomal recombination in the setting of induced Cre recombinase in the adult cardiomyocytes. Further iteration of MADM for more efficient recombination would improve our understanding of adult cardiomyocyte proliferation in homeostasis and after injuries. The study by Ali et al. demonstrated for the first time how cardiomyocytes can be labeled through MADM and how their fate can be tracked both in response to injury and through several stages of development. MADM has also been used to study cardiomyocyte division stimulated by cell- cycle gene induction. Mohamed and colleagues used a combination of four cell cycle regulators (CDK1:CCNB and CDK4:CCND complexes) to induce cardio- myocyte proliferation and growth in vitro and in vivo [18]. Particularly, they used MADM lineage tracing and demonstrated that adult cardiomyocyte division could be induced in vivo at an efficiency of at least 15%. Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
- 32. 20 2.3 Utility of MADM in Other Organ Systems Zong et al. first reported the development of the MADM system [12]. In their groundbreaking report, they demonstrated that Cre-dependent inter-chromosomal recombination can be induced efficiently in vivo in mitotic and post-mitotic cells (Fig. 1d). They used this system for conditional gene knockout, lineage analysis, and neural connection tracing. To illustrate the utility of MADM, they studied the fate of granule cell progenitors in the cerebellar cortex. Using the MADM system, they identified 26 distinct subclusters of granule cells in the cerebellar cortex. They showed that these granule cell clusters exhibit limited dispersion, that there was a low frequency of generating these clusters, and that there was a small chance that clusters were generated from two separate clonal lineages. They reasoned that each cluster was likely the progeny of a single-labeled clone. Thus, Zong et al. success- fully used the MADM system for the first time to demonstrate that granule cell progenitors are fated to give rise to adult granule cells which distinctly localize and project axons to specific sublayers of the cerebellar cortex. MADM has been used extensively for developmental studies in the field of neu- roscience. Mihalas and Hevner used MADM to study the differentiation of early intermediate progenitors (IP) and their role in the developing cerebral cortex [16]. IP cells are derived from radial glial progenitor cells and give rise to pyramidal projection neurons in the cerebral cortex. Their data suggested three main models for IP cell differentiation. Their analysis revealed that IP cells can have asymmetric fates and generate multilayered clones, or undergo rapid or delayed terminal dif- ferentiation to produce either upper or lower-layer cortical neurons, respectively. In all of the suggested processes, the authors observed asymmetric cell death. MADM has since been used in other systems, particularly cancer. Liu et al. used a MADM-based model for glioma to lineage trace neural stem cells (NSC) and distinguish between cell-of-mutation and cell-of-origin [15]. The lineage tracing feature of MADM allowed this group to track clones throughout the process of tumorigenesis. The mutant cells were labeled GFP+ , whereas the wild-type cells, which served as internal controls, were labeled RFP+ . Importantly, the MADM sys- tem was utilized to trace the cells at pre-malignant stages. They induced p53 and NF1 mutations in NSCs and observed that oligodendrocyte progenitor cells exhib- ited higher proliferative capacity, thus pointing to these as the cell-of-origin in their glioma model. By using MADM to differentiate between mutant and wild type cells, Liu et al. were able to conduct a detailed analysis of the precise physiological changes that occur during tumor formation and identify a cell-of-origin in their glioma model. The MADM system has also been used in a variety of genetic imprinting studies. A particular advantage is that MADM can be used to study uniparental disomy (UPD) by indelibly and unambiguously labeling either unimaternal or unipaternal disomic cells. Hippenmeyer et al. studied the effects of genomic imprinting in chro- mosome 7 and 12 and explored chromosome and cell-type specific imprinting [19]. In addition, Laukoter et al. found that UPD in the neocortex results in highly K. Kolluri et al.
- 33. 21 cell-type specific genome-wide changes [20]. They also used MADM to reveal dif- ferences in paternal dominant and maternal dominant UPD within cortical astrocytes. 2.4 Limitations/Future Directions The major limitation of traditional systems for conditional gene knockout has been the difficulty to achieve strict coupling of knockout and labeling. Since there is a single chromosomal exchange event in MADM, generation of homozygous cells and labeling is coupled, hence leaving little chance for ambiguity. However, there are several limitations with MADM. First, the efficiency of interchromosomal recombination is much less than intrachromosomal recombination used in tradi- tional knockout systems. Although this could be a desirable feature for analyzing single-cell autonomous gene function, it may become a problem where high fre- quency of gene knockout is desired. Also, since MADM is based on the availability of a pair of MADM knock-ins between the gene of interest and the centromere, there is a need to generate knock-in cassettes for other chromosomes, an effort that has been realized in recent publications [12, 15, 19, 20]. For lineage tracing and clonal analysis studies, a potential limitation of the MADM system is performing event quantification. A binucleated daughter cell resulting from G2-Z segregation without cytokinesis would be double-labeled, an observation that is frequently encountered when analyzing heart tissue during development (Fig. 1e). However, if cytokinesis occurs, G2-Z segregation could lead to one double-labeled daughter cell while its sibling would be unlabeled (Fig. 1f). This ultimately could cause underestimation of the number of proliferated cells. Another limitation is the fact that MADM cannot be used as effectively in post- mitotic cells as it depends on mitosis in order to label cells [12]. Terminally differ- ential cells in G0 can also undergo recombination without cytokinesis, leading to the presence of a double-labeled cell. 3 Rainbow Reporter The rainbow reporter system is a novel stochastic four-color Cre-dependent reporter system that has been used for clonal analysis studies. The advantage of this system lies in its ability to randomly assign different fluorescent labels to cells of interest, allowing for retrospective tracing of their progeny with easily distinguishable clones in vivo. When used in combination with an inducible tissue-specific Cre mouse line, recombination events can be controlled in a spatiotemporal manner. Random recom- bination events in proliferating cells will result in clones of cells that retain the same fluorescent label as the parental cell. When rare recombination events occur (i.e. as a result of limited amount of tamoxifen administration), sparse clones are direct Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
- 34. 22 evidence of cell proliferation, whereas the size of clones is suggestive of their pro- liferative capacity through a specific time window. 3.1 Methodology The Rainbow system relies on Cre-dependent recombination to induce indelible labeling of one of three random fluorescent markers. Rainbow mice carry a cassette of four fluorescent genes (GFP, mCerulean, mOrange, and mCherry) inserted in the Rosa26 (R26) locus. Without Cre-mediated recombination, all tissues express the default GFP reporter label. When the Rainbow mouse is crossed with a tissue- specific inducible Cre line, tamoxifen administration leads to random excision of a pair of the mutated LoxP sites, resulting in permanent and exclusive expression of one of the three fluorescent proteins. Rainbow can be used to retrospectively trace cell lineages by identifying and counting same-colored clones that arise from a common progenitor. When crossed with a mouse line that expresses Cre/CreER under the promoter of a certain marker gene, this model can be used to label and track the fate of a population of cells of interest. The permanent labeling feature also allows for analysis over a long period of time in processes such as cellular dynamics and symmetry of cell divisions in a single cell lineage [21]. Figure 2a illustrates a schematic of how the Rainbow reporter works. We previously reported the utility of the Rainbow system to retrospectively iden- tify the source of new cardiomyocytes during fetal and neonatal development, as well as in adult hearts after injury [23]. Through 3D clonal analysis of cardiovascu- lar progenitors and cardiomyocytes, we demonstrated that cardiac progenitors are the main source of cardiomyocytes during murine cardiac development. The lineage tracing experiments revealed that immature cardiomyocytes maintain their prolif- erative potential throughout embryonic development, however, there is a decline in their proliferation as they progress to more mature cardiomyocytes. In this study, several inducible mouse models were used in combination with the Rainbow system which allowed for distinguishable reporter expression in the heart as opposed to a mosaic pattern generated by a non-inducible Cre model. Clones of cells were per- manently labeled with one of the three fluorescent proteins and further staining for α-sarcomeric actinin confirmed their cardiomyocyte identity. In order to determine the size of the generated clonal clusters, the cell counter tool on ImageJ software was used to quantify the number of cells per clone (Fig. 2b). Additionally, for a more detailed three-dimensional clone volume analysis and anatomical localization, a modified CLARITY technique was used in which the heart was transformed into an optically translucent but structurally preserved organ. The cleared hearts were subsequently imaged by confocal and light-sheet fluorescence microscopy. This advanced imaging modality facilitated an accurate measurement of clone volumes at different time points during heart development. K. Kolluri et al.
- 35. 23 Fig. 2 Rainbow Reporter: (a) Schematic of how Rainbow works. When a Rosa26 Rainbow mouse is crossed with a tissue-specific CreER mouse, expression of the tissue-specific marker results in Cre expression, which allows for the excision of a random pair of loxP sites and results in expres- sion of one of three fluorescent proteins (mCerulean, mOrange, or mCherry). Cells that do not undergo recombination express GFP. In the absence of cell proliferation, the tissue gives a mosaic appearance. When cell proliferation occurs, a clear abundance of same-colored cells is visible. (b) Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
- 36. 24 3.2 Labeling of Rare Cell Populations and Lineage Tracing Sereti et al. were the first to use Rainbow mice to study the regenerative capacity of the heart [22]. In order to first demonstrate the utility of Rainbow in the heart, they utilized 3 different transgenic Cre mouse models, under the control of either the cardiovascular progenitor genes Mesp1 and Nkx2.5 or the more mature cardiomyo- cyte marker αMHC. Analysis of hearts at embryonic day 14.5 (E14.5), postnatal day 1 (P1) and P21 revealed that the hearts marked by progenitor markers formed clonal clusters while those marked by αMHC showed a mosaic pattern of singletons (Fig. 2c). The use of constitutively active Cre lines, although very informative, nor- mally produces high levels of recombination, and one cannot exclude the possibility that the observed single-color cell clusters are the results of random recombination events. On the other hand, a tamoxifen-inducible Cre line permits spatiotemporal control of recombination events. By administering a limiting amount of tamoxifen, one can achieve a handful of labeled cells in an organ and follow their fate retrospectively. The authors next crossed Rainbow mice with mice harboring an inducible Cre under the control of a βactin, Nkx2.5, or αMHC promoter. This approach allowed for distinction between non-cardiomyocyte-derived clonal expansion (βactinCreER ; R26VT2/GK ), cardiac progenitor-derived clonal expansion (Nkx2.5CreER ; R26VT2/GK ), or mature cardiomyocyte (αMHCCreER ; R26VT2/GK ) clonal expansion. When a limiting amount of tamoxifen was administered at E9.5 or E12.5 to βactinCreER ; R26VT2/GK mice, postnatal heart analysis revealed clear clones of cardiomyocytes, fibroblasts, endothelial and vascular smooth muscle cells (Fig. 2d). Clonal analysis of Nkx2.5CreER ; R26VT2/GK mice labeled at E9.5 or E12.5, and analyzed postnatally, also revealed a similar pattern of clonal expansion with comparable clone size and vol- ume to those observed in βactinCreER ; R26VT2/GK mice. These findings supported the Fig. 2 (continued) Marking a cardiomyocyte clone for counting purposes. Cells are pseudo-col- ored red and WGA staining is used to mark cell boundaries (Scale bar, 100 μm) [22]. (c) Longitudinal sections from E14.5, P1 and P21 transgenic mice under the control of the progenitor markers Mesp1 (i-iii) and Nkx2.5 (iv-vi) or the adult CM marker αMHC (vii-ix). Fluorescent microscope images from hearts under the control of progenitor markers (ii, v) revealed the pres- ence of clear clonal clusters. Images from hearts under the control of αMHC (viii) revealed a mosaic pattern of singletons, with no definite clonal clusters (Scale bar (iii, vi, ix), 500 μm; all others 50 μm) [22]. (d) The utility of the βactinCreER ; R26VT2/GK model in marking other cell types in the heart. (i) shows a close up confocal image of a clone (blue) containing vascular smooth muscle cells in a P7 heart stained for smooth muscle Myosin Heavy Chain (smMHC). (ii) shows a confocal image of cardiomyocyte clones in a P15 Nkx2.5CreER ; R26VT2/GK heart stained for α-sarcomeric actinin (Scale bar (i), 50 μm; (ii), 100 μm) [22]. (e) Limited tamoxifen adminis- tration allows for rare recombination events. Section of a βactinCreER ; R26VT2/GK adult heart, in which recombination was induced at E9.5, shows the presence of sparse single-colored clones labeled with mOrange and mCherry (Scale bar, 500 μm) [22]. (f) Representative confocal images of sections from βactinCreER ; R26VT2/GK (i), Nkx2.5CreER ; R26VT2/GK (ii) and αMHCCreER ; R26VT2/GK (iii) neonatal mice that received LAD ligation at P0. Clonal analysis performed 21 days after MI reveals the presence of sparse single-labeled clones, suggesting that neonatal mice undergo cardio- myocyte regeneration in response to injury [22] K. Kolluri et al.
- 37. 25 proliferative capacity of progenitor cells to generate cardiomyocytes during early fetal development (Fig. 2e). In order to explore the proliferative capacity of cardiomyocytes during fetal development, recombination was induced in cardiomyocytes at different embryonic time points using αMHCCreER ; R26VT2/GK mice. When tamoxifen was administered at E12.5, postnatal analysis revealed mostly singleton cardiomyocytes with few small size clones. However, interrogation of αMHCCreER ; R26VT2/GK mice labeled at E9.5 revealed similar size cardiomyocyte clones compared to βactinCreER ; R26VT2/GK or Nkx2.5CreER ; R26VT2/GK mice. These data suggest that αMHC-expressing cardiomyo- cytes at E9.5 retain the ability to proliferate and that this capacity is significantly diminished by E12.5. They went on to perform single cell transcriptional analysis of αMHC-expressing cardiomyocytes at E9.5, E12.5 and P1. Their investigation demonstrated the exis- tence of a heterogeneous population of cardiomyocytes within the early stages of cardiac development and their transition into a mature, less proliferative, and homogenous population by the early postnatal period. Overall, the use of the Rainbow model revealed that clonal dominance of differentiating progenitors medi- ates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late fetal and early postnatal life. Such precise analyses at a single cell resolution would be challenging and prone to inaccurate interpretations if traditional lineage tracing experiments were utilized. It would be important to incorporate new technology in future, eg. DNA barcoding, for high-throughput analysis of a large number of individual cardiomyocytes and their progenies in developing hearts. Sereti et al. also used Rainbow to study clonal expansion in neonatal and adult cardiomyocytes in response to cardiac injury. Newborn (P0) αMHCCreER ; R26VT2/GK , βactinCreER ; R26VT2/GK and Nkx2.5CreER ; R26VT2/GK mice received tamoxifen followed by LAD ligation or Sham operation at P1. At 21 days after MI, hearts were analyzed and frequent clones of cardiomyocytes were observed in the infarct and border zone areas of αMHCCreER ; R26VT2/GK mice (Fig. 2f). Similar observations were made with βactinCreER ; R26VT2/GK and Nkx2.5CreER ; R26VT2/GK mice after injury. This suggested that regeneration of the heart after injury in neonates was largely due to cardiomyo- cyte proliferation, which was confirmed by recent fate mapping studies of cardio- myocytes and non-cardiomyocytes in neonates [24]. In adults, LAD ligation was performed at 8 weeks of age and analysis at 21 days post-MI revealed the presence of sparse single-labeled clones in the infarct and border zone areas. These observa- tions suggested that injury induces cardiomyocyte proliferation in the neonatal heart but not in the adult heart. Wang et al. used the Rainbow system to study the clonal expansion of smooth muscle cells (SMC) in atherosclerosis [25]. Myh11CreERT2 ; R26VT2/GK ; ApoE−/− mice were fed a high-fat Western diet to induce atherogenesis. The authors observed that during early atherogenesis, there was a distinct subpopulation of SMCs that de- differentiated and upregulated Sca1 (a stem cell marker). Sca1 staining was most intense within the core of the dominant clone and Sca1+ cells appeared to colocalize near the necrotic core of the atherosclerotic plaque. Single cell RNA sequencing Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
- 38. 26 analysis of this Sca1+ population revealed that these cells down-regulated SMC marker genes and upregulated genes relating to inflammation and the complement cascade (complement C3 was one of the most significantly up-regulated factors). These findings translated to human specimens as well. Histological analysis of post- mortem carotid artery specimens revealed localization of C3 expression to the necrotic core of human atherosclerotic plaque, similar to mice. Furthermore, this Sca1+ SMC signature was found in humans and was associated with coronary artery disease, with an enriched expression of inflammatory genes (including C3). Production of C3 by the Sca1+ SMC population may play a role in triggering further SMC proliferation and vascular inflammation, which could explain the rapid expan- sion of SMCs during early atherogenesis. Overall, Wang et. al’s study reveals the potential of the Rainbow system to identify rapidly proliferating and differentiating populations of cells to gain insight into the pathophysiology of complex diseases such as atherosclerosis. 3.3 Utility of Rainbow in Other Organ Systems Rinkevich et al. used an inducible lineage tracing mouse model (βactinCreER ; R26VT2/ GK ) to perform lineage tracing and clonal analysis of individual cells of mouse hind limb tissue during regeneration of the digit tip, cutaneous wound healing, and nor- mal maintenance [26]. They removed nerve supply and observed clonal expansion, revealing that cellular regeneration remains largely intact in the absence of nerve supply. In a study of the kidney, Rinkevich et al. used the Rainbow mouse model for clonal analysis and lineage tracing of cells that contribute to the development, main- tenance and regeneration of the kidney [27]. In all three processes, they found that cells generating distinct parts of the nephron (i.e. Proximal, Distal tubules or col- lecting duct) were fate-restricted and stayed within their lineage. Furthermore, they used an Axin2CreER ; R26VT2/GK3 mouse line to track Wnt pathway responsive cells (WRC). They showed that WRCs increased their proliferative capacity and that their clones were restricted to either a proximal tubule or collecting duct fate. Recent studies using dual recombinases and Confetti reporter (also random labeling by one of the three fluorescences) demonstrated that bronchioalveolar stem cells residing in the bronchioalveolar duct junction could clonally expand to form bronchial epithelial cells and/or alveolar type I and II cells during lung repair and regeneration [28, 29]. This demonstrates the utility of clonal analysis by rainbow/ confetti reporters for resolving the uni- or bi-differentiation potential of stem cells in tissue regeneration. Interestingly, Rainbow has also been used in cancer models. For example, Corey et al. lineage traced endothelial cells within the tumor microenvironment and con- cluded that clonal expansion within the microvasculature is crucial to an invasive melanoma phenotype [30]. Particularly, their studies showed that there is a dimin- ishing of founder clones to produce subclones, with tumor blood vessels upregulat- ing genes associated with angiogenesis and a downregulation of lymphocyte K. Kolluri et al.
- 39. 27 adhesion molecules. Thus, the Rainbow mouse model allowed lineage tracing of single cells and their progeny to conclude that clonal evolution within melanoma can induce changes within the microvasculature to confer cancer cells an advantage. 3.4 Limitations/Future Directions The ability of Rainbow to precisely track the fate of certain cells across multiple organ systems makes it a strong candidate for clonal analysis and lineage tracing within the heart. This model allows researchers to differentiate between proliferat- ing and quiescent cardiomyocytes, a phenomenon that remains controversial in the field of cardiac development and regeneration. However, it is not without limita- tions. A major consideration when using the Rainbow mouse model is the efficiency of recombination events that partially depends on the Cre line used. While in many mouse lines, Rainbow faithfully induces equal expression of fluorescent proteins in labeled cells, some Cre lines have been reported to show uneven expression of the markers. Additionally, the model’s dependence on tamoxifen could leave the pos- sibility that a smaller dose does not induce enough recombination to mark all pos- sible clones, thus underestimating the number of clones [22]. Furthermore, since in clonal expansion studies, only a small number of cells are labeled and their fate is monitored retrospectively, it is possible that rare populations of cells cannot be labeled by this strategy. The Rainbow system can be used for clonal analysis studies and the presence of clusters of single-colored cells confidently supports the exis- tence of proliferating cells. However, the absence of an observation does not con- firm its lack of existence. References 1. Kretzschmar, K., Watt, F.M.: Lineage tracing. Cell. 148(1-2), 33–45 (2012). https://doi. org/10.1016/j.cell.2012.01.002 2. Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabé-Heider, F., Walsh, S., et al.: Evidence for cardiomyocyte renewal in humans. Science. 324(5923), 98–102 (2009). https:// doi.org/10.1126/science.1164680 3. Bergmann, O., Zdunek, S., Felker, A., Salehpour, M., Alkass, K., Bernard, S., et al.: Dynamics of cell generation and turnover in the human heart. Cell. 161(7), 1566–1575 (2015). https:// doi.org/10.1016/j.cell.2015.05.026 4. Cai, C.L., Molkentin, J.D.: The elusive progenitor cell in cardiac regeneration: slip Slidin’ away. Circ. Res. 120(2), 400–406 (2017). https://doi.org/10.1161/CIRCRESAHA.116.309710 5. Eschenhagen, T., Bolli, R., Braun, T., Field, L.J., Fleischmann, B.K., Frisén, J., et al.: Cardiomyocyte regeneration: a consensus statement. Circulation. 136(7), 680–686 (2017). https://doi.org/10.1161/CIRCULATIONAHA.117.029343 6. Vagnozzi, R.J., Molkentin, J.D., Houser, S.R.: New myocyte formation in the adult heart: endogenous sources and therapeutic implications. Circ. Res. 123(2), 159–176 (2018). https:// doi.org/10.1161/CIRCRESAHA.118.311208 Lineage Tracing Models to Study Cardiomyocyte Generation During Cardiac…
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- 42. 31 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. Zhang, V. Serpooshan (eds.), Advanced Technologies in Cardiovascular Bioengineering, https://doi.org/10.1007/978-3-030-86140-7_3 Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis Daniel J. Garry and Javier E. Sierra-Pagan Abbreviations AGM Aorta-gonad-mesonephros Cas9 CRISPR-associated protein 9 CD31 Cluster of differentiation 31 CD41 Integrin alpha chain 2b CD44 Cluster of differentiation 44 CD45 Leukocyte common antigen Cdh5 Vascular endothelial cadherin CRISPR Clustered regularly interspaced short palindromic repeats ES/EB Embryonic stem cells/embryoid bodies ETV2 Ets variant transcription factor 2 FLK1 Fetal liver kinase 1 Gata4 GATA transcription factor 4 HE hematoendothelial hiPSC Human induced pluripotent stem cell D. J. Garry (*) Department of Medicine, University of Minnesota, Minneapolis, MN, USA Developmental Biology Center, University of Minnesota, Minneapolis, MN, USA Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, USA Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA e-mail: garry@umn.edu J. E. Sierra-Pagan Department of Medicine, University of Minnesota, Minneapolis, MN, USA Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, USA
- 43. 32 HSC Hematopoietic stem cell Mlc2v Myosin light chain 2v Myf5 Myogenic factor 5 MyHC Myosin heavy chain Myod Myoblast determination protein 1 Nkx2-5 Homeobox Protein Nkx2–5 OE Overexpression Pdx1 Pancreatic and duodenal homeobox 1 qPCR Quantitative polymerase chain reaction scRNA-seq Single cell RNA sequencing Tie2 Endothelial cell specific receptor tyrosine kinase 2 Ve-Cad Vascular endothelial cadherin 1 Cardiovascular Diseases Are Common and Have Considerable Morbidity and Mortality Peripheral artery disease affects more than 10 M Americans resulting in more than 150,000 limb amputations each year in the U.S. In addition, more than 300,000 patients have coronary artery bypass grafting (surgical revascularization) [1]. These diseases collectively are amplified by the rising incidence of diabetes, obesity and cardiovascular disease. These complications result in considerable morbidity and mortality [1, 2]. Current medical therapies for vascular diseases include limb ampu- tation and vascular bypass grafting. However, these therapeutic interventions have significant limitations. These diseases are chronic, debilitating, lethal and they war- rant new and novel therapies. The definition of the molecular mechanisms that gov- ern the endothelial lineage and vascular development will provide a platform to modulate these pathways and promote vasculogenesis as a therapeutic initiative. The overall goal for this chapter is to highlight the key regulators that govern endo- thelial and vascular development. 2 Master Regulators Govern Fate Decisions and Lineage Development Loss of function and gain of function genetic studies have defined essential factors that govern cell fate and lineage development (Fig. 1a). These factors also known as master regulators occupy or sit at the top of a regulatory hierarchy [3, 4]. Perhaps a prototypic example are members of the MYOD family of transcription factors. The MYOD family consists of bHLH transcription factors that have distinct and over- lapping functional roles for the regulation of the myogenic lineage [5]. These mas- ter regulators also have the capacity to convert another differentiated cell type (usually a fibroblast) to a specific lineage (i.e. skeletal muscle) using a D. J. Garry and J. Sierra-Pagan
- 44. 33 promoter- reporter construct that demonstrates expression with lineage specific dif- ferentiation (Fig. 1) [6–9]. These assays were initially referred to as conversion assays. Using conversion assays in combination with gene disruption and transgenic technologies, hundreds of master regulators have been described [3]. In addition to the MYOD family, PDX1 (pancreas), OCT4/SOX2/NANOG (pluripotency), SCL/ TAL1 (blood), HIF1 (hypoxia), and others have been identified (Fig. 1b) [6–8, 10– 12]. ETV2 is a recently identified master regulator that has been shown to be essen- tial for the specification and development of the endothelial lineage (Fig. 1b) [13, 14]. Fig. 1 Master regulators specify lineage-specific development. (a) Adaptation of Waddington’s landscape that outlines the role of master regulators to govern fate decisions during embryogene- sis. (b) Schematic outlining examples of master regulators for specific lineages. PDX1 is a master regulator for pancreatogenesis and ETV2 is master regulator for hematoendothelial lineages Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 45. 34 3 Developmental Milestones for Endothelial Development The initial developmental stage for vasculogenesis occurs as specified progeni- tors (i.e. angioblasts) that migrate from the primitive streak in the developing mouse embryo to the yolk sac [15]. Later, these angioblasts migrate from the extraembry- onic yolk sac to the embryo proper to form cord like structures, lumens (a process known as tubulogenesis) and ultimately form a vascular plexus (Fig. 2) [15]. This process continues and is associated with the onset of cardiac contractility in the E8.25 heart tube, the appearance of primitive blood cells associated with the primi- tive circulation (E8.5) and the establishment of a complete circulation with the propagation of blood throughout the E10-E10.25 mouse embryo [16]. This circula- tion is impacted by the growth of the embryo and the transition from diffusion to the circulation of blood in response to hypoxic signals (HIF1 and HIF2) and signaling pathways (VEGF1-FLK1/FLT1/FLT2, SHH-GLI1/2/3, SRC-CDH5, ANG1/2- TIE2, etc.) [16, 17]. This latter process is termed angiogenesis, which is character- ized by the formation of new vessels that originate or sprout from pre-established or Fig. 2 Overview of hematoendothelial development. (a) Schematic highlighting the role of angio- blasts being recruited to form a vascular plexus, tubulogenesis and sprouts to generate vascular networks and remodeling. (b) Schematic highlighting the role of hemogenic endothelium during primitive (yolk sac) and definitive (AGM) hematopoiesis D. J. Garry and J. Sierra-Pagan
- 46. 35 pre-existing vessels [17]. The vasculature continues to architecturally evolve and mature with the determination of venous and arterial endothelial fates. This matura- tion phase is marked by the expression of integrins, the balance of apoptosis and cell proliferation and the impact of the extracellular matrix [15]. Overall, the endothelial and vascular lineages are coordinated and responsive to microenvironmental cues and signals. The endothelial-endocardial relationship is established early during embryogen- esis. The endothelial lineage is characterized by a single cell thickness of develop- ing vascular networks that form a tight syncytium separating the luminal space with the underlying vascular wall. This endothelial lining is contiguous with the endocar- dium, which lines the four-chambered heart. The ontogeny of the endocardium is distinct from the endothelial lineage and is reflected in the expression of a lineage specific molecular program [18]. 4 The Common Origin of Endothelial and Hematopoietic Lineages The endothelial and hematopoietic lineages are both derivatives of the mesodermal germ layer [19]. As both lineages develop in close proximity (i.e., blood islands of the yolk sac and the blood containing vessels) and express overlapping molecular programs, studies support a common origin for the lineages [19]. The extraembry- onic yolk sac is the source for primitive hematopoiesis (Fig. 2b) [20]. Indeed, stud- ies have demonstrated that multilineage hematopoietic stem cells are derivatives of hemogenic endothelium. Hemogenic endothelium, despite its endothelial gene expression profile, loses its endothelial potential early in development and only gives rise to blood [21]. Hemogenic endothelium are flat shaped cells that undergo endothelial-to-hematopoietic transition (EHT and marked by CD44 expression) and acquire a spherical shape characteristic of blood cells [20, 22]. These hemogenic endothelial cells are found within the allantois, the yolk sac, the endocardium and the aorta-gonad-mesonephros (AGM) of the developing embryo [20, 23]. The AGM is closely associated with the ventral wall of the dorsal aorta (~E9.5-E11.5) and has been shown to produce hematopoietic stem cells that are capable of engrafting and reconstituting the irradiated bone marrow. Therefore, the AGM is recognized for its critical role in definitive hematopoiesis (Fig. 2b) [23]. Single cell RNA-seq of the AGM in the developing mouse have identified multiple cell types and defined the expression of GATA2, RUNX1, LYL1, ERG, FLI1, LMO2 and TAL1 transcription factors, which result in the loss of endothelial gene expression and acquisition of hematopoietic gene expression [22]. Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 47. 36 5 ETV2 Is Necessary and Sufficient for Endothelial Lineage Development ETV2 (Ets Variant Transcription Factor 2) was initially sequenced from the testis by Steve McKnight’s laboratory [24]. Later, ETV2 was co-discovered as an essential factor for hematoendothelial (HE) lineage development by the Choi laboratory and the Garry laboratory [25, 26]. These independent efforts resulted in studies by the Choi laboratory, which defined a BMP/NOTCH/WNT-ETV2 axis during mouse embryogenesis and demonstrated that Etv2 null embryos were lethal and lacked hematopoietic and vascular lineages [25]. Similarly, the Garry laboratory used a Nkx2-5-reporter transgenic strategy and identified Etv2 as a putative downstream target. Further, they demonstrated that Etv2 null embryos were nonviable at E9-E9.5 and had an absence of blood and endothelial lineages and defined that ETV2 was a direct upstream regulator of the Tie2 gene [26]. Studies further demonstrated the essential role for ETV2 in zebrafish, xenopus, pig and human [13, 14, 27–35]. These latter results support the evolutionary conserved role for ETV2 as an essential factor for hematoendothelial development. Furthermore, forced overexpression (OE) of ETV2 using mouse embryonic stem cells/embryoid bodies (ES/EB) differentiation assays demonstrated that it was sufficient for HE development (Fig. 3) [25, 35–37]. Collectively, these studies provided a foundation for ETV2 as a master regulator for the HE lineages. 6 ETV2 Expression during Mouse Embryogenesis and the Postnatal Period Using in situ hybridization, PCR, immunohistochemistry and the 3.9Kb Etv2-EYFP reporter transgenic expression, ETV2 expression was restricted to the angioblasts at E6.5, hematoendothelial cells and endocardium until ~E10-E10.5 after which it was rapidly extinguished with the exception of a small subpopulation of cells associated with the dorsal aorta (Fig. 4) [25, 26, 36–41]. Similarly, using the ES/EB differen- tiation assay, ETV2 was robustly expressed on Days 3–4 following differentiation after which it was extinguished (Fig. 3) [25, 42]. These expression studies support the notion that ETV2 functions as a “rheostat” to tightly regulate HE lineage devel- opment similar to other master regulators for the specification of other lineages. While the mechanisms that regulate extinguished expression of ETV2 are incom- pletely defined, the feedback mechanisms involving FLT1 contribute, in part, to the negative regulation of Etv2 gene expression [42]. Future studies will need to focus on the definition of these mechanisms to enhance our understanding of ETV2 expression. ETV2 is expressed postnatally in the testis and in the HSC population (Lin- Sca1+ cKit+ cells) in adult mouse bone marrow [44, 45]. ETV2 is induced and upreg- ulated following tissue and vascular injury without sustained long-term or persistent D. J. Garry and J. Sierra-Pagan
- 48. 37 expression [46]. Furthermore, recent studies support the notion that ETV2 is expressed in tumorigenic tissues and may be associated with the angiogenic response observed with various solid tumors (as outlined below) [47–49]. These expression patterns and its role as a master regulator suggest that ETV2 may be an important target to promote or repress angiogenesis depending on the physiological context. 7 ETV2 Is Dynamically and Transcriptionally Regulated by Upstream Factors Previous studies by our laboratory demonstrated that the 3.9 kb upstream fragment of the Etv2 gene harbored all the modules and motifs necessary for the spatial and temporal expression pattern of endogenous ETV2 (Fig. 5) [26, 31, 36, 41, 42, 50, 51]. These studies established that the EYFP reporter expression pattern recapitu- lated endogenous ETV2 activity demonstrating the onset of expression and Fig. 3 Embryoid body (EBs) differentiation assays recapitulate developmental mechanisms. (a) Schematic highlighting ES cells differentiating to EBs and forming mesodermal derivatives including: hematoendothelial, cardiac and skeletal muscle lineages. (b) FACS profile of dissoci- ated EBs stained for FLK1 and PDGFR-a demonstrate the HE (FLK1+ / PDGFR-a− ), cardiac (FLK1+ /PDGFR-a+ ) and skeletal muscle (FLK1− /PDGFR-a+ ) lineages. (c) Schematic of the expression profile of Etv2 during mesodermal EB differentiation showing its transient pattern of expression Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 49. 38 extinguished activity (Fig. 4) [41]. Bioinformatics analysis revealed evolutionary conservation of two modules (CRI and CRII) within the 3.9 kb fragment and the deletion of this fragment phenocopied the global deletion of Etv2 (Fig. 5b) [42]. Furthermore, the characterization of the Etv2 cis-regulatory modules in vitro revealed the importance of the CRI and CRII modules using luciferase assays [26, 42, 52]. Collectively, these studies confirmed that the upstream regulation of this gene utilized the binding motifs contained within the 3.9 kb fragment. Using gene disruption models, transcriptional assays, EMSAs and mutagenesis, transcriptional regulators of Etv2 gene expression included: ETV2, GATA2, VEFGF/FLK1- Calcineurin- NFAT, CREB1, MESP1, NKX2-5 and other signaling pathways (BMP, Notch and Wnt signaling pathways) (Fig. 5c) [25, 26, 39, 42, 52, 53]. While all Fig. 4 ETV2 fate mapping identifies ETV2 expressing lineages and its descendants in the heart. Using the 3.9 kb Etv2-Cre transgenic mouse model, the ETV2 contribution to embryogenesis was mapped during cardiogenesis in the developing mouse. E12.5 heart co-stained with NKX2–5 (red), DAPI (blue), and ETV2 cells/descendants (green) [43] demonstrate that every endothelial and endocardial cell is labeled with GFP including the developing valves (arrowhead) and aorta (open arrowhead) (a, atria and v, ventricle) D. J. Garry and J. Sierra-Pagan
- 50. 39 Fig. 5 The regulatory mechanisms that govern the Etv2 gene. (a) Schematic highlighting the ATG start site and seven exons associated with the Etv2 gene. (b) Schematic demonstrating the CRI and CRII modules that regulate Etv2 gene expression. Deletion of the CRI and CRII modules pheno- copies the Etv2 global gene KO with embryonic lethality and absence of blood and vasculature. (c) Upstream cis transcriptional regulators of the Etv2 gene and their binding motifs are outlined. (d) Schematic highlighting the ETV2 protein with its transcriptional activation domain (TAD) and DNA binding or Ets domain Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 51. 40 these factors were important regulators, it appeared that MESP1-CREB1 may initi- ate Etv2 gene expression in the mesodermal lineage. Gene activation may also be context dependent, as in zebrafish, the overexpression of Nkx2-5 functions to repress Etv2 gene activity whereas in the mouse it appears to function as a direct upstream regulator of Etv2 in the endocardium [54]. 8 Definition of Transcriptional Targets for ETV2 Mediate Distinct Developmental Events Master regulators have a number of functions that are mediated by their respective downstream targets. ETV2 binds a canonical GGAA/T Ets motif and transactivates the hematoendothelial molecular program including: Scl/Tal1, Lmo2, Tie2/Tek, VE-Cadherin/Cdh5, Pecam1/CD31, Gata1, Gata2, Flk1, Elk3, Fli1, Sox7, Cepd, and others (Fig. 6) [14, 26, 34, 37, 42, 43, 53, 55–61]. These targets were identified and confirmed using CHiP-seq, transcriptional assays, EMSA, Standard ChIP and other molecular techniques [14, 55, 62]. More recent studies have demonstrated that ETV2 promotes cell proliferation by regulating Yes1 gene expression, which inter- acts with the Hippo signaling pathway (Fig. 6) [63]. ETV2 also transcriptionally regulates Rhoj gene expression to modulate endothelial progenitor cell migration during embryogenesis (Fig. 6) [64]. Finally, ETV2 regulates microRNAs (i.e. miR130a), which govern fate determination by promoting endothelial differentia- tion but not hematopoietic differentiation (Fig. 6) [59, 60]. Context specific gene regulation is observed in the testis where ETV2 is a direct upstream regulator of Sox9 gene expression. In a positive feed-back loop, SOX9 binds to the Etv2 pro- moter and serves to transactivate its regulator thereby maintaining the sertoli cell phenotype [65]. ETV2-Chip-seq datasets are publicly available and are continuing to be mined to further define and explore additional targets and functional roles for this master regulator. 9 Protein-Protein Interacting Factors for ETV2 Are Important Coregulators The Etv2 gene harbors seven exons and encodes a protein that has carboxy terminal domain, a DNA binding domain (amino acids 316–336 which overlaps with the ETS domain that spans from 231 to 315 aa) and an amino terminal domain (amino acids 1–157) (Fig. 5d) [44]. Using an array of biochemical and molecular tech- niques (i.e. mass spectrometry, yeast two hybrid screening assay, FRET, etc.) inter- acting factors have been identified for ETV2. Early studies identified FOXC2 as an interacting factor for ETV2 and suggested that adjacent Ets-Fox binding motifs (Ets-Fox enhancer motif) in more than 20% of endothelial specific genes were D. J. Garry and J. Sierra-Pagan
- 52. 41 potent coactivators of gene expression [66]. Furthermore, GATA2 has been shown using multiple assays to interact with ETV2 and coactivate hematoendothelial gene expression [53]. Other factors (OVol11311) have also been shown to cooperate with ETV2 [67]. For example, forced overexpression of ETV2 and GATA2 in hiPSCs promoted a hematopoietic fate [68]. These interacting factors are important context dependent cofactors that function to amplify and modify the functional role of ETV2. 10 ETV2 Is a Master Regulator for Hematoendothelial Lineages Using Conversion Assays Previous studies have demonstrated that forced overexpression of ETV2 promotes a hematoendothelial cell fate (Fig. 7) [9, 36, 37]. Forced overexpression of ETV2 alone or in combination with other factors (i.e. GATA2, SCL, etc.) during murine EB differentiation promotes a hematoendothelial fate (FLK1+ cells) (Fig. 7) [14, 36, 37, 55, 68]. Furthermore, the delivery of ETV2 converted cell populations into Fig. 6 ETV2 functions as an upstream regulator of gene expression. Schematic which demon- strates direct downstream targets of ETV2 and their functional role during vasculogenesis and angiogenesis Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 53. 42 ischemic hindlimbs demonstrated that these converted cells were endothelial cells as they participated in the repair of ischemic hindlimb mouse models [9]. Additionally, the delivery of lentivirus overexpressing ETV2 promoted repair in response to ischemic injury and reduced the fibroproliferative response in mouse hearts [69]. Collectively, these preliminary studies provide an important platform for therapeutic initiatives focused on the overexpression of ETV2. 11 ETV2 Overexpression in Tumor Angiogenesis Previous studies have demonstrated that master regulators not only promote fate determination during embryogenesis but also are overexpressed in the context of cancer [70]. Solid tumors require an enhanced vascular supply for growth, cell pro- liferation and metastasis. Therefore, if ETV2 is at the top of the transcriptional hierarchy for vasculogenesis/angiogenesis, then ETV2 should be expressed at dis- tinct time-periods during tumorigenesis. Expression analysis demonstrated coex- pression of ETV2 and the histone dymethylase, Junonji domain containing 2A in Fig. 7 The Etv2 network regulates hematoendothelial lineage development. Upstream and down- stream regulators and ETV2 effectors specify HE lineage development. ETV2 also represses non- hematoendothelial lineages D. J. Garry and J. Sierra-Pagan
- 54. 43 neuroendocrine prostate tumors [49]. In a separate study, the knockdown of Etv2 using siRNA nanoparticles resulted in the inhibition of tumor angiogenesis and the inhibition of tumor growth [48]. Furthermore, using a zebrafish xenotransplantation model, ETV2 and FLI1b were shown to have redundant roles in promoting tumor angiogenesis [71–73]. Collectively, these initial studies support the conclusion that strategies focused on the inhibition of ETV2 may be effective therapies that target tumor growth and angiogenesis. 12 ETV2 Functions to Repress Nonhematoendothelial Fate Decisions Master regulators function to reprogram the fate of differentiated cells [3, 4]. They also have the capacity to repress other lineages and direct progenitor cell popula- tions down a specified pathway as outlined in the Waddington’s landscape (Fig. 1) [4, 74]. In the Etv2 global knockout mouse model, mesodermal progenitors that typically are destined for the hematoendothelial lineage are redirected to the cardio- myocyte lineage using genetic fate mapping techniques (Fig. 7) [26, 41, 75]. Similarly, the Etv2 null zebrafish endothelial progenitors are redirected to the skel- etal muscle lineage (Fig. 7) [29, 30]. These studies using a gene disruption strategy emphasize that ETV2 represses nonhematopoietic lineage formation during early stages of embryogenesis. 13 Summary The endothelial and vascular lineages require a complex network of gene expres- sion that governs fate determination. ETV2 is an important master regulator for the endothelial and vascular lineages. Definition of the ETV2 mediated molecular net- works provide a platform for future therapeutic interventions. Acknowledgements The authors acknowledge Cynthia Faraday for her assistance with the prep- aration of the figures. References 1. Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Benjamin, E.J., Berry, J.D., Borden, W.B., et al.: Executive summary: heart disease and stroke statistics–2012 update: a report from the American Heart Association. Circulation. 125(1), 188–197 (2012). https://doi.org/10.1161/ CIR.0b013e3182456d46 Mechanisms that Govern Endothelial Lineage Development and Vasculogenesis
- 55. Another Random Scribd Document with Unrelated Content
- 59. The Project Gutenberg eBook of An Unsentimental Journey through Cornwall
- 60. This ebook is for the use of anyone anywhere in the United States and most other parts of the world at no cost and with almost no restrictions whatsoever. You may copy it, give it away or re-use it under the terms of the Project Gutenberg License included with this ebook or online at www.gutenberg.org. If you are not located in the United States, you will have to check the laws of the country where you are located before using this eBook. Title: An Unsentimental Journey through Cornwall Author: Dinah Maria Mulock Craik Illustrator: Charles Napier Hemy Release date: January 1, 2014 [eBook #44557] Most recently updated: October 23, 2024 Language: English Credits: Produced by Chris Curnow and the Online Distributed Proofreading Team at http://www.pgdp.net (This file was produced from images generously made available by The Internet Archive) *** START OF THE PROJECT GUTENBERG EBOOK AN UNSENTIMENTAL JOURNEY THROUGH CORNWALL ***
- 62. ST. MICHAEL'S MOUNT. BY The Author of John Halifax, Gentleman
- 63. WITH ILLUSTRATIONS BY C. NAPIER HEMY London MACMILLAN AND CO. 1884 The Right of Translation and Reproduction is Reserved LONDON: R. Clay, Sons, and Taylor, BREAD STREET HILL, E.C.
- 64. CONTENTS PAGE Day the First 1 Day the Second 9 Day the Third 25 Day the Fourth 45 Day the Fifth 53 Day the Sixth 59 Day the Seventh 67 Day the Eighth 75 Day the Ninth 86 Day the Tenth 101 Day the Eleventh 110 Day the Twelfth 118 Day the Thirteenth 127 Days Fourteenth, Fifteenth, and Sixteenth 133
- 65. LIST OF ILLUSTRATIONS PAGE ST. MICHAEL'S MOUNT Frontispiece FALMOUTH, FROM FLUSHING 1 ST. MAWE'S CASTLE, FALMOUTH BAY 5 VIEW OF FLUSHING FROM THE GREEN BANK HOTEL, FALMOUTH 7 A FISHERMAN'S CELLAR NEAR THE LIZARD 11 THE CORNISH COAST: FROM YNYS HEAD TO BEAST POINT 15 THE LIZARD LIGHTS BY NIGHT 23 CORNISH FISH 24 POLTESCO 29 CADGWITH COVE 32 THE DEVIL'S FRYING PAN, NEAR CADGWITH 34 MULLION COVE, CORNWALL 38 A CRABBER'S HOLE, GERRAN'S BAY 41 STEAM SEINE BOATS GOING OUT 46 HAULING IN THE BOATS—EVENING 50 HAULING IN THE LINES 55 THE LIZARD LIGHTS BY DAY 60 THE FISHERMAN'S DAUGHTER—A CORNISH STUDY 63 KYNANCE COVE, CORNWALL 68 THE STEEPLE ROCK, KYNANCE COVE 71 THE LION ROCKS—A SEA IN WHICH NOTHING CAN LIVE 76 HAULING IN THE BOATS 79 ENYS DODNAN AND PARDENICK POINTS 83 JOHN CURGENVEN FISHING 87
- 66. THE ARMED KNIGHT AND THE LONG SHIP'S LIGHTHOUSE 94 CORNISH FISHERMAN 100 THE SEINE BOAT—A PERILOUS MOMENT 103 ST. IVES 108 THE LAND'S END AND THE LOGAN ROCK 114 SENNEN COVE, WAITING FOR THE BOATS 119 ON THE ROAD TO ST. NIGHTON'S KEEVE 124 TINTAGEL 128 CRESWICK'S MILL IN THE ROCKY VALLEY 135 BOSCASTLE 139 THE OLD POST-OFFICE, TREVENA 145 AN UNSENTIMENTAL JOURNEY THROUGH CORNWALL
- 68. DAY THE FIRST I believe in holidays. Not in a frantic rushing about from place to place, glancing at everything and observing nothing; flying from town to town, from hotel to hotel, eager to do and to see a country, in order that when they get home they may say they have done it, and seen it. Only to say;—as for any real vision of eye, heart, and brain, they might as well go through the world blindfold. It is not the things we see, but the mind we see them with, which makes the real interest of travelling. Eyes and No Eyes,—an old- fashioned story about two little children taking a walk; one seeing everything, and enjoying everything, and the other seeing nothing, and thinking the expedition the dullest imaginable. This simple tale, which the present generation has probably never read, contains the essence of all rational travelling. So when, as the old hen, (which I am sometimes called, from my habit of going about with a brood of chickens, my own or other people's) I planned a brief tour with two of them, one just entered upon her teens, the other in her twenties, I premised that it must be a tour after my own heart. In the first place, my children, you must obey orders implicitly. I shall collect opinions, and do my best to please everybody; but in travelling one only must decide, the others coincide. It will save them a world of trouble, and their 'conductor' also; who, if competent to be trusted at all, should be trusted absolutely. Secondly, take as little luggage as possible. No sensible people travel with their point-lace and diamonds. Two 'changes of raiment,' good, useful dresses, prudent boots, shawls, and waterproofs—these I shall insist upon, and nothing more. Nothing for show, as I shall take you to no place where you can show off. We will avoid all huge hotels, all fashionable towns; we will study life in its simplicity, and
- 69. make ourselves happy in our own humble, feminine way. Not 'roughing it' in any needless or reckless fashion—the 'old hen' is too old for that; yet doing everything with reasonable economy. Above all, rushing into no foolhardy exploits, and taking every precaution to keep well and strong, so as to enjoy the journey from beginning to end, and hinder no one else from enjoying it. There are four things which travellers ought never to lose: their luggage, their temper, their health, and their spirits. I will make you as happy as I possibly can, but you must also make me happy by following my rules: especially the one golden rule, Obey orders. So preached the old hen, with a vague fear that her chickens might turn out to be ducklings, which would be a little awkward in the region whither she proposed to take them. For if there is one place more risky than another for adventurous young people with a talent for perpetuating themselves down prejudices, as Mrs. Malaprop would say, it is that grandest, wildest, most dangerous coast, the coast of Cornwall. I had always wished to investigate Cornwall. This desire had existed ever since, at five years old, I made acquaintance with Jack the Giantkiller, and afterwards, at fifteen or so, fell in love with my life's one hero, King Arthur. Between these two illustrious Cornishmen,—equally mythical, practical folk would say—there exists more similarity than at first appears. The aim of both was to uphold right and to redress wrong. Patience, self-denial; tenderness to the weak and helpless, dauntless courage against the wicked and the strong: these, the essential elements of true manliness, characterise both the humble Jack and the kingly Arthur. And the qualities seem to have descended to more modern times. The well-known ballad:— And shall they scorn Tre, Pol, and Pen? And shall Trelawny die? There's twenty thousand Cornishmen Will know the reason why,
- 70. has a ring of the same tone, indicating the love of justice, the spirit of fidelity and bravery, as well as of that common sense which is at the root of all useful valour. I wanted to see if the same spirit lingered yet, as I had heard it did among Cornish folk, which, it was said, were a race by themselves, honest, simple, shrewd, and kind. Also, I wished to see the Cornish land, and especially the Land's End, which I had many a time beheld in fancy, for it was a favourite landscape-dream of my rather imaginative childhood, recurring again and again, till I could almost have painted it from memory. And as year after year every chance of seeing it in its reality seemed to melt away, the desire grew into an actual craving. After waiting patiently for nearly half a century, I said to myself, I will conquer Fate; I will go and see the Land's End. And it was there that, after making a circuit round the coast, I proposed finally to take my chickens. We concocted a plan, definite yet movable, as all travelling plans should be, clear in its dates, its outline, and intentions, but subject to modifications, according to the exigency of the times and circumstances. And with that prudent persistency, without which all travelling is a mere muddle, all discomfort, disappointment, and distaste—for on whatever terms you may be with your travelling companions when you start, you are quite sure either to love them or hate them when you get home—we succeeded in carrying it out. The 1st of September, 1881, and one of the loveliest of September days, was the day we started from Exeter, where we had agreed to meet and stay the night. There, the previous afternoon, we had whiled away an hour in the dim cathedral, and watched, not without anxiety, the flood of evening sunshine which poured through the great west window, lighting the tombs, old and new, from the Crusader, cross-legged and broken-nosed, to the white marble bas- relief which tells the story of a not less noble Knight of the Cross, Bishop Patteson. Then we wandered round the quaint old town, in
- 71. such a lovely twilight, such a starry night! But—will it be a fine day to-morrow? We could but live in hope: and hope did not deceive us. To start on a journey in sunshine feels like beginning life well. Clouds may come—are sure to come: I think no one past earliest youth goes forth into a strange region without a feeling akin to Saint Paul's not knowing what things may befall me there. But it is always best for each to keep to himself all the shadows, and give his companions the brightness, especially if they be young companions. And very bright were the eyes that watched the swift-moving landscape on either side of the railway: the estuary of Exe; Dawlish, with its various colouring of rock and cliff, and its pretty little sea- side houses, where family groups stood photographing themselves on our vision, as the train rushed unceremoniously between the beach and their parlour windows; then Plymouth and Saltash, where the magnificent bridge reminded us of the one over the Tay, which we had once crossed, not long before that Sunday night when, sitting in a quiet sick-room in Edinburgh, we heard the howl outside of the fearful blast which destroyed such a wonderful work of engineering art, and whirled so many human beings into eternity. But this Saltash bridge, spanning placidly a smiling country, how pretty and safe it looked! There was a general turning to carriage- windows, and then a courteous drawing back, that we, the strangers, should see it, which broke the ice with our fellow- travellers. To whom we soon began to talk, as is our conscientious custom when we see no tangible objection thereto, and gained, now, as many a time before, much pleasant as well as useful information. Every one evinced an eager politeness to show us the country, and an innocent anxiety that we should admire it; which we could honestly do. I shall long remember, as a dream of sunshiny beauty and peace, this journey between Plymouth and Falmouth, passing Liskeard, Lostwithiel, St. Austell, c. The green-wooded valleys, the rounded hills, on one of which we were shown the remains of the old castle of Ristormel, noted among the three castles of Cornwall; all this,
- 72. familiar to so many, was to us absolutely new, and we enjoyed it and the kindly interest that was taken in pointing it out to us, as happy- minded simple folk do always enjoy the sight of a new country. ST. MAWE'S CASTLE, FALMOUTH BAY. Our pleasure seemed to amuse an old gentleman who sat in the corner. He at last addressed us, with an unctuous west-country accent which suited well his comfortable stoutness. He might have fed all his life upon Dorset butter and Devonshire cream, to one of
- 73. which counties he certainly belonged. Not, I think, to the one we were now passing through, and admiring so heartily. So you're going to travel in Cornwall. Well, take care, they're sharp folk, the Cornish folk. They'll take you in if they can. (Then, he must be a Devon man. It is so easy to sit in judgment upon next-door neighbours.) I don't mean to say they'll actually cheat you, but they'll take you in, and they'll be careful that you don't take them in —no, not to the extent of a brass farthing. We explained, smiling, that we had not the slightest intention of taking anybody in, that we liked justice, and blamed no man, Cornishman or otherwise, for trying to do the best he could for himself, so that it was not to the injury of other people. Well, well, perhaps you're right. But they are sharp, for all that, especially in the towns. We replied that we meant to escape towns, whenever possible, and encamp in some quiet places, quite out of the world. Our friend opened his eyes, evidently thinking this a most singular taste. Well, if you really want a quiet place, I can tell you of one, almost as quiet as your grave. I ought to know, for I lived there sixteen years. (At any rate, it seemed to have agreed with him.) Gerrans is its name—a fishing village. You get there from Falmouth by boat. The fare is —(I regret to say my memory is not so accurate as his in the matter of pennies), and mind you don't pay one farthing more. Then you have to drive across country; the distance is—and the fare per mile— (Alas! again I have totally forgotten.) They'll be sure to ask you double the money, but never you mind! refuse to pay it, and they'll give in. You must always hold your own against extortion in Cornwall. I thanked him, with a slightly troubled mind. But I have always noticed that in travelling with such measure as ye mete it shall be meted to you again, and that those who come to a country expecting to be cheated generally are cheated. Having still a
- 74. lingering belief in human nature, and especially in Cornish nature, I determined to set down the old gentleman's well-meant advice for what it was worth, no more, and cease to perplex myself about it. For which resolve I have since been exceedingly thankful. He gave us, however, much supplementary advice which was rather useful, and parted from us in the friendliest fashion, with that air of bland complaisance natural to those who assume the character of adviser in general. Mind you go to Gerrans. They'll not take you in more than they do everywhere else, and you'll find it a healthy place, and a quiet place —as quiet, I say, as your grave. It will make you feel exactly as if you were dead and buried. That not being the prominent object of our tour in Cornwall, we thanked him again, but as soon as he left the carriage determined among ourselves to take no further steps about visiting Gerrans. VIEW OF FLUSHING FROM THE GREEN BANK HOTEL, FALMOUTH.
- 75. However, in spite of the urgency of another fellow-traveller—it is always good to hear everybody's advice, and follow your own—we carried our love of quietness so far that we eschewed the magnificent new Falmouth Hotel, with its table d'hôte, lawn tennis ground, sea baths and promenade, for the old-fashioned Green Bank, which though it had no green banks, boasted, we had been told, a pleasant little sea view and bay view, and was a resting-place full of comfort and homely peace. Which we found true, and would have liked to stay longer in its pleasant shelter, which almost conquered our horror of hotels; but we had now fairly weighed anchor and must sail on. You ought to go at once to the Lizard, said the friend who met us, and did everything for us at Falmouth—and the remembrance of whom, and of all that happened in our brief stay, will make the very name of the place sound sweet in our ears for ever. The Lizard is the real point for sightseers, almost better than the Land's End. Let us see if we can hear of lodgings. She made inquiries, and within half an hour we did hear of some most satisfactory ones. The very thing! We will telegraph at once— answer paid, said this good genius of practicality, as sitting in her carriage she herself wrote the telegram and despatched it. Telegrams to the Lizard! We were not then at the Ultima Thule of civilisation. Still, she said, you had better provide yourself with some food, such as groceries and hams. You can't always get what you want at the Lizard. So, having the very dimmest idea what the Lizard was—whether a town, a village, or a bare rock—when we had secured the desired lodgings (quite ideal lodgings, remarked our guardian angel), I proceeded to lay in a store of provisions, doing it as carefully as if fitting out a ship for the North Pole—and afterwards found out it was a work of supererogation entirely.
- 76. The next thing to secure was an ideal carriage, horse, and man, which our good genius also succeeded in providing. And now, our minds being at rest, we were able to write home a fixed address for a week, and assure our expectant and anxious friends that all was going well with us. Then, after a twilight wander round the quaint old town—so like a foreign town—and other keen enjoyments, which, as belonging to the sanctity of private life I here perforce omit, we laid us down to sleep, and slept in peace, having really achieved much; considering it was only the first day of our journey.
- 77. DAY THE SECOND Is there anything more delightful than to start on a smiling morning in a comfortable carriage, with all one's impedimenta (happily not much!) safely stowed away under one's eyes, with a good horse, over which one's feelings of humanity need not be always agonising, and a man to drive, whom one can trust to have as much sense as the brute, especially in the matter of refreshment. Our letters that morning had brought us a comico-tragic story of a family we knew, who, migrating with a lot of children and luggage, and requiring to catch a train thirteen miles off, had engaged a driver who refreshed himself so successfully at every public-house on the way, that he took five hours to accomplish the journey, and finally had to be left at the road-side, and the luggage transferred to another vehicle, which of course lost the train. We congratulated ourselves that no such disaster was likely to happen to us. Yes; I've been a teetotaller all my life, said our driver, a bright- looking, intelligent young fellow, whom, as he became rather a prominent adjunct to our life and decidedly to our comfort, I shall individualise by calling him Charles. I had good need to avoid drinking. My father drank through a small property. No fear of me, ma'am. So at once between him and us, or him and we, according to the Cornish habit of transposing pronouns, was established a feeling of fraternity, which, during the six days that we had to do with him, deepened into real regard. Never failing when wanted, never presuming when not wanted, straightforward, independent, yet full of that respectful kindliness which servants can always show and masters should always appreciate, giving us a chivalrous care, which, being unprotected females, was to us extremely valuable, I here record that much of the pleasure of our tour was owing to this
- 78. honest Cornishman, who served us, his horse, and his master—he was one of the employés of a livery-stable keeper—with equal fidelity. Certainly, numerous as were the parties he had driven—(I go to the Lizard about three times a week, he said)—Charles could seldom have driven a merrier trio than that which leisurely mounted the upland road from Falmouth, leading to the village of Constantine. Just turn and look behind you, ladies (we had begged to be shown everything and told everything); isn't that a pretty view? It certainly was. From the high ground we could see Falmouth with its sheltered bay and glittering sea beyond. Landward were the villages of Mabe and Constantine, with their great quarries of granite, and in the distance lay wide sweeps of undulating land, barren and treeless, but still beautiful—not with the rich pastoral beauty of our own Kent, yet having a charm of its own. And the air, so fresh and pure, yet soft and balmy, it felt to tender lungs like the difference between milk and cream. To breathe became a pleasure instead of a pain. I could quite understand how the semi-tropical plants that we had seen in a lovely garden below, grew and flourished, how the hydrangeas became huge bushes, and the eucalyptus an actual forest tree. But this was in the sheltered valley, and we had gained the hill-top, emerging out of one of those deep-cut lanes peculiar to Devon and Cornwall, and so pretty in themselves, a perfect garden of wild flowers and ferns, except that they completely shut out the view. This did not much afflict the practical minds of my two juniors. Half an hour before they had set up a shout— Stop the carriage! Do stop the carriage! Just look there! Did you ever see such big blackberries? and what a quantity! Let us get out; we'll gather them for to-morrow's pudding. Undoubtedly a dinner earned is the sweetest of all dinners. I remember once thinking that our cowslip tea (I should not like to drink it now) was better than our grandmother's best Bohea or
- 79. something out of her lovely old tea-caddy. So the carriage, lightened of all but myself, crawled leisurely up and waited on the hill-top for the busy blackberry-gatherers. While our horse stood cropping an extempore meal, I and his driver began to talk about him and other cognate topics, including the permanent one of the great advantage to both body and soul in being freed all one's life long from the necessity of getting something to drink stronger than water. Yes, he said, I find I can do as much upon tea or coffee as other men upon beer. I'm just as strong and as active, and can stand weather quite as well. It's a pretty hard life, winter and summer, driving all day, coming in soaked, sometimes in the middle of the night, having to turn in for an hour or two, and then turn out again. And you must look after your horse, of course, before you think of yourself. Still, I stand it well, and that without a drop of beer from years end to years end. I congratulated and sympathised; in return for which Charles entered heart and soul into the blackberry question, pointed out where the biggest blackberries hung, and looked indeed—he was still such a young fellow!—as if he would have liked to go blackberry- hunting himself. I put, smiling, the careless question, Have you any little folks of your own? Are you married? How cautious one should be over an idle word! All of a sudden the cheerful face clouded, the mouth began to quiver, with difficulty I saw he kept back the tears. It was a version in every-day life of Longfellow's most pathetic little poem, The Two Locks of Hair. My wife broke her heart after the baby, I think. It died. She went off in consumption. It's fifteen months now—(he had evidently counted them)—fifteen months since I have been alone. I didn't like to give up my home and my bits of things; still, when a man has to come in wet and tired to an empty house——
- 80. A FISHERMAN'S CELLAR NEAR THE LIZARD. He turned suddenly away and busied himself over his horse, for just that minute the two girls came running back, laughing heartily, and showing their baskets full of the very biggest blackberries you ever saw! I took them back into the carriage; the driver mounted his box, and drove on for some miles in total silence. As, when I had whispered that little episode to my two companions, so did we. There are two ways of going from Falmouth to the Lizard—the regular route through the town of Helstone, and another, a trifle longer, through the woods of Trelowarren, the seat of the old Cornish family of Vyvyan.
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