Thesis
Reference
Identifying new regulators of cardiovascular development
LINNERZ, Tanja
Abstract
This thesis provides the first in vivo characterization of dlc1, dlc3 and cndp2 during embryonic development in zebrafish. Dlc1 and dlc3 play a crucial role in vasculogenic, angiogenic and cardiogenic processes. Both genes impair the growth of intersegmental vessels and the common cardinal veins, indicating an important role for dlc's in endothelial cell migration during angiogenic sprouting. The observed phenotypes could be linked to dlc1's RhoGAP activity on RhoA and the VEGF-A/flk1/Nrp1 signaling axis. Moreover, dlc1 negatively affects the lumenization of the first aortic arch arteries, the lateral dorsal aortae and proper outflow tract development. Cndp2 is enriched in endothelial cells in the CHT and in macrophages.
Whereas overepression of cndp2 showed an impaired caudal vein plexus formation, loss of cndp2 impaired HSC survival in the endothelial niche and reduced HSC numbers in the thymus. This decrease in HSC survival was independent of nitric oxide or reactive oxygen species-dependent mechanisms and requires further investigation.
LINNERZ, Tanja. Identifying new regulators of cardiovascular development. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5215
DOI : 10.13097/archive-ouverte/unige:106444 URN : urn:nbn:ch:unige-1064448
Available at:
http://archive-ouverte.unige.ch/unige:106444
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITE DE GENEVE
Département de biologie cellulaire FACULTE DES SCIENCES Professeure Sandra Citi Département de pathologie et immunologie FACULTE DE MEDECINE
Professeur Julien Bertrand
Identifying new regulators of cardiovascular development
THESE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Tanja LINNERZ de
Worms, Allemagne
Thèse n° 5215
Centre d'impression de l'UNIGE 2018
UNIVERSITE DE GENEVE
Département de biologie cellulaire FACULTE DES SCIENCES Professeure Sandra Citi Département de pathologie et immunologie FACULTE DE MEDECINE
Professeur Julien Bertrand
Identifying new regulators of cardiovascular development
THESE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Tanja LINNERZ de
Worms, Allemagne
Thèse n° 5215
Centre d'impression de l'UNIGE 2018
4
1 Abstract ... 6
1 Résumé ... 8
2 Introduction ... 10
2.1 The importance of the cardiovascular system ... 10
2.2 The zebrafish to study vascular development ... 10
2.3 The development of the cardiovascular system ... 13
2.3.1 Lateral plate mesoderm ... 13
2.3.1.1 Anterior lateral plate mesoderm ... 14
2.3.1.2 The cardiac field... 15
2.3.1.3 Posterior lateral plate mesoderm ... 16
2.4 Heart development ... 17
2.5 Vasculogenesis ... 18
2.5.1 Cranial vasculogenesis ... 20
2.5.2 The formation of the aorta and the vein ... 23
2.6 Angiogenesis ... 24
2.6.1 Angiogenesis in the head and brain ... 25
2.6.2 Angiogenesis in the trunk/tail and lymphangiogenesis ... 26
2.6.1 Important signaling pathways during angiogenesis ... 28
2.7 Cell migration ... 33
2.7.1 Regulators of the cytoskeleton ... 34
2.7.2 Directed endothelial cell migration ... 36
2.8 The “Deleted in Liver Cancer” (DLC) family of proteins ... 37
2.8.1 DLC1 ... 38
2.8.2 DLC2 and DLC3 ... 40
2.9 Carnosine dipeptidases (cndp1 and cndp2) ... 40
2.10 Aims of the thesis ... 42
3 Materials and Methods... 43
3.1 Zebrafish husbandry and maintenance ... 43
3.2 Generation of full length mRNA ... 43
3.3 Whole mount in situ hybridization ... 43
3.4 Cryosections ... 44
3.5 Generation of transgenic lines (tol2) and mutant lines (CRISPR/Cas9) ... 44
3.6 Microinjection ... 44
3.7 Heatshocks and chemical inhibitor treatments ... 45
3.8 Acridine orange staining ... 45
3.9 Microangiography ... 45
3.10 Confocal microscopy ... 45
3.11 Cell sorting using FACS ... 46
3.12 Heart isolation... 46
3.13 Quantitative real-time PCR and analysis ... 46
3.14 HUVEC culture and immunofluorescence ... 46
4 Results ... 49
4.1 The role of the dlc family in zebrafish development ... 49
4.1.1 dlc1 and dlc3 are expressed in the LPM and in endothelial cells ... 49
4.1.2 Global overexpression of dlc1 and dlc3 is embryonically lethal and affects ... ISV growth... 52
4.1.3 Temporal overexpression of dlc1 induces local ISV misbranching ... 52
4.1.4 Temporal overexpression of dlc1 affects the growth of the common cardinal vein (CCV) ... 54
4.1.5 Endogenous hDLC1, hNRP1 and hPLXND1 are localized in close proximity ... in forming protrusions after VEGF-A and Sema3E stimulation ... 57
5
4.1.6 dlc1 acts downstream of VEGF-A/flk1/Nrp1 signaling ... 59
4.1.7 dlc1 activity is modulated by phosphorylation ... 61
4.1.8 CRISPR/Cas9-mediated knockout (KO) and Morpholino (MO) design ... 62
4.1.9 The dlc1 mutation is partially embryonic lethal and shows differential phenotypes ... 64
4.1.10 Loss of dlc1 affects the formation of the LDA and AA1s ... 64
4.1.11 Transient knockdown of dlc1 and dlc3 greatly impairs ISV growth ... 66
4.1.12 The severe phenotype in the dlc1 mutant as a model of congenital heart defects (CHDs) ... 68
4.2 The role of cndp2 in hemato-vascular development ... 71
4.2.1 Cndp2 is expressed by macrophages and in the vascular hematopoietic niche 71 4.2.2 Overexpression of cndp2 causes branching defects in the CVP ... 73
4.2.3 The cndp2 mutant has a defect in definitive hematopoiesis ... 75
4.2.4 The action of cndp2 on HSC expansion is independent of NO- and ROS- related pathways ... 77
5 Discussion ... 79
5.1 Cndp2 plays a role in hemato-vascular development ... 79
5.2 Dlc1 plays a crucial role during cardiovascular development... 80
6 References ... 88
7 Abbreviations ... 104
8 Acknowledgements ... 106
Abstract 6
1 Abstract
In my thesis, I provided the first characterization of dlc1, dlc3 and cndp2 during in vivo zebrafish embryonic development.
I was able to demonstrate that dlc1 and dlc3 play a crucial role in vasculogenic, angiogenic and cardiogenic processes. Both genes induced misbranching intersegmental vessels (ISV) in the trunk of the embryo in gain-of-function experiments and conversely stalling ISVs in MO- mediated knockdown experiments, indicating an important role for dlc’s in orchestrated endothelial cell migration during angiogenic sprouting. Moreover, the growth behavior of the forming common cardinal vein was also affected and showed an uneven leading edge of the endothelial cell sheet and alterations in the cellular integrity of the sheet, which was a result of RhoA inactivation due to dlc1’s enzymatic RhoGAP activity. I was able to show that dlc1 acts downstream of VEGF-A/flk1/Nrp1 signaling by rescueing arrested ISV growth with heat shock-induced dlc1 overexpression after chemical VEGFR inhibition. Moreover, I could show in vivo that dlc1’s activity is regulated by its phosphorylation status, which I could mediate using a PP2A inhibitor and rescue the gain-of-function phenotypes. In the course of this thesis, I generated stable dlc KO lines that provide the first in vivo animal model to investigate the impact of dlc KO on embryonic development, as the Dlc1 KO mouse dies before birth. I could demonstrate that dlc1 negatively affects the lumenization of the first aortic arch arteries, the formation of the lateral dorsal aortae and proper outflow tract development of the early zebrafish heart. Loss of dlc3 had only mild or transient impacts on embryonic development and analysis of the combinatorial loss of dlc1 and dlc3 suggested a certain degree of redundancy of these genes during cardiovascular development. However, only dlc1 mutants and dlc1/dlc3 double mutants displayed the defective OFT formation, which resulted in a blocked blood circulation and was embryonical lethal. In concordance with a study that described the loss of rare variants of dlc1 is associated to human congenital heart defects (CHD), the zebrafish dlc1 mutant recapitulates this phenotype and hence provides an in vivo model to study the etiology of genetic sporadic CHD.
The dipetidase cndp2 has not been studied in animal models so far, which prompted me to examine cndp2’s functional role during zebrafish development. I could show that cndp2 is enriched in endothelial cells (especially in the future endothelial hematopoietic stem cell (HSC) niche) and in macrophages. Overepression of cndp2 showed an impaired caudal vein plexus formation, which could be linked to cndp2’s involvement in L-histidine and histamine metabolism. Moreover, loss of cndp2 strikingly impaired HSC survival in the endothelial niche and reduced HSC numbers in the thymus, which was independent of nitric oxide (NO)-
Abstract 7
or reactive oxygen species (ROS)-dependent mechanisms. Future experiments will determine if a possible link to the L-histidine/histamine metabolism exists and how cndp2 can modulate HSC survival in the niche.
Résumé 8
1 Résumé
Pendant ma thèse, j’ai réalisé la première caractérisation in vivo des gènes dlc1, dlc3 et cndp2 au cours du développement embryonnaire du poisson-zèbre. J’ai démontré le rôle crucial de dlc1 et dlc3 au cours de la vasculogenèse, de l’angiogenèse et de la cardiogenèse. Ces deux gènes induisent, après gain de fonction, une mauvaise arborisation des vaisseaux inter segmentaires (ISVs), et à l’opposé, leur absence induit un arrêt de la croissance vasculaire, indiquant un rôle crucial de ces deux gènes dans la migration des cellules endothéliales. De plus, la formation de la veine commune cardinale est aussi influencée par dlc1, ce qui résulte de l’inactivation de RhoA qui est contrôlée par dlc1. J’ai pu montrer que dlc1 agit sous le contrôle de la voie VEGFA/flk1/nrp1 en compensant le défaut d’angiogenèse du a l’inhibiteur de la voie VEGF par une surexpression de dlc1. J’ai enfin pu montrer que l’activation de dlc1 dépend de son état de phosphorylation in vivo en utilisant des inhibiteurs de la phosphatase PP2A. Au cours de ma thèse, j’ai établi des lignées de poisson-zèbre mutantes pour le gène dlc1, établissant le premier model in vivo ou ce gène est invalide, puisque la mutation dlc1 est létale à des stades précoces d’embryogenèse chez la souris. Ce model m’a permis de démontrer que dlc1 est important pour la formation de plusieurs vaisseaux (artères aortiques, aortes dorsales latérales) ainsi que pour la structure qui donnera naissance à la crosse aortique adulte. La mutation dlc3, quant à elle, n’a qu’un effet très faible et transitoire sur le developpement cardio-vasculaire, bien que des études plus poussées m’ont permis de déterminer un certain degré de redondance entre les deux gènes. Le phénotype cardiaque est propre aux mutants dlc1, et concorde avec une étude de patients souffrant de maladie congénitale cardiaque qui a montré la présence de mutations dlc1 dans ces patients. En résumé, notre model pourrait être un excellent model pour les malformations cardiaques liées à dlc1.
En parallèle, j’ai aussi étudié le rôle du gène cndp2, pour lequel aucune étude n’a été menée dans aucun modèle animal. Cndp2 est un gène exprime par les macrophages mais aussi un gène endothélial, plus particulièrement exprime par les cellules endothéliales qui forment la niche hématopoïétique, nécessaire à l’expansion des cellules souches hématopoïétiques. Le gain de fonction montre une désorganisation du plexus caudal veineux, qui peut être relié au métabolisme de l’histidine et de l’histamine. De plus, le mutant cndp2 a un phénotype hématopoïétique important puisque toutes l’hématopoïèse est perdue au niveau des organes hématopoïétiques définitifs que sont le thymus et le tissu hématopoïétique caudal. Ce phénotype est indépendant du NO ou des ROS. Il faudra établir d’autres expériences pour
Résumé 9
totalement comprendre le rôle de cndp2 dans l’expansion des cellules souches hématopoïétiques.
Introduction 10
2 Introduction
2.1 The importance of the cardiovascular system
The establishment of a cardiovascular system is crucial for proper vertebrate embryonic development and survival through adulthood and consists of the heart, blood vessels and the lymphatic system. All vertebrates are dependent on a functional blood flow that is transporting oxygen, nutrients and waste products throughout the body, as well as circulating immune cells and signaling molecules. The cardiovascular system is one of the first organ systems to form during embryonic development 28: the first blood circulation loop appears in the zebrafish embryo by 24 hours post fertilization (hpf), in the mouse embryo between embryonic day (E)8 and E8.5 29 and in the human embryo between 10-13 weeks of development (uteroplacental circulation) 30, 31. The vascular system plays additional important roles in living organisms, as it represents the first source of hematopoietic stem cells (HSCs) that derive from the dorsal aorta 32-34 and it forms a major component of the embryonic and adult hematopoeitic niche, crucial for HSC expansion and survival 35-37. The vascular system is very plastic and dynamic, which allows (re)growth and remodeling also in the adult organism, which can be an advantage or disadvantage. Stimulating its regrowing capacity can aid in revascularizing engraftments after transplantations or undersupplied areas after ischemic events or strokes 3839, but this characteristic can also present a serious aggravement in the development of diseases, such as cancer and age-related macular degeneration 40. Thus, understanding the development of the (cardio)vascular system can be applied to many health- and disease-related issues.
2.2 The zebrafish to study vascular development
There are many models existing to study vascular development, in particular angiogenesis, ranging from in vitro models to ex vivo and in vivo animal models. In vitro models provide the opportunity to study certain aspects in great details, such as cell migration (scratch wound assays or Boyden chambers/Transfilter assays) 4142 43 and capillary/tube formation in 2D and 3D matrigel setups 44, 45. Major issues with the in vitro experimental setup consist in the fact that the results can be variable depending on which endothelial cell (EC) type is used, as well as the composition or amount of matrix being used. Primary ECs (e.g. Human umbilical vein endothelial cells (HUVECs)) usually lose their physiological properties while proliferating, as they are quiescent in natural conditions, which is why they can only be used for a certain number of passages 46. To overcome this problem, many immortalized EC lines exist, but the
Introduction 11
immortalization process can also change their angiogenic potential or gene expression/physiological profile 47. Despite the development of many different in vitro models
48, there is still a lack of a microenvironment and organ or organism context, which makes it difficult to study complex physiological interactions. Ex vivo studies usually consist of parts of organs, which contain ECs as well as surrounding cells or tissues. Those explants are then cultured in vitro in 3D matrices and angiogenic responses, such as microvessel outgrowth, are measured and quantified 49, 50. The most commonly used explants are rat and mice aortae or porcine carotid arteries to perform aortic ring assays. Usually several experiments can be performed with one animal and one aorta, limiting the amount of animals which need to be used. One big drawback of this method is the variation between different animals, which is a limiting factor in how many experiments can be performed using only one aorta from one animal (e.g. 30 rings can be obtained from one rat aorta) 51, which makes it more difficult to draw representative conclusions. Even though much information can be obtained using in vitro and ex vivo models, it is necessary to validate these findings in vivo, as e.g. new substances need to be tested and approved in whole organisms to allow a translation into clinics. Several in vivo models exist using different species, such as chick, rodents, xenopus and zebrafish. The chick embryo can be studied as whole mounts and is subsequently stained, which implies that most studies are endpoint experiments and provide snapshots of the vascular development. A more dynamic angiogenesis model is provided by the chick chorioallantoic membrane assay (CAM) 52, during which extraembryonic vascular growth can be studied with or without the addition of factors that can influence the formation of the vasculature. It is possible to perform the experiments in ovo, as well as to culture the chick embryos in petridishes 53, which facilitates the analysis. Overall, CAM assays are only short- term, and the fact that the CAM is already highly vascularized, makes it difficult to properly observe neovascularization. Many different rodent models exist to study angiogenesis, using mice, rats and rabbits. Mouse embryos are used to study vasculogenesis and angiogenesis, however as they develop in utero, surgical manipulation is often needed and complicates intravital imaging, although possible 54. Many studies also use endpoint experiments to perform staining techniques after removal of the embryo from the uterus and subsequent fixation. Another commonly used angiogenesis assay uses the mouse postnatal retina, as the retinal vasculature is still immature in rodents at birth 55. A combined use of transgenic animals with staining techniques allows a detailed study of many important physiological and pathological angiogenic processes 56, 57. Disadvantages comprise the fact that the eye is a specialized organ and not all findings can be generalized to other sites of vascular
Introduction 12
development within the body. Moreover, these experiments represent endpoint experiments, which do not allow a dynamic in vivo analysis/observation. Many other rodent models exist, which often use implantation of (matrigel) plugs, discs or sponges to investigate angiogenesis
58-60, usually in a tumor context. These models require invasive surgical manipulation of the animals and can also lead to inflammation, a factor known to influence angiogenesis, and therefore have to be carefully interpreted. The sites of implantation often prove to be non- physiological (subcutaneous vs. organ system) and can thus alter the outcome of the experiment. Besides the zebrafish, xenopus also provides a whole animal model organism that allows in vivo observation of vasculogenesis and angiogenesis. Xenopus share many advantages with the zebrafish embryo, although much fewer transgenic lines exist 61, 62, which limits the observation of the developing vascular system without invasive manipulation (such as injection of reporter dyes). Additionally, transgenic lines exist mostly for xenopus laevis that have a long generation time (1-2 years) and a pseudotetraploid genome. The zebrafish embryo is a great model organism to study cardiovascular development. A pair of adult zebrafish can easily generate 100-200 eggs per week and the embryos develop externally, which allows easy access to the growing animal throughout its rapid life cycle. As previously mentioned, a primitive cardiovascular system is established by 24 hpf and mostly completed after 3 days of development. Zebrafish embryos are entirely optically transparent until 2 days post fertilization (dpf) and subsequent pigment formation can be chemically inhibited until larval stages or piment-defective mutant lines can be used (casper, nacre, crystal), allowing for a non-invasive observation and imaging of the developing cardiovascular system 63-65. Due to the existence of numerous transgenic lines, every aspect of cardiovascular development can be easily studied, ranging from heart development to vasculogenesis (the de novo formation of blood vessels) 66-68, angiogenesis (sprouting vessels from pre-existing vessels) 69 and lymphatic development 70, 71. Zebrafish embryos are particularly suitable to study vascular development, because they can survive the first five days of their life through diffusion of oxygen and nutrients and are hence not dependent on a functional vascular system (cloche mutation: npas4l) 72, 73 and blood flow (silent heart mutation: tnnt2) 74. As zebrafish and mammals share high conservation in genes governing cardiovascular development, findings from zebrafish studies can easily be translated into mammalian and human research. Besides its transparency, the external fertilization and subsequent development provide additional advantages, as this renders the embryos accessible to mutagenesis approaches, such as full- length mRNA and morpholino injections, as well as small molecule applications.
Introduction 13
2.3 The development of the cardiovascular system
The cardiac system, as well as the vascular system and the limbs, originate from mesoderm (specifically the lateral plate mesoderm), which is one of the three germ layers forming after gastrulation 75, 76. The other two germ layers constitute the ectoderm and the endoderm. The ectoderm gives rise to the nervous system and skin, whereas the endoderm develops into the epithelial inner lining of multiple organs, such as the gastrointestinal and respiratory tract, as well as endocrine organs and the auditory system. Besides lateral plate mesoderm (LPM), additional distinct mesodermal sub layers exist, which give rise to other tissues and organs, such as the somites/muscles, bone and cartilage deriving from paraxial mesoderm and the urogenital system (kidneys and gonads) from intermediate mesoderm (see Fig.1A and B).
2.3.1 Lateral plate mesoderm
The LPM can be subdivided into discrete regions according to their location along the anterior-posterior axis in the embryo and their gene expression profile. As such, the LPM is further compartmentalized into the anterior lateral plate mesoderm (ALPM), the cardiac field and the posterior lateral plate mesoderm (PLPM) (see Fig. 2A). The sites of primitive hematopoiesis and vascular development are located in close proximity in the ALPM and the PLPM and share a common transcription factor signature before acquiring a definitive cell fate. Cells co-expressing scl/tal1, gata2, lmo2, fli1 and etsrp/etv2 (see Fig. 2B) have the potential to become either hematopoietic cells or angioblasts 77-80. The existence of a clonal
PM LPM
IM
A B
Fig. 1: Schematic representation of mesodermal layers in zebrafish embryos
A) shows a brightfield image of a 12 hpf embryo. To flatmount (B), the yolk is incised and removed. B) shows the schematic distribution of the different mesodermal layers in the latmounted embryo and C) in cross-section: IM intermediate mesoderm (dark rosé), LPM lateral plate mesoderm (red), PM paraxial mesoderm (light rosé), axial mesoderm (bright red);
neuroectoderm in blue and endoderm in green.
Introduction 14
bipotent precursor for the hematopoietic and vascular system, the “hemangioblast”, was already described very early by Murray and colleagues in chick embryos 81. First evidences supporting this theory came from in vitro experiments, where blast-colony-forming cells derived from either mouse embryonic stem cells (ESCs) 82, 83 or human ESCs 84 could give rise to both endothelium and blood. In vivo evidence of this precursor cell was shown so far in drosophila 85, xenopus 86, 87, zebrafish 88, 89, chicken 90 and mouse embryos 91. The latter three models demonstrated through fate mapping experiments that only a rare subset of cells have the potential to be true bipotent hemangioblasts and most blood and endothelial cells (ECs) derive from independent polyclonal progenitors 89, 90, 92, arguing against the hypothesis of a hemangioblast population.
2.3.1.1 Anterior lateral plate mesoderm
The ALPM harbors the rostral blood island (RBI), the first site of primitive myelopoiesis, and the origin of the cephalic vasculature. The first wave of primitive hematopoiesis in the RBI initiates through the expression of the myeloid-specific transcription factor pu.1 at the 5 somite stage (ss). The predominant cell type that is produced during primitive myelopoiesis are macrophages that start to migrate at 18 hpf from the head to the yolk ball and disperse in surrounding tissues or enter circulation at 25 hpf 93-95. Immature neutrophils emerge slightly later (33-35 hpf) and become functionally mature by 48 hpf 96. The angioblast population
lmo2 scl /gata1 etsrp/krox20 fli1/krox20
Introduc%on*:*LPM!
Trunk!vasculature!/!HSCs! drl$
flk1$
Primi've!erythropoiesis! drl$
gata1%
Primi've!myelopoiesis!
+!Head!vasculature!
drl$flk1$
pu.1%
Cardiac!field! drl$
nkx2.5%
A*
P! 12hpf!
Primitive myelopoiesis head vasculatureCardiac
field
Primitive erythropoiesis
Trunk vasculature
HSCs
12hpf
ALPM
PLPM
Fig. 2: Schematic representation of lateral plate mesoderm territories and example ISH for putative hemangioblast markers
A) schematic map of the main LPM territories at 12 hpf, showing the location of the anterior LPM, cardiac field and posterior LPM B) shows example images of genes expressed in angioblasts and hematopoietic progenitors, from left to right: lmo2; double in situ for scl (red) and gata1 (blue); double in situs for etsrp (blue) and krox20 (red) to mark rhombomeres and fli1 (blue)/krox20 (red) Adapted from Davidson and Zon, 2004; Simoes, 2011 12, 13
B A
Introduction 15
within the ALPM emerges from fli1+ and etsrp/etv2+ hemato-vascular progenitors 97, which are further committed towards the EC fate through the onset of expression of cadherin-5 (Cdh5)/vascular endothelial cadherin (VE-Cdh) and fetal-liver-kinase 1 (flk1)/vascular endothelial growth factor receptor 2 (VEGFR2) by etsrp/etv2 transcriptional activity at 5 ss 80,
98. The endothelial precursors proliferate and migrate medially to form first the primitive cords (see paragraph 2.3.3). The fate choice of the hemato-vascular cell population to become either myeloid or endothelial is hereby governed through a combination of etsrp and Bmp-signaling through alk8, which is subsequently followed by a downregulation of etspr in the myeloid lineage 99, 100.
2.3.1.2 The cardiac field
The cardiac field is located adjacent to the ALPM in the anterior part of the embryo (see Fig.
2A). The first cardiogenic transcription factors are induced by multiple signals, including BMP, Notch, FGF, Hh and WNT that activate cardiac specification and differentiation 101-104. The process of cardiogenesis and the cardiac transcription program are evolutionarily conserved in vertebrates 105, the specification of cardiac progenitors is hereby inititated with the expression of nkx2.5, GATA factors, hand2 and T-box proteins 106, 107. The embryonic linear heart tube consists of two cell types: cardiomyocytes that form the myocardium and ECs that line the inner side of the myocardium (endocardium) and connect the heart to the vascular system. The endocardial cells derive from the ALPM and start to migrate at 16 hpf, prior to the onset of cardiomyocyte migration 26, 108. They form a sheet medial to the myocardial cells and migrate posteriorly until they fuse with the cardiomyocytes into a bilayered disc at 18 hpf (Fig. 3A, D and E). The endocardium stays connected to the lateral dorsal aortae 20 through the first aortic arches (AA1s) throughout cardiogenesis (Fig. 3B). Cardiomyocyte differentiation and migration occur in two waves from distinct regions within the cardiac field. At 15 hpf, all cardiomyocytes start to express cardiac contractile genes, such as myl7 and can be further subdivided into lateral atrial cardiomyocytes (myh6) and medial ventricular cardiomyocytes (myh7) prior to migration and heart tube formation 27, 106 (Fig. 3C and E). In the first wave, cardiac progenitors from the first heart field (FHF) migrate at 17 hpf to the midline, fuse to form the myocardial layer and connect with the endocardial cells (18 hpf) and subsequently establish the linear heart tube at 22 hpf 72, 109 (Fig. 3B). To ensure growth and elongation of the heart tube, progenitors located in the medially second heart field (SHF) proliferate and differentiate into myocardial and smooth muscle cells. The SHF-derived progenitors migrate in a second wave and contribute to the ventricle and outflow tract (OFT) 110-114. FHF and SHF
