As an animal embryo develops, it establishes a circulatory system that includes the heart, vessels and blood. Vessels and blood initially form in the yolk sac, a membrane that surrounds the embryo. These yolk sac vessels act as a rudimentary circulatory system, connecting to the heart and blood vessels within the embryo itself. In older embryos, cells in the inner layer of the largest blood vessel (known as the dorsal aorta) generate blood stem cells that give rise to the different types of blood cells.

A gene called Nkx2.5 encodes a protein that controls the activity of a number of complex genetic programs and has been long studied as a key player in the development of the heart. Nkx2.5 is essential for forming normal heart muscle cells and for shaping the primitive heart and its surrounding vessels into a working organ. Interfering with the normal activity of the Nkx2.5 gene results in severe defects in blood vessels and the heart. However, many details are missing on the role played by Nkx2.5 in specifying the different cellular components of the circulatory system and heart.

Zamir et al. genetically engineered chick and mouse embryos to produce fluorescent markers that could be used to trace the cells that become part of blood vessels and heart. The experiments found that some of the cells that form the blood and vessels in the yolk sac originate from within the membranes surrounding the embryo, outside of the areas previously reported to give rise to the heart. The Nkx2.5 gene is active in these cells for only a short period of time as they migrate toward the heart and dorsal aorta, where they give rise to blood stem cells

These findings suggest that Nkx2.5 plays an important role in triggering developmental processes that eventually give rise to blood vessels and blood cells. The next step following on from this work will be to find out what genes the protein encoded by Nkx2.5 regulates to drive these processes. Mapping the genes that control the early origins of blood and blood-forming vessels will help biologists understand this complex and vital tissue system, and develop new treatments for patients with conditions that affect their circulatory system. In the future, this knowledge may also help to engineer synthetic blood and blood products for use in trauma and genetic diseases.

Development of the cardiovascular system takes place during the early stages of embryogenesis. Cardiac progenitors residing in the cardiac crescent are formed from the first heart field (FHF) located in the anterior lateral plate mesoderm (LPM). As the embryo develops, FHF progenitors fuse at the midline to form the primitive heart tube, which begins to beat and, as a consequence, blood begins to circulate (DeRuiter et al., 1992; Stalsberg and DeHaan, 1969). Second heart field (SHF) progenitors residing within the pharyngeal mesoderm (Diogo et al., 2015) contribute to subsequent growth and elongation of the heart tube (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). In both chick and mouse embryos, the FHF gives rise to myocytes of the left ventricle and parts of the atria, whereas the SHF contributes to myocardium of the outflow tract, right ventricle, and atria (Buckingham et al., 2005). Recent studies suggest that these heart fields contain both unipotent and multipotent mesodermal progenitors that give rise to the diverse lineage types within the heart (Kattman et al., 2006; Lescroart et al., 2014; Meilhac et al., 2004; Moretti et al., 2006; Wu et al., 2006). For example, bipotent SHF progenitors generate endocardium or smooth muscle cells as well as cardiomyocytes (Lescroart et al., 2014; Moretti et al., 2006). Cardiovascular progenitors sequentially express the cardiac transcription factors Mesp1/2, Islet1 (Isl1) and Nkx2.5, and, in response to cues from the microenvironment, undergo lineage diversification and differentiation (Laugwitz et al., 2008; Prall et al., 2007; Saga et al., 1999).

The formation of blood vessels begins with the appearance of blood islands in the extraembryonic region. In the chick embryo, this occurs in the area vasculosa around St. 3–5. Sabin first proposed that some blood cells differentiate directly from endothelial cells (Sabin, 1920). Indeed, endothelial and blood cells that form the rudimentary circulatory system have long been thought to originate from bipotent mesoderm progenitors termed ‘hemangioblasts’ (Choi et al., 1998). Recent experimental advances revealed the existence of a specialized cell, hemogenic endothelium, that harbours the potential to generate hematopoietic progenitors (Boisset et al., 2010; Jaffredo et al., 1998). These cells arise early in embryonic development and migrate from extra- to intra-embryonic locations (Tanaka et al., 2014). However, while blood cell formation from hemogenic endothelium has been visualised directly in multiple animal models (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010; Lam et al., 2010), and in vitro during ES cells differentiation (Eilken et al., 2009; Lancrin et al., 2009), the existence in vivo of a bipotential hemangioblast that contributes to both blood and endothelial cells remains controversial (Hirschi, 2012). Recent lineage tracing and live imaging studies addressing the ontogenic origins of hemoangiogenic cells in mouse embryos suggest that all hemogenic cells derive from a Flk1⁺/Runx1⁺/Gata1^- ‘hemogenic angioblast’ population located in extraembryonic mesoderm bordering the region forming blood islands (Tanaka et al., 2014). As for angioblasts that contribute to the embryonic vasculature, these cells actively migrate to embryonic sites of hemopoiesis before the establishment of circulation. Runx1 activation in the aorta-gonad-mesonephros (AGM) region is essential at this early stage for the specification of hemogenic endothelial cell fate and for definitive hemopoiesis (Tanaka et al., 2012).

The origins of the myocardium have been studied intensively, although that of the endocardium remains largely obscure (Harris and Black, 2010). Early lineage tracing studies in chick have shown that myocardial and endocardial progenitors segregate at primitive streak stages (Wei and Mikawa, 2000). In line with this view, we previously demonstrated that the endocardium originates, in part, from committed vascular endothelial progenitors that segregate medially from myocardial progenitors within the cardiac crescent (Milgrom-Hoffman et al., 2011). Numerous studies have demonstrated the close ontological relationship between the blood and vascular lineages, and the myocardium and endocardium of the forming heart. Heart progenitor cells in the embryo, or those derived from differentiating embryonic stem cells, co-express markers of blood (Tal1, Gata1) and endothelial (Flk1) lineages; conversely, vascular progenitors in the yolk sac express at some point in their lineage histories multiple cardiac transcription factors including Nkx2.5, Isl1 and Tbx20 (Keenan et al., 2012; Stanley et al., 2002; Stennard et al., 2005). Studies in both fish and mouse show that the blood and heart-forming territories develop in close proximity, and are mutually antagonistic (Bussmann et al., 2007; Schoenebeck et al., 2007; Van Handel et al., 2012). These territories appear to represent distinct spatio-temporal domains of the nascent mesoderm regulated in common by the transcription factor Mesp1 (Chan et al., 2013).

Nkx2.5 has long been known as a key transcription factor for cardiac development, specifically in the specification and differentiation of the myocardial lineage (Evans et al., 2010; Lyons et al., 1995; Turbay et al., 1996). However, the mis-patterning of the heart and arrest of cardiac development in Nkx2-5 null mutant mouse embryos has precluded a deep analysis of the role of this transcription factor in other mesodermal lineages. Cre recombinase-based lineage tracing with Nkx2-5^irescre driver mice revealed a contribution of labelled cells to endothelial and blood cells in the yolk sac blood islands (Stanley et al., 2002), consistent with severe defects reported in remodelling of the yolk sac vasculature in Nkx2-5 null embryos (Tanaka et al., 1999). However, it was not determined whether this was a primary affect of loss of Nkx2-5 in yolk sac, or secondary to hemodynamic effects arising from arrested heart development.

Recent studies have begun to shed new light on the role of Nkx2-5 in hemoangiogenic lineage specification. Nkx2-5 was found to directly bind a cis-regulatory element of the Etv2 gene, encoding an ETS domain family transcription factor essential for formation of the embryonic and extra-embryonic endothelium, endocardium and blood lineages, and Nkx2-5 induces its expression in vitro (Ferdous et al., 2009). In zebrafish, clusters of nkx2.5⁺ cells segregate from the main nkx2.5⁺ myogenic fields prior to heart formation, giving rise to the pharyngeal arch arteries (Paffett-Lugassy et al., 2013). Here, nkx2.5 is essential for endothelial cell fate specification and expression of etsrp/etv2 and scl/tal1. Consistent with these results, genetic lineage tracing in the mouse using the Nkx2-5^irescre driver showed a contribution of Nkx2-5⁺ cells to the pharyngeal arch arteries and aortic sac, and analysis of Nkx2-5 null embryos demonstrated that Nkx2-5 is required for pharyngeal arch artery formation (Paffett-Lugassy et al., 2013). Nkx2-5 and Isl1 are expressed in and required for a distinct subset of endocardial cells defined as the hemogenic endocardium, proposed to contribute to primitive and transient definitive hematopoiesis in the embryo (Nakano et al., 2013). Finally, expression of the human NKX2-5 gene occurs in a subset of paediatric T and B cell acute lymphoblastic leukaemias defined by a t(5;14) translocation (Nagel et al., 2003; Su et al., 2008).

Despite this knowledge, the origins of hemogenic endothelium and endocardium, and the role of Nkx2.5 in their lineage networks in vertebrates, are poorly understood. In this study, we used chick and mouse embryonic models to investigate the origins and plasticity of early cardiovascular progenitors. Using defined early Nkx2-5 and Isl1 embryonic enhancers driving expression of GFP and RFP as surrogate lineage tracing tools in the chick, we revealed the origin of the cardiac hemogenic endocardium to be hemogenic angioblasts in the extraembryonic/lateral plate mesoderm. These mesodermal cells express Nkx2.5 already in the primitive streak before migrating to the posterior LPM and extraembryonic regions, and, subsequently, into the endocardium and DA, where they form hemogenic endothelial clusters. We further demonstrated in chick, through gain-of-function experiments, that Nkx2.5 induces the expression of hemoangiogenic markers in nascent mesoderm. In mouse, Nkx2-5^irescre lineage tracing demonstrated activation of Nkx2-5 in extraembryonic tissue proximal to the cardiac crescent, and Nkx2-5⁺ cells make a substantial contribution to Runx1⁺ and CD41⁺ hemogenic endothelium in the DA and to a lesser extent endocardium. Our data provide the first in vivo evidence that Nkx2.5 has a role in initiating the hemoangiogenic program and that Nkx2.5⁺ progenitors contribute via the LPM/extraembryonic region to hemogenic endothelium of the endocardium and AGM region, and subsequently blood. Furthermore, we identify a novel population of endocardial progenitors outside the classical FHF and SHF domains.

The present study provides novel insights into early cardiogenesis and hemangiogenesis in vertebrate embryos. First, we revealed an early contribution of mesodermal cells residing outside the classical cardiac crescent region, to the developing heart (Figure 10). Cellular and molecular analyses indicate that a subset of extra-embryonic/LPM-derived hemogenic angioblasts travels via the inflow tract to populate the heart endocardium. Moreover, our results in both chick and mouse models demonstrate that the hemogenic endothelium in the DA and endocardium derives, at least in part, from Nkx2.5 lineage⁺ angioblast progenitors that may also contribute to hematopoietic stem cells (HSCs) in the fetal liver (Figure 10). Based on these studies and gain of function experiments, we propose a broader role for Nkx2.5 in the cardiovascular and blood lineage diversification programs than currently appreciated.

Figure 10

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The cardiovascular and hematopoietic lineage specification programs begin at gastrulation, when nascent mesodermal cells ingress through the primitive streak. In the chick, we demonstrated that Nkx2.5 is transiently expressed in the mid-primitive streak and the posterior LPM/extra-embryonic tissues at St. 4–6. Moreover, overexpression of Nkx2.5 induced endothelial/hematopoietic gene expression. In the mouse, Nkx2-5 activation in extra-embryonic tissue occurred later in gastrulation than in the chick, at the time of cardiac crescent formation, but positive cells nonetheless contributed extensively to the yolk sac vasculature and blood. Expression was also seen in hemogenic endothelium of the AGM, although we have not yet shown definitively in mouse whether positive cells migrate to the AGM from extra-embryonic tissue or whether Nkx2-5 is expressed de novo in hemogenic angioblasts arising in embryonic LPM. Confirming findings of Nakano and colleagues (Nakano et al., 2013), we found Nkx2-5 lineage⁺ blood cells in the yolk sac and embryonic vessels. Nakano et al. found that Nkx2-5 lineage⁺ blood cells expressed haemoglobins typical of primitive then definitive erythropoiesis, although no positive blood cells were present in adults. These results resonate with those of Yoder et al., who demonstrated that the yolk sac contains HSCs capable of sustaining long-term multi-lineage hemopoiesis when transferred to conditioned hemopoietic organs of neonates although not adults (Yoder et al., 1997). Taken together our findings suggest that Nkx2.5 plays a conserved role in the establishment of the cardiovascular and early blood cell lineages, in apparent agreement with the severe defects observed in the yolk sac vasculature and hematopoiesis seen in Nkx2-5 mutant mouse embryos (Tanaka et al., 1999).

The electroporation technique in early chick embryos enabled us to follow nascent mesoderm progenitors with hemogenic angioblastic characteristics. What makes these cells hemangioblast-like? First, these extra-embryonic/LPM mesoderm progenitors were labelled by both Nkx2-5-en and hemangioblast enhancers. Morphologically, we observed the expression of these two enhancers within blood islands, and could track their migration into the DA (and other vessels), as well as to the endocardium. These cells broadly expressed hematopoietic and endothelial lineage markers such as Runx1, Flk1, Tal1, Ets1, Gata1, Lmo2 and Gata4. When cultured in vitro, these cells differentiated into endothelial and blood progenitors, although we have not yet shown a bipotent differentiation potential in a clonal assay. Finally, we showed that in vivo, these cells contributed to the hemogenic endothelium in the DA and heart endocardium.

Tight molecular and cellular relationships exist between myocardial, endocardial, endothelial, and hematopoietic lineages in different organisms. A number of transcription factors control these cell fate choices, primarily through cross-inhibition mechanisms (Caprioli et al., 2011; Schoenebeck et al., 2007; Van Handel et al., 2012). Heart, vessels and blood likely represent an ancient lineage triad controlled by Nkx2-5 that has contributed to cardiovascular and hemogenic developmental plasticity in vertebrates. This plasticity is highlighted by activation of the hematopoietic program within hemogenic endothelium at various embryonic sites, including the yolk sac (Palis et al., 1999), DA (Jaffredo et al., 1998), extra-embryonic arteries (Gordon-Keylock et al., 2013), and endocardium (Nakano et al., 2013).

The mouse enhancer for Nkx2-5 labels a broad cardiovascular mesoderm population in the chick, including FHF cardiac progenitors, endothelial and hematopoietic progenitors. Because of the demonstrated early expression of Nkx2.5 protein and mRNA in the nascent mesoderm at St. 4–6, we suggest that the reporter expression provides a valid readout of the transient expression of Nkx2.5 in these cells, which is later downregulated in extra-cardiac lineages by endothelial and hematopoietic transcription factors. In the mouse, we showed that Nkx2-5 lineage⁺ cells arise in extra-embryonic tissues later in gastrulation associated with cardiac crescent formation. Linage⁺ cells also contributed substantially to the hemogenic endothelium within the AGM, liver, and, to a lesser degree, the endocardium. The extra-cardiac Nkx2-5 lineage⁺ cells that contribute to mouse hemopoiesis in the embryo from E8.5-E15.5 (Nakano et al., 2013), likely derive from the yolk sac or AGM region via the liver, rather than the endocardium as proposed (Nakano et al., 2013). However, whether there is a contribution of Nkx2-5 lineage⁺ cells to primitive hemopoiesis in the mouse yolk sac, and how Nkx2-5 lineage⁺ cell arrive in the AGM region, remain to be explored. Moreover, how Nkx2-5 lineage⁺ cells relate to the Runx1⁺ Gata1^- Etv2⁺ VE-cadherin⁺ hemogenic angioblasts which are already present in the yolk sac at E7.5 (Eliades et al., 2016; Lie-A-Ling et al., 2014; Tanaka et al., 2014), needs detailed analysis. Notwithstanding the differences in the timing of activation of Nkx2-5 in extraembryonic tissue in chick and mouse, our findings on the Nkx2-5 lineage in these two models seem highly concordant.

Bioinformatics and in vitro analyses demonstrated that several Gata and Ets sites are present and active within the Nkx2-5 enhancer (data not shown and Lien et al., 1999). Our ISH and RT-PCR analysis revealed the expression of Gata4 in both LPM and cardiac crescent (and also nascent mesoderm) corresponding to the expression of the Nkx2-5-en. Hence, Gata factors could drive the early expression of Nkx2.5. Scl/Tal1 was previously shown to be a key regulator of hemangioblasts (Bussmann et al., 2007; Schoenebeck et al., 2007; Van Handel et al., 2012). Our findings demonstrating the ectopic activation of Tal1 by Nkx2.5 suggest that the latter acts upstream of Tal1 in the hemangioblast hierarchy and may have a role in priming hemopoiesis. Activation of Tal1 in the chick inhibited cardiac gene expression, in line with the repressive role of Scl/Tal1 on cardiogenesis, as demonstrated in Scl/Tal1 mutant mouse embryos in which ectopic cardiomyogenesis was observed in the yolk sac vasculature and endocardium (Van Handel et al., 2012). Similar to Scl/Tal1, loss of etsrp/etv2 induced cardiac gene expression in endocardial progenitors (Palencia-Desai et al., 2011; Rasmussen et al., 2011). Taken together, these studies and our data highlight the dynamic developmental interplay between embryonic mesoderm progenitors for cardiac, endothelial and blood lineages.

Endocardial cells are known to be heterogeneous in origins (Harris and Black, 2010; Milgrom-Hoffman et al., 2011; Vincent and Buckingham, 2010). In vitro studies in chick and mouse provided evidence for bipotential progenitors for myocardial and endocardial cells that exist in the SHF (Hutson et al., 2010; Kattman et al., 2006; Lescroart et al., 2014; Moretti et al., 2006), but probably not in the FHF in which myocardial progenitors are unipotent (Lescroart et al., 2014; Später et al., 2013). Our findings are consistent with the idea that endocardial and myocardial lineage separation occurs very early during embryogenesis, in line with other studies (Li et al., 2015; Milgrom-Hoffman et al., 2011; Paffett-Lugassy et al., 2013).

Understanding the origin and regulation of definitive HSCs is a subject of great interest and contention. It is clear that HSCs are mesoderm-derived and their genesis involves differentiation from endothelial cells. In the chick, zebrafish and mouse embryos, the splanchnic mesoderm has been considered to be the source of hemogenic endothelium-derived HSCs (Childs et al., 2002; Lancrin et al., 2009; Zovein et al., 2010). More recent data suggest that hemogenic endothelium at embryonic sites have their origins in the yolk sac (Tanaka et al., 2014). It is likely that multiple sources of endothelial cells contribute to the formation and establishment of HSCs including extra-embryonic hemogenic angioblasts (Tanaka et al., 2014) and intra-embryonic angioblasts that are induced to form HSCs by local signalling (Nguyen et al., 2014; Richard et al., 2013). Consistent with these findings we suggest that part of the hemogenic endothelium in both DA and endocardium derives from extra-embryonic/LPM hemogenic angioblast progenitors previously expressing Nkx2.5. The detection of Nkx2.5 lineage⁺ hematopoietic progenitors in DA, liver and endocardium, suggests that these progenitors derive from a common embryonic origin, although this remains to be formally tested. Potentially relevant to this work, a lineage tracing study with Nfatc1-Cre mice suggests that endocardial cells, which are in contact with liver epithelium, contribute to the liver vasculature (Zhang et al., 2016).

As in the DA, endocardial cells have multiple origins, some of which derive from specialized angioblasts with hemogenic properties. Indeed, the existence of specialized angioblasts has now been documented in the floor of the cardinal vein that generates lymph, arterial and venous cell fates (Nicenboim et al., 2015). Thus, analysis of heart development has revealed on the one hand major contributions from the well-characterised cardiac cell populations (e.g. FHF, SHF, cardiac neural crest, epicardium), along with minor contributions from other mesodermal progenitors that provide further developmental plasticity and functionality.

Our studies and those of others (see Introduction) suggest a conserved role for Nkx2.5 in both heart and hemoangiogenic lineage development. In addition to its role in the specification of cardiac mesoderm, the Drosophila Nkx2.5 homologue, tinman, is essential for formation of the fly larval lymph gland, a hemopoietic organ proposed to be similar to the hemogenic endothelium of the mammalian AGM region (Mandal et al., 2004). Thus, cardio-hemoangiogenic lineage development may be one of the ancestral functions of Nkx2.5. In summary, the role of Nkx2.5 in endothelial and blood specification programs is evolutionarily conserved from fly to human. In vertebrates, Nkx2.5 expression is mostly restricted to cardiac progenitors, but in a narrow window of time may prime vascular and blood development, as shown already in pharyngeal arch arteries and endocardium (Nakano et al., 2013; Paffett-Lugassy et al., 2013). The definitive vasculature and blood networks ultimately utilise other transcription factors for lineage specialisation, which also prevent ectopic heart formation in these highly plastic mesodermal cells (Van Handel et al., 2012). Unravelling the dynamic roles of Nkx2.5 in cardiac, endothelial, and blood lineage specification will lead to a deeper understanding of the cardiovascular/blood regulatory networks, and may point the way toward novel regenerative therapies for cardiovascular disease.

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Author details

1. Lyad Zamir

   Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   LZ, Conceptualization, Data curation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Reena Singh

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-3442-2952

2. Reena Singh

   Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   RS, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Lyad Zamir

   Competing interests

   The authors declare that no competing interests exist.

3. Elisha Nathan

   Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   EN, Methodology

   Competing interests

   The authors declare that no competing interests exist.

4. Ralph Patrick

   Victor Chang Cardiac Research Institute, Sydney, Australia

   Contribution

   RP, Data analysis

   Competing interests

   The authors declare that no competing interests exist.

5. Oren Yifa

   Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   OY, Performed experiments and analyzed data

   Competing interests

   The authors declare that no competing interests exist.

6. Yfat Yahalom-Ronen

   Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   YY-R, Methodology

   Competing interests

   The authors declare that no competing interests exist.

7. Alaa A Arraf

   Department of Genetics and Developmental Biology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   AAA, Resources

   Competing interests

   The authors declare that no competing interests exist.

8. Thomas M Schultheiss

   Department of Genetics and Developmental Biology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   TMS, Resources

   Competing interests

   The authors declare that no competing interests exist.

9. Shengbao Suo

   Key Laboratory of Computational Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   SS, Resources

   Competing interests

   The authors declare that no competing interests exist.

10. Jing-Dong Jackie Han

    Key Laboratory of Computational Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Contribution

    J-DJH, Resources

    Competing interests

    The authors declare that no competing interests exist.

11. Guangdun Peng

    State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Contribution

    GP, Resources

    Competing interests

    The authors declare that no competing interests exist.

12. Naihe Jing

    State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Contribution

    NJ, Resources

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0003-1509-6378

13. Yuliang Wang

    Institute for Stem Cell and Regenerative Medicine, The University of Washington, Seattle, United States

    Contribution

    YW, Analyzing RNA-seq data

    Competing interests

    The authors declare that no competing interests exist.

14. Nathan Palpant

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

    Contribution

    NP, Analyzing RNA-seq data

    Competing interests

    The authors declare that no competing interests exist.

15. Patrick PL Tam

    1. School of Medical Sciences, Sydney Medical School, The University of Sydney, Westmead, Australia
    2. Embryology Unit, Children’s Medical Research Institute, Westmead, Australia

    Contribution

    PPLT, Resources

    Competing interests

    The authors declare that no competing interests exist.

16. Richard P Harvey

    1. Victor Chang Cardiac Research Institute, Sydney, Australia
    2. St. Vincent’s Clinical School, School of Biological and Biomolecular Sciences, University of New South Wales, Kensington, Australia

    Contribution

    RPH, Conceptualization, Resources, Data curation, Supervision, Investigation, Methodology, Writing—original draft, Writing—review and editing

    For correspondence

    r.harvey@victorchang.edu.au

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-9950-9792

17. Eldad Tzahor

    Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

    Contribution

    ET, Data curation, Methodology, Writing—original draft, Writing—review and editing

    For correspondence

    eldad.tzahor@weizmann.ac.il

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-5212-9426

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