Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.

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The individual contributions of the three vertebrate GATA factors to endoderm formation have been unclear. Here we detail the early expression of GATA4, 5 and 6 in presumptive endoderm in Xenopus embryos and their induction of endodermal markers in presumptive ectoderm. Induction of HNF3β by all three GATA factors was abolished when protein synthesis was inhibited, showing that these inductions are indirect. In contrast, whereas induction of Sox17α and HNF1β by GATA4 and 5 was substantially reduced when protein synthesis was inhibited, induction by GATA6 was minimally affected, suggesting that GATA6 is a direct activator of these early endodermal genes. GATA4 induced GATA6 expression in the same assay and antisense morpholino oligonucleotides (MOs), designed to knock down translation of GATA6, blocked induction of Sox17α and HNF1β by GATA4, suggesting that GATA4 induces these genes via GATA6 in this assay. All three GATA factors were induced by activin, although GATA4 and 6 required lower concentrations. GATA MOs inhibited Sox17α and HNF1β induction by activin at low and high concentrations in the order:GATA6>GATA4>GATA5. Together with the timing of their expression and the effects of GATA MOs in vivo, these observations identify GATA6 as the predominant GATA factor in the maintenance of endodermal gene expression by TGFβ signaling in gastrulating embryos. In addition, examination of gene expression and morphology in later embryos, revealed GATA5 and 6 as the most critical for the development of the gut and the liver.

Cardiogenesis is inhibited by canonical Wnt/β-catenin signalling and stimulated by non-canonical Wnt11/JNK signalling, but how these two signalling pathways crosstalk is currently unknown. Here, we show that Wnt/β-catenin signalling restricts cardiogenesis via inhibition of GATA gene expression, as experimentally reinstating GATA function overrides β-catenin-mediated inhibition and restores cardiogenesis. Furthermore, we show that GATA transcription factors in turn directly regulate Wnt11 gene expression, and that Wnt11 is required to a significant degree for mediating the cardiogenesis-promoting function of GATA transcription factors. These results demonstrate that GATA factors occupy a central position between canonical and non-canonical Wnt signalling in regulating heart muscle formation.

Hematopoietic stem cells (HSCs) sustain blood production throughout life and are of pivotal importance in regenerative medicine. Although HSC generation from pluripotent stem cells would resolve their shortage for clinical applications, this has not yet been achieved mainly because of the poor mechanistic understanding of their programming. Bone marrow HSCs are first created during embryogenesis in the dorsal aorta (DA) of the midgestation conceptus, from where they migrate to the fetal liver and, eventually, the bone marrow. It is currently accepted that HSCs emerge from specialized endothelium, the hemogenic endothelium, localized in the ventral wall of the DA through an evolutionarily conserved process called the endothelial-to-hematopoietic transition. However, the endothelial-to-hematopoietic transition represents one of the last steps in HSC creation, and an understanding of earlier events in the specification of their progenitors is required if we are to create them from naïve pluripotent cells. Because of their ready availability and external development, zebrafish and Xenopus embryos have enormously facilitated our understanding of the early developmental processes leading to the programming of HSCs from nascent lateral plate mesoderm to hemogenic endothelium in the DA. The amenity of the Xenopus model to lineage tracing experiments has also contributed to the establishment of the distinct origins of embryonic (yolk sac) and adult (HSC) hematopoiesis, whereas the transparency of the zebrafish has allowed in vivo imaging of developing blood cells, particularly during and after the emergence of HSCs in the DA. Here, we discuss the key contributions of these model organisms to our understanding of developmental hematopoiesis.

Blood development has been highly conserved during evolution. Hematopoietic cells in amphibian and fish embryos, as in mammalian embryos, emerge and progressively differentiate in several locations. Hematopoiesis, including of the immune system, is similar in the amphibian, Xenopus, to mammals and the embryos are ideal for tissue transplantation and lineage labelling experiments, which have enabled the elucidation of the distinct origins of embryonic and adult hematopoietic cells, as well as their migration pathways and organ colonisation behaviours. The zebrafish hematopoietic system is less well understood, but these embryos have recently emerged as a powerful system for both genetic analysis and imaging. In this review, we summarise our current knowledge of the cellular and genetic basis of ontogeny of the hematopoietic system in Xenopus and zebrafish embryos.

The regulation of stem cell ontogeny is poorly understood. We show that the leukemia-associated Ets transcription factor, Tel1/ETV6, specifies the first hematopoietic stem cells (HSCs) in the dorsal aorta (DA). In contrast, Tel1/ETV6 has little effect on embryonic blood formation, further distinguishing the programming of the long- and short-term blood populations. Consistent with the notion of concordance of arterial and HSC programs, we show that Tel1/ETV6 is also required for the specification of the DA as an artery. We further show that Tel1/ETV6 acts by regulating the transcription of VegfA in both the lateral plate mesoderm and also in the somites. Exogenous VEGFA rescues Tel1/ETV6 morphants, and depletion of VEGFA or its receptor, Flk1, largely phenocopies Tel1/ETV6 depletion. Few such links between intrinsic and extrinsic programming of stem cells have been reported previously. Our data place Tel1/ETV6 at the apex of the genetic regulatory cascade leading to HSC production.

Hematopoietic stem cells (HSCs) emerge during embryogenesis from hemogenic endothelium, but it remains unclear how the HSC lineage is initially established from mesoderm during ontogeny. In Xenopus, the definitive hemangioblast precursors of the HSC lineage have been identified in dorsal lateral plate (DLP) mesoderm, and a transcriptional gene regulatory network (GRN) controlling hemangioblast programming has been elucidated. Herein, we identify an essential role for microRNAs (miRNAs) in establishing the mesodermal lineage leading to both HSC emergence and vasculogenesis and determine that a single miRNA, miR-142-3p, is primarily responsible for initiation of definitive hemangioblast specification. miR-142-3p forms a double-negative gate unlocking entry into the hemangioblast program, in part by inhibiting TGFβ signaling. Our results table miR-142-3p as a master regulator of HSC lineage specification, sitting at the apex of the hierarchy programming the adult hemangioblast, thus illustrating that miRNAs can act as instructive determinants of cell fate during development.