The appearance of cells having HSC characteristics has been observed in close association with arterial endothelium^5,7. In particular, the ventral floor of the dorsal aorta (DA) has been suggested by a number of investigators to be the primary birthplace of HSCs⁸. In the zebrafish embryo, we⁹ and others¹⁰ previously demonstrated that expression of a cmyb:eGFP transgene marks nascent HSCs along the ventral aspect of the DA between 28–48 hpf. To determine whether these cells arise directly from vascular precursors, we generated cmyb:eGFP; kdrl:memCherry¹¹ double transgenic animals and performed confocal timelapse imaging. Between 28–32 hpf, expression of the kdrl trangene (also known as flk1 and vegfr2) within the zebrafish equivalent of the AGM region is localized to the aorta, vein and developing intersomitic vessels; hematopoietic expression of the cmyb transgene initiates in cells along the DA around this time (Figure 1a,b). Four-dimensional imaging demonstrated that cmyb:eGFP⁺ cells arose directly from kdrl:memCherry⁺ cells specifically along the ventral aspect of the DA (Supplemental Movies 1, 2). As shown in Figure 1, kdrl:memCherry⁺ ECs displaying typical flattened morphology were occasionally observed to transform into spherical shapes, forming buds that extended into the lumen of the DA. By virtue of the membrane-specific expression of mCherry, buds were observed to initiate as kdrl⁺cmyb⁻ cells transitioned to kdrl⁺cmyb⁺ cells (Figure 1). In contrast to the proposed budding of mammalian HSCs into aortic circulation⁵, we almost always observed HSCs to migrate ventrally towards the caudal vein (CV; Supplementary Movies 1, 2). This is consistent with the observations of Kissa et al.¹², which suggested that AGM HSCs enter circulation via the dorsal wall of the CV in the zebrafish.

To confirm the hematopoietic nature of these budding AGM cells, we performed flow cytometry on dissociated kdrl:RFP¹³; cmyb:eGFP embryos at 36 hpf, the timepoint at which we observed the peak in number of kdrl⁺cmyb⁺ cells. Embryos were dissected to separate anterior, head tissues from the posterior trunk/tail region that contains the AGM. In accordance with our microscopic observations, no kdrl⁺cmyb⁺ cells were observed in anterior regions (Figure 2a) above background. By contrast, 0.25% of posterior cells were kdrl⁺cmyb⁺ cells (Figure 2b). We thus reasoned that these double positive cells represented the nascent HSCs observed in our imaging experiments (Figure 2c). kdrl⁺cmyb⁺ cells could be subdivided based upon differing levels of the cmyb:eGFP transgene (Figure 2b); each subset, along with single positive posterior fractions were highly purified by FACS and queried for expression of hematopoietic and vascular genes by qPCR. As expected, expression of endothelial genes, including kdrl, cdh5, and lmo2, were highly expressed in kdrl⁺cmyb⁻ cells (Figure 2d). In general, early kdrl⁺cmyb^lo precursors maintained similar expression levels of these vascular markers. As cmyb:eGFP levels increased in maturing kdrl⁺cmyb⁺ cells, however, expression of most endothelial genes dropped dramatically. By contrast, expression of cd41, one of the earliest markers of mesodermal commitment to definitive hematopoiesis^14,15, initiated in kdrl⁺cmyb⁻ cells and increased as kdrl⁺ cells became cmyb⁺ (Figure 2d). We observed little to no cd45 expression in either kdrl⁺cmyb⁻ or kdrl⁺cmyb^lo cells. As these precursors matured, however, we observed concomitant upregulation of cd45 in kdrl⁺cmyb⁺ and kdrl⁻cmyb⁺ subsets (Figure 2d). These results are consistent with findings in the mouse. On embryonic day 10, murine AGM HSCs do not express CD45 (encoded by ptprc)^14,15. By day 11, however, embryonic HSCs become CD45^+14,15. Collectively, these results support the hypothesis that the kdrl⁺cmyb⁺ cells observed to arise from the ventral wall of the DA are definitive hematopoietic precursors.

In order to trace the progeny of ECs in the zebrafish embryo, we employed an indelible marking system utilizing a floxed reporter transgene and a Cre driver that is specific to endothelium via kdrl upstream promoter/enhancer elements¹⁶. In the zebrafish, there exist two orthologues of the mammalian flk1 gene, kdr and kdrl. The former exhibits a pan mesodermal expression pattern¹⁷, whereas kdrl and Tg(kdrl:Cre)^s898 (Supplementary Figure 1) is expressed only in endothelium in a manner nearly identical to that of the murine cdh5 gene^18,19. Thus, the Tg(kdrl:Cre)^s898 line used in our studies mimics the cdh5:Cre knock-in mouse line employed in previous studies^1,2. We crossed kdrl:Cre animals to animals carrying a Tg(βactin2:loxP-STOP-loxP-DsRed-express)^sd5 “switch” reporter transgene in which 10.5kb of upstream βactin promoter/enhancer sequence is followed by a 5.7kb floxed “superstop” cassette. Immediately downstream is a DsRed^express gene that serves as a reporter for Cre-based removal of the superstop cassette. As presented in Supplementary Figure 2, reporter gene expression was never observed in the absence of Cre, and induction of Cre in 24 hpf embryos led to stable reporter expression within nearly all hematopoietic cells for one year.

To test whether the kdrl⁺; cmyb⁺ hematopoietic precursors observed in the AGM are bona fide HSCs, we generated kdrl:Cre; switch animals for long-term studies. Compared to whole kidney marrow (WKM) isolated from single transgenic switch animals that showed no expression of DsRed (Figure 3a), double transgenic kdrl:Cre; switch animals showed the vast majority of leukocytes to be labeled at six months of age (Figure 3b, c). Analysis of a large cohort of double transgenic animals showed that over 90% of WKM cells were marked at 3 months of age (Figure 3b). At 6 months of age, over 96% of WKM myeloid cells expressed the DsRed^express lineage tracer (Figure 3c). Since this cellular subset is comprised of over 90% neutrophils, which are characterized by lifespans restricted to a few days, this result suggests that the vast majority of, if not all, HSCs were marked by the kdrl:Cre transgene during their embryonic formation. Finally, marked WKM was sorted by DsRed expression level and subjected to qPCR for lineage-affiliated genes. As presented in Figure 3d, pax5 (B lymphocyte-affiliated) and pu.1 (myeloid-affiliated) expressing cells were contained within DsRed^high cells, and gata1 expressing cells within the DsRed^low fraction. These data are consistent with our previous demonstration that the βactin promoter is highly expressed in leukocytes, but is silenced upon erythropoietic differentiation²⁰. Thus, lineage tracing of kdrl⁺ hemogenic endothelium demonstrates robust, multilineage, long-term population of the adult hematopoietic organ. Furthermore, since expression of kdrl is rapidly extinguished as HSCs arise from hemogenic endothelium (Figure 2d), and since kdrl⁺cmyb⁺ transitional intermediates were no longer detectable in larval or adult stages (Supplementary Figure 3), our lineage tracing results support the hypothesis that HSCs no longer arise de novo following their specification in the embryo.

In summary, our imaging and lineage tracing studies demonstrate that the first HSCs born in the zebrafish embryo arise directly from hemogenic endothelium lining the ventral wall of the DA. These results complement previous studies in the avian²¹, amphibian²², and mammalian embryo^1,2,7 and suggest that the cellular mechanisms of HSC generation have been highly conserved across vertebrate evolution. The finding that HSC development requires transition through a hemogenic endothelial intermediate should aid efforts to instruct HSC formation in vitro from pluripotent precursors, a necessity for therapies designed to replace the adult blood cell lineages.

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