In this study, we report characterization and functional analysis of a novel zebrafish Ets1-related protein, Etsrp. Knockdown of Etsrp resulted in the complete absence of functional blood vessels and downregulation of all endothelial-specific markers analyzed. In zebrafish, hedgehog
signaling is necessary for migration of angioblasts from the lateral mesoderm toward the midline, where they subsequently differentiate within the ICM region [4
]. Such migration is not apparent in the anterior region which gives rise to the anterior vessels including head vessels. Interestingly, vasculogenesis defects in etsrp
morphants were more pronounced in the posterior region of the embryo which was particularly evident at lower downregulation levels. At high MO doses, endothelial cells were nearly completely absent from axial and intersegmental vessels. However, head expression of endothelial markers was not completely eliminated. It is possible that the remaining amount of Etsrp protein activity is sufficient for the head vasculogenesis. Upregulation of etsrp
RNA was particularly strong in the head region in etsrp
morphants, which may partially compensate for the MO-mediated translation inhibition. Alternatively, etsrp
function may be redundant in the vasculogenesis of head vessels.
Among the vascular-specific genes analyzed, expression of only fli1
was not globally downregulated in the early embryos. The early fli1
expression, which overlaps with the hematopoietic factor gata2
expression, is unaffected in the clo
mutants and scl
morphants and has been suggested to mark the common blood and vascular precursors, hemangioblasts [13
is later expressed specifically in the vascular endothelial cell precursors, and this expression was reduced but not completely eliminated in etsrp
expression may not be directly regulated by etsrp,
therefore undifferentiated angioblasts retain some of fli1
expression in etsrp
is expressed in both hematopoietic and vascular progenitors throughout most of the early development. Although we did not see global downregulation of scl
expression in etsrp
morphants, it is likely that scl
expression is lost in angioblasts but retained in hematopoietic precursors. Interestingly, expression of both fli1
genes was lost from the anterior and trunk domains of the lateral mesoderm. Anterior domain contains angioblast and myeloid progenitors, which appear to be absent from the etsrp
morphants. The difference between the trunk and posterior lateral mesoderm has not been previously analyzed in great detail. Possibly, the trunk region contains mostly angioblasts, while the tail region contains both angioblasts and hematopoietic precursors.
Overexpression of etsrp
induced strong ectopic expression of vascular markers in multiple cell types, including even tissues that normally commit to a very different fate such as neuroectoderm. Furthermore, overexpression of etsrp
specifically initiated vascular development without affecting related hematopoiesis, which argues that the observed effects are not caused by an early ventralization of the whole embryo. These results indicate that etsrp
is sufficient to initiate vasculogenesis. As a contrast, overexpression of other regulators of vasculogenesis such as vegf
induced expression of vascular markers only within the lateral or somitic mesoderm [14
Overexpression of vegf and scl did not have an effect on vascular development in the absence of etsrp function, which suggests that etsrp is an essential mediator of vegf and scl signaling, at least in the vascular induction. Loss of vegf or scl did not affect early expression of etsrp within angioblasts. Overexpression of etsrp caused strong scl induction, indicating that etsrp plays an important role in controlling scl expression, at least within angioblasts. This is also supported by the loss of scl expression in the anterior and trunk regions in etsrp morphants. Our data suggest that etsrp is necessary for scl expression within angioblasts, and both genes are then required for induction of multiple vascular endothelial genes.
The current study shows for the first time that a single gene can be necessary and sufficient for initiating vascular development in vertebrates. Our results demonstrate that Etsrp acts as a very early regulator of vasculogenesis. These findings will greatly advance our understanding of vascular development and the general mechanisms of cell fate specification.