Identification of Novel Transcription Factors Regulating Recovery of the Endothelial Lineage in Avascular Mutants

Multiple mesodermal tissues are known to give rise to endothelial cells during development. Furthermore, markers for functionally distinct endothelium are well established. Therefore, it is likely that multiple distinct populations of endothelial cells exist during development and into adulthood. Two zebrafish mutants, cloche and groom of cloche provide a unique opportunity to interrogate the heterogeneous origin of the endothelial lineage. Homozygous mutant embryos lack the majority of the endothelial lineage at early developmental stages, however, generate rudimentary vessels at later stages, indicating that they retain the ability to generate endothelial cells despite this initial lack of early endothelial progenitors. To delineate the developmental source of the endothelial cells in these avascular mutant embryos, we first performed lineage tracing from early gastrula to determine the fate of mesoderm. Consistent with their phenotype, we found an increase of kdrl- unspecified mesoderm, indicating that much of the mesoderm apportioned to become angioblasts fails to become specified. Conversely, endothelial differentiation from the tailbud, a proposed secondary source of endothelial cells during development, was largely unperturbed. Consistent with this finding, the majority of the early kdrl+ cells found in avascular mutant embryos are specified from tailbud. To better elucidate the molecular basis of endothelial recovery in avascular mutant embryos, we analyzed the gene expression profile using microarray analysis on isolated mutant endothelial cells. We find that the expression of the genes characteristic of other mesodermal lineages are substantially elevated in kdrl+ cells isolated from avascular mutant embryos. Subsequent validation identified two transcription factors, sox11b and pax9, both of which have not previously implicated in vascular development. Yet, we confirmed that both genes are expressed in the vasculature and knockdown of either gene results in a vascular phenotype. Additionally, we found that the function of Pax9 appears to be evolutionarily conserved. Taken together, our analyses illustrate a complex regulation of endothelial specification and differentiation during vertebrate development. Furthermore, we have identified two new key transcription factors involved in vascular development.