VEGFA Controls Haematopoietic Stem Cell Specification In a Dose-Dependent, Isoform-Specific Manner

Abstract 406

VEGFA signalling is critical for endothelial and haematopoietic development during embryogenesis. Perturbation of the VEGFA pathway results in defective haemopoietic stem cell (HSC) specification and vascular/arterial formation. Because of the intimate relationship between arterial and blood development, the contribution of VEGFA to HSC specification is unclear. Characterising the exact function of VEGFA is of primary interest for understanding mechanisms regulating haematopoietic specification. Here, we show that morpholino-induced knock-down of the co-repressor and oncoprotein ETO2 in Xenopus embryos leads to a failure of HSC specification via a VEGFA–dependent mechanism, independently from dorsal aorta (DA) formation and arterial specification. Knock-down of ETO2 represses Vegfa levels in somitic tissues along the migratory pathway from the dorsal lateral plate to the midline. This, in turn, leads to absence of Notch1 expression in the hemogenic endothelim of the DA and failure to activate the HSC transcriptional programme. DA formation and arterial specification occur normally, thus uncoupling VEGFA requirements for DA versus HSC formation. Then, we examined expression of the 3 VEGFA isoforms that differ in their physical and biological properties. In Eto2 morphant embryos, there was reduction (∼50%) in mRNA expression of the medium and long Vegfa variants in the somites. Importantly, Vegfa hypomorph embryos phenocopy the Eto2 morphant phenotype; this further demonstrates that reduction in VEGFA levels is sufficient for failure of HSC specification. Furthermore, restoration of medium Vegfa isoform (Vegfa₁₇₀) levels alone in either Eto2 morphants or Vegfa hypomorph embryos was sufficient to re-establish the HSC programme. In summary, this is the first report of a dose-dependent, isoform-specific function of VEGFA at the onset of the HSC programme, independent from its functions in vessel formation and specification of the DA. We propose that signalling from VEGFA₁₇₀ in somites instructs the HSC programme of the DA/HSC progenitors as they migrate towards the midline, in a paracrine manner. This VEGFA₁₇₀ signal is critically required later in the haemogenic endothelium of the newly formed DA to initiate Notch1 expression and trigger the haematopoietic transcriptional programme. This work also reveals for the first time that ETO2 controls the onset of the HSC programme in a non cell-autonomous manner. This work furthers our understanding of the complex interplay between the cell populations controlling key signaling events leading to HSC specification. Manipulation of these signaling cascades may allow us to better develop strategies to produce and expand HSCs for therapeutic and regenerative purposes.