Another developmental regulator Hedgehog Hh acts as a morpho

Another developmental regulator, Hedgehog (Hh), acts as a morphogen in many developing tissues. Visceral endoderm is instructive to the development of endothelial and hematopoietic buy A 779 through Hh signaling early in mouse gastrulation (Belaoussoff et al., 1998; Dyer et al., 2001). Hh protein can replace endodermal tissue (gut) to induce HSCs in AGM explant cultures before the normal onset of HSC generation (Peeters et al., 2009). Zebrafish Hh pathway mutants display significant defects in HSC formation, and Hh factors act upstream of VEGF to regulate definitive hematopoiesis in the embryo (Gering and Patient, 2005). Although BMP4 and Hh, when studied individually, have been shown to influence HSC growth, it is unknown whether these signaling pathways intersect in the same HSCs. In this study, we make use of BMP Responsive Element (BRE) GFP transgenic mice to study the BMP signaling pathway and the effects of Hh simultaneously on AGM HSC development. We show in explant cultures that the AGM contains two types of HSCs, BMP-activated and non-BMP-activated HSCs, with distinct but overlapping genetic programs. The non-BMP-activated HSC type is lost when the Hh signaling pathway is inhibited, but can be partially rescued by VEGF. We reveal here the signaling pathway regulation involved in the bifurcation of HSC types during development.
Results
Discussion We initiated this study to examine the interface of two developmental signaling pathways in AGM HSCs. Rather than converging on the same HSCs, BMP and Hh pathways were found to regulate two distinct HSC types: BMP-activated and non-BMP-activated Hh/VEGF-responsive HSCs. The appearance of non-BMP-activated HSCs in this culture system was unexpected, since we found the percentage of BMP-activated cells was increased in AGM explants. It is likely that the explant culture conditions affect both the generation and growth of AGM HSCs. The absence of the tissues surrounding the AGM dorsally and ventrally, such as the neural tube and gut, respectively, may contribute to changes in the HSC composition by altering the positional information. Normally the dorsal microenvironment is restrictive to HSC growth in vivo (Peeters et al., 2009; Pardanaud and Dieterlen-Lievre, 1999) and its absence in AGM explants may explain the appearance of the non-BMP-activated HSC type. Our recent demonstration of the two HSC types in the FL (Crisan et al., 2015) suggests that the explant culture of AGM induces a bifurcation of HSCs into an FL-like state. In the AGM explant, the non-BMP-activated HSCs are exclusively sensitive to cyclopamine treatment and provide no hematopoietic engraftment upon transplantation, whereas the BMP-activated HSCs are unaffected. Importantly, HSC activity can be partially restored in cyclopamine conditions when VEGF is exogenously added. It would be interesting to know whether the non-BMP-activated HSCs in the FL are also controlled by Hh/VEGF signaling, and an appropriate culture system should be developed to test this. However, Lagging strand of DNA is known that at E11 both Hh and Gli1 (Cridland et al., 2009; Hirose et al., 2009) are expressed in the FL, and thus may influence the second type of HSC in this tissue. The role of Hh signaling in HSC development is unclear, and published data regarding this have been in conflict (reviewed in Lim and Matsui, 2010; Mar et al., 2011). Fifty percent of germline Ihh−/− mouse embryos die at E13.5 from hematological disorders, the stage when HSCs are highly expanding in the FL microenvironment (reviewed in Kaimakis et al., 2013). In these mice, erythroid lineage differentiation is profoundly affected (Cridland et al., 2009), but surprisingly phenotypic (LSK) HSC numbers remain unchanged. In contrast, Gli1−/− BM LSK HSCs transplanted into adult recipients show a higher engraftment efficiency compared with wild-type HSCs, and myeloid development is adversely affected (Merchant et al., 2010). Both these results could be explained by our results showing that not all HSCs are regulated by Hh, and we suggest that BMP-activated HSCs fill in for the Hh-regulated HSCs in the deficient mice. However, downstream of the HSC, Hh is necessary in progenitors and for erythroid lineage differentiation.