Abstract

The hematopoietic morphogen BMP4 has been implicated in early blood development, however the precise role and the underlying genetic pathways that link BMP action to blood fate specification remain poorly understood. In this study, we have employed the in vitro system of murine embryonic stem (mES) cell differentiating into embryoid bodies (EBs) as a model to explore mesoderm induction and hematopoietic specification during mammalian embryogenesis. We show that BMP plays a role in blood fate specification in two sequential phases: initially, BMP acts as a dorsoventral patterning agent, inducing ventroposterior mesoderm; at a later phase, BMP is required for hematopoietic fate specification from mesoderm. In dissecting this second specification phase, we discovered a previously unsuspected coordination between BMP4 and Wnt3a signaling, and linked these morphogens directly to Cdx and Hox genes. Using chromatin immunoprecipitation assays performed on EB-derived cells, we detected the association of the Wnt signaling effector LEF1 with the proximal Cdx1 promoter. This association was modulated by BMP signaling. In promoter reporter assays both BMP4 and Wnt3a activated the Cdx1 promoter while Wnt inhibitors or point mutations in the LEF1 responsive elements of the Cdx1 promoter abrogated this response. Together, these results suggest that BMP4 activates the Cdx1 promoter via LEF1 and the Wnt pathway. In functional assays, inhibition of Wnt signaling strongly suppressed the formation of hematopoietic colony initiating cells, suggesting that activation of the Wnt-Cdx-Hox pathway is a central downstream component of the BMP dependent blood fate specification program. Using ES cell lines engineered for conditonal Cdx1 or Cdx4 gene overexpression, we demonstrate that ectopic activation of Cdx genes is sufficient to establish hematopoietic fate decisions in the absence of BMP signaling during mesoderm specification to blood. Finally, to explore the relevance of our findings in vivo, we studied the effects of conditional BMP overexpression on cdx4 expression and embryonic blood formation during zebrafish embryogenesis. Matching our results with EBs, we find that overexpression of bmp2b at midgastrulation expands the cdx4 expression domain and strongly enhances blood formation. Taken together our results reveal an interaction between the BMP and Wnt signaling pathways, upstream of the Cdx genes, that is critical for the induction of the embryonic blood cell program. Given the evolutionary conservation of the BMP-Wnt-Cdx-Hox effect on blood formation from fish to mice, we anticipate that similar pathways can be exploited to direct the differentiation of human ES cells into blood.

Author notes

Disclosure: No relevant conflicts of interest to declare.