Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5m1/m1 amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4+) and alkaline phosphatase-positive (AP+) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4+, AP+ cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.

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