Investigation of the Role of Interdigit BMP Signaling in Programmed Cell Death and Mouse Limb Development

Shaping of the embryonic limb involves many processes including growth, differentiation, and programmed cell death (PCD). Furthermore, these processes integrate complex information from multiple signaling cascades such as the BMP and FGF pathways. Previous work shows that BMP signaling regulates interdigit (ID) PCD indirectly by affecting expression of Fgfs, which encode cell survival factors secreted from the apical ectodermal ridge. Nevertheless, this indirect model does not exclude a direct role for BMPs in PCD. Due to lack of genetic evidence, it is still unclear whether ID BMPs directly trigger PCD. To test whether BMPs act as direct triggers of ID PCD, the gene encoding receptor BMPR1A was inactivated within the ID tissue with Osr1-Cre. The resulting mutants show a decrease of ID PCD. To test redundancy between BMPR1A and BMPR1B, ID-specific Bmpr1a was inactivated in a Bmpr1b null background. This compound mutant has a further decrease in ID PCD. Analysis of BMPR1A mutants reveals a reduction in ID BMP signaling as observed through a decrease in Msx2 expression and phosphorylated SMAD 1 and 5 levels. Furthermore, a decrease in ID Dkk1 expression and a lack of ID webbing in Osr1-Cre; Smad4 mutants suggests both SMAD independent and dependent components in the regulation of ID PCD. During this analysis a potential role of the ID in digit formation was serendipitously discovered. Bmpr1b null mice have brachydactyly, which is completely rescued in digit one with the ID-specific inactivation of Bmpr1a. Furthermore, preliminary analysis of mice with both Bmp7 and Bmpr1a inactivated in the ID of Bmpr1b mutants suggests that brachydactyly is reproduced and ID BMP7 secretion is necessary to rescue digit development. To fully understand the role of ID BMP signaling on normal limb development, ID Bmp2, 4, and 7 were inactivated in various combinations with Osr1-Cre. Analysis of these mutants reveals that BMP7 is necessary for ID BMP signaling and PCD during normal development while BMP2 and BMP4 play redundant roles in ID PCD regulation. This work establishes that BMP signaling directly regulates ID PCD and that ID BMP secretion can regulate digit development