The Role of BMP Signaling in Embryonic Limb Development and Postnatal Bone Formation
Date of Award
Doctor of Philosophy (PhD)
Bone formation requires differentiation and increased function of osteoblast lineage cells, which are influenced by major developmental signals. Detailed insight into the molecular mechanisms that regulate osteoblast differentiation and function is valuable as they may provide the fundamental basis for therapeutic intervention of numerous skeletal diseases. In this dissertation, I have investigated the role of BMP signaling in the osteoblast lineage during embryonic limb development and in postnatal bone formation. Furthermore, I investigated the mechanisms underlying the effects of BMPs on osteoblast differentiation and function.
Bone formation is initiated by aggregation and compaction of mesenchymal progenitors, a process known as mesenchymal condensation, which is followed by differentiation into chondrogenic cells. As BMPs are known to induce ectopic bone formation, we hypothesized that Smad-dependent BMP signaling is required for mesenchymal condensation. To directly test the requirement of BMP signaling in mesenchymal condensation, we conditionally deleted the type I BMP receptors (Alk2, Alk3 and Alk6) or the obligatory transcription factor Smad4 in the limb bud mesenchyme (with Prx1-Cre). Deletion of the type I BMP receptors or Smad4 resulted in a similar limbless phenotype characterized by the failure to complete mesenchymal condensation, indicating that BMP-Smad signaling is required for the early steps of chondrogenesis. Earlier studies have reported that BMP promotes chondrogenesis by stimulating Sox9 expression in vitro, raising the possibility that Sox9 may be the critical downstream target of Smad signaling. However, Sox9 was expressed at normal levels in the proximal mesenchyme of the limb bud in the absence of Smad4. Furthermore, forced-expression of Sox9 did not restore cartilage formation in the Smad4-deficient embryo. Thus, BMP-Smad signaling is indispensable for mesenchymal condensation to initiate skeletal development but the mechanism appears to be independent of Sox9 induction.
While we and others have shown that BMP signaling is essential for embryonic bone formation, the physiological effect of BMP signaling in osteoblast lineage cells in postnatal stages remains unclear. To directly assess the role of BMP signaling in osteoblast lineage cells in vivo, we deleted the type I BMP receptor Alk3 in Osx-positive cells (with Sp7-Cre) in the postnatal mouse. Unexpectedly, postnatal removal of Alk3 rapidly increased the cancellous bone mass. Similarly, deletion of Alk3 with Dmp1-Cre that also target osteoblast-lineage cells markedly increased the cancellous bone mass. In addition, the Alk3 mutant mediated by Dmp1- Cre possessed thinner long bones by one month of age due to reduced osteoblast activity at the periosteal surface. The increase in cancellous bone mass was associated with a significant increase in osteoblast numbers, which may be due to the enhanced proliferation of preosteoblasts detected at the chondro-osseous junction. Remarkably, deletion of Smad4 using either of the aforementioned approaches caused a notably lesser effect than the Alk3 deletion. Thus, Alk3 appears to regulate postnatal bone formation partly through Smad-independent mechanisms.
In summary, my thesis work demonstrates that BMP signaling is indispensable for pre- chondrogenic condensation in a Smad4-dependent manner in the mouse embryo, but it controls postnatal bone formation partly independent of Smad4. I further show that overall effect of BMP signaling on bone formation varies between cancellous and cortical bone compartments.
Chair and Committee
David C Beebe, Deborah V Novack, David M Ornitz, Matthew J Silva
Lim, Joohyun, "The Role of BMP Signaling in Embryonic Limb Development and Postnatal Bone Formation" (2015). Arts & Sciences Electronic Theses and Dissertations. 478.