UCLA

Endochondral bone formation involves a highly coordinated program of chondrocyte differentiation, proliferation, maturation, and hypertrophy. The Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGFβ) and Activin signaling pathways are regulators of these processes. The importance of the BMP type I receptors had been well documented; however, the physiological role of the BMP type I receptor Activin Like Kinase 2 (ALK2/ACVR1) during the process of mammalian endochondral bone formation was unknown. The ALK2 receptor gained its infamy when activating mutations in it were discovered to cause the devastating disease fibrodysplasia ossificans progressiva (FOP), an autosomal dominant condition that causes paralysis and early death due to ossification of soft connective tissue. Early in vivo and in vitro chick studies revealed that the ALK2 receptor plays an important role in the formation of cartilage and bone. However, the physiological relevance of ALK2 in mammalian skeletal tissues remained unknown. Furthermore, whether ALK2 receptor impacts BMP signaling in the development of skeletal tissues as strongly as and/or has overlapping functions with those of the other BMP Type I receptors ALK3/BMPR1A and ALK6/BMPR1B remained unclear. My studies addressed these unknowns.

Previous studies in the laboratory of Professor Karen Lyons had shown that the main transducers of the BMP signaling pathways in cartilage are the receptor regulated Smads (R-Smads) 1, 5, and 8. This was demonstrated by the observation that cartilage-specific knockouts of all three Smads recapitulated the phenotype seen in mice lacking the BMP receptors ALK3 and ALK6. Smad4, a co-Smad that complexes with R-Smads, was thought to be required for all transcriptional R-Smad activity. However, cartilage specific knockout of Smad4 reported by another group surprisingly rendered a viable mouse. The phenotypic analysis of pre- and postnatal Smad4 mutants was characterized, however, the mechanism by which BMP signaling persisted in the absence of Smad4 remained unclear. The studies in this thesis address the mechanisms underlying this apparent R-Smad-dependent, Smad4-independent signaling. Cartilage specific knockout of Smad4 was found to result in an accumulation of phosphorylated Smads 1/5/8 in the cell that could not be targeted for degradation. Overall these studies showed that the requirement of Smad4 for Smad1/5/8-mediated transcription is, in part, obsolete in systems with persistent BMP signaling such as the developing cartilage growth plate. However, Smad4 appears to be required in events that require temporal, rapid, and potent signaling, like that of limb bud development. Therefore, Smad4 plays a role in regulating the duration and strength of BMP signaling.

This thesis includes one published review and two original articles (one of which has been published). The known mechanisms by which BMP signaling regulates chondrogenesis and cartilage maintenance will be highlighted in Chapter One, “BMP signaling in Skeletogenesis” and Chapter Two “TGFβ signaling in Cartilage Development and Maintenance”. The roles of the BMP type I receptors ALK2/ALK3/ALK6, with a focus on ALK2, will be highlighted in Chapter Three. This work has been published. The novel Smad4-independent, Smad1/5/8-dependent signaling pathway will be the subject of Chapter Four “ Challenging the Dogma of Canonical BMP signaling in the absence of Smad4 during endochondral bone formation”. Next, a short methods paper highlighting the art of skeletal tissue staining will be outlined in Chapter Five “Whole-Mount Skeletal Staining”. This work has been published. Finally, Chapter Six will discuss the overall conclusions and future directions of my thesis project.