Endochondral ossification is the process of skeletal bone growth via the formation of a cartilage template that subsequently undergoes mineralization to form trabecular bone. Genetic mutations affecting the proliferation or differentiation of chondrocytes result in skeletal abnormalities. Activating transcription factor-2 (ATF-2) modulates expression of cell cycle regulatory genes in chondrocytes, and mutation of ATF-2 results in a dwarfed phenotype. Here we investigate the regulatory role that ATF-2 plays in expression of the pocket proteins, cell cycle regulators important in cellular proliferation and differentiation. The spatial and temporal pattern of pocket protein expression was identified in wild type and mutant growth plates. Expression of retinoblastoma (pRb) mRNA and protein were decreased in ATF-2 mutant primary chondrocytes. pRb mRNA expression was coordinated with chondrogenic differentiation and cell cycle exit in ATDC5 cells. Type X collagen immunohistochemistry was performed to visualize a delay in differentiation in response to loss of ATF-2 signaling. Chondrocyte proliferation was also affected by loss of ATF-2. These studies suggest pRb plays a role in chondrocyte proliferation, differentiation and growth plate development by modulating cell cycle progression. ATF-2 regulates expression of pRb within the developing growth plate, contributing to the skeletal phenotype of ATF-2 mutant mice through the regulation of chondrocyte proliferation and differentiation.

Pocket proteins (pRb, p107 and p130) are well studied in the role of regulating cell proliferation by controlling progression through the G1/S phase of the cell cycle. Increasing genetic and anatomical evidence suggests that these proteins also control early differentiation and even later stages of cell maturation including neural migration. However, the multifaceted functions of pocket proteins in the regulation of cell proliferation and cell death has complicated our interpretation of their role during development. As a result, the mechanisms through which pocket proteins regulate neuronal migration and neural maturation remain unknown. Using a pRb and p107 double knock out model, we show that a population of upper layer cortical neurons fails to pass through the intermediate zone into the cortical plate. Importantly, these neurons are born at the appropriate time and have exited the cell cycle. In addition, the role of pocket proteins in radial migration is independent cell death, since this migration defect cannot be rescued by eliminating ectopic cell death through Bax deletion. We also show a novel role of pRb and p107 in development of the dorsal midline and guidance of callosal axons. In the absence of pRb and p107, the structures of the commissural plate are highly disorganized and the callosal axons fail to cross the midline. We identify primary defects in axon extension and expression of multiple guidance cues, which can be observed prior to the disorganization of the midline axon guidance structures. Through the use of in vitro cortical explants and in utero electroporation, we identify defects in the rate of axon extension and directional guidance independent from the midline. In addition, protein levels of Netrin and Neuropilin-1 are decreased in the absence of pRb and p107, which could mediate the function of pocket proteins in guiding callosal axons. Indeed, we identify a previously undescribed population of Netrin expressing cells in the cingulate cortex of control embryos which is lost in the pRb/p107 deficient littermates. We propose that these cells play a significant role in callosal axon guidance during normal development. The results presented in this dissertation define multiple novel roles of pRb and p107 in the regulation of radial migration and axon guidance, independent from the role of these pocket proteins in cell death and proliferation.

A complex regulatory framework underlies the generation of newborn neurons in the adult mammalian brain, including the lifelong maintenance of neural stem cell (NSC) quiescence and instructing NSC entry to and exit from quiescence. Future therapies targeting endogenous repair of the aging or afflicted brain, including neurodegenerative pathologies, rely on present efforts to define and characterize the mechanisms underlying the regulation of adult NSC fate. In this dissertation, we demonstrate a requirement for the Rb/E2F axis in the regulation of the molecular program instructing adult NSC quiescence and activation, with a potential role in the impaired hippocampal function observed in Alzheimer's disease pathology. While Rb plays a role in the production and survival of hippocampal newborn neurons, we identify a collective requirement for Rb family proteins — pRb, p107 and p130 — as well as their targets, E2F family transcriptional activators E2F1 and E2F3, in the regulation of NSC quiescence and activation. We further demonstrate that this is mediated through pivotal factors REST and ASCL1, identified as direct molecular targets of the Rb/E2F axis, and that REST inactivation can partially rescue NSC depletion following Rb family loss. We finally demonstrate impaired NSC activation and a return to quiescence in the 3xTG-AD model of Alzheimer's disease, with altered expression of Rb/E2F genes observed within cell population-specific defects. Ultimately, this work addresses the key issue of how transcriptional signatures of quiescence and activation among adult NSCs are co- ordinated with cell cycle control, and demonstrates that Rb family proteins serve as master regulators of the molecular program instructing adult NSC exit from and re-entry into quiescence.