Hapln1a-ECM mediates Cx43 dependent skeletal phenotypes during zebrafish fin regeneration

Abstract Skeletal development is a tightly regulated process and requires proper communication between the cells for efficient exchange of information. Cell–cell communication, facilitating the exchange of small metabolites, ions and second messengers, takes place via aqueous proteinaceous channels called gap junctions. Connexins (Cx) are the subunits of a gap junction channel, and Cx43 is the major Cx expressed in bone cells. Mutations in human and mouse Cx43 result in a severe skeletal disorder called oculodentaldigital dysplasia (ODDD) characterized by craniofacial abnormalities and limb deformities. Mutations in zebrafish cx43 produces the short fin (sof b123) phenotype and is characterized by short fins due to reduced segment length of the bony fin rays and reduced cell proliferation. The mechanism by which CX43-based mutations cause skeletal defect phenotypes is largely unknown. However, it is apparent that the function of Cx43 in the vertebrate skeleton is conserved. Hence, it is important to understand the role of Cx43 during skeletal development.The zebrafish caudal fin is an excellent model for studying bone/skeletal morphogenesis during fin regeneration for several reasons. The fin has a simple architecture with few tissue types. The bony fin rays made up of joints and segments are clearly visible, allowing for easy genetic manipulation, and the fin can completely regenerate within 2 weeks after amputation. An important, yet poorly understood, question with respect to mutations in connexin genes, in general, is how does gap-junctional intercellular communication (GJIC) impact tangible cellular events like cell division and differentiation? One hypothesis is that Cx43-based GJIC can influence gene expression patterns. Our lab exploited the availability of the two mutants, sof b123 and alf dty86, in order to identify those genes whose expression depends on Cx43. Previously established results from our lab using the fin mutants demonstrate that Cx43 plays a dual role, regulating both cell proliferation (growth) and joint formation (patterning) during the process of skeletal morphogenesis. Thus, we utilized a novel microarray strategy to identify a set of candidate genes, which are both downregulated in sof b123 and upregulated in alf dty86. Hapln1a (Hyaluronan and Proteoglycan Link Protein 1a) is one among the several genes identified by the microarray analysis. The focus of this thesis was to elucidate the role and mechanism of Hapln1a in mediating Cx43 function during skeletal development in the regenerating zebrafish fin. Hapln1a belongs to the family of link proteins that play an important role in stabilizing the extracellular matrix (ECM) by linking the aggregates of hyaluronan (HA) and proteoglycans (PGs). In the first part of this study, we have shown that Hapln1a is molecularly and functionally downstream of Cx43, and knockdown of hapln1a resulted in reduced segment length, cell proliferation, and reduced HA. In the second part of the study, we have shown that besides destabilization of HA, hapln1a knockdown results in reduced aggrecan (Acan) and provides evidence that both HA and Acan are required for skeletal growth and patterning. Additionally, we show that the Hapln1a–ECM stabilizes the secreted growth factor Semaphorin3d (Sema3d) and Hapln1a-dependent ECM provides the required conditions for Sema3d stabilization and function. This study demonstrates the requirement for components of the Hapln1a–ECM for Sema3d signal transduction. ECM plays a dynamic role during the process of wound healing, embryogenesis, and tissue regeneration, and in the final part of the study, we have shown that a transitional matrix analogous to the one formed during newt skeletal and heart muscle regeneration is synthesized during fin regeneration. We have demonstrated that a provisional matrix rich in hyaluronic acid, tenascin C, and fibronectin is synthesized following amputation. Additionally, we observed that the link protein Hapln1a-dependent ECM, consisting of Hapln1a, HA, and PG aggrecan, is upregulated during fin regeneration. Our findings on zebrafish fin regeneration implicates that changes in the extracellular milieu represent an evolutionarily conserved mechanism that proceeds during tissue regeneration, yet with distinct players depending on the type of tissue that is involved. Collectively data from this dissertation provide evidence that Cx43 and Hapln1a–ECM function in a common pathway to coordinate skeletal growth and patterning. Moreover, this study provides novel insights into the mechanistic role of the ECM and, in particular, the role of Hapln1a–ECM during vertebrate skeleton regeneration that has not yet been elucidated in any other mammalian systems.