A Molecular Analysis of Skeletal Morphogenesis in the Sea Urchin Embryo

Cell migration and differentiation are fundamental aspects of embryogenesis, essential to the development of any complex multicellular organism. Like most biological processes, the directional migration of different cell types and their differentiation into various specified cells with unique functions are regulated by intricate mechanisms, many details of which remain unresolved. The sea urchin embryo, which is optically clear and amenable to a wide variety of experimental manipulations, is an excellent model system to study these processes. Of specific significance is the formation of the embryonic endoskeleton, in which early cell migration and differentiation events can be observed in vivo. The sea urchin embryonic endoskeleton is formed by the sequential ingression, directed migration, and fusion of the primary mesenchyme cells (PMCs). The fused PMCs then secrete a calcareous matrix, forming the characteristic rigid endoskeleton of the embryo. The mechanisms governing skeletogenesis have been of interest to researchers for decades. However, several aspects of its regulation are still unclear.

The work described in this thesis details progress made in understanding cell migration and differentiation using skeletogenesis in the sea urchin embryo as a model. Skeletogenesis is regulated by a complex gene regulatory network (GRN) which is arguably the most complete developmental GRN presently available. The aim of this work was to build linkages between the components of this GRN and observable morphological events during skeletogenesis. Recent research into skeletogenesis has been mainly focused on deciphering the roles that upstream transcription factors play in the specification of PMCs. Hence, a significant gap exists in our knowledge of the functions of downstream morphoeffector genes regulated by these well-studied transcription factors. To this end, we have analyzed the roles of two novel morphoeffector genes, p58-a and p58-b, which encode similar type 1 transmembrane proteins. These two genes are expressed specifically in the PMCs throughout development. We find that the knockdown of either p58-a or p58-b results in defects in skeletogenesis, though PMC specification, migration and fusion occur unperturbed. We conclude that p58-a and p58-b most likely play a role in biomineralization.

Additionally, we describe progress made in understanding the role that ectodermal cues play during skeletogenesis, another poorly understood aspect of this process. The precise and extremely replicable pattern of PMC migration to specific sites within the blastocoel during skeletogenesis has long been of interest to researchers. However, the molecular mechanisms controlling this process have remained mostly elusive. Recent studies have identified the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling pathways as playing significant roles in regulating cell migration and differentiation during skeletogenesis in the sea urchin species Paracentrotus lividus, though these studies provided few details on the specific roles each of these pathways play. The FGF and VEGF pathways have long been shown to play complex, sometimes interacting roles in cell migration during development, and our research aimed at revealing the fine details of their functions in the sea urchin embryo. We have found that in the sea urchin species Lytechinus variegatus, VEGF signaling plays a more significant role in regulating skeletogenesis than the FGF pathway. Blocking VEGF signaling leads to profound defects in skeletogenesis: all aspects of PMC migration are abolished in these morphants, and the extension of filopodia from the PMCs is compromised. We have also identified a separate role for VEGF signaling in the synthesis of the endoskeleton and in regulating the expression of several morphoeffector genes in the PMC gene regulatory network. Conversely, we observed that inhibiting FGF signaling does not lead to severe defects in skeletogenesis, as FGF morphant embryos form extensive skeletal elements. Lastly, we document the presence of reciprocal signals from the PMCs regulating gene expression in the ectoderm, a phenomenon not previously described. These findings significantly expand our understanding of the regulation of directional cell migration and differentiation during embryonic development.