This project will provide the applicant with Ph.D. training in cell and developmental biology. Thesis research will further our understanding how cell signaling networks regulate skeletal pattering of zebrafish fin appendages during development and regeneration. The applicant also will pursue professional development, mentorship, and advocacy activities during the fellowship period. Zebrafish fins robustly develop and regenerate with a branched skeleton of bony rays, which are likely analogous to tetrapod digits. Ray bifurcation occurs by the splitting of progenitor osteoblast (pOb) pools that progressively produce extending skeletal rays. Sonic hedgehog signaling is specifically required for ray branching during development and regeneration. In both cases, shha is uniquely expressed by small basal epidermis domains overlying pOb pools found at the distal aspect of outgrowing fins. Basal epidermal cells constantly migrate distally, upregulating shha only when moving into the pOb-defined growth zone. Importantly, ray branching morphogenesis appears to initiate by the lateral splitting of the shha+-epidermal domain followed by Shh-responsive pObs. Therefore, a key unresolved upstream step in ray branching is the transcriptional activation of basal epidermal shha. This proposal aims to uncover mechanisms of skeletal morphogenesis by investigating how shha is induced in pOb-neighboring basal epidermis. Previous studies and the applicant?s preliminary data suggest canonical Wnt/b-catenin, well known to be required for fin regenerative outgrowth, is upstream of basal epidermal shha. Further, the applicant has identified candidate conserved binding sites for the canonical Wnt effector, Lef1, within a functionally important shha regulatory region. Collectively, these and other results support the applicant?s hypothesis that a pOb-produced Wnt activates canonical Wnt/b-catenin signaling in nearby basal epidermal cells to induce shha expression. The applicant will explore this hypothesis with two specific aims.
Aim 1 will determine how Wnt signaling acts upstream of Shh signaling for ray branching morphogenesis using inducible in vivo manipulations of canonical Wnt activity and CRISPR/Cas9 mutagenesis of a canonical pOb-expressed Wnt.
Aim 2 will explore functional requirements of the candidate Lef1 binding sites within the shha locus. Further, the applicant will use comparative genomics and mutagenized transgenic reports to identify cis- regulatory elements specifically driving shha expression in distal fin basal epidermis. Together, these aims will provide a detailed understanding how intersecting signaling pathways direct skeletal patterning of fin appendages. The proposed research will uncover Shh signaling mechanisms of potential relevance in many biomedical contexts and support novel regenerative medicine approaches for bone damage and disease.
Humans possess limited capacity to repair skeletal damage from trauma and disease. The proposed research will study zebrafish fin development and its remarkably robust regeneration to uncover conserved signaling mechanisms that direct bone cells to organize into precise skeletal patterns. Insights will motivate novel regenerative medicine approaches that restore bones to their original sizes and shapes.
