Tag: Scott Stewart

Short Range Sonic Hedgehog Signaling Promotes Heterotypic Cell Interactions Underlying Branching Morphogenesis of the Zebrafish Fin Skeleton

Presenter(s): Joshua Braunstein – Biology

Faculty Mentor(s): Kryn Stankunas, Scott Stewart

Poster 83

Research Area: Developmental Biology

Funding: ESPRIT
IMB Summer Scholarship Award
Alden Award

Zebrafish remarkably regenerate severed fins, perfectly restoring their original size and branched skeletal pattern. Sonic hedgehog a (Shha)-expressing epidermal cells mediate ray branching during regeneration by guiding localization of the pre-osteoblasts (pObs) while migrating and splitting into two populations. However, mechanisms of shha induction, the splitting of shha+ epidermal cells, and the mechanisms underlying epidermal to pObs interactions remain unresolved. Towards answering these questions, we explored if Hh/Smo signaling and epidermal dynamics also underlie developmental ray branching. We found that shha is expressed initially in basal epidermal cells along the entire length of forming fin bones in juvenile fish. As bones progressively mature, shha becomes distally restricted to epidermal cells neighboring Runx2+ pObs. We used TgBAC(ptch2:Kaede) fish and photoconversion to show Hh/Smo signaling is restricted to these pObs and immediately adjacent epidermal cells. shha+ epidermal cells split into two groups immediately preceding ray branching. By live imaging, we found these basal epidermal cells migrate distally over the pObs, cease Hh/Smo signaling, and are then shed. Small molecule inhibition of Hh/Smo using BMS-833923 increased epidermal migration speed, suggesting Hh/Smo signaling typically restricts the rate of migration by adhering epidermal cells to the pObs. Additional small molecule trials show the pathway is largely dedicated to ray branching during fin development. We conclude that instructive shha+ epidermal movements and Shh/Smo-promoted adhesion between epidermal cells and pObs direct branching morphogenesis to pattern the fin skeleton during both development and regeneration.

Mutational Analysis of dach Genes During Zebrafish Fin Regeneration

Presenter(s): George Deardorff

Co Presenter(s): Bryson Ramona

Faculty Mentor(s): Kryn Stankunas & Scott Stewart

Poster 44

Session: Sciences

Following amputation, zebrafish fins, comprised of intricate skeletal rays and other tissues, perfectly regenerate to their original size and shape regardless of the nature or position of injury. A cell population observed in the regenerating fin, termed “niche”, produces Wnt signals that promote fin outgrowth. As a known transcriptional regulator of the niche, dach plays a role in maintaining proper regeneration. Depending upon the extent of regenerative demand, dach becomes enriched at the distal region of the regenerating fin, and is eventually downregulated once the fin has stopped regenerating. A mechanistic explanation for dach induction, in addition to a thorough understanding of how it regulates the niche, is lacking. To explore the role of dach we used CRISPR/Cas9 gene editing to mutate two isoforms of the dach gene, dachc and dacha. By utilizing high-resolution fin imaging, our data showed that dachc mutants display improper morphology, including: abnormal joint segmentation, fusion of rays, and trident-shaped branching patterns. Surprisingly, this observation was not seen during development, but rather only after amputation and subsequent regeneration. Further, while dacha single mutants exhibited normal regeneration, dachc;dacha double mutants died off at an appreciable rate during development. Our data shows a novel role that dach has in promoting correct branching patterns, in addition to its unique regulation in development versus regeneration. Demonstrating how the misregulation of certain genes like dach can lead to the disruption of growth control mechanisms is critical for understanding the basis of a range of diseases.

Polycomb Repressive Complex 2 Ensures Robust Skeletal Growth and Patterning During Zebrafish Fin Regeneration

Presenter(s): Bryson Tyler Ricamona—Biology

Faculty Mentor(s): Scott Stewart, Kryn Stankunas

Session 5: The Bonds that Make Us

After amputation zebrafish regenerate their fins back to the correct size and shape . Fin bone regeneration is driven by an endogenous “stem cell” population generated by dedifferentiation of mature osteoblasts at the amputation site . The resulting osteo-progenitors both self-renew and re-differentiate until regeneration is complete . Yet it is unknown how mature osteoblasts reprogram and change gene expression patterns upon dedifferentiation . Recent in mammal work links chromatin function and covalent modification of histones to cellular potency and differentiation . Ezh1 and Ezh2 are key subunits of Polycomb Repressive Complex 2 (PRC2) that tri-methylates lysine 27 of histone H3 (H3K27me3) to maintain repressed states of developmental regulatory genes in mammals . To test if PRC2 is required for dedifferentiation during fin regeneration we analyzed regeneration in ezh1 and ezh2 mutant zebrafish . Here we show that, although ezh1-/-; ezh2+/- mutant fins regenerated largely to the same size as wildtype, they display notable defects in bone patterning . These defects, including the formation of large bony plates and the fusion of adjacent rays occur within 5 days post-amputation suggesting PRC2 is needed for a relatively early phase of regeneration . Such defects are exacerbated when PRC2 mutants are subjected to a second round of amputation in the regenerated region, possibly due to an increased amount of cells with abnormal H3K27me3 levels leading to dysregulation of gene expression . This suggests that PRC2 is a necessary regulator in the lineage specific osteoblast pathway during regeneration due to observations of abnormal bony ray morphology .