Plastic Surgery Research Council
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Presenter: Michael Grimaldi, Student
Co-Authors: Kong Y; Liao EC
Massachusetts General Hospital Harvard Medical School

Background: It has long been hypothesized that neural crest (NC) stem or progenitor cells exist and they are able to undergo self-renewal, proliferation and multi-lineage differentiation. Most of the NC stem-like properties described to date were gleaned from in vitro assays, such as neural tube explants. In order to generate an in vivo regeneration assay, we develped a lineage-restricted inducible transgenic model where NC cells can be selectively ablated and their regenerative potential be assessed.

Methods: sox10:NTR-GFP transgenic animal was generated by Tol2 transposase mediated germline integration, where sox10 promoter drives the expression of bacterial nitroreductase (NTR), which catalyzes the production of cytotoxin from innocuous prodrug metrodinazole (Mtz), thereby inducing specific cell death in the NC cells. 8 mM Mtz was administrated to developmental zebrafish embryos from 10-somites to 96 hpf. Craniofacial sturcture was assayed by Alcian blue staining. Wholemount RNA in situ hybridization (WISH) was used for gene expression analysis.

Results: Progeny of stable transgenics was screened by GFP fluoresence and subjected to Mtz exposure. Compared to WT control, NTR/Mtz embryos showed severe disruption of maxillary and mandible skeleton by D4, demonstrating the robust ablation of NCC-derived chondrocytes. Meanwhile, NTR/Mtz embryos exhibit small head with dramatic loss of pharynx tissues, and severe reductions in NCC-derived melanophores. WISH showed that the expression of NCC markers sox9a, mitf, and foxd3 were dramatically reduced.

Conclusion: We have generated a novel transgenic model where NCC can be identified and conditionally ablated throughout embryogenesis. This animal model provides us a useful substrate to interrogate NC regenerative capacity by cell transplantation either from in vivo isolated or in vitro cultured NC. This system also allows us to identify the NC regenerative niche in the adult animal.

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