Graduate Thesis Or Dissertation
 

Unraveling tissue regeneration using chemical genetics

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  • The emerging field of regenerative medicine is mainly approached by two different aspects. First is the use of stem cell based models to generate a suite of differentiated cells for therapeutic applications and the alternative approach is to utilize the non-mammalian models that have the inherent capacity to regenerate their body parts. Zebrafish caudal fin regeneration is a well established research system to understand the basic principles of tissue regeneration. We combined a toxicological, a chemical genetic and a candidate gene approach to define the molecular signaling pathways important for regeneration. TCDD, an aryl hydrocarbon receptor (AHR) ligand was used as a chemical probe to impair regeneration and we identified that AHR2 and ARNT1 are the in vivo molecular partners for TCDD-mediated inhibition of regeneration. We further performed a global genomic analysis in the regenerating fin tissue after TCDD exposure to identify the downstream target genes modulated by AHR activation. Functional grouping of the differentially expressed genes by TCDD revealed misexpression of Wnt signaling genes as well as Wnt target genes, suggestive of a cross talk between AHR and Wnt signaling pathways. We hypothesized that, mis-expression of R-Spondin1, a TCDD-induced gene as well as a novel ligand for Wnt co-receptor LRP6 was responsible for the differential expression of the Wnt target genes. Partial antisense repression of R-Spondin1 or LRP6 prevented the inhibition of regeneration by TCDD, indicating that mis-induction of R-Spondin1 which mediates through LRP6 is absolutely required for TCDD-mediated inhibitory effect on fin regeneration. Understanding the advantages of chemicals to probe tissue regeneration, we developed a rapid throughput regeneration assay to identify additional small molecules that modulated regeneration. Glucocorticoids were identified as inhibitors of regeneration and we demonstrated that glucocorticoid receptor activation is absolutely required for mediating the inhibition of regeneration. We further illustrated that, signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capability in vertebrates through an acute inflammation-independent mechanism and also report that, neutrophils and macrophages are not required for fin regeneration. Finally, we performed a comparative global genomic analysis between different zebrafish regeneration models and identified raldh2, a rate limiting enzyme for retinoic acid (RA) synthesis as a candidate gene across the distinct regeneration models. We demonstrated that, in addition to the well established role of RA signaling during the later phase of regenerative outgrowth, this signaling pathway is also critical for the initiation of regeneration, suggesting a dual phase of RA signaling during fin regeneration. Collectively, our results obtained through different experimental approaches suggest that, epimorphic regeneration is completed by a well orchestrated process of multiple molecular signaling events.
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