Unraveling tissue regeneration using chemical genetics

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

The Anti-Inflammatory Drug Leflunomide Is an Agonist of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases.During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR.These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders

Glucocorticoid receptor-dependent induction of () inhibits zebrafish caudal fin regeneration.

We previously used a chemical genetics approach with the larval zebrafish to identify small molecule inhibitors of tissue regeneration. This led to the discovery that glucocorticoids (GC) block early stages of tissue regeneration by the inappropriate activation of the glucocorticoid receptor (GR). We performed a microarray analysis to identify the changes in gene expression associated with beclomethasone dipropionate (BDP) exposure during epimorphic fin regeneration. Oncofetal cripto-1 showed > eight-fold increased expression in BDP-treated regenerates. We hypothesized that the mis-expression of cripto-1 was essential for BDP to block regeneration. Expression of cripto-1 was not elevated in GR morphants in the presence of BDP indicating that cripto-1 induction was GR-dependent. Partial translational suppression of Cripto-1 in the presence of BDP restored tissue regeneration. Retinoic acid exposure prevented increased cripto-1 expression and permitted regeneration in the presence of BDP. We demonstrated that BDP exposure increased cripto-1 expression in mouse embryonic stem cells and that regulation of cripto-1 by GCs is conserved in mammals

Glucocorticoid receptor-dependent induction of () inhibits zebrafish caudal fin regeneration.

We previously used a chemical genetics approach with the larval zebrafish to identify small molecule inhibitors of tissue regeneration. This led to the discovery that glucocorticoids (GC) block early stages of tissue regeneration by the inappropriate activation of the glucocorticoid receptor (GR). We performed a microarray analysis to identify the changes in gene expression associated with beclomethasone dipropionate (BDP) exposure during epimorphic fin regeneration. Oncofetal cripto-1 showed > eight-fold increased expression in BDP-treated regenerates. We hypothesized that the mis-expression of cripto-1 was essential for BDP to block regeneration. Expression of cripto-1 was not elevated in GR morphants in the presence of BDP indicating that cripto-1 induction was GR-dependent. Partial translational suppression of Cripto-1 in the presence of BDP restored tissue regeneration. Retinoic acid exposure prevented increased cripto-1 expression and permitted regeneration in the presence of BDP. We demonstrated that BDP exposure increased cripto-1 expression in mouse embryonic stem cells and that regulation of cripto-1 by GCs is conserved in mammals

Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration*

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling