Lijoy K. Mathew

Unraveling tissue regeneration pathways using chemical genetics.

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.

Aryl hydrocarbon receptor activation inhibits regenerative growth.

There is considerable literature supporting the conclusion that inappropriate activation of the aryl hydrocarbon receptor (AHR) alters cellular signaling. We have established previously that fin regeneration is specifically inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult zebrafish and have used this in vivo endpoint to evaluate interactions between AHR and growth-controlling pathways. Because there are experimental limitations in studying regeneration in adult animals, we have developed a larval model to evaluate the effect of AHR activation on tissue regeneration. Two-day-old zebrafish regenerate their amputated caudal fins within 3 days. Here, we demonstrate that TCDD specifically blocks regenerative growth in larvae. The AHR pathway in zebrafish is considerably more complex than in mammals, with at least three zebrafish AHR genes (zfAHR1a, zfAHR1b, and zfAHR2) and two ARNT genes (zfARNT1 and zfARNT2). Although it was presumed that the block in regeneration was mediated by AHR activation, it had not been experimentally demonstrated. Using antisense morpholinos and mutant fish lines, we report that zfAHR2 and zfARNT1 are the in vivo dimerization partners that are required for inhibition of regeneration by TCDD. Several pathways including fibroblast growth factor (FGF) signaling are essential for fin regeneration. Even though impaired FGF signaling and TCDD exposure both inhibit fin regeneration, their morphometric response is distinct, suggesting that the mechanisms of impairment are different. With the plethora of molecular and genetic techniques that can be applied to larval-stage embryos, this in vivo regeneration system can be further exploited to understand cross-talk between AHR and other signaling pathways.

Crosstalk between AHR and Wnt signaling through R-Spondin1 impairs tissue regeneration in zebrafish

Exposure to dioxins, including 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), causes a wide array of toxicities in vertebrates, which are mostly considered to be mediated through the inappropriate activation of the aryl hydrocarbon receptor (AHR) signaling pathway. Although transcriptional regulation by AHR is widely studied, the molecular mechanisms responsible for the adverse outcomes after AHR activation are largely unknown. To identify the important downstream events of AHR activation, we employed the zebrafish caudal fin regeneration model, where AHR activation blocks the regenerative process. Comparative toxicogenomic analysis revealed that both adult and larval fins respond to TCDD during regeneration with misexpression of Wnt signaling pathway members and Wnt target genes. R‐Spondin1, a novel ligand for the Wnt coreceptor, was highly induced, and we hypothesized that misexpression of R‐Spondin1 is necessary for AHR activation to block regeneration. Partial antisense repression of R‐Spondin1 reversed the inhibitory effect of TCDD, and tissue regeneration was restored. This finding demonstrates that inhibition of regeneration by TCDD is mediated by misinduction of R‐Spondin1. Because R‐Spondin1 signals through the Wnt coreceptor LRP6, we further demonstrated that the TCDD‐mediated block in regeneration is also LRP6 dependent. Collectively, these results indicate that inappropriate regulation of R‐Spondin/LRP6 is absolutely required for TCDD to inhibit fin regeneration.—Mathew, L. K., Sengupta, S. S., LaDu, J., Andreasen, E. A., Tanguay, R. L. Crosstalk between AHR and Wnt signaling through R‐Spondin1 impairs tissue regeneration in zebrafish. FASEB J. 22, 3087–3096 (2008)

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.

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

Background 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. Methodology/Principal Findings 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. Conclusions 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.

AHR-dependent misregulation of Wnt signaling disrupts tissue regeneration.

The origins of molecular toxicology can be traced to understanding the interactions between halogenated aromatic hydrocarbons and the aryl hydrocarbon receptor (AHR). The physiological consequences of activation of the aryl hydrocarbon receptor are diverse, and we are just beginning to understand the importance of the AHR signal transduction pathway in homeostasis and disease. The many downstream targets that mediate these biological responses remain undefined. Studies have exploited the power of the zebrafish model to elucidate the mechanisms by which AHR activation disrupts biological signaling. Recent genomic analysis performed in a zebrafish tissue regeneration model revealed functional cross talk between AHR and the well-established Wnt/beta-catenin signal transduction pathway. This review focuses on the development of the zebrafish model of AHR biology and the application of in vivo toxicogenomics to unravel molecular mechanisms.

Alternate glucocorticoid receptor ligand binding structures influence outcomes in an in vivo tissue regeneration model.

Since their characterization, glucocorticoids (GCs), the most commonly prescribed immunomodulatory drugs, have undergone numerous structural modifications designed to enhance their activity. In vivo assessment of these corticosteroid analogs is essential to understand the difference in molecular signaling of the ligands that share the corticosteroid backbone. Our research identified a novel function of GCs as modulators of tissue regeneration and demonstrated that GCs activate the glucocorticoid receptor (GR) to inhibit early stages of tissue regeneration in zebrafish (Danio rerio). We utilized this phenomenon to assess the effect of different GC analogs on tissue regeneration and identified that some GCs such as beclomethasone dipropionate (BDP) possess inhibitory properties, while others, such as dexamethasone and hydrocortisone have no effect on regeneration. We performed in silico molecular docking and dynamic studies and demonstrated that type and size of substitution at the C17 position of the cortisol backbone confer a unique stable conformation to GR on ligand binding that is critical for inhibitory activity. In the field of tissue regeneration, our study is one of the first Structure Activity Relationship (SAR) investigations performed in vertebrates demonstrating that the in vivo tissue regeneration model is a powerful tool to probe structure function relationships, to understand regenerative biology, and to assist in rational drug design.