Chemical and biological approaches to identify vertebrate tissue regeneration pathways

Numerous human conditions would be improved if therapies to encourage tissue regeneration were available. The goal of regenerative medicine is to encourage the body's intrinsic ability to repair and restore tissues lost by disease, injury or aging. While certain vertebrates have the inherent capacity to regenerate, mammals do not. To study tissue regeneration we developed an early life stage zebrafish model. Through comparative global mRNA expression analysis in regenerating tissues isolated from adult caudal fins, hearts and larval fins, we discovered that raldh2 is induced across all regenerating platforms and its expression is critical during early stages of regeneration. Our studies determined the role of Wnt and Fgf in larval regenerating tissue, establishing the early life stage model as a powerful platform to study regeneration. Utilizing this model we developed a rapid in vivo larval regeneration assay to identify small molecule modulators of regeneration. Our initial screening of a 2000 member FDA approved drug library identified eight glucocorticoids (GCs) that inhibited regeneration. We chose beclomethasone dipropionate (BDP) as a representative glucocorticoid receptor (GR) ligand and performed mRNA expression analysis in BDP exposed fin regenerates to identify downstream effectors of GR that are required to block tissue regeneration. Bioinformatic analysis revealed that Cripto-1 mRNA expression increased significantly following BDP exposure. We hypothesized that misexpression of Cripto-1, an Activin inhibitor, was necessary for GR ligands to block tissue regeneration. Suppression of Cripto-1 by morpholino or retinoic acid exposure restored regeneration in the presence of BDP supporting our hypothesis. Our chemical biological screen also identified 21 glucocorticoids that activated GR but did not impact regeneration. We hypothesized that differences in ligand structure induced alternate GR conformational changes and these structural differences resulted in distinct regenerative activity. Docking studies identified that ligands with large substitutions at position17 induce an energetically stable active GR confirmation that correlates with the blocking of tissue regeneration. Our research identified novel GR ligands with cortisol backbones and bulky C17 substitutions that confirmed our hypothesis. Collectively, our results demonstrate the power of the larval zebrafish regeneration model to understand the pathways that permit tissue regeneration.