RESUMO
We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/fisiologia , Células Endoteliais/fisiologia , Regeneração Tecidual Guiada/métodos , Heterozigoto , Microscopia de Fluorescência , Mutação , Neurônios/metabolismo , Fosfolipase C gama/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regeneração/fisiologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Engenharia TecidualRESUMO
Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. Zebrafish fin regeneration is mediated by the creation of blastema cells. Here, we characterize the devoid of blastema (dob) mutant that fails fin regeneration during initial steps, forms abnormal regeneration epithelium, and does not form blastema. This mutation has no impact on embryonic survival. Dob results from an fgf20a null mutation, Y148S. Fgf20a is expressed during initiation of fin regeneration at the epithelial-mesenchymal boundary and later overlaps with the blastema marker msxb. Thus, fgf20a has a regeneration-specific requirement, initiating fin regeneration, and controlling blastema formation.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Regeneração/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Extremidades , Proteínas de Homeodomínio/biossíntese , Masculino , Mesoderma , Mutação , Regeneração/genética , Temperatura , Cicatrização , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
Zebrafish fin regeneration requires the formation and maintenance of blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for blastema formation and maintenance. We used a chemical mutagenesis screen to identify no blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue.