Abstract
Purpose: :
Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we tested the hypothesis that heparin binding epidermal-like growth factor (HB-EGF) is a secreted MG-derived factor that stimulates MG dedifferentiation and retina regeneration.
Methods: :
Eye lesions were performed by needle poke. Dividing cells were labeled with BrdU. Growth factors were delivered into the uninjured eyes' vitreous through the cornea. RT-PCR was combined with in situ hybridization and immunohistochemistry to identify the expression of regeneration-associated genes and regenerated cell types. The function of proteins induced during regeneration was explored by knocking down their expression with morpholino-modified antisense oligonucleotides introduced into the retina by electroporation.
Results: :
Within 1 hr post retinal injury, HB-EGF was induced in MG residing at the injury site. Morpholino-mediated HB-EGF knockdown suppressed injury-dependent retina regeneration. Metalloproteinase inhibition suggested proHB-EGF ectodomain shedding was necessary for HB-EGF’s action in the injured retina. Remarkably, HB-EGF stimulated the formation of multipotent MG-derived progenitors in the uninjured retina. HB-EGF mediated its effects via an EGFR/MAPK signal transduction cascade that regulated the expression of regeneration-associated genes, like ascl1a and pax6b. We uncovered an HB-EGF/Ascl1a/Notch/hb-egfa signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/b-catenin signaling cascade that controls progenitor proliferation.
Conclusions: :
These results suggest that HB-EGF may be master regulators of MG dedifferentiation following retinal injury and MG themselves influence their regenerative capacity. This study provides the first link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggests strategies for stimulating retina regeneration in mammals.
Keywords: regeneration • growth factors/growth factor receptors • glia