Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration

Abstract

The light-damaged zebrafish retina results in the death of photoreceptor cells and the subsequent regeneration of the missing rod and cone cells. Photoreceptor regeneration initiates with asymmetric Müller glial cell division to produce neuronal progenitor cells, which amplify, migrate to the outer nuclear layer (ONL), and differentiate into both classes of photoreceptor cells. In this study, we examined the role of the Pax6 protein in regeneration. In zebrafish, there are two Pax6 proteins, one encoded by the pax6a gene and the other encoded by the pax6b gene. We intravitreally injected and electroporated morpholinos that were complementary to either the pax6a or pax6b mRNA to knockdown the translation of the corresponding protein. Loss of Pax6b expression did not affect Müller glial cell division, but blocked the subsequent first cell division of the neuronal progenitors. In contrast, the paralogous Pax6a protein was required for later neuronal progenitor cell divisions, which maximized the number of neuronal progenitors. Without neuronal progenitor cell amplification, proliferation of resident ONL rod precursor cells, which can only regenerate rods, increased inversely proportional to the number of INL neuronal progenitor cells. This confirmed that Müller glial-derived neuronal progenitor cells are necessary to regenerate cones and that distinct mechanisms selectively regenerate rod and cone photoreceptors. This work also defines distinct roles for Pax6a and Pax6b in regulating neuronal progenitor cell proliferation in the adult zebrafish retina and increases our understanding of the molecular pathways required for photoreceptor cell regeneration.

Analysis of DNA methylation reveals a partial reprogramming of the Müller glia genome during retina regeneration

“…S1A). MGPC formation is accompanied by the increased expression of pluripotency factors (14) and other key signaling molecules (15)(16)(17)(18)(19)(20)(21). The induction of pluripotency genes, along with the finding that the putative cytidine deaminases Apobec2a and Apobec2b (Apobec2a,2b) are necessary for MGPC formation (22), is consistent with the idea that active modification of the DNA methylation landscape may underlie the reprogramming of MG to MGPCs.…”

Tissue regeneration after injury in adult zebrafish: The regenerative potential of the caudal fin

“…During blastema formation, fgf20a co-expresses with msxb. Increase of msxb expression is required for tissue outgrowth during regeneration (Thummel et al, 2010). Genetic control of organ regeneration has been reported in other zebrafish organs, such as heart, spinal cord, and retina (Curado et al, 2007;Schoenebeck et al, 2007;Qin et al, 2009;Jopling et al, 2010;Montgomery et al, 2010).…”

“…While amphibians have long been the central characters employed in studies on tissue or organ regeneration (Brockes, 1997;Beck et al, 2009;Contreras et al, 2009;Kragl et al, 2009;Calve et al, 2010), the zebrafish (Danio rerio) have recently emerged as a new vertebrate model for genetic studies of tissue/ organ regeneration. Like amphibians, zebrafish exhibit an enhanced capability of regenerating adult tissues, which include retina, spinal cord, kidney, heart, and fin (Poss et al, 2000a(Poss et al, ,b, 2002aNechiporuk and Keating, 2002;Nechiporuk et al, 2003;Jazwinska et al, 2007;Schoenebeck et al, 2007;Tsai et al, 2007;Qin et al, 2009;Jopling et al, 2010;Thummel et al, 2010). Fin regeneration is a particularly efficient model for studying tissue regeneration.…”

Ascl1a/Dkk/β-catenin signaling pathway is necessary and glycogen synthase kinase-3β inhibition is sufficient for zebrafish retina regeneration

“…Ascl1a may also regulate the proliferation of dedifferentiated MG (16). In addition, injury-dependent induction of Pax6 appears to control the expansion of MG-derived progenitors, but not their initial entry into the cell cycle (17). Although injury-dependent induction of Ascl1a and Pax6 are necessary for proliferation of MG-derived progenitors, it is not clear how they are activated or what signaling pathways underlie their effects.…”

“…To investigate whether the GSK-3β inhibitor actually stimulated MG dedifferentiation similar to retinal injury or simply forced MG into the cell cycle, we assayed for regeneration-associated genes such as ascl1a, lin-28, pax6 b , c-myc b , wnt4a, and fzd2 (15)(16)(17). Indeed, the GSK-3β inhibitor stimulated a pattern of gene expression in the uninjured retina that was very similar to that after retinal injury (Fig.…”