Study of regulatory genes in retinal pigmented epithelium cell reprogramming and differentiation of retinal anlage during eye regeneration in the newt

摘要

Adult newts (Urodela) are able of eye regeneration even after complete removal of the original neural retina (NR). Because of this, visual system of the newt is a unique in vivo model for study of molecular mechanisms regulating retinal pigmented epithelium (RPE) cell proliferation and differentiation, from one side, and morphogenesis of newly forming NR, from another. At the basis of NR regeneration in the newt is RPE cell transdifferentiation, the process resembling a lot of the acquiring of "stemnessy" by iPSCs, what makes the model quite relevant in the present. After retina removal, RPE cells loose cell type identity, stop to synthesize melanin, loose pigment granules and epithelial morphology, re enter cell cycle with altered parameters, change cytoskeleton protein range and cell microsurrounding. Then, dedifferentiated RPE cells form transitory, neuroblast like cell population enable to produce all cell types of definitive NR. In parallel, in the newt, throughout their life, there is a slow growth of the cells at the periphery edge of the retina, in the ciliary marginal zone (CMZ) that consisting a small population of stem cells. Multipotent cells of the CMZ are also involved in NR regeneration in the newt. We study on the role of regulatory genes and signaling pathways which control RPE cell reprogramming to a stem cell like state, subsequent differentiation to cell types of the NR and morphogenesis of the latter in the newt. Using PCR, real time PCR and immunochemistry we analyzed the expression patterns of genes encode to signaling FGF 2 FGFR, transcriptional factors Pax6, Otx2, Prox1, Six3, Sox2, Pitx1, Pitx2, and nucleostemin, a multiplex regulator of cell cycle progression. Cell proliferation was studied also by means of several markers: [3H] TdR, PCNA and BrdU. Cell phenotypes of transdifferentiating RPE and regenerating NR were determined by means of cell type specific markers: RPE65; b II tubulin, rhodopsin, and recoverin. We found the expression of FGFRs in the RPE and their activation by FGF2 signals in early and late time courses of NR regeneration. The co expression of the proteins FGF2, FGFR2, Pax6, Otx2, Prox1, Six3, Sox2, Pitx2 and nucleostemin was detected at early stages of NR regeneration in proliferating multipotent stem like neuroblasts from which the NR primordium was formed. The vivid e xpression of regulatory proteins we studied (FGF2, FGFR2, Pax6, Prox1, Six3, Rx, So x2, Pitx2, nucleostemin and PCNA) were localized also in CMZ. In other words, the early NR regenerate and CMZ of the adult newt maintains regulative network described during embryonic eye development. Differences in the expression patterns of studied molecules may arise at the level of interactions of similar molecular components of the network, determining both, NR development and regeneration