Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

The roles of Pax6 in the cornea,retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.     

Early and late changes in Pax6 expression accompany eye degeneration during cavefish development

We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.     

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.     

Cre-loxp fate-mapping of Pax6 enhancer active retinal and pancreatic progenitors

Pax6 plays important roles in the control of ocular and pancreatic development. We identified a 450 bp Pax6 enhancer that contains two interacting sequences: a 274 bp fragment sufficient for expression in retinal progenitors and an adjacent 156 bp fragment required for expression in pancreatic progenitors. Since this enhancer is only transiently expressed during embryogenesis, a Cre-loxP fate-mapping strategy was used to investigate the developmental potential of these progenitors. Surprisingly, the labeled retinal precursors predominantly gave rise to horizontal cells, indicating a cell lineage role in horizontal cell differentiation. In the pancreas, all enhancer-specific cells were restricted to endocrine and ductal cell lineages. This result lends support to a model whereby Pax6-expressing progenitors contribute to the adult pancreatic islets and ducts. The progenitor cell-specificity of this enhancer will be useful in studies that require either cell-specific expression or conditional gene inactivation in these cell populations.