Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spa(pol), a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a "master" control gene for eye development. Our data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently.     

Pitx2c modulates Pax3+/Pax7+ cell populations and regulates Pax3 expression by repressing miR27 expression during myogenesis

Pitx2 is a paired-related homeobox gene that is expressed in muscle progenitors during myogenesis. We have previously demonstrated that overexpression of Pitx2c isoform in myoblasts maintained these cells with a high proliferative capacity and completely blocked terminal differentiation by inducing high Pax3 expression levels (Martinez et al., 2006). We now report that Pitx2c-mediated proliferation vs. differentiation effect is maintained during in vivo myogenesis. In vivo Pitx2c loss of function leads to a decrease in Pax3+/Pax7− cell population in the embryo accompanied by an increase of Pax3+/Pax7+ cells. Pitx2c transient-transfection experiments further supported the notion that Pitx2c can modulate Pax3/Pax7 expression. Pitx2c but not Pitx3 controls Pax3/Pax7 expression, although redundant roles are elicited at the terminal myoblast differentiation. Contrary to Pitx2c, Pitx3 does not regulate cell proliferation or Pax3 expression, demonstrating the specificity of Pitx2c mediating these actions in myoblasts. Furthermore we demonstrated that Pitx2c modulates Pax3 by repressing miR27 expression and that Pax3-miR-27 modulation mediated by Pitx2c is independent of Pitx2c effects on cell proliferation. Therefore, this study sheds light on previously unknown function of Pitx2c balancing the different myogenic progenitor populations during myogenesis.     

Pax6 and eye development in Arthropoda

The arthropod compound eye is one of the three main types of eyes observed in the animal kingdom. Comparison of the eyes seen in Insecta, Crustacea, Myriapoda and Chelicerata reveals considerable variation in terms of overall cell number, cell positioning, and photoreceptor rhabdomeres, yet, molecular data suggest there may be unexpected similarities. We review here the role of Pax6 in eye development and evolution and the relationship of Pax6 with other retinal determination genes and signaling pathways. We then discuss how the study of changes in Pax6 primary structure, in the gene networks controlled by Pax6 and in the relationship of Pax6 with signaling pathways may contribute to our insight into the relative role of conserved molecular-genetic mechanisms and emergence of evolutionary novelty in shaping the ommatidial eyes seen in the Arthropoda.     

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.     

Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6

We have studied the molecular basis of the Pax2 and Pax6 function in the establishment of visual system territories. Loss-of-function mutants have revealed crucial roles for Pax2 in the generation of the optic stalk and for Pax6 in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under control of Pax2 promoter elements resulted in a shift of the optic cup/optic stalk boundary indicated by the presence of retinal pigmented cells on the optic stalk. By studying mouse embryos at early developmental stages we detected an expansion of Pax2 expression domain in the Pax6(-/-) mutant and of Pax6 expression domain in the Pax2(-/-) embryo. These results suggest that the position of the optic cup/optic stalk boundary depends on Pax2 and Pax6 expression, hinting at a possible molecular interaction. Using gel shift experiments, we confirmed the presence of Pax2- and Pax6-binding sites on the retina enhancer of the Pax6 gene and on the Pax2 upstream control region, respectively. Co-transfection experiments revealed a reciprocal inhibition of Pax2 promoter/enhancer activity by Pax6 protein and vice versa. Based on our findings, we propose a model for Pax gene regulation that establishes the proper spatial regionalization of the mammalian visual system.     

Differential expression of insulin genes 1 and 2 in MIN6 cells and pseudoislets

There is some evidence that the two rodent insulin genes are differentially regulated in mice, although there is no satisfactory consensus on the relative levels and patterns of expression for the two genes. Using the mouse insulinoma cell line MIN6, we have demonstrated by quantitative RT-PCR, differential patterns of expression for the two genes. In mouse islets and early passage MIN6 cells, expression of ins 1 and ins 2 were found to be approximately equal, but levels of ins 1 mRNA diminished rapidly with continued passage. Furthermore, the ins 1 gene was found to be up-regulated in response to glucose stimulation and as a result of increased cell-cell contact, but no effect on the ins 2 gene was observed. Since the MIN6 cell line is frequently used as a beta-cell model for gene expression studies, consideration should be given to both insulin genes.     

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.     

A conserved regulatory element present in all Drosophila rhodopsin genes mediates Pax6 functions and participates in the fine-tuning of cell-specific expression

The Drosophila rhodopsin genes (rh's) represent a unique family of highly regulated cell-specific genes, where each member has its own expression pattern in the visual system. Extensive analysis of the rh's has revealed several functional elements that are involved in cell-specificity. We have investigated the functional role of the RCSI/P3 site that is found in the proximal promoter of all Drosophila rh genes. This sequence is remarkably conserved in evolution and is located 15-30 bp upstream of the TATA box. We have previously shown that, in the context of the rh1 promoter, this element is recognized in vivo by a Pax6 protein, the master regulator of eye development. Thus, rh regulation might represent the ancestral function of Pax6. Here, we investigated the role of the RCSI/P3 sequence in the other rh genes and show that they also mediate Pax6 function. We also tested the potential impact of the various RCSI/P3 sequences on the precise cell-specific expression of rh genes. Our results demonstrate that, even though all RCSI/P3 sequences bind Pax6, they are clearly distinct in various rh promoters and these differences are conserved throughout evolution: RCSI/P3 appears to participate in the fine-tuning of cell-specificity. We also show that Pax6 or a related Pax protein may be involved in the regulation of olfactory genes. Therefore, in addition to performing a global photoreceptor-specific function, RCSI also appears to mediate the combined action of Pax6 and other factors and to contribute to rh regulation in subsets of photoreceptors.