Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.     

CHX10 targets a subset of photoreceptor genes

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.     

Expression of Chx10 and Chx10-1 in the developing chicken retina

We have isolated full-length cDNAs of chick Chx10 and Chx10-1, two members of the paired type homeobox/CVC gene family. A comparison of sequences suggests that Chx10 is closely related to Alx/Vsx-2 and Vsx-2 of zebrafish and goldfish, respectively; while Chx10-1 is closely related to Vsx-1 of zebrafish and goldfish. Chx10 and Chx10-1 are expressed in the early retinal neuroepithelium, but not in the pigment epithelium and lens. The expression of Chx10 is present in most retinal neuroblasts, while Chx10-1 exhibits a novel pattern along the nasotemporal border. In the differentiating retina, both Chx10 and Chx10-1 are restricted to bipolar cells and are maintained at a low level in bipolar cells of the mature retina.     

Roles of homeobox and bHLH genes in specification of a retinal cell type

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.     

Expression of Vsx transcription factors in the morphogenesis of retina in the chicken <Emphasis Type="Italic">Gallus domesticus</Emphasis>

Using PCR analysis and immunofluorescence staining, we have investigated the expression of homeobox genes Vsx1/Chx10-1 and Vsx2/Chx10 from the Vsx family (visual system homeobox) during retinal morphogenesis in the chicken Gallus domesticus. It was found that the expression of the studied genes starts at the early stages of embryogenesis. It was shown that the proteins of Vsx1 and Vsx2 are localized in the bipolar cells of the inner nuclear layer of the forming retina. The participation of Vsx1/Chx10-1 and Vsx2/Chx1 in the regulation of retinal cell differentiation in various species of vertebrates and in humans was discussed.     

A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina

Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.     

Partial rescue of the ocular retardation phenotype by genetic modifiers

The or(J) allele of the murine ocular retardation mutation is caused by a premature stop codon in the homeodomain of the Chx10 gene. When expressed on an inbred 129/Sv strain, the or(J) phenotype is characterized by microphthalmia and a thin, poorly differentiated retina in which the peripheral portion is affected to a greater extent than the central portion. Such mutant retinae lack differentiated bipolar cells and the optic nerve typically fails to form, leading to blindness. Here, we show that progeny from an outcrossed backcross between 129/Sv-or(J) /or(J) and Mus musculus castaneus produce animals that are homozygous for the or(J) mutation and exhibit a much ameliorated eye phenotype. Although not of normal size, such modified or(J) eyes are significantly larger than those in 129/Sv-or(J) /or(J) mice, and contain a better organized retina which includes bipolar cells. Furthermore, optic nerves are frequently present, and the eyes show a degree of function as reflected by electroretinogram and pupillary response. As in 129/Sv-or(J) /or(J) mice, however, modified or(J) eyes show incomplete growth and a lack of cell differentiation in the periphery of the retina. The selective, and apparently nonmodifiable, effect of the ocular retardation phenotype on the periphery of the retina indicates that Chx10 plays an important role in the central-to-peripheral gradient of retinal development. These findings demonstrate that the ocular retardation phenotype can be greatly modified by the genetic background, and help to define a role for Chx10 in ocular development.