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Background

Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells.

Methodology/Principal Findings

Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease.

Conclusions

Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE.

The homeodomain protein, Otx2, is a critical regulator of vertebrate photoreceptor genesis. However, the genetic elements that define the expression of Otx2 during photoreceptor development are unknown. Therefore, we sought to identify an Otx2 enhancer element that functions in photoreceptor development in order to better understand this specification event. Using the technique of electroporation, we tested a number of evolutionarily conserved elements (ECRs) for expression in the developing retina, and identified ECR2 as having robust activity in the retina. We have characterized this element using a number of assays, including Cre-fate mapping experiments. We found that ECR2 recapitulates expression/function of Otx2 primarily in newly postmitotic photoreceptor cells (PRs), as well as in a subset of retinal progenitor cells (RPCs). ECR2 was also found to be expressed in a subset of horizontal cells (HCs), in keeping with the role of Otx2 in HC development. Furthermore, we determined that the ECR2 element is not active in other Otx2-positive cells such as retinal bipolar cells (BPs), retinal pigmented epithelium (RPE), or the tectum, suggesting that the transcriptional networks controlling Otx2 expression in these cells are unique from those of developing PRs and HCs. These results reveal a distinct molecular state in dividing retinal cells and their newly postmitotic progeny, and provide genetic access to an early and critical transcriptional node involved in the genesis of vertebrate PRs.

In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease.

Background

Histone deacetylases (HDACs) play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins.

Results

To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA), was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells.

Conclusion

HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells.

Crx is a member of the Otx family of homeobox genes with expression restricted to vertebrate retinal photoreceptor and bipolar cells as well as the pinealocytes of the pineal organ. To facilitate the visualization of Crx-expressing cells, we generated transgenic mice expressing several reporters under the control of the Crx regulatory sequences present within a bacterial artificial chromosome (BAC). These mice expand the transgenic mouse collection, which uses photoreceptor regulatory elements for reporter gene expression by providing a broader repertoire of reporter genes. In addition, since Crx is expressed very soon after a cell fated to be a photoreceptor cell becomes postmitotic, they provide a means for early identification of immature photoreceptor cells.

Background

Otd-related transcription factors are evolutionarily conserved to control anterior patterning and neurogenesis. In humans, two such factors, OTX2 and CRX, are expressed in all photoreceptors from early specification through adulthood and associate with several photoreceptor-specific retinopathies. It is not well understood how these factors function independently vs. redundantly, or how specific mutations lead to different disease outcomes. It is also unclear how OTX1 and OTX2 functionally overlap during other aspects of neurogenesis and ocular development. Drosophila encodes a single Otd factor that has multiple functions during eye development. Using the Drosophila eye as a model, we tested the ability of the human OTX1, OTX2, and CRX genes, as well as several disease-associated CRX alleles, to rescue the different functions of Otd.

Results

Our results indicate the following: OTX2 and CRX display overlapping, yet distinct subfunctions of Otd during photoreceptor differentiation; CRX disease alleles can be functionally distinguished based on their rescue properties; and all three factors are able to rescue rhabdomeric photoreceptor morphogenesis.

Conclusions

Our findings have important implications for understanding how Otx proteins have subfunctionalized during evolution, and cement Drosophila as an effective tool to unravel the molecular bases of photoreceptor pathogenesis.

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins CRX, OTX1, and OTX2 fulfill these functions. In Drosophila, the single founding-member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.

Retinal stem cells (RSCs) are present in the ciliary margin of the adult human eye and can give rise to all retinal cell types. Here we show that modulation of retinal transcription factor gene expression in human RSCs greatly enriches photoreceptor progeny, and that strong enrichment was obtained with the combined transduction of OTX2 and CRX together with the modulation of CHX10. When these genetically modified human RSC progeny are transplanted into mouse eyes, their retinal integration and differentiation is superior to unmodified RSC progeny. Moreover, electrophysiologic and behavioral tests show that these transplanted cells promote functional recovery in transducin mutant mice. This study suggests that gene modulation in human RSCs may provide a source of photoreceptor cells for the treatment of photoreceptor disease.

The homeobox transcription factor OTX2 plays an essential role during embryonic brain development. It is normally silenced in the adult brain, but is overexpressed by genomic amplification or other mechanisms in the majority of medulloblastomas (MBs). Retinoic acids (RAs) can suppress OTX2 expression and inhibit MB growth. In this study, 9-cis RA most potently inhibited MB cell growth. 9-cis RA functions through the downregulation of OTX2 expression, which subsequently induces neuronal differentiation of OTX2-expressing cells. Treatment with 9-cis RA reduced the growth of D425 flank xenograft tumors in mice. In an intracranial model, however, MB tumors showed resistance to 9-cis RA treatment, and we implicated fibroblast growth factor (FGF) as a potential mediator of resistance to RA therapy. These findings suggest a mechanism for RA-mediated anti-tumor effect on OTX2-positive MB cells and indicate that therapeutic targeting of OTX2 might be effective if FGF pathway-mediated resistance can be overcome.

Otx and Mitf transcription factors have been implicated in the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific roles in the development of the RPE have not been fully defined. The role of the three Otx transcription factors (Otx1a, Otx1b, and Otx2) and two Mitf transcription factors (Mitfa and Mitfb) in the development of the zebrafish RPE was explored in these experiments. The loss of Otx activity through morpholino knockdown produced variable eye defects, ranging from delayed RPE pigmentation to severe coloboma, depending on the combination of Otx factors that were targeted. Expression analysis through in situ hybridization demonstrates that otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcription factors are not required for the expression of otx genes. Surprisingly, the loss of Mitf activity in mitfa, mitfb, or double mitf mutant zebrafish had no effect on RPE pigmentation or development. Moreover, histological analysis revealed that retinal lamination is unaffected in mitf mutants, as well as in otx morphants, even in regions lacking RPE. Otx and Mitf combined loss of function experiments suggest that mitfa and mitfb may still influence zebrafish RPE development. This is further supported by the ability of mitfa to induce pigmentation in the zebrafish retina when misexpressed. These findings suggest that one or more Otx targets in addition to mitfa and mitfb, possibly another mitf family member, are necessary for development of the RPE in zebrafish.

Cadherins are Ca++-dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g.rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.

Background

Homeobox genes of the orthodenticle (otd)/Otx family have conserved roles in the embryogenesis of head and brain. Gene replacement experiments show that the Drosophila otd gene and orthologous mammalian Otx genes are functionally equivalent, in that overexpression of either gene in null mutants of Drosophila or mouse can restore defects in cephalic and brain development. This suggests that otd and Otx genes control a comparable subset of downstream target genes in either organism. Here we use quantitative transcript imaging to analyze this equivalence of otd and Otx gene action at a genomic level.

Results

Oligonucleotide arrays representing 13,400 annotated Drosophila genes were used to study differential gene expression in flies in which either the Drosophila otd gene or the human Otx2 gene was overexpressed. Two hundred and eighty-seven identified transcripts showed highly significant changes in expression levels in response to otd overexpression, and 682 identified transcripts showed highly significant changes in expression levels in response to Otx2 overexpression. Among these, 93 showed differential expression changes following overexpression of either otd or Otx2, and for 90 of these, comparable changes were observed under both experimental conditions. We postulate that these transcripts are common downstream targets of the fly otd gene and the human Otx2 gene in Drosophila.

Conclusion

Our experiments indicate that approximately one third of the otd-regulated transcripts also respond to overexpression of the human Otx2 gene in Drosophila. These common otd/Otx2 downstream genes are likely to represent the molecular basis of the functional equivalence of otd and Otx2 gene action in Drosophila.

The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type.

The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.

Background

Otx genes, orthologues of the Drosophila orthodenticle gene (otd), play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by Drosophila OTD, we performed an in vivo molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd.

Results

We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and vice versa. Moreover, the insertion of this box into Drosophila OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box.

Conclusion

Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of Drosophila into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.

Rod and cone photoreceptors in the mammalian retina are special types of neurons that are responsible for phototransduction, the first step of vision. Development and maintenance of photoreceptors require precisely regulated gene expression. This regulation is mediated by a network of photoreceptor transcription factors centered on Crx, an Otx-like homeodomain transcription factor. The cell type (subtype) specificity of this network is governed by factors that are preferentially expressed by rods or cones or both, including the rod-determining factors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; and cone-determining factors, mostly nuclear receptor family members. The best-documented of these include thyroid hormone receptor β2 (Trβ2), retinoid related orphan receptor Rorβ, and retinoid X receptor Rxrγ. The appropriate function of this network also depends on general transcription factors and co-factors that are ubiquitously expressed, such as the Sp zinc finger transcription factors and STAGA coactivator complexes. These cell type-specific and general transcription regulators form complex interactomes; mutations that interfere with any of the interactions can cause photoreceptor development defects or degeneration. In this manuscript, we review recent progress on the roles of various photoreceptor transcription factors and interactions in photoreceptor subtype development. We also provide evidence of auto-, para-, and feedback regulation among these factors at the transcriptional level. These protein-protein and protein-promoter interactions provide precision and specificity in controlling photoreceptor subtype-specific gene expression, development and survival. Understanding these interactions may provide insights to more effective therapeutic interventions for photoreceptor diseases.

Background

In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx.

Results

A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer.

Conclusions

This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA.

Purpose

Tyrosinase is the rate-limiting enzyme responsible for melanin biosynthesis in the retinal pigment epithelium (RPE) of the eye. Melanin has an important role in retinal development, function, and protection against light-induced oxidative stress, and melanin levels are associated with age-related macular degeneration (AMD). Because the levels of and protection afforded by melanin seem to decline with increasing age, proper regulation of the human tyrosinase gene (TYR) in the RPE is an important but insufficiently understood process. Our purpose was to obtain detailed information on regulation of the TYR gene promoter in the human RPE and to specify the role of orthodenticle homeobox 2 (OTX2) and microphthalmia-associated transcription factor (MITF).

Methods

We used luciferase reporter constructs to study regulation of the human TYR gene promoter in cultured human RPE cells. We further studied the role of OTX2 and MITF, their binding sites, and endogenous expression by using mutagenesis, electrophoretic mobility shift assay, yeast two-hybrid assay, RNA interference, and gene expression analyses.

Results

In the RPE, OTX2 activated the human TYR gene promoter via direct trans-activation of novel OTX2 binding elements. In addition, we found that indirect activation by OTX2 via more proximal MITF binding sites, even in the absence of OTX2 sites, took place. These results are consistent with the physical interaction observed between OTX2 and MITF. Overexpression or knockdown of OTX2 in RPE cells resulted in corresponding changes in tyrosinase mRNA expression. Finally, we found that a single nucleotide polymorphism (SNP rs4547091) at the most proximal OTX2 binding site is associated with altered nuclear protein binding and a remarkable decrease in TYR promoter activity in RPE cells. This single nucleotide polymorphism (SNP) is more common in the European population in which AMD is also more prevalent.

Conclusions

In the RPE, OTX2 activates the human TYR gene promoter by direct DNA binding and by interaction with MITF. Such synergistic interaction highlights the role of OTX2 as a potential coregulator of numerous MITF target genes in the eye. Genetic differences in OTX2 binding sites affect tyrosinase regulation. Collectively, these findings emphasize the role of OTX2 in regulating the human TYR gene, with implications for inter-individual differences in melanin synthesis, retinal development, and function as well as susceptibility to retinal degeneration associated with aging.

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human ES and iPS cells, potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ES cells with defined factors. In this study we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ES cell-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx+ and Rhodopsin+ photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ES cell-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ES cell-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ES cell-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ES cell-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.

The present study shows, by electroporation of chick embryonic eyes, that co-transfection of activated β-catenin and Otx2 can convert retinal progenitor cells into presumptive RPE cells expressing Mitf.

Purpose.

Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/β-catenin pathway, respectively. Otx2 and β-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and β-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.

Methods.

The sufficiency of Wnt/β-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and β-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers.

Results.

In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/β-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both β-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro.

Conclusions.

The present study provides evidence that β-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as β-catenin.

Here, we report the identification and expression analysis of the zebrafish G protein gamma T1 subunit gene (gngT1) during development. Similar to its human and mouse homologs, we confirm zebrafish gngT1 is expressed in the developing retina, where its transcription overlaps with the photoreceptor cell-specific marker, rhodopsin (rho). Surprisingly, we also show zebrafish gngT1 is expressed in the dorsal diencephalon, where its transcription overlaps with the pineal specific markers, arylalkylamine N-acetyltransferase-2 (annat-2) and extra-ocular rhodopsin (exorh). Analysis of the proximal promoter sequence of the zebrafish gngT1 gene identifies several conserved binding sites for the cone-rod homeobox/orthodenticle (Crx/Otx) homeodomain family of transcription factors. Using a morpholino antisense approach in zebrafish, we show that targeted knockdown of otx5 potently suppresses gngT1 expression in the pineal gland, whereas knockdown of crx markedly reduces gngT1 expression in the retina. Taken together, these data indicate that pineal- and retinal-specific expression of the gngT1 gene are controlled by different transcription factors and exogenous signals.

The Retinal Homeobox (Rx/rax) gene is essential for the development of the eye. Rax is among the earliest genes expressed during eye development, beginning in the prospective eye fields in the anterior neural plate. Additionally Rax expression persists in retinal progenitor cells and in differentiated photoreceptors. We have isolated and characterized a 2.8 kb genomic DNA fragment that regulates expression of Rax in the developing and maturing retina. We have discovered and characterized cis-acting elements that function to specifically control spatial and temporal Rax expression during retinal development. We have found that the regulation of Rax2A promoter activity requires cooperative interactions between positive and negative regulatory elements. Further, a highly conserved genomic element containing SOX, OTX, and POU transcription factor binding sites is necessary but not sufficient for promoter activity in retinal progenitor or stem cells. Finally, a putative binding element for forkhead transcription factors is necessary for promoter activity and can cooperate with other cis-acting elements to drive Rax2A promoter activity.

Background

Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing.

Results

We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type.

Conclusion

The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.

Specific transfer of Otx2 homeoprotein into GABAergic interneurons expressing parvalbumin (PV) is necessary and sufficient to open, then close, a critical period (CP) of plasticity in the developing mouse visual cortex. The accumulation of endogenous Otx2 in PV-cells suggests the presence of specific Otx2 binding sites. Here, we find that perineuronal nets (PNNs) on the surface of PV-cells permit the specific, constitutive capture of Otx2. We identify a 15 amino-acid domain containing an arginine-lysine doublet (RK-peptide) within Otx2, bearing prototypic traits of a glycosaminoglycan (GAG) binding sequence that mediates Otx2 binding to PNNs and specifically Chondroitin sulfate D and E with high affinity. Accordingly, PNN hydrolysis by Chondroitinase ABC reduces the amount of endogenous Otx2 in PV-cells. Direct infusion of RK-peptide similarly disrupts endogenous Otx2 localization to PV-cells, reduces PV and PNN expression and reopens plasticity in adult mice. The closure of one eye during this transient window reduces cortical acuity and is specific to the RK motif, as an AA variant or scrambled peptide fail to reactivate plasticity. Conversely, this transient reopening of plasticity in the adult restores binocular vision in amblyopic mice. Thus, one function of PNNs is to facilitate the persistent internalization of Otx2 by PV-cells to maintain CP closure. The pharmacological use of the Otx2 GAG-binding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain.

Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor-like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0 ± 5.9%) began to differentiate towards photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRγ, and phototransduction pathway components, including transducin, cGMP-gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca2+) levels and responded to 9-cis-retinal by increasing their Ca2+ levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies.