During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.
The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.
PMID: 19686387 CAMSID: cams3215
bipolar; cone-rod homeobox; orthodenticle homeobox 2; photoreceptor; proliferation; retina; retinoblastoma
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.
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.
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.
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.
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.
During retinal development, photoreceptors and bipolar cells express the transcription factor Otx2. Blimp1 is transiently expressed in Otx2+ cells. Blimp1 deletion results in excess bipolar cell formation at the expense of photoreceptors. In principle, Blimp1 could be expressed only in Otx2+ cells that are committed to photoreceptor fate. Alternatively, Blimp1 could be expressed broadly in Otx2+ cells and silenced to allow bipolar cell development. To distinguish between these alternatives, we followed the fate of Blimp1 expressing cells using Blimp1-Cre mice and Lox-Stop-Lox reporter strains. We observed that Blimp1+ cells gave rise to all photoreceptors, but also to one third of bipolar cells, consistent with the latter alternative: that Blimp1 inhibits bipolar competence in Otx2+ cells and must be silenced to allow bipolar cell generation. To further test this hypothesis, we looked for transitioning rod photoreceptors in Blimp1 conditional knock-out (CKO) mice carrying the NRL-GFP transgene, which specifically labels rods. Control animals lacked NRL-GFP+ bipolar cells. In contrast, about half of the precociously generated bipolar cells in Blimp1 CKO mice co-expressed GFP, suggesting that rods become re-specified as bipolar cells. Birthdating analyses in control and Blimp1 CKO mice showed that bipolar cells were birthdated as early as E13.5 in Blimp1 CKO mice, five days before this cell type was generated in the wild-type retina. Taken together, our data suggest that early Otx2+ cells upregulate photoreceptor and bipolar genes, existing in a bistable state. Blimp1 likely forms a cross-repressive network with pro-bipolar factors such that the winner of this interaction stabilizes the photoreceptor or bipolar state, respectively.
Retina; Photoreceptor; Bipolar Cell; Cell Fate Specification; Transdifferentiation; Competence
Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.
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).
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.
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.
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.
Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.
•Transcription factor Otx2 drives enhancer activation in differentiating mouse ESCs•Oct4 controls Otx2 expression levels•Otx2 collaborates with Oct4 in enhancer activation•Otx2 contributes to enhancer maintenance and de novo activation
The transcription factor Otx2 plays an important role in neural development and in the exit of embryonic stem cells from the pluripotent ground state. In this study, Yang et al. demonstrate that Otx2 drives enhancer activation and maintenance during the early cell-fate transition away from ground state pluripotency. Otx2 is involved in a regulatory partnership with Oct4 wherein Oct4 initially promotes Otx2 expression. Otx2 subsequently recruits Oct4 to a subset of enhancers and establishes a regulatory chromatin landscape.
Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.
We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.
In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.
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.
retina; rods and cones; development; cell adhesion molecules; Danio rerio
Cone-rod homeobox (CRX) protein is a “paired-like” homeodomain transcription factor that is essential for regulating rod and cone photoreceptor transcription. Mutations in human CRX are associated with the dominant retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congenital Amaurosis (LCA), with variable severity. Heterozygous Crx Knock-Out (KO) mice (“+/−”) have normal vision as adults and fail to model the dominant human disease. To investigate how different mutant CRX proteins produce distinct disease pathologies, we generated two Crx Knock-IN (K-IN) mouse models: CrxE168d2 (“E168d2”) and CrxR90W (“R90W”). E168d2 mice carry a frameshift mutation in the CRX activation domain, Glu168del2, which is associated with severe dominant CoRD or LCA in humans. R90W mice carry a substitution mutation in the CRX homeodomain, Arg90Trp, which is associated with dominant mild late-onset CoRD and recessive LCA. As seen in human patients, heterozygous E168d2 (“E168d2/+”) but not R90W (“R90W/+”) mice show severely impaired retinal function, while mice homozygous for either mutation are blind and undergo rapid photoreceptor degeneration. E168d2/+ mice also display abnormal rod/cone morphology, greater impairment of CRX target gene expression than R90W/+ or +/− mice, and undergo progressive photoreceptor degeneration. Surprisingly, E168d2/+ mice express more mutant CRX protein than wild-type CRX. E168d2neo/+, a subline of E168d2 with reduced mutant allele expression, displays a much milder retinal phenotype, demonstrating the impact of Crx expression level on disease severity. Both CRX[E168d2] and CRX[R90W] proteins fail to activate transcription in vitro, but CRX[E168d2] interferes more strongly with the function of wild type (WT) CRX, supporting an antimorphic mechanism. E168d2 and R90W are mechanistically distinct mouse models for CRX-associated disease that will allow the elucidation of molecular mechanisms and testing of novel therapeutic approaches for different forms of CRX-associated disease.
The transcription factor Cone-Rod Homeobox (CRX) plays a central role in regulating gene expression of rod and cone photoreceptors, the primary light sensing cells of the retina. Mutations in the human CRX gene have been associated with the retinal degeneration diseases Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congential Amaurosis (LCA). These diseases cause progressive and permanent loss of vision, vary widely in age of onset and severity, and are currently untreatable. To understand how mutations in CRX cause distinct forms of retinal disease, we have genetically engineered mice to carry human disease-causing mutations in their Crx gene. These mouse lines accurately recapitulate distinct forms of CRX-associated disease, demonstrating that different classes of CRX mutations are responsible for phenotype variability in humans. We have characterized the pathology of these mice and identified critical mechanisms of disease. In addition, we have discovered that modifying the level of mutant protein had a dramatic effect on disease pathology in one mutant model, suggesting that targeted therapy against the mutant CRX could be an effective treatment strategy. These mouse models will allow for the testing of novel therapeutic strategies for retinal diseases caused by CRX mutations.
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.
FIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes.
FIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.
FIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.
Degeneration of retinal neurons is an underlying cause of several major types of blinding diseases, and effective therapies remain to be developed. The suppositive strategy of repopulating a degenerative retina with new cells generated onsite faces serious challenges, because the mammalian retina seems to lack the ability to regenerate itself or replace its lost neurons. We investigated the possibility of using a transcriptional factor with proneural activities to reprogram ocular tissue with regenerative capability to give rise to retinal cells. Transgenic mice were generated with DNA constructs that targeted the expression in the retinal pigment epithelium of proneural gene neurogenin1 from the promoter of Bestrophin1, or neurogenin3 from RPE65 promoter. Here we report the presence of ectopic retina-like tissue in some of the transgenic mice, young and aged. The ectopic retina-like tissue contained cells positive for photoreceptor proteins Crx, recoverin, red opsin, and rhodopsin, and cells positive for proteins that label other types of retinal neurons, including AP2α and Pax6 for amacrine cells, Otx2 for bipolar cells, and Brn3A for ganglion cells. The retina-like tissue often co-existed with darkly pigmented tissue positive for RPE proteins: cytokeratin 18, Otx2, and RPE65. The ectopic retina-like tissue was detected in the subretinal space, including two retinae co-existing in the same eye, and/or in the optic nerve or in the vicinity of the optic nerve head. On rare occasions, it was detected in the choroid and in the vicinity of the ciliary body. The presence of ectopic retina-like tissue in the transgenic mouse supports the possibility of inducing retinal regeneration in the mammalian eyes through gene-directed reprograming.
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.
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.
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.
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.
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.
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.
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.
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.
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.
rhodopsin; orthodenticle; eye; sense organ; congenital disease; retina
Thyroid hormone (TH) is an important developmental regulator in many tissues, including the retina. TH is activated locally via deiodinase 2 (Dio2), and it is destroyed by deiodinase 3 (Dio3). The TH receptors, TRa and TRb, mediate TH activity through hormone and DNA binding, and interactions with transcription regulators.
In the current work, the expression of these TH components was examined in the chick retina over time. Three waves of expression were characterized and found to be correlated with critical developmental events. The first wave occurred as progenitor cells began to make photoreceptors, the second as some cell types adopted a more mature location and differentiation state, and the third as Müller glia were generated. The cell types expressing the components, as well as the kinetics of expression within the cell cycle, were defined. TRb expression initiated during G2 in progenitor cells, concomitant with NeuroD and Otx2, which are expressed in early photoreceptor cells. TRb was expressed in photoreceptor cells for several days and then was reduced in expression level, as the expression of Crx, a later photoreceptor gene, became more evident. Dio3 was expressed throughout the cell cycle in progenitor cells. TRa was in most, if not all, retinal cells. Dio2 appeared transiently in a ventral (high) to dorsal gradient, likely in a subset of photoreceptor cells.
Multiple TH components were expressed in dynamic patterns in cycling progenitor cells and photoreceptors cells across the developing chick retina. These dynamic patterns suggest that TH is playing several roles in retinal development, both within the cycling progenitor cells and possibly with respect to the timing of differentiation of photoreceptor cells.
The diversity of cell types found within the vertebrate central nervous system arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200-bp Grm6 enhancer sequence, a 445-bp Cabp5 promoter sequence, and a 164-bp Chx10 enhancer sequence were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites (TFBSs), which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs), Crx and Otx2, and POU homeodomain factor, Brn2, are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay (EMSA). Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and RT-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs, Otx2 and Brn2, together appear to play a common role in regulating gene expression in retinal bipolar cells.
retina; bipolar cells; transcription factor; metabotropic glutamate receptor 6; calcium-binding protein 5; Chx10
In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.
We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.
We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap’s TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1-mediated transactivation of photoreceptor cell genes during zebrafish retinogenesis.
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.
phototransduction; photoreceptor; circadian rhythms; orthodenticle; ocelliless; oc; visual transduction; eye evolution; photoreceptor development
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.
OTX2 is a developmentally regulated transcription factor that plays important roles in early morphogenesis of the central nervous system. We previously identified OTX2 amplification and overexpression in medulloblastoma cell lines. However, the nature and frequency of OTX2 genetic alterations have not been determined in large sample sizes of primary tumors. To refine our understanding of genomic events affecting OTX2, we analyzed genomic copy number and mRNA expression of OTX2 in a large cohort of primary medulloblastomas. We found that the most frequent focal copy number gain in these primary medulloblastomas (gained in 21% of the tumors, n=201) contained the single gene OTX2, indicating that copy number gain of OTX2 is a driver event in this tumor. In this cohort of tumors, we found that the event was restricted to tumor subtypes that do not express a molecular signature of either Wnt or Shh pathway activation. FISH analysis revealed that OTX2 copy number gain is present in a subset of cells within medulloblastoma samples, suggesting that it is a late event contributing to tumor progression. Gain of OTX2 copy number is associated with the presence of anaplastic histological features and shorter survival in medulloblastoma patients. To verify a functional role of the observed genomic events, we demonstrate that ectopic OTX2 expression enhances proliferation and tumorigenicity of immortalized primary cells and that OTX2 knockdown in medulloblastoma xenografts prolongs survival of recipient animals. Finally, we demonstrate that OTX2 transcriptionally upregulates the medulloblastoma oncogene MYC as a potential mechanism whereby OTX2 promotes tumor progression.
OTX2; medulloblastoma; MYC