Table of contents

Orthodenticle homologs: eye and pineal development

Transcription of the human interphotoreceptor retinoid binding protein (IRBP) gene is strictly tissue specific, being restricted to retinal photoreceptors and pinealocytes. A sequence named A element in the IRBP promoter is essential for IRBP gene transcription in vivo. The human homeodomain protein OTX2 is present in nuclear extracts of IRBP expressing cells and specifically interacts with the IRBP A promoter element in vitro. OTX2, as well as CRX, a homeodomain protein very similar to OTX2, activates the human IRBP promoter in co-transfection experiments (Bobola, 1999).

In the embryonic mouse eye Otx2, a paired homeodomain transcription factor, is found in retinal pigment epithelial cells and a restricted subset of retinal neurons, including ganglion cells. In the postnatal and adult eye, however, both the cellular and subcellular distribution of the Otx2 protein are cell type-specific. Otx2 is detected only in the nuclei of retinal pigment epithelial and bipolar cells, but is present in the cytoplasm of rod photoreceptors. Immunohistochemical studies of retinal explants and transfected cell lines both suggest that the retention of Otx2 in the cytoplasm of immature rods is a developmentally regulated process. The differential distribution of Otx2 in the cytoplasm of rods and the nucleus of other cell types, suggests that subcellular localization of this transcription factor may participate cell fate determination during specific phases of retinal development (Baas, 2000).

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Both Otx1 and Otx2 might contribute to the establishment of the different eye territories. The ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 has been analyzed and it has been show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1-/-; Otx2+/- and 30% of Otx1+/-; Otx2+/- genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding is severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, is lost. Lack of pigment epithelium specification is associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquires features of mature neural retina, including the generation of Islet1-positive neurons. Furthermore, in Otx1-/-; Otx2+/- mice neural retina cell proliferation, cell differentiation and apoptotic cell death are also severely affected. Based on these findings a model is proposed in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialized tissues that will constitute the mature vertebrate eye (Martinez-Morales, 2001).

Evidence from several sources supports the idea that Otx genes might be involved at different steps of vertebrate eye formation, including the determination of a field permissive for eye formation, the morphogenesis of the lens and optic cup (OC), and the generation of retina-specific neurons. In all bilaterian species so far examined, the formation of the anterior neuroectoderm and, therefore, that of the eye field, which lies within the Otx expression domain, depends upon Otx2 function. Only in this Otx2-positive anterior neuroectoderm, can the overexpression of Pax6 and Six3 induce the formation of ectopic eyes in vertebrates, indicating that Otx activity provides the anterior neuroectoderm with the necessary competence for ocular specification. Despite of the subsequent gross eye malformations observed in Otx1-/-; Otx2+/- mice, optic vesicle (OV) outpocketing occurs normally, suggesting that a single copy of Otx2 is sufficient to establish the eye morphogenetic field and to allow its evagination (Martinez-Morales, 2001).

Subsequent to eye field specification, the morphogenesis of the vertebrate eye takes place by the infolding of the undifferentiated eye primordia into a bi-layered cup. In this structure, both the basic architecture of the adult organ and the identity of the different tissues become established by the coordinated action of autonomously expressed transcription factors and inductive signals. In the presence of low levels of Otx activity, folding of the OV is severely affected. In Otx mutants, the delayed or abnormal development of the lens placode and/or the low levels of OTX protein in the lens ectoderm could be in part responsible for the morphological defect of the OV. Similar abnormal OV invagination has indeed been observed in a lens-specific Pax6 conditional mouse mutation, where the absence of the lens placode has permitted the analysis of its effects on vesicle morphogenesis. Alternatively, OTX protein activity could be directly implicated in the control of morphogenetic movements of the cells. Association between cell migration and Otx2 expression has been proposed in the mouse olfactory system and gastrulation movements are severely affected in Otx2-/- mice. Furthermore, Otx2 appears to control the expression of molecules implicated in cytoskeleton organization and cell-cell interaction, such as calponins, R-cadherin or ephrinA2 and the ectopic expression of Otx1 and otd results in strong cell aggregation in zebrafish. Therefore, it is possible that alterations of similar molecular pathways are responsible for aberrant morphogenesis of the OC in Otx1-/-; Otx2+/- and Otx1+/-; Otx2+/- embryos (Martinez-Morales, 2001).

In addition to morphogenetic defects, Otx-deficient mice show altered expression of genes involved in the proper subdivision of the OV territory into RPE, neural retina (NR) and optic stalk (OS). Soluble molecules derived from adjacent tissues (the surface ectoderm and the surrounding mesenchyme) appear to influence NR versus RPE fate decision. Fibroblast growth factors, normally expressed in the presumptive lens ectoderm (PLE), can determine neural retina identity even in tissue fated to give rise to RPE. In contrast, extraocular mesenchyme is the source of molecules, possibly activin-like signals, capable of repressing NR while activating RPE markers. The regulation of tissue-specific genes might therefore be the results of these (positive and negative) inductive signals. However, the suppression of PLE development does not affect the formation and the separation of NR and RPE progenitor cell, giving strong support to the finding that the loss of functional alleles in Otx mutants is the direct cause of the failure of RPE determination. Interestingly, in Otx mutants the expression of Mitf and tyrosinase is also largely absent and maintained only in little patches of tissue where OTX2 is also localized. In Mitf mutants the expression of Otx2 is specifically down regulated in those areas where RPE has not differentiated. This suggests that both transcription factors cooperate to determine the identity and possibly maintain the function of the RPE. However, it is believed that Otx genes have a prevalent role in this process, because the expression of Otx precedes that of Mitf. In addition, in most cases the entire RPE territory is affected in Otx-deficient mice, whereas only patches of the dorsal RPE is affected when the entire Mitf gene is lost (Martinez-Morales, 2001).

The enlargement of the expression domains of OS- (Pax2) and NR- (Pax6 and Six3) specific genes further suggests the importance of Otx activity in the proper subdivision of the OV. Whether OTX proteins repress the expression of Pax2, Six3 or Pax6 directly or indirectly needs to be determined. However, a recent report shows that the identity of the OS and OC depends on the specific expression and reciprocal transcriptional repression of Pax2 and Pax6. Similar mechanisms may be acting in the segregation of other genes involved in the establishment of RPE/OS and RPE/NR boundaries. A hypothetical model that takes into account all the data described above is presented. In this simplified scheme, the expression, regulated respectively by TGFß-like and Shh signaling, and the reciprocal repression of Otx and Pax2 will determine, in coordination with other genes: for instance, Vax1 and Vax2, the initial dorsal and ventral patterning of the OV. In this respect, initially only the expression of Pax2, but not that of either Six3 or Pax6 is significantly altered in Otx-deficient mice. In addition, in Pax2 null mice, pigmented cells invade the optic stalk. In support of a possible antagonistic interaction between Otx and Pax2, it is also worth mentioning that the two genes may be acting in a similar antagonistic manner to establish the pattern of the inner ear primordium. Otx and Pax2 genes are expressed in the otic vesicle in the presumptive vestibular and cochlear domains, respectively. Furthermore, Pax2-/- and Otx mutants (Otx1-/-, and Otx1-/-;Otx2+/-) show divergent otic phenotypes, lacking the auditory (cochlea and spiral ganglion) and the vestibular (semicircular canal, lateral ampulla, utricosaccular and cochleosaccular duct) parts of the organ, respectively (Martinez-Morales, 2001).

Later, as the RPE, NR and OS progenitor cells acquire their identity, the mutual positive interaction between Otx and Mitf in the dorsal portion of the eye will induce and probably maintain the network of genes necessary for the establishment RPE identity. Complete segregation of eye territories would also imply the mutual regulation of different OS, NR and RPE specific genes. Thus, regulation of Otx2 and Pax2 expression in cells located at the RPE/OS border will finally establish the separation of the two territories with a mechanism similar to that proposed for Pax2 and Pax6 interaction at the OS/OC boundary. In Otx mutants, Pax6 expression appeared unaffected in spite of the expansion of the Pax2-positive territory. Whether this is due to an independent autoregulation of Pax6 expression or whether OTX proteins could also contribute to the direct regulation of Pax6 expression is not clear. Indeed, the precise hierarchical interactions among all the genes required for the OV subdivision are just beginning to be elucidated (Martinez-Morales, 2001).

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus -- the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. These results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex (Bamse, 2002).

Photoreceptor and bipolar cells are molecularly related cell types in the vertebrate retina. XOtx5b is expressed in both photoreceptors and bipolars, while a closely related member of the same family of transcription factors, XOtx2, is expressed in bipolar cells only. Lipofection of retinal precursors with XOtx5b biases them toward photoreceptor fates whereas a similar experiment with XOtx2 promotes bipolar cell fates. Domain swap experiments show that the ability to specify different cell fates is largely contained in the divergent sequence C-terminal to the homeodomain, while the more homologous N-terminal and homeodomain regions of both genes, when fused to VP16 activators, promote only photoreceptor fates. XOtx5b is closely related to Crx and like Crx it drives expression from an opsin reporter in vivo. XOtx2 suppresses this XOtx5b-driven reporter activity providing a possible explanation for why bipolars do not express opsin. Similarly, co-lipofection of XOtx2 with XOtx5b overrides the latter's ability to promote photoreceptor fates and the combination drives bipolar fates. The results suggest that the shared and divergent parts of these homologous genes may be involved in specifying the shared and distinct characters of related cell types in the vertebrate retina (Viczia, 2003).

Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Using conditional gene ablation in mice it has been shown that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converts differentiating photoreceptor cells to amacrine-like neurons and leads to a total lack of pinealocytes in the pineal gland. Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. These results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development (Nishida, 2003).

It remains unclear which gene induction effectively generates photoreceptor-specific phenotypes from nonretinal tissues. The purpose of this study was to determine whether Crx and Otx2, homeobox genes related to photoreceptor development, can induce the generation of these phenotypes in cells derived from adult ciliary and iris tissue and in mesencephalon-derived neural stem cells. Crx and Otx2 were transferred into adult rat ciliary- and embryonic mesencephalon-derived neurospheres and adult rat iris-derived cells with the aid of a recombinant retrovirus. The presence of photoreceptor-specific phenotypes was confirmed by immunocytochemistry and Western blot analysis. More than 90% of the Crx- and Otx2-transfected ciliary- and iris-derived cells exhibited rod opsin immunoreactivity, whereas few of the similarly transfected mesencephalon-derived neural stem cells expressed rod opsin. At least two additional key components of the phototransduction cascade, recoverin and Gdeltat1, were expressed by Crx- and Otx2-transfected iris-derived cells. It is concluded that Crx and Otx2 effectively induce the generation of photoreceptor-specific phenotypes from ciliary- and iris-derived cells. That both Crx and Otx2 induced phenotype generation in cells derived from iris or ciliary tissue may suggest an approach to photoreceptor cell preparation for retinal transplantation (Akagi, 2004).

The diversity of cell types found within the vertebrate CNS 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 metabotropic glutamate receptor 6 (Grm6) enhancer sequence, a 445 bp calcium-binding protein 5 (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, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. 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 reverse transcription-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 (Kim, 2008).

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, the fate of Blimp1 expressing cells was followed using Blimp1-Cre mice and Lox-Stop-Lox reporter strains. It was 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, transitioning rod photoreceptors were sought 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, these 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 (Brzezinski, 2013).

β-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression

The retinal pigment epithelium (RPE) consists of a monolayer of cuboidal, pigmented cells that is located between the retina and the choroid. The RPE is vital for growth and function of the vertebrate eye and improper development results in congenital defects, such as microphthalmia or anophthalmia, or a change of cell fate into neural retina called transdifferentiation. The transcription factors microphthalmia-associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2) are crucial for RPE development and function; however, very little is known about their regulation. By using a Wnt-responsive reporter, this study shows that the Wnt/β-catenin pathway is activated in the differentiating mouse RPE. Cre-mediated, RPE-specific disruption of β-catenin after the onset of RPE specification causes severe defects, resulting in microphthalmia with coloboma, disturbed lamination, and mislocalization of adherens junction proteins. Upon β-catenin deletion, the RPE transforms into a multilayered tissue in which the expression of Mitf and Otx2 is downregulated, while retina-specific gene expression is induced, which results in the transdifferentiation of RPE into retina. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that β-catenin binds near to and activates potential TCF/LEF sites in the Mitf and Otx2 enhancers. It is concluded that Wnt/β-catenin signaling is required for differentiation of the RPE by directly regulating the expression of Mitf and Otx2. This study is the first to show that an extracellular signaling pathway directly regulates the expression of RPE-specific genes such as Mitf and Otx2, and elucidates a new role for the Wnt/β-catenin pathway in organ formation and development (Westenskow, 2009).

The RPE originates from the optic neuroepithelium of the ventral forebrain, which undergoes morphogenetic movements leading to formation of the optic cup. The resulting inner layer of the optic cup develops into the neural retina and the outer layer differentiates into RPE. Both retina and RPE are specified early, prior to optic cup formation. Subsequent to RPE specification, a period of differentiation and maturation follows, resulting in dramatic morphological, structural and functional changes. Interestingly, the RPE fate is reversible for several days following the initial activation of differentiation, as evidenced by a propensity to downregulate RPE-specific genes, to hyperproliferate and to differentiate into retina, a process considered to be transdifferentiation. Thus, it is crucial that mechanisms exist to maintain RPE differentiation in the optic cup (Westenskow, 2009).

RPE specification and differentiation are regulated by two key regulatory transcription factors, Mitf and Otx2. Disruption of either gene, similar to genetic ablation of the RPE, results in microphthalmia and coloboma during murine eye development. Mitf isoforms and Otx2 transactivate essential genes for terminal pigment differentiation in the RPE and neural crest (e.g. tyrosinase-related protein 1; Tyrp1) and for RPE-specific functions. Initiation and maintenance of Mitf and Otx2 expression is controlled by interaction with surrounding extraocular tissues, including the extraocular mesenchyme. A few candidate regulators have been identified; however, the exact mechanisms controlling the expression of Mitf and Otx2 are not known. The Wnt/β-catenin pathway is an excellent candidate because it is active in the developing RPE; activation results in cytoplasmic stabilization of β-catenin, which then translocates into the nucleus and associates with TCF/LEF transcription factors. Interestingly, Wnt/β-catenin signaling promotes differentiation of neural crest-derived pigmented cells by direct transactivation of the Mitf-M promoter. Although melanocytes and RPE cells originate from different tissues, some aspects of the mechanisms regulating pigment cell differentiation in different lineages could be similar. This report is the first to show a direct role for an extracellular signaling pathway in controlling development of the mammalian RPE. Although it cannot be ruled out that cell adhesion defects can independently interfere with RPE differentiation, the results strongly suggest that β-catenin, via TCF/LEF activation, is essential for maintaining cell fate in the developing RPE by the direct regulation of Mitf and Otx2 expression. Thus, this mechanism of Mitf regulation appears to be evolutionary conserved between the RPE and neural crest-derived melanocytes. It remains to be determined what the source(s) of the actual ligand(s) is and how Wnt/β-catenin signaling integrates with other putative regulatory pathways to control RPE development (Westenskow, 2009).

Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity

Neural circuits are shaped by experience in early postnatal life. Distinct GABAergic connections within visual cortex determine the timing of the critical period for rewiring ocular dominance to establish visual acuity. Maturation of the parvalbumin (PV)-cell network that controls plasticity onset is regulated by a selective re-expression of the embryonic Otx2 homeoprotein. Visual experience promoted the accumulation of non-cell-autonomous Otx2 in PV-cells, and cortical infusion of exogenous Otx2 accelerated both PV-cell development and critical period timing. Conversely, conditional removal of Otx2 from non-PV cells or from the visual pathway abolished plasticity. Thus, the experience-dependent transfer of a homeoprotein may establish the physiological milieu for postnatal plasticity of a neural circuit (Sugiyama, 2008).

Direct Otx2 gain- or loss-of-function, respectively, promoted or hindered both the expression of PV-cell molecular constituents and ocular dominance plasticity. Surprisingly, these bidirectional actions were non-cell-autonomous. Selective deletion of the Otx2 gene outside cortical interneurons successfully removed Otx2 protein from PV-cells. Moreover, intercepting Otx2 by infusion of antibodies or blocking its synthesis by siRNA injection into the eye prevented critical period plasticity. It is intriguing that passage of extra-cortical factors may establish the milieu for cortical plasticity in vivo. Robust Otx2 mRNA expression is observed in the retina, LGN and superior colliculus, demarcating a possible conduit for propagation along the visual pathway. Homeoprotein family members contain secretion and internalization sequences in the homeodomain, allowing potential transfer from cell to cell as observed in vitro. The mechanisms of transport and transfer in vivo will be an important line of investigation (Sugiyama, 2008).

Crx, an Otx like homeodomain protein involved in eye and pineal development

A novel otx-like homeobox gene, Crx, has been isolated from the mouse retina. Crx expression is restricted to developing and mature photoreceptor cells. CRX binds and transactivates the sequence TAATCC/A, which is found upstream of several photoreceptor-specific genes, including the opsin genes from many species. Overexpression of Crx using a retroviral vector increases the frequency of clones containing exclusively rod photoreceptors and reduces the frequency of clones containing amacrine interneurons and Muller glial cells. Presumptive photoreceptor cells expressing a dominant-negative form of CRX fail to form proper photoreceptor outer segments and terminals. Crx is a novel photoreceptor-specific transcription factor and plays a crucial role in the differentiation of photoreceptor cells (Furukawa, 1997).

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, a new member of the otd family, Crx (Cone rod homeobox) has been isolated. Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases (Chen, 1997).

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors (Freund, 1997).

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD has revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregates with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution shows decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function (Swain, 1997).

The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein is produced as a truncated form of the Wilson disease gene (Atp7b) product. To identify the regulatory elements required for pineal-specific gene expression, sequences upstream of the rat PINA gene were isolated and a cis-acting element was identified that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5' regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE (Li, 1998).

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. Mice were generated carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice (Furukawa, 1999).

The paired-like homeodomain transcription factor CRX (cone-rod homeobox) is involved in regulating photoreceptor gene expression and rod outer segment development. Mutations in CRX have been associated with several retinal degenerative diseases. These conditions range from Leber congenital amaurosis (a severe cone and rod degeneration of childhood onset) to adult onset cone-rod dystrophy and retinitis pigmentosa (an adult onset condition that primarily affects rods). The goal of this study is to better understand the molecular basis of CRX function and to provide insight into how mutations in CRX cause such a variety of clinical phenotypes. Deletion analysis in conjunction with DNA binding and transient transfection-based transactivation studies were performed to identify the functional domains within CRX. DNA binding requires a complete homeodomain. Furthermore, truncated proteins that do not contain an intact homeodomain fail to demonstrate detectable expression in tissue culture upon transfection. Transactivation analysis indicates that both the OTX tail and the WSP domain are important for controlling positive regulatory activity of CRX. Interestingly, the mapped CRX transactivation domains are also critical when coexpressed with NRL. Specifically, the synergy between CRX and NRL is constant regardless of which CRX variant is used (Chau, 2000).

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis- acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, it has been demonstrated that the leucine zipper of NRL can physically interact with CRX. Deletion analysis reveals that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone is weak, a strong interaction is detected when flanking regions including the glutamine-rich and the basic regions that follow the HD are included. A reciprocal deletion analysis shows that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function (Mitton, 2000).

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. Transgenic mice have been generated using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and the promoter function of the cell-specific and developmentally regulated expression of Crx has been investigated. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. The transgenic lines were characterized in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells (Furukawa, 2002).

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. 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. 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 understanding of the basic pathology of LCA (Morrow, 2005).

Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL (Drosophila homolog: Traffic Jam ) determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. This study reports an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data with global expression profiling and in vivo knockdown studies. Approximately 300 direct NRL target genes were identified. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX, an Orthodenticle homolog. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. Histone demethylase Kdm5b (Drosophila homolog: Little imaginal discs) was identified as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. These studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis (Hao, 2012).

Expression of Otx2 in the adult: Otx2 is restricted to dopaminergic neurons of the ventral tegmental area

Mesencephalic-diencephalic dopaminergic (mdDA) neurons control motor, sensorimotor and motivated behaviour and their degeneration or abnormal functioning is associated with important pathologies, such as Parkinsons disease and psychiatric disorders. Despite great efforts, the molecular basis and the genetic factors differentially controlling identity, survival and vulnerability to neurodegeneration of mdDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA) are poorly understood. Otx2 has been shown to be required for identity, fate and proliferation of mesencephalic DA (mesDA) progenitors. By using mouse models and immunohistochemistry, whether Otx2 is expressed also in post-mitotic mdDA neurons was investigated. The data reveal that Otx2 is expressed in post-mitotic mesDA neurons during mid-late gestation and in the adult brain. Remarkably, Otx2 expression is sharply excluded from mdDA neurons of the SN and is restricted to a relevant fraction of VTA neurons. Otx2+-TH+ neurons are concentrated to the ventral part of the VTA. Combined expression with other regionalized VTA markers shows that Otx2+-TH+ neurons are prevalently Girk2- and Calb+ and among these, those located in the medial and ventralmost portion of the VTA are also Ahd2+. These findings indicate that Otx2 represents the first transcription factor with a proven role in mdDA neurogenesis whose expression discriminates between SN and a relevant proportion of VTA neurons. This supports the possibility that Otx2 may act as a post-mitotic selector controlling functional features (e.g. identity and/or survival) of a relevant fraction of VTA neurons in the adult (Di Salvio, 2010).

Expression of OTX1 in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma

Studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. This study investigated OTX1 and OTX2 expression in Non-Hodgkin Lymphoma (NHL) and multiple myeloma. A combination of semiquantitative RT-PCR, Western blot, and immunohistochemical analyses was used to measure OTX1 and OTX2 levels in normal lymphoid tissues and in 184 tumor specimens representative of various forms of NHL and multiple myeloma. OTX1 expression was activated in 94% of diffuse large B-cell lymphomas, in all Burkitt lymphomas, and in 90% of high-grade follicular lymphomas. OTX1 was undetectable in precursor-B lymphoblastic lymphoma, chronic lymphocytic leukemia, and in most marginal zone and mantle cell lymphomas and multiple myeloma. OTX2 was undetectable in all analyzed malignancies. Analysis of OTX1 expression in normal lymphoid tissues identified a subset of resting germinal center (GC) B cells lacking PAX5 and BCL6 and expressing cytoplasmic IgG and syndecan. About 50% of OTX1(+) GC B cells co-expressed CD10 and CD20. This study identifies OTX1 as a molecular marker for high-grade GC-derived NHL and suggests an involvement of this transcription factor in B-cell lymphomagenesis. Furthermore, OTX1 expression in a subset of normal GC B cells carrying plasma cell markers suggests its possible contribution to terminal B-cell differentiation (Omodei, 2009).

Table of contents

orthodenticle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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