Eye Degeneration and Loss of otx5b Expression in the Cavefish Sinocyclocheilus tileihornes.

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.

OTX1 and OTX2 as possible molecular markers of sinonasal carcinomas and olfactory neuroblastomas.

OTX Homeobox genes are involved in embryonic morphogenesis and in the development of olfactory epithelium in adult. Mutations occurring in the OTX genes are reported to be associated to tumorigenisis in human. No reports correlate the expression of OTX genes and neoplasms of the nasal cavity. Thus, through immunohistochemical and Real-time PCR analysis we investigated OTX1 and OTX2 expression in the more frequent types of nasal and sinonasal tumours. Variable expression of both genes were found in normal sinonasal mucosa and in tumours. Interestingly, no expression of both OTX genes were detected in sinonasal intestinal-type adenocarcinomas; only OTX1 was found in non-intestinal-type adenocarcinomas and OTX2 was selectively expressed in olfactory neuroblastomas. In conclusion, OTX1 and OTX2 genes might have a role in the pathogenesis of different types of sinonasal neoplasms.

Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells.

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. A microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

OTX2 Defines a Subgroup of Atypical Teratoid Rhabdoid Tumors With Close Relationship to Choroid Plexus Tumors.

Atypical teratoid rhabdoid tumors (ATRT) are highly malignant brain tumors of early childhood that have been regarded as a homogenous entity characterized by inactivation of the SMARCB1/INI1 or SMARCA4/BRG1 genes as the only characteristic alteration. Recent studies suggest that similar to other embryonal tumors ATRT can also be divided into subgroups based on their mRNA or methylation profiles. Using microarray-based expression analysis of 12 patient ATRT specimens we demonstrated the existence of 2 subgroups of ATRT. One subgroup is characterized by high expression of OTX2, encoding a transcription factor involved in brain development. OTX2 expression was verified by immunohistochemistry and might function as a novel therapeutic target for this fatal tumor. High expression of OTX2 as well as expression of Kir7.1/KCNJ13, TRPM3 and ENPP2, which have all previously been linked to either choroid plexus epithelium or choroid plexus tumors (CPTs), suggests a close histogenetic relation of this subgroup to CPTs.

OTX2 is a therapeutic target for retinoblastoma and may function as a common factor between C-MYC, CRX, and phosphorylated RB pathways.

The homeobox transcription factor orthodenticle homeobox 2 (OTX2) plays a critical role in very early neurogenesis, but can become oncogenic when aberrantly expressed later in life. We previously discovered its novel oncogenic role in the malignant childhood brain tumor medulloblastoma and hypothesize an oncogenic role in retinoblastoma. Primary retinoblastoma tumors and cell lines were analyzed by quantitative-PCR, immunoblotting and immunohistochemistry for OTX2. The effect of modulating OTX2 expression on tumorigenesis was tested pharmacologically and by siRNA. A lentiviral shRNA-engineered vector was used for conditional knockdown studies on tumor growth in vivo. A luciferase reporter assay was used to analyze ATRA's effect on OTX2's promoter. In this study on retinoblastoma, OTX2 was frequently amplified and/or overexpressed in primary tumors and cell lines. Knockdown of OTX2 expression by siRNA or pharmacologic inhibition by all-trans retinoic acid (ATRA) repressed OTX2 expression and cell proliferation and significantly decreased tumor growth in vivo. Loss of OTX2 expression also resulted in decreased expression of C-MYC and CRX, genes previously implicated in retinoblastoma tumorigenesis. Loss of OTX2 expression increased the phosphorylation of RB, a potential mechanism of modulating cell proliferation. Aberrant expression of OTX2 may contribute to the development of retinoblastoma. OTX2 may serve as a common transcription factor that interlinks multiple tumor-driving pathways. These results also show that OTX2 can be genetically and pharmacologically targeted, providing an exciting new therapeutic option that may be less toxic and more efficacious than current treatments.

CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

miR-410 Inhibition Induces RPE Differentiation of Amniotic Epithelial Stem Cells via Overexpression of OTX2 and RPE65.

The retinal pigment epithelium (RPE) is a highly specialized cell type located between the choroid and neural retina of the eye. RPE degeneration causes irreversible visual impairment, extending to blindness. Cell therapy has recently emerged as a potential therapeutic approach for retinal degeneration. MicroRNA-based differentiation of stem cells is a new strategy for producing tissue-specific cell types. In this study, we developed a novel microRNA-based strategy for RPE induction from human amniotic epithelial stem cells (AESCs). We identified microRNAs involved in RPE development in AESCs. Of 29 putative human RPE-relevant microRNAs, microRNA-410 (miR-410) was predicted to target multiple RPE development-relevant genes. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including OTX2, RPE65, Bestrophin and EMMPRIN. These RPE-like cells were morphologically altered toward a cobblestone-like shape and were able to phagocytize microbeads. We showed that miR-410 directly regulates predicted target genes OTX2 and RPE65. Our microRNA-based strategy demonstrated RPE differentiation in AESCs by treatment of an antisense microRNA-410 (anti-miR-410), without the use of additional factors or exogenous transduction. These findings suggest that miR-410 inhibition can be a useful tool for directed cell differentiation and an attractive method for cell therapy in human retinal degenerative diseases.

An otx/nodal regulatory signature for posterior neural development in ascidians.

In chordates, neural induction is the first step of a complex developmental process through which ectodermal cells acquire a neural identity. In ascidians, FGF-mediated neural induction occurs at the 32-cell stage in two blastomere pairs, precursors respectively of anterior and posterior neural tissue. We combined molecular embryology and cis-regulatory analysis to unveil in the ascidian Ciona intestinalis the remarkably simple proximal genetic network that controls posterior neural fate acquisition downstream of FGF. We report that the combined action of two direct FGF targets, the TGFß factor Nodal, acting via Smad- and Fox-binding sites, and the transcription factor Otx suffices to trigger ascidian posterior neural tissue formation. Moreover, we found that this strategy is conserved in the distantly related ascidian Phallusia mammillata, in spite of extreme sequence divergence in the cis-regulatory sequences involved. Our results thus highlight that the modes of gene regulatory network evolution differ with the evolutionary scale considered. Within ascidians, developmental regulatory networks are remarkably robust to genome sequence divergence. Between ascidians and vertebrates, major fate determinants, such as Otx and Nodal, can be co-opted into different networks. Comparative developmental studies in ascidians with divergent genomes will thus uncover shared ascidian strategies, and contribute to a better understanding of the diversity of developmental strategies within chordates.

Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency.

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.

Xnr3 affects brain patterning via cell migration in the neural-epidermal tissue boundary during early Xenopus embryogenesis.

Neural induction and anteroposterior neural patterning occur simultaneously during Xenopus gastrulation by the inhibition of BMP and Wnt signaling, respectively. However, other processes might be necessary for determining the neural-epidermal boundary. Xenopus nodal-related-3 (Xnr3) is expressed in dorsal blastula and plays a role in neural formation. In this study, we analyzed how Xnr3 affects neural patterning to identify novel mechanisms of neural-epidermal-boundary determination. In situ hybridization revealed that ventro-animal injection with Xnr3 shifted the lateral krox20 expression domain anteriorly and reduced Otx2 expression. The mature region of Xnr3 is necessary for these effects to occur, and the pro-region accelerated them. Phalloidin labeling revealed that cells around the neural-epidermal boundary lost their slender shape following Xnr3 injection. Moreover, we analyzed the cell migration of ectodermal cells and found specific Xnr3-induced effects at the neural-epidermal boundary. These findings together suggested that Xnr3 affects anterior ectoderm migration around the neural-epidermal boundary to induce a specific neural pattern abnormality. Change of the shape of surrounding ectodermal cells and the specific migratory pattern might therefore reflect the novel mechanism of neural-epidermal boundary.

Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition.

Mouse embryonic stem cells (ESCs) represent the naïve ground state of the preimplantation epiblast and epiblast stem cells (EpiSCs) represent the primed state of the postimplantation epiblast. Studies have revealed that the ESC state is maintained by a dynamic mechanism characterized by cell-to-cell spontaneous and reversible differences in sensitivity to self-renewal and susceptibility to differentiation. This metastable condition ensures indefinite self-renewal and, at the same time, predisposes ESCs for differentiation to EpiSCs. Despite considerable advances, the molecular mechanism controlling the ESC state and pluripotency transition from ESCs to EpiSCs have not been fully elucidated. Here we show that Otx2, a transcription factor essential for brain development, plays a crucial role in ESCs and EpiSCs. Otx2 is required to maintain the ESC metastable state by antagonizing ground state pluripotency and promoting commitment to differentiation. Furthermore, Otx2 is required for ESC transition into EpiSCs and, subsequently, to stabilize the EpiSC state by suppressing, in pluripotent cells, the mesendoderm-to-neural fate switch in cooperation with BMP4 and Fgf2. However, according to its central role in neural development and differentiation, Otx2 is crucially required for the specification of ESC-derived neural precursors fated to generate telencephalic and mesencephalic neurons. We propose that Otx2 is a novel intrinsic determinant controlling the functional integrity of ESCs and EpiSCs.

OTX2 represses myogenic and neuronal differentiation in medulloblastoma cells.

The brain development transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas, but the mechanistic basis for its contributions in this setting are not understood. In this study, we identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. OTX2 binds to the MyoD1 core enhancer through its homeobox domain, and the remarkable repressor activity exhibited by the homeobox domain renders OTX2 transcriptionally repressive. RNA interference-mediated attenuation of OTX2 expression triggered myogenic and neuronal differentiation in vitro and prolonged the survival in an orthotopic medulloblastoma mouse model. Conversely, inducing myogenic conversion of medulloblastoma cells led to the loss of OTX2 expression. In medullomyoblastoma, a medulloblastoma subtype containing muscle elements, myogenic cells share cytogenetic signatures with the primitive tumor cells and OTX2 expression was lost in the differentiated myogenic cells. Thus, OTX2 functions via its homeobox domain as a suppressor of differentiation, and the loss of OTX2 expression is linked to the myogenesis in medullomyoblastoma. Together, our findings illustrate the origin of muscle cells in medullomyoblastomas and the oncogenic mechanism of OTX2 as a repressor of diverse differentiating potential.

Neuronal induction and regional identity by co-culture of adherent human embryonic stem cells with chicken notochords and somites.

The role of somites and notochords in neuroectoderm differentiation from the embryonic ectoderm and its subsequent patterning into regional compartments along rostro-caudal and dorso-ventral axes, especially in humans, remains elusive. Here, we demonstrate the co-culture effect of somites and notochords isolated from chicken embryos on the neuronal differentiation and regional identity of an adherent culture of human embryonic stem cells (hESCs). Notochord increased the efficiency and speed of neuronal induction, whereas somites had a weak neuronal inducing effect on hESCs. However, a synergistic effect was not observed when notochords and somites were used together. Moreover, in somite and notochord co-culture groups, hESCs-derived neuronal cells expressed HOXB4, OTX2, IRX3 and PAX6, indicative of dorsal hindbrain and ventral anterior identities, respectively. Our results reveal the influence of embryonic notochord and somite co-culture in providing neuronal induction as well as rostro-caudal and dorso-ventral regional identity of hESCs-derived neuronal cells. This study provides a model through which in vivo neuronal induction events may be imitated.

Regulation of cell cycle genes and induction of senescence by overexpression of OTX2 in medulloblastoma cell lines.

The transcription factor orthodenticle homeobox 2 (OTX2) has been implicated in the pathogenesis of medulloblastoma, as it is often highly expressed and sometimes amplified in these tumors. Little is known of the downstream pathways regulated by OTX2. We therefore generated MED8A and DAOY medulloblastoma cell lines with doxycycline-inducible OTX2 expression. In both cell lines, OTX2 inhibited proliferation and induced a senescence-like phenotype with senescence-associated ß-galactosidase activity. Expression profiles of time series after OTX2 induction in MED8A showed early upregulation of cell cycle genes related to the G(2)-M phase, such as AURKA, CDC25C, and CCNG2. Paradoxically, G(1)-S phase genes such as MYC, CDK4, CDK6, CCND1, and CCND2 were strongly downregulated, in line with the observed G(1) arrest. ChIP-on-chip analyses of OTX2 binding to promoter regions in MED8A and DAOY showed a strong enrichment for binding to the G(2)-M genes, suggesting a direct activation. Their mRNA expression correlated with OTX2 expression in primary tumors, underscoring the in vivo relevance of this regulation. OTX2 induction activated the P53 pathway in MED8A, but not in DAOY, which carries a mutated P53 gene. In DAOY cells, senescence-associated secretory factors, such as interleukin-6 and insulin-like growth factor binding protein 7, were strongly upregulated after OTX2 induction. We hypothesize that the imbalance in cell cycle stimulation by OTX2 leads to cellular senescence either by activating the P53 pathway or through the induction of secretory factors. Our data indicate that OTX2 directly induces a series of cell cycle genes but requires cooperating genes for an oncogenic acceleration of the cell cycle.

Evaluation of retinoic acid therapy for OTX2-positive medulloblastomas.

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.

Expression of the brain transcription factor OTX1 occurs in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma.

The roles in brain development. Previous studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. Here, we 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.

A core paired-type and POU homeodomain-containing transcription factor program drives retinal bipolar cell gene expression.

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 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, 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.

GABA immunoreactivity in the developing rat thalamus and Otx2 homeoprotein expression in migrating neurons.

We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.

In vivo identification of a 107-base pair promoter element mediating neuron-specific expression of mouse gonadotropin-releasing hormone.

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.

Developmental malformations of the eye: the role of PAX6, SOX2 and OTX2.

Eye development initiates as an evagination of the early neural plate, before the closure of the neural tube. Structural malformations of the eye such as anophthalmia and microphthalmia arise very early in development. It is not surprising therefore that three of the genes currently identified to play a significant role in these developmental eye anomalies are also major players in brain development and regionalization. However, as has been emerging for a high proportion of transcriptional regulators studied, these genes have evolved to play multiple roles throughout development, and perhaps even in adult tissue maintenance. This complex spatiotemporal expression pattern requires elaborate regulatory systems which we are beginning to unravel. A major component of these complex regulatory networks is a series of cis-acting elements, highly conserved through evolution, which spread large distances from the coding region of each gene. We describe how cross regulation for PAX6, SOX2 and perhaps OTX2 has now been uncovered, pointing to the mechanisms that can fine-tune the expression of such essential developmental components. These interactions also help us understand why there is significant phenotypic overlap between mutations at these three loci.