Otx2 Expression Research Articles

Overexpression of OTX1 promotes tumorigenesis in patients with esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a multifactorial disease and the sixth leading cause of death from cancer worldwide. Patients with ESCC usually have a short survival period due to the late stage at diagnosis. Incidence rates of ESCC remain high among the elderly. With recent advances, it has been demonstrated that ESCC tumors display a unique genetic profile. This study aimed to examine the possible function of OTX1 in ESCC. MethodsWe collected paraffin-embedded tumor tissues from 107 patients originally diagnosed with ESCC at Xijing Hospital and fresh-harvested and paired adjacent normal esophageal tissues from 15 ESCC patients undergoing curative resection. The expression level of OTX1 was evaluated through immunohistochemistry and western blot. Prognostic and survival analyses were conducted using univariate/multivariate analysis and log-rank analysis with SPSS 23.0. Cell models and xenograft models were used to examine the overexpression of OTX1 in vivo and in vitro. ResultsOTX1 was overexpressed in ESCC tissues compared with normal esophageal tissues. Both the mRNA expression level and protein level of OTX1 were higher than they were in paired normal tissue. Prognostic and OS analyses showed that the OTX1 expression level was an individual prognostic factor in ESCC patients. Cell viability was significantly promoted when OTX1 was overexpressed in ESCC cell, Furthermore, downregulating OTX1 in EC109 cell significantly attenuated the cell proliferation migration and invasion. Flow cytometric detection showed that cells overexpressing OTX1 were predominantly in the S and G2&M phases. In the xenograft model, both tumor size and weight in the OTX1 overexpression group were significantly larger than those in the control group. ConclusionOTX1 is an independent prognostic factor of ESCC and contributes to tumorigenesis both in vivo and in vitro.

Extracellular Matrix Expression in Human Induced Pluripotent Stem Cell-Derived Optic Vesicles

Background: Human tissue/organ development is a complex, highly orchestrated process, regulated in part by the surrounding extracellular matrix (ECM). Every complex tissue, including the retina, has a unique ECM configuration that plays a critical role in cellular differentiation, adhesion, migration, and maturation. Aim: To characterize ECM expression of human induced pluripotent stem cell-derived optic vesicles (iPSC-OVs). Methods: A 3- dimensional (3D) in vitro suspension culture system was used to direct differentiation of human induced pluripotent stem cells (iPSCs) into optic vesicles (OVs). Stepwise differentiation of iPSCs into retinal progenitor cells was confirmed by sequential expression of OTX2, SOX1, SIX6, LHX2, PAX6, and CHX10. Expression of ECM genes in iPSC-derived OVs was analyzed by RT2 ProfilerTM PCR Array, whereas immunofluorescence staining was performed to detect ECM proteins in the OVs. Results: A number of cell adhesion molecules (CAMs) previously reported to be abundantly expressed in iPSCs such as E-cadherin, Intercellular adhesion molecule-1 (ICAM1), Integrin-α L, Integrin-α M, Integrin-α 6 were downregulated while neural and retina specific CAMs including neural cell adhesion molecule 1 (NCAM1), neural plakophilin-related armadillo repeat protein (NPRAP), Integrin-α 1 and Integrin-α 4 were upregulated. Several glycoproteins that have been reported to play key roles during retinogenesis, namely CD44, Tenascin C, Tenascin R, Neurocan, Neuroglycan C, Delta 2 Catenin, Vitronectin, and Reelin were also present. Conclusion: We have identified an array of ECM proteins that were expressed during retinogenesis. Further characterization of these proteins will lead to a better understanding of retinal development.

OTX2 Homeoprotein Functions in Adult Choroid Plexus.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

Initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity.

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Non-cell-autonomous OTX2 transcription factor regulates anxiety-related behavior in the mouse

The OTX2 homeoprotein transcription factor is expressed in the dopaminergic neurons of the ventral tegmental area, which projects to limbic structures controlling complex behaviors. OTX2 is also produced in choroid plexus epithelium, from which it is secreted into cerebrospinal fluid and transferred to limbic structure parvalbumin interneurons. Previously, adult male mice subjected to early-life stress were found susceptible to anxiety-like behaviors, with accompanying OTX2 expression changes in ventral tegmental area or choroid plexus. Here, we investigated the consequences of reduced OTX2 levels in Otx2 heterozygote mice, as well as in Otx2+/AA and scFvOtx2tg/0 mouse models for decreasing OTX2 transfer from choroid plexus to parvalbumin interneurons. Both male and female adult mice show anxiolysis-like phenotypes in all three models. In Otx2 heterozygote mice, we observed no changes in dopaminergic neuron numbers and morphology in ventral tegmental area, nor in their metabolic output and projections to target structures. However, we found reduced expression of parvalbumin in medial prefrontal cortex, which could be rescued in part by adult overexpression of Otx2 specifically in choroid plexus, resulting in increased anxiety-like behavior. Taken together, OTX2 synthesis by the choroid plexus followed by its secretion into the cerebrospinal fluid is an important regulator of anxiety-related phenotypes in the mouse.

The phenotypic spectrum associated with OTX2 mutations in humans.

Objective The transcription factor OTX2is implicated in ocular, craniofacial, and pituitary development.DesignWe aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action.MethodsWe screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants.ResultsTwo chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary.ConclusionsOTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

Molecular mechanisms of hearing loss in Nager syndrome

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.

Response of Human Retinal Pigment Epithelial Cells to the Effect of the Conditioned Media of Newt Retinal Regenerates

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Choroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

Metabolic control of DNA methylation in naive pluripotent cells.

The naïve epiblast and embryonic stem cells (ESCs) give rise to all cells of the adult. Such developmental plasticity is associated with genome hypomethylation. Here we show that LIF/Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased alpha-ketoglutarate production from glutamine, leading to increased Dnmt3a/b expression and DNA methylation. Notably, genome methylation is dynamically controlled by modulating alpha-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome, by reducing the expression of Otx2 and its targets Dnmt3a/b. Genetic inactivation of Otx2 or Dnmt3a/b results in genomic hypomethylation even in the absence of active LIF/Stat3. Stat3-/- ESCs show increased methylation at Imprinting Control Regions and altered expression of cognate transcripts. Single-cell analysis of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a/b and imprinted genes. Several cancers display Stat3-overactivation and abnormal DNA methylation, therefore the molecular module we described might be exploited under pathological conditions.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

SummaryRecent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

ATRT-27. COST-EFFECTIVE ASSAYS TO SUBGROUP ATRT IN THE DAILY ROUTINE

Three atypical teratoid rhabdoid tumors (ATRT) molecular subgroups with different bio-clinical characteristics have been reported (TYR, SHH and MYC). Molecular subgrouping relies on either methylation profiling (reference methods), or expression profiling. However, the cost-effectiveness of such pangenomic screening is questionable. This work aims to study the reliability of alternative techniques for subgroup classification in the daily routine. Illumina EPIC-arrays were performed on 46 samples. Among those cases, expression profiling were analysed by RNAseq (n=30). We designed a 26-gene panel to assess expression profiling using the Nanostring technology; this was applied to 35 tumors. Immunohistochemistry (IHC) was used for 20 samples; it relied on the expression of MITF, TYR, OTX2 and MYC. We first assessed the concordance between DNA methylation and RNAseq based profilings; then, between RNAseq and Nanostring and, finally, between methylation profiling and Nanostring or IHC, the two rapidest and cheapest tools. The concordance between the two expression-based profiling was 19/21. EPIC-arrays and RNAseq or Nanostring were concordant in 26/30 and 30/35 samples, respectively. The concordance was perfect for methylation-defined MYC subtype. Finally, 17/20 tumor samples were classified in the same subgroup by EPIC-arrays and IHC; the 3/20 misclassified tumors were SHH by methylation but consistently MYC by IHC, Nanostring and RNAseq. There was 90–100% of concordance for TYR subgroup (all techniques). We have designed a gene panel-based expression signature that shows promising concordance with RNAseq and methylation profiling. Nanostring assay and IHC well predict ATRT subgroup classification for MYC and TYR subclass, but less so for methylation-defined SHH ones.

Long noncoding RNA MAFG-AS1 facilitates the progression of hepatocellular carcinoma via targeting miR-3196/OTX1 axis.

Hepatocellular carcinoma (HCC) is an invasive malignant tumor with high mortality rate. Long non-coding RNA (lncRNA) MAFG-AS1 has been showed to play an oncogenic role in several malignant tumors. Nonetheless, the exact role of MAFG-AS1 in the progression of HCC has not been fully elucidated. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the mRNA and protein expression of MAFG-AS1 in HCC tissues and cells. Cell counting kit-8 (CCK-8), transwell and tubule formation assays were applied to uncover the proliferation, migration, invasion and tumor angiogenesis of HCC cells, respectively. RNA binding protein immunoprecipitation (RIP) assay and Luciferase reporter gene assay were employed to explore the molecular mechanism. In addition, Xenograft assay was used to investigate the effect of MAFG-AS1 in vivo. MAFG-AS1 was highly expressed in HCC tissues and cells. Attenuation of MAFG-AS1 evidently suppressed the proliferation, migration, invasion and tumor angiogenesis of HCC cells, suggesting that MAFG-AS1 played an oncogenic role in HCC. MiR-3196 was sponged by MAFG-AS1, and OTX1 was a downstream target of miR-3196 in HCC. In addition, OTX1 expression was negatively associated with miR-3196 but positively associated with MAFG-AS1 in HCC tissues. Overexpression of OTX1 could abolish the repressive influence of MAFG-AS1 inhibition on the proliferation, migration, invasion and tumor angiogenesis of HCC cells. MAFG-AS1 facilitated the progression of HCC via targeting miR-3196/OTX1 axis, which might be used as a new insight for HCC treatment.

Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) are valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies.

OTX1 exerts an oncogenic role and is negatively regulated by miR129-5p in laryngeal squamous cell carcinoma

BackgroundOrthodenticle homeobox 1 (OTX1) is a transcription factor that plays an important role in various human cancers. However, the function of OTX1 in laryngeal squamous cell carcinoma (LSCC) is largely unknown. We aimed to explore the roles of OTX1 in LSCC and its possible molecular mechanism.MethodsThe expression levels of OTX1 were assessed in LSCC cell lines and tissue samples. We further examined the effect of OTX1 on LSCC progression. The upstream regulator of OTX1 was identified using a computer algorithm and confirmed experimentally.ResultsOTX1 was highly expressed in 70.7% (70/99) of LSCC tissue samples. The OTX1 expression in LSCC was significantly correlated with lymph node metastasis. High OTX1 expression in patients with LSCC was correlated with poor prognosis. Knockdown of OTX1 inhibited proliferation, colony formation, migration and invasion in LSCC cells. Knockdown of OTX1 inhibited tumor growth in a xenograft mouse model. Mechanistically, OTX1 might act as a direct target of miR-129-5p. OTX1 enhanced tumorigenicity and tumor growth both in vitro and in vivo.ConclusionsOur findings support that OTX1 is an oncogene in LSCC tumorigenesis and progression. Furthermore, OTX1 is a direct target of miR-129-5p in LSCC cells. Taken together, OTX1 is a promising diagnostic and therapeutic marker for LSCC.

Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina.

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.

OR16-04 OTX2 Mutations in Congenital Hypopituitarism Patients

The transcription factor OTX2 is implicated in pituitary, ocular and craniofacial development. Mutations have been described in patients with variable congenital hypopituitarism (CH) ranging from isolated growth hormone deficiency (IGHD) to combined pituitary hormone deficiency (CPHD) with/without an ectopic posterior pituitary (EPP).We aimed (i) to establish the contribution of OTX2 mutations in the etiology of CH in a sub-cohort of patients and to study their functional consequences and (ii) establish a detailed human OTX2 expression profile in a hypothalamo-pituitary (HP) context. We screened 127 patients from national (n=103) and international centers (n=24) on the septo-optic dysplasia (SOD) spectrum with variable eye abnormalities. Eye abnormalities included micro/anophthalmia, retinal dystrophy and/or coloboma in 29 of these patients, with the rest having optic nerve hypoplasia (ONH). An EPP was reported on MRI in 35 patients. The cohort previously tested negative for mutations in HESX1, SOX2, SOX3, PROKR2 and GH1. Transactivation assays involved a dual-luciferase reporter in murine hypothalamic GT1-7 neurons transiently transfected with OTX2 constructs. In situ hybridization was performed to analyze human brain OTX2 expression during embryogenesis. Seven heterozygous OTX2 changes were identified: two chromosomal deletions spanning OTX2 in patients with micro/anophthalmia, GHD and an EPP, with one patient having cerebellar hypoplasia. Three missense substitutions resulting in truncated proteins: (i) the previously reported p.S138* and (ii) p.C170*, in two patients with retinal dystrophy, EPP and IGHD respectively, and (iii) the novel p.E79* in a patient with micro/anophthalmia, EPP and CPHD. A novel insertion-deletion resulting in a truncated protein p.S167* in a patient with microphthalmia, GHD, ONH, EPP and an enlarged abnormal pituitary, and a novel deletion resulting in a frameshift p.Val139Aspfs*39 in a patient with microcephaly, microphthalmia, ONH and an EPP were also identified. The human OTX2 variants caused a significant reduction in transactivation compared to wild type. Our gene expression data identified human OTX2 transcripts in the posterior pituitary, retina, ear, thalamus, choroid plexus, and in the hypothalamus during embryogenesis, but not in RP. To conclude, we identified OTX2 variants in 7 unrelated CH patients with eye abnormalities including 3 with retinal dystrophy and one with a cerebellar abnormality. As OTX2 is involved at multiple levels during HP development, these patients should be monitored for evolving endocrinopathies. Human OTX2 is expressed in the posterior pituitary, the retina and the ear at CS19 and 20 (between 6-7 weeks gestation), and in areas of the hindbrain at CS23, but not in RP at any stage analyzed in this study. The endocrine phenotypes in patients with OTX2 mutations are most likely of hypothalamic origin.

Long non-coding RNA HNF1A-AS1 upregulates OTX1 to enhance angiogenesis in colon cancer via the binding of transcription factor PBX3

Colon cancer shows characteristics of metastasis, which is associated with angiogenesis. Increasing evidence highlights long non-coding RNAs (lncRNAs) as important participants in angiogenesis of cancers, including colon cancer. Hence, this study investigated the role of HNF1A-AS1 in angiogenesis of colon cancer. RT-qPCR and Western blot analysis were applied to detect HNF1A-AS1 and OTX1 expression in colon cancer tissues and cell lines. Then the interactions among HNF1A-AS1, PBX3, OTX1 and ERK/MAPK pathway were evaluated with RNA pull-down, RIP, ChIP and dual-luciferase reporter gene assays. Next, HCT116 and SW620 cells were treated with si-HNF1A-AS1 and/or oe-OTX1 plasmids to assess the effects of HNF1A-AS1 and OTX1 on angiogenesis, which was further evaluated in nude mice injected with SW620 cells transfected with sh-HNF1A-AS1 or sh-OTX1 lentivirus. HNF1A-AS1 and OTX1 were highly expressed in colon cancer. Silencing of HNF1A-AS1 inhibited angiogenesis of colon cancer in vivo and in vitro. HNF1A-AS1 increased the OTX1 expression by binding to transcription factor PBX3 to promote angiogenesis in colon cancer. Further, HNF1A-AS1 upregulated OTX1 to activate the ERK/MAPK pathway. Altogether, our findings identified HNF1A-AS1 as a tumor-promoting RNA in colon cancer, which could serve as a potential therapeutic target for colon cancer treatment.