Central role of the habenulo-interpeduncular system in the neurodevelopmental basis of susceptibility and resilience to anxiety in mice.

Having experienced stress during sensitive periods of brain development strongly influences how individuals cope with later stress. Some are prone to develop anxiety or depression, while others appear resilient. The as-yet-unknown mechanisms underlying these differences may lie in how genes and environmental stress interact to shape the circuits that control emotions. Here, we investigated the role of the habenulo-interpeduncular system (HIPS), a critical node in reward circuits, in early stress-induced anxiety in mice. We found that habenular and IPN components characterized by the expression of Otx2 are synaptically connected and particularly sensitive to chronic stress (CS) during the peripubertal period. Stress-induced peripubertal activation of this HIPS subcircuit elicits both HIPS hypersensitivity to later stress and susceptibility to develop anxiety. We also show that HIPS silencing through conditional Otx2 knockout counteracts these effects of stress. Together, these results demonstrate that a genetic factor, Otx2, and stress interact during the peripubertal period to shape the stress sensitivity of the HIPS, which is shown to be a key modulator of susceptibility or resilience to develop anxiety.

The complete cell lineage and MAPK- and Otx-dependent specification of the dopaminergic cells in the Ciona brain.

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.

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.

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.

Cis-regulatory dissection of cone development reveals a broad role for Otx2 and Oc transcription factors.

The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

MiR-497-5p ameliorates the oxyhemoglobin-induced subarachnoid hemorrhage injury in vitro by targeting orthodenticle homeobox protein 1 (Otx1) to activate the Nrf2/HO-1 pathway.

Subarachnoid hemorrhage (SAH) is a neurological disorder that severely damages the brain and causes cognitive impairment. MicroRNAs are critical regulators in a variety of neurological diseases. MiR-497-5p has been found to be downregulated in the aneurysm vessel walls obtained from patients with aneurysmal subarachnoid hemorrhage, but its functions and mechanisms in SAH have not been reported. Therefore, this study was designed to investigate the effect of miR-497-5p and its related mechanisms in SAH. We established an in vitro SAH model by exposing PC12 cells to oxyhemoglobin (oxyHb). We found that miR-497-5p was downregulated in SAH serum and oxyHb-treated PC12 cells, and its overexpression inhibited the oxyHb-induced apoptosis, inflammatory response and oxidative stress via activation of the Nrf2 pathway. Mechanistically, the targeting relationship between miR-497-5p and Otx1 was verified by luciferase reporter assays. Moreover, Otx1 upregulation abolished the protective effects of miR-497-5p upregulation against oxyHb-induced apoptosis, inflammation and oxidative stress in PC12 cells. Collectively, our findings indicate that miR-497-5p could inhibit the oxyHb-induced SAH damage by targeting Otx1 to activate the Nrf2/HO-1 pathway, which provides a potential therapeutic target for SAH treatment.

14q22.3 duplication including OTX2 in a girl with medulloblastoma: A case report with literature review.

Orthodenticle homeobox 2 (OTX2) is a known oncogenic driver of medulloblastoma. Germline duplication of 14q22.3 including OTX2 is a rare condition reported in patients with combined pituitary hormone deficiency, oculo-auriculo-vertebral spectrum, and hemifacial microsomia. There has been one previously published case of a patient carrying a 14q22.3 duplication that included OTX2 with hemifacial microsomia who also developed medulloblastoma. Here, we present a case of a 6-year-old girl with a history of delayed development who was diagnosed with medulloblastoma. Genetic evaluations revealed that she inherited a germline duplication of 14q22.3, which included OTX2. This genetic alteration was passed down from her mother, who also had a history of delayed development. Results from other genetic testing, including exome sequencing, fragile X syndrome, and mtDNA testing, were negative/normal. This is the second report of a 14q22.3 duplication that included OTX2 in a patient with medulloblastoma. Further studies are necessary to establish a clear association.

The homeodomain transcription factor CEH-37 regulates PMK-1/p38 MAPK pathway to protect against intestinal infection via the phosphatase VHP-1.

Increasing evidence indicate that the expression of defense genes at the right place and the right time are regulated by host-defense transcription factors. However, the precise mechanisms of this regulation are not well understood. Homeodomain transcription factors, encoded by homeobox genes, play crucial role for the development of multicellular eukaryotes. In this study, we demonstrated that homeodomain transcription factor CEH-37 (known as OTX2 in mammals) was a key transcription factor for host defense in Caenorhabditis elegans. Meanwhile, CEH-37 acted in the intestine to protect C. elegans against pathogen infection. We further showed that the homeodomain transcription factor CEH-37 positively regulated PMK-1/ p38 MAPK activity to promote the intestinal immunity via suppression phosphatase VHP-1. Furthermore, we demonstrated that this function was conserved, because the human homeodomain transcription factor OTX2 also exhibited protective function in lung epithelial cells during Pseudomonas aeruginosa infection. Thus, our work reveal that CEH-37/OTX2 is a evolutionarily conserved transcription factor for defense against pathogen infection. The finding provides a model in which CEH-37 decreases VHP-1 phosphatase activity, allowing increased stimulation of PMK-1/p38 MAPK phosphorylation cascade in the intestine for pathogen resistance.

MicroRNA-195-5p Attenuates Pregnancy-Induced Hypertension by Inhibiting Oxidative Stress via OTX1/MAPK Signaling Pathway.

Pregnancy-induced hypertension (PIH) is a hypertensive disorder during pregnancy and can induce perinatal death of human infants. MicroRNA (miR)-195-5p was validated to display low expression in severe preeclampsia placentas, but the role of miR-195-5p in pregnancy-induced hypertension (PIH) has not been investigated. The study emphasized on the functions and mechanism of miR-195-5p in PIH. A reduced uterine perfusion pressure (RUPP) rat model was established to mimic PIH in vivo. Adenovirus (Ad)-miR-195-5p agomir and/or Ad-OTX1 were further injected into some model rats. RT-qPCR was conducted to assess the expression of miR-195-5p and orthodenticle homeobox 1 (OTX1) in rat placental tissues, the isolated aortic endothelial cells (AECs), and in serum samples of PIH patients. Western blot analysis was implemented to measure the protein levels of OTX1, VEGFA, and key factors involved in the MAPK signaling pathway. The concentrations of oxidative stress markers (superoxide dismutase, catalase, and lipid hydroperoxide) in AECs and placental tissues of RUPP rats were measured by corresponding kits. The binding relation between miR-195-5p and OTX1 was verified using the dual-luciferase reporter assay. Hematoxylin-eosin staining was conducted to evaluate the pathological features of rat placental tissues. MiR-195-5p was downregulated, while OTX1 was upregulated in rat placental tissues and human serum samples of PIH patients. MiR-195-5p could target OTX1 and inversely regulate OTX1 expression in AECs and rat placental tissues. In addition, miR-195-5p can negatively regulate VEGFA level. Furthermore, miR-195-5p inactivates oxidative stress and the MAPK signaling by downregulating OTX1 in AECs. In vivo experiments revealed that OTX1 overexpression reversed the protective effect of miR-195-5p overexpression on placental damage and oxidative stress. MiR-195-5p alleviates PIH by inhibiting oxidative stress via targeting OTX1 and inactivating MAPK signaling.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

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 homeobox 2 (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.

BRD4: New hope in the battle against glioblastoma.

The BET family proteins, comprising BRD2, BRD3 and BRD4, represent epigenetic readers of acetylated histone marks that play pleiotropic roles in the tumorigenesis and growth of multiple human malignancies, including glioblastoma (GBM). A growing body of investigation has proven BET proteins as valuable therapeutic targets for cancer treatment. Recently, several BRD4 inhibitors and degraders have been reported to successfully suppress GBM in preclinical and clinical studies. However, the precise role and mechanism of BRD4 in the pathogenesis of GBM have not been fully elucidated or summarized. This review focuses on summarizing the roles and mechanisms of BRD4 in the context of the initiation and development of GBM. In addition, several BRD4 inhibitors have been evaluated for therapeutic purposes as monotherapy or in combination with chemotherapy, radiotherapy, and immune therapies. Here, we provide a critical appraisal of studies evaluating various BRD4 inhibitors and degraders as novel treatment strategies against GBM.

The long non-coding RNA OTX2-AS1 promotes tumor growth and predicts response to BCL-2 inhibition in medulloblastoma.

PURPOSE: Primary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma. METHODS: Analyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action. RESULTS: We detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro. CONCLUSIONS: Our study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.

HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch-Nyhan disease.

In Lesch-Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

OTX Genes in Adult Tissues.

OTX homeobox genes have been extensively studied for their role in development, especially in neuroectoderm formation. Recently, their expression has also been reported in adult physiological and pathological tissues, including retina, mammary and pituitary glands, sinonasal mucosa, in several types of cancer, and in response to inflammatory, ischemic, and hypoxic stimuli. Reactivation of OTX genes in adult tissues supports the notion of the evolutionary amplification of functions of genes by varying their temporal expression, with the selection of homeobox genes from the "toolbox" to drive or contribute to different processes at different stages of life. OTX involvement in pathologies points toward these genes as potential diagnostic and/or prognostic markers as well as possible therapeutic targets.

The Midbrain Preisthmus: A Poorly Known Effect of the Isthmic Organizer.

This essay reexamines molecular evidence supporting the existence of the 'preisthmus', a caudal midbrain domain present in vertebrates (studied here in the mouse). It is thought to derive from the embryonic m2 mesomere and appears intercalated between the isthmus (caudally) and the inferior colliculus (rostrally). Among a substantial list of gene expression mappings examined from the Allen Developing and Adult Brain Atlases, a number of quite consistent selective positive markers, plus some neatly negative markers, were followed across embryonic stages E11.5, E13.5, E15.5, E18.5, and several postnatal stages up to the adult brain. Both alar and basal subdomains of this transverse territory were explored and illustrated. It is argued that the peculiar molecular and structural profile of the preisthmus is due to its position as rostrally adjacent to the isthmic organizer, where high levels of both FGF8 and WNT1 morphogens must exist at early embryonic stages. Isthmic patterning of the midbrain is discussed in this context. Studies of the effects of the isthmic morphogens usually do not attend to the largely unknown preisthmic complex. The adult alar derivatives of the preisthmus were confirmed to comprise a specific preisthmic sector of the periaqueductal gray, an intermediate stratum represented by the classic cuneiform nucleus, and a superficial stratum containing the subbrachial nucleus. The basal derivatives, occupying a narrow retrorubral domain intercalated between the oculomotor and trochlear motor nuclei, include dopaminergic and serotonergic neurons, as well as a variety of peptidergic neuron types.

Knockdown of circMYOF inhibits cell growth, metastasis, and glycolysis through miR-145-5p/OTX1 regulatory axis in laryngeal squamous cell carcinoma.

New evidence suggests that abnormal expression of circular RNA (circRNA) is associated with the development of human cancers. This study aims to reveal circMYOF roles in the malignant phenotype of laryngeal squamous cell carcinoma (LSCC). The expression of circMYOF, microRNA (miR)-145-5p, and orthodenticle homeobox 1 (OTX1) was detected by quantitative real-time PCR. Cell proliferation, migration, invasion, and apoptosis were determined using colony formation assay and EdU assay, wound healing assay, transwell assay, and flow cytometry, respectively. Protein expression was examined by western blot analysis. Cell glycolysis was assessed by detecting glucose consumption and lactate production. Mice xenograft models were constructed to evaluate the regulation of circMYOF on LSCC tumorigenesis. The regulatory relationships among circMYOF, miR-145-5p, and OTX1 were identified using dual-luciferase reporter assay and RIP assay. Serum exosomes were isolated to confirm the existence of circMYOF in LSCC patients. CircMYOF was upregulated in LSCC tissues and cells, and its knockdown suppressed LSCC cell growth, metastasis, and glycolysis, as well as inhibited LSCC tumor growth. MiR-145-5p had decreased expression in LSCC, and it could be sponged by circMYOF. The inhibition effect of circMYOF lentivirus short hairpin RNA (sh-circMYOF) on LSCC progression was restored by the inhibitor of miR-145-5p (in-miR-145-5p). Also, OTX1 was targeted by miR-145-5p and was positively regulated by circMYOF. MiR-145-5p could repress LSCC progression, and OTX1 overexpression also eliminated this effect. In addition, we found that circMYOF was significantly overexpressed in the serum exosomes of LSCC patients. Our data revealed that circMYOF contributed to LSCC progression by promoting cell growth, metastasis, and glycolysis through miR-145-5p/OTX1 axis.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.