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.

OTX1 regulates tumorigenesis and metastasis in glioma.

The most prevalent kind of primary brain tumors, gliomas, have a dismal prognosis. Recent advances in the tumor-promoting ability of OTX1 have drawn increasing attention. The overexpression of OTX1 has been reported to be associated with tumor-promoting effects in several malignancies, but its expression in gliomas is unknown. The oncogene OTX1 is increased in gliomas and is linked to a poor prognosis, as we show here. The degree of OTX1 positive expression is doubtlessly concomitant with the grade of glioma. We observed that OTX1 was up-regulated in gliomas, influenced the epithelial-mesenchymal transition (EMT), encouraged glioma cell growth and proliferation, and was linked to a poor clinical outcome for patients. At present, the prognosis of glioma is still not optimistic, and further research is needed to find a new target for treatment. According to our research, OTX1 is anticipated to emerge as a novel biological target for determining glioma prognosis and treatment.

Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

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.

Orthodenticle Homeobox OTX1 Promotes Papillary Thyroid Carcinoma Progression and Is a Potential Prognostic Biomarker.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid neoplasms, characterized by evidence of follicular cell differentiation. Orthodenticle homeobox 1 (OTX1) is a transcription factor which has been implicated in numerous diseases, including malignancies. The objective of this research was to explore the function of OTX1 in PTC. Immunohistochemistry (IHC) was employed to determine the protein level of OTX1 in PTC specimens. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, a xenograft model on nude mice was established to investigate in vivo effects of OTX1. Our results revealed that OTX1 was significantly upregulated within specific PTC tissues and was remarkably correlated with unfavorable clinical outcomes in PTC. Silencing OTX1 resulted in a significant inhibition in cell viability and suppressed cell proliferation. In addition, in vivo experiments demonstrated that OTX1 silencing resulted in a significant suppression of tumor growth in nude mice. Collectively, these results suggest that OTX1 may play crucial roles in promoting PTC progression.

Taurodeoxycholic acid-YAP1 upregulates OTX1 in promoting gallbladder cancer malignancy through IFITM3-dependent AKT activation.

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.

Upregulation of OTX2-AS1 is Associated With Immune Infiltration and Predicts Prognosis of Gastric Cancer.

BACKGROUND: It is unclear whether the long non-coding RNA (lncRNA) OTX2 antisense RNA 1 (OTX2-AS1) plays a pivotal role in gastric cancer (GC). An analysis of The Cancer Genome Atlas (TCGA) database data and bioinformatics was used to explore the relationship between OTX2-AS1 and GC in the current study. METHODS: We evaluated the relationship between clinical features and OTX2-AS1 expression, prognostic factors, and the significant involvement of OTX2-AS1 in function using various statistical methods, such as Kaplan-Meier method, Cox regression analysis, Gene Set Enrichment Analysis (GSEA), and immune infiltration analysis. GC cell lines were tested for OTX2-AS1 expression using qRT-PCR. RESULTS: A high level of OTX2-AS1 expression was significantly and negatively associated with Helicobacter pylori (H pylori) infection in GC patients (P = .006) and predicted a poorer overall survival (OS) (HR: 1.54; 95% CI: 1.10-2.14; P = .011), progression-free interval (PFI) (HR: 1.75; 95% CI: 1.22-2.51; P = .002) and disease-specific survival (DSS) (HR: 1.85; 95% CI: 1.21-2.85; P = .005) in GC patients. There was an independent correlation between OTX2-AS1 expression (HR: 1.771; 95% CI: 1.164-2.696; P = .008) and OS in patients with GC. There were differential enrichments for the OTX2-AS1 high expression phenotype in the olfactory transduction, G alpha (s) signaling events, keratinization, olfactory signaling pathway, and preimplantation embryo. OTX2-AS1 expression may be related to certain immune-infiltrating cells. Compared to gastric epithelial cells (GES-1), GC cell lines showed a significant increase in OTX2-AS1 expression. CONCLUSION: There was a significant association between OTX2-AS1 expression in GC patients and poor survival, suggesting that it may be a useful biomarker for prognosis and immunotherapy outcome of stomach adenocarcinoma (STAD) in GC.

The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration.

Tissue-specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.

c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor that regulates early retinal development.

BACKGROUND: The transcription factor orthodenticle homeobox 2 (OTX2) has critical functions in brain and eye development, and its mutations in humans are related to retinal diseases, such as ocular coloboma and microphthalmia. However, the regulatory mechanisms of OTX2 are poorly identified. OBJECTIVE: The identification of JNK1 as an OTX2 regulatory protein through the protein interaction and phosphorylation. METHODS: To identify the binding partner of OTX2, we performed co-immunoprecipitation and detected with a pooled antibody that targeted effective kinases. The protein interaction between JNK1 and OTX2 was identified with the co-immunoprecipitation and immunocytochemistry. In vivo and in vitro kinase assay of JNK1 was performed to detect the phosphorylation of OTX2 by JNK1. RESULTS: JNK1 directly interacted with OTX2 through the transactivation domain at the c-terminal region. The protein-protein interaction and co-localization between JNK1 and OTX2 were further validated in the developing P0 mouse retina. In addition, we confirmed that the inactivation of JNK1 K55N mutant significantly reduced the JNK1-mediated phosphorylation of OTX2 by performing an immune complex protein kinase assay. CONCLUSION: c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor through the protein-protein interaction.

MiR-195-5p suppresses gastric adenocarcinoma cell progression via targeting OTX1.

Gastric adenocarcinoma (GAC) caused by malignant transformation of gastric adenocytes is a malignancy with high incidence. MiR-195-5p modulates a variety of cancers. One of its target genes, orthodenticle homeobox 1 (OTX1), is believed to be a key modulator of tumor progression. We aim to analyze the mechanism of miR-195-5p and OTX1 in GAC. MiR-195-5p and OTX1 mRNA levels in GAC cells were tested via qRT-PCR. OTX1 protein and EMT-related protein levels were examined through western blot. Several cell functional assays were designed to measure changes in cell malignant behaviors. Dual luciferase assay verified the targeting relation of miR-195-5p and OTX1. These experimental results showed significantly low miR-195-5p expression and significantly high OTX1 expression in GAC cells. Enforced miR-195-5p level repressed cell malignant progression and accelerated cell apoptosis in GAC. Increased OTX1 weakened the above-mentioned effect caused by overexpressing miR-195-5p. Thus, miR-195-5p restrained migration, proliferation, invasion and epithelial-mesenchymal transition process of GAC cells, and promoted cell apoptosis through regulating OTX1. A new insight is provided for searching for biomarkers or therapeutic targets of GAC.

OTX1 promotes tumorigenesis and progression of cervical cancer by regulating the Wnt signaling pathway.

Cervical cancer is a common malignant tumor in females. Orthodenticle homolog 1 (OTX1) serves a key role in the occurrence and progression of tumors. The present study aimed to investigate the role and potential mechanism of OTX1 in cervical cancer. OTX1 expression was analyzed by western blotting, reverse transcription­quantitative PCR and immunohistochemistry. MTT assay was performed to assess cell viability. EdU and colony formation assay were used to measure cell proliferation. Wound healing and Transwell assays were performed to measure cell migration and invasion. Western blot assay was performed for the assessment of protein expression. Gene set enrichment analysis (GSEA) was performed to analyze signaling pathways regulated by OTX1. Co­Immunoprecipitation assay was performed to confirm the interaction between OTX1 and Wnt9b. In cervical cancer tissue and cells, OTX1 was significantly upregulated. OTX1 overexpression promoted proliferation, migration and invasion of cervical cancer cells. OTX1 silencing significantly decreased cell proliferation, migration and invasion of cervical cancer. GSEA showed that OTX1 activated the Wnt signaling pathway. OTX1 silencing inhibited the increased levels of adenomatous polyposis coli (APC), glycogen synthase kinase (GSK)­3ß and axis inhibition protein (AXIN)2 and decreased levels of Wnt9b and ß­catenin. OTX1 overexpression decreased the levels of APC, GSK­3ß and AXIN2 and increased levels of Wnt9b and ß­catenin. However, XAV939 (a Wnt signaling inhibitor) and ß­catenin silencing partly eliminated the effect of OTX1 overexpression on cervical cancer cells. OTX1 promoted the progression of cervical cancer by activating the Wnt signaling pathway.

Exogenous Otx2 protects midbrain dopaminergic neurons from MPP+ by interacting with ATP5a1 and promoting ATP synthesis.

Mitochondrial dysfunction is the main pathological mechanism responsible for the death of midbrain dopaminergic (mDA) neurons. Thus, mitochondria-targeting therapy is a potential therapeutic strategy for Parkinson's disease (PD). Homeodomain transcription factors such as Otx2 can translocate between cells and exert non-cellular autonomous functions in recipient cells to stimulate neuronal survival. In this study, we investigated if exogenous Otx2 acts as a survival factor for mDA neurons by protecting them against MPP+-induced neurotoxicity in vitro. We show that subacute MPTP dosing regimen induces significant reduction in the levels of Otx2 homeoprotein in the ventral midbrain of PD mice. We also show that exogenous Otx2-myc recombinant protein protected primary mDA neurons against MPP+ by interacting with ATP5a1and promoting ATP synthesis.

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

BACKGROUND: Esophageal 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. METHODS: We 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. RESULTS: OTX1 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. CONCLUSION: OTX1 is an independent prognostic factor of ESCC and contributes to tumorigenesis both in vivo and in vitro.

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry.

Primordial germ cells (PGCs) arise from cells of the post-implantation epiblast in response to cytokine signaling. PGC development can be recapitulated in vitro by differentiating epiblast-like cells (EpiLCs) into PGC-like cells (PGCLCs) through cytokine exposure. Interestingly, the cytokine requirement for PGCLC induction can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that NANOG mediates Otx2 downregulation in the absence of cytokines and that this is essential for PGCLC induction by NANOG. Moreover, the direct NANOG target gene Esrrb, which can substitute for several NANOG functions, does not downregulate Otx2 when overexpressed in EpiLCs and cannot promote PGCLC specification. However, expression of ESRRB in Otx2+/- EpiLCs rescues emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification.

Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

Pluripotent stem cell derived dopaminergic subpopulations model the selective neuron degeneration in Parkinson's disease.

In Parkinson's disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery.

Orthodenticle homeobox OTX1 is a potential prognostic biomarker for bladder cancer.

Bladder cancer (BC) is one of the most aggressive tumors worldwide. OTX1 (orthodenticle homeobox 1) is an important transcription factor involved in various diseases, such as cancers. The aim of this study was to further investigate the role of OTX1 in BC. In this study, differentially expressed genes (DEGs) were screened from tumor tissues and para-cancerous tissues by bioinformatics. The expression of protein and RNA was separately detected by western blotting and immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR); cell viability and cell growth were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays, respectively; cell motility was measured by transwell and wound healing assays; cell cycle was measured by flow cytometry. In this study, 9 DEGs were screened out, and OTX1 was employed as a candidate gene for subsequent study. Results found that OTX1 was highly expressed in BC cells and BC tissues, which was significantly associated with poor prognosis of patients. In addition, OTX1 silencing significantly reduced cell viability, and inhibited cell growth and motility, while OTX1 overexpression got opposite results. Moreover, OTX1 co-expressed genes were enriched in cell cycle-related pathways, suggesting that the role of OTX1 in BC may be related to cell cycle, which was confirmed by flow cytometry analysis. Furthermore, in vivo experiments showed that OTX1 silencing significantly inhibited tumor growth in tumor-bearing mice. Taken together, our findings suggested that OTX1 may play a promotional role in BC progression.

miR-223-5p Suppresses OTX1 to Mediate Malignant Progression of Lung Squamous Cell Carcinoma Cells.

BACKGROUND: Lung squamous cell carcinoma (LUSC) features high morbidity and mortality as a worldwide malignant tumor. This study mainly explored a miR-223-5p-dependent mechanism that affected proliferation, invasion, and migration of LUSC cells. METHODS: Expression data of mature miRNAs and sequencing data of total RNA of LUSC were downloaded from TCGA database. Differentially expressed mRNAs were obtained. Function of miR-223-5p in LUSC cells was detected by assays like qRT-PCR, MTT, wound healing assay, Western blot, and Transwell assay. Western blot was performed to analyze the relationship between OTX1 and JAK/STAT signaling pathways. Dual-luciferase assay detected the relationship between miR-223-5p and OTX1. The way how miR-223-5p regulated LUSC cell biological functions via OTX1 was further explored. RESULTS: It was noted that miR-223-5p expression in LUSC tissue and cells was significantly reduced. Overexpression of miR-223-5p negatively regulated the proliferation, invasion, and migration of LUSC cells. The downstream target gene OTX1 was detected to be notably elevated in LUSC cells. A negative correlation between OTX1 and miR-223-5p was also found. As analyzed by GSEA, OTX1 was significantly enriched in the JAK/STAT signaling pathway and activated the pathway. Dual-luciferase assay demonstrated that OTX1 was a direct molecular target of miR-223-5p in LUSC cells. Rescue experiment verified that miR-223-5p regulated the malignant phenotypes of LUSC cells by pairing with OTX1. CONCLUSION: This study indicated that miR-223-5p was lowly expressed in LUSC cells. The impact of miR-223-5p on cell proliferation, invasion, and migration was realized by targeting OTX1. It is likely that miR-223-5p can be a novel target for LUSC treatment, which provides new ideas for future LUSC treatment.

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.