TGF-ß modulates cell fate in human ES cell-derived foregut endoderm by inhibiting Wnt and BMP signaling.

Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.

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.

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.

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.

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 transcription activity of OTX2 on p16 expression is significantly blocked by methylation of CpG shore in non-promoter of lung cancer cell lines.

Background: The aberrant expression of the classical tumor suppressor gene p16 is a frequent event in lung cancer mainly due to the hypermethylation of its 5'-cytosine-phosphate-guanine-3' island (Cgi). However, whether methylation happens in other regions and how p16 expression and function are affected are largely unknown. Methods: Clustered Regularly Interspaced Short Palindromic Repeats/dCas9 (CRISPR/dCas9) technology was used for methylation editing at specific site of p16. The effects of methylation editing were detected by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS), transwell migration and wound healing tests. Chromatin immnoprecipitation-quantitative polymerase chain reaction (CHIP-qPCR) was performed to explore the impact of Cgi shore methylation on the binding abilities of transcription factors (TFs) including YY1, SP1, ZNF148 and OTX2 to p16 gene. A rescue experiment was performed to verify the regulatory effect of OTX2 on p16. The negative relationship between p16 expression and the methylation level of Cgi shore in non-promoter region was further verified with datasets from The Cancer Genome Atlas (TCGA) program and lung adenocarcinoma (LUAD) patients' samples. Results: The suppressive effect of p16 Cgi shore methylation on its expression was demonstrated in both HEK293 and A549 cells using CRISPR/dCas9-mediated specific site methylation editing. Methylation of the Cgi shore in the p16 non-promoter region significantly decreased its expression and promoted cell growth and migration. The ability of OTX2 bound to p16 was significantly reduced by 19.35% after methylation modification. Over-expression of OTX2 in A549 cells partly reversed the inhibitory effect of methylation on p16 expression by 19.04%. The verification results with TCGA and LUAD patients' samples supported that the p16 Cgi shore is a key methylation regulatory region. Conclusions: Our findings suggested that methylation of the Cgi shore in the p16 non-promoter region can hamper the transcriptional activity of OTX2, leading to a reduction in the expression of p16, which might contribute to the development of lung cancer.

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.

The mesodermal and myogenic specification of hESCs depend on ZEB1 and are inhibited by ZEB2.

Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1, ZEB2, or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy.

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.

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.

Temporal transcriptional control of neural induction in human induced pluripotent stem cells.

Introduction: Neural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition. Methods: Here, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction. Results: In addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules. Discussion: We infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs.

The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development.

Testis-specific transcript 10 (Tex10) is a critical factor for pluripotent stem cell maintenance and preimplantation development. Here, we dissect its late developmental roles in primordial germ cell (PGC) specification and spermatogenesis using cellular and animal models. We discover that Tex10 binds the Wnt negative regulator genes, marked by H3K4me3, at the PGC-like cell (PGCLC) stage in restraining Wnt signaling. Depletion and overexpression of Tex10 hyperactivate and attenuate the Wnt signaling, resulting in compromised and enhanced PGCLC specification efficiency, respectively. Using the Tex10 conditional knockout mouse models combined with single-cell RNA sequencing, we further uncover critical roles of Tex10 in spermatogenesis with Tex10 loss causing reduced sperm number and motility associated with compromised round spermatid formation. Notably, defective spermatogenesis in Tex10 knockout mice correlates with aberrant Wnt signaling upregulation. Therefore, our study establishes Tex10 as a previously unappreciated player in PGC specification and male germline development by fine-tuning Wnt signaling.

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.

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.

Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos).

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.

An Orthodenticle Homeobox 2 (OTX2) Mutation in a Patient With Combined Pituitary Hormone Deficiency, Pituitary Malformation, and Retinitis Pigmentosa.

Heterozygous mutations of orthodenticle homeobox 2 (OTX2)can result in ocular malformations, pituitary abnormalities, or hypopituitarism spanning from isolated growth hormone (GH) deficiency to combined pituitary hormone deficiency. We present a patient exhibiting growth and pubertal disturbances, developmental delay, and pigmentary retinopathy. Further examination revealed deficiencies in GH following clonidine stimulation, hypogonadism, and, subsequently, central hypothyroidism. Brain magnetic resonance imaging uncovered hypoplasia of the pituitary and an ectopic pituitary tissue. Sequence analysis of OTX2 identified a novel heterozygous mutation c.555_556dup, p.(Ser186Ilefs*21), indicative of a frameshift mutation. Replacement therapy with recombinant human GH, testosterone enanthate, and levothyroxine was started. Notably, GH therapy resulted in significant catch-up growth. This case report contributes to our comprehension of the molecular and clinical findings, particularly highlighting endocrine manifestations and a rare ophthalmologic manifestation associated with mutations in the OTX2 gene.

Agnathia-Otocephaly Complex Due to a De Novo Deletion in the OTX2 Gene.

Agnathia-otocephaly complex (AOC) is a rare and usually lethal malformation typically characterized by hypoplasia or the absence of the mandible, ventromedial and caudal displacement of the ears with or without the fusion of the ears, a small oral aperture with or without a tongue hypoplasia. Its incidence is reported as 1 in 70,000 births and its etiology has been attributed to both genetic and teratogenic causes. AOC is characterized by a wide severity clinical spectrum even when occurring within the same family, ranging from a mild mandibular defect to an extreme facial aberration incompatible with life. Most AOC cases are due to a de novo sporadic mutation. Given the genetic heterogeneity, many genes have been reported to be implicated in this disease but to date, the link to only two genes has been confirmed in the development of this complex: the orthodenticle homeobox 2 (OTX2) gene and the paired related homeobox 1 (PRRX1) gene. In this article, we report a case of a fetus with severe AOC, diagnosed in routine ultrasound scan in the first trimester of pregnancy. The genetic analysis showed a novel 10 bp deletion mutation c.766_775delTTGGGTTTTA in the OTX2 gene, which has never been reported before, together with a missense variant c.778T>C in cis conformation.

Genetic alterations of TP53 and OTX2 indicate increased risk of relapse in WNT medulloblastomas.

This study aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in WNT-activated medulloblastoma (WNT-MB). In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by DNA sequencing. Chromosomal copy-number aberrations were assessed by molecular inversion probe technology (MIP), SNP6, or 850k methylation array hybridization. Prognostic impact was evaluated in 120 patients with follow-up data from the HIT2000 medulloblastoma trial or HIT registries. CTNNB1 mutations were present in 92.2%, and APC mutations in 6.8% of samples. One CTNNB1 wild-type tumor gained WNT activation due to homozygous FBXW7 deletion. Monosomy 6 was present in 78.6%, and more frequent in children than adults. 16.1% of tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. Loss of heterozygosity at the TP53 locus (chromosome 17p13.1) was found in 40.7% (11/27) of TP53 mutant tumor samples and in 12.6% of TP53 wild-type cases (13/103). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; 5-year-PFS 68% versus 93%, p = 0.001), and were enriched for chromosomes 17p (p = 0.001), 10, and 13 losses. Gains of OTX2 (14q22.3) occurred in 38.9% of samples and were associated with poor PFS and OS (5-year-PFS 72% versus 93%, p = 0.017 resp. 5-year-OS 83% versus 97%, p = 0.006). Multivariable Cox regression analysis for PFS/OS identified both genetic alterations as independent prognostic markers. Our data suggest that patients with WNT-MB carrying TP53 mutations or OTX2 gains (58.1%) are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Cell fate decisions, transcription factors and signaling during early retinal development.

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.

Brevican, Neurocan, Tenascin-C, and Tenascin-R Act as Important Regulators of the Interplay Between Perineuronal Nets, Synaptic Integrity, Inhibitory Interneurons, and Otx2.

Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix (ECM) structure called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons, and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C, and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. In addition, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Furthermore, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs, did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin interneuron and PNN maturation, and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C, and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution, and Otx2 in the V1.