Neuroepithelium Research Articles

Clinicopathological significance of SOX2 and FOXG1 expression patterns in ovarian immature teratomas

Objective: To investigate the relationship between the expression patterns of SOX2 and FOXG1 and the differentiation/development level of neural components in immature teratoma and to determine the clinical significance and potential application of this correlation in a clinical setting. Methods: We conducted a comprehensive whole transcriptome sequencing analysis to identify differentially expressed genes (DEGs) across various subtypes of ovarian germ cell tumors. Additionally, immunohistochemical staining of paraffin-embedded tissue sections was employed to assess the nuclear staining pattern of SOX2 and FOXG1 proteins within the tumor tissues. Results: The transcriptome sequencing data showed that transcription factors SOX2 and FOXG1 exhibited high levels of expression typically in immature teratoma and occupied a pivotal position within the protein-protein interaction network. Immunohistochemical staining revealed the absence of both SOX2 and FOXG1 protein expression in dysgerminoma and yolk sac tumor samples. In immature teratoma, immunohistochemical staining demonstrated diffuse expression of SOX2 and FOXG1 proteins within the inner layer of densely-arranged primitive neuroepithelial tubules. This pattern of expression suggested the presence of stem cell-like properties within these tumor cells. In the sparsely peripheral neurogliocytes, FOXG1 maintained a diffuse nuclear staining pattern resembling that of neuroepithelial cells, while SOX2 exhibited a scattered pattern of positive staining, hinting at a neural lineage differentiation potential. This spatial differential expression pattern of SOX2 and FOXG1 proteins in immature teratoma suggested that primitive neural components within these tumors often recapitulated the trajectory of neural formation and cortical development that was typically observed during embryogenesis. The primitive neural tube acted as the center that constantly moved from inside to outside, with a dynamic shift from the interior to the exterior, paralleled by the sequential differentiation of cell lineages from primitive neuroepithelial stem cells to radial glia, intermediate progenitor cells, and ultimately to precursor glia. Conclusions: This spatial expression pattern of SOX2 and FOXG1 proteins observed in immature teratoma mirrors the lineage differentiation and migration trajectories of primitive neuroepithelial components typically seen in embryonic neurogenesis and cortical development. In daily practice, the combined application of SOX2 and FOXG1 SOX2 and FOXG1 helps identify the primitive neuroepithelial components in immature teratoma, avoid misjudgment of similar morphologies, and thereby assist in the histological grading and clinical decision-making.

Erythropoietin Production in Embryonic Neural Cells is Controlled by Hypoxia Signaling and Histone Deacetylases with an Undifferentiated Cellular State.

During mammalian development, production sites of the erythroid growth factor erythropoietin (EPO) shift from the neural tissues to the liver in embryos and to the kidneys in adults. Embryonic neural EPO-producing (NEP) cells, a subpopulation of neuroepithelial and neural crest cells, express the Epo gene between embryonic day (E) 8.5 and E11.5 to promote primitive erythropoiesis in mice. While Epo gene expression in the liver and kidneys is induced under hypoxic conditions through hypoxia-inducible transcription factors (HIFs), the Epo gene regulatory mechanisms in NEP cells remain to be elucidated. Here, we confirmed the presence of cells co-expressing EPO and HIFs in mouse neural tubes, where the hypoxic microenvironment activates HIFs. Chemical activation and inhibition of HIFs demonstrated the hypoxic regulation of EPO expression in human fetal neural progenitors and mouse embryonic neural tissues. In addition, we found that histone deacetylase inhibitors can reactivate EPO production in cell lines derived from NEP cells and human neuroblastoma, as well as in mouse primary neural crest cells, while rejuvenating these cells. Furthermore, the ability of the rejuvenated cells to produce EPO was maintained in hypoxia. Thus, EPO production is controlled by epigenetic mechanisms and hypoxia signaling in the immature state of hypoxic NEP cells.

SLMAP3 is essential for neurulation through mechanisms involving cytoskeletal elements, ABP, and PCP.

SLMAP3 is a tail-anchored membrane protein that targets subcellular organelles and is believed to regulate Hippo signaling. The global loss of SLMAP3 causes late embryonic lethality in mice, with some embryos exhibiting neural tube defects such as craniorachischisis. We show here that SLMAP3 -/- embryos display reduced length and increased width of neural plates, signifying arrested convergent extension. The expression of planar cell polarity (PCP) components Dvl2/3 and the activity of the downstream targets ROCK2, cofilin, and JNK1/2 were dysregulated in SLMAP3 -/- E12.5 brains. Furthermore, the cytoskeletal proteins (γ-tubulin, actin, and nestin) and apical components (PKCζ and ZO-1) were mislocalized in neural tubes of SLMAP3 -/- embryos, with a subsequent decrease in colocalization of PCP proteins (Fzd6 and pDvl2). However, no changes in PCP or cytoskeleton proteins were found in cultured neuroepithelial cells depleted of SLMAP3, suggesting an essential requirement for SLMAP3 for these processes in vivo for neurulation. The loss of SLMAP3 had no impact on Hippo signaling in SLMAP3 -/- embryos, brains, and neural tubes. Proteomic analysis revealed SLMAP3 in an interactome with cytoskeletal components, including nestin, tropomyosin 4, intermediate filaments, plectin, the PCP protein SCRIB, and STRIPAK members in embryonic brains. These results reveal a crucial role of SLMAP3 in neural tube development by regulating the cytoskeleton organization and PCP pathway.

Quantitative in toto live imaging analysis of apical nuclear migration in the mouse telencephalic neuroepithelium.

In the embryonic neuroepithelium (NE), neural progenitor cells undergo cell cycle-dependent interkinetic nuclear migration (IKNM) along the apicobasal axis. Extensive IKNM supports increasing cell production rates per unit apical surface, as typically observed in the mammalian telencephalic NE. Apical nucleokinesis during the G2 phase is an essential premitotic event, but its occurrence has not yet been quantitatively analyzed at a large 3D-scale with sufficient spatiotemporal resolution. Here, we comprehensively analyzed apically migrating nuclei/somata in reference to their surroundings from embryonic day (E)11 to E13 in the mouse telencephalon. The velocity of apical nucleokinesis decreased, with more frequent nuclear pausing occurring at E12 and E13, whereas the nuclear density in the middle NE zone (20-40-μm deep) increased. This result, together with the results of Shh-mediated overproliferation experiments in which the nuclear density was increased in vivo at E11, suggests that apical nucleokinesis is physically influenced by the surrounding nuclei. Mean square displacement analysis for nuclei being passed by the apically migrating nuclei via horizontal sectioning in toto-recorded movies revealed that the "tissue fluidity" or physical permissiveness of the NE to apical nucleokinesis gradually decreased (E11 > E12 > E13). To further investigate the spatial relationship between preexisting mitoses and subsequent premitotic apical nucleokinesis, the horizontal distribution of mitoses was cumulatively (~3 hr) analyzed under in toto monitoring. The four-dimensional cumulative apical mitoses presented a "random", not "clustered" or "regular", distribution pattern throughout the period examined. These methodologies provide a basis for future comparative studies of interspecies differences.

NCOG-16. A PROSPECTIVE OBSERVATIONAL STUDY OF CLINICAL PROFILE, COURSE AND OUTCOME OF PRIMARY BRAIN TUMOR AT A TERTIARY CARE CENTER

Abstract Primary brain tumors include tumor arising from neuro-epithelial cells, cranial nerves, meninges, germ cells, pituitary gland, and blood-forming organs. These tumors can present with either generalized or focal signs and symptoms.General symptoms are due to raised intracranial pressure (headache, vomiting). Focal symptoms are due to the compression of a structure or destruction (focal neurological deficits). The aim of this study is to analyze the clinical profiles of pediatric and adult brain tumor patients. Data of primary brain tumor patients who were diagnosed from 1st December 2019 to December 2021 were prospectively collected and analysed. In our study of 61 patients, the majority of patients had GBM (32.8%) and astrocytoma (31.1%) and medulloblastoma (11.5%). Less common histologies ependymomas (9.8%), meningioma (6.6%), brain stem glioma (3.3%), oligodendrogliomas (3.3%) and germinoma (1.6%). Out of 19 astrocytomas, 10 were of low grade (1 or 2), while 9 were of high grade (3 or 4).The majority (61.7%) of gliomas were of high grade, while the remaining 38.3% were of low grade. The majority (57.5%) of patients received multimodal treatment,26.2%received dual modality (surgery and RT), while the remaining 16.3% received single modality, either surgery or RT. Most (86.9%) of the patients treated with radiotherapy as a part of a single or multimodal treatment. A significant percentage of patients (77.1%) received concurrent and adjuvant temozolamide-based chemotherapy. Primary brain tumors have a different profile in pediatric patients. Among pediatric patients, the median age was 11.8 years, with male predominance. This study concludes that primary brain tumors have a different profile among adults and pediatric patients, with the majority of patients achieving a response to the first line therapy with an overall good tolerance for therapy.

Neuroepithelial cell transforming 1 as a key regulator in non-small cell lung cancer: unveiling causal links and therapeutic potentials

Neuroepithelial cell transforming 1 as a key regulator in non-small cell lung cancer: unveiling causal links and therapeutic potentials

8134 Hypothalamic Dlk1 Expression is Not Essential for Preventing Early Maturation of the Reproductive Axis in Female and Male Mice

Abstract Disclosure: D.B. Macedo: None. H. Kim Kyeol: None. A. Abreu: None. A. Latronico: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Inactivating mutations in Delta-like homolog 1 (DLK1), a maternally imprinted gene on chromosome 14, have been recognized as a genetic cause of central precocious puberty (CPP) in humans. It is well established that the timing of puberty, especially in females, is highly sensitive to energy stores and metabolic cues, with lower body fat negatively correlated with age at puberty and menarche. Mice lacking Dlk1 have pre- and postnatal growth retardation. Despite their considerably lower body weight (BW), we found that Dlk1 deficient females achieved puberty at the same age as controls (‘relative precocious puberty’). Moreover, Dlk1 deficiency led to activation of the reproductive axis despite lower levels of kisspeptin and leptin, an adipose tissue hormone with a permissive/stimulatory effect on metabolic control of reproduction. Dlk1 is a transmembrane protein that plays an essential role in inhibiting adipocyte differentiation and its biologically active form is soluble. Increased hypothalamic expression postnatally suggests a neuroendocrine function, but the precise mechanism by which DLK1 deficiency leads to CPP is yet to be deciphered. In this study, we aimed to investigate the role of hypothalamic Dlk1 expression on pubertal onset without the interference of diminished BW, using a conditional knockout (cKO) mouse model. Methods and Results: We generated a targeted Dlk1 knockout mouse model by crossing mice expressing Cre recombinase under the control of Nestin gene (Nes Cre), expressed primarily in neuro-epithelial cells, with mice harboring the Dlk1 gene flanked by loxP sites (Dlk1fl). Since Dlk1 is imprinted and expressed only by the paternal allele, we bred heterozygous Dlk1fl/+ male mice with Nes Cre females to obtain Dlk1fl/Cre (Dlk1 cKO), Dlk1+/Cre (Nes Cre) and Dlk1+/+ (wild type - WT). We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus of Dlk1 cKO adult male mice, whereas it was present in Nes Cre and WT mice. We noted a lower BW in Nes Cre compared to WT mice, as previously reported. However, no difference in BW was observed between Dlk1 cKO and Nes Cre mice at the timing of puberty (females: Dlk1 cKO 12.6 ± 0.8 g vs. Nes Cre 13.8 ± 0.2 g, P = 0.18; males: Dlk1 cKO 12.4 ± 0.2 g vs. Nes Cre 13.1 ± 0.5 g, P = 0.26). Puberty onset, assessed by age at vaginal opening (females) and at preputial separation (males), was not affected by hypothalamic Dlk1 deletion (Dlk1 cKO: 35.6 ± 2.7 days vs. Nes Cre 34.6 ± 0.7 days, P = 0.73; Dlk1 cKO 29.0 ± 0.6 vs. Nes Cre: 30.3 ± 0.6 days, P = 0.19). Conclusion: In contrast to global Dlk1 deficiency, targeted deletion of Dlk1 from neuronal and glial cells did not affect body weight or timing of pubertal onset. These findings suggest that Dlk1 originates from peripheral tissues, such as adipocytes, to regulate reproduction and metabolism. Presentation: 6/1/2024

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

Cycloleucine induces neural tube defects by reducing Pax3 expression and impairing the balance of proliferation and apoptosis in early neurulation

S-adenosylmethionine (SAM) plays a critical role in the development of neural tube defects (NTDs). Studies have shown that the paired box 3 (Pax3) gene is involved in neural tube closure. However, the exact mechanism between Pax3 and NTDs induced by SAM deficiency remains unclear. Here, The NTD mouse model was induced using cycloleucine (CL), an inhibitor of SAM biosynthesis, to determine the effect of Pax3 on NTDs. The effect of CL on NTD occurrence was assessed by 5-ethynyl-2′-deoxyuridine (EdU) staining, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and Western blot in NTD embryonic brain tissues and immortalized hippocampal neuron cells (HT-22). A high incidence of NTDs was observed when CL was administered at a dose of 200 mg/kg body weight. The levels of SAM and Pax3 were significantly reduced in NTD embryonic brain tissues and HT-22 cells after CL exposure. Decreased proliferation and excessive apoptosis were observed in neuroepithelial cells of NTD embryos and HT-22 cells under SAM deficiency, but these effects were reversed by overexpression of Pax3. These results suggest that decreased expression of Pax3 impairs the dynamic balance between cellular proliferation and apoptosis, contributing to NTDs induced by SAM deficiency, which would provide new insights for clarifying the underlying mechanism of NTDs.

Collagen protein-chitosan nerve conduits with neuroepithelial stem cells promote peripheral nerve regeneration

The peripheral nervous system consists of ganglia, nerve trunks, plexuses, and nerve endings, that transmit afferent and efferent information. Regeneration after a peripheral nerve damage is sluggish and imperfect. Peripheral nerve injury frequently causes partial or complete loss of motor and sensory function, physical impairment, and neuropathic pain, all of which have a negative impact on patients’ quality of life. Because the mechanism of peripheral nerve injury and healing is still unclear, the therapeutic efficacy is limited. As peripheral nerve injury research has processed, an increasing number of studies have revealed that biological scaffolds work in tandem with progenitor cells to repair peripheral nerve injury. Here, we fabricated collagen chitosan nerve conduit bioscaffolds together with collagen and then filled neuroepithelial stem cells (NESCs). Scanning electron microscopy showed that the NESCs grew well on the scaffold surface. Compared to the control group, the NESCs group contained more cells with bigger diameters and myelinated structures around the axons. Our findings indicated that a combination of chitosan-collagen bioscaffold and neural stem cell transplantation can facilitate the functional restoration of peripheral nerve tissue, with promising future applications and research implications.

Changes in gill neuroepithelial cells and morphology of threespine stickleback (Gasterosteus aculeatus) to hypoxia and simulated ocean acidification.

Coastal marine environments are characterized by daily, seasonal and long-term changes in both O2 and CO2, driven by local biotic and abiotic factors. The neuroepithelial cells (NECs) of fish are thought to be the putative chemoreceptors for sensing oxygen and CO2, and, thus, NECs play a key role in detecting these environmental changes. However, the role of NECs as chemosensors in marine fish remains largely understudied. In this study, the NECs of marine threespine sticklebacks (Gasterosteus aculeatus) were characterized using immunohistochemistry. We then determined if there were changes in NEC size and density, and in gill morphology in response to either mild (10kPa) or moderate (6.8kPa) hypoxia and two levels of elevated CO2 (1,500 and 3,000 µatm). We found that the NECs of stickleback contained synaptic vesicles and were innervated, and were 50-300% larger and 2 to 4 times more abundant than in other similar sized freshwater fishes. NEC size and density were largely unaffected by exposure to hypoxia, but there was a 50% decrease in interlamellar cell mass (ILCM) in response to mild and moderate hypoxia. NECs increased in size, but not abundance in response to elevated CO2. Moreover, fish exposed to moderate or elevated CO2 had 53-78% larger ILCMs compared to control fish. Our results demonstrated that adult marine sticklebacks have NECs that can respond to environmentally relevant pCO2 and likely hypoxia, which highlights the importance of NECs in marine fishes under the heterogeneity of environmental conditions in coastal areas.

Bibliometric analysis of neuroepithelial stem cells and brain ischemia

Objective: This bibliometric analysis aims to summarize the progress of neuroepithelial stem cells (NESCs) therapy in brain ischemia and provide insights for future research. Methods: Relevant literature was first screened from the Web of Science Core Collection with keywords “neuroepithelial stem cells” and “brain ischemia”, Then the research trends in these fields were analyzed by using VOSviewer, Pajek, Microsoft Excel, and bibliometric online analysis platform. Results: In total, 12 publications on NESCs and brain ischemia were identified, and “stem cells” is the most frequent keyword. A total of 6 countries and regions have published articles in this field, among which the UK has the largest number of publications (5 Articles), followed by the USA and China. The University of Florida is the institution with the highest number of publications. Regarding author contribution, Hodges H published the highest number of articles, with 69.8 citations. The highest-ranking journal was Neurobiology of Disease with 2 publications, while the most cited journal was Neuroscience with 4.00 average citations per item. Conclusions: This study provides a comprehensive analysis of NESC therapy in brain ischemia. In the past decade, the number of articles in this field has decreased slowly, but at the same time, the application of NESCs and ischemia has provided new methodological ideas for the clinical treatment of brain ischemia.

PRDM6 promotes medulloblastoma by repressing chromatin accessibility and altering gene expression

SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

Single-cell RNA sequencing of the holothurian regenerating intestine reveals the pluripotency of the coelomic epithelium.

In holothurians, the regenerative process following evisceration involves the development of a "rudiment" or "anlage" at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and HCR-FISH analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified thirteen distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.

Melanocortin-receptor 4 activation modulates proliferation and differentiation of rat postnatal hippocampal neural precursor cells

Postnatal hippocampal neurogenesis is essential for learning and memory. Hippocampal neural precursor cells (NPCs) can be induced to proliferate and differentiate into either glial cells or dentate granule cells. Notably, hippocampal neurogenesis decreases dramatically with age, partly due to a reduction in the NPC pool and a decrease in their proliferative activity. Alpha-melanocyte-stimulating hormone (α-MSH) improves learning, memory, neuronal survival and plasticity. Here, we used postnatally-isolated hippocampal NPCs from Wistar rat pups (male and female combined) to determine the role of the melanocortin analog [Nle4, D-Phe7]-α-MSH (NDP-MSH) in proliferation and fate acquisition of NPCs. Incubation of growth-factor deprived NPCs with 10 nM NDP-MSH for 6 days increased the proportion of Ki-67- and 5-bromo-2′-deoxyuridine (BrdU)-positive cells, compared to the control group, and these effects were blocked by the MC4R antagonist JKC-363. NDP-MSH also increased the proportion of glial fibrillar acidic protein (GFAP)/Ki-67, GFAP/sex-determining region Y-box2 (SOX2) and neuroepithelial stem cell protein (NESTIN)/Ki-67-double positive cells (type-1 and type-2 precursors). Finally, NDP-MSH induced peroxisome proliferator-activated receptor (PPAR)-γ protein expression, and co-incubation with the PPAR-γ inhibitor GW9662 prevented the effect of NDP-MSH on NPC proliferation and differentiation. Our results indicate that in vitro activation of MC4R in growth-factor-deprived postnatal hippocampal NPCs induces proliferation and promotes the relative expansion of the type-1 and type-2 NPC pool through a PPAR-γ-dependent mechanism. These results shed new light on the mechanisms underlying the beneficial effects of melanocortins in hippocampal plasticity and provide evidence linking the MC4R and PPAR-γ pathways in modulation of hippocampal NPC proliferation and differentiation.

Multiple isoforms of the Activin-like receptor baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain.

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor β (TGF-β) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.

Gill morphology adapted to oxygen-limited caves in Astyanax mexicanus.

Sensing and acquiring dissolved oxygen is crucial for nearly all aquatic life. This may become even more vital as dissolved oxygen concentrations continue to decline in many aquatic environments. While certain phenotypes that enable fish to live in low oxygen have been characterized, adaptations that arise following sudden, drastic reductions in dissolved oxygen are relatively unknown. Here, we assessed the blind Mexican cavefish, Astyanax mexicanus, for alterations to gill morphology that may be adaptive for life in hypoxic caves. The Astyanax system provides the unique opportunity to compare gill morphology between stereotypical "surface" adapted morphotypes and obligate cave-dwelling conspecifics. While the surface environment is well-oxygenated, cavefish must cope with significantly reduced oxygen. We began by quantifying traditional morphological gill traits including filament number and length as well as lamellar density and height in surface fish and two distinct cave populations, Pachón and Tinaja. This enabled us to estimate total lamellar height, a proxy for gill surface area. We then used immunohistochemical staining to label 5-HT-positive neuroepithelial cells (NECs), which serve as key oxygen sensors in fish. We discovered an increase in gill surface area for both cavefish populations compared to surface, which may enable a higher capacity of oxygen acquisition. Additionally, we found more NECs in Pachón cavefish compared to both surface fish and Tinaja cavefish, suggesting certain selective pressures may be cave-specific. Collectively, this work provides evidence that cavefish have adapted to low oxygen conditions via alterations to gill morphology and oxygen sensing, and informs evolutionary mechanisms of rapid adaptation to dramatic, chronic hypoxia.

Drosophila medulla neuroblast termination via apoptosis, differentiation, and gliogenic switch is scheduled by the depletion of the neuroepithelial stem cell pool.

The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.

MDB-32. PRDM6 PROMOTES MEDULLOBLASTOMA FORMATION IN NEUROEPITHELIAL STEM CELLS AND IS A CHROMATIN REPRESSOR BY BINDING TO HISTONE H3 LYSINE 27 TRIMETHYLATION MARKS

Abstract A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. PRDM6 is a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and its potential role as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

MDB-08. MYC AND TGFΒ PROMOTE GROUP 3 MEDULLOBLASTOMA TUMOR RESISTANCE THROUGH DEREGULATION OF POLYCOMB TARGETS

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We have generated new humanized models for G3MB by transforming neuroepithelial stem cells (NESC) derived from human induced pluripotent stem cells with TGFβ effectors alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors and MYC leading to more aggressive tumors. Moreover, NESC-derived tumors clustered with human G3/G4MB samples, indicating our models recapitulates human disease. We next found that NESCs expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT&RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis and discovered a subset of genes upregulated in MYC and TGFβ-driven lines that are targets of the histone demethylase KDM2B and Polycomb Repressive Complex 2 (PRC2) member SUZ12. Moreover, we found that MYC is bound to regions normally regulated by PRC interactors. We postulate that chromatin remodeling via MYC and recruitment of other MYC-interacting cofactors to PRC targets culminates in transcriptional changes that lead to aggressive disease. Work from others suggests that PRC-mediated chromatin regulation is critical in many adult and pediatric brain tumors, underscoring its significance in MB. Our studies provide important insight on identifying new therapeutic avenues with a focus on the epigenetic landscape for patients with MYC and TGFβ driven G3MB.