Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

CCL2/CCR2 system in neuroepithelial radial glia progenitor cells: involvement in stimulatory, sexually dimorphic effects of maternal ethanol on embryonic development of hypothalamic peptide neurons.

BACKGROUND: Clinical and animal studies show that alcohol consumption during pregnancy produces lasting behavioral disturbances in offspring, including increased alcohol drinking, which are linked to inflammation in the brain and disturbances in neurochemical systems that promote these behaviors. These include the neuropeptide, melanin-concentrating hormone (MCH), which is mostly expressed in the lateral hypothalamus (LH). Maternal ethanol administration at low-to-moderate doses, while stimulating MCH neurons without affecting apoptosis or gliogenesis, increases in LH the density of neurons expressing the inflammatory chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 and their colocalization with MCH. These neural effects associated with behavioral changes are reproduced by maternal CCL2 administration, reversed by a CCR2 antagonist, and consistently stronger in females than males. The present study investigates in the embryo the developmental origins of this CCL2/CCR2-mediated stimulatory effect of maternal ethanol exposure on MCH neurons. METHODS: Pregnant rats from embryonic day 10 (E10) to E15 during peak neurogenesis were orally administered ethanol at a moderate dose (2 g/kg/day) or peripherally injected with CCL2 or CCR2 antagonist to test this neuroimmune system's role in ethanol's actions. Using real-time quantitative PCR, immunofluorescence histochemistry, in situ hybridization, and confocal microscopy, we examined in embryos at E19 the CCL2/CCR2 system and MCH neurons in relation to radial glia progenitor cells in the hypothalamic neuroepithelium where neurons are born and radial glia processes projecting laterally through the medial hypothalamus that provide scaffolds for neuronal migration into LH. RESULTS: We demonstrate that maternal ethanol increases radial glia cell density and their processes while stimulating the CCL2/CCR2 system and these effects are mimicked by maternal administration of CCL2 and blocked by a CCR2 antagonist. While stimulating CCL2 colocalization with radial glia and neurons but not microglia, ethanol increases MCH neuronal number near radial glia cells and making contact along their processes projecting into LH. Further tests identify the CCL2/CCR2 system in NEP as a primary source of ethanol's sexually dimorphic actions. CONCLUSIONS: These findings provide new evidence for how an inflammatory chemokine pathway functions within neuroprogenitor cells to mediate ethanol's long-lasting, stimulatory effects on peptide neurons linked to adolescent drinking behavior.

Yin Yang 1 is critical for mid-hindbrain neuroepithelium development and involved in cerebellar agenesis.

The highly conserved and ubiquitously expressed transcription factor Yin Yang 1 (Yy1), was named after its dual functions of both activating and repressing gene transcription. Yy1 plays complex roles in various fundamental biological processes such as the cell cycle progression, cell proliferation, survival, and differentiation. Patients with dominant Yy1 mutations suffer from central nervous system (CNS) developmental defects. However, the role of Yy1 in mammalian CNS development remains to be fully elucidated. The isthmus organizer locates to the mid-hindbrain (MHB) boundary region and serves as the critical signaling center during midbrain and cerebellar early patterning. To study the function of Yy1 in mesencephalon/ rhombomere 1 (mes/r1) neuroepithelium development, we utilized the tissue-specific Cre-LoxP system and generated a conditional knockout mouse line to inactivate Yy1 in the MHB region. Mice with Yy1 deletion in the mes/r1 region displayed cerebellar agenesis and dorsal midbrain hypoplasia. The Yy1 deleted neuroepithelial cells underwent cell cycle arrest and apoptosis, with the concurrent changes of cell cycle regulatory genes expression, as well as activation of the p53 pathway. Moreover, we found that Yy1 is involved in the transcriptional activation of Wnt1 in neural stem cells. Thus, our work demonstrates the involvement of Yy1 in cerebellar agenesis and the critical function of Yy1 in mouse early MHB neuroepithelium maintenance and development.

A genomic atlas of mouse hypothalamic development.

The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.

Mash1 is required for generic and subtype differentiation of hypothalamic neuroendocrine cells.

The neuroendocrine hypothalamus regulates a number of critical biological processes and underlies a range of diseases from growth failure to obesity. Although the elucidation of hypothalamic function has progressed well, knowledge of hypothalamic development is poor. In particular, little is known about the processes underlying the neurogenesis and specification of neurons of the ventral nuclei, the arcuate and ventromedial nuclei. The proneural gene Mash1 is expressed throughout the basal retrochiasmatic neuroepithelium and loss of Mash1 results in hypoplasia of both the arcuate and ventromedial nuclei. These defects are due to a failure of neurogenesis and apoptosis, a defect that can be rescued by ectopic Ngn2 under the control of the Mash1 promoter. In addition to its role in neurogenesis, analysis of Mash1(-/-), Mash1(+/-), Mash1(KINgn2/KINgn2), and Mash1(KINgn2/+) mice demonstrates that Mash1 is specifically required for Gsh1 expression and subsequent GHRH expression, positively regulates SF1 expression, and suppresses both tyrosine hydroxylase (TH) and neuropeptide Y (NPY) expression. Although Mash1 is not required for propiomelanocortin (POMC) expression, it is required for normal development of POMC(+) neurons. These data demonstrate that Mash1 is both required for the generation of ventral neuroendocrine neurons as well as playing a central role in subtype specification of these neurons.