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Highlights • The septal island in dromedaries is a distinctive anatomical structure. • It has a curiously rostral location and innervated by the trigeminal nerve. • It has an unusual ultrastructure and may be specialized for nociception., The septal organs are islands or patches of sensory epithelium, located in the ventral parts of the nasal septum and innervated by the olfactory nerve. The septal island in dromedaries (Camelus dromedarius) was unusually located in the rostro-dorsal part of the nasal septum, where the ethmoidal branch of the trigeminal nerve provides innervation to the island mucosa. Therefore, the objectives of this study were to reveal the microscopic and ultrastructure of this island and to explain the probable functions. Twelve septal islands from 12 healthy male camels were used. Unlike the olfactory epithelium, which has a pseudostratified structure, the island neuroepithelium had a true neural lamination. Furthermore, in electron micrographs, the receptor, bipolar, and basal cells were connected with an orderly, organized network of cell–cell communication, which had some spine synapses. This network substituted the absence of supporting cells, maintained the shape of the tissue, and held the cells together. Moreover, the receptor cells were not similar to any of the different types of olfactory sensory neurons. Instead, they possessed the apical domain that might be specialized for the detection of chemical stimuli. Interestingly, a resident population of immune cells, namely mast cells and macrophages, was observed. The probable functions were discussed based on the cellular context and architecture. The nasal septal island in dromedaries may have a role in pain perception. The receptor cells most probably work as nociceptive cells that interact with the resident immune cells to coordinate pain signaling with immune response.

The highly conserved and ubiquitously expressed transcription factorYin Yang 1(Yy1), was named after its dual functions of both activating and repressing gene transcription.Yy1plays complex roles in various fundamental biological processes such as the cell cycle progression, cell proliferation, survival, and differentiation. Patients with dominantYy1mutations suffer from central nervous system (CNS) developmental defects. However, the role ofYy1in 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 ofYy1in mesencephalon/ rhombomere 1 (mes/r1) neuroepithelium development, we utilized the tissue-specificCre-LoxPsystem and generated a conditional knockout mouse line to inactivateYy1in the MHB region. Mice withYy1deletion in the mes/r1 region displayed cerebellar agenesis and dorsal midbrain hypoplasia. TheYy1deleted 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 thatYy1is involved in the transcriptional activation ofWnt1in neural stem cells. Thus, our work demonstrates the involvement ofYy1in cerebellar agenesis and the critical function ofYy1in mouse early MHB neuroepithelium maintenance and development.

The generation of structurally standardized human pluripotent stem cell (hPSC)‐derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio‐temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly‐organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination‐like folding, where primitive ectodermal cells undergo E‐to‐N‐cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio‐temporal patterning of the mesoendoderm, which guides the development and self‐organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFβ signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient‐derived hPSCs as well as differentiating between different classes of neural tube defect‐inducing drugs., This paper reports the generation of a reproducible and highly organized neuroepithelial (NE) tissue by combining human pluripotent stem cell (hPSC) micropatterning and a temporally sequenced induction protocol to specify NE cells in spatial juxtaposition to mesoendoderm cells. By evaluating NE tissue structural dysmorphia in the micropatterned NE model, we can successfully model gene‐ and drug‐induced neurodevelopmental defects.

Summary The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion., Graphical abstract, Highlights • Human brain organoids are expanded relative to nonhuman apes prior to neurogenesis • Ape neural progenitors go through a newly identified transition morphotype state • Delayed morphological transition with shorter cell cycles underlie human expansion • ZEB2 is as an evolutionary regulator of this transition, Cerebral organoid models reveal that differences in the duration of a developmental transitional state driven by the factor ZEB2 underlie the basis of brain expansion in humans in comparison to great apes.

BACKGROUND Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.

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.

BACKGROUND Immature teratoma (IT) is a tumor containing immature neuroectodermal tissue, primarily in the form of neuroepithelial tubules. However, the diagnosis of tumors containing only cellular neuroglial tissue (CNT) without distinct neuroepithelial tubules is often difficult, since the histological characteristics of immature neuroectodermal tissues remain unclear. Here, we examined the significance of CNT and tried to define immature neuroectodermal tissues by comparing the histological features of neuroglial tissues between mature teratoma (MT) and IT. METHODS The histological features of neuroglial tissue, including the cellularity, border between the neuroglial and adjacent tissues, cellular composition, mitotic index, Ki-67 proliferation rate, presence or absence of tissue necrosis, vascularity, and endothelial hyperplasia, were compared between 91 MT and 35 IT cases. RESULTS CNTs with a cellularity grade of ≥ 2 were observed in 96% of IT cases and 4% of MT cases (p < .001); however, CNT with a cellularity grade of 3 in MT cases was confined to the histologically distinct granular layer of mature cerebellar tissue. Moreover, CNT in IT exhibited significantly higher rates of Ki-67 proliferation, mitoses, and necrosis than those in MT (p < .001). Furthermore, an infiltrative border of neuroglial tissue and glomeruloid endothelial hyperplasia were significantly more frequent in IT cases than in MT cases (p < .001). CONCLUSIONS Our results suggest that if CNT with a cellularity grade of ≥ 2 is not a component of cerebellar tissue, such cases should be diagnosed as IT containing immature neuroectodermal tissue, particularly if they exhibit an infiltrative border, mitoses, necrosis, and increased Ki-67 proliferation.

Embryonic neural stem cells (NSCs), comprising neuroepithelial and radial glial cells, are indispensable precursors of neurons and glia in the mammalian developing brain. Since the process of neurogenesis occurs in a hypoxic environment, the question arises of how NSCs deal with low oxygen tension and whether it affects their stemness. Genes from the hypoxia-inducible factors (HIF) family are well known factors governing cellular response to hypoxic conditions. In this study, we have discovered that the endogenous stabilization of hypoxia-inducible factor 1α (Hif1α) during neural induction is critical for the normal development of the NSCs pool by preventing its premature depletion and differentiation. The knock-out of the Hif1α gene in mESC-derived neurospheres led to a decrease in self-renewal of NSCs, paralleled by an increase in neuronal differentiation. Similarly, neuroepithelial cells differentiated in hypoxia exhibited accelerated neurogenesis soon after Hif1α knock-down. In both models, the loss of Hif1α was accompanied by an immediate drop in neural repressor Hes1 levels while changes in Notch signaling were not observed. We found that active Hif1α/Arnt1 transcription complex bound to the evolutionarily conserved site in Hes1 gene promoter in both neuroepithelial cells and neural tissue of E8.5 - 9.5 embryos. Taken together, these results emphasize the novel role of Hif1α in the regulation of early NSCs population through the activation of neural repressor Hes1, independently of Notch signaling.

Phosphatidylinositol 3-kinases (PI3Ks) are signal transducers of many biological processes. Class 1 A PI3Ks are hetero dimers formed by a regulatory and a catalytic subunit. We have used the developing chicken neural tube (NT) to study the roles played by PI3K during the process of cell proliferation and differentiation. Notably, we have observed that in addition to its well characterized anti apoptotic activity, PI3K also plays a crucial role in intra epithelial cell positioning, and unlike its role in survival that mainly depends on AKT, the activity in cell positioning is mediated by Rho GTPase family members. Additionally, we have observed that activating mutations of PI3K that are remarkably frequent in many human cancers, cause an unrestrained basal migration of the neuroepithelial cells that end up breaking through the basal membrane invading the surrounding mesenchymal tissue. The mechanism described in this work contribute not only to acquire a greater knowledge of the intraepithelial cell positioning process, but also give new clues on how activating mutations of PI3K contribute to cell invasion during the first stages of tumour dissemination, The Work in the S.P.’s laboratory was supported by grants BFU2011–24099 and BFU2014–53633-P from the Spanish Ministry of Science and Competitiveness.

The folding of epithelial tissues is crucial for development of three-dimensional structure and function. Understanding this process can assist in determining the etiology of developmental disease and engineering of tissues for the future of regenerative medicine. Folding of epithelial tissues towards the apical surface has long been studied, but the molecular mechanisms that mediate epithelial folding towards the basal surface are just emerging. Here, we utilize zebrafish neuroepithelium to identify mechanisms that mediate basal tissue folding to form the highly conserved embryonic midbrain-hindbrain boundary. Live imaging revealed Wnt5b as a mediator of anisotropic epithelial cell shape, both apically and basally. In addition, we uncovered a Wnt5b-mediated mechanism for specific regulation of basal anisotropic cell shape that is microtubule dependent and likely to involve JNK signaling. We propose a model in which a single morphogen can differentially regulate apical versus basal cell shape during tissue morphogenesis., Summary: Examination of cell shape changes during zebrafish neuroepithelium tissue folding reveals that Wnt5b specifically regulates basal anisotropic cell shape via a microtubule-dependent mechanism, likely involving JNK signaling.

Oligodendrocyte precursor cells (OPC), neurons and astrocytes share a neural progenitor cell (NPC) in the early ventricular zone (VZ) of the embryonic neuroepithelium. Both switch to produce either of the three cell types and the generation of the right number of them undergo complex genetic regulation. The components of these regulatory cascades vary between brain regions giving rise to the unique morphological and functional heterogeneity of this organ. Forkhead b1 (Foxb1) is a transcription factor gene expressed by NPCs in specific regions of the embryonic neuroepithelium. We used the mutant mouse line Foxb1-Cre to analyze the cell types derived from Fobx1-expressing NPCs (the Foxb1 cell lineage) from two restricted regions, the medulla oblongata (MO; hindbrain) and the thalamus (forebrain), of normal and Foxb1-deficient mice. Foxb1 cell lineage derivatives appear as clusters in restricted regions, including the MO (hindbrain) and the thalamus (forebrain). Foxb1-expressing NPCs produce mostly oligodendrocytes (OL), some neurons and few astrocytes. Foxb1-deficient NPCs generate mostly OPC and immature OL to the detriment of neurons, astrocytes and mature OL. The axonal G-ratio however is not changed. We reveal Foxb1 as a novel modulator of neuronal and OL generation in certain restricted CNS regions. Foxb1 biases NPCs towards neuronal generation and inhibits OPC proliferation while promoting their differentiation.

Geminin is a nucleoprotein that can directly bind chromatin regulatory complexes to modulate gene expression during development. Geminin knockout mouse embryos are preimplantation lethal by the 32-cell stage, precluding in vivo study of Geminin׳s role in neural development. Therefore, here we used a conditional Geminin allele in combination with several Cre-driver lines to define an essential role for Geminin during mammalian neural tube (NT) formation and patterning. Geminin was required in the NT within a critical developmental time window (embryonic day 8.5–10.5), when NT patterning and closure occurs. Geminin excision at these stages resulted in strongly diminished expression of genes that mark and promote dorsal NT identities and decreased differentiation of ventral motor neurons, resulting in completely penetrant NT defects, while excision after embryonic day 10.5 did not result in NT defects. When Geminin was deleted specifically in the spinal NT, both NT defects and axial skeleton defects were observed, but neither defect occurred when Geminin was excised in paraxial mesenchyme, indicating a tissue autonomous requirement for Geminin in developing neuroectoderm. Despite a potential role for Geminin in cell cycle control, we found no evidence of proliferation defects or altered apoptosis. Comparisons of gene expression in the NT of Geminin mutant versus wild-type siblings at embryonic day 10.5 revealed decreased expression of key regulators of neurogenesis, including neurogenic bHLH transcription factors and dorsal interneuron progenitor markers. Together, these data demonstrate a requirement for Geminin for NT patterning and neuronal differentiation during mammalian neurulation in vivo.

Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In theDrosophilaventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. Here, we find that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing ofchinmoby ecdysone, the main steroid hormone, therefore allowing coordination of NSC self-renewal with organismal growth. In contrast,chinmosilencing in NBs is post-transcriptional and does not require ecdysone. Thus, duringDrosophiladevelopment, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor.SUMMARY STATEMENTHere, we demonstrate that the transcription factorchinmoacts as a master gene of NSC self-renewal in the different regions of the developingDrosophilabrain where it is controlled by distinct regulatory strategies.

Cell-producing events in developing tissues are mechanically dynamic throughout the cell cycle. In many epithelial systems, cells are apicobasally tall, with nuclei and somata that adopt different apicobasal positions because nuclei and somata move in a cell cycle–dependent manner. This movement is apical during G2 phase and basal during G1 phase, whereas mitosis occurs at the apical surface. These movements are collectively referred to as interkinetic nuclear migration, and such epithelia are called “pseudostratified”. The embryonic mammalian cerebral cortical neuroepithelium is a good model for highly pseudostratified epithelia, and we previously found differences between mice and ferrets in both horizontal cellular density (greater in ferrets) and nuclear/somal movements (slower during G2 and faster during G1 in ferrets). These differences suggest that neuroepithelial cells alter their nucleokinetic behavior in response to physical factors that they encounter, which may form the basis for evolutionary transitions towards more abundant brain-cell production from mice to ferrets and primates. To address how mouse and ferret neuroepithelia may differ physically in a quantitative manner, we used atomic force microscopy to determine that the vertical stiffness of their apical surface is greater in ferrets (Young’s modulus = 1700 Pa) than in mice (1400 Pa). We systematically analyzed factors underlying the apical-surface stiffness through experiments to pharmacologically inhibit actomyosin or microtubules and to examine recoiling behaviors of the apical surface upon laser ablation and also through electron microscopy to observe adherens junction. We found that although both actomyosin and microtubules are partly responsible for the apical-surface stiffness, the mouse

Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na+ increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function: formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF.

Epigenetic modification such as DNA methylation and histone acetylation plays essential roles in many aspects of cellular function and development of animals. There is an increasing amounts of evidence for dynamic changes in the histone acetylation of specific gene segments, but little attempt was made to examine global pattern changes in the histone acetylation in developing nervous system. In this study, we found that acetylated histone H3 and H4 immunoreactivities were relatively weak in neuroepithelial cells in the ventricular zone of developing rat cerebral cortex or chick spinal cord, compared to the immature young neurons in the cortical plate of a rat embryo or lateral motor column in chick spinal cord. On the other hand, adult neural stem cells in the dentate gyrus (DG) of rat hippocampal formation did not exhibit such diminished histone acetylation, compared to neuroblasts and mature DG neurons. These results suggest that the level of histone acetylation is highly dynamic and tightly linked to the neuronal types and the differentiation stages.

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell–cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.

Adult neurogenesis, generation of new functional cells in the mature central nervous system (CNS), has been documented in a number of diverse organisms, ranging from humans to invertebrates. However, the origin and evolution of this phenomenon is still poorly understood for many of the key phylogenetic groups. Echinoderms are one such phylum, positioned as a sister group to chordates within the monophyletic clade Deuterostomia. They are well known for the ability of their adult organs, including the CNS, to completely regenerate after injury. Nothing is known, however, about production of new cells in the nervous tissue under normal physiological conditions in these animals. In this study, we show that new cells are continuously generated in the mature radial nerve cord (RNC) of the sea cucumber Holothuria glaberrima. Importantly, this neurogenic activity is not evenly distributed, but is significantly more extensive in the lateral regions of the RNC than along the midline. Some of the new cells generated in the apical region of the ectoneural neuroepithelium leave their place of origin and migrate basally to populate the neural parenchyma. Gene expression analysis showed that generation of new cells in the adult sea cucumber CNS is associated with transcriptional activity of genes known to be involved in regulation of various aspects of neurogenesis in other animals. Further analysis of one of those genes, the transcription factor Myc showed that it is expressed, in some, but not all radial glial cells, suggesting heterogeneity of this CNS progenitor cell population in echinoderms

The cell division axis determines the position of daughter cells and is therefore critical for cell fate. During vertebrate neurogenesis, most cell divisions take place within the plane of the neuroepithelium (Das, T., Payer, B., Cayouette, M., and Harris, W.A. (2003). In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. Neuron 37, 597–609. Haydar, T.F., Ang, E., Jr., and Rakic, P. (2003). Mitotic spindle rotation and mode of cell division in the developing telencephalon. Proc Natl Acad Sci U S A 100, 2890–5. Kosodo, Y., Roper, K., Haubensak, W., Marzesco, A. M., Corbeil, D., and Huttner, W. B. (2004). Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J. 23, 2314–24). The cellular constraints responsible for this preferential orientation are poorly understood. Combining electroporation and time-lapse confocal imaging of chick neural progenitors, the events responsible for positioning the mitotic spindle and their dependence on RhoA were investigated. The results indicate that the spindle forms with a random orientation. However, the final orientation of cell divisions is dependent on two main factors: (i) an early rotation of the spindle that aligns it within the plane of the neuroepithelium, and (ii) a specific limitation of spindle oscillations, despite free rotation around the apico-basal axis. Expressing a dominant-negative RhoA leads to apico-basal cell divisions after a correct initial rotation of the spindle. Our data reveal a specific role for RhoA in the maintenance of spindle orientation, prior to anaphase. Thus, RhoA could be a key player potentially regulated by the neurogenic program or by the neural stem cell environment to control the balance between planar and apico-basal divisions, during normal or pathological development.

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L(-1)) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan-neuronal labelling with anti-Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of Gα olf-, TRPC2- and TrkA-expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and Gα olf-immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.

International audience; The early developing retinal neuroepithelium is composed of multipotent retinal progenitor cells that differentiate in a time specific manner, giving rise to six major types of neuronal and one type of glial cells. These cells migrate and organize in three distinct nuclear layers divided by two plexiform layers. Apical and adherens junction complexes have a crucial role in this process by the establishment of polarity and adhesion. Changes in these complexes disturb the spatiotemporal aspects of retinogenesis, leading to retinal degeneration resulting in mild or severe impairment of retinal function and vision. In this review, we summarize the mouse models for the different members of the apical and adherens junction protein complexes and describe the main features of their retinal phenotypes. The knowledge acquired from the different mutant animals for these proteins corroborate their importance in retina development and maintenance of normal retinal structure and function. More recently, several studies have tried to unravel the connection between the apical proteins, important cellular signaling pathways and their relation in retina development. Still, the mechanisms by which these proteins function remain largely unknown. Here, we hypothesize how the mammalian apical CRB1 complex might control retinogenesis and prevents onset of Leber congenital amaurosis or retinitis pigmentosa.

Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS, while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory element. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations.

The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF) and surrounded by neuroepithelial cells. Several lines of evidence suggest that the eCSF contains diffusible factors regulating the survival, proliferation, and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. In addition, SCO-spondin is required for the correct morphogenesis of the posterior commissure and pineal gland. Because SCO-spondin is secreted by the diencephalon, we sought to corroborate the long-range function of this protein in vitro by performing gain and loss of function experiments on mesencephalic explants. We find that culture medium enriched in SCO-spondin causes an increased neurodifferentiation of explanted mesencephalic region. Conversely, inhibitory antibodies against SCO-spondin cause a reduction in neurodifferentiation and an increase of mitosis when such explants are cultured in eCSF. Our results suggest that SCO-spondin is a crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. Our findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions.

The generation of new neurons involves a great variety of cell-extrinsic and cell-intrinsic signals. The primary cilium, long regarded as an "evolutionary vestige," has emerged as an essential signaling hub in many cells, including neural progenitors and differentiating neurons. Most progenitors harbor an apically-localized primary cilium, which is assembled and disassembled following the cell cycle, while the presence, position and length of this organelle appears to be even more variable in differentiating neurons. One of the main extracellular cues acting through the cilium is Sonic Hedgehog, which modulates spatial patterning, the progression of the cell cycle and the timing of neurogenesis. Other extracellular signals appear to bind to cilia-localized receptors and affect processes such as dendritogenesis. All the observed dynamics, as well as the many signaling pathways depending on cilia, indicate this organelle as an important structure involved in neurogenesis. Agencia Nacional de Investigación e Innovación PEDECIBA Institut Pasteur de Montevideo–FOCEM Mercosur

We recently showed that Ambra 1, a WD40-containing approximately 130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra 1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra 1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra 1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this article, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.

The retinal pigment epithelium (RPE) is essential for eye development by interacting with the overlaying neuroepithelium. Regulatory sequences of the gene encoding for tyrosinase-related protein 1 (TRP- 1), linked to the lacZ reporter gene, lead to strong and specific beta- galactosidase expression in the RPE. We asked how the oncogene ret would affect this epithelial cell type during mouse development. We used the TRP-1 promoter to express ret in the developing RPE, and obtained transgenic mouse lines, which showed mild to severe microphthalmia. During development, the RPE changed to a stratified epithelium with reduced or absent pigmentation from E10.5 onward. In addition, proliferation of RPE cells and tumor formation were observed from E12.5 onward. These early events prevent closure of choroid fissure and lead to microphthalmia and secondary malformations after birth. We conclude that ret transgene expression in the RPE prevents normal differentiation of this epithelial layer and induces proliferation and tumor formation. The appearance of the microphthalmic phenotype underlines the requirement of a normally developed RPE for eye development.

Cultured spinal cord neuroepithelial (NEP) cells can differentiate into neurons, oligodendrocytes and astrocytes and are morphologically and antigenically distinct from neural crest stem cells (NCSCs) that generate the PNS. NEP cells, however, can generate p75/nestin-immunoreactive cells that are morphologically and antigenically similar to previously characterized NCSCs. NEP-derived p75-immunoreactive cells differentiate into peripheral neurons, smooth muscle, and Schwann cells in mass and clonal culture. Clonal analysis of NEP cells demonstrates that a common NEP progenitor cell generated both CNS and PNS phenotypes. Differentiation into NCSCs was promoted by BMP-2/4 and differentiation did not require cells to divide, indicating that BMP played an instructive role in the differentiation process. Thus, individual NEP cells are multipotent and can differentiate into most major types of cell in the CNS and PNS and that PNS differentiation involves a transition from a NEP stem to another more limited, p75-immunoreactive, neural crest stem cell.

Genetic screens in zebrafish identified several loci that play essential roles in the patterning of retinal architecture. Here, we show that one of them, glass onion, encodes the N-cadherin gene. The glom117 mutant allele contains a substitution of the Trp2 residue known for its essential role in the adhesive properties of classic cadherins. Both the glom117 and pactm101b mutant N-cadherin alleles affect the polarity of the retinal neuroepithelial sheet and, unexpectedly, both result in cell-nonautonomous phenotypes in retinal patterning. The late onset of mutant N-cadherin phenotypes may be due to the ability of classic cadherins to substitute each other’s function.

Cerebral cortical precursor cells reside in a neuroepithelial cell layer that regulates their proliferation and differentiation. Global disruptions in epithelial architecture induced by loss of the adherens junction component alphaE-catenin lead to hyperproliferation. Here we show that cell autonomous reduction of alphaE-catenin in the background of normal precursors in vivo causes cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate, while normal neighboring precursors are unaffected. Mechanistically, alphaE-catenin likely regulates cortical precursor differentiation by maintaining beta-catenin signaling, as reduction of alphaE-catenin leads to reduction of beta-catenin signaling in vivo. These results demonstrate that, at the cellular level, alphaE-catenin serves to maintain precursors in the proliferative ventricular zone, and suggest an unexpected function for alphaE-catenin in preserving beta-catenin signaling during cortical development.

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface β1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis.

The zebrafish curly fry (cfy) mutation leads to embryonic lethality and abnormal cell divisions starting at 12–14 h postfertilization (hpf) during neural tube formation. The mitotic defect is seen in a variety of tissues including the central nervous system (CNS). In homozygous mutant embryos, mitoses are disorganized with an increase in mitotic figures throughout the developing neural tube. One consequence of aberrant mitoses in cfy embryos is an increase in cell death. Despite this, patterning of the early CNS is relatively unperturbed with distribution of the early, primary neurons indistinguishable from that of wild-type embryos. At later stages, however, the number of neurons was dramatically decreased throughout the CNS. The effect on neurons in older cfy embryos but not young ones correlates with the time of birth of neurons: primary neurons are born before the action of the cfy gene and later neurons after. Presumably, death of neuronal progenitors that divide beginning at the neural keel stage or death of their neuronal progeny accounts for the diminution of neurons in older mutant embryos. In addition, oligodendrocytes, which also develop late in the CNS, are greatly reduced in number in cfy embryos due to an apparent decrease in oligodendrocyte precursors. Genetic mosaic analysis demonstrates that the mutant phenotype is cell-autonomous. Furthermore, there are no obvious defects in apical/basal polarity within the neuroepithelium, suggesting that the cfy gene is not critical for epithelial polarity and that polarity defects are unlikely to account for the increased mitotic figures in mutants. These results suggest that the cfy gene regulates mitosis perhaps in a stage-dependent manner in vertebrate embryos.