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BackgroundDiverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa.ResultsTo that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelidCapitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC,Ct-soxB1andCt-neurogeninwere expressed in a large proportion of apically-localized, EdU+NPCs. In contrast,Ct-ash1was expressed in a small subset of apically-localized, EdU+NPCs and subsurface, EdU−cells, but not inCt-neuroD+orCt-elav1+cells, which also were subsurface.ConclusionsOur data suggest a putative genetic hierarchy withCt-soxB1andCt-neurogeninat the top, followed byCt-ash1, thenCt-neuroD, and finallyCt-elav1. Comparison of our data with that fromPlatynereis dumeriliirevealed expression ofneurogeninhomologs in proliferating NPCs in annelids, which appears different than the expression of vertebrateneurogeninhomologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk ofC. teletasuggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.

Peripheral Primitive NeuroEctodermal Tumors (pPNETs) are rare highly malignant tumors; originating from the neuroectoderm. Although PNETs may arise in various locations (most commonly in the extremities), very few cases have been reported in the pelvis. There is still poor evidence about the management of these tumors in the literature. We present a rare case of pelvic PNET in a 20-year-old male. The patient presented with symptoms mimicking a lumbar disk hernia, which delayed the diagnosis. He was managed with a combination of a debulking procedure, adjuvant chemotherapy, radiotherapy; and has been in remission for 2 years upon follow-up. This case highlights the importance of diagnosing such aggressive tumors as early as possible (as prognosis may vary significantly), and the challenge in the management of PNETs due to poor evidence., Highlights • Primitive NeuroEctodermal Tumors (PNET) are highly malignant tumors that generally manifest in the extremities. • Clinical symptoms are usually due to mass effect and depend on the site of presentation, but can sometimes be misleading. • There is still poor evidence about the management of PNET in the existing literature.

In the sea urchin larva, most neurons lie within an ectodermal region called the ciliary band. Our understanding of the mechanisms of specification and patterning of these peripheral ciliary band neurons is incomplete. Here, we first examine the gene regulatory landscape from which this population of neural progenitors arise in the neuroectoderm. We show that ciliary band neural progenitors first appear in a bilaterally symmetric pattern on the lateral edges of chordin expression in the neuroectoderm. Later in development, these progenitors appear in a salt-and-pepper pattern in the ciliary band where they express soxC, and prox, which are markers of neural specification, and begin to express synaptotagminB, a marker of differentiated neurons. We show that the ciliary band expresses the acid sensing ion channel gene asicl, which suggests that ciliary band neurons control the larva's ability to discern touch sensitivity. Using a chemical inhibitor of MAPK signaling, we show that this signaling pathway is required for proper specification and patterning of ciliary band neurons. Using live imaging, we show that these neural progenitors undergo small distance migrations in the embryo. We then show that the normal swimming behavior of the larvae is compromised if the neurogenesis pathway is perturbed. The developmental sequence of ciliary band neurons is very similar to that of neural crest-derived sensory neurons in vertebrates and may provide insights into the evolution of sensory neurons in deuterostomes.

Pluripotent stem cells recapitulate many aspects of embryogenesis in vitro. Here, we established a novel culture system to differentiate human embryonic stem cell aggregates (HESCA), and evaluated its utility for teratogenicity assessment. Culture of HESCA with modulators of developmental signals induced morphogenetic and molecular changes associated with differentiation of the paraxial mesoderm and neuroectoderm. To examine impact of teratogenic exposures on HESCA differentiation, 18 compounds were tested, for which adequate information on in vivo plasma concentrations is available. HESCA treated with each compound were examined for gross morphology and transcript levels of 15 embryogenesis regulator genes. Significant alterations in the transcript levels were observed for 94% (15/16) of the teratogenic exposures within 5-fold margin, whereas no alteration was observed for 92% (11/12) of the non-teratogenic exposures. Our study demonstrates that transcriptional changes in HESCA serve as predictive indicator of teratogenicity in a manner comparable to in vivo exposure levels.

SUMMARY How ubiquitous transcription factors (TFs) coordinate temporal inputs from broadly expressed epigenetic factors to control cell fate remains poorly understood. Here, we uncover a molecular relationship between p53, an abundant embryonic TF, and WDR5, an essential member of the MLL chromatin modifying complex, that regulates mouse embryonic stem cell fate. Wild-type Wdr5 or transient Wdr5 knockout promotes a distinct pattern of global chromatin accessibility and spurs neuroectodermal differentiation through an RbBP5-dependent process in which WDR5 binds to, and activates transcription of, neural genes. Wdr5 rescue after its prolonged inhibition targets WDR5 to mesoderm lineage-specifying genes, stimulating differentiation toward mesoderm fates in a p53-dependent fashion. Finally, we identify a direct interaction between WDR5 and p53 that enables their co-recruitment to, and regulation of, genes known to control cell proliferation and fate. Our results unmask p53-dependent mechanisms that temporally integrate epigenetic WDR5 inputs to drive neuroectoderm and mesoderm differentiation from pluripotent cells., In Brief How ubiquitous chromatin-associated proteins and transcription factors (TFs) regulate cell fate determination is poorly understood. Li et al. show that regulation of the broadly expressed TF p53 by the chromatin-associated protein WDR5 is required for neuroectoderm versus mesoderm lineage determination in mouse embryonic stem cells (ESCs)., Graphical Abstract

The chromatin remodeler CHD8, which belongs to the ATP-dependent chromatin remodelers CHD family, is one of the most high-risk mutated genes in autism spectrum disorders. However, the role of CHD8 in neural differentiation and the mechanism of CHD8 in autism remains unclear, despite there are a few studies based on the CHD8 haploinsufficient models. Here, we generate the CHD8 knockout human ESCs by CRISPR/Cas9 technology and characterize the effect of loss-of-function of CHD8 on pluripotency maintenance and lineage determination by utilizing efficient directed differentiation protocols. The results show loss-of-function of CHD8 does not affect human ESC maintenance although having slight effect on proliferation and cell cycle. Interestingly, CHD8 depletion results in defective neuroectoderm differentiation, along with severe cell death in neural progenitor stage. Transcriptome analysis also indicates CHD8 does not alter the expression of pluripotent genes in ESC stage, but in neural progenitor cells depletion of CHD8 induces the abnormal expression of the apoptosis genes and suppresses neuroectoderm-related genes. These results provide the evidence that CHD8 plays an essential role in the pluripotency exit and neuroectoderm differentiation as well as the regulation of apoptosis during neurogenesis.

Despite numerous efforts to generate vascular tissues that recapitulate the physiological characteristics of native vessels, vascular cell source remains one of the principal challenges in the construction of tissue-engineered vascular grafts (TEVGs). Human pluripotent stem cells, therefore, represent an indispensable source to supply a large production of vascular smooth muscle cells (VSMCs) for cell-based therapy. In particular, human induced pluripotent stem cells (hiPSCs) generated from the same individual have opened up new avenues of achieving patient specificity through the derivation of autologous and immunocompatible VSMCs. This book chapter will detail three representative methods of differentiating hiPSCs into VSMCs that are structurally and functionally mature for TEVG engineering. Luo et al. reported an embryoid body (EB)-based approach to generate a robust, large-scale production of mature, functional hiPSC-derived VSMCs as a cell replacement for vascular tissue engineering. EB formation has an advantage of resembling early embryonic development and allowing cellular interactions in three dimensions. Cheung et al. established a system to produce embryological origin-specific hiPSC-derived VSMCs from the neuroectoderm, lateral plate mesoderm, and paraxial mesoderm lineages in a chemically defined manner. This allows site-specific vascular disease modeling. Moreover, Eoh et al. followed Wanjare et al.'s method to construct hiPSC-derived VSMCs using monolayer cultures of extracellular matrix proteins, with the addition of a pulsatile flow for the secretion of mature, organized elastic fibers. The generation of TEVGs, powered by the unlimited supply of hiPSC-derived VSMCs, has begun a new era in cellular therapy for vascular bypass and defective vessel segment replacement, aimed at addressing millions of cases of cardiovascular diseases across the globe.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

Polycomb repressive complexes (PRCs) are essential in mouse gastrulation and specify neural ectoderm in human embryonic stem cells (hESCs), but the underlying molecular basis remains unclear. Here in this study, by employing an array of different approaches, such as gene knock-out, RNA-seq, ChIP-seq, et al., we uncover that EZH2, an important PRC factor, specifies the normal neural fate decision through repressing the competing meso/endoderm program. EZH2−/− hESCs show an aberrant re-activation of meso/endoderm genes during neural induction. At the molecular level, EZH2 represses meso/endoderm genes while SOX2 activates the neural genes to coordinately specify the normal neural fate. Moreover, EZH2 also supports the proliferation of human neural progenitor cells (NPCs) through repressing the aberrant expression of meso/endoderm program during culture. Together, our findings uncover the coordination of epigenetic regulators such as EZH2 and lineage factors like SOX2 in normal neural fate decision. Supplementary Information The online version contains supplementary material available at 10.1186/s13619-021-00092-6.

Despite the numerous attempts in nerve tissue engineering, no ideal strategy has been translated into effective therapy for neuronal regeneration yet. Here, we designed a novel nerve regeneration scaffold combining aligned laminin-immobilized polyethersulfone (PES) nanofibers and human-induced pluripotent stem cells (hiPSCs) for transplantation strategies. Aligned and random PES nanofibers were fabricated by electrospinning method with a diameter of 95-500 nm and were then modified with covalent laminin bounding subsequent to O2 plasma treatment. PES-functionalized fibers found to induce a remarkable higher rate of neuronal genes expression as compared to nontreated group. In addition, hiPSCs cultured on aligned pure fibers exhibited the extension of neurites along with fibers direction and an exponentially elevated expression of neuron specific enolase (early neuroectoderm marker), Tuj-1 (axonal marker), and microtubule-associated protein 2 (dendritic marker) in comparison with random pure fibers. The concomitant of increased hydrophilicity and biocompatibility along with exploiting topographical cues and directional guidance make aligned PES-plasma-laminin a versatile scaffold for adhesion, proliferation, spreading, and differentiation of hiPSCs into nerve cells.

Concurrent with the ‘3R’ principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

Nervous system development has been intensely studied in insects (especially Drosophila melanogaster), providing detailed insights into the genetic regulatory network governing the formation and maintenance of the neural stem cells (neuroblasts) and the differentiation of their progeny. Despite notable advances over the last two decades, neurogenesis in other arthropod groups remains by comparison less well understood, hampering finer resolution of evolutionary cell type transformations and changes in the genetic regulatory network in some branches of the arthropod tree of life. While the neurogenic cellular machinery in malacostracan crustaceans is well-described morphologically, its genetic molecular characterization is pending. To address this, we established an in-situ hybridization protocol for the crayfish Procambarus virginalis and studied embryonic expression patterns of a suite of key genes, encompassing three SoxB group transcription factors, two achaete-scute homologs, a Snail family member, the differentiation determinants Prospero and Brain tumor, and the neuron marker Elav. We document cell type expression patterns with notable similarities to insects and branchiopod crustaceans, lending further support to the homology of hexapod-crustacean neuroblasts and their cell lineages. Remarkably, in the crayfish head region, cell emigration from the neuroectoderm coupled with gene expression data point to a neuroblast-independent initial phase of brain neurogenesis. Further, SoxB group expression patterns suggest an involvement of Dichaete in segmentation, in concordance with insects. Our target gene set is a promising starting point for further embryonic studies, as well as for the molecular genetic characterization of sub-regions and cell types in the neurogenic systems in the adult crayfish brain. This article is protected by copyright. All rights reserved.

Early embryogenesis requires tightly controlled temporal and spatial coordination of cellular behavior and signaling. Modulations are achieved at multiple levels, from cellular transcription to tissue-scale behavior. Intracellularly, the endolysosomal system emerges as an important regulator at different levels, but in vivo studies are rare. In the frog Xenopus, little is known about the developmental roles of endosomal regulators, or their potential involvement in signaling, especially for late endosomes. Here, we analyzed a hypothesized role of Rab7 in this context, a small GTPase known for its role as a late endosomal regulator. First, rab7 showed strong maternal expression. Following localized zygotic transcript enrichment in the mesodermal ring and neural plate, it was found in tailbud-stage neural ectoderm, notochord, pronephros, eyes and neural crest tissues. Inhibition resulted in strong axis defects caused by a requirement of rab7 for mesodermal patterning and correct gastrulation movements. To test a potential involvement in growth factor signaling, we analyzed early Wnt-dependent processes in the mesoderm. Our results suggest a selective requirement for ligand-induced Wnt activation, implicating a context-dependent role of Rab7., Summary: The late endosomal regulator Rab7 is required for gastrulation movements and axis elongation in Xenopus by regulating early mesoderm patterning.

Coordinated polarization of cells in the tissue plane, known as planar cell polarity (PCP), is associated with a signaling pathway critical for the control of morphogenetic processes. Although the segregation of PCP components to opposite cell borders is believed to play a critical role in this pathway, whether PCP derives from egg polarity or preexistent long-range gradient, or forms in response to a localized cue, remains a challenging question. Here we investigate the Xenopus neural plate, a tissue that has been previously shown to exhibit PCP. By imaging Vangl2 and Prickle3, we show that PCP is progressively acquired in the neural plate and requires a signal from the posterior region of the embryo. Tissue transplantations indicated that PCP is triggered in the neural plate by a planar cue from the dorsal blastopore lip. The PCP cue did not depend on the orientation of the graft and was distinct from neural inducers. These observations suggest that neuroectodermal PCP is not instructed by a preexisting molecular gradient but induced by a signal from the dorsal blastopore lip., Summary: The blastopore lip contains a cue that induces the progressive establishment of planar polarity in the neural plate in the posterior to anterior direction.

Optic development involves sequential interactions between several different tissue types, including the overlying ectoderm, adjacent mesoderm, and neural crest mesenchyme and the neuroectoderm. In an ongoing expression screen, we identified that Tfap2β, Casq2, Penk, Zic1, and Zic3 are expressed in unique cell types in and around the developing eye. Tfap2β, Zic1, and Zic3 are transcription factors, Casq2 is a calcium binding protein and Penk is a neurotransmitter. Tfap2β, Zic1, and Zic3 have reported roles in brain and craniofacial development, while Casq2 and Penk have unknown roles. These five genes are expressed in the major tissue types in the eye, including the muscles, nerves, cornea, and sclera. Penk expression is found in the sclera and perichondrium. At E12.5 and E15.5, the extra-ocular muscles express Casq2, the entire neural retina expresses Zic1, and Zic3 is expressed in the optic disk and lip of the optic cup. The expression of Tfap2β expanded from corneal epithelium to the neural retina between E12.5 to E15.5. These genes are expressed in similar domains as Hedgehog ( Gli1, and Ptch1) and the Wnt ( Lef1) pathways. The expression patterns of these five genes warrant further study to determine their role in eye morphogenesis

In its early stages, an embryo polarizes to form cell subpopulations that subsequently produce specific organ cell types. These cell subpopulations are defined by transcription factors (TFs) that activate or repress specific genes. Although an embryo comprises thousands of TFs, surprisingly few are needed to determine the fate of a given cell. The ectoderm divides into the neuroectoderm and surface ectoderm, the latter of which gives rise to epidermal keratinocytes and corneal epithelial cells (CECs). Meanwhile, neuroectoderm cells give rise to other parts of the eye such as the corneal endothelium and retina. To investigate the regulatory role of TFs in CECs, we overexpressed the "core TFs" (PAX6, OVOL2, and KLF4) in human fibroblasts and found that the cells adopted a CEC-like quality. OVOL2 overexpression was even able to directly induce cells with a neuroectoderm fate toward a surface ectoderm fate, designated "direct reprogramming." Conversely, suppression of OVOL2 or PAX6 expression induced CECs to show qualities consistent with neural lineage cells or epidermal keratinocytes, respectively. This suggests that these core TFs can maintain the CEC phenotype through reciprocal gene regulation. Direct reprogramming has important implications for cell therapies. The potential benefits of cells derived by direct reprogramming compared with induced pluripotent stem cells include the fact that it requires less time than reprogramming a cell back to the pluripotent state and then to another cell type. Further understanding of the reciprocally repressive mechanism of action for core TFs could lead to alternative treatments for regenerative medicine not requiring cell transplantation.

Bisphenol A (BPA) is a very versatile industrial chemical. Many reports have associated BPA with several health effects. Some bisphenol alternatives have been introduced to replace BPA in its many applications. However, comprehensive toxicological evaluations for these replacements are still lacking. In this study, we examined the potential effects of BPA, bisphenol F (BPF) and bisphenol S (BPS), on embryonic development with an in vitro stem cell toxicology system and transcriptomics analyses. Mouse embryonic stem cells (mESCs) were differentiated via embryoid body formation, either globally towards the three primary germ layers and their lineages, or specifically into neuroectoderm/neural progenitor cells. During the differentiation, cells were treated with BPA, BPF, BPS, or DMSO control. Samples were collected at different time points, for qRT-PCR and RNA-seq analyses. BPA, BPF and BPS disrupted many processes, during mESC global and neural differentiations, in very similar manners. In fact, at each time point the three chemicals differentially regulated analogous gene categories, particularly the ones involved in cell-matrix and cell-cell adhesion, signal transduction pathways, and medical conditions such as cardiovascular diseases and cancer. Our findings demonstrate once more then BPA substitutes may not be very safe. They potentially have a very complex developmental toxicity, similarly to BPA, and seem more toxic than BPA itself. In addition, our results reveal that stem cell-based developmental toxicity assays can be very comprehensive.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1–3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.—Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Threshold responses to an activity gradient allow a single signaling pathway to yield multiple outcomes. Extracellular signal-regulated kinase (ERK) is one such signal, which couples receptor tyrosine kinase signaling with multiple cellular responses in various developmental processes. Recent advances in the development of fluorescent biosensors for live imaging have enabled the signaling activities accompanying embryonic development to be monitored in real time. Here, we used an automated computational program to quantify the signals of a fluorescence resonance energy transfer (FRET) reporter for activated ERK, and we used this system to monitor the spatio-temporal dynamics of ERK during neuroectoderm patterning in Drosophila embryos. We found that the cytoplasmic and nuclear ERK activity gradients show distinct kinetics in response to epidermal growth factor receptor activation. The ERK activation patterns implied that the cytoplasmic ERK activity is modulated into a threshold response in the nucleus.

Recently, stem-cell therapy as a promising therapeutic alternative is considered to treat retinal degenerative diseases. Here, we used small molecules and concentrated conditioned medium selectively enriched with Amicon filter units from human adipose-derived stem cells (hADSC-CM) containing various neurotrophic factors to induce hADSCs toward eye field neuroectoderm (EFN). For induction of stem cells, hADSC-CM and small molecules CKI-7, SB431542 and LDN193189 as inhibitors of Wnt, Nodal and BMP4 signaling pathways were used, respectively. We found the highest expression of β-TUB III as a neural marker in the group in which small molecules and conditioned medium were applied simultaneously. Moreover, EFN markers SIX3, PAX6 and RAX had higher expression in the presence of a conditioned medium. However, the superior expression of ENF marker OTX2 was seen in the small molecules group. Our results indicated that neurotrophic factors present in hADSCs-CM could induce hADSCs into EFN cells. Therefore, a more thorough study of these factors and their effects in hADSC-CM might pave the way for cellular and non-cellular therapy in retinal degenerative diseases.

The migration of neuroblasts at the stage of neurulation and the formation of a primary pattern of neural connections of the brain in a spur frog (Xenopus laevis) were studied. In the experiments, 129 embryos from the stage of the formation of the neural plate before the start of active feeding of the hatched larvae were used. To study the migration of neuroepithelial cells during neurulation, intracellular labeling of individual neuroblasts was performed using procyon dyes RS and B2BS. After the formation of the neural tube and the start of differentiation of neurons, cells containing the dye were detected using a fluorescent microscope. Using a microelectrode, a second dye, luciferous yellow CH, was introduced into the detected cells using a microelectrode. It filled the bodies and processes of neurons, which made it possible to reveal the pattern of connections of the brain. Is established that primary bonds are formed between cells originating from a different clones, which disintegrates due to the intercalation of the epidermal and hypodermal layers of the neuroectoderm, proliferation and migration of neuroblasts. Key words: neuroblasts, cell migration, pattern formation, brain development, neuronal connections

SUMMARYThe orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here we show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. Proximity biotinylation and crosslinking studies revealed that the Vangl2-Pk3 interaction is suppressed by overexpressed Fz3, but enhanced in Fz3 morphants. In addition, Fz3 induced Vangl2 phosphorylation on T76 and T78, and this phosphorylation was required for Fz3-mediated inhibition of Vangl2-Pk3 complex formation. Consistent with this observation, the complex of Pk3 with nonphosphorylatable Vangl2 was not polarized in the neural plate. These findings provide evidence for in vivo regulation of the Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

During early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type" (Waddington in Nature 183: 1654-1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772-774, 1988; Lander in Cell 144: 955-969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed.

Epigenetic modifications play a crucial role in neurogenesis, learning, and memory, but the study of their role in early neuroectoderm commitment from pluripotent inner cell mass is relatively lacking. Here we utilized the system of directed neuroectoderm differentiation from human embryonic stem cells and identified that KDM6B, an enzyme responsible to erase H3K27me3, was the most upregulated enzyme of histone methylation during neuroectoderm differentiation by transcriptome analysis. We then constructed KDM6B-null embryonic stem cells and found strikingly that the pluripotent stem cells with KDM6B knockout exhibited much higher neuroectoderm induction efficiency. Furthermore, we constructed a series of embryonic stem cell lines knocking out the other H3K27 demethylase KDM6A, and depleting both KDM6A and KDM6B, respectively. These cell lines together confirmed that KDM6 impeded early neuroectoderm commitment. By RNA-seq, we found that the expression levels of a panel of WNT genes were significantly affected upon depletion of KDM6. Importantly, the result that WNT agonist and antagonist could abolish the differential neuroectoderm induction due to manipulating KDM6 further demonstrated that WNT was the major downstream of KDM6 during early neural induction. Moreover, we found that the chemical GSK-J1, an inhibitor of KDM6, could enhance neuroectoderm induction from both embryonic stem cells and induced pluripotent stem cells. Taken together, our findings not only illustrated the important role of the histone methylation modifier KDM6 in early neurogenesis, providing insights into the precise epigenetic regulation in cell fate determination, but also showed that the inhibitor of KDM6 could facilitate neuroectoderm differentiation from human pluripotent stem cells.

Coordinated polarization of cells in the tissue plane, known as planar cell polarity (PCP), is associated with a signaling pathway critical for the control of morphogenetic processes. Although the segregation of PCP components to opposite cell borders is believed to play a critical role in this pathway, whether PCP derives from egg polarity or preexistent long-range gradient, or forms in response to a localized cue remains a challenging question. Here we investigate the Xenopus neural plate, a tissue that has been previously shown to exhibit PCP. By imaging Vangl2 and Prickle3, we show that PCP is progressively acquired in the neural plate and requires a signal from the posterior region of the embryo. Tissue transplantations indicated that PCP is triggered in the neural plate by a planar cue from the dorsal blastopore lip. The PCP cue did not depend on the orientation of the graft and was distinct from neural inducers. These observations suggest that neuroectodermal PCP is not instructed by a preexisting molecular gradient, but induced by a signal from the dorsal blastopore lip.HighlightsThe Xenopus neural plate progressively acquires PCP in a posterior-to-anterior direction.The dorsal blastopore lip is likely the source of the PCP-instructing signal for the Xenopus neural plate.The PCP cue is distinct from neural inducers and has a planar mode of transmission.

The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the signalling requirements of naïve human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naïve hPSCs. The downstream effector proteins – SMAD2/3 – bind common sites in naïve and primed hPSCs, including shared pluripotency genes. In naïve hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naïve pluripotency genes. Inhibiting TGFβ signalling in naïve hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naïve and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naïve hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naïve to primed states.

Genetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 majortrans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. Onetrans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

Fibroblast growth factor (FGF) is a highly conserved polypeptide in biological evolution. It has been found that 23 FGF family members have high amino acid sequence homology. FGF has a wide range of biological effects on tissue cells derived from mesoderm and neuroectoderm. FGF promotes cell proliferation, migration, survival, and differentiation through FGF receptor (FGFR); participates in embryonic development, damaged tissue repair, neovascularization, stem cell proliferation and differentiation, nerve regeneration, calcium and phosphorus metabolism, etc.; plays an important role in wound repair, cardiovascular system diseases, nervous system diseases, and osteochondral regeneration; and have important clinical application value and broad market prospects.

Wnt/&beta, catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/&beta, catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/&beta, catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/&beta, catenin signaling during development.

The self-renewal and pluripotency features of mouse embryonic stem cells (mESCs) make them a great tool to study early mammalian development. Various signaling pathways that shape early mammalian development can be mimicked for in vitro mESC differentiation toward primitive lineages first and more specialized cell types later. Since the precise nature of the molecular mechanisms and the crosstalk between these signaling pathways is yet to be fully understood, there is a high level of variability in the efficiency and synchronicity among available differentiation protocols. Commitment of mESCs toward mesoderm, endoderm, or neuroectoderm lineages happens over only a few days and is highly efficient. Here, we provide protocols for the directed differentiation of mESCs toward these lineages in vitro.

Planar cell polarity (PCP) refers to the coordinated polarization of cells within the plane of a tissue. PCP is a controlled by a group of conserved proteins organized in a specific signaling pathway known as the PCP pathway. A hallmark of PCP signaling is the asymmetric localization of "core" PCP protein complexes at the cell cortex, although endogenous PCP cues needed to establish this asymmetry remain unknown. While the PCP pathway was originally discovered as a mechanism directing the planar organization of Drosophila epithelial tissues, subsequent studies in Xenopus and other vertebrates demonstrated a critical role for this pathway in the regulation of actomyosin-dependent morphogenetic processes, such as neural tube closure. Large size and external development of amphibian embryos allows live cell imaging, placing Xenopus among the best models of vertebrate neurulation at the molecular, cellular and organismal level. This review describes cross-talk between core PCP proteins and actomyosin contractility that ultimately leads to tissue-scale movement during neural tube closure.

This chapter examines the development, structure, and function of the central and peripheral nervous systems within the context of caring for critically ill children. The development of the nervous system begins with the establishment of the neuroectoderm and continues through neurogenesis, neuronal migration, synaptogenesis, and programmed cell death. This chapter describes these processes using both normal and pathologic consequences of various developmental stages. An illustrated overview of the structures and function of the nervous system is then provided using clinical correlates to emphasize key concepts.

The hypothalamus and pituitary have been identified to play essential roles in maintaining homeostasis. Various diseases can disrupt the functions of these systems, which can often result in serious lifelong symptoms. The current treatment for hypopituitarism involves hormone replacement therapy. However, exogenous drug administration cannot mimic the physiological changes that are a result of hormone requirements. Therefore, patients are at a high risk of severe hormone deficiency, including adrenal crisis. Pluripotent stem cells (PSCs) self-proliferate and differentiate into all types of cells. The generation of endocrine tissues from PSCs has been considered as another new treatment for hypopituitarism. Our colleagues established a 3-dimensional (3D) culture method for embryonic stem cells (ESCs). In this culture, the ESC-derived aggregates exhibit self-organization and spontaneous formation of highly ordered patterning. Recent results have shown that strict removal of exogenous patterning factors during early differentiation efficiently induces rostral hypothalamic progenitors from mouse ESCs. These hypothalamic progenitors generate vasopressinergic neurons, which release neuropeptides upon exogenous stimulation. Subsequently, we reported adenohypophysis tissue self-formation in 3D cultures of mouse ESCs. The ESCs were found to differentiate into both nonneural oral ectoderm and hypothalamic neuroectoderm in adjacent layers. Interactions between the 2 tissues appear to be critically important for in vitro induction of a Rathke’s pouch-like developing embryo. Various endocrine cells were differentiated from nonneural ectoderm. The induced corticotrophs efficiently secreted adrenocorticotropic hormone when engrafted in vivo, which rescued hypopituitary hosts. For future regenerative medicine, generation of hypothalamic and pituitary tissues from human PSCs is necessary. We and other groups succeeded in establishing a differentiation method with the use of human PSCs. Researchers could use these methods for models of human diseases to elucidate disease pathology or screen potential therapeutics.

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (~Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d + 12h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue did not appear to be greatly affected. Our results compared to those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

There is increasing evidence that long noncoding RNAs (lncRNAs) are among the main regulatory factors of stem cell maintenance and differentiation. They act through various mechanisms and interactions with proteins, DNA, and RNA. This heterogeneity in function increases the capabilities of the lncRNome toolkit but also makes it difficult to predict the function of novel lncRNAs or even rely on biological information produced in animal models. As lncRNAs are species- and tissue-specific, the recent technical advances in self-renewal and differentiation of human embryonic stem cells (ESCs) make these cells the ideal system to identify key regulatory lncRNAs and study their molecular functions. Here we provide an overview of the functional versatility of lncRNA mechanistic heterogeneity in regulating pluripotency maintenance and human differentiation.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TR&alpha, and TR&beta, ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.