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Summary p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice., Graphical abstract, Highlights • H3K4me defective WDR5 supports self-renewal and GCM differentiation in p53-null mESCs • WDR5 regulates H3K4me-independent stemness and GCM gene expression in p53-null mESCs • MAX and WDR5 repress GCM-related gene chromatin accessibility upon differentiation, In this article, Rao and colleagues show that an H3K4 methylation-independent, WDR5-driven mechanism maintains self-renewal and represses germ cell/meiosis (GCM)-specific transcription in p53-null mESCs. WDR5 inactivation triggers enhanced chromatin accessibility on most gene targets bound by MAX and WDR5. Thus, WDR5, a component of polycomb repressive complex 1.6, controls cell type-specific gene repression during mESC fate determination in a non-canonical fashion.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior–posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease. Stem cells cultured in a micropattern-constrained platform form a quantitative and robust model of human neural tube morphogenesis.

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

Intellectual disability (ID) is a neurodevelopmental disorder defined by below-average intelligence (intelligence quotient of

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.

Background Light is essential for various biological activities. In particular, visual information through eyes or eyespots is very important for most of animals, and thus, the functions and developmental mechanisms of visual systems have been well studied to date. In addition, light-dependent non-visual systems expressing photoreceptor Opsins have been used to study the effects of light on diverse animal behaviors. However, it remains unclear how light-dependent systems were acquired and diversified during deuterostome evolution due to an almost complete lack of knowledge on the light-response signaling pathway in Ambulacraria, one of the major groups of deuterostomes and a sister group of chordates. Results Here, we show that sea urchin larvae utilize light for digestive tract activity. We found that photoirradiation of larvae induces pyloric opening even without addition of food stimuli. Micro-surgical and knockdown experiments revealed that this stimulating light is received and mediated by Go(/RGR)-Opsin (Opsin3.2 in sea urchin genomes) cells around the anterior neuroectoderm. Furthermore, we found that the anterior neuroectodermal serotoninergic neurons near Go-Opsin-expressing cells are essential for mediating light stimuli-induced nitric oxide (NO) release at the pylorus. Our results demonstrate that the light>Go-Opsin>serotonin>NO pathway functions in pyloric opening during larval stages. Conclusions The results shown here will lead us to understand how light-dependent systems of pyloric opening functioning via neurotransmitters were acquired and established during animal evolution. Based on the similarity of nervous system patterns and the gut proportions among Ambulacraria, we suggest the light>pyloric opening pathway may be conserved in the clade, although the light signaling pathway has so far not been reported in other members of the group. In light of brain-gut interactions previously found in vertebrates, we speculate that one primitive function of anterior neuroectodermal neurons (brain neurons) may have been to regulate the function of the digestive tract in the common ancestor of deuterostomes. Given that food consumption and nutrient absorption are essential for animals, the acquirement and development of brain-based sophisticated gut regulatory system might have been important for deuterostome evolution.

This paper overviews the development and differentiation of the hypothalamus and pituitary gland from embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is important to replicate the developmental process in vivo to create specific cells/organoids from ES/iPS cells. We also introduce the latest findings and discuss future issues for clinical application. Neuroectodermal progenitors are induced from pluripotent stem cells by strictly removing exogenous patterning factors during the early differentiation period. The induced progenitors differentiate into rostral hypothalamic neurons, in particular magnocellular vasopressin+ neurons. In three-dimensional cultures, the ES/iPS cells differentiate into hypothalamic neuroectoderm as well as non-neural head ectoderm back to back. Rathke's pouch-like structures self-organize at the interface between the two layers and generated various endocrine cells, including corticotrophs and somatotrophs from the Rathke's pouch-like structures. Our next objective is to sophisticate our stepwise methodology to establish the novel transplantation treatment for hypopituitarism and to apply it to developmental disease models.

LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.

Olfactory neuroblastoma (ONB) or esthesioneuroblastoma is a rare malignancy of the nose and paranasal sinuses that originates from the upper nasal vault. We report a rare case of ONB in a 27-year-old female patient with a 2-year history of nasal obstruction, sneezing, nasal discharge, and left eye protrusion. The patient was successfully treated with endoscopic sinus surgery and adjuvant radiotherapy (RT) with 5-year follow-up duration. Studying ONB cases is of great importance due to its rare occurrence, and the lack of literature regarding its management often creates a dilemma leading to nonstandardized treatment methods. Prolonged surveillance is essential due to its late recurrence and endoscopic resection followed by adjuvant RT, which may be a feasible and effective treatment. There is no single case of ONB published yet in Saudi Arabia, and to our knowledge, this is the first case report.

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.

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 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.

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.

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.

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.

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.

Graphical abstract, Highlights • Ocular surface ectodermal fate is determined in the early stage of human eye development. • p63+ cells which co-express PAX6, are seen in the early eye development. • Combined WNT inhibition and exogenous BMP4 promote ocular surface ectodermal differentiation. • SFRP2 and DKK1 act as WNT inhibitors to initiate ocular surface ectodermal development., We sought to elucidate how and when the ocular surface ectoderm commits to its differentiation into the corneal epithelium in eye development from human induced pluripotent stem cells (hiPSCs) under the influence of WNT signaling and the actions of BMP4. These signals are key drivers ocular surface ectodermal cell fate determination. It was discovered that secreted frizzled related protein-2 (SFRP2) and Dickkopf1 (DKK1), which are expressed in neural ectoderm, are both influential in the differentiation of hiPSCs, where they act as canonical WNT antagonists. BMP4, moreover, was found to simultaneously initiate non-neural ectodermal differentiation into a corneal epithelial lineage. Combined treatment of hiPSCs with exogenous BMP4 aligned to WNT inhibition for the initial four days of differentiation increased the ocular surface ectodermal cell population and induced a corneal epithelial phenotype. Specification of a surface ectodermal lineage and its fate is thus determined by a fine balance of BMP4 exposure and WNT inhibition in the very earliest stages of human eye development.

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired‐less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification. Induced expression of PAX6D but not PAX6A in a PAX6‐null background restored the NR specification capacity. ChIP‐Seq, confirmed by functional assays, revealed a set of retinal genes and non‐retinal neural genes that are potential targets of PAX6D, including WNT8B. Inhibition of WNTs or knocking down of WNT8B restored the NR specification capacity of neuroepithelia with PAX6D knockout, whereas activation of WNTs blocked NR specification even when PAX6D was induced. Thus, PAX6D specifies neuroepithelia to NR cells via the regulation of WNT8B.

Advances in the development of three-dimensional (3D) brain organoids maintained in vitro have provided excellent opportunities to study brain development and neurodegenerative disorders, including Alzheimer's disease (AD). However, there remains a need to generate AD organoids bearing patient-specific genomic backgrounds that can functionally recapitulate the key features observed in the AD patient's brain. To address this need, we described a strategy to generate self-organizing 3D cerebral organoids which develop a functional neuronal network connectivity. This was achieved by neuroectoderm induction of human pluripotent stem cell (hPSCs) aggregates and subsequent differentiation into desired neuroepithelia and mature neurons in a 3D Matrigel matrix. Using this approach, we successfully generated AD cerebral organoids from human pluripotent stem cells (hPSCs) derived from a familial AD patient with a common mutation in presenilin 2 (PSEN2N141I). An isogenic control with an identical genetic background but wild-type PSEN2 was generated using CRISPR/Cas9 technology. Both control and AD organoids were characterized by analyzing their morphology, the Aβ42/Aβ40 ratio, functional neuronal network activity, drug sensitivity, and the extent of neural apoptosis. The spontaneous activity of the network and its synchronization was measured in the organoids via calcium imaging. We found that compared with the mutation-corrected control organoids, AD organoids had a higher Aβ42/Aβ40 ratio, asynchronous calcium transients, and enhanced neuronal hyperactivity, successfully recapitulating an AD-like pathology at the molecular, cellular, and network level in a human genetic context. Moreover, two drugs which increase neuronal activity, 4-aminopyridine (4-AP) and bicuculline methochloride, induced high-frequency synchronized network bursting to a similar extent in both organoids. Therefore, our study presents a promising organoid-based biosystem for the study of the pathophysiology of AD and a platform for AD drug development.

Fibroblast growth factor (Fgf) and anti-bone morphogenetic protein (Bmp) signals are derived from the organiser of mesoderm origin and cooperate to promoteXenopusneural development from the gastrula ectoderm. Using antisense oligos to Fgf2 and Fgf8 and dominant-negative Ets transcription factors, we showed that the expression of Fgf2, Fgf8, and Ets in ectoderm cells is essential to initiate neural induction bothin vivoandin vitro. Our findings show that neural induction is initiated primarily by autonomous signalling in ectoderm cells, rather than by paracrine signalling from organiser cells. The signalling in ectoderm cells is transduced via the Fgf/Ras/Mapk/Ets pathway, independent of Bmp signal inhibition via the Fgf/Ras/Mapk/Smad1 route, as indicated by earlier studies. Through the same pathway, Fgfs activated position-specific neural genes dose-dependently along the anteroposterior axis in cultured ectoderm cells. The expression of these genes coincides with the establishment of the activated Ets gradient within the gastrula ectoderm. Organiser cells, being located posteriorly to the ectoderm, secrete Fgfs as gastrulation proceeds, which among several candidate molecules initially promote neural patterning of the induced neuroectoderm as morphogens.Summary statementFgf/Ets signalling in ectodermal cells is required to initiate the expression of both anterior and posterior neural genes from the late blastula to gastrula stages, independent of anti-Bmp signalling.

Melanocytes are neuroectoderm-derived pigment-producing cells with highly polarized dendritic morphology. They protect the skin against ultraviolet radiation by providing melanin to neighbouring keratinocytes. However, the mechanisms underlying melanocyte polarization and its relevance for diseases remain mostly elusive. Numerous studies have instead revealed roles for polarity regulators in other neuroectoderm-derived lineages including different neuronal cell types. Considering the shared ontogeny and morphological similarities, these lineages may be used as reference models for the exploration of melanocyte polarity, for example, regarding dendrite formation, spine morphogenesis and polarized organelle transport. In this review, we summarize and compare the latest progress in understanding polarity regulation in neuronal cells and melanocytes and project key open questions for future work.

The internalization of the central nervous system, termed neurulation in vertebrates, is a critical step in embryogenesis. Open questions remain as to how force propels coordinated tissue movement during the process, and little is known as to how internalization happens in invertebrates. We show that inC. elegansmorphogenesis, apical constriction in the retracting pharynx drives involution of the adjacent neuroectoderm. Localized HMR-1/Cadherin mediates the inter-tissue attachment, as well as within the neuroectoderm to maintain intratissue cohesion. Our results demonstrate that localized HMR-1 is capable of mediating embryo wide reorganization driven by a centrally located force generator, and indicate a non-canonical use of Cadherin on the basal side of an epithelium that may apply to vertebrate neurulation. Additionally, we highlight shared morphology and gene expression in tissues driving involution, which suggests that neuroectoderm involution inC. elegansis potentially homologous with vertebrate neurulation and thus may help elucidate the evolutionary origin of the brain.

During vertebrate gastrulation, convergence and extension (C and E) of the primary anteroposterior (AP) embryonic axis is driven by polarized mediolateral (ML) cell intercalations and is influenced by AP axial patterning. Nodal signaling is essential for patterning of the AP axis while planar cell polarity (PCP) signaling polarizes cells with respect to this axis, but how these two signaling systems interact during C and E is unclear. We find that the neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C and E cell behaviors, which require Nodal signaling in both cell- and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient embryos and its inhibition exacerbates their C and E defects. Within otherwise naïve zebrafish blastoderm explants, however, Nodal induces C and E in a largely PCP-dependent manner, arguing that Nodal acts both upstream of and in parallel with PCP during gastrulation to regulate embryonic axis extension cooperatively.

XBF-1 is an anterior neural plate-specific, winged helix transcription factor that affects neural development in a concentration-dependent manner. A high concentration of XBF-1 results in suppression of endogenous neuronal differentiation and an expansion of undifferentiated neuroectoderm. Here we investigate the mechanism by which this expansion is achieved. Our findings suggest that XBF-1 converts ectoderm to a neural fate and it does so independently of any effects on the mesoderm. In addition, we show that a high dose of XBF-1 promotes the proliferation of neuroectodermal cells while a low dose inhibits ectodermal proliferation. Thus, the neural expansion observed after high dose XBF-1 misexpression is due both to an increase in the number of ectodermal cells devoted to a neural fate and an increase in their proliferation. We show that the effect on cell proliferation is likely to be mediated by p27(XIC1), a cyclin-dependent kinase (cdk) inhibitor. We show that p27(XIC1) is expressed in a spatially restricted pattern in the embryo, including the anterior neural plate, and when misexpressed it is sufficient to block the cell cycle in vivo. We find that p27(XIC1)is transcriptionally regulated by XBF-1 in a dose-dependent manner such that it is suppressed or ectopically induced by a high or low dose of XBF-1, respectively. However, while a low dose of XBF-1 induces ectopic p27(XIC1)and ectopic neurons, misexpression of p27(XIC1)does not induce ectopic neurons, suggesting that the effects of XBF-1 on cell fate and cell proliferation are distinct. Finally, we show that p27(XIC1)is suppressed by XBF-1 in the absence of protein synthesis, suggesting that at least one component of p27(XIC1)regulation by XBF-1 may be direct. Thus, XBF-1 is a neural-specific transcription factor that can independently affect both the cell fate choice and the proliferative status of the cells in which it is expressed.

Summary Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity., Graphical abstract, Highlights • Low Sox2 expression is sufficient for naive pluripotent stem cell self-renewal • Low Sox2 expression does not impair neurectoderm differentiation in vitro • Low Sox2 expression impairs Oct4 genomic occupancy • Low Sox2 increases naive pluripotent stem cell plasticity in vitro and in vivo, Biological Sciences; Cell Biology; Stem Cells Research

Neuroectoderm formation is the first step in development of a proper nervous system for vertebrates. The developmental decision to form a non-neural ectoderm versus a neural one involves the regulation of BMP signaling, first reported many decades ago. However, the precise regulatory mechanism by which this is accomplished has not been fully elucidated, particularly for transcriptional regulation of certain key transcription factors. BMP4 inhibition is a required step in eliciting neuroectoderm from ectoderm and Foxd4l1.1 is one of the earliest neural genes highly expressed in the neuroectoderm and conserved across vertebrates, including humans. In this work, we focused on how Foxd4l1.1 downregulates the neural repressive pathway. Foxd4l1.1 inhibited BMP4/Smad1 signaling and triggered neuroectoderm formation in animal cap explants of Xenopus embryos. Foxd4l1.1 directly bound within the promoter of endogenous neural repressor ventx1.1 and inhibited ventx1.1 transcription. Foxd4l1.1 also physically interacted with Xbra in the nucleus and inhibited Xbra-induced ventx1.1 transcription. In addition, Foxd4l1.1 also reduced nuclear localization of Smad1 to inhibit Smad1-mediated ventx1.1 transcription. Foxd4l1.1 reduced the direct binding of Xbra and Smad1 on ventx1.1 promoter regions to block Xbra/Smad1-induced synergistic activation of ventx1.1 transcription. Collectively, Foxd4l1.1 negatively regulates transcription of a neural repressor ventx1.1 by multiple mechanisms in its exclusively occupied territory of neuroectoderm, and thus leading to primary neurogenesis. In conjunction with the results of our previous findings that ventx1.1 directly represses foxd4l1.1, the reciprocal repression of ventx1.1 and foxd4l1.1 is significant in at least in part specifying the mechanism for the non-neural versus neural ectoderm fate determination in Xenopus embryos.

Over the past 15 years, extraordinary strides have demonstrated that stem cell-based therapy is a promising treatment for retinal diseases. Retinal degenerative diseases that cause irreversible dysfunction and death of retinal pigment epithelium (RPE) and photoreceptor cells may lead to blindness. The retina, which is derived from the neuroectoderm, has little regenerative potential. Stem cell-based therapies that introduce healthy tissue into the eye have been shown to restore or rescue the degenerated retina and improve vision in animal models.

The origin(s) of smooth muscle-like cells contributing to the development of subclinical arteriosclerosis remains controversial. Using Sca1-eGFP and S100β-eGFP transgenic mice together with genetic lineage tracing of S100β+ vascular stem cells (vSCs), we identified a S100β+/Sca1+ population, originating from a perivascular non-smooth muscle cell (SMC) S100β+ parent population in lesions developed following iatrogenic flow restriction. Neuroectoderm S100β+ vSCs isolated from atheroprone murine vessels were Hedgehog (Hh) responsive, enriched for the trimethylation of lysine 27 at histone 3 (H3K27me3) repressive mark at the Myh11 locus, and underwent myogenic differentiation in vitro. Inhibition of Hh signalling reduced the number of S100β+/Sca1+ cells and attenuated lesion formation. Hh promoted myogenic differentiation of S100β+ vSCs derived from human induced pluripotent stem cells (iPSC) in vitro, while S100β+ cells were enriched in human lesions. We conclude that Hh-responsive S100β vSC-derived progeny contribute to adaptive lesions and support targeting Hh signalling to treat subclinical arteriosclerosis.

The Drosophila embryonic central nervous system (CNS) is a powerful model system for addressing basic mechanisms of nervous system development. Studies during the last three decades have resulted in a detailed description of the basic anatomical features of the CNS, as well as an in-depth understanding of the molecular genetic mechanisms acting to control its development. This has revealed a complex multistep process, from early patterning events to the selection of neural progenitor cells, neuroblasts, with unique identities, onward to lineage progression and the establishment of unique neuronal and glial identities. In this chapter we will review our current understanding of these processes and end by highlighting a number of outstanding issues.

The formation of the iris is an interaction between neural ectoderm, neural crest and mesoderm. It starts from formation of the optic cup from neural ectoderm which invaginates into itself to form the posterior double layer of iris pigment epithelium. The neural crest origin melanoblasts, will migrate into the stromal tissue that is formed by the periocular mesenchyme. The stroma of the iris is formed from mesoderm as part of anterior chamber formation and regression and disappearance of the endothelium on the front surface of the iris by birth.

Since the discovery of methylcytosine oxidase ten-eleven translocation (TET) proteins, we have witnessed an exponential increase in studies examining their roles in epigenetic regulation. TET family proteins catalyze the sequential oxidation of 5-methylcytosine (5mC) to oxidized methylcytosines including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine. TETs contribute to the regulation of lineage-specific gene expression via modulating DNA 5mC/5hmC balances at the proximal and distal regulatory elements of cell identity genes, and therefore enhance chromatin accessibility and gene transcription. Emerging evidence suggests that TET dioxygenases participate in the establishment and/or maintenance of hypomethylated bivalent domains at multiple differentiation-associated genes, and thus ensure developmental plasticity. Here, we review the current state of knowledge concerning TET family proteins, DNA hydroxymethylation, their distribution, and function in endoderm, mesoderm, and neuroectoderm specification. We will summarize the evidence pertaining to their crucial regulatory roles in lineage commitment and development.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have attracted attention as a novel tool for drug safety screening and several differentiation protocols of hiPSC lines into cardiomyocytes have been reported; the standardization of these protocols will expand their applications for safety assessments such as “clinical safety trial-on-dish”. Bone morphogenetic protein 4 (BMP4) is an important factor in promoting mesoderm differentiation and BMP4 treatment has been used at the early stage of cardiac differentiation into different hiPSCs. In the present study, we evaluated the effects of BMP4 treatment at the early stage of cardiac differentiation. We performed gene expression profiling of the germ layer during mesoderm differentiation of hiPSCs derived from three different donors. The expression of T (a mesoderm marker) and GATA6 (an endoderm marker) increased and that of PAX6 (a neuroectoderm marker) decreased in pooled embryoid bodies (EBs) after BMP4 treatment. Single-cell gene expression analysis revealed that mesodermal and mesendodermal populations increased in EBs derived from 253G1. Finally, BMP4 treatment increased mesodermal and mesendodermal populations compared with that without BMP4 in two other hiPSC lines, confirming the reproducibility of multiple hiPSC lines. Thus, our results suggest that BMP4 treatment increases mesodermal and mesendodermal populations at the early stage of cardiac differentiation in different hiPSC lines.

International audience; Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.

Summary The molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm., Highlights • Self-renewing epiblast stem cells can be maintained under Nodal inhibition • Nodal-inhibited epiblast stem cells and the ectoderm display similar transcriptome • Blocking Nodal changes the epigenome to that associated with ectoderm potency • Nodal-inhibited epiblast stem cells differentiate preferentially to ectodermal cells, In this article, Liu and colleagues show that Nodal-inhibited epiblast stem cells can self-renew in vitro and display similar transcriptome compared with the ectoderm of late-gastrula embryo. Further analysis shows that inhibition of Nodal signaling predisposes the epigenome to ectoderm-associated status. Moreover, these Nodal-inhibited epiblast stem cells efficiently differentiate to ectodermal cells but not mesendoderm lineages.