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Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation. Electronic supplementary material The online version of this article (10.1007/s00018-017-2700-0) contains supplementary material, which is available to authorized users.

In the deuterostomes and ecdysozoans that have been studied (e.g. chordates and insects), neural fate specification relies on signaling from surrounding cells. However, very little is known about mechanisms of neural specification in the third major bilaterian clade, spiralians. Using blastomere isolation in the annelid Capitella teleta, a spiralian, we studied to what extent extrinsic versus intrinsic signals are involved in early neural specification of the brain and ventral nerve cord. For the first time in any bilaterian, we found that brain neural ectoderm is autonomously specified. This occurs in the daughters of first-quartet micromeres, which also generate anterior neural ectoderm in other spiralians. In contrast, isolation of the animal cap, including the 2d micromere, which makes the trunk ectoderm and ventral nerve cord, blocked ventral nerve cord formation. When the animal cap was isolated with the 2D macromere, the resulting partial larvae had a ventral nerve cord. These data suggest that extrinsic signals from second-quartet macromeres or their daughters, which form mesoderm and endoderm, are required for nerve cord specification in C. teleta and that the 2D macromere or its daughters are sufficient to provide the inductive signal. We propose that autonomous specification of anterior neural ectoderm evolved in spiralians in order to enable them to quickly respond to environmental cues encountered by swimming larvae in the water column. In contrast, a variety of signaling pathways could have been co-opted to conditionally specify the nerve cord. This flexibility of nerve cord development may be linked to the large diversity of trunk nervous systems present in Spiralia.

Cleft lip with or without palate (CLP) and isolated cleft palate (CP) are common human developmental malformations with a complex etiology that reflects a failure of normal facial development. VAX1 encodes a homeobox-containing transcription factor identified as a candidate gene for CLP in human populations, with targeted deletion in mice associated with multiple anomalies, including disruption of the visual apparatus and basal forebrain, lobar holoprosencephaly, and CP. We have investigated Vax1 function during murine palatogenesis but found no evidence for a direct role in this process. Vax1 is not expressed in the developing palate and mutant palatal shelves elevate above the tongue, demonstrating morphology and proliferation indices indistinguishable from wild type. However, mutant mice did have a large midline cavity originating from the embryonic forebrain situated beneath the floor of the hypothalamus and extending through the nasal cavity to expand this region and prevent approximation of the palatal shelves. Interestingly, despite strong expression of Vax1 in ectoderm of the medial nasal processes, the upper lip remained intact in mutant mice. We found further evidence of disrupted craniofacial morphology in Vax1 mutants, including truncation of the midface associated with reduced cell proliferation in forebrain neuroectoderm and frontonasal mesenchyme. Sonic hedgehog (Shh) signal transduction was downregulated in the mutant forebrain, consistent with a role for Vax1 in mediating transduction of this pathway. However, Shh was also reduced in this region, suggestive of a Shh-Vax1 feedback loop during early development of the forebrain and a likely mechanism for the underlying lobar holoprosencephaly. Despite significant associations between VAX1 and human forms of CLP, we find no evidence of a direct role for this transcription factor in development of this region in a mutant mouse model.

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3, whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4, a direct regulator of pou5f3, in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s. Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4.

Summary During gastrulation, the neuroectoderm cells form the neural tube and neural crest. The nervous system contains significantly more microRNAs than other tissues, but the role of microRNAs in controlling the differentiation of neuroectodermal cells into neural tube epithelial (NTE) cells and neural crest cells (NCCs) remains unknown. Using embryonic stem cell (ESC) neural differentiation systems, we found that miR-29b was upregulated in NTE cells and downregulated in NCCs. MiR-29b promoted the differentiation of ESCs into NTE cells and inhibited their differentiation into NCCs. Accordingly, the inhibition of miR-29b significantly inhibited the differentiation of NTE cells. A mechanistic study revealed that miR-29b targets DNA methyltransferase 3a (Dnmt3a) to regulate neural differentiation. Moreover, miR-29b mediated the function of Pou3f1, a critical neural transcription factor. Therefore, our study showed that the Pou3f1-miR-29b-Dnmt3a regulatory axis was active at the initial stage of neural differentiation and regulated the determination of cell fate., Graphical Abstract, Highlights • MiR-29b promoted NTE differentiation and inhibited NCC differentiation from ESCs • MiR-29b targeted Dnmt3a to regulate neural differentiation • MiR-29b mediated the function of Pou3f1 • The Pou3f1-miR-29b-Dnmt3a axis regulated the cell fate determination, In this article, Kang and colleagues demonstrated that miR-29b mediated the function of a critical neural transcription factor Pou3f1 to promote the differentiation of ESCs into NTE cells and inhibit their differentiation into NCCs by targeting Dnmt3a. This study showed that the Pou3f1-miR-29b-Dnmt3a regulatory axis was active at the initial stage of neural differentiation and regulated cell fate determination.

Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein-protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.

Background: In the avian embryo, neural crest (NC) progenitors arise in the neuroectoderm during gastrulation, long before their dissemination. Although the gene regulatory network involved in NC specification has been deciphered, the mechanisms involved in their segregation from the other neuroectoderm-derived progenitors, notably the epidermis and neural tube, are unknown. Because cadherins mediate cell recognition and sorting, we scrutinized their expression profiles during NC specification and delamination. Results: We found that the NC territory is defined precociously by the robust expression of Cadherin-6B in cells initially scattered among other cells uniformly expressing E-cadherin, and that NC progenitors are progressively sorted and regrouped into a discrete domain between the prospective epidermis and neural tube. At completion of NC specification, the epidermis, NC and neural tube are fully segregated in contiguous compartments characterized by distinct cadherin repertoires. We also found that Cadherin-6B downregulation constitutes a major event during NC delamination and that, with the exception of the caudal part of the embryo, N-cadherin is unlikely to control NC emigration. Conclusions: Our results indicate that partition of the neuroectoderm is mediated by cadherin interplays and ascribe a key role to Cadherin-6B in the specification and delamination of the NC population. This article is protected by copyright. All rights reserved.

We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm. To investigate the translational potential of the SEAM, cells within it that resemble ocular-surface epithelia can be isolated by pipetting and FACS sorting into a population of corneal epithelial-like progenitor cells. These can be expanded and differentiated to form an epithelial layer expressing K12 and PAX6, and able to recover function in an animal model of corneal epithelial dysfunction after surgical transplantation. The whole protocol, encompassing human iPS cell preparation, autonomous differentiation, purification, and subsequent differentiation, takes between 100 and 120 d, and is of potential use to researchers with an interest in eye development and/or ocular-surface regeneration. Experience with human iPS cell culture and sorting via FACS will be of benefit for researchers performing this protocol.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation.

Research into regulation of the differentiation of stem cells is critical to understanding early developmental decisions and later development growth. The transcription factor ARID3A previously was shown to be critical for trophectoderm and hematopoetic development. Expression of ARID3A increases during embryonic differentiation, but the underlying reason remained unclear. Here we show that Arid3a null embryonic stem (ES) cells maintain an undifferentiated gene expression pattern and form teratomas in immune-compromised mice. However, Arid3a null ES cells differentiated in vitro into embryoid bodies (EBs) significantly faster than control ES cells, and the majority forming large cystic embryoid EBs. Analysis of gene expression during this transition indicated that Arid3a nulls differentiated spontaneously into mesoderm and neuroectoderm lineages. While young ARID3A-deficient mice showed no gross tissue morphology, proliferative and structural abnormalities were observed in the kidneys of older null mice. Together these data suggest that ARID3A is not only required hematopoiesis, but is critical for early mesoderm differentiation.

Macroevolutionary gains and losses in Bilateria are to a large extent controlled by the architecture of the gene regulatory network, including Hox- and other regulatory genes and also posttranscriptional regulation of microRNA. The concepts of evolutionary biology and reproduction strategy allow the recognition of different evolutionary strategy in Ecdysozoa and Deuterostomia. The emergence of numerous new populations of stem cells was a key evolutionary acquisition in Metazoa, especially in the evolutionary lineage of deuterostomes–chordates–vertebrates. The emergence of the neural plate interpreted as the fourth germ layer (neuroectoderm), with subsequent development of a hollow neural tube, was an aromorphic evolutionary innovation of vertebrates that provided the excess of neuroblasts and rapid evolution of the brain.

Nature, 574 (7778), ISSN:0028-0836, ISSN:1476-4687

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

ADNP (Activity Dependent Neuroprotective Protein) is a neuroprotective protein whose aberrant expression has been frequently linked to neural developmental disorders, including the Helsmoortel-Van der Aa syndrome (also called the ADNP syndrome). However, its role in neural development and pathology remains unclear. Here, we show that ADNP is required for neural induction and differentiation by enhancing Wnt signaling. Mechanistically, ADNP functions to stabilize β-Catenin through binding to its armadillo domain which prevents its association with key components of the degradation complex: Axin and APC. Loss of ADNP promotes the formation of the degradation complex and β-Catenin degradation via ubiquitin-proteasome pathway, resulting in down-regulation of key neuroectoderm developmental genes. In addition, adnp gene disruption in zebrafish leads to defective neurogenesis and reduced Wnt signaling. Our work provides important insights into the role of ADNP in neural development and the pathology of the Helsmoortel-Van der Aa syndrome caused by ADNP gene mutation., ADNP has been connected to neural developmental disorders. Here, the authors uncover a role for ADNP in neural induction and differentiation via β-Catenin stabilization, with ADNP disruption in zebrafish leading to defective neurogenesis and decreased Wnt signaling.

The ellipsoid body (EB) of the Drosophila central complex mediates sensorimotor 33 integration and action selection for adaptive behaviours. Insights into its physiological 34 function are steadily accumulating, however the developmental origin and genetic 35 specification have remained largely elusive. Here we identify two stem cells in the 36 embryonic neuroectoderm as precursor cells of neuronal progeny that establish EB 37 circuits in the adult brain. Genetic tracing of embryonic neuroblasts ppd5 and mosaic 38 analysis with a repressible cell marker identified lineage-related progeny as Pox neuro 39 (Poxn)-expressing EB ring neurons, R1-R4. During embryonic brain development, 40 engrailed function is required for the initial formation of Poxn-expressing ppd5-derived 41 progeny. Postembryonic determination of R1-R4 identity depends on lineage-specific 42 Poxn function that separates neuronal subtypes of ppd5-derived progeny into hemi-43 lineages with projections either terminating in the EB ring neuropil or the superior 44 protocerebrum (SP). Poxn knockdown in ppd5-derived progeny results in identity 45 transformation of engrailed-expressing hemi-lineages from SP to EB-specific circuits. In 46 contrast, lineage-specific knockdown of engrailed leads to reduced numbers of Poxn-47 expressing ring neurons. These findings establish neuroblasts ppd5-derived ring 48 neurons as lineage-related sister cells that require engrailed and Poxn function for the 49 proper formation of EB circuitry in the adult central complex of Drosophila.

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes1–5. However, the genetic and developmental programs underlying this divergence are not fully understood6–8. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

During embryogenesis, the distinct morphogenetic cell behavior programs that shape tissues are influenced both by the fate of cells and their position with respect to the embryonic axes, making embryonic patterning a prerequisite for morphogenesis. These two essential processes must therefore be coordinated in space and time to ensure proper development, but mechanisms by which patterning information is translated to the cellular machinery that drives morphogenesis remain poorly understood. Here, we address the role of Nodal morphogen signaling at the intersection of cell fate specification, patterning, and anteroposterior (AP) axis extension in zebrafish gastrulae and embryonic explants. AP axis extension is impaired in Nodal-deficient embryos, but it is unclear whether this defect is strictly secondary to their severe mesendoderm deficiencies or also results from loss of Nodal signalingper se. We find that convergence & extension (C&E) gastrulation movements and underlying mediolateral (ML) cell polarization are reduced in the neuroectoderm of Nodal-deficient mutants and exacerbated by simultaneous disruption of Planar Cell Polarity (PCP) signaling, demonstrating at least partially parallel functions of Nodal and PCP. ML polarity of mutant neuroectoderm cells is not fully restored upon transplantation into wild-type gastrulae, demonstrating a cell autonomous, mesoderm-independent role for Nodal in neural cell polarization. This is further demonstrated by the ability of Nodal ligands to promote neuroectoderm-driven C&E of naïve blastoderm explants in a tissue-autonomous fashion. Finally, temporal manipulation of signaling reveals that Nodal contributes to neural C&E in explants after mesoderm is specified and promotes C&E even in the absence of mesoderm. Together these results reveal a mesoderm-independent, cell-autonomous role for Nodal signaling in neural C&E that may cooperate with previously-described mesoderm-dependent mechanisms to drive AP embryonic axis extension.

Unlike the Hox genes in arthropods and vertebrates, those in molluscs show diverse expression patterns and, with some exceptions, have generally been described as lacking the canonical staggered pattern along the anterior-posterior (AP) axis. This difference is unexpected given that almost all molluscs share highly conserved early development. Here, we show that molluscan Hox expression can undergo dynamic changes, which may explain why previous research observed different expression patterns. Moreover, we reveal that a key character of molluscan Hox expression is that the dorsal and ventral expression is dissociated. We then deduce a generalized molluscan Hox expression model, including conserved staggered Hox expression in the neuroectoderm on the ventral side and lineage-specific dorsal expression that strongly correlates with shell formation. This generalized model clarifies a long-standing debate over whether molluscs possess staggered Hox expression and it can be used to explain the diversification of molluscs. In this scenario, the dorsoventral dissociation of Hox expression allows lineage-specific dorsal and ventral patterning in different clades, which may have permitted the evolution of diverse body plans in different molluscan clades.

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis-regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis-regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half-life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4-9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension-class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.

Eye is a complex organ with a highly specialized tissue structure. The establishment of human pluripotent stem cells (hPSCs) has allowed the simulation of eye development in vitro. Most differentiation works of hPSC-derived ocular cells focus on a single, tissue-specific lineage, however, that faces difficulty in reflecting the complexity of eye development. Recently, the generation of a self-formed ectodermal autonomous multi-zone of ocular cells availably mimics the process of whole-eye development. In this study, we developed a rapid defined method to induce the differentiation of multi-zone ocular cells (MZOCs) from human induced pluripotent stem cells, which specifically experienced the key progenitor stages of anterior neuroectoderm and eye field stem cells by a 2.5-dimensional culture. These differentiated cell types spanned neural retina, retinal pigment epithelium, surface ectoderm, and neural crest and lens cells. In addition, the surface ectoderm zone of MZOCs could be mechanically isolated and induced into corneal epithelial cells, and the isolated neural crest zone could be directed into corneal endothelial cells. This in vitro differentiation process vividly mimics the development of vertebrate eye, and it provides a promising model for the study of ocular morphogenesis, as well as an ideal resource of seed cells for corneal regenerative medicine.

Human embryonic hypothalamo-pituitary development is a complex process controlled by a spatio-temporal genetic cascade of transcription factors and signalling molecules within the hypothalamus and Rathke’s pouch, the primordium of the anterior pituitary (AP). The anterior and intermediate lobes of the pituitary derive from the oral ectoderm and the posterior lobe from the overlying neural ectoderm. The tightly regulated processes of cell proliferation and differentiation give rise to five different specialized AP cell types that secrete six hormones. Disordered embryogenesis can give rise to congenital hypopituitarism (CH), characterized by deficiencies in one or more of these six hormones in isolation or as combined pituitary hormone deficiency, with a wide variation in severity. CH may present early in the neonatal period or later in childhood, with midline and craniofacial structural abnormalities often accompanying the phenotype, giving rise to a range of highly variable disorders that will be discussed in this chapter.

Human induced pluripotent stem cells (iPSCs) hold substantial promise for modeling diseases in the relevant human genetic backgrounds. Here, we leverage the shared developmental pathways of spinal neuroectoderm and paraxial mesoderm via neuromesodermal precursors to produce a sensorimotor organoid model, which we validate with single-cell RNA sequencing and physiology in multiple control and diseased human stem cell lines. The organoids contain motor neurons and skeletal muscle connected by physiologically functional neuromuscular junctions. We also generate additional neuronal and mesodermal derivatives, identified by RNA-seq cluster analysis and verified by staining, electron microscopy, and physiology. These cell types include astrocytes, endothelial cells, microglia, and sensory neurons, the last from which we record tetrodotoxin-resistant sodium currents and capsaicin-elicited calcium flux. The physiological resolution of the neuromuscular junction synapse combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases may prove valuable for more comprehensive disease modeling.

The retina is a thin membrane derived from the neuroectoderm, it is the physical morphological substrate in which the transformation of light energy into electrical impulses, that later will be led to the cerebral cortex, is performed. Due to its prosencephalic embryological origin, the retina is normally considered a specially di erentiated part of the brain. It is a very complex tissue, formed by multiple cell layers and by several types of neuronal cells (ganglion, bipolar, horizontal, amacrine, and photoreceptor cells), microglia (macrophages), macroglia (Müller cells, astrocytes), and vascular cells (endothelium and pericytes). Under physiological conditions, the retina is characterized by a high oxygen consumption rate, intense exposition to pro-oxidizing agents (i.e., light) and a high content of polyunsaturated fatty acids (especially in the photoreceptor membranes). Therefore, retina is especially susceptible to oxidative stress. Sin financiación 5.014 JCR (2019) Q1, 56/297 Biochemistry & Molecular Biology, 7/61 Chemistry, Medicinal, 10/139 Food Science & Technology 1.100 SJR (2019) Q1, 23/130 Clinical Biochemistry; Q2, 133/456 Biochemistry, 140/300 Cell Biology, 176/414 Molecular Biology, 61/186 Physiology No data IDR 2019 UEM

A Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/β-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/β-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation., Summary: Non-canonical Wnt16 signaling is essential for establishing the position of the early germ layer gene regulatory networks along the anterior-posterior axis, and activates molecular mechanisms necessary for gastrulation and mesenchyme morphogenesis.

During embryogenesis, a stringent regulation of Wnt activity is critical for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor, Dkk1, results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorization of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be critical for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSC) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.

Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...

Anterior–posterior (A–P) specification of the neural tube involves initial acquisition of anterior fate followed by the induction of posterior characteristics in the primitive anterior neuroectoderm. Several morphogens have been implicated in the regulation of A–P neural patterning; however, our understanding of the upstream regulators of these morphogens remains incomplete. Here, we show that the Krüppel-like zinc finger transcription factor GLI-Similar 3 (GLIS3) can direct differentiation of human embryonic stem cells (hESCs) into posterior neural progenitor cells in lieu of the default anterior pathway. Transcriptomic analyses reveal that this switch in cell fate is due to rapid activation of Wingless/Integrated (WNT) signaling pathway. Mechanistically, through genome-wide RNA-Seq, ChIP-Seq, and functional analyses, we show that GLIS3 binds to and directly regulates the transcription of several WNT genes, including the strong posteriorizing factor WNT3A, and that inhibition of WNT signaling is sufficient to abrogate GLIS3-induced posterior specification. Our findings suggest a potential role for GLIS3 in the regulation of A–P specification through direct transcriptional activation of WNT genes. Stem Cells 2018 Stem Cells 2019;37:202–215

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry–based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.