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Epidermoid cysts are infrequent lesions occupying the intracranial space, comprising approximately 1–2% of all intracranial tumors. Brainstem epidermoids are exceptionally uncommon in children; up until now, only a few scattered case reports have been documented in the literature regarding this unique location. These cysts commonly arise from the inclusion of ectodermal elements during neural tube closure. Complete excision of these cysts is challenging due to their close proximity and adherence to the brainstem, which makes it difficult to achieve. As a result, recurrence of the cysts is not uncommon. We have reported a rare case of a 3-year-old with a 5-month history of progressive headache, imbalance while walking and progressive weakness in his right upper limb and lower limb along with difficulty in swallowing. On MRI Brain imaging study he had a pre-pontine epidermoid with intra-axial extension in the pons. The patient underwent retro-sigmoid/suboccipital craniotomy and microsurgical excision of the tumor, including the tumor capsule. After completing the surgery, the cavity was irrigated using a solution containing hydrocortisone and Ringer lactate to prevent the occurrence of aseptic meningitis. In the postoperative, the patient recovered without any complications, as all symptoms showed immediate improvement, and the lower cranial nerves returned to normal functioning. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells (PSCs) are a unique cell type that can differentiate into all cell types in the body. In PSC culture, subpopulations with different levels of pluripotency may exist, which leads to different results during their differentiation. One of the key factors that determine the state of pluripotency and influence the differentiation potential of PSCs is the epigenetic state of cells, including the level of histone deacetylation. Activation of histone deacetylase (HDAC) in human and mouse PSCs increases the percentage of heterochromatin. In this work, we used a protocol for the differentiation of embryoid bodies from induced human pluripotent hiPSC cells designed for the formation of ectoderm and neuroectoderm with their subsequent development into skin organoids. However, after hiPSCs were exposed to HDAC inhibitors (sodium butyrate and valproic acid), the direction of their differentiation changed: mesoderm was formed, which subsequently developed into contracting cardiospheres. [ABSTRACT FROM AUTHOR]

Background: Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. Methods: Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. Results: We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. Conclusions: Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms. [ABSTRACT FROM AUTHOR]

Ovarian immature teratoma is a rare subtype of germ cell tumour that can be pure or associated with non-teratomatous germ cell tumour elements and is graded based on extent of the immature neuroectodermal component. Immature teratoma (IT) can also be associated with somatic differentiation in the form of sarcoma, carcinoma, or extensive immature neuroectodermal elements and may produce low levels of serum alpha-fetoprotein. Variable interpretation of these issues underlies diagnostic and management dilemmas, resulting in substantial practice differences between paediatric and adult women with IT. The Malignant Germ Cell International Consortium (MaGIC) convened oncologists, surgeons, and pathologists to address the following crucial clinicopathologic issues related to IT: (1) grading of IT, (2) definition and significance of 'microscopic' yolk sac tumour, (3) transformation to a somatic malignancy, and (4) interpretation of serum tumour biomarkers. This review highlights the discussion, conclusions, and suggested next steps from this clinicopathologic conference. • Diagnosis and grading (three- vs. two-tier) of immature teratoma (IT) remains challenging. • IT grade significantly affects treatment options. • Treatment options for IT differ between children and adults. • IT was discussed at a MaGIC-sponsored multidisciplinary conference. • This review presents conference discussion, conclusions, and proposed next steps. [ABSTRACT FROM AUTHOR]

Melanotic neuroectodermal tumor of infancy (MNTI) is a rare, rapidly growing, and pigmented neoplasm of neural crest origin. It predominantly affects the maxilla of infants during the first year of life. A seven-month-old boy presented with a mass approximately 5 cm in diameter in the right oral cavity. On computerized tomography, a lytic expansile lesion was detected in the right maxilla. Microscopically, the tumor consisted of two different neoplastic cell proliferation, located peripherally and centrally, arranged in alveolar clusters within the fibrous connective tissue. Immunohistochemically, peripheral tumor cells showed diffuse staining for Pancytokeratin and HMB-45; the central cells were positive for CD56 and Synaptophysin. MNTI is a rare tumor that can be easily confused with malign small round cell tumors, especially in small biopsies. It has characteristic histomorphological and immunohistochemical findings. Its biological behavior is not fully understood. These tumors can present locally aggressive behavior and a high recurrence rate. [ABSTRACT FROM AUTHOR]

Planar cell polarity (PCP) is the aligned cell polarity within a tissue plane. Mechanical signals are known to act as a global cue for PCP, yet their exact role is still unclear. In this study, we focused on PCP in the posterior neuroectoderm of Xenopus laevis and investigated how mechanical signals regulate polarity. We reveal that the neuroectoderm is under a greater tension in the anterior-posterior direction and that perturbation of this tension causes PCP disappearance. We show that application of uniaxial stretch to explant tissues can control the orientation of PCP and that cells sense the tissue stretch indirectly through a change in their shape, rather than directly through detection of anisotropic tension. Furthermore, we reveal that PCP is most strongly established when the orientation of tissue stretch coincides with that of diffusion of locally expressed Wnt ligands, suggesting a cooperative relationship between these two PCP regulators. [ABSTRACT FROM AUTHOR]

Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates. [Display omitted] • ESCs naturally differ in their mitochondrial ROS level • ESC fate decisions at the onset of gastrulation are governed by mitochondrial ROS • A high mitochondrial ROS level biases ESCs toward mesendodermal (ME) fates • ME specification is controlled by redox-sensitive ROS/p53-Wnt3/Nrf2-H3K4me3 axis How pluripotent embryonic stem cells (ESCs) choose distinct cell fates during embryonic development is still a mystery. Ulfig and Jakob now demonstrate that ESCs naturally differ in their mitochondrial reactive oxygen species levels that individualize their nuclear redox state, H3K4me3 landscape, and ultimately, cell fate. [ABSTRACT FROM AUTHOR]

The 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 use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3, also known as Fzd3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. This inhibition required Fz3-dependent Vangl2 phosphorylaton. Consistent with our observations, the complex of Pk3 with nonphosphorylatable Vangl2 did not polarize in the neural plate. These findings provide evidence for in vivo regulation of Vangl2-Pk3 complex formation and localization by a Frizzled receptor. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Embryonic differentiation at the gastrulation stage is a very tightly regulated process in which many long noncoding RNAs (lncRNAs) have been demonstrated to play a crucial role in gene expression modulation. lncRNAs can regulate cellular functions at the transcriptional, post-transcriptional, translational, and post-translational levels. lncRNAs exert a large repertoire of regulatory functions both in the nucleus and in the cytoplasm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these diseases are associated with heteroplasmic mutations in the mitochondrial DNA (mtDNA). The proportion of mutated mtDNA must exceed a critical threshold to produce disease. Therefore, understanding how embryonic development determines the heteroplasmy level in each tissue could explain the organ susceptibility and the clinical heterogeneity observed in these patients. In this report, the dynamics of heteroplasmy and the influence in cardiac commitment of the mutational load of the m.13513G>A mutation has been analyzed. This mutation has been reported as a frequent cause of Leigh syndrome (LS) and is commonly associated with cardiac problems. In this report, induced pluripotent stem cell (iPSc) technology has been used to delve into the molecular mechanisms underlying cardiac disease in LS. When mutation m.13513G>A is above a threshold, iPSc‐derived cardiomyocytes (iPSc‐CMs) could not be obtained due to an inefficient epithelial‐mesenchymal transition. Surprisingly, these cells are redirected toward neuroectodermal lineages that would give rise to the brain. However, when mutation is below that threshold, dysfunctional CM are generated in a mutant‐load dependent way. We suggest that distribution of the m.13513G>A mutation during cardiac differentiation is not at random. We propose a possible explanation of why neuropathology is a frequent feature of MD, but cardiac involvement is not always present. [ABSTRACT FROM AUTHOR]

F-53B, as an alternative to the persistent organic pollutant perfluorooctane sulfonate (PFOS), is amply used in the electric plating industry. F-53B and PFOS have similar physicochemical, biochemical and physiological properties, due to the similarity in their chemical structure. Thus, they may also possess similar toxicities. Although epidemiological studies and in vivo assays have shown that prenatal exposure to PFOS may impair the development of the nervous system, toxicity data for F-53B are still scarce. In this study, we employed an embryonic stem cell (ESC) in vitro differentiation system, to detect the potential developmental neural toxicity of F-53B and PFOS, at human exposure relevant doses. We demonstrated that during early mouse ESC (mESC) neural differentiation, F-53B and PFOS disrupted the expression of neural marker genes and affected the morphology of the differentiated cells. However, the very same treatments did not cause any cytotoxic effects. In conclusion, our ESC in vitro differentiation system was able to prove for the first time that F-53B and PFOS at human exposure relevant concentrations, could alter the expression of differentiation biomarkers, indicating a potential developmental neural toxicity. Based on our findings, it is reasonable to deduce that excessive exposure to F-53B and PFOS may cause severe dysfunctions during early stages of embryo development. [ABSTRACT FROM AUTHOR]

Characterizing endogenous protein expression, interaction and function, this study identifies in vivo interactions and competitive balance between N-cadherin and E-cadherin in developing avian ( Gallus gallus ) neural and neural crest cells. Numerous cadherin proteins, including neural cadherin (Ncad) and epithelial cadherin (Ecad), are expressed in the developing neural plate as well as in neural crest cells as they delaminate from the newly closed neural tube. To clarify independent or coordinate function during development, we examined their expression in the cranial region. The results revealed surprising overlap and distinct localization of Ecad and Ncad in the neural tube. Using a proximity ligation assay and co-immunoprecipitation, we found that Ncad and Ecad formed heterotypic complexes in the developing neural tube, and that modulation of Ncad levels led to reciprocal gain or reduction of Ecad protein, which then alters ectodermal cell fate. Here, we demonstrate that the balance of Ecad and Ncad is dependent upon the availability of β-catenin proteins, and that alteration of either classical cadherin modifies the proportions of the neural crest and neuroectodermal cells that are specified. [ABSTRACT FROM AUTHOR]

Transcription factor PRH/Hhex suppresses cell proliferation and contributes to regulation of prenatal and postnatal ontogeny. Neurons of the peripheral nervous system and chromaffin cells were previously considered as non-expressing PRH/Hhex in postnatal development. In our study, the expression of PRH/Hhex in chromaffin cells of rat adrenal glands and association between the decrease of proliferation and activation of PRH/Hhex expression were demonstrated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To observe simultaneous differentiation and analyse possible interactions between co‐cultured human oral mucosal stem cells (hOMSC) and mouse neural stem cells (mNSC). Materials and Methods: hOMSC and mNSC were co‐cultured in mouse and in human medium, and their immunocytochemical characterization to detect survival rate and differentiation pattern was performed. Co‐cultures in different media were compared to hOMSC in human medium and mNSC in mouse medium as controls. Results: Co‐culture of hOMSC and mNSC in medium for human cells led to normal differentiation pattern of human cells, while mNSC were directed towards astrocytes. When the same cells were cultivated in the mouse medium, both cell types succeeded to form neurons, although mNSC showed a tendency to overgrow hOMSC. hOMSC alone in the human‐specific medium differentiated towards ectodermal (Oct4, Map2) and mesodermal (Osterix) cell populations. mNSC in the mouse‐specific medium differentiated towards Map2‐, β3‐tubulin‐ and NeuN‐positive neurons. Conclusions: hOMSC and mNSC can form co‐cultures. Different media considerably affected the differentiation pattern of co‐cultures, whereas one cell population itself modestly influenced differentiation of the other cell type. The in vitro differentiation pattern of hOMSC in the mouse neural tissue environment suggested that hOMSC could be beneficial in the brain tissue affected by ischaemia. [ABSTRACT FROM AUTHOR]

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1-3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions. [ABSTRACT FROM AUTHOR]

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases. [ABSTRACT FROM AUTHOR]

Objective: Protein O-GlcNAcylation, a monosaccharide posttranslational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and OGlcNAcase (OGA), is a major nutrient sensor integrating key metabolic pathways. Recently, mutations in OGT have been genetically associated with X-linked intellectual disability (XLID). However, the underlying mechanisms remain unclear. This talk aims to summarize current genetic evidence for the involvement of OGT mutation in XLID, and showcase our recent findings with the Drosophila intellectual disability model to highlight the importance of a balanced OGlcNAcylation level for the development of the nervous system. Methods: We combined Live-imaging and genomics approaches to delineate the function of protein OGlcNAcylation during embryogenesis. Results: We develop a new fluorescent probe to visualize OGlcNAcylation levels in live Drosophila early embryos. Our study shows that protein O-GlcNAcylation declines as the embryos develop to the mid-blastula transition when the facultative heterochromatin makes its first appearance. Lowering O-GlcNAcylation levels by exogenous OGA activity promotes the polycomb group O-GlcNAc protein Polyhomeotic (Ph) to form nuclear foci and K27 trimethylation of histone H3. This enhanced facultative heterochromatin formation influences the expression of several neurodevelopmental genes including short of gastrulation (sog). Conclusion: We provide evidence that perturbation of OGlcNAcylation during early embryogenesis affects learning ability in adulthood, highlighting the importance of OGlcNAcylation homeostasis for the development of the nervous system. [ABSTRACT FROM AUTHOR]

Directed neural differentiation of human embryonic stem cells (ESCs) enables researchers to generate diverse neuronal populations for human neural development study and cell replacement therapy. To realize this potential, it is critical to precisely understand the role of various endogenous and exogenous factors involved in neural differentiation. Cell density, one of the endogenous factors, is involved in the differentiation of human ESCs. Seeding cell density can result in variable terminal cell densities or localized cell densities (LCDs), giving rise to various outcomes of differentiation. Thus, understanding how LCD determines the differentiation potential of human ESCs is important. The aim of this study is to highlight the role of LCD in the differentiation of H9 human ESCs into neuroectoderm (NE), the primordium of the nervous system. We found the initially seeded cells form derived cells with variable LCDs and subsequently affect the NE differentiation. Using a newly established method for the quantitative examination of LCD, we demonstrated that in the presence of induction medium supplemented with or without SMAD signaling blockers, high LCD promotes the differentiation of NE. Moreover, SMAD signaling blockade promotes the differentiation of NE but not non-NE germ layers, which is dependent on high LCDs. Taken together, this study highlights the need to develop innovative strategies or techniques based on LCDs for generating neural progenies from human ESCs. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) have been used extensively as in vitro models of neural development and disease, with special efforts towards their conversion into forebrain progenitors and neurons. The forebrain is the most complex brain region, giving rise to several fundamental structures, such as the cerebral cortex, the hypothalamus, and the retina. Due to the multiplicity of signaling pathways playing different roles at distinct times of embryonic development, the specification and patterning of forebrain has been difficult to study in vivo. Research performed on ESCs in vitro has provided a large body of evidence to complement work in model organisms, but these studies have often been focused more on cell type production than on cell fate regulation. In this review, we systematically reassess the current literature in the field of forebrain development in mouse and human ESCs with a focus on the molecular mechanisms of early cell fate decisions, taking into consideration the specific culture conditions, exogenous and endogenous molecular cues as described in the original studies. The resulting model of early forebrain induction and patterning provides a useful framework for further studies aimed at reconstructing forebrain development in vitro for basic research or therapy. [ABSTRACT FROM AUTHOR]

Several transcription factors ( TFs) have been implicated in neuroectoderm ( NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, micro RNA networks regulating human NE development have been poorly documented. We hypothesized that micro RNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells ( hESC). PAX6 binding to active enhancers was found in the proximity of several micro RNAs, including hsa-mi R-135b. Mi R-135b was activated during NE development, and ectopic expression of mi R-135b in hESC promoted differentiation toward NE. Mi R-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-mi RNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. [ABSTRACT FROM AUTHOR]

The goal of coaxial methods is to direct pluripotent stem cells toward specific phenotypes, limiting the appearance of tumorigenic or apoptotic cells. Beside culture medium components, other parameters, such as incubator atmosphere composition, temperature or pH of the culture medium, may have an influence on culture progression. In this study, the influence of different CO2 concentrations (1·5 and 5%) was analyzed in the commitment of D3 mouse embryonic stem cells to a neuroectodermal lineage. Real-time polymerase chain reaction (PCR) experiments showed that the expression level of three neuroectoderm marker genes (Neurofilament middle, Nestin and Acetylcholine esterase) was three times higher at 1·5% CO2 than at 5%, suggesting a favored commitment to neuroectoderm at low CO2 concentration. No change was observed in the expression of other markers such as Oct4, Nanog (pluripotency), Sox1 (neuroectoderm and gonads) and Pax6 (neuroectoderm and definitive endoderm) at the two CO2 conditions. Markers for primitive endoderm (Amnionless), mesendoderm (Brachyury) and germinal lineage (Vasa) were not detected. The percentage of cells in the G0 phase of cell cycle is slightly higher in cells growing at 1·5%CO2. Therefore, incubation in the presence of 1·5%CO2 seems to favor neuroectoderm commitment of mouse D3-cells modulating cell cycle progression. [ABSTRACT FROM AUTHOR]

Background: Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established. Methods: D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon+ cells. Results: Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon+ cells co-expressed neurofilament middle and some glucagon-like peptide-1+ cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1+ cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon+ cells did not display Ca2+ oscillations typical of pancreatic α-cells. Discussion: These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins. [ABSTRACT FROM AUTHOR]

Abstract: A suitable culture condition using advanced biomaterials has the potential to improve stem cell differentiation into selective lineages. In this study, we evaluated the effects of recombinant extracellular matrix (ECM) components on the mouse embryonic stem (mES) and induced pluripotent stem (miPS) cells'' self-renewal and differentiation into neural progenitors, comparing conventional culture substrata. The recombinant ECMs were established by immobilizing two chimera proteins of cadherin molecules, E-cadherin-Fc and N-cadherin-Fc, either alone or in combination. We report that the completely homogeneous population of mES and miPS cells could be maintained on E-cadherin-based substrata under feeder- and serum-free culture conditions to initiate neural differentiation. Using defined monolayer differentiation conditions on E-cadherin and N-cadherin (E-/N-cad-Fc) hybrid substratum, we routinely obtained highly homogeneous population of primitive ectoderm and neural progenitor cells. Moreover, the differentiated cells with higher expression of βIII-tubulin, Pax6, and tyrosine hydroxylase (TH) in absence of GFAP (a glial cell marker) expression suggesting the presence of a lineage restricted to neural cells. Our improved culture method should provide a homogeneous microenvironment for differentiation and obviate the need for protocols based on stromal feeders or embryoid bodies. [Copyright &y& Elsevier]

Abstract: The Drosophila neuroectoderm is initially subdivided into three longitudinal domains that give rise to columns of neuroblasts. This subdivision is coordinately accomplished by the action of the signaling pathways, Dorsal and Epidermal Growth Factor Receptor (EGFR), in conjunction with the homeodomain proteins, Ventral nervous system defective, Intermediate neuroblasts defective (Ind) and Muscle Segment Homeobox. We previously demonstrated that Ind expression is activated in response to the EGFR pathway. Here we show that EGF signaling subsequently mediates the direct phosphorylation of Ind by MAP kinase, which enhances the capacity of Ind to repress target genes, such as achaete. Specifically, we show that reduced EGF signaling results in diminished repression of achaete in the intermediate column, despite the presence of high levels of Ind protein. We also demonstrate that ectopic activation of MAP kinase results in the lateral expansion of the Ind expression domain with a corresponding reduction in achaete expression. This regulation is also dependent on the co-repressor, Dichaete. Our data indicate that EGF signaling, acting through MAP kinase, impinges on multiple aspects of Ind regulatory activity. While it has been often demonstrated that MAP kinase phosphorylation of transcriptional repressors attenuates their repressor activity, here we provide an example of phosphorylation enhancing repressor activity. [Copyright &y& Elsevier]

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency-associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by realtime gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct-4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct-4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines. [ABSTRACT FROM AUTHOR]

Abstract: Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-β signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis. [Copyright &y& Elsevier]

Abstract: Neuroectoderm development is a milestone of vertebrate neurogenesis. However, the molecular mechanism underlying the differentiation of neuroectoderm is still unclear, especially in mammals. ES cells co-cultured with PA6 cells can differentiate to neuroectoderm by the stromal cell-derived inducing activity method (SDIA method), but contamination of PA6 cells is an obstacle to the analysis of molecular mechanisms of differentiation. Here we describe a novel method by which differentiated ES cells are easily isolated from PA6 cells. We attempted to induce the differentiation of ES cells using paraformaldehyde-fixed PA6 cells. RT-PCR and DNA microarray analysis revealed that the background noise derived from contaminated PA6 cells disappeared when fixed PA6 cells were used. Furthermore, genes up-regulated during the differentiation of ES cells were expressed in a developing mouse embryo. Thus, our newly developed method will be very useful for identifying novel genes associated with mouse neuroectoderm development in vitro and in vivo. [Copyright &y& Elsevier]

Abstract: Embryonic stem cells differentiated on M15 cells have previously been shown to give rise to cells of the mesendodermal and definitive endodermal lineages. Here we demonstrate that neuroectodermal and mesodermal lineages can be derived from ES cells cultured on M15 cells and subsequently subjected to specific culture conditions, as confirmed by the expression of molecular markers. Prospective isolation and microarray analyses showed that neuroectodermal cells expressed anterior-to-posterior, as well as dorso-ventral regional markers, suggesting that this procedure could be used for the induction of cells belonging to a wide variety of neural lineages. Lateral mesoderm and paraxial mesoderm cells were also produced and their gene expression profiles were confirmed by microarray analyses. These results indicate that the M15 cell system provides a valuable tool for generating ES cell-derived lineage-specific cell types belonging to the three germ layers, namely neuroectoderm, mesoderm, and definitive endoderm. [Copyright &y& Elsevier]

Abstract: Chemokines regulate proliferation and migration of various types of normal stem and progenitor cells, including precursor cells of neuroectodermal origin. Based on this it is conceivable that the established role of chemokines in cancer cell proliferation and organ-specific metastasis might also be associated with stem cell-like cells present in the tumor. Such cancer stem cells (CSCs) represent a small subpopulation of tumor cells that are thought to initiate and sustain tumor formation. More recently, characteristics of stem cells have also been observed in metastatic cancer cells, and it has been suggested that CSCs might play a crucial role in the metastatic process as such. Intriguingly, first evidence has been provided that the metastatic spread of specific CSCs is driven by chemokine signaling. Thus it is possible that chemokine-mediated CSC regulation might be a general feature of metastasis formation. [Copyright &y& Elsevier]

Nervous system development takes place after positional information has been established along the dorsal-ventral (D/V) axis. The initial subdivision provided by a gradient of nuclear dorsal protein is maintained by the zygotic genes expressed along the D/V axis. In this study, an investigation was conducted to determine the range of Dpp function in repressing the expression of eagle (eg) that is present in intermediate neuroblasts defective (ind) and muscle specific homeobox (msh) gene domain, eg is expressed in neuroblast (NB) 2-4, 3-3 and 6-4 of the msh domain, and NB7-3 of the ind domain at the embryonic stage 11. In de-capentaplegic (dpp) loss-of-function mutant embryos, eg was ectopically expressed in the dorsal region, while in dpp gain-of-function mutants produced by sog or sca-GAL4AJAS-dpp, eg was repressed by Dpp. It is worthy of note that Dpp produced from sim;;dpp embryos showed that Dpp could function at long range. However, Dpp produced from en-GAL4AJAS-dpp or wg-GAL4/UAS-dpp primarily acted at short-range. This result demonstrated mat this discrepancy seems to be due to the repression of Dpp to EGFR signaling in sim;;dpp embryos. Taken together, these results suggest that Dpp signaling works at short-range, but can function indirectly at long-range by way of repression of EGFR signaling during embryonic neurogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Nodal, a member of the TGF-β family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403–421], a finding that seems paradoxical given Nodal''s central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of β-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of β-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells. [Copyright &y& Elsevier]

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein (BMP) signaling plays a major role in dorsoventral patterning in vertebrates and in Drosophila. Remarkably, in Tribolium, a beetle with an ancestral type of insect development, early BMP/dpp exhibits differential expression along the anteroposterior axis. However, the BMP/Dpp inhibitor Sog/chordin is expressed ventrally and establishes a dorsal domain of BMP/Dpp activity by transporting BMPs toward the dorsal side, like in Drosophila. Loss of Tribolium Sog not only abolishes dorsoventral polarity in the ectoderm, but also leads to the complete absence of the CNS. This phenotype suggests that sog is the main BMP antagonist in Tribolium, in contrast to vertebrates and Drosophila, which possess redundant antagonists. Surprisingly, Sog also is required for head formation in Tribolium, as are the BMP antagonists in vertebrates. Thus, in Tribolium, the system of BMP and its antagonists is less complex than in Drosophila or vertebrates and combines features from both, suggesting that it might represent an ancestral state. [ABSTRACT FROM AUTHOR]

Abstract: The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model. [Copyright &y& Elsevier]

Abstract: The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-β. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early–mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis. [Copyright &y& Elsevier]

Abstract: The hypocerebral organs of Euperipatoides kanangrensis are a pair of spherical vesicles located ventral to the cerebral ganglia. They develop in the embryo from the most anterior pair of ventral organs, in the antennal segment. The wall of each hypocerebral organ is a dense epithelium of elongate cells with peripheral nuclei. The cytoplasm of the cells includes numerous mitochondria, Golgi bodies and microtubules. The small lumen, located eccentrically within the organ, contains concentrically layered electron-dense material resembling cuticle. Each hypocerebral organ is enclosed by a layer of extracellular matrix continuous with that surrounding the adjacent cerebral ganglion. There are no nerve connections between ganglion and organ, but cellular connections traverse the intervening matrix and could serve as a communication pathway. The ultrastructure of the hypocerebral organs indicates that they are glands. [Copyright &y& Elsevier]

Abstract: Fate maps are essential to understand embryonic development; they provide a background for deducing maps of differential cellular specification in the context of other experimental data and molecular expression patterns. Due to its accessibility, the chick neural plate has been fate-mapped many times, albeit without complete agreement with respect to its shape, extent and fated subdivisions. In this review, we first comment about avian neural plate fate maps reported since the early period of experimental embryology, referring to the different methods followed. We next review a perfected fate-mapping methodology, which recently allowed us rather precise delimitation of the chick neural plate at stages 3d/4. This leads to a general discussion about the apparent border of the neural plate and the prospective main rostrocaudal and longitudinal divisions of the neural tube. [Copyright &y& Elsevier]

Abstract: From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face. [Copyright &y& Elsevier]

Abstract: Studies in amphibian embryos have suggested that retinoic acid (RA) may function as a signal that stimulates posterior differentiation of the nervous system as postulated by the activation-transformation model for anteroposterior patterning of the nervous system. We have tested this hypothesis in retinaldehyde dehydrogenase-2 (Raldh2) null mutant mice lacking RA synthesis in the somitic mesoderm. Raldh2-/- embryos exhibited neural induction (activation) as evidenced by expression of Sox1 and Sox2 along the neural plate, but differentiation of spinal cord neuroectodermal progenitor cells (posterior transformation) did not occur as demonstrated by a loss of Pax6 and Olig2 expression along the posterior neural plate. Spinal cord differentiation in Raldh2-/- embryos was rescued by maternal RA administration, and during the rescue RA was found to act directly in the neuroectoderm but not the somitic mesoderm. RA generated by Raldh2 in the somitic mesoderm was found to normally travel as a signal throughout the mesoderm and neuroectoderm of the trunk and into tailbud neuroectoderm, but not into tailbud mesoderm. Raldh2-/- embryos also exhibited increased Fgf8 expression in the tailbud, and decreased cell proliferation in tailbud neuroectoderm. Our findings demonstrate that RA synthesized in the somitic mesoderm is necessary for posterior neural transformation in the mouse and that Raldh2 provides the only source of RA for posterior development. An important concept to emerge from our studies is that the somitic mesodermal RA signal acts in the neuroectoderm but not mesoderm to generate a spinal cord fate. [Copyright &y& Elsevier]

Abstract: Krox-20, originally identified as a member of “immediate-early” genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. Here we cloned a genomic sequence of Xenopus Krox-20 (XKrox-20) and studied functions of a promoter element in the flanking sequence and associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, we showed that the 5′ flanking sequence was sufficient to induce luciferase activities when the reporter construct was injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5′ flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (−72 to −51 from the transcription initiation site), both of which were essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bound to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts were serum response factor (SRF) and a member of CRE binding protein (CREB) family proteins that bound the CArG-box and CRE, respectively. They appeared to assemble on the minimal promoter element to produce a novel ternary complex. When we injected mRNA of a dominant-negative version of Xenopus SRF (XSRFΔC) into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity was strongly suppressed. Suppression by XSRFΔC was counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element. [Copyright &y& Elsevier]

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-β) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development. [Copyright &y& Elsevier]

Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Here, we demonstrate that leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural proper. ties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (Fgfs) play important roles in the pattern formation of early vertebrate embryos. We have identified a zebrafish ortholog of human FGF17, named fgf17b. The first phase of fgf17b expression occurs in the blastodermal margin of late blastulae and in the embryonic shield of early gastrulae. The second phase starts after the onset of segmentation, mainly in the presomitic mesoderm and newly formed somites. Injection of fgf17b mRNA into one-cell embryos induces expression of the mesodermal marker no tail (ntl) and rescues ntl expression suppressed by overexpression of lefty1 (lft1). Overexpression of fgf17b dorsalizes zebrafish gastrulae by enhancing expression of chordin (chd), which is an antagonist of the ventralizing signals BMPs. In addition, overexpression of fgf17b posteriorizes the neuroectoderm. Simultaneous knockdown of fgf17b and fgf8 with antisense morpholinos results in reduction of chd and ntl. Knockdown of fgf17b can alleviate inhibitory effect of ectopic expression of fgf3 on otx1. These data together suggest that Fgf17b plays a role in early embryonic patterning. We also demonstrate that fgf17b and fgf8 have stronger mesoderm inducting activity than fgf3, whereas fgf17b and fgf3 have stronger activity in posteriorizing the neuroectoderm than fgf8. Like fgf8, activation of fgf17b expression depends on Nodal signaling. [Copyright &y& Elsevier]

sax1/nkx1.2 and sax2/nkx1.1 are members of the evolutionally conserved NK-1 homeobox gene family. sax1/nkx1.2 is reported to be expressed in the central nervous system during early and late neurogenesis in the chick and mouse, but the expression of sax2/nkx1.1 has not been reported. We isolated zebrafish cDNAs for sax1/nkx1.2 and sax2/nkx1.1 and examined their expression. In zebrafish, unlike chick and mouse, sax1/nkx1.2 was expressed in the prospective medial floor plate from the mid-gastrula period and was dependent on Nodal signaling. From the early segmentation period, sax1/nkx1.2 was also expressed in the posterior neuroectoderm. sax2/nkx1.1 was expressed in the prospective extraocular muscles, mesencephalic neurons residing along the tract of the posterior commissure, ventral neurons in the hindbrain, and interneurons in the spinal cord. [Copyright &y& Elsevier]