Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis.

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.

A regulatory sub-circuit downstream of Wnt signaling controls developmental transitions in neural crest formation.

The process of cell fate commitment involves sequential changes in the gene expression profiles of embryonic progenitors. This is exemplified in the development of the neural crest, a migratory stem cell population derived from the ectoderm of vertebrate embryos. During neural crest formation, cells transition through distinct transcriptional states in a stepwise manner. The mechanisms underpinning these shifts in cell identity are still poorly understood. Here we employ enhancer analysis to identify a genetic sub-circuit that controls developmental transitions in the nascent neural crest. This sub-circuit links Wnt target genes in an incoherent feedforward loop that controls the sequential activation of genes in the neural crest lineage. By examining the cis-regulatory apparatus of Wnt effector gene AXUD1, we found that multipotency factor SP5 directly promotes neural plate border identity, while inhibiting premature expression of specification genes. Our results highlight the importance of repressive interactions in the neural crest gene regulatory network and illustrate how genes activated by the same upstream signal become temporally segregated during progressive fate restriction.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

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

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm.

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

The histone methyltransferase KMT2D, mutated in Kabuki syndrome patients, is required for neural crest cell formation and migration.

Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.

Ocular Neural Heterotopia in a Moroccan Uromastyx (Uromastyx acanthinurus nigriventris).

A juvenile female Moroccan uromastyx (Uromastyx acanthinurus nigriventris) that died unexpectedly was necropsied. Necropsy examination revealed minimal intracoelomic fat, small numbers of intestinal nematodes and intraocular masses within the vitreous chamber of both eyes. One of the intraocular masses was focally contiguous with the optic nerve and composed of neuroparenchyma with rare glial cells, consistent with a diagnosis of neural heterotopia. This condition is considered a neuroectodermal malformation, readily recognized in human medicine but rarely reported in animals. To the authors' knowledge, this is the first case of intraocular neural heterotopia reported in a reptile.

Znf703 is a novel RA target in the neural plate border.

Znf703 is an RAR- and Wnt-inducible transcription factor that exhibits a complex expression pattern in the developing embryo: Znf703 mRNA is found in the early circumblastoporal ring, then later throughout the neural plate and its border, and subsequently in the mid/hindbrain and somites. We show that Znf703 has a different and separable function in early mesoderm versus neural crest and placode development. Independent of its early knockdown phenotype on Gdf3 and Wnt8, Znf703 disrupts patterning of distinct neural crest migratory streams normally delineated by Sox10, Twist, and Foxd3 and inhibits otocyst formation and otic expression of Sox10 and Eya1. Furthermore, Znf703 promotes massive overgrowth of SOX2+ cells, disrupting the SoxB1 balance at the neural plate border. Despite prominent expression in other neural plate border-derived cranial and sensory domains, Znf703 is selectively absent from the otocyst, suggesting that Znf703 must be specifically cleared or down-regulated for proper otic development. We show that mutation of the putative Groucho-repression domain does not ameliorate Znf703 effects on mesoderm, neural crest, and placodes. We instead provide evidence that Znf703 requires the Buttonhead domain for transcriptional repression.

The mutation m.13513G>A impairs cardiac function, favoring a neuroectoderm commitment, in a mutant-load dependent way.

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.

Spatial and temporal analysis of PCP protein dynamics during neural tube closure.

Planar cell polarity (PCP) controls convergent extension and axis elongation in all vertebrates. Although asymmetric localization of PCP proteins is central to their function, we understand little about PCP protein localization during convergent extension. Here, we use quantitative live imaging to simultaneously monitor cell intercalation behaviors and PCP protein dynamics in the Xenopus laevis neural plate epithelium. We observed asymmetric enrichment of PCP proteins, but more interestingly, we observed tight correlation of PCP protein enrichment with actomyosin-driven contractile behavior of cell-cell junctions. Moreover, we found that the turnover rates of junctional PCP proteins also correlated with the contractile behavior of individual junctions. All these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation in the closing vertebrate neural tube.

A catenin-dependent balance between N-cadherin and E-cadherin controls neuroectodermal cell fate choices.

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 ß-catenin proteins, and that alteration of either classical cadherin modifies the proportions of the neural crest and neuroectodermal cells that are specified.

NMDA Receptor Signaling Is Important for Neural Tube Formation and for Preventing Antiepileptic Drug-Induced Neural Tube Defects.

Failure of neural tube closure leads to neural tube defects (NTDs), which can have serious neurological consequences or be lethal. Use of antiepileptic drugs (AEDs) during pregnancy increases the incidence of NTDs in offspring by unknown mechanisms. Here we show that during Xenopus laevis neural tube formation, neural plate cells exhibit spontaneous calcium dynamics that are partially mediated by glutamate signaling. We demonstrate that NMDA receptors are important for the formation of the neural tube and that the loss of their function induces an increase in neural plate cell proliferation and impairs neural cell migration, which result in NTDs. We present evidence that the AED valproic acid perturbs glutamate signaling, leading to NTDs that are rescued with varied efficacy by preventing DNA synthesis, activating NMDA receptors, or recruiting the NMDA receptor target ERK1/2. These findings may prompt mechanistic identification of AEDs that do not interfere with neural tube formation.SIGNIFICANCE STATEMENT Neural tube defects are one of the most common birth defects. Clinical investigations have determined that the use of antiepileptic drugs during pregnancy increases the incidence of these defects in the offspring by unknown mechanisms. This study discovers that glutamate signaling regulates neural plate cell proliferation and oriented migration and is necessary for neural tube formation. We demonstrate that the widely used antiepileptic drug valproic acid interferes with glutamate signaling and consequently induces neural tube defects, challenging the current hypotheses arguing that they are side effects of this antiepileptic drug that cause the increased incidence of these defects. Understanding the mechanisms of neurotransmitter signaling during neural tube formation may contribute to the identification and development of antiepileptic drugs that are safer during pregnancy.

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity.

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.

A Nonredundant Role for the TRPM6 Channel in Neural Tube Closure.

In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6's close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.

Xenopus as a model organism to study heterotrimeric G-protein pathway during collective cell migration of neural crest.

Collective cell migration is essential in many fundamental aspects of normal development, like morphogenesis, organ formation, wound healing, and immune responses, as well as in the etiology of severe pathologies, like cancer metastasis. In spite of the huge amount of data accumulated on cell migration, such a complex process involves many molecular actors, some of which still remain to be functionally characterized. One of these signals is the heterotrimeric G-protein pathway that has been studied mainly in gastrulation movements. Recently we have reported that Ric-8A, a GEF for Gα proteins, plays an important role in neural crest migration in Xenopus development. Xenopus neural crest cells, a highly migratory embryonic cell population induced at the border of the neural plate that migrates extensively in order to differentiate in other tissues during development, have become a good model to understand the dynamics that regulate cell migration. In this review, we aim to provide sufficient evidence supporting how useful Xenopus model with its different tools, such as explants and transplants, paired with improved in vivo imaging techniques, will allow us to tackle the multiple signaling mechanisms involved in neural crest cell migration.

Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1.

Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment.

Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm.

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc.

IGF-2/IGF-1R signaling has distinct effects on Sox1, Irx3, and Six3 expressions during ES cell derived-neuroectoderm development in vitro.

Insulin-like growth factors (IGFs) are involved in growth and tissue development, including diseases such as type-2 diabetes and cancers. However, their roles in lineage specification, especially in early mammalian neural development, are poorly understood. Here, we analyzed the protein expression of IGF-2 in early mouse embryo, and it was preferentially detected in anterior mesodermal tissue, adjacent to the neural plate. We utilized a self-organizing neural tissue culture system and analyzed the direct effect of IGF-2 on the general neural marker Sox1. Interestingly, using recombinant IGF-2 and a chemical inhibitor of its receptor (IGF-1R), we found that the IGF-2/IGF-1R pathway positively regulated Sox1 expression in embryonic stem (ES) cell-derived neural tissue. Furthermore, to visualize the expression patterns of other neural markers, we used reporter ES cell lines and we found that the IGF-2/IGF-1R signaling upregulated the expression of the posterior neural marker Irx3. In contrast, the anterior neural marker Six3 was downregulated by IGF-2/IGF-1R signaling. Together, our results demonstrate that IGF-2/IGF-1R signaling has different effects on neural marker expression, which may influence the early regional identity of ES cell-derived neural tissues.

Chondroitin sulfate effects on neural stem cell differentiation.

We have investigated the role chondroitin sulfate has on cell interactions during neural plate formation in the early chick embryo. Using tissue culture isolates from the prospective neural plate, we have measured neural gene expression profiles associated with neural stem cell differentiation. Removal of chondroitin sulfate from stage 4 neural plate tissue leads to altered associations of N-cadherin-positive neural progenitors and causes changes in the normal sequence of neural marker gene expression. Absence of chondroitin sulfate in the neural plate leads to reduced Sox2 expression and is accompanied by an increase in the expression of anterior markers of neural regionalization. Results obtained in this study suggest that the presence of chondroitin sulfate in the anterior chick embryo is instrumental in maintaining cells in the neural precursor state.

Genetic, epigenetic, and environmental contributions to neural tube closure.

The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences.

Morphogenetic movements in the neural plate and neural tube: mouse.

The neural tube (NT), the embryonic precursor of the vertebrate brain and spinal cord, is generated by a complex and highly dynamic morphological process. In mammals, the initially flat neural plate bends and lifts bilaterally to generate the neural folds followed by fusion of the folds at the midline during the process of neural tube closure (NTC). Failures in any step of this process can lead to neural tube defects (NTDs), a common class of birth defects that occur in approximately 1 in 1000 live births. These severe birth abnormalities include spina bifida, a failure of closure at the spinal level; craniorachischisis, a failure of NTC along the entire body axis; and exencephaly, a failure of the cranial neural folds to close which leads to degeneration of the exposed brain tissue termed anencephaly. The mouse embryo presents excellent opportunities to explore the genetic basis of NTC in mammals; however, its in utero development has also presented great challenges in generating a deeper understanding of how gene function regulates the cell and tissue behaviors that drive this highly dynamic process. Recent technological advances are now allowing researchers to address these questions through visualization of NTC dynamics in the mouse embryo in real time, thus offering new insights into the morphogenesis of mammalian NTC.