Neural Plate Stage Research Articles

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Single Cell transcriptomes highlight the temporal hierarchy of fate decisions of the developmental tree from Neural Border Stages to Migration of NC Cells

Gene regulatory networks govern the steps of embryonic development and control cell transcriptional states. The neural crest cells are an archetypal example of cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices governing the emergence of neural crest heterogeneity from the ectoderm state remains elusive. Here we combine ultra‐dense single cell transcriptomes with machine‐learning strategies and experimental validation to provide a comprehensive gene regulatory network driving vertebrate neural crest fate diversification from induction to early migration stages. Transcription factor connectome and bifurcation analyses discover that several early neural crest fates emerge at the neural plate stage, alongside an unbiased multipotent neural crest lineage persisting until after epithelial‐mesenchymal transition. We also define a new and transient neural border zone state, preceding choice between neural crest and placodes during gastrulation. Our comprehensive experimental approach combined to several ML strategies broadly applicable to single cell transcriptomics deciphers the circuits driving cranial and vagal neural crest formation and provides a general model for investigating vertebrate GRNs in development, evolution and disease

Repressive Interactions Between Transcription Factors Separate Different Embryonic Ectodermal Domains.

The embryonic ectoderm is composed of four domains: neural plate, neural crest, pre-placodal region (PPR) and epidermis. Their formation is initiated during early gastrulation by dorsal-ventral and anterior-posterior gradients of signaling factors that first divide the embryonic ectoderm into neural and non-neural domains. Next, the neural crest and PPR domains arise, either via differential competence of the neural and non-neural ectoderm (binary competence model) or via interactions between the neural and non-neural ectoderm tissues to produce an intermediate neural border zone (NB) (border state model) that subsequently separates into neural crest and PPR. Many previous gain- and loss-of-function experiments demonstrate that numerous TFs are expressed in initially overlapping zones that gradually resolve into patterns that by late neurula stages are characteristic of each of the four domains. Several of these studies suggested that this is accomplished by a combination of repressive TF interactions and competence to respond to local signals. In this study, we ectopically expressed TFs that at neural plate stages are characteristic of one domain in a different domain to test whether they act cell autonomously as repressors. We found that almost all tested TFs caused reduced expression of the other TFs. At gastrulation these effects were strictly within the lineage-labeled cells, indicating that the effects were cell autonomous, i.e., due to TF interactions within individual cells. Analysis of previously published single cell RNAseq datasets showed that at the end of gastrulation, and continuing to neural tube closure stages, many ectodermal cells express TFs characteristic of more than one neural plate stage domain, indicating that different TFs have the opportunity to interact within the same cell. At neurula stages repression was observed both in the lineage-labeled cells and in adjacent cells not bearing detectable lineage label, suggesting that cell-to-cell signaling has begun to contribute to the separation of the domains. Together, these observations directly demonstrate previous suggestions in the literature that the segregation of embryonic ectodermal domains initially involves cell autonomous, repressive TF interactions within an individual cell followed by the subsequent advent of non-cell autonomous signaling to neighbors.

Eya1 protein distribution during embryonic development of Xenopus laevis

Eya1 and other Eya proteins are important regulators of progenitor proliferation, cell differentiation and morphogenesis in all three germ layers. At present, most of our knowledge of Eya1 distribution is based on in situ hybridization for Eya1 mRNA. However, to begin to dissect the mechanisms underlying Eya1 functions, we need a better understanding of the spatiotemporal distribution of Eya1 proteins during embryonic development, their subcellular localization and their levels of expression in various tissues. Here we report the localization of Eya1 protein throughout embryonic development from neural plate stages to tadpole stages of Xenopus laevis using a specific antibody for Xenopus Eya1. Our study confirms the expression of Eya1 protein in cranial placodes, placodally derived sensory primordia (olfactory epithelium, otic vesicle, lateral line primordia) and cranial ganglia, as well as in somites, secondary heart field and pharyngeal endoderm. In addition, we report here a novel expression of Eya1 proteins in scattered epidermal cells in Xenopus. Our findings also reveal that, while being predominantly expressed in nuclei in most expression domains, Eya1 protein is also localized to the cytoplasm, in particular in the early preplacodal ectoderm, some placode-derived ganglia and a subset of epidermal cells. While some cytoplasmic roles of Eya1 have been previously described in other contexts, the functions of cytoplasmic Eya1 in the preplacodal ectoderm, cranial ganglia and epidermal cells remain to be investigated.

Sobp modulates the transcriptional activation of Six1 target genes and is required during craniofacial development.

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR.

Zmym2 and Zmym4 Act as Co‐Factors of Six1 During Craniofacial Development

Branchio-oto-renal syndrome (BOR) is an autosomal dominant condition that presents with variable cranial, otic, and renal malformations. However, only half of BOR cases have a known genetic cause: mutations in the genes encoding Six1 and its co-factor Eya1. Therefore, it is important to identify other proteins that may interact with either Six1 or Eya1 that could be involved in the craniofacial phenotypes involved in BOR patients. In a screen for potential new cofactors, we identified Zinc Finger MYM-containing 2 (Zmym2) and Zmym4, whose expression overlaps with Six1 the pre-placodal ectoderm and otic vesicle; they also are expressed in the cranial neural crest and branchial arches. Knockdown of Zmym2 or Zmym4 resulted in a broader but less intense expression of msx1, pax3 and tfap2 in the neural border zone at neural plate stages, and expanded neural crest (foxD3, sox9) and placode (six1) genes at neurula stages. At larval stages, dlx5 expression in the neural crest and otic vesicles were reduced. Luciferase assays demonstrate that Zmym2 reduces Six1-Eya1 transcriptional activation whereas Zmym4 enhances it. These data indicate that Zmym2 and Zmym4 serve an important role in the development of craniofacial tissue, including the otic vesicle, and suggest they may have a role in BOR.

Sobp is a novel Six1 co‐factor during inner ear development

According to the CDC, the prevalence of hearing loss is 1.4 per 1000 babies screened in 2009 in the USA. Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by hearing loss, branchial fistulas/cysts, and renal anomalies in about a third of patients. Mutations in the SIX1 gene and the SIX1 co-factor EYA1 gene are present in half of BOR patients. We are using Xenopus laevis and mouse as models to discover novel genes involved in this syndrome. Based on a screen in fly of proteins shown to interact with So (Drosophila Six1 homologue), sobp was identified as potential Six1 co-factor. Expression data show that this gene is expressed in patterns that overlap with six1 during craniofacial development. Co-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes with Six1 in the cell nucleus. Additionally, Six1 is able to bind and translocate to the cell nucleus an Sobp construct lacking its nuclear localization signal further demonstrating interaction. Luciferase assays to test if this factor is able to modify Six1 function show that Sobp represses Six1+Eya1 transcriptional activity. Knockdown experiments using morpholino antisense oligonucleotides and CRISPR/Cas9 followed by ISH and qPCR analyses of embryos at late neural plate stages show that Sobp is required for neural crest (foxd3) and placode (six1) formation. These changes are accompanied by an expansion of the neural plate (sox2) and reduction of the embryonic epidermis (krt12.4). Sobp overexpression confirmed that Sobp is required for neural crest (foxd3) and placode (six1) formation; changes in those two genes were accompanied by a slight expansion of the krt12.4-expressing domain. With further development, Sobp knockdown and overexpression disrupt otocyst development shown by disrupted expression of dlx5, pax2, irx1 and sox9 at tailbud stages, and cause severe craniofacial defects in the tadpole. These results indicate that Sobp is required for proper craniofacial development, and that it modifies Six1 function, suggesting this protein may be a potential candidate gene for BOR and other craniofacial defects.

A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate Stages Following In Situ Hybridization.

The technique of in situ hybridization can be used to visualize the spatial and temporal pattern of gene expression during development. Ascidians are invertebrate chordates that develop with a fixed cell cleavage patterninto a tadpole larvae. The knowledge of the cell lineage allows the earliest steps of cell fate specification to be followed at a single cell resolution. This protocol describes preparation of Ciona intestinalis embryos, classical in situ hybridization protocol coupled with nuclear staining, and a guide to identify gene expression in specific precursors of the developing brain at neural plate stages of development.

Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner.

Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.

Long-term self-renewal of naïve neural stem cells in a defined condition

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

Ketamine Modulates Zic5 Expression via the Notch Signaling Pathway in Neural Crest Induction.

Ketamine is a potent dissociative anesthetic and the most commonly used illicit drug. Many addicts are women at childbearing age. Although ketamine has been extensively studied as a clinical anesthetic, its effects on embryonic development are poorly understood. Here, we applied the Xenopus model to study the effects of ketamine on development. We found that exposure to ketamine from pre-gastrulation (stage 7) to early neural plate (stage 13.5) resulted in disruption of neural crest (NC) derivatives. Ketamine exposure did not affect mesoderm development as indicated by the normal expression of Chordin, Xbra, Wnt8, and Fgf8. However, ketamine treatment significantly inhibited Zic5 and Slug expression at early neural plate stage. Overexpression of Zic5 rescued ketamine-induced Slug inhibition, suggesting the blockage of NC induction was mediated by Zic5. Furthermore, we found Notch signaling was altered by ketamine. Ketamine inhibited the expression of Notch targeted genes including Hes5.2a, Hes5.2b, and ESR1 and ketamine-treated embryos exhibited Notch-deficient somite phenotypes. A 15 bp core binding element upstream of Zic5 was induced by Notch signaling and caused transcriptional activation. These results demonstrated that Zic5 works as a downstream target gene of Notch signaling in Xenopus NC induction. Our study provides a novel teratogenic mechanism whereby ketamine disrupts NC induction via targeting a Notch-Zic5 signaling pathway.

Developmental evolution of the forebrain in cavefish, from natural variations in neuropeptides to behavior.

The fish Astyanax mexicanus comes in two forms: the normal surface-dwelling and the blind depigmented cave-adapted morphs. Comparing the development of their basal forebrain, we found quantitative differences in numbers of cells in specific clusters for six out of nine studied neuropeptidergic cell types. Investigating the origins of these differences, we showed that early Shh and Fgf signaling impact on the development of NPY and Hypocretin clusters, via effect on Lhx7 and Lhx9 transcription factors, respectively. Finally, we demonstrated that such neurodevelopmental evolution underlies behavioral evolution, linking a higher number of Hypocretin cells with hyperactivity in cavefish. Early embryonic modifications in signaling/patterning at neural plate stage therefore impact neuronal development and later larval behavior, bridging developmental evolution of a neuronal system and the adaptive behavior it governs. This work uncovers novel variations underlying the evolution and adaptation of cavefish to their extreme environment.

Brain maps 4.0-Structure of the rat brain: An open access atlas with global nervous system nomenclature ontology and flatmaps.

The fourth edition (following editions in 1992, 1998, 2004) of Brain maps: structure of the rat brain is presented here as an open access internet resource for the neuroscience community. One new feature is a set of 10 hierarchical nomenclature tables that define and describe all parts of the rat nervous system within the framework of a strictly topographic system devised previously for the human nervous system. These tables constitute a global ontology for knowledge management systems dealing with neural circuitry. A second new feature is an aligned atlas of bilateral flatmaps illustrating rat nervous system development from the neural plate stage to the adult stage, where most gray matter regions, white matter tracts, ganglia, and nerves listed in the nomenclature tables are illustrated schematically. These flatmaps are convenient for future development of online applications analogous to “Google Maps” for systems neuroscience. The third new feature is a completely revised Atlas of the rat brain in spatially aligned transverse sections that can serve as a framework for 3‐D modeling. Atlas parcellation is little changed from the preceding edition, but the nomenclature for rat is now aligned with an emerging panmammalian neuroanatomical nomenclature. All figures are presented in Adobe Illustrator vector graphics format that can be manipulated, modified, and resized as desired, and freely used with a Creative Commons license.

Early embryonic development and spatiotemporal localization of mammalian primordial germ cell-associated proteins in the basal rodent Lagostomus maximus

The gene network controlling primordial germ cell (PGC) specification in eutherian mammals has been exhaustively investigated in mice. The egg-cylinder morphology of the mouse embryo is the key event enabling inductive signals from the extra-embryonic ectoderm (ExE) to specify epiblast cells as PGCs early on. We investigated the embryonic development and the spatiotemporal localization of PGC-associated proteins in the basal Hystricognathi rodent Lagostomus maximus. L. maximus develops through a flat-disc epiblast far apart from the ExE. In the primitive streak stage, OCT4-positive cells are detected in the posterior pole of the embryo disc in the mesoderm of the proximal epiblast. In the neural plate stage, a reduced 8 to 12 OCT4-positive cell population transiently expresses FRAGILIS, STELLA and SOX17 in the posterior streak. Soon after translocation to the hindgut, pluripotent OCT4 cells start expressing VASA, and then, STELLA and FRAGILIS are turned on during migration toward the genital ridge. L. maximus shows a spatiotemporal pattern of PGC-associated markers divergent from the early PGC restriction model seen in mice. This pattern conforms to alternative models that are based on a pluripotent population in the embryonic axis, where PGCs are specified later during development.

PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate n-cadherin and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

Sensory evolution in blind cavefish is driven by early embryonic events during gastrulation and neurulation.

Natural variations in sensory systems constitute adaptive responses to the environment. Here, we compared sensory placode development in the blind cave-adapted morph and the eyed river-dwelling morph of Astyanax mexicanus Focusing on the lens and olfactory placodes, we found a trade-off between these two sensory components in the two morphs: from neural plate stage onwards, cavefish have larger olfactory placodes and smaller lens placodes. In a search for developmental mechanisms underlying cavefish sensory evolution, we analyzed the roles of Shh, Fgf8 and Bmp4 signaling, which are known to be fundamental in patterning the vertebrate head and are subtly modulated in space and time during cavefish embryogenesis. Modulating these signaling systems at the end of gastrulation shifted the balance toward a larger olfactory derivative. Olfactory tests to assess potential behavioral outcomes of such developmental evolution revealed that Astyanax cavefish are able to respond to a 105-fold lower concentration of amino acids than their surface-dwelling counterparts. We suggest that similar evolutionary developmental mechanisms may be used throughout vertebrates to drive adaptive sensory specializations according to lifestyle and habitat.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

Functional Cloning Using a <em>Xenopus</em> Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.