The patterning of the CNS relies on the interaction of multiple signaling molecules such as Sonic Hedgehog, Wnts, and BMPs and their antagonists Chordin and Noggin. The identification of the secreted molecule Tiarin (Tsuda et al., 2002, this issue of Neuron), produced by the nonneural ectoderm at border of the anterior and lateral neural plate, now introduces a novel signaling pathway participating in CNS development.

We have investigated the lineage and tissue interactions that result in avian neural crest cell formation from the ectoderm. Presumptive neural plate was grafted adjacent to non-neural ectoderm in whole embryo culture to examine the role of tissue interactions in ontogeny of the neural crest. Our results show that juxtaposition of non-neural ectoderm and presumptive neural plate induces the formation of neural crest cells. Quail/chick recombinations demonstrate that both the prospective neural plate and the prospective epidermis can contribute to the neural crest. When similar neural plate/epidermal confrontations are performed in tissue culture to look at the formation of neural crest derivatives, juxtaposition of epidermis with either early (stages 4-5) or later (stages 6-10) neural plate results in the generation of both melanocytes and sympathoadrenal cells. Interestingly, neural plates isolated from early stages form no neural crest cells, whereas those isolated later give rise to melanocytes but not crest-derived sympathoadrenal cells. Single cell lineage analysis was performed to determine the time at which the neural crest lineage diverges from the epidermal lineage and to elucidate the timing of neural plate/epidermis interactions during normal development. Our results from stage 8 to 10+ embryos show that the neural plate/neural crest lineage segregates from the epidermis around the time of neural tube closure, suggesting that neural induction is still underway at open neural plate stages.

In vertebrates, the neural plate border (NPB) is established by a group of transcription factors including Dlx3, Msx1 and Zic1. The crosstalk between these NPB specifiers governs the separation of the NPB region into placode and neural crest (NC) territories and also their further differentiation. Understanding the mechanisms of NPB formation and NC development is critical for our knowledge of related human diseases. Here we identified Nkx6.3, a transcription factor of the Nkx family, as a new NPB specifier required for neural crest development in Xenopus embryos. XNkx6.3 is expressed in the ectoderm of the neural plate border region at neurula stages, covering the epidermis, placode and neural crest territories, but not the neural plate. Inhibition of Nkx6.3 by dominant negative construct or specific morpholino leads to neural crest defects, while overexpression of Nkx6.3 induces ectopic neural crest in the anterior neural fold. In animal caps, Nkx6.3 alone is able to initiate the whole neural crest regulatory network and induces neural crest fate robustly. We showed that overexpression of Nkx6.3 affects multiple signaling pathways, creating a high-Wnt, low-BMP environment required for neural crest development. Gain- and loss-of-function of Nkx6.3 have compound effects on the expression of known NPB genes, which is largely opposite to that of Dlx3. Overexpression of Dlx3 blocks the NC inducing activity of Nkx6.3. The crosstalk between Nkx6.3, Dlx3 and Msx1 is likely crucial for proper NPB formation and neural crest development in Xenopus.

The COMMD (copper metabolism MURR1 domain containing) family includes ten structurally conserved proteins (COMMD1 to COMMD10) in eukaryotic multicellular organisms that are involved in a diverse array of cellular and physiological processes, including endosomal trafficking, copper homeostasis, and cholesterol metabolism, among others. To understand the role of COMMD10 in embryonic development, we used Commd10Tg(Vav1-icre)A2Kio/J mice, where the Vav1-cre transgene is integrated into an intron of the Commd10 gene, creating a functional knockout of Commd10 in homozygous mice. Breeding heterozygous mice produced no COMMD10-deficient (Commd10Null) offspring, suggesting that COMMD10 is required for embryogenesis. Analysis of Commd10Null embryos demonstrated that they displayed stalled development by embryonic day 8.5 (E8.5). Transcriptome analysis revealed that numerous neural crest-specific gene markers had lower expression in mutant versus wild-type (WT) embryos. Specifically, Commd10Null embryos displayed significantly lower expression levels of a number of transcription factors, including a major regulator of the neural crest, Sox10. Moreover, several cytokines/growth factors involved in early embryonic neurogenesis were also lower in mutant embryos. On the other hand, Commd10Null embryos demonstrated higher expression of genes involved in tissue remodeling and regression processes. Taken together, our findings show that Commd10Null embryos die by day E8.5 due to COMMD10-dependent neural crest failure, revealing a new and critical role for COMMD10 in neural development.

Vertebrate neural tube closure is associated with complex changes in cell shape and behavior, however, the relative contribution of these processes to tissue folding is not well understood. At the onset of Xenopus neural tube folding, we observed alternation of apically constricted and apically expanded cells. This apical domain heterogeneity was accompanied by biased cell orientation along the anteroposterior axis, especially at neural plate hinges, and required planar cell polarity signaling. Vertex models suggested that dispersed isotropically constricting cells can cause the elongation of adjacent cells. Consistently, in ectoderm, cell-autonomous apical constriction was accompanied by neighbor expansion. Thus, a subset of isotropically constricting cells may initiate neural plate bending, whereas a 'tug-of-war' contest between the force-generating and responding cells reduces its shrinking along the body axis. This mechanism is an alternative to anisotropic shrinking of cell junctions that are perpendicular to the body axis. We propose that apical domain changes reflect planar polarity-dependent mechanical forces operating during neural folding.

With the rapid rise of private vehicles around the world, License Plate Recognition (LPR) plays a vital role in supporting the government to manage vehicles effectively. However, an introduction of new types of license plate (LP) or slight changes in the LP format can break previous LPR systems, as they fail to recognize the LP. Moreover, the LPR system is extremely sensitive to the conditions of the surrounding environment. Thus, this paper introduces a novel deep learning-based Korean LPR system that can effectively deal with existing challenges. The main contributions of this study include (1) a robust LPR system with the integration of three pre-processing techniques (defogging, low-light enhancement, and super-resolution) that can effectively recognize the LP under various conditions, (2) the establishment of two original Korean LPR approaches for different scenarios, including whole license plate recognition (W-LPR) and single-character license plate recognition (SC-LPR), and (3) the introduction of two Korean LPR datasets (synthetic data and real data) involving a new type of LP introduced by the Korean government. Through several experiments, the proposed LPR framework achieved the highest recognition accuracy of 98.94%.

Introduction: Neural tube defects (NTDs) are among the most debilitating and common developmental defects in humans. The induction of NTDs has been attributed to abnormal folic acid (vitamin B9) metabolism, Wnt and BMP signaling, excess retinoic acid (RA), dietary components, environmental factors, and many others. In the present study we show that reduced RA signaling, including alcohol exposure, induces NTDs. Methods: Xenopus embryos were exposed to pharmacological RA biosynthesis inhibitors to study the induction of NTDs. Embryos were treated with DEAB, citral, or ethanol, all of which inhibit the biosynthesis of RA, or injected to overexpress Cyp26a1 to reduce RA. NTD induction was studied using neural plate and notochord markers together with morphological analysis. Expression of the neuroectodermal regulatory network and cell proliferation were analyzed to understand the morphological malformations of the neural plate. Results: Reducing RA signaling levels using retinaldehyde dehydrogenase inhibitors (ethanol, DEAB, and citral) or Cyp26a1-driven degradation efficiently induce NTDs. These NTDs can be rescued by providing precursors of RA. We mapped this RA requirement to early gastrula stages during the induction of neural plate precursors. This reduced RA signaling results in abnormal expression of neural network genes, including the neural plate stem cell maintenance genes, geminin, and foxd4l1.1. This abnormal expression of neural network genes results in increased proliferation of neural precursors giving rise to an expanded neural plate. Conclusion: We show that RA signaling is required for neural tube closure during embryogenesis. RA signaling plays a very early role in the regulation of proliferation and differentiation of the neural plate soon after the induction of neural progenitors during gastrulation. RA signaling disruption leads to the induction of NTDs through the mis regulation of the early neuroectodermal network, leading to increased proliferation resulting in the expansion of the neural plate. Ethanol exposure induces NTDs through this mechanism involving reduced RA levels.

Neural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail and Pax3/7 and generate melanin-containing pigment cells, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate several cell types. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural-crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs.

Tissue internalisation is a key morphogenetic mechanism by which embryonic tissues generate complex internal organs and a number of studies of epithelia have outlined a general view of tissue internalisation. Here we have used quantitative live imaging and mutant analysis to determine whether similar mechanisms are responsible for internalisation in a tissue that apparently does not have a typical epithelial organisation - the zebrafish neural plate. We found that although zebrafish embryos begin neurulation without a conventional epithelium, medially located neural plate cells adopt strategies typical of epithelia in order to constrict their dorsal surface membrane during cell internalisation. Furthermore, we show that Myosin-II activity is a significant driver of this transient cell remodeling which also depends on Cdh2 (N-cadherin). Abrogation of Cdh2 results in defective Myosin-II distribution, mislocalised internalisation events and defective neural plate morphogenesis. Our work suggests Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

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 ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

In previous work (Elul, T., Keller, R., 2000. Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus. Dev. Biol. 224, 3-19; Ezin, A.M., Skoglund, P. Keller, R. 2003. The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate. Dev. Biol. 256, 100-114), the midline tissues of notochord and overlying notoplate were found to induce the monopolar, medially directed protrusive activity of deep neural cells. This behavior is thought to drive the mediolateral intercalation and convergent extension of the neural plate in Xenopus. Here we address the issue of whether the notochord, the notoplate, or both is essential for this induction. Our strategy was to remove the notochord, leaving the overlying notoplate intact, and determine whether it alone can induce the monopolar, medially directed cell behavior. We first establish that the notoplate (presumptive floor plate), when separated from the underlying notochord in the early neurula (stages 13-14), will independently mature into a floor plate as assayed three criteria: (1) continued expression of an early marker, sonic hedgehog, and a later, marker, F-spondin; (2) the display of the notoplate/floor plate-specific randomly oriented protrusive activity; (3) the characteristic lack of mixing of cells between the notoplate and lateral neural plate. Under these conditions, in the presence of a mature notoplate/floor plate and in the absence of the notochord, the characteristic monopolar, medially directed behavior occurred, but only locally near the midline. These results show that the notoplate/floor plate capacity to induce the medially directed motility is limited in range, and they suggest that the notochord is necessary for the normally observed longer range induction in lateral neural plate cells. This work helps to further the understanding of molecular and tissue interactions required for convergent extension.

Neural tube defects (NTDs), one of the most common birth defects in human, present a multifactorial etiology with a poorly defined genetic component. The Dlx5 and Dlx6 bigenic cluster encodes two evolutionary conserved homeodomain transcription factors, which are necessary for proper vertebrate development. It has been shown that Dlx5/6 genes are essential for anterior neural tube closure, however their role in the formation of the posterior structures has never been described. Here, we show that Dlx5/6 expression is required during vertebrate posterior axis formation. Dlx5 presents a similar expression pattern in neural plate border cells during posterior neurulation of zebrafish and mouse. Dlx5/6-inactivation in the mouse results in a phenotype reminiscent of NTDs characterized by open thoracic and lumbar vertebral arches and failure of epaxial muscle formation at the dorsal midline. The dlx5a/6a zebrafish morphants present posterior NTDs associated with abnormal delamination of neural crest cells showing altered expression of cell adhesion molecules and defects of motoneuronal development. Our findings provide new molecular leads to decipher the mechanisms of vertebrate posterior neurulation and might help to gather a better understanding of human congenital NTDs etiology.

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

The evolutionarily conserved Dbx homeodomain-containing proteins play important roles in the development of vertebrate central nervous system. In mouse, Dbx and Nkx6 have been suggested to be cross-repressive partners involved in the patterning of ventral neural tube. Here, we have isolated Xenopus Dbx2 and studied its developmental expression and function during neural development. Like XDbx1, from mid-neurula stage on, XDbx2 is expressed in stripes between the primary motoneurons and interneurons. At the tailbud stages, it is detected in the middle region of the neural tube. XDbx2 acts as a transcriptional repressor in vitro and over-expression of XDbx2 inhibits primary neurogenesis in Xenopus embryos. Over-expression of XDbx genes represses the expression of XNkx6.2 and vise versa. Knockdown of either XDbx1, XDbx2 or both by specific morpholinos induces lateral expansion of XNkx6.2 expression domains. These data reveal conserved roles for Dbx in primary neurogenesis and dorsoventral neural patterning in Xenopus.

During primary neurulation, the separation of a single-layered ectodermal sheet into the surface ectoderm (SE) and neural tube specifies SE and neural ectoderm (NE) cell fates. The mechanisms underlying fate specification in conjunction with neural tube closure are poorly understood. Here, by comparing expression profiles between SE and NE lineages, we observed that uncommitted progenitor cells, expressing stem cell markers, are present in the neural plate border/neural fold prior to neural tube closure. Our results also demonstrated that canonical Wnt and its antagonists, DKK1/KREMEN1, progressively specify these progenitors into SE or NE fates in accord with the progress of neural tube closure. Additionally, SE specification of the neural plate border via canonical Wnt signaling is directed by the grainyhead-like 3 (Grhl3) transcription factor. Thus, we propose that the fate specification of uncommitted progenitors in the neural plate border by canonical Wnt signaling and its downstream effector Grhl3 is crucial for neural tube closure. This study implicates that failure in critical genetic factors controlling fate specification of progenitor cells in the neural plate border/neural fold coordinated with neural tube closure may be potential causes of human neural tube defects.

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.

Neural crest and cranial sensory placodes arise from ectodermal epithelium lying between the neural plate and non-neural ectoderm (neural border). BMP signaling is important for both an induction of the neural border and a subsequent induction of the neural crest within the neural border. In contrast, FGF signaling is important for the neural border induction and the following induction of the pre-placodal ectoderm (PPE), which later gives rise to the cranial sensory placodes. While previous studies have demonstrated that the neural plate explants could be converted to the neural crest cells by adding BMP4 in a culture medium, there is no report showing a similar conversion of the neural plate to the PPE. We therefore examined the effect of FGF2 along with BMP4 on the rostral neural plate explants and found that the explants became the simple squamous epithelia, which were characterized by the desmosomes/tonofilaments in membranes of adjacent cells. Such epithelia expressed sets of neural border markers and the PPE genes, suggesting that the neural plate explants were converted to a PPE-like tissue. This method will be useful for further studying mechanisms of PPE induction and subsequent specifications of the cranial placodes.

In vertebrate embryos, the earliest definitive marker for the neural plate, which will give rise to the entire central nervous system, is the transcription factor Sox2. Although some of the extracellular signals that regulate neural plate fate have been identified, we know very little about the mechanisms controlling Sox2 expression and thus neural plate identity. Here, we use electroporation for gain- and loss-of-function in the chick embryo, in combination with bimolecular fluorescence complementation, two-hybrid screens, chromatin immunoprecipitation, and reporter assays to study protein interactions that regulate expression of N2, the earliest enhancer of Sox2 to be activated and which directs expression to the largest part of the neural plate. We show that interactions between three coiled-coil domain proteins (ERNI, Geminin, and BERT), the heterochromatin proteins HP1alpha and HP1gamma acting as repressors, and the chromatin-remodeling enzyme Brm acting as activator control the N2 enhancer. We propose that this mechanism regulates the timing of Sox2 expression as part of the process of establishing neural plate identity.