RESUMEN
Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.
Asunto(s)
Tubo Neural , Neurulación , Tomografía de Coherencia Óptica , Animales , Femenino , Ratones , Fenómenos Biomecánicos , Embrión de Mamíferos/metabolismo , Formiato-Tetrahidrofolato Ligasa/genética , Formiato-Tetrahidrofolato Ligasa/metabolismo , Formiatos/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ratones Noqueados , Microscopía Confocal , Mutación/genética , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patología , Neurulación/genética , Tomografía de Coherencia Óptica/métodosRESUMEN
This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.
Asunto(s)
Xenopus , Animales , Xenopus/embriología , Xenopus/genética , Gastrulación , Embrión no Mamífero/citología , Neurulación/genética , Neurulación/fisiología , Análisis de la Célula Individual/métodos , Regulación del Desarrollo de la Expresión GénicaRESUMEN
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.
Asunto(s)
Cresta Neural , Análisis de la Célula Individual , Xenopus laevis , Animales , Cresta Neural/citología , Cresta Neural/metabolismo , Análisis de la Célula Individual/métodos , Xenopus laevis/embriología , Regulación del Desarrollo de la Expresión Génica , Movimiento Celular , Redes Reguladoras de Genes , Transcriptoma , Gastrulación , Placa Neural/metabolismo , Placa Neural/embriología , Placa Neural/citología , Transición Epitelial-Mesenquimal/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/citología , Neurulación/genética , Neurulación/fisiología , Diferenciación CelularRESUMEN
Congenital vertebral malformations (CVMs) and neural tube defects (NTDs) are common birth defects affecting the spine and nervous system, respectively, due to defects in somitogenesis and neurulation. Somitogenesis and neurulation rely on factors secreted from neighbouring tissues and the integrity of the axial structure. Crucial signalling pathways like Wnt, Notch and planar cell polarity regulate somitogenesis and neurulation with significant crosstalk. While previous studies suggest an association between CVMs and NTDs, the exact mechanism underlying this relationship remains unclear. In this review, we explore embryonic development, signalling pathways and clinical phenotypes involved in the association between CVMs and NTDs. Moreover, we provide a summary of syndromes that exhibit occurrences of both CVMs and NTDs. We aim to provide insights into the potential mechanisms underlying the association between CVMs and NTDs, thereby facilitating clinical diagnosis and management of these anomalies.
Asunto(s)
Defectos del Tubo Neural , Femenino , Embarazo , Humanos , Defectos del Tubo Neural/epidemiología , Defectos del Tubo Neural/genética , Columna Vertebral/metabolismo , Desarrollo Embrionario , Neurulación/genética , Transducción de Señal/genéticaRESUMEN
Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.
Asunto(s)
Defectos del Tubo Neural , Neurulación , Humanos , Neurulación/genética , Gastrulación/genética , Polaridad Celular/genética , Vía de Señalización Wnt/genética , Tubo Neural/metabolismo , Morfogénesis/genética , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismoRESUMEN
Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.
Asunto(s)
Defectos del Tubo Neural , Disrafia Espinal , Embarazo , Femenino , Ratones , Animales , Factores de Transcripción/metabolismo , Neurulación/genética , Tubo Neural/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismo , Defectos del Tubo Neural/metabolismo , Ratones Noqueados , Columna Vertebral/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Neural tube closure (NTC) is crucial for proper development of the brain and spinal cord and requires precise morphogenesis from a sheet of cells to an intact three-dimensional structure. NTC is dependent on successful regulation of hundreds of genes, a myriad of signaling pathways, concentration gradients, and is influenced by epigenetic and environmental cues. Failure of NTC is termed a neural tube defect (NTD) and is a leading class of congenital defects in the United States and worldwide. Though NTDs are all defined as incomplete closure of the neural tube, the pathogenesis of an NTD determines the type, severity, positioning, and accompanying phenotypes. In this review, we survey pathogenesis of NTDs relating to disruption of cellular processes arising from genetic mutations, altered epigenetic regulation, and environmental influences by micronutrients and maternal condition. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Stem Cells and Development.
Asunto(s)
Defectos del Tubo Neural , Tubo Neural , Epigénesis Genética , Ácido Fólico/metabolismo , Humanos , Tubo Neural/anomalías , Defectos del Tubo Neural/genética , Neurulación/genéticaRESUMEN
Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.
Asunto(s)
Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Movimiento Celular/genética , Pollos/genética , Coturnix/embriología , Coturnix/genética , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/metabolismo , Neurulación/genética , Animales , Embrión de Pollo , Pollos/metabolismo , Coturnix/metabolismo , Femenino , Redes Reguladoras de Genes , Cresta Neural/embriología , Tubo Neural/embriología , Tubo Neural/metabolismo , Oviparidad/genética , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes/métodos , MicroARNs/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Hibridación in Situ/métodos , Morfolinos/genética , Cresta Neural/embriología , Cresta Neural/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Neurulación/genética , Fenotipo , ARN Guía de Kinetoplastida/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.
Asunto(s)
Caveolina 3/genética , Adhesión Celular/fisiología , Ciona/metabolismo , Animales , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Caveolina 3/metabolismo , Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Morfogénesis/genética , Placa Neural/metabolismo , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Neurulación/genéticaRESUMEN
The neural crest (NC) is a transient multipotent cell population that migrates extensively to produce a remarkable array of vertebrate cell types. NC cell specification progresses in an anterior to posterior fashion, resulting in distinct, axial-restricted subpopulations. The anterior-most, cranial, population of NC is specified as gastrulation concludes and neurulation begins, while more posterior populations become specified as the body elongates. The mechanisms that govern development of the more posterior NC cells remain incompletely understood. Here, we report a key role for zebrafish Cdx4, a homeodomain transcription factor, in the development of posterior NC cells. We demonstrate that cdx4 is expressed in trunk NC cell progenitors, directly binds NC cell-specific enhancers in the NC GRN, and regulates expression of the key NC development gene foxd3 in the posterior body. Moreover, cdx4 mutants show disruptions to the segmental pattern of trunk NC cell migration due to loss of normal leader/follower cell dynamics. Finally, using cell transplantation to generate chimeric specimens, we show that Cdx4 does not function in the paraxial mesoderm-the environment adjacent to which crest migrates-to influence migratory behaviors. We conclude that cdx4 plays a critical, and likely tissue autonomous, role in the establishment of trunk NC migratory behaviors. Together, our results indicate that cdx4 functions as an early NC specifier gene in the posterior body of zebrafish embryos.
Asunto(s)
Proteínas de Homeodominio/genética , Cresta Neural/metabolismo , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Factores de Transcripción Forkhead/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis/genética , Placa Neural/metabolismo , Tubo Neural/metabolismo , Neurulación/genética , Factores de Transcripción/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Defectos del Tubo Neural/genética , Neurulación/genética , Neurulación/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Polaridad Celular/fisiología , Modelos Animales de Enfermedad , Eliminación de Gen , Ratones , Morfogénesis/genética , Morfogénesis/fisiología , Mutación , Miosina Tipo II/metabolismo , Cresta Neural/metabolismo , Células Neuroepiteliales/metabolismo , Células Neuroepiteliales/patología , TranscriptomaRESUMEN
BACKGROUND: Microexons, exons that are ≤ 30 nucleotides, are a highly conserved and dynamically regulated set of cassette exons. They have key roles in nervous system development and function, as evidenced by recent results demonstrating the impact of microexons on behaviour and cognition. However, microexons are often overlooked due to the difficulty of detecting them using standard RNA-seq aligners. RESULTS: Here, we present MicroExonator, a novel pipeline for reproducible de novo discovery and quantification of microexons. We process 289 RNA-seq datasets from eighteen mouse tissues corresponding to nine embryonic and postnatal stages, providing the most comprehensive survey of microexons available for mice. We detect 2984 microexons, 332 of which are differentially spliced throughout mouse embryonic brain development, including 29 that are not present in mouse transcript annotation databases. Unsupervised clustering of microexons based on their inclusion patterns segregates brain tissues by developmental time, and further analysis suggests a key function for microexons in axon growth and synapse formation. Finally, we analyse single-cell RNA-seq data from the mouse visual cortex, and for the first time, we report differential inclusion between neuronal subpopulations, suggesting that some microexons could be cell type-specific. CONCLUSIONS: MicroExonator facilitates the investigation of microexons in transcriptome studies, particularly when analysing large volumes of data. As a proof of principle, we use MicroExonator to analyse a large collection of both mouse bulk and single-cell RNA-seq datasets. The analyses enabled the discovery of previously uncharacterized microexons, and our study provides a comprehensive microexon inclusion catalogue during mouse development.
Asunto(s)
Desarrollo Embrionario/genética , Exones , Neuronas/metabolismo , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Ratones , Neurogénesis/genética , Neurulación/genética , Neurulación/fisiología , Empalme del ARN , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Transcriptoma , Corteza Visual , Pez CebraRESUMEN
BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Embrión de Mamíferos/efectos de los fármacos , Etanol/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Tubo Neural/efectos de los fármacos , Neurulación/efectos de los fármacos , Animales , Proliferación Celular/genética , Cilios/genética , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Ratones , Tubo Neural/metabolismo , Neurulación/genética , Biogénesis de Organelos , Embarazo , RNA-Seq , Ribosomas/genética , Proteína p53 Supresora de TumorRESUMEN
BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.
Asunto(s)
Autofagia/genética , Estrés del Retículo Endoplásmico/genética , Enfermedades Fetales/genética , Células-Madre Neurales/metabolismo , Defectos del Tubo Neural/genética , Embarazo en Diabéticas/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Respuesta de Proteína Desplegada/genética , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Glucemia/metabolismo , Óxidos N-Cíclicos/farmacología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Enfermedades Fetales/etiología , Enfermedades Fetales/metabolismo , Glucosa/farmacología , Técnicas In Vitro , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Células-Madre Neurales/efectos de los fármacos , Defectos del Tubo Neural/embriología , Defectos del Tubo Neural/metabolismo , Células Neuroepiteliales/efectos de los fármacos , Células Neuroepiteliales/metabolismo , Neurulación/genética , Estrés Oxidativo , Embarazo , Embarazo en Diabéticas/metabolismo , Marcadores de Spin , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Every year nearly 6 percent of children worldwide are born with a serious congenital malformation, resulting in death or lifelong disability. In the United States, birth defects remain one of the leading causes of infant mortality. Among the common structural congenital defects are conditions known as neural tube defects (NTDs). These are a class of malformation of the brain and spinal cord where the neural tube fails to close during the neurulation. Although NTDs remain among the most pervasive and debilitating of all human developmental anomalies, there is insufficient understanding of their etiology. Previous studies have proposed that complex birth defects like NTDs are likely omnigenic, involving interconnected gene regulatory networks with associated signals throughout the genome. Advances in technologies have allowed researchers to more critically investigate regulatory gene networks in ever increasing detail, informing our understanding of the genetic basis of NTDs. Employing a systematic analysis of these complex birth defects using massively parallel DNA sequencing with stringent bioinformatic algorithms, it is possible to approach a greater level of understanding of the genomic architecture underlying NTDs. Herein, we present a brief overview of different approaches undertaken in our laboratory to dissect out the genetics of susceptibility to NTDs. This involves the use of mouse models to identify candidate genes, as well as large scale whole genome/whole exome (WGS/WES) studies to interrogate the genomic landscape of NTDs. The goal of this research is to elucidate the gene-environment interactions contributing to NTDs, thus encouraging global research efforts in their prevention.
Asunto(s)
Genómica/métodos , Defectos del Tubo Neural/genética , Animales , Anticonvulsivantes/efectos adversos , Anomalías Congénitas/epidemiología , Anomalías Congénitas/etiología , Anomalías Congénitas/genética , Modelos Animales de Enfermedad , Femenino , Genómica/tendencias , Humanos , Tubo Neural/embriología , Tubo Neural/metabolismo , Defectos del Tubo Neural/epidemiología , Neurulación/genética , Embarazo , Efectos Tardíos de la Exposición Prenatal/epidemiología , Efectos Tardíos de la Exposición Prenatal/genética , Secuenciación del ExomaRESUMEN
Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.
Asunto(s)
Proteínas Hedgehog/metabolismo , Tubo Neural/embriología , Neurulación/genética , Notocorda/metabolismo , Codorniz/embriología , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Mesodermo/metabolismo , Neuronas Motoras/metabolismo , Placa Neural/metabolismo , Tubo Neural/metabolismo , Neurogénesis/genética , Receptor Patched-1/metabolismo , Transducción de Señal/genética , TransfecciónRESUMEN
In amniotes, unlike primary neurulation in the anterior body, secondary neurulation (SN) proceeds along with axial elongation by the mesenchymal-to-epithelial transition of SN precursors in the tail bud. It has been under debate whether the SN is generated by neuromesodermal common progenitor cells (NMPs) or neural restricted lineage. Our direct cell labeling and serial transplantations identify uni-fated (neural) precursors in the early tail bud. The uni-fated SN precursor territory is further divided into two subpopulations, neural-differentiating and self-renewing cells, which are regulated by high- and low levels of Sox2, respectively. Unexpectedly, uni-fated SN precursors change their fate at later stages to produce both SN and mesoderm. Thus, chicken embryos adopt a previously unappreciated prolonged phase with uni-fated SN stem cells in the early tail bud, which is absent or very limited in mouse embryos.
Asunto(s)
Autorrenovación de las Células/fisiología , Pollos/genética , Células-Madre Neurales/citología , Tubo Neural/embriología , Neurulación/fisiología , Factores de Transcripción SOXB1/fisiología , Cola (estructura animal)/embriología , Animales , Linaje de la Célula , Embrión de Pollo , Genes Reporteros , Mesodermo/citología , Tubo Neural/citología , Neurulación/genética , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/genética , Cola (estructura animal)/citologíaRESUMEN
The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 âkb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Sitios Genéticos , Neurulación/genética , Hueso Paladar/embriología , Factores de Transcripción/genética , Alelos , Animales , Femenino , Eliminación de Gen , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tubo Neural/embriología , Defectos del Tubo Neural/genética , Factores de Transcripción/metabolismoRESUMEN
Attempts to constitutively knockout HTT in rodents resulted in embryonic lethality, curtailing efforts to study HTT function later in development. Here we show that HTT is dispensable for early zebrafish development, contrasting published zebrafish morpholino experiment results. Homozygous HTT knockouts were embryonically viable and appeared developmentally normal through juvenile stages. Comparison of adult fish revealed significant reduction in body size and fitness in knockouts compared to hemizygotes and wildtype fish, indicating an important role for wildtype HTT in postnatal development. Our zebrafish model provides an opportunity to understand the function of wildtype HTT later in development.