RESUMEN
Kabuki syndrome (KS) is a congenital craniofacial disorder resulting from mutations in the KMT2D histone methylase (KS1) or the UTX histone demethylase (KS2). With small cohorts of KS2 patients, it is not clear whether differences exist in clinical manifestations relative to KS1. We mutated KMT2D in neural crest cells (NCCs) to study cellular and molecular functions in craniofacial development with respect to UTX. Similar to UTX, KMT2D NCC knockout mice demonstrate hypoplasia with reductions in frontonasal bone lengths. We have traced the onset of KMT2D and UTX mutant NCC frontal dysfunction to a stage of altered osteochondral progenitor differentiation. KMT2D NCC loss-of-function does exhibit unique phenotypes distinct from UTX mutation, including fully penetrant cleft palate, mandible hypoplasia and deficits in cranial base ossification. KMT2D mutant NCCs lead to defective secondary palatal shelf elevation with reduced expression of extracellular matrix components. KMT2D mutant chondrocytes in the cranial base fail to properly differentiate, leading to defective endochondral ossification. We conclude that KMT2D is required for appropriate cranial NCC differentiation and KMT2D-specific phenotypes may underlie differences between Kabuki syndrome subtypes.
Asunto(s)
Anomalías Múltiples/enzimología , Anomalías Múltiples/patología , Diferenciación Celular , Cara/anomalías , Enfermedades Hematológicas/enzimología , Enfermedades Hematológicas/patología , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Cresta Neural/enzimología , Cresta Neural/patología , Enfermedades Vestibulares/enzimología , Enfermedades Vestibulares/patología , Alelos , Animales , Linaje de la Célula , Movimiento Celular , Condrocitos/patología , Cara/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Mutación/genética , Osteogénesis , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Hueso Paladar/patología , Fenotipo , Cráneo/patologíaRESUMEN
Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.
Asunto(s)
Anomalías Múltiples/enzimología , Cara/anomalías , Enfermedades Hematológicas/enzimología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cresta Neural/metabolismo , Enfermedades Vestibulares/enzimología , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Acetilación , Animales , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Cara/patología , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Enfermedades Hematológicas/patología , Histonas/metabolismo , Mutación con Pérdida de Función , Metilación , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Cresta Neural/enzimología , Cresta Neural/patología , Placa Neural/crecimiento & desarrollo , Placa Neural/metabolismo , Placa Neural/patología , Semaforinas/genética , Semaforinas/metabolismo , Enfermedades Vestibulares/genética , Enfermedades Vestibulares/metabolismo , Enfermedades Vestibulares/patología , Xenopus/embriología , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/fisiologíaRESUMEN
BACKGROUND: Cells derived from the neural crest colonize the developing gut and give rise to the enteric nervous system. The rate at which the ENCC population advances along the bowel will be affected by both the speed and directionality of individual ENCCs. The aim of the study was to use time-lapse imaging and pharmacological activators and inhibitors to examine the role of several intracellular signalling pathways in both the speed and the directionality of individual enteric neural crest-derived cells in intact explants of E12.5 mouse gut. Drugs that activate or inhibit intracellular components proposed to be involved in GDNF-RET and EDN3-ETB signalling in ENCCs were used. FINDINGS: Pharmacological inhibition of JNK significantly reduced ENCC speed but did not affect ENCC directionality. MEK inhibition did not affect ENCC speed or directionality. Pharmacological activation of adenylyl cyclase or PKA (a downstream cAMP-dependent kinase) resulted in a significant decrease in ENCC speed and an increase in caudal directionality of ENCCs. In addition, adenylyl cyclase activation also resulted in reduced cell-cell contact between ENCCs, however this was not observed following PKA activation, suggesting that the effects of cAMP on adhesion are not mediated by PKA. CONCLUSIONS: JNK is required for normal ENCC migration speed, but not directionality, while cAMP signalling appears to regulate ENCC migration speed, directionality and adhesion. Collectively, our data demonstrate that intracellular signalling pathways can differentially affect the speed and directionality of migrating ENCCs.
Asunto(s)
Adenilil Ciclasas/metabolismo , Movimiento Celular , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Cresta Neural/citología , Animales , Inducción Embrionaria , Sistema Nervioso Entérico/embriología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Cresta Neural/enzimología , Cresta Neural/metabolismo , Factores de TiempoRESUMEN
Alternative splicing (AS) creates proteomic diversity from a limited size genome by generating numerous transcripts from a single protein-coding gene. Tissue-specific regulators of AS are essential components of the gene regulatory network, required for normal cellular function, tissue patterning, and embryonic development. However, their cell-autonomous function in neural crest development has not been explored. Here, we demonstrate that splicing factor Rbfox2 is expressed in the neural crest cells (NCCs), and deletion of Rbfox2 in NCCs leads to cleft palate and defects in craniofacial bone development. RNA-Seq analysis revealed that Rbfox2 regulates splicing and expression of numerous genes essential for neural crest/craniofacial development. We demonstrate that Rbfox2-TGF-ß-Tak1 signaling axis is deregulated by Rbfox2 deletion. Furthermore, restoration of TGF-ß signaling by Tak1 overexpression can rescue the proliferation defect seen in Rbfox2 mutants. We also identified a positive feedback loop in which TGF-ß signaling promotes expression of Rbfox2 in NCCs.
Asunto(s)
Anomalías Craneofaciales/patología , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/embriología , Cresta Neural/enzimología , Factores de Empalme de ARN/deficiencia , Animales , Modelos Animales de Enfermedad , Ratones , Análisis de Secuencia de ARNRESUMEN
Neural crest cells migrate long distances throughout the embryo and rely on extracellular signals that attract, repel and/or stimulate survival to ensure proper contribution to target derivatives. Here, we show that leukocyte receptor tyrosine kinase (LTK), an ALK-type receptor tyrosine kinase, is expressed by neural crest cells during early migratory stages in chicken embryos. Loss of LTK in the cranial neural crest impairs migration and results in increased levels of apoptosis. Conversely, midkine, previously proposed as a ligand for ALK, is secreted by the non-neural ectoderm during early neural crest migratory stages and internalized by neural crest cells in vivo Similar to loss of LTK, loss of midkine reduces survival of the migratory neural crest. Moreover, we show by proximity ligation and co-immunoprecipitation assays that midkine binds to LTK. Taken together, these results suggest that LTK in neural crest cells interacts with midkine emanating from the non-neural ectoderm to promote cell survival, revealing a new signaling pathway that is essential for neural crest development.
Asunto(s)
Movimiento Celular , Midkina/metabolismo , Cresta Neural/citología , Cresta Neural/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Tipificación del Cuerpo , Supervivencia Celular , Embrión de Pollo , Ectodermo/metabolismo , Unión ProteicaRESUMEN
The neural crest, a progenitor population that drove vertebrate evolution, retains the broad developmental potential of the blastula cells it is derived from, even as neighboring cells undergo lineage restriction. The mechanisms that enable these cells to preserve their developmental potential remain poorly understood. Here, we explore the role of histone deacetylase (HDAC) activity in this process in Xenopus We show that HDAC activity is essential for the formation of neural crest, as well as for proper patterning of the early ectoderm. The requirement for HDAC activity initiates in naïve blastula cells; HDAC inhibition causes loss of pluripotency gene expression and blocks the ability of blastula stem cells to contribute to lineages of the three embryonic germ layers. We find that pluripotent naïve blastula cells and neural crest cells are both characterized by low levels of histone acetylation, and show that increasing HDAC1 levels enhance the ability of blastula cells to be reprogrammed to a neural crest state. Together, these findings elucidate a previously uncharacterized role for HDAC activity in establishing the neural crest stem cell state.
Asunto(s)
Histona Desacetilasa 1/metabolismo , Cresta Neural/embriología , Cresta Neural/enzimología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Acetilación , Animales , Biomarcadores/metabolismo , Blástula/citología , Blástula/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Cresta Neural/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Xenopus laevis/genéticaRESUMEN
Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGFß, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.
Asunto(s)
Fisura del Paladar/enzimología , Cresta Neural/enzimología , Proteína-Arginina N-Metiltransferasas/fisiología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Eliminación de Gen , Células Madre Mesenquimatosas/enzimología , Ratones , Ratones Transgénicos , Fenotipo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteína Wnt1/metabolismoRESUMEN
Neural crest migration is critical to its physiological function. Mechanisms controlling mammalian neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here we report requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest in Xenopus and mouse models. We demonstrate that GSK3 is tyrosine phosphorylated (pY) in mouse neural crest cells and that loss of GSK3 leads to increased pFAK and misregulation of Rac1 and lamellipodin, key regulators of cell migration. Genetic reduction of GSK3 results in failure of migration. We find that pY-GSK3 phosphorylation depends on anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma. Consistent with this, neuroblastoma cells with increased ALK activity express high levels of pY-GSK3, and blockade of GSK3 or ALK can affect migration of these cells. Altogether, this work identifies a role for GSK3 in cell migration during neural crest development and cancer.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Cresta Neural/citología , Cresta Neural/enzimología , Proteínas de Xenopus/química , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/metabolismo , Animales , Línea Celular Tumoral , Linaje de la Célula , Movimiento Celular/fisiología , Femenino , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta/deficiencia , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Ratones , Ratones Noqueados , Cresta Neural/embriología , Neuroblastoma/enzimología , Fosforilación , Embarazo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismoRESUMEN
Nonobstructive hydronephrosis, defined as dilatation of the renal pelvis with or without dilatation of the ureter, is the most common antenatal abnormality detected by fetal ultrasound. Yet, the etiology of nonobstructive hydronephrosis is poorly defined. We previously demonstrated that defective development of urinary tract pacemaker cells (utPMCs) expressing hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) and the stem cell marker cKIT causes abnormal ureteric peristalsis and nonobstructive hydronephrosis. However, further investigation of utPMC development and function is limited by lack of knowledge regarding the embryonic derivation, development, and molecular apparatus of these cells. Here, we used lineage tracing in mice to identify cells that give rise to utPMCs. Neural crest cells (NCCs) indelibly labeled with tdTomato expressed HCN3 and cKIT. Furthermore, purified HCN3+ and cKIT+ utPMCs were enriched in Sox10 and Tfap-2α, markers of NCCs. Sequencing of purified RNA from HCN3+ cells revealed enrichment of a small subset of RNAs, including RNA encoding protein kinase 2ß (PTK2ß), a Ca2+-dependent tyrosine kinase that regulates ion channel activity in neurons. Immunofluorescence analysis in situ revealed PTK2ß expression in NCCs as early as embryonic day 12.5 and in HCN3+ and cKIT+ utPMCs as early as embryonic day 15.5, with sustained expression in HCN3+ utPMCs until postnatal week 8. Pharmacologic inhibition of PTK2ß in murine pyeloureteral tissue explants inhibited contraction frequency. Together, these results demonstrate that utPMCs are derived from NCCs, identify new markers of utPMCs, and demonstrate a functional contribution of PTK2ß to utPMC function.
Asunto(s)
Quinasa 2 de Adhesión Focal/fisiología , Regulación del Desarrollo de la Expresión Génica , Células Intersticiales de Cajal/enzimología , Pelvis Renal/fisiología , Cresta Neural/enzimología , Peristaltismo/fisiología , Uréter/fisiología , Animales , Antígenos de Diferenciación/análisis , Quinasa 2 de Adhesión Focal/biosíntesis , Quinasa 2 de Adhesión Focal/genética , Genes Reporteros , Edad Gestacional , Hidronefrosis/enzimología , Hidronefrosis/fisiopatología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/análisis , Células Intersticiales de Cajal/fisiología , Pelvis Renal/citología , Pelvis Renal/embriología , Pelvis Renal/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cresta Neural/fisiología , Canales de Potasio/análisis , Proteínas Proto-Oncogénicas c-kit/análisis , ARN Mensajero/biosíntesis , Factores de Transcripción SOXE/análisis , Transducción de Señal , Factor de Transcripción AP-2/análisis , Uréter/citología , Uréter/embriología , Uréter/crecimiento & desarrolloRESUMEN
Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.
Asunto(s)
Citocinas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Drosophila , Ojo/metabolismo , Humanos , Linfoma/enzimología , Cresta Neural/enzimología , Células PC12 , Pigmentación , Ratas , Pez CebraRESUMEN
Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.
Asunto(s)
Nucléolo Celular/metabolismo , Nucléolo Celular/patología , Daño del ADN , ADN Ribosómico/metabolismo , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/patología , Estrés Fisiológico , Animales , Apoptosis , Benzotiazoles/farmacología , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patología , Cromatina/metabolismo , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Ribosómico/genética , ARN Polimerasas Dirigidas por ADN/deficiencia , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Disostosis Mandibulofacial/embriología , Ratones , Naftiridinas/farmacología , Cresta Neural/enzimología , Cresta Neural/patología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Fenotipo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Helicasas/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , ARN Ribosómico/biosíntesis , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Ribosomas/genética , Ribosomas/metabolismo , Cráneo/patología , Estrés Fisiológico/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Xenopus , Pez Cebra/embriología , Proteínas de Pez Cebra/deficienciaRESUMEN
BACKGROUND: To understand the basis of nervous system development, we must learn how multipotent progenitors generate diverse neuronal and glial lineages. We addressed this issue in the zebrafish enteric nervous system (ENS), a complex neuronal and glial network that regulates essential intestinal functions. Little is currently known about how ENS progenitor subpopulations generate enteric neuronal and glial diversity. RESULTS: We identified temporally and spatially dependent progenitor subpopulations based on coexpression of three genes essential for normal ENS development: phox2bb, sox10, and ret. Our data suggest that combinatorial expression of these genes delineates three major ENS progenitor subpopulations, (1) phox2bb + /ret- /sox10-, (2) phox2bb + /ret + /sox10-, and (3) phox2bb + /ret + /sox10+, that reflect temporal progression of progenitor maturation during migration. We also found that differentiating zebrafish neurons maintain phox2bb and ret expression, and lose sox10 expression. CONCLUSIONS: Our data show that zebrafish enteric progenitors constitute a heterogeneous population at both early and late stages of ENS development and suggest that marker gene expression is indicative of a progenitor's fate. We propose that a progenitor's expression profile reveals its developmental state: "younger" wave front progenitors express all three genes, whereas more mature progenitors behind the wave front selectively lose sox10 and/or ret expression, which may indicate developmental restriction. Developmental Dynamics 245:1081-1096, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Sistema Nervioso Entérico/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/embriología , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Cresta Neural/enzimología , Cresta Neural/metabolismo , ARN Mensajero/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5' monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.
Asunto(s)
Sistema Nervioso Entérico/irrigación sanguínea , Sistema Nervioso Entérico/embriología , Cara/embriología , IMP Deshidrogenasa/metabolismo , Cresta Neural/embriología , Cresta Neural/enzimología , Cráneo/embriología , Alelos , Animales , Bromodesoxiuridina/metabolismo , Sistema Nervioso Entérico/enzimología , Sistema Nervioso Entérico/patología , Femenino , Feto/anomalías , Feto/embriología , Eliminación de Gen , Genes Reporteros , Enfermedad de Hirschsprung/patología , IMP Deshidrogenasa/deficiencia , Etiquetado Corte-Fin in Situ , Integrasas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Especificidad de Órganos , ARN no Traducido/metabolismo , Recombinación Genética/genética , Cráneo/metabolismo , Proteína Wnt1/metabolismoRESUMEN
BACKGROUND: The small GTPase Rac1 regulates diverse cellular functions, including both apicobasal and planar cell polarity pathways; however, its role in cardiac outflow tract (OFT) development remains unknown. In the present study, we aimed to examine the role of Rac1 in the anterior second heart field (SHF) splanchnic mesoderm and subsequent OFT development during heart morphogenesis. METHODS AND RESULTS: Using the Cre/loxP system, mice with an anterior SHF-specific deletion of Rac1 (Rac1(SHF)) were generated. Embryos were collected at various developmental time points for immunostaining and histological analysis. Intrauterine echocardiography was also performed to assess aortic valve blood flow in embryos at embryonic day 18.5. The Rac1(SHF) splanchnic mesoderm exhibited disruptions in SHF progenitor cellular organization and proliferation. Consequently, this led to a spectrum of OFT defects along with aortic valve defects in Rac1(SHF) embryos. Mechanistically, it was found that the ability of the Rac1(SHF) OFT myocardial cells to migrate into the proximal OFT cushion was severely reduced. In addition, expression of the neural crest chemoattractant semaphorin 3c was decreased. Lineage tracing showed that anterior SHF contribution to the OFT myocardium and aortic valves was deficient in Rac1(SHF) hearts. Furthermore, functional analysis with intrauterine echocardiography at embryonic day 18.5 showed aortic valve regurgitation in Rac1(SHF) hearts, which was not seen in control hearts. CONCLUSIONS: Disruptions of Rac1 signaling in the anterior SHF results in aberrant progenitor cellular organization and defects in OFT development. Our data show Rac1 signaling to be a critical regulator of cardiac OFT formation during embryonic heart development.
Asunto(s)
Insuficiencia de la Válvula Aórtica/enzimología , Válvula Aórtica/enzimología , Cardiopatías Congénitas/enzimología , Miocardio/enzimología , Neuropéptidos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Válvula Aórtica/anomalías , Insuficiencia de la Válvula Aórtica/diagnóstico , Insuficiencia de la Válvula Aórtica/genética , Linaje de la Célula , Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Edad Gestacional , Cardiopatías Congénitas/diagnóstico , Cardiopatías Congénitas/genética , Ratones Noqueados , Morfogénesis , Miocardio/patología , Cresta Neural/anomalías , Cresta Neural/enzimología , Neuropéptidos/deficiencia , Neuropéptidos/genética , Fenotipo , Semaforinas/genética , Semaforinas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genéticaRESUMEN
Prenatal ethanol exposure causes persistent neurodevelopmental deficits by inducing apoptosis within neuronal progenitors including the neural crest. The cellular signaling events underlying this apoptosis are unclear. Using an established chick embryo model, we previously identified ethanol's activation of calmodulin-dependent protein kinase II (CaMKII) as a crucial early step in this pathway. Here, we report that CaMKII is pro-apoptotic because it mediates the loss of transcriptionally active ß-catenin, which normally provides trophic support to these cells. ß-catenin over-expression normalized cell survival in ethanol's presence. CaMKII inhibition similarly restored ß-catenin content and transcriptional activity within ethanol-treated cells and prevented their cell death. In contrast, inhibition of alternative effectors known to destabilize ß-catenin, including glycogen synthase kinase-3ß, Protein Kinase C, JNK, and calpain, failed to normalize cell survival and ß-catenin activity in ethanol's presence. Importantly, we found that purified CaMKII can directly phosphorylate ß-catenin. Using targeted mutagenesis we identified CaMKII phosphorylation sites within human ß-catenin at T332, T472, and S552. This is the first demonstration that ß-catenin is a phosphorylation target of CaMKII and represents a novel mechanism by which calcium signals could regulate ß-catenin-dependent transcription. These results inform ethanol's neurotoxicity and offer unexpected insights into other neurodevelopmental and neurodegenerative disorders having dysregulated calcium or ß-catenin signaling.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/farmacología , Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , Embrión de Pollo , Electroporación , Inmunohistoquímica , Cresta Neural/enzimología , Cresta Neural/metabolismo , Fosforilación , Factor 1 de Transcripción de Linfocitos T/genética , Transcripción Genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: Neural crest cells (NC cells) are highly migratory multipotent cells. Their multipotency is transient at the early stage of their generation; soon after emerging from the neural tube, these cells turn into lineage-restricted precursors. However, recent studies have disputed this conventionally believed paradigm. In this study, we analyzed the differentiation potency of NC-derived cells after their arrival at target tissues. RESULTS: Using Sox10-IRES-Venus mice, we found that the NC-derived cells in the skin, DRG, and inner ear could be divided into two populations: Sox10-positive/Kit-negative cells (Sox10+/Kit- cells) and Sox10- and Kit-positive cells (Sox10+/Kit+ cells). Only the Sox10+/Kit- cells were detected in the intestines. Unexpectedly, the Sox10+/Kit+ cells differentiated into neurons, glial cells, and melanocytes, showing that they had maintained their multipotency even after having entered the target tissues. The Sox10+/Kit+ cells in the DRG maintained their multipotency for a restricted period during the earlier embryonic stages, whereas those in the skin and inner ear were multipotent yet even in later embryonic stages. CONCLUSIONS: We showed that NC-derived Sox10+/Kit+ cells maintained their multipotency even after entry into the target tissues. This unexpected differentiation potency of these cells in tissues seems to have been strictly restricted by the tissue microenvironment.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias , Células Madre Multipotentes , Cresta Neural , Animales , Antígenos de Diferenciación/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Ratones , Ratones Transgénicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Cresta Neural/citología , Cresta Neural/enzimología , Especificidad de Órganos/fisiologíaRESUMEN
Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.
Asunto(s)
Anomalías Cardiovasculares/embriología , Anomalías Cardiovasculares/enzimología , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/enzimología , Morfogénesis , Cresta Neural/patología , Proteína de Unión al GTP cdc42/metabolismo , Actinas/metabolismo , Animales , Proteína Morfogenética Ósea 2/farmacología , Anomalías Cardiovasculares/patología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Anomalías Craneofaciales/patología , Cruzamientos Genéticos , Citoesqueleto/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/patología , Activación Enzimática/efectos de los fármacos , Femenino , Eliminación de Gen , Genotipo , Masculino , Ratones , Ratones Noqueados , Morfogénesis/efectos de los fármacos , Cresta Neural/efectos de los fármacos , Cresta Neural/enzimología , Osteogénesis/efectos de los fármacos , Fenotipo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Timo/anomalías , Timo/efectos de los fármacos , Timo/patologíaRESUMEN
Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.
Asunto(s)
Encéfalo/anomalías , Labio Leporino/genética , Fisura del Paladar/genética , Cresta Neural/metabolismo , Receptores de Superficie Celular/genética , Animales , Encéfalo/metabolismo , Muerte Celular/genética , Proliferación Celular , Forma de la Célula/genética , Labio Leporino/metabolismo , Fisura del Paladar/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Estudios de Asociación Genética , Proteínas Hedgehog/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/genética , Mucosa Nasal/metabolismo , Cresta Neural/enzimología , Nariz/embriología , Receptores Patched , Receptor Patched-1 , Fenotipo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Integrin-linked kinase (ILK) is a serine/threonine kinase and a major regulator of integrin signaling. It links integrins to the actin cytoskeleton and recruits other adaptor molecules into a large complex to regulate actin dynamics and integrin function. Using the Cre-lox system, we deleted Ilk from NCCs of mice to investigate its role in NCC morphogenesis. The resulting mutants developed a severe aneurysmal arterial trunk that resulted in embryonic lethality during late gestation. Ilk mutants showed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and the outflow tract. Within the conotruncal cushions, Ilk-deficient NCCs exhibited disorganization of F-actin stress fibers and a significantly rounder morphology, with shorter cellular projections. Additionally, absence of ILK resulted in reduced in vivo phosphorylation of Smad3 in NCCs, which correlated with reduced αSMA levels. Our findings resemble those seen in Pinch1 and ß1 integrin conditional mutant mice, and therefore support that, in neural crest-derived cells, ILK and Pinch1 act as cytoplasmic effectors of ß1 integrin in a pathway that protects against aneurysms. In addition, our conditional Ilk mutant mice might prove useful as a model to study aortic aneurysms caused by reduced Smad3 signaling, as occurs in the newly described aneurysms-osteoarthritis syndrome, for example.
Asunto(s)
Aneurisma de la Aorta/enzimología , Aneurisma de la Aorta/patología , Pérdida del Embrión/enzimología , Eliminación de Gen , Cresta Neural/enzimología , Cresta Neural/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Citoesqueleto de Actina/metabolismo , Animales , Aorta Torácica/anomalías , Aorta Torácica/embriología , Aorta Torácica/patología , Anomalías Cardiovasculares/embriología , Anomalías Cardiovasculares/patología , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/patología , Pérdida del Embrión/patología , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Integrasas/metabolismo , Ratones , Ratones Mutantes , Morfogénesis , Especificidad de Órganos , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMEN
A subset of transcription factors classified as neural crest 'specifiers' are also core epithelial-mesenchymal transition regulatory factors, both in the neural crest and in tumour progression. The bHLH factor Twist is among the least well studied of these factors. Here we demonstrate that Twist is required for cranial neural crest formation and fate determination in Xenopus. We further show that Twist function in the neural crest is dependent upon its carboxy-terminal WR domain. The WR domain mediates physical interactions between Twist and other core epithelial-mesenchymal transition factors, including Snail1 and Snail2, which are essential for proper function. Interaction with Snail1/2, and Twist function more generally, is regulated by GSK-3-ß-mediated phosphorylation of conserved sites in the WR domain. Together, these findings elucidate a mechanism for coordinated control of a group of structurally diverse factors that function as a regulatory unit in both developmental and pathological epithelial-mesenchymal transitions.