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1.
Development ; 149(17)2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35950911

RESUMEN

Coordinated migration of the mesoderm is essential for accurate organization of the body plan during embryogenesis. However, little is known about how mesoderm migration influences posterior neural tube closure in mammals. Here, we show that spinal neural tube closure and lateral migration of the caudal paraxial mesoderm depend on transmembrane protein 132A (TMEM132A), a single-pass type I transmembrane protein, the function of which is not fully understood. Our study in Tmem132a-null mice and cell models demonstrates that TMEM132A regulates several integrins and downstream integrin pathway activation as well as cell migration behaviors. Our data also implicates mesoderm migration in elevation of the caudal neural folds and successful closure of the caudal neural tube. These results suggest a requirement for paraxial mesodermal cell migration during spinal neural tube closure, disruption of which may lead to spina bifida.


Asunto(s)
Proteínas de la Membrana , Defectos del Tubo Neural , Tubo Neural , Animales , Integrinas/metabolismo , Proteínas de la Membrana/genética , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Tubo Neural/metabolismo , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo
2.
Development ; 147(22)2020 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-33214242

RESUMEN

Disruptions in neural tube (NT) closure result in neural tube defects (NTDs). To understand the molecular processes required for mammalian NT closure, we investigated the role of Snx3, a sorting nexin gene. Snx3-/- mutant mouse embryos display a fully-penetrant cranial NTD. In vivo, we observed decreased canonical WNT target gene expression in the cranial neural epithelium of the Snx3-/- embryos and a defect in convergent extension of the neural epithelium. Snx3-/- cells show decreased WNT secretion, and live cell imaging reveals aberrant recycling of the WNT ligand-binding protein WLS and mis-trafficking to the lysosome for degradation. The importance of SNX3 in WNT signaling regulation is demonstrated by rescue of NT closure in Snx3-/- embryos with a WNT agonist. The potential for SNX3 to function in human neurulation is revealed by a point mutation identified in an NTD-affected individual that results in functionally impaired SNX3 that does not colocalize with WLS and the degradation of WLS in the lysosome. These data indicate that Snx3 is crucial for NT closure via its role in recycling WLS in order to control levels of WNT signaling.


Asunto(s)
Lisosomas/metabolismo , Defectos del Tubo Neural/embriología , Tubo Neural/embriología , Receptores Acoplados a Proteínas G/metabolismo , Nexinas de Clasificación/metabolismo , Vía de Señalización Wnt , Animales , Humanos , Lisosomas/genética , Lisosomas/patología , Ratones , Ratones Noqueados , Tubo Neural/patología , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/patología , Receptores Acoplados a Proteínas G/genética , Nexinas de Clasificación/genética
3.
Hum Mutat ; 42(4): 392-407, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33382518

RESUMEN

Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.


Asunto(s)
Cinesinas , Escoliosis , Pez Cebra , Animales , Cilios/metabolismo , Humanos , Cinesinas/genética , Mutación , Escoliosis/genética , Pez Cebra/genética , Proteínas de Pez Cebra
4.
Dev Biol ; 464(1): 24-34, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32446700

RESUMEN

Development of the craniofacial structures requires the precise differentiation of cranial neural crest cells into osteoblasts or chondrocytes. Here, we explore the epigenetic and non-epigenetic mechanisms that are required for the development of craniofacial chondrocytes. We previously demonstrated that the acetyltransferase activity of the highly conserved acetyltransferase GCN5, or KAT2A, is required for murine craniofacial development. We show that Gcn5 is required cell autonomously in the cranial neural crest. Moreover, GCN5 is required for chondrocyte development following the arrival of the cranial neural crest within the pharyngeal arches. Using a combination of in vivo and in vitro inhibition of GCN5 acetyltransferase activity, we demonstrate that GCN5 is a potent activator of chondrocyte maturation, acting to control chondrocyte maturation and size increase during pre-hypertrophic maturation to hypertrophic chondrocytes. Rather than acting as an epigenetic regulator of histone H3K9 acetylation, our findings suggest GCN5 primarily acts as a non-histone acetyltransferase to regulate chondrocyte development. Here, we investigate the contribution of GCN5 acetylation to the activity of the mTORC1 pathway. Our findings indicate that GCN5 acetylation is required for activation of this pathway, either via direct activation of mTORC1 or through indirect mechanisms. We also investigate one possibility of how mTORC1 activity is regulated through RAPTOR acetylation, which is hypothesized to enhance mTORC1 downstream phosphorylation. This study contributes to our understanding of the specificity of acetyltransferases, and the cell type specific roles in which these enzymes function.


Asunto(s)
Movimiento Celular , Condrocitos/enzimología , Transducción de Señal , Cráneo/embriología , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Condrocitos/citología , Histonas/genética , Histonas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/citología , Cresta Neural/embriología , Cráneo/citología , Factores de Transcripción p300-CBP/genética
5.
Development ; 143(7): 1192-204, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-26903501

RESUMEN

The transcription factor grainyhead-like 2 (GRHL2) is expressed in non-neural ectoderm (NNE) and Grhl2 loss results in fully penetrant cranial neural tube defects (NTDs) in mice. GRHL2 activates expression of several epithelial genes; however, additional molecular targets and functional processes regulated by GRHL2 in the NNE remain to be determined, as well as the underlying cause of the NTDs in Grhl2 mutants. Here, we find that Grhl2 loss results in abnormal mesenchymal phenotypes in the NNE, including aberrant vimentin expression and increased cellular dynamics that affects the NNE and neural crest cells. The resulting loss of NNE integrity contributes to an inability of the cranial neural folds to move toward the midline and results in NTD. Further, we identified Esrp1, Sostdc1, Fermt1, Tmprss2 and Lamc2 as novel NNE-expressed genes that are downregulated in Grhl2 mutants. Our in vitro assays show that they act as suppressors of the epithelial-to-mesenchymal transition (EMT). Thus, GRHL2 promotes the epithelial nature of the NNE during the dynamic events of neural tube formation by both activating key epithelial genes and actively suppressing EMT through novel downstream EMT suppressors.


Asunto(s)
Transición Epitelial-Mesenquimal/genética , Cresta Neural/embriología , Tubo Neural/embriología , Factores de Transcripción/genética , Animales , Cadherinas/metabolismo , Línea Celular , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Ectodermo/embriología , Ectodermo/metabolismo , Técnicas de Cultivo de Embriones , Mesodermo/citología , Mesodermo/embriología , Ratones , Ratones Noqueados , Defectos del Tubo Neural/genética , Neurulación/fisiología , Factores de Transcripción/biosíntesis , Vimentina/biosíntesis
6.
J Neurosci ; 37(10): 2565-2579, 2017 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-28154153

RESUMEN

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.


Asunto(s)
Diencéfalo/anatomía & histología , Diencéfalo/metabolismo , Transducción de Señal/fisiología , Tretinoina/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Diencéfalo/embriología , Activación Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/fisiología
7.
Dev Biol ; 416(2): 279-85, 2016 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-27343896

RESUMEN

The embryonic brain and spinal cord initially form through the process of neural tube closure (NTC). NTC is thought to be highly similar between rodents and humans, and studies of mouse genetic mutants have greatly increased our understanding of the molecular basis of NTC with relevance for human neural tube defects. In addition, studies using amphibian and chick embryos have shed light into the cellular and tissue dynamics underlying NTC. However, the dynamics of mammalian NTC has been difficult to study due to in utero development until recently when advances in mouse embryo ex vivo culture techniques along with confocal microscopy have allowed for imaging of mouse NTC in real time. Here, we have performed live imaging of mouse embryos with a particular focus on the non-neural ectoderm (NNE). Previous studies in multiple model systems have found that the NNE is important for proper NTC, but little is known about the behavior of these cells during mammalian NTC. Here we utilized a NNE-specific genetic labeling system to assess NNE dynamics during murine NTC and identified different NNE cell behaviors as the cranial region undergoes NTC. These results bring valuable new insight into regional differences in cellular behavior during NTC that may be driven by different molecular regulators and which may underlie the various positional disruptions of NTC observed in humans with neural tube defects.


Asunto(s)
Ectodermo/fisiología , Tubo Neural/embriología , Animales , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Técnicas de Cultivo de Embriones , Células Epiteliales/ultraestructura , Femenino , Microscopía Intravital , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neurulación/fisiología , Seudópodos/ultraestructura
8.
Dev Biol ; 404(2): 76-87, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26025922

RESUMEN

Locomotion and respiration require motor axon connectivity and activation of the neuromuscular junction (NMJ). Through a forward genetic screen for muscle weakness, we recently reported an allele of ryanodine receptor type 1 (Ryr1(AG)). Here we reveal a role for functional RyR1 during acetylcholine receptor (AChR) cluster formation and embryonic synaptic transmission. Ryr1(AG) homozygous embryos are non-motile. Motor axons extend past AChR clusters and enlarged AChR clusters are found under fasciculated nerves. Using physiological and pharmacological methods, we show that contractility can be resumed through the masking of a potassium leak, and evoked vesicular release can be resumed via bypassing the defect in RyR1 induced calcium release. Moreover, we show the involvement of ryanodine receptors in presynaptic release at the NMJ. This data provides evidence of a role for RyR1 on both the pre- and postsynaptic sides of the NMJ.


Asunto(s)
Músculo Esquelético/fisiología , Unión Neuromuscular/embriología , Receptores Colinérgicos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Transmisión Sináptica/fisiología , Animales , Calcio/metabolismo , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/metabolismo , Contracción Muscular/fisiología , Debilidad Muscular/patología , Músculo Esquelético/embriología , Tejido Nervioso/metabolismo , Unión Neuromuscular/fisiología , Potasio/metabolismo , Respiración
9.
Dev Dyn ; 244(6): 736-47, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25774014

RESUMEN

BACKGROUND: Cilia are important for Hedgehog signaling in vertebrates and many genes that encode proteins involved in ciliogenesis have been studied for their roles in embryonic development. Null mutations in many of these genes cause early embryonic lethality, hence an understanding of their roles in postnatal development is limited. RESULTS: The Inturned (Intu) gene is required for ciliogenesis and here we report a recessive hypomorphic mutation, resulting in substitution of a conserved hydrophobic residue (I813N) near the C-terminus, that sheds light on later functions of Intu. Mice homozygous for this Double-thumb (Intu(Dtm)) allele exhibit polydactyly, retarded growth, and reduced survival. There is a moderate loss of cilia in Intu(Dtm/Dtm) mutants, and Intu(I813N) exhibits compromised ability to increase ciliogenesis in cultured Intu null mutant cells. Intu(Dtm) mutants show rib defects and delay of endochondral ossification in long bones, digits, vertebrae, and the sternum. These skeletal defects correlate with a decrease in Hh signaling. However, patterning of the neural tube and planar cell polarity appear to be normal. CONCLUSIONS: This hypomorphic Intu allele highlights an important role of Intu in mouse skeletal development.


Asunto(s)
Anomalías Múltiples/genética , Proteínas de la Membrana/fisiología , Mutación Missense , Osteogénesis/genética , Mutación Puntual , Anomalías Múltiples/embriología , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Huesos/anomalías , Huesos/embriología , Polaridad Celular , Células Cultivadas , Cilios/ultraestructura , Trastornos del Crecimiento/genética , Proteínas Hedgehog/fisiología , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Defectos del Tubo Neural/genética , Receptores Patched , Polidactilia/embriología , Polidactilia/genética , Estructura Terciaria de Proteína , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/genética
10.
Dev Biol ; 395(1): 84-95, 2014 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-25176044

RESUMEN

Peroxisome biogenesis disorders (PBD) are autosomal recessive disorders in humans characterized by skeletal, eye and brain abnormalities. Despite the fact that neurological deficits, including peripheral nervous system (PNS) defects, can be observed at birth in some PBD patients including those with PEX10 mutations, the embryological basis of the PNS defects is unclear. Using a forward genetic screen, we identified a mouse model for Pex10 deficiency that exhibits neurological abnormalities during fetal development. Homozygous Pex10 mutant mouse embryos display biochemical abnormalities related to a PBD deficiency. During late embryogenesis, Pex10 homozygous mutant mice experience progressive loss of movement and at birth they become cyanotic and die shortly thereafter. Homozygous Pex10 mutant fetuses display decreased integrity of axons and synapses, over-extension of axons in the diaphragm and decreased Schwann cell numbers. Our neuropathological, molecular and electrophysiological studies provide new insights into the embryological basis of the PNS deficits in a PBD model. Our findings identify PEX10 function, and likely other PEX proteins, as an essential component of the spinal locomotor circuit.


Asunto(s)
Modelos Animales de Enfermedad , Embrión de Mamíferos/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Trastorno Peroxisomal/genética , Receptores Citoplasmáticos y Nucleares/genética , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/ultraestructura , Humanos , Inmunohistoquímica , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Actividad Motora/genética , Placa Motora/fisiología , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Enfermedades del Sistema Nervioso Periférico/embriología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Peroxinas , Trastorno Peroxisomal/embriología , Trastorno Peroxisomal/metabolismo , Peroxisomas/metabolismo , Peroxisomas/ultraestructura , Receptores Citoplasmáticos y Nucleares/metabolismo , Nervio Ciático/embriología , Nervio Ciático/metabolismo , Homología de Secuencia de Aminoácido , Enfermedades de la Médula Espinal/embriología , Enfermedades de la Médula Espinal/genética , Enfermedades de la Médula Espinal/metabolismo
11.
Dev Biol ; 373(2): 267-80, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-23195221

RESUMEN

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with ß-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell-cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.


Asunto(s)
Comunicación Celular , Células Epiteliales/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/citología , Pulmón/embriología , Mamíferos/embriología , Morfogénesis , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Animales , Comunicación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Epitelio/embriología , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen , Imagenología Tridimensional , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Modelos Biológicos , Morfogénesis/efectos de los fármacos , Morfolinos/farmacología , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , beta Catenina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
12.
Nat Commun ; 15(1): 5136, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38879605

RESUMEN

Coordination of neuronal differentiation with expansion of the neuroepithelial/neural progenitor cell (NEPC/NPC) pool is essential in early brain development. Our in vitro and in vivo studies identify independent and opposing roles for two neural-specific and differentially expressed non-coding RNAs derived from the same locus: the evolutionarily conserved lncRNA Rncr3 and the embedded microRNA miR124a-1. Rncr3 regulates NEPC/NPC proliferation and controls the biogenesis of miR124a, which determines neuronal differentiation. Rncr3 conserved exons 2/3 are cytosine methylated and bound by methyl-CpG binding protein MeCP2, which restricts expression of miR124a embedded in exon 4 to prevent premature neuronal differentiation, and to orchestrate proper brain growth. MeCP2 directly binds cytosine-methylated Rncr3 through previously unrecognized lysine residues and suppresses miR124a processing by recruiting PTBP1 to block access of DROSHA-DGCR8. Thus, miRNA processing is controlled by lncRNA m5C methylation along with the defined m5C epitranscriptomic RNA reader protein MeCP2 to coordinate brain development.


Asunto(s)
Proteína 2 de Unión a Metil-CpG , MicroARNs , Células-Madre Neurales , Neurogénesis , ARN Largo no Codificante , MicroARNs/metabolismo , MicroARNs/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Neurogénesis/genética , Animales , Ratones , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Encéfalo/metabolismo , Encéfalo/embriología , Humanos , Diferenciación Celular , Metilación de ADN , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proliferación Celular , Ratones Endogámicos C57BL , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Masculino , Exones/genética , Neuronas/metabolismo , Ribonucleasa III
13.
Hum Mol Genet ; 19(11): 2251-67, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20223754

RESUMEN

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.


Asunto(s)
Pulmón/embriología , Morfogénesis/genética , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Mucosa Respiratoria/metabolismo , Transducción de Señal/genética , Animales , Polaridad Celular/genética , Polaridad Celular/fisiología , Immunoblotting , Inmunohistoquímica , Ratones , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Oligonucleótidos/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Mucosa Respiratoria/embriología
14.
Hum Mol Genet ; 18(23): 4565-75, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19776033

RESUMEN

Meckel syndrome (MKS) is a rare autosomal recessive disease causing perinatal lethality associated with a complex syndrome that includes occipital meningoencephalocele, hepatic biliary ductal plate malformation, postaxial polydactyly and polycystic kidneys. The gene mutated in type 1 MKS encodes a protein associated with the base of the cilium in vertebrates and nematodes. However, shRNA knockdown studies in cell culture have reported conflicting results on the role of Mks1 in ciliogenesis. Here we show that loss of function of mouse Mks1 results in an accurate model of human MKS, with structural abnormalities in the neural tube, biliary duct, limb patterning, bone development and the kidney that mirror the human syndrome. In contrast to cell culture studies, loss of Mks1 in vivo does not interfere with apical localization of epithelial basal bodies but rather leads to defective cilia formation in most, but not all, tissues. Analysis of patterning in the neural tube and the limb demonstrates altered Hedgehog (Hh) pathway signaling underlies some MKS defects, although both tissues show an expansion of the domain of response to Shh signaling, unlike the phenotypes seen in other mutants with cilia loss. Other defects in the skull, lung, rib cage and long bones are likely to be the result of the disruption of Hh signaling, and the basis of defects in the liver and kidney require further analysis. Thus the disruption of Hh signaling can explain many, but not all, of the defects caused by loss of Mks1.


Asunto(s)
Anomalías Múltiples/metabolismo , Cilios/fisiología , Modelos Animales de Enfermedad , Proteínas Hedgehog/metabolismo , Ratones , Proteínas/metabolismo , Transducción de Señal , Anomalías Múltiples/embriología , Anomalías Múltiples/genética , Anomalías Múltiples/fisiopatología , Animales , Cilios/genética , Femenino , Técnicas de Silenciamiento del Gen , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas/genética
15.
Front Cell Dev Biol ; 8: 599890, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33324648

RESUMEN

Wnt signaling pathway plays indispensable roles in embryonic development and adult tissue homeostasis. However, the regulatory mechanisms involved in Wnt ligand trafficking within and secretion from the signal sending cells is still relatively uncharacterized. Here, we discover a novel regulator of Wnt signaling pathway called transmembrane protein 132A (TMEM132A). Our evidence shows a physical and functional interaction of TMEM132A with the Wnt ligand transporting protein Wntless (WLS). We show that TMEM132A stabilizes Wnt ligand, enhances WLS-Wnt ligand interaction, and activates the Wnt signaling pathway. Our results shed new light on the cellular mechanism underlying the fundamental aspect of WNT secretion from Wnt signal sending cells.

16.
J Dev Biol ; 6(4)2018 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-30424580

RESUMEN

Cranial neural crest cells undergo cellular growth, patterning, and differentiation within the branchial arches to form cartilage and bone, resulting in a precise pattern of skeletal elements forming the craniofacial skeleton. However, it is unclear how cranial neural crest cells are regulated to give rise to the different shapes and sizes of the bone and cartilage. Epigenetic regulators are good candidates to be involved in this regulation, since they can exert both broad as well as precise control on pattern formation. Here, we investigated the role of the histone acetyltransferases Kat2a and Kat2b in craniofacial development using TALEN/CRISPR/Cas9 mutagenesis in zebrafish and the Kat2ahat/hat (also called Gcn5) allele in mice. kat2a and kat2b are broadly expressed during embryogenesis within the central nervous system and craniofacial region. Single and double kat2a and kat2b zebrafish mutants have an overall shortening and hypoplastic nature of the cartilage elements and disruption of the posterior ceratobranchial cartilages, likely due to smaller domains of expression of both cartilage- and bone-specific markers, including sox9a and col2a1, and runx2a and runx2b, respectively. Similarly, in mice we observe defects in the craniofacial skeleton, including hypoplastic bone and cartilage and altered expression of Runx2 and cartilage markers (Sox9, Col2a1). In addition, we determined that following the loss of Kat2a activity, overall histone 3 lysine 9 (H3K9) acetylation, the main epigenetic target of Kat2a/Kat2b, was decreased. These results suggest that Kat2a and Kat2b are required for growth and differentiation of craniofacial cartilage and bone in both zebrafish and mice by regulating H3K9 acetylation.

17.
G3 (Bethesda) ; 8(8): 2663-2672, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-29930198

RESUMEN

Idiopathic scoliosis (IS) is a structural lateral spinal curvature of ≥10° that affects up to 3% of otherwise healthy children and can lead to life-long problems in severe cases. It is well-established that IS is a genetic disorder. Previous studies have identified genes that may contribute to the IS phenotype, but the overall genetic etiology of IS is not well understood. We used exome sequencing to study five multigenerational families with IS. Bioinformatic analyses identified unique and low frequency variants (minor allele frequency ≤5%) that were present in all sequenced members of the family. Across the five families, we identified a total of 270 variants with predicted functional consequences in 246 genes, and found that eight genes were shared by two families. We performed GO term enrichment analyses, with the hypothesis that certain functional annotations or pathways would be enriched in the 246 genes identified in our IS families. Using three complementary programs to complete these analyses, we identified enriched categories that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.


Asunto(s)
Citoesqueleto de Actina/genética , Matriz Extracelular/genética , Microtúbulos/genética , Escoliosis/genética , Adulto , Niño , Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Microtúbulos/metabolismo , Linaje , Polimorfismo Genético , Escoliosis/etiología , Escoliosis/patología
18.
Am Nat ; 169(1): 105-17, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17206589

RESUMEN

Understanding the mechanisms by which parallel evolution occurs has the potential to clarify the complex relationship between evolution and development. In this study, we examine the role of development in the repeated reduction of zeugopod elements during mammalian evolution, a functionally important phenomenon enabling locomotor specialization. By completing a morphometric study (incorporating both analyses of variation and phylogenetics) of mammalian limbs, we are able to demonstrate an evolutionary trend toward width reduction in posterior zeugopod elements of the forelimbs and hindlimbs, the ulna and fibula, respectively. We also examine the developmental basis of limb reduction in three test cases, the bat Carollia perspicillata ulna and fibula and the mouse Mus musculus fibula. The most common pattern of reduction, that of reduced element width, was achieved via the same developmental process in both bat and mouse limbs (i.e., by a slower growth rate relative to other skeletal elements), suggesting that the parallel reduction of the posterior zeugopod element within mammals could have occurred primarily by the repeated evolution of the same developmental mechanism. However, our findings also suggest that the developmental mechanisms behind the parallel evolution of other, more taxon-specific characteristics of limb reduction (i.e., element fusion) are not conserved.


Asunto(s)
Quirópteros/anatomía & histología , Extremidades/anatomía & histología , Ratones/anatomía & histología , Animales , Evolución Biológica , Quirópteros/embriología , Ratones/embriología
19.
J Orthop Res ; 23(5): 1112-9, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15936916

RESUMEN

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, "patched" (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.


Asunto(s)
Disco Intervertebral/embriología , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/fisiología , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Femenino , Proteínas Hedgehog , Ratones , Factores de Transcripción Paired Box , ARN Mensajero/análisis , Transactivadores/genética , Factores de Transcripción/genética
20.
J Neurosci Methods ; 238: 95-104, 2014 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-25251554

RESUMEN

Signals from nerve and muscle regulate the formation of synapses. Transgenic mouse models and muscle cell cultures have elucidated the molecular mechanisms required for aggregation and stabilization of synaptic structures. However, far less is known about the molecular pathways involved in redistribution of muscle synaptic components. Here we established a physiologically viable whole-muscle embryonic explant system, in the presence or absence of the nerve, which demonstrates the synaptic landscape is dynamic and malleable. Manipulations of factors intrinsic to the muscle or extrinsically provided by the nerve illustrate vital functions during formation, redistribution and elimination of acetylcholine receptor (AChR) clusters. In particular, RyR1 activity is an important mediator of these functions. This physiologically relevant and readily accessible explant system provides a new approach to genetically uncouple nerve-derived signals and for manipulation via signaling molecules, drugs, and electrical stimulation to examine early formation of the neuromuscular circuit.


Asunto(s)
Diafragma/embriología , Diafragma/fisiología , Sinapsis/fisiología , Animales , Estimulación Eléctrica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones Transgénicos , Nervio Frénico/embriología , Nervio Frénico/fisiología , Receptores Colinérgicos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Wnt3A/metabolismo
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