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1.
FASEB J ; 36(3): e22176, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35129866

RESUMEN

To gain further insight into chromatin-mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome-associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male-to-female sex reversal, and that both of these proteins regulate co-transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre-Sertoli cells again suggests a role at the chromatin-spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a-mutant pre-Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7-mutant pre-Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway-associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin-mediated regulatory mechanisms in these processes.


Asunto(s)
Empalme Alternativo , Síndrome CHARGE/genética , Proteínas de Unión al ADN/metabolismo , Proteínas/metabolismo , Procesos de Determinación del Sexo , Transcriptoma , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas/genética , Células de Sertoli/metabolismo , Espermatogénesis , Porcinos
2.
EMBO Rep ; 22(6): e50958, 2021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-33900016

RESUMEN

Mutations in the chromatin remodeller-coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention-deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7-/- model. chd7-/- mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype-based screen in chd7-/- zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies.


Asunto(s)
Trastorno del Espectro Autista , Animales , Caenorhabditis elegans , Cromatina , ADN Helicasas , Proteínas de Unión al ADN/genética , Neuronas GABAérgicas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Mutación , Pez Cebra
3.
PLoS Genet ; 16(9): e1009008, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32898154

RESUMEN

Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.


Asunto(s)
ARN Helicasas DEAD-box/genética , Enfermedad de Hirschsprung/genética , Antígenos de Histocompatibilidad Menor/genética , Animales , ARN Helicasas DEAD-box/metabolismo , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/metabolismo , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Enfermedad de Hirschsprung/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Ratones , Antígenos de Histocompatibilidad Menor/metabolismo , Mutagénesis Insercional , Mutación , Cresta Neural/metabolismo , Factores Sexuales , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba
4.
Int J Mol Sci ; 24(15)2023 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-37569849

RESUMEN

The enteric nervous system (ENS), known as the intrinsic nervous system of the gastrointestinal tract, is composed of a diverse array of neuronal and glial cell subtypes. Fascinating questions surrounding the generation of cellular diversity in the ENS have captivated ENS biologists for a considerable time, particularly with recent advancements in cell type-specific transcriptomics at both population and single-cell levels. However, the current focus of research in this field is predominantly restricted to the study of enteric neuron subtypes, while the investigation of enteric glia subtypes significantly lags behind. Despite this, enteric glial cells (EGCs) are increasingly recognized as equally important regulators of numerous bowel functions. Moreover, a subset of postnatal EGCs exhibits remarkable plasticity and multipotency, distinguishing them as critical entities in the context of advancing regenerative medicine. In this review, we aim to provide an updated overview of the current knowledge on this subject, while also identifying key questions that necessitate future exploration.

5.
Int J Mol Sci ; 23(22)2022 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-36430161

RESUMEN

The first volume of this Special Issue met its goal of covering several aspects regarding both the normal and abnormal development of neural crest cells, which form a truly unique multipotent and highly migratory cell population that only exists in vertebrates [...].


Asunto(s)
Cresta Neural , Neurogénesis , Animales , Movimiento Celular , Vertebrados
6.
Gastroenterology ; 159(5): 1824-1838.e17, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32687927

RESUMEN

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. METHODS: We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. RESULTS: GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. CONCLUSIONS: GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.


Asunto(s)
Colon/efectos de los fármacos , Colon/inervación , Sistema Nervioso Entérico/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Enfermedad de Hirschsprung/tratamiento farmacológico , Regeneración Nerviosa/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Animales , Colon/microbiología , Colon/patología , Modelos Animales de Enfermedad , Disbiosis , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/fisiopatología , Microbioma Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Enfermedad de Hirschsprung/fisiopatología , Humanos , Absorción Intestinal/efectos de los fármacos , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Permeabilidad , Recuperación de la Función , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Células de Schwann/patología , Técnicas de Cultivo de Tejidos
7.
Proc Natl Acad Sci U S A ; 115(4): E620-E629, 2018 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-29311329

RESUMEN

CHARGE syndrome-which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies-is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in CHD7 (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for CHD7 mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for CHD7 mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of Fam172a (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both CHD7 mutation-positive and CHD7 mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.


Asunto(s)
Empalme Alternativo , Síndrome CHARGE/etiología , Modelos Animales de Enfermedad , Proteínas/genética , Animales , Antibióticos Antineoplásicos/uso terapéutico , Proteínas Argonautas/metabolismo , Síndrome CHARGE/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Cresta Neural/embriología , Embarazo , Conejos , Ratas , Sirolimus/uso terapéutico
8.
Int J Mol Sci ; 22(23)2021 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-34884944

RESUMEN

Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.


Asunto(s)
Colágeno Tipo VI/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Enfermedad de Hirschsprung/tratamiento farmacológico , Enfermedad de Hirschsprung/genética , Animales , Modelos Animales de Enfermedad , Enema , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Medicina Regenerativa , Resultado del Tratamiento
9.
Development ; 143(8): 1363-74, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26952979

RESUMEN

Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of post-head tissues. In urochordate embryos, Cdx loss-of-function has been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that circumvents Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we provide in vivo evidence that murine Cdx proteins impact melanocyte and enteric nervous system development by, at least in part, directly controlling the expression of the key early regulators of NC ontogenesis Pax3,Msx1 and Foxd3 Our work thus reveals a novel role for Cdx proteins at the top of the trunk NC gene regulatory network in the mouse, which appears to have been inherited from their ancestral ortholog.


Asunto(s)
Redes Reguladoras de Genes , Cresta Neural/citología , Animales , Diferenciación Celular/genética , Factores de Transcripción Forkhead/genética , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1/genética , Melanocitos/citología , Ratones , Modelos Biológicos , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/genética
10.
PLoS Genet ; 11(3): e1005093, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25786024

RESUMEN

Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line-named TashT-that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung's disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a "tipping point" of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung's disease but also provides important new insights into its male sex bias.


Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/patología , Ratones , Mutagénesis Insercional , Animales , Cromosomas de los Mamíferos , Sistema Nervioso Entérico/anomalías , Enfermedad de Hirschsprung/embriología , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/metabolismo , Elementos Silenciadores Transcripcionales , Transcriptoma
11.
Am J Med Genet A ; 173(11): 3070-3074, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28898547

RESUMEN

Isolated congenital diaphragmatic hernia is often a sporadic event with a low recurrence risk. However, underlying genetic etiologies, such as chromosome anomalies or single gene disorders, are identified in a small number of individuals. We describe two fetuses with a unique pattern of multiple congenital anomalies, including diaphragmatic hernia, short bowel and asplenia, born to first-cousin parents. Whole exome sequencing showed that both were homozygous for a missense variant, c.950A>C, predicting p.Asp317Ala, in the H.20-Like Homeobox 1 (HLX1) gene. HLX is a homeobox transcription factor gene which is relatively conserved across species. Hlx homozygous null mice have a short bowel and reduced muscle cells in the diaphragm, closely resembling the anomalies in the two fetuses and we therefore suggest that the HLX mutation in this family could explain the fetal findings.


Asunto(s)
Hernias Diafragmáticas Congénitas/genética , Síndrome de Heterotaxia/genética , Proteínas de Homeodominio/genética , Síndrome del Intestino Corto/genética , Factores de Transcripción/genética , Anomalías Múltiples/genética , Anomalías Múltiples/fisiopatología , Animales , Anomalías del Sistema Digestivo/genética , Anomalías del Sistema Digestivo/fisiopatología , Predisposición Genética a la Enfermedad , Hernias Diafragmáticas Congénitas/fisiopatología , Síndrome de Heterotaxia/fisiopatología , Humanos , Ratones , Mutación , Análisis de Secuencia de ADN , Síndrome del Intestino Corto/fisiopatología , Secuenciación del Exoma
12.
Biochim Biophys Acta ; 1839(7): 546-58, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24815547

RESUMEN

Pax3 encodes a paired-box transcription factor with key roles in neural crest and neural tube ontogenesis. Robust control of Pax3 neural expression is ensured by two redundant sets of cis-regulatory modules (CRMs) that integrate anterior-posterior (such as Wnt-ßCatenin signaling) as well as dorsal-ventral (such as Shh-Gli signaling) instructive cues. In previous work, we sought to characterize the Wnt-mediated regulation of Pax3 expression and identified the Cdx transcription factors (Cdx1/2/4) as critical intermediates in this process. We identified the neural crest enhancer-2 (NCE2) from the 5'-flanking region of Pax3 as a Cdx-dependent CRM that recapitulates the restricted expression of Pax3 in the mouse caudal neuroectoderm. While this is consistent with a key role in relaying the inductive signal from posteriorizing Wnt ligands, the broad expression of Cdx proteins in the tailbud region is not consistent with the restricted activity of NCE2. This implies that other positive and/or negative inputs are required and, here, we report a novel role for the transcription factor Zic2 in this regulation. Our data strongly suggests that Zic2 is involved in the induction (as a direct Pax3NCE2 activator and Cdx neural cofactor) as well as the maintenance of Pax3 dorsal restriction (as a target of the ventral Shh repressive input). We also provide evidence that the inductive Cdx-Zic2 interaction is integrated on NCE2 with a positive input from the neural-specific transcription factor Sox2. Altogether, our data provide important mechanistic insights into the coordinated integration of different signaling pathways on a short Pax3 CRM.


Asunto(s)
Elementos de Facilitación Genéticos , Tubo Neural/crecimiento & desarrollo , Factores de Transcripción Paired Box/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Humanos , Ratones , Cresta Neural/crecimiento & desarrollo , Tubo Neural/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/biosíntesis , Factores de Transcripción SOXB1/genética , Transducción de Señal/genética , Vía de Señalización Wnt/genética
13.
Sci Rep ; 14(1): 11063, 2024 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-38744932

RESUMEN

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.


Asunto(s)
Inmunidad Adaptativa , Colon , Citometría de Flujo , Inmunidad Innata , Animales , Colon/inmunología , Colon/metabolismo , Ratones , Citometría de Flujo/métodos , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/metabolismo
14.
J Bone Miner Res ; 39(4): 498-512, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38477756

RESUMEN

Mutations in the Chromodomain helicase DNA-binding protein 7 - coding gene (CHD7) cause CHARGE syndrome (CS). Although craniofacial and skeletal abnormalities are major features of CS patients, the role of CHD7 in bone and cartilage development remain largely unexplored. Here, using a zebrafish (Danio rerio) CS model, we show that chd7-/- larvae display abnormal craniofacial cartilage development and spinal deformities. The craniofacial and spine defects are accompanied by a marked reduction of bone mineralization. At the molecular level, we show that these phenotypes are associated with significant reduction in the expression levels of osteoblast differentiation markers. Additionally, we detected a marked depletion of collagen 2α1 in the cartilage of craniofacial regions and vertebrae, along with significantly reduced number of chondrocytes. Chondrogenesis defects are at least in part due to downregulation of htr2b, which we found to be also dysregulated in human cells derived from an individual with CHD7 mutation-positive CS. Overall, this study thus unveils an essential role for CHD7 in cartilage and bone development, with potential clinical relevance for the craniofacial defects associated with CS.


Patients with CHARGE syndrome exhibit skeletal defects. CHARGE syndrome is primarily caused by mutations in the chromatin remodeler-coding gene CHD7. To investigate the poorly characterized role of CHD7 in cartilage and bone development, here, we examine the craniofacial and bone anomalies in a zebrafish chd7-/- mutant model. We find that zebrafish mutant larvae exhibit striking dysmorphism of craniofacial structures and spinal deformities. Notably, we find a significant reduction in osteoblast, chondrocyte, and collagen matrix markers. This work provides important insights to improve our understanding of the role of chd7 in skeletal development.


Asunto(s)
Cartílago , ADN Helicasas , Proteínas de Pez Cebra , Pez Cebra , Animales , Humanos , Cartílago/metabolismo , Síndrome CHARGE/genética , Síndrome CHARGE/metabolismo , Síndrome CHARGE/patología , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Cráneo/metabolismo , Pez Cebra/metabolismo , Pez Cebra/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
15.
Genesis ; 51(11): 777-84, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23913642

RESUMEN

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal-related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R-YFP reporter mice have indicated that the Cdx2NSE-Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double-transgenic embryos has revealed that this novel Cre-driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso-ventral axis (including pre-migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non-cranial neural crest derivatives of double-transgenic embryos. Therefore, we believe that the Cdx2NSE-Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems.


Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Ratones Transgénicos , Placa Neural/embriología , Tubo Neural/embriología , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Factor de Transcripción CDX2 , Embrión de Mamíferos , Genes Reporteros , Proteínas de Homeodominio/genética , Integrasas/genética , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Animales , Cresta Neural/citología , Cresta Neural/embriología , Placa Neural/metabolismo , Factores de Transcripción/genética
16.
J Biol Chem ; 287(20): 16623-35, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22457346

RESUMEN

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·ß-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.


Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Proteínas de Homeodominio/metabolismo , Cresta Neural/embriología , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Factor de Transcripción CDX2 , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/genética , Factores de Transcripción/genética
17.
Dev Dyn ; 241(7): 1192-204, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22539273

RESUMEN

BACKGROUND: The SRY/Sry gene is expressed in pre-Sertoli cells of the male genital ridge and functions as the mammalian testis determining factor (TDF). In addition, expression of SRY/Sry outside the genital ridge has been reported, including preimplantation embryos, although the functional significance of this is not well understood. RESULTS: Using Cre-mediated lineage studies and transgenic reporter mouse models, we now show that promoter sequences of human, pig and mouse SRY drive robust reporter gene expression in epiblast cells of peri-implantation embryos between embryonic day (E) 4.5 and E6.5. Analysis of endogenous Sry expression revealed that linear transcripts are produced by means of multiple polyadenylation sites in E4.5 embryos. Within the epiblast, SRY reporter expression mimics the expression seen using a Gata4 reporter model, but is dissimilar to that seen using an Oct4 reporter model. In addition, we report that overexpression of mouse Sry in embryonic stem cells leads to down-regulation of the core pluripotency markers Sox2 and Nanog. CONCLUSION: We propose that SRY/Sry may function as a male-specific maturation factor in the peri-implantation mammalian embryo, providing a genetic mechanism to help explain the observation that male embryos are developmentally more advanced compared with female embryos, and suggesting a role for SRY beyond that of TDF.


Asunto(s)
Blastocisto/metabolismo , Proteína de la Región Y Determinante del Sexo/metabolismo , Animales , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Proteína de la Región Y Determinante del Sexo/genética , Porcinos
18.
Life Sci Alliance ; 6(8)2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37221016

RESUMEN

CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7 Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on the FAM172A-AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/ß pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.


Asunto(s)
Síndrome CHARGE , Humanos , Transporte Activo de Núcleo Celular , Cromatina , Mutación Missense , Proteínas
19.
Pigment Cell Melanoma Res ; 35(5): 506-516, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35816394

RESUMEN

Waardenburg syndrome type 4 (WS4) combines abnormal development of neural crest cell (NCC)-derived melanocytes (causing depigmentation and inner ear dysfunction) and enteric nervous system (causing aganglionic megacolon). The full spectrum of WS4 phenotype is present in Spot mice, in which an insertional mutation close to a silencer element leads to NCC-specific upregulation of the transcription factor-coding gene Nr2f1. These mice were previously found to develop aganglionic megacolon because of NR2F1-induced premature differentiation of enteric neural progenitors into enteric glia. Intriguingly, this prior work also showed that inner ear dysfunction in Spot mutants specifically affects balance but not hearing, consistent with the absence of melanocytes in the vestibule only. Here, we report an analysis of the effect of Nr2f1 upregulation on the development of both inner ear and skin melanocytes, also taking in consideration their origin relative to the dorsolateral and ventral NCC migration pathways. In the trunk, we found that NR2F1 overabundance in Spot NCCs forces dorso-laterally migrating melanoblasts to abnormally adopt a Schwann cell precursor (SCP) fate and conversely prevents ventrally migrating SCPs to normally adopt a melanoblast fate. In the head, Nr2f1 upregulation appears not to be uniform, which might explain why SCP-derived melanocytes do colonize the cochlea while non-SCP-derived melanocytes cannot reach the vestibule. Collectively, these data point to a key role for NR2F1 in the control of SCP-vs-melanocyte fate choice and unveil a new pathogenic mechanism for WS4. Moreover, our data argue against the proposed existence of a transit-amplifying compartment of melanocyte precursors in hair follicles.


Asunto(s)
Enfermedad de Hirschsprung , Síndrome de Waardenburg , Animales , Diferenciación Celular/genética , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Melanocitos/metabolismo , Ratones , Cresta Neural/metabolismo , Células de Schwann , Síndrome de Waardenburg/genética
20.
Trends Mol Med ; 27(5): 451-468, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33627291

RESUMEN

Neurocristopathies form a heterogeneous group of rare diseases caused by abnormal development of neural crest cells. Heterogeneity of neurocristopathies directly relates to the nature of these migratory and multipotent cells, which generate dozens of specialized cell types throughout the body. Neurocristopathies are thus characterized by congenital malformations of tissues/organs that otherwise appear to have very little in common, such as the craniofacial skeleton and enteric nervous system. Treatment options are currently very limited, mainly consisting of corrective surgeries. Yet, as reviewed here, analyses of normal and pathological neural crest development in model organisms have opened up the possibility for better treatment options involving cellular and molecular approaches. These approaches provide hope that some neurocristopathies might soon be curable or preventable.


Asunto(s)
Malformaciones del Sistema Nervioso/prevención & control , Cresta Neural , Animales , Trasplante de Células , Anomalías Congénitas , Humanos , Modelos Animales , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Cresta Neural/patología , Medicina Regenerativa/métodos , Células Madre/patología
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