RESUMEN
The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.
Asunto(s)
Cromatina/química , Elementos de Facilitación Genéticos/fisiología , Miembro Posterior/embriología , Conformación Molecular , Morfogénesis/genética , Factores de Transcripción Paired Box/fisiología , Animales , Sistemas CRISPR-Cas , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , ADN/química , ADN/metabolismo , Embrión de Mamíferos , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Miembro Posterior/metabolismo , Ratones , Ratones Transgénicos , Conformación de Ácido Nucleico , Factores de Transcripción Paired Box/genéticaRESUMEN
Forelimbs (FLs) and hindlimbs (HLs) develop under the instructive and integrated guidance of signaling centers and transcription factor (TF) action. The development of structures specific to each limb type depends on the limb-specific modulation of these integrated components. Pitx1 is a transcription factor gene expressed in HL, absent in FL, and required for HL-specific patterning and development, in particular for formation of anterior HL skeletal elements. Pitx1 achieves this function by direct TF action on the core limb program, which is largely shared between FL and HL. Shh signaling plays a crucial role in anterior-posterior (AP) patterning in both FL and HL. The present work assessed the relationship between Shh signaling and Pitx1 action for AP patterning. We found that reducing the gene dosage of Shh in the context of the Pitx1-/- HL decreases the severity of the Pitx1-/- phenotype, in particular, the loss of anterior limb structures and the shortening of femur length. However, this did not rescue HL-specific patterning features. Thus, Pitx1 action integrates Shh signaling but not for limb-type-specific patterning.
Asunto(s)
Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiología , Factores de Transcripción Paired Box/metabolismo , Animales , Tipificación del Cuerpo/genética , Extremidades/embriología , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/fisiología , Fenotipo , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Rhabdomyosarcoma is the most common soft-tissue sarcoma in childhood and histologically resembles developing skeletal muscle. Alveolar rhabdomyosarcoma (ARMS) is an aggressive subtype with a higher rate of metastasis and poorer prognosis. The majority of ARMS tumors (80%) harbor a PAX3-FOXO1 or less commonly a PAX7-FOXO1 fusion gene. The presence of either the PAX3-FOXO1 or PAX7-FOXO1 fusion gene foretells a poorer prognosis resulting in clinical re-classification as either fusion-positive (FP-RMS) or fusion-negative RMS (FN-RMS). The PAX3/7-FOXO1 fusion genes result in the production of a rogue transcription factors that drive FP-RMS pathogenesis and block myogenic differentiation. Despite knowing the molecular driver of FP-RMS, targeted therapies have yet to make an impact for patients, highlighting the need for a greater understanding of the molecular consequences of PAX3-FOXO1 and its target genes including microRNAs. Here we show FP-RMS patient-derived xenografts and cell lines display a distinct microRNA expression pattern. We utilized both loss- and gain-of function approaches in human cell lines with knockdown of PAX3-FOXO1 in FP-RMS cell lines and expression of PAX3-FOXO1 in human myoblasts and identified microRNAs both positively and negatively regulated by the PAX3-FOXO1 fusion protein. We demonstrate PAX3-FOXO1 represses miR-221/222 that functions as a tumor suppressing microRNA through the negative regulation of CCND2, CDK6, and ERBB3. In contrast, miR-486-5p is transcriptionally activated by PAX3-FOXO1 and promotes FP-RMS proliferation, invasion, and clonogenic growth. Inhibition of miR-486-5p in FP-RMS xenografts decreased tumor growth, illustrating a proof of principle for future therapeutic intervention. Therefore, PAX3-FOXO1 regulates key microRNAs that may represent novel therapeutic vulnerabilities in FP-RMS.
Asunto(s)
MicroARNs/genética , Neoplasias de los Músculos/genética , Proteínas de Fusión Oncogénica/fisiología , Factores de Transcripción Paired Box/fisiología , Rabdomiosarcoma Alveolar/genética , Animales , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Niño , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Ratones SCID , Análisis por Micromatrices , Neoplasias de los Músculos/patología , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Rabdomiosarcoma Alveolar/patologíaRESUMEN
Complement factor B (cfB) is an essential component of the alternative pathway (AP) and plays an important role in the pathogenesis of polymicrobial sepsis. However, the mechanism leading to cfB production and AP activation during sepsis remains poorly understood. In this study, we found that plasma cell-free RNA was significantly increased following cecal ligation and puncture (CLP), an animal model of polymicrobial sepsis, and was closely associated with sepsis severity. Quantitative RT-PCR and microRNA (miRNA) array analysis revealed an increase in bacterial RNA and multiple host miRNAs (miR-145, miR-146a, miR-122, miR-210) in the blood following CLP. Treatment with tissue RNA or synthetic miRNA mimics (miR-145, miR-146a, miR-122, miR-34a) induced a marked increase in cfB production in cardiomyocytes or macrophages. The newly synthesized cfB released into medium was biologically active because it participated in AP activation initiated by cobra venom factor. Genetic deletion of TLR7 or MyD88, but not TLR3, and inhibition of the MAPKs (JNK and p38) or NF-κB abolished miR-146a-induced cfB production. In vivo, CLP led to a significant increase in splenic cfB expression that correlated with the plasma RNA or miRNA levels. Peritoneal injection of RNA or miR-146a led to an increase in cfB expression in the peritoneal space that was attenuated in MyD88-knockout or TLR7-knockout mice, respectively. These findings demonstrate that host cellular RNA and specific miRNAs are released into the circulation during polymicrobial sepsis and may function as extracellular mediators capable of promoting cfB production and AP activation through specific TLR7 and MyD88 signaling.
Asunto(s)
Factor B del Complemento/metabolismo , Vía Alternativa del Complemento , MicroARNs/administración & dosificación , ARN/administración & dosificación , Sepsis/inmunología , Animales , Biomimética , Ciego/cirugía , Células Cultivadas , Factor B del Complemento/genética , Vía Alternativa del Complemento/genética , Modelos Animales de Enfermedad , Venenos Elapídicos/metabolismo , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/inmunología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/fisiología , ARN/inmunología , Ratas , Transducción de Señal/genética , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
During development of the CNS, stem and progenitor cell proliferation, cell fate designation, and patterning decisions are tightly regulated by interdependent networks of key transcriptional regulators. In a genetic approach we analyzed divergent functionality of the PAI and RED sub-domains of the Pax6 Paired domain (PD) during progenitor zone formation, motor and interneuron development, and peripheral connectivity at distinct levels within the neural tube: within the hindbrain, mutation of the PAI sub-domain severely affected patterning of the p3 and pMN domains and establishment of the corresponding motor neurons. Exit point designation of hypoglossal axons was disturbed in embryos harboring either mutations in the PD sub-domains or containing a functional Pax6 Null allele. At brachial spinal levels, we propose a selective involvement of the PAI sub-domain during patterning of ventral p2 and pMN domains, critically disturbing generation of specific motor neuron subtypes and increasing V2 interneuron numbers. Our findings present a novel aspect of how Pax6 not only utilizes its modular structure to perform distinct functions via its paired and homeodomain. Individual sub-domains can exert distinct functions, generating a new level of complexity for transcriptional regulation by one single transcription factor not only in dorso-ventral, but also rostro-caudal neural tube patterning.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Tubo Neural/embriología , Factores de Transcripción Paired Box/genética , Sistema Nervioso Periférico/embriología , Proteínas Represoras/genética , Alelos , Animales , Axones/metabolismo , Axones/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Linaje de la Célula , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas del Ojo/fisiología , Regulación del Desarrollo de la Expresión Génica , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/fisiología , Inmunohistoquímica , Hibridación in Situ , Interneuronas/metabolismo , Ratones , Neuronas Motoras/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/fisiología , Fenotipo , Estructura Terciaria de Proteína , Proteínas Represoras/fisiología , Rombencéfalo/metabolismo , Células Madre/citología , Factores de Transcripción/genéticaRESUMEN
Like other subclasses within the PAX transcription factor family, PAX3 and PAX7 play important roles in the emergence of a number of different tissues during development. PAX3 regulates neural crest and, together with its orthologue PAX7, is also expressed in parts of the central nervous system. In this chapter we will focus on their role in skeletal muscle. Both factors are key regulators of myogenesis where Pax3 plays a major role during early skeletal muscle formation in the embryo while Pax7 predominates during post-natal growth and muscle regeneration in the adult. We review the expression and functions of these factors in the myogenic context. We also discuss mechanistic aspects of PAX3/7 function and modulation of their activity by interaction with other proteins, as well as the post-transcriptional and transcriptional regulation of their expression.
Asunto(s)
Desarrollo de Músculos/fisiología , Factor de Transcripción PAX7/fisiología , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Humanos , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/fisiologíaRESUMEN
The execution of developmental programs entails specific spatio-temporal expression of transcriptional regulators that ultimately control tissue morphogenesis and embryo patterning. Pax transcription factors are sequence-specific DNA-binding proteins exerting such regulatory activity in several tissues. In the urogenital system, Pax2 and Pax8 have emerged as crucial players at multiple steps of kidney and urinary tract development. They are involved in important processes such as cell survival, cell lineage decisions and tissue interactions through the regulation of sophisticated gene regulatory networks. Pax2/8 have additionally been directly associated with Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) and renal cancers in human. In this review, we provide an overview of landmark contributions to the understanding of Pax gene function in urinary tract development and disease with an emphasis on recent advances in the field.
Asunto(s)
Riñón/fisiología , Factores de Transcripción Paired Box/fisiología , Animales , Humanos , Riñón/embriología , Riñón/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismoRESUMEN
The nine vertebrate PAX transcription factors (PAX1-PAX9) play essential roles during early development and organogenesis. Pax genes were identified in vertebrates using their homology with the Drosophila melanogaster paired gene DNA-binding domain. PAX1-9 functions are largely conserved throughout vertebrate evolution, in particular during central nervous system and neural crest development. The neural crest is a vertebrate invention, which gives rise to numerous derivatives during organogenesis, including neurons and glia of the peripheral nervous system, craniofacial skeleton and mesenchyme, the heart outflow tract, endocrine and pigment cells. Human and mouse spontaneous mutations as well as experimental analyses have evidenced the critical and diverse functions of PAX factors during neural crest development. Recent studies have highlighted the role of PAX3 and PAX7 in neural crest induction. Additionally, several PAX proteins - PAX1, 3, 7, 9 - regulate cell proliferation, migration and determination in multiple neural crest-derived lineages, such as cardiac, sensory, and enteric neural crest, pigment cells, glia, craniofacial skeleton and teeth, or in organs developing in close relationship with the neural crest such as the thymus and parathyroids. The diverse PAX molecular functions during neural crest formation rely on fine-tuned modulations of their transcriptional transactivation properties. These modulations are generated by multiple means, such as different roles for the various isoforms (formed by alternative splicing), or posttranslational modifications which alter protein-DNA binding, or carefully orchestrated protein-protein interactions with various co-factors which control PAX proteins activity. Understanding these regulations is the key to decipher the versatile roles of PAX transcription factors in neural crest development, differentiation and disease.
Asunto(s)
Cresta Neural/fisiología , Factores de Transcripción Paired Box/fisiología , Animales , Humanos , Cresta Neural/embriología , Cresta Neural/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismoAsunto(s)
Anomalías Inducidas por Medicamentos , Contaminantes Ambientales/efectos adversos , Factor de Transcripción PAX7/fisiología , Factores de Transcripción Paired Box/fisiología , Anomalías Inducidas por Medicamentos/etiología , Anomalías Inducidas por Medicamentos/prevención & control , Humanos , Factor de Transcripción PAX3RESUMEN
The esophagus links the oral cavity to the stomach and facilitates the transfer of bolus. Using genetic tracing and mouse mutants, we demonstrate that esophagus striated muscles (ESMs) are not derived from somites but are of cranial origin. Tbx1 and Isl1 act as key regulators of ESMs, which we now identify as a third derivative of cardiopharyngeal mesoderm that contributes to second heart field derivatives and head muscles. Isl1-derived ESM progenitors colonize the mouse esophagus in an anterior-posterior direction but are absent in the developing chick esophagus, thus providing evolutionary insight into the lack of ESMs in avians. Strikingly, different from other myogenic regions, in which embryonic myogenesis establishes a scaffold for fetal fiber formation, ESMs are established directly by fetal myofibers. We propose that ESM progenitors use smooth muscle as a scaffold, thereby bypassing the embryonic program. These findings have important implications in understanding esophageal dysfunctions, including dysphagia, and congenital disorders, such as DiGeorge syndrome.
Asunto(s)
Embrión de Mamíferos/citología , Esófago/embriología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Desarrollo de Músculos/fisiología , Músculo Estriado/embriología , Cráneo/embriología , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Pollos , Embrión de Mamíferos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Corazón/embriología , Técnicas para Inmunoenzimas , Proteínas con Homeodominio LIM/fisiología , Masculino , Ratones , Ratones Noqueados , Cresta Neural/citología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/fisiología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somitos/citología , Proteínas de Dominio T Box/fisiología , Factores de Transcripción/fisiologíaRESUMEN
The embryonic development of the pancreas is orchestrated by a complex and coordinated transcription factor network. Neurogenin3 (Neurog3) initiates the endocrine program by activating the expression of additional transcription factors driving survival, proliferation, maturation and lineage allocation of endocrine precursors. Among the direct targets of Neurog3, Pax4 appears as one of the key regulators of ß-cell specification. Indeed, mice lacking Pax4 die a few days postpartum, as they develop severe hyperglycemia due to the absence of mature pancreatic ß-cells. Pax4 also directly regulates the expression of Arx, a gene that plays a crucial role in α-cell specification. Comparative analysis of Pax4 and Arx mutants, as well as Arx/Pax4 double mutants, showed that islet subtype destiny is mainly directed by cross-repression of the Pax4 and Arx factors. Importantly, the ectopic expression of Pax4 in α-cells was found sufficient to induce their neogenesis and conversion into ß-like cells, not only during development but also in adult rodents. Therefore, differentiated endocrine α-cells can be considered as a putative source for insulin-producing ß-like cells. These findings have clearly widened our understanding regarding pancreatic development, but they also open new research avenues in the context of diabetes research.
Asunto(s)
Proteínas de Homeodominio/fisiología , Factores de Transcripción Paired Box/fisiología , Animales , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Páncreas/embriología , Páncreas/metabolismo , Páncreas/fisiologíaRESUMEN
The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3'-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.
Asunto(s)
Carcinoma Papilar/metabolismo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Yoduros/metabolismo , MicroARNs/fisiología , Proteínas de Neoplasias/fisiología , Factores de Transcripción Paired Box/fisiología , ARN Neoplásico/fisiología , Simportadores/fisiología , Neoplasias de la Tiroides/metabolismo , Regiones no Traducidas 3' , Transporte Biológico , Carcinoma Papilar/patología , Diferenciación Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Humanos , MicroARNs/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/antagonistas & inhibidores , Factores de Transcripción Paired Box/genética , Fenotipo , ARN/metabolismo , Interferencia de ARN , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/patología , TransfecciónRESUMEN
BACKGROUND/AIMS: The lack of available beta cells greatly limits the use of beta cell transplantation as a therapy for diabetes. Thus, generation of beta cells from other sources is substantially required. Pax4 has been shown to induce reprograming of alpha cells into beta cells during embryogenesis. Nevertheless, whether expression of Pax4 in adult alpha cells could trigger this alpha-to-beta cell reprogramming is unknown. METHODS: Here we generated an adeno-associated virus carrying Pax4 and GFP under a CMV promoter (AAV-Pax4). We used AAV-Pax4 to transduce a mouse alpha cell line in vitro, and to transduce primary alpha cells in diabetic mice. Reprogramming was examined by double immunostaining and by changes in beta cell number. The effects on blood glucose were evaluated by fasting blood glucose and glucose response. RESULTS: In vitro, Pax4 overexpression neither induced insulin expression, nor suppressed glucagon expression in alpha cells. In vivo, Pax4 overexpression failed to increase beta cell number, and did not alter hyperglycemia and glucose response in diabetic mice. CONCLUSION: Pax4 expression is not sufficient to transduce pancreatic alpha cells into beta cells. Overexpression of Pax4 in alpha cells may not increase functional beta cell number in diabetic patients.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Homeodominio/fisiología , Factores de Transcripción Paired Box/fisiología , Páncreas/citología , Animales , Glucemia/metabolismo , Línea Celular , Dependovirus/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/genética , Ratones , Factores de Transcripción Paired Box/genética , Páncreas/metabolismoRESUMEN
Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene's function in neuronal development.
Asunto(s)
Proliferación Celular/fisiología , Cerebelo/embriología , Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/fisiología , Animales , Muerte Celular/fisiología , Proteínas del Ojo/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Proteínas de Homeodominio/biosíntesis , Factor 4 Similar a Kruppel , Ratones , Células-Madre Neurales/fisiología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/biosíntesisRESUMEN
PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058-1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-ß signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue.
Asunto(s)
Linaje de la Célula/fisiología , Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Sistema Límbico/citología , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/fisiología , Células Madre/citología , Animales , Córnea/embriología , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Sistema Límbico/metabolismo , Proteínas de la Membrana/genética , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Transducción de Señal , Células Madre/metabolismoRESUMEN
BACKGROUND: Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. RESULTS: Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. CONCLUSIONS: Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes.
Asunto(s)
Corteza Cerebral/citología , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/fisiología , Células-Madre Neurales/citología , Neurogénesis/fisiología , Factores de Transcripción/fisiología , Animales , Recuento de Células , Ciclo Celular , Linaje de la Célula , Corteza Cerebral/anomalías , Corteza Cerebral/embriología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas del Ojo/fisiología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/fisiología , Ratones , Ratones Noqueados , Neurogénesis/genética , Neuronas/clasificación , Neuronas/citología , Nucleosomas/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/fisiología , Proteínas de Dominio T Box/análisis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , TransgenesRESUMEN
Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.
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Aniridia/embriología , Aniridia/genética , Proteínas del Ojo/genética , Proteínas del Ojo/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Mutación , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Xenopus/embriología , Xenopus/genética , Animales , Aniridia/patología , Secuencia de Bases , Codón sin Sentido , ADN/genética , Modelos Animales de Enfermedad , Exones , Ojo/embriología , Ojo/crecimiento & desarrollo , Marcación de Gen , Humanos , Datos de Secuencia Molecular , Mutagénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/deficiencia , Fenotipo , Proteínas Represoras/deficiencia , Especificidad de la EspecieRESUMEN
Exposure to environmental teratogenic pollutant leads to severe birth defects. However, the biological events underlying these developmental abnormalities remain undefined. Here, we report a molecular link between an environmental stress response pathway and key developmental genes during craniofacial development. Strikingly, mutant mice with impaired Pax3/7 function display severe craniofacial defects. We show that these are associated with an upregulation of the signaling pathway mediated by the Aryl hydrocarbon receptor (AHR), the receptor to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), revealing a genetic interaction between Pax3 and AHR signaling. Activation of AHR signaling in Pax3-deficient embryos drives facial mesenchymal cells out of the cell cycle through the upregulation of p21 expression. Accordingly, inhibiting AHR activity rescues the cycling status of these cells and the facial closure of Pax3/7 mutants. Together, our findings demonstrate that the regulation of AHR signaling by Pax3/7 is required to protect against TCDD/AHR-mediated teratogenesis during craniofacial development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Anomalías Congénitas/prevención & control , Anomalías Craneofaciales/prevención & control , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Contaminantes Ambientales/toxicidad , Factor de Transcripción PAX7/fisiología , Factores de Transcripción Paired Box/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Anomalías Congénitas/etiología , Anomalías Craneofaciales/inducido químicamente , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción PAX3 , Dibenzodioxinas Policloradas/análogos & derivados , Dibenzodioxinas Policloradas/toxicidad , Embarazo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Hidrocarburo de Aril/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Idiopathic scoliosis (IS) is a common paediatric musculoskeletal disease that displays a strong female bias. By performing a genome-wide association study (GWAS) of 3,102 individuals, we identify significant associations with 20p11.22 SNPs for females (P=6.89 × 10(-9)) but not males (P=0.71). This association with IS is also found in independent female cohorts from the United States of America and Japan (overall P=2.15 × 10(-10), OR=1.30 (rs6137473)). Unexpectedly, the 20p11.22 IS risk alleles were previously associated with protection from early-onset alopecia, another sexually dimorphic condition. The 174-kb associated locus is distal to PAX1, which encodes paired box 1, a transcription factor involved in spine development. We identify a sequence in the associated locus with enhancer activity in zebrafish somitic muscle and spinal cord, an activity that is abolished by IS-associated SNPs. We thus identify a sexually dimorphic IS susceptibility locus, and propose the first functionally defined candidate mutations in an enhancer that may regulate expression in specific spinal cells.
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Elementos de Facilitación Genéticos , Predisposición Genética a la Enfermedad , Factores de Transcripción Paired Box/genética , Escoliosis/genética , Alelos , Animales , Mapeo Cromosómico , Estudios de Cohortes , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Japón , Desequilibrio de Ligamiento , Masculino , Mutación , Factores de Transcripción Paired Box/fisiología , Fenotipo , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Análisis de Secuencia de ADN , Factores Sexuales , Estados Unidos , Pez CebraRESUMEN
Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox-Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.