RESUMEN
During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.
Asunto(s)
Proteínas Portadoras/fisiología , Movimiento Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/embriología , Neuronas/fisiología , Proteínas Nucleares/fisiología , Semaforinas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Portadoras/genética , Diferenciación Celular/genética , Polaridad Celular/genética , Proteínas de Unión al ADN , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Técnicas de Cultivo de Órganos , Proteínas Represoras , Semaforinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Dorsal horn neurons express many different neuropeptides that modulate sensory perception like the sensation of pain. Inhibitory neurons of the dorsal horn derive from postmitotic neurons that express Pax2, Lbx1 and Lhx1/5, and diversify during maturation. In particular, fractions of maturing inhibitory neurons express various neuropeptides. We demonstrate here that a coordinate molecular mechanism determines inhibitory and peptidergic fate in the developing dorsal horn. A bHLH factor complex that contains Ptf1a acts as upstream regulator and initiates the expression of several downstream transcription factors in the future inhibitory neurons, of which Pax2 is known to determine the neurotransmitter phenotype. We demonstrate here that dynorphin, galanin, NPY, nociceptin and enkephalin expression depends on Ptf1a, indicating that these neuropeptides are expressed in inhibitory neurons. Furthermore, we show that Neurod1/2/6 and Lhx1/5, which act downstream of Ptf1a, control distinct aspects of peptidergic differentiation. In particular, the Neurod1/2/6 factors are essential for dynorphin and galanin expression, whereas the Lhx1/5 factors are essential for NPY expression. We conclude that a transcriptional network operates in maturing dorsal horn neurons that coordinately determines transmitter and peptidergic fate.
Asunto(s)
Neuropéptidos/biosíntesis , Células del Asta Posterior/embriología , Médula Espinal/embriología , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/metabolismo , Células del Asta Posterior/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Insm1 (IA-1) encodes a Zn-finger factor that is expressed in the developing nervous system. We demonstrate here that the development of the sympatho-adrenal lineage is severely impaired in Insm1 mutant mice. Differentiation of sympatho-adrenal precursors, as assessed by the expression of neuronal subtype-specific genes such as Th and Dbh, is delayed in a pronounced manner, which is accompanied by a reduced proliferation. Sympathetic neurons eventually overcome the differentiation blockade and mature correctly, but sympathetic ganglia remain small. By contrast, terminal differentiation of adrenal chromaffin cells does not occur. The transcription factors Mash1 (Ascl1), Phox2a, Gata3 and Hand2 (previously dHand) control the differentiation of sympatho-adrenal precursor cells, and their deregulated expression in Insm1 mutant mice demonstrates that Insm1 acts in the transcriptional network that controls differentiation of this lineage. Pronounced similarities between Mash1 and Insm1 phenotypes are apparent, which suggests that Insm1 might mediate aspects of Mash1 function in the subtype-specific differentiation of sympatho-adrenal precursors. Noradrenaline is the major catecholamine produced by developing sympatho-adrenal cells and is required for fetal survival. We demonstrate that the fetal lethality of Insm1 mutant mice is caused by catecholamine deficiency, which highlights the importance of Insm1 in the development of the sympatho-adrenal lineage.
Asunto(s)
Glándulas Suprarrenales/citología , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Sistema Nervioso Simpático/citología , Factores de Transcripción/metabolismo , Glándulas Suprarrenales/embriología , Glándulas Suprarrenales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Células Cromafines/citología , Proteínas de Unión al ADN/genética , Pérdida del Embrión , Embrión de Mamíferos/patología , Epistasis Genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Mutantes , Norepinefrina/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras , Células Madre/citología , Sistema Nervioso Simpático/embriología , Sistema Nervioso Simpático/metabolismo , Factores de Transcripción/genéticaRESUMEN
The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.
Asunto(s)
Encéfalo/metabolismo , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Animales , Northern Blotting , Encéfalo/citología , Encéfalo/embriología , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/citología , Humanos , Hibridación in Situ , Ratones , Transcripción GenéticaRESUMEN
The pancreatic and intestinal primordia contain epithelial progenitor cells that generate many cell types. During development, specific programs of gene expression restrict the developmental potential of such progenitors and promote their differentiation. The Insm1 (insulinoma-associated 1, IA-1) gene encodes a Zinc-finger factor that was discovered in an insulinoma cDNA library. We show that pancreatic and intestinal endocrine cells express Insm1 and require Insm1 for their development. In the pancreas of Insm1 mutant mice, endocrine precursors are formed, but only few insulin-positive beta cells are generated. Instead, endocrine precursor cells accumulate that express none of the pancreatic hormones. A similar change is observed in the development of intestine, where endocrine precursor cells are formed but do not differentiate correctly. A hallmark of endocrine cell differentiation is the accumulation of proteins that participate in secretion and vesicle transport, and we find many of the corresponding genes to be down-regulated in Insm1 mutant mice. Insm1 thus controls a gene expression program that comprises hormones and proteins of the secretory machinery. Our genetic analysis has revealed a key role of Insm1 in differentiation of pancreatic and intestinal endocrine cells.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Células Enteroendocrinas/metabolismo , Células Secretoras de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Factores de Transcripción/fisiología , Dedos de Zinc , Animales , Diferenciación Celular/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células Enteroendocrinas/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Secretoras de Insulina/citología , Mucosa Intestinal/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Represoras , Factores de Transcripción/biosíntesis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
E2F transcription factors play a pivotal role in the regulation of cellular proliferation and can be subdivided into activating and repressing family members [1]. Like other E2Fs, E2F6 binds to E2F consensus sites, but in contrast to E2F1-5, it lacks an Rb binding domain and functions as an Rb-independent transcriptional repressor [2, 3, 4 and 5]. Instead, E2F6 has been shown to complex with Polycomb (PcG) group proteins [6 and 7], which have a well-established role in gene silencing. Here, we show that E2F6 plays an unexpected and essential role in the tissue specificity of gene expression. E2F6-deficient mice ubiquitously express the alpha-tubulin 3 and 7 genes, which are expressed strictly testis-specifically in control mice. Like an additional E2F6 target gene, Tex12, that we identified, tubulin 3 and 7 are normally expressed in male germ cells only. The promoters of the alpha-tubulin and Tex12 genes share a perfectly conserved E2F site, which E2F6 binds to. Mechanistically, E2F6-mediated repression involves CpG hypermethylation locking target promoters in an inactive state. Thus, E2F6 is essential for the long-term somatic silencing of certain male-germ-cell-specific genes, but it is dispensable for cell-cycle regulation.
Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica , Testículo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , Cromatografía de Afinidad , Huella de ADN , Cartilla de ADN , Factor de Transcripción E2F6 , Ensayo de Cambio de Movilidad Electroforética , Masculino , Ratones , Ratones Noqueados , Análisis por Micromatrices , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Genetic analysis in mice has demonstrated a crucial role of the Met tyrosine kinase receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in development of the liver, muscle, and placenta. Here, we use conditional mutagenesis in mice to analyze the function of Met during liver regeneration, using the Mx-cre transgene to introduce the mutation in the adult. After partial hepatectomy in mice carrying the Mx-cre-induced Met mutation, regeneration of the liver is impaired. Comparison of signal transduction pathways in control and mutant livers indicates that Met and other signaling receptors cooperate to fully activate particular signaling molecules, for instance, the protein kinase Akt. However, activation of the Erk1/2 kinase during liver regeneration depends exclusively on Met. Signaling crosstalk is thus an important aspect of the regulation of liver regeneration. Analysis of cell cycle progression of hepatocytes in conditional Met mutant mice indicates a defective exit from quiescence and diminished entry into S phase. Impaired liver regeneration is accompanied by compensatory physiological responses that include prolonged up-regulation of HGF/SF and IL-6 in peripheral blood. Our data demonstrate that the HGF/SF/Met signaling system is essential not only during liver development but also for the regeneration of the organ in the adult.