RESUMEN
Mutations in SLITRK1 are found in patients with Tourette's syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an alpha2-adrenergic agonist that is frequently used to treat patients with Tourette's syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourette's syndrome and trichotillomania.
Asunto(s)
Ansiedad/genética , Ansiedad/metabolismo , Encéfalo/metabolismo , Norepinefrina/metabolismo , Agonistas alfa-Adrenérgicos/uso terapéutico , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/patología , Conducta Animal , Cromatografía Líquida de Alta Presión , Clonidina/uso terapéutico , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Modelos Animales de Enfermedad , Electroquímica/métodos , Conducta Exploratoria/fisiología , Miedo/fisiología , Suspensión Trasera/fisiología , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Estadísticas no Paramétricas , Natación/psicologíaRESUMEN
Zic1 and Zic2 encode closely related zinc finger proteins expressed in dorsal neural tube and its derivatives. In previous studies, we showed that the homozygous Zic1 null mutation (Zic1-/-) results in cerebellar malformation with severe ataxia and that holoprosencephaly and spina bifida occur in homozygotes for Zic2 knockdown mutation (Zic2kd/kd). Since human ZIC2 haploinsufficiency is a cause of holoprosencephaly, the Zic2kd/kd mice are regarded as an animal model for holoprosencephaly in humans. In this study, the behavioral characteristics of the Zic1 and Zic2 mutant mice were investigated in heterozygotes (Zic1-/+ or Zic2kd/+), and significant abnormalities were found in the hanging, spontaneous locomotor activity, stationary rod (Zic1-/+), acoustic startle response, and prepulse inhibition tests (Zic2kd/+). The abnormalities in the Zic1-/+ mice may be explained in part by the hypotonia caused by hypoplasia of the cerebellar anterior vermis, and these mice are regarded as a model of Joubert syndrome. In contrast, the sensorimotor gating abnormality in the Zic2kd/+ mice may be attributable to the presumed abnormality in the dorsomedial forebrain, which was strongly affected in the Zic2kd/kd mice. Zic2kd/+ mice can serve as a model for diseases involving sensorimotor gating abnormalities, such as schizophrenia.
Asunto(s)
Conducta Animal/fisiología , Modelos Animales de Enfermedad , Holoprosencefalia/genética , Ratones Mutantes Neurológicos/genética , Hipotonía Muscular/genética , Esquizofrenia/genética , Factores de Transcripción/genética , Animales , Encéfalo/anomalías , Humanos , Ratones , Ratones Noqueados , Modelos Genéticos , Proteínas Nucleares , SíndromeRESUMEN
Inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) is a ubiquitously expressed IP(3)R gene in the IP(3)R gene family. We identified an upstream region of the mouse IP(3)R3 genomic DNA. Transcription start points for the IP(3)R3 gene were found to be located mainly at four sites between nucleotide position -325 and -285 relative to the first ATG codon. The major start point was mapped around -325. Transcription promotion ability was detected between -325 and -285 in an IP(3)R3 proximal promoter sequence. The promoter had no TATA-box but was highly GC-rich and contained two putative Sp1-binding sites. There was no sequence similarity between promoter regions of IP(3)R3 and IP(3)R2, another ubiquitous gene, except for GC-boxes. By using a series of 5'-truncation versions and a transient luciferase assay, we detected multiple common and cell-type-dependent regulatory regions within the distal promoter sequence downstream from -4.0 kb that function positively or negatively. The IP(3)R3 gene was highly transcribed in the kidney, spleen, heart, and skeletal muscle, and this tissue distribution pattern was nearly complementary to that of IP(3)R2. We found that IP(3)R3 gene expression was repressed in retinoic acid-treated and neural differentiated P19 mouse embryonic carcinoma cells.
Asunto(s)
Canales de Calcio/genética , Regiones Promotoras Genéticas/genética , Receptores Citoplasmáticos y Nucleares/genética , Células 3T3 , Región de Flanqueo 5'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Diferenciación Celular/genética , Línea Celular , Codón Iniciador/genética , ADN/química , ADN/genética , Regulación hacia Abajo , Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Distribución Tisular , Sitio de Iniciación de la Transcripción , Células Tumorales CultivadasRESUMEN
A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.
Asunto(s)
Tipificación del Cuerpo , Ciclinas/biosíntesis , Ciclinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso , Neuronas/metabolismo , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Cromatina/metabolismo , Secuencia Conservada , ADN Complementario/metabolismo , Drosophila , Embrión no Mamífero/metabolismo , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Cresta Neural/embriología , Filogenia , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rombencéfalo/embriología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Homología de Secuencia de Aminoácido , Médula Espinal/embriología , Factores de Tiempo , Factores de Transcripción/biosíntesis , XenopusRESUMEN
Meis1 (Myeloid Ecotropic viral Integration Site 1) is a homeobox gene that was originally isolated as a common site of viral integration in myeloid tumors of the BXH-2 recombinant inbred mice strain. We previously isolated a Xenopus homolog of Meis1 (Xmeis1). Here we show that Xmeis1 may play a significant role in neural crest development. In developing Xenopus embryos, Xmeis1 displays a broad expression pattern, but strong expression is observed in tissue of neural cell fate, such as midbrain, hindbrain, the dorsal portion of the neural tube, and neural crest derived branchial arches. In animal cap explants, overexpression of Xmeis1b, an alternatively spliced form of Xmeis1, induces expression of neural crest marker genes in the absence of mesoderm. Moreover, Xmeis1b induces XGli-3 and XZic3, pre-pattern genes involved at the earliest stages of neural crest development, and like these two genes, can induce ectopic pigmented cell masses when overexpressed in developing embryos. Misexpression of Xmeis1b also induces ectopic expression of neural crest markers along the antero-posterior axis of the neural tube in developing Xenopus embryos. In contrast, Xmeis1a, another splice variant, is much less effective at inducing these effects. These data suggest that Xmeis1b is involved in neural crest cell fate specification during embryogenesis, and can functionally intersect with the Gli/Zic signal transduction pathway.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Sistema Nervioso/embriología , Cresta Neural/embriología , Proto-Oncogenes , Proteínas de Xenopus , Empalme Alternativo , Animales , Antígenos de Diferenciación , Ectodermo/trasplante , Embrión no Mamífero , Inducción Embrionaria , Proteínas de Homeodominio/genética , Mesodermo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Proteínas Oncogénicas , Unión Proteica , ARN Mensajero/metabolismo , Transactivadores , Factores de Transcripción , Xenopus , Proteína con Dedos de Zinc GLI1RESUMEN
Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. We provide evidence that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. Our result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway.
Asunto(s)
Proteínas Oncogénicas/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Línea Celular , Núcleo Celular/metabolismo , Eliminación de Gen , Glutatión Transferasa/metabolismo , Proteínas Hedgehog , Proteínas de Homeodominio , Humanos , Luciferasas/metabolismo , Ratones , Proteínas Nucleares , Proteínas Oncogénicas/química , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas/metabolismo , Transducción de Señal , Factores de Transcripción/química , Transcripción Genética , Transfección , Proteína con Dedos de Zinc GLI1 , Dedos de ZincRESUMEN
We have used the yeast one-hybrid system to identify transcription factors that bind to specific sequences in proximal regions of the apolipoprotein E gene promoter. The sequence between -163 and -124, that has been previously defined as a functional promoter element, was used as a bait to screen a human brain cDNA library. Ten cDNA clones that encoded portions of the human Zic1 (five clones) and Zic2 (five clones) transcription factors were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for Zic1 and Zic2 in the -136/-125 region. Displacement of binding with oligonucleotides derived from adjacent sequences within the APOE promoter revealed the existence of two additional Zic-binding sequences in this promoter. These sequences were identified by electrophoretic mobility shift assays and mutational analysis in regions -65/-54 and -185/-174. Cotransfection of Zic1 and Zic2 expression vector and different APOE promoter-luciferase reporter constructs in U87 glioblastoma cell line showed that the three binding sites partially contributed to the trans-stimulation of the luciferase reporter. Ectopic expression of Zic1 and Zic2 in U87 cells also trans-stimulated the expression of the endogenous gene, increasing the amount of apolipoprotein E produced by glial cells. These data indicate that Zic proteins might contribute to the transcriptional activity of the apolipoprotein E gene and suggest that apolipoprotein E could mediate some of the developmental processes in which Zic proteins are involved.
Asunto(s)
Apolipoproteínas E/genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Proteínas Nucleares , Unión Proteica , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into cultured cells, Zic proteins enhanced or suppressed sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas Represoras , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Dedos de Zinc , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Proteínas/genética , Factores de Transcripción/genética , Transcripción Genética , Proteína Gli3 con Dedos de ZincRESUMEN
We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Cresta Neural/embriología , Proteínas de Xenopus , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Genes Dominantes , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Filogenia , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.
Asunto(s)
Proteínas de Homeodominio/fisiología , Proteínas Nucleares , Factores de Transcripción/fisiología , Proteínas de Xenopus , Xenopus laevis/embriología , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Cartilla de ADN/genética , Sistema Digestivo/embriología , Gástrula/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Mesodermo/metabolismo , Factores de Transcripción Paired Box , Estructura Terciaria de Proteína , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Xenopus laevis/genética , Dedos de Zinc , Proteína del Homeodomínio PITX2RESUMEN
Zic1 encodes a zinc finger protein, which is required for the development of the dorsal neural tissue. The gene is a mammalian homologue of the Drosophila odd-paired. We examined the regulatory elements in the 5' flanking region of the Zic1 gene as an initial step to understanding how the Zic1 expression is restricted to the dorsal neural tissue. When a 2.9-kb fragment of the 5' flanking segment of the mouse Zic1 gene was linked to the E. coli beta-galactosidase gene, the enzyme was consistently expressed in the dorsal half of the embryonic spinal cord and in the vestibulocochlear nucleus in all four transgenic mouse lines. The transgene expression mimics the Zic1 expression with respect to the region where it occurs. But this is not so for the neuronal cell types. This suggests that the segment contains a region-specific enhancer. In vivo and in vitro deletion analyses indicated that there are essential regions between -2.0 and -0.9 kb and within the proximal 0.9 kb. The distal element is necessary for the transgene expression in the embryonic dorsal spinal cord whereas the adult vestibulocochlear nucleus expression is regulated by both elements. In these regions, there are sequences similar to the binding sequences for potential regulatory proteins.
Asunto(s)
Núcleo Coclear/fisiología , Elementos de Facilitación Genéticos/genética , Médula Espinal/fisiología , Factores de Transcripción/genética , Núcleos Vestibulares/fisiología , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Química Encefálica/genética , Núcleo Coclear/química , Núcleo Coclear/embriología , Regulación del Desarrollo de la Expresión Génica , Genotipo , Operón Lac , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Médula Espinal/química , Médula Espinal/embriología , Núcleos Vestibulares/química , Núcleos Vestibulares/embriologíaRESUMEN
Mutation in human ZIC2, a zinc finger protein homologous to Drosophila odd-paired, causes holoprosencephaly (HPE), which is a common, severe malformation of the brain in humans. However, the pathogenesis is largely unknown. Here we show that reduced expression (knockdown) of mouse Zic2 causes neurulation delay, resulting in HPE and spina bifida. Differentiation of the most dorsal neural plate, which gives rise to both roof plate and neural crest cells, also was delayed as indicated by the expression lag of a roof plate marker, Wnt3a. In addition the development of neural crest derivatives such as dorsal root ganglion was impaired. These results suggest that the Zic2 expression level is crucial for the timing of neurulation. Because the Zic2 knockdown mouse is the first mutant with HPE and spina bifida to survive to the perinatal period, the mouse will promote analyses of not only the neurulation but also the pathogenesis of human HPE.
Asunto(s)
Sistema Nervioso Central/embriología , Factores de Transcripción/genética , Animales , Huesos/embriología , Huesos/patología , Diferenciación Celular , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Holoprosencefalia/embriología , Humanos , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Mutación , Defectos del Tubo Neural/genética , Proteínas Nucleares , Proteínas/genética , Disrafia Espinal/embriología , Factores de Transcripción/farmacología , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , Dedos de ZincRESUMEN
Skeletal abnormalities are described that appeared in Zic1-deficient mice. These mice show multiple abnormalities in the axial skeleton. The deformities are severe in the dorsal parts of the vertebrae, vertebral arches, but less so in the vertebral bodies (spina bifida occulta). The proximal ribs are deformed having ectopic processes. The abnormalities found in the vertebral arches can be traced back to disturbed segmental patterns of dorsal sclerotome. The Zic1/Gli3 double mutants showed severe abnormalities of vertebral arches not found in single mutants. The abnormalities in the vertebral arches were less severe in Zic1/Pax1 mutants than Zic1/Gli3 mutants, but significantly more pronounced than in Zic1 single mutants. The three genes may act synergistically in the development of the vertebral arches.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Proteínas del Tejido Nervioso , Proteínas Represoras , Columna Vertebral/embriología , Factores de Transcripción/genética , Proteínas de Xenopus , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Masculino , Mesodermo , Ratones , Ratones Endogámicos , Ratones Mutantes , Factores de Transcripción Paired Box , Costillas/anomalías , Costillas/embriología , Columna Vertebral/anomalías , Factores de Transcripción/metabolismo , Proteína Gli3 con Dedos de ZincRESUMEN
We characterized two new members of the Zic family, Xenopus Zic1 and Zic2. They are very similar to mouse Zic1 and Zic2 in the protein coding region including the zinc finger domain. In early gastrula, Zic1 expression was restricted to the prospective neural plate region whereas Zic2 was expressed widely in the ectoderm. We observed enhanced neural and neural crest-derived tissue formation in the Zic1 or Zic2 overexpressed embryos and neural and neural crest marker induction in the Zic1 or Zic2 overexpressed animal cap explants. Our findings suggest that Zic1 and Zic2 have essentially the same properties as Zic3 and that the Xenopus Zic family may act cooperatively in the initial phase of neural and neural crest development.
Asunto(s)
Sistema Nervioso/embriología , Cresta Neural/embriología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Xenopus laevis/embriología , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Embrión no Mamífero/química , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Xenopus laevis/genética , Dedos de Zinc/genéticaRESUMEN
Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generated mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.
Asunto(s)
Cerebelo/embriología , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Ratones Mutantes Neurológicos , Dedos de Zinc/genética , Animales , Conducta Animal/fisiología , Cerebelo/anomalías , Cerebelo/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Electrónica , Mutagénesis/fisiología , Neuronas/química , Neuronas/ultraestructura , Células Madre/química , Células Madre/fisiología , Factores de Transcripción/genéticaRESUMEN
Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.
Asunto(s)
Inducción Embrionaria , Proteínas de Homeodominio/genética , Sistema Nervioso/embriología , Cresta Neural/embriología , Factores de Transcripción/genética , Proteínas de Xenopus , Xenopus/embriología , Animales , Antígenos de Diferenciación , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Clonación Molecular , Ectodermo , Gástrula , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Transducción de Señal , Factores de Transcripción/biosíntesisRESUMEN
We examined the expression of Zic1, Zic2, and Zic3 genes in the mouse embryo by means of in situ hybridization. Zic genes were found as a group of genes coding for zinc finger proteins that are expressed in a restricted manner in the adult mouse cerebellum. We showed that the genes are the vertebrate homologues of Drosophila odd-paired, which may play an essential role in parasegmental subdivision and in visceral mesoderm development. The expression of the three Zic genes was first detected at gastrulation in a spatially restricted manner. At neurulation, the expression became restricted to the dorsal neural ectoderm and dorsal paraxial mesoderm. During organogenesis, the three genes were expressed in specific regions of several developing organs, including dorsal areas of the brain, spinal cord, paraxial mesenchyme, and epidermis, the marginal zone of the neural retina and distal regions of the developing limb. For all stages, significant differences in the spatial expression of Zic1, Zic2, and Zic3 were observed. Furthermore, the expression of Zic genes in Pax3, Wnt-1, and Wnt-3a mutant embryos suggested that Zic genes are not primarily regulated by the three genes which were expressed in dorsal areas similar to Zic genes. However, in open brain, a mutant with severe neural tube defects, and in the Wnt-3a mutant mice, the expression of Zic genes was changed. The changed expression pattern in Wnt-3a mutant mice suggests that Zic genes in the neural tube are regulated by the factors from notochord. Our findings suggest that Zic genes are involved in many developmental processes. Furthermore, analysis of gene expression patterns in different mouse mutants indicated that Zic genes may act upstream of many known developmental regulatory genes.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra , Dedos de Zinc/genética , Animales , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/genética , Desarrollo Embrionario y Fetal/genética , Extremidades/embriología , Ojo/embriología , Gástrula , Expresión Génica , Proteínas de Homeodominio/genética , Hibridación in Situ , Ratones , Defectos del Tubo Neural/genética , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Proteínas Wnt , Proteína Wnt1 , Proteína Wnt3 , Proteína Wnt3A , Dedos de Zinc/fisiologíaRESUMEN
Rabphilin 3A and Doc2 alpha are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2 alpha plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2 beta) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2 alpha (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (M(r)) of 45,837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2 beta is expressed in all cell lines and tissues tested. Ca(2+)-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2 beta binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca(2+)-dependent, with an EC50 value of approximately 1 microM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2 alpha. Our results suggest that Doc2 beta is involved in constitutive membrane trafficking.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Cerebelo/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Fosfolípidos/metabolismo , Proteínas de Unión al GTP rab , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Calcio/farmacología , Proteínas de Unión al Calcio/biosíntesis , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Humanos , Cinética , Liposomas , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Sinaptotagminas , Proteínas de Transporte Vesicular , Rabfilina-3ARESUMEN
We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.
Asunto(s)
Animales Recién Nacidos/crecimiento & desarrollo , Antígenos/metabolismo , Cerebelo/crecimiento & desarrollo , Neuronas/inmunología , Animales , Inmunohistoquímica , Ratones , Ratones MutantesRESUMEN
There is increasing interest in the role of RNA-binding proteins during neural development. Drosophila Musashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the Drosophila adult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology to Drosophila Musashi and Xenopus NRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of a Drosophila neuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS. In vitro studies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.