RESUMO
A balanced chromosomal translocation segregating with schizophrenia and affective disorders in a large Scottish family disrupting DISC1 implicated this gene as a susceptibility gene for major mental illness. Here we study neurons derived from a genetically engineered mouse strain with a truncating lesion disrupting the endogenous Disc1 ortholog. We provide a detailed account of the consequences of this mutation on axonal and dendritic morphogenesis as well as dendritic spine development in cultured hippocampal and cortical neurons. We show that the mutation has distinct effects on these two types of neurons, supporting a cell-type specific role of Disc1 in establishing structural connections among neurons. Moreover, using a validated antibody we provide evidence indicating that Disc1 localizes primarily to Golgi apparatus-related vesicles. Our results support the notion that in vitro cultures derived from Disc1(Tm1Kara) mice provide a valuable model for future mechanistic analysis of the cellular and biochemical effects of this mutation, and can thus serve as a platform for drug discovery efforts.
Assuntos
Córtex Cerebral/citologia , Espinhas Dendríticas/metabolismo , Hipocampo/citologia , Mutação , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Vesículas Citoplasmáticas/metabolismo , Complexo de Golgi/metabolismo , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Especificidade de Órgãos , Transporte ProteicoRESUMO
Novel molecular genetic approaches, at genome-scale in different species allowed characterizing genes that have undergone recent selection. The interest in this research field is not limited to the natural curiosity about our evolutionary past, but it is also to identify novel susceptibility genes for neuropsychiatic disorders by pointing specific human traits, such as behavioral and cognitive abilities. Hypotheses have been proposed to relate specific psychiatric disorders to the origin of modern humans, as evidenced by the theory of Crow about schizophrenia. In the present review, we will focus on genes that underwent positive selection in humans or displayed a human specific evolutionary pattern and which were reported as associated with psychiatric disorders. This will include the (1) DRD4 gene associated with attentiondeficit/ hyperactivity disorder, located in a locus that underwent a positive selection; the (2) GABRB2 gene, a gene associated with schizophrenia and recently reported as the target of a positive selection; (3) MARK1, a candidate gene for autism that was reported as displaying a signature of adaptative evolution in the human lineage, and (4) the ADH and ALDH2 genes which are associated with alcoholism, and for which evidence of positive selection was identified in the human lineage since the divergence between humans and chimpanzees. Identification of novel candidate genes based on recent evolution selection, coupled to genome-wide strategies designed to detect rare structural variants, could lead to a better knowledge of the molecular mechanisms of neurodevelopmental disorders and might therefore help to develop new medical chemistry.
Assuntos
Evolução Molecular , Transtornos Psicóticos/tratamento farmacológico , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Animais , Antipsicóticos/uso terapêutico , Descoberta de Drogas , Humanos , Neurofisinas/genética , Neurofisinas/metabolismo , Primatas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transtornos Psicóticos/genética , Interferência de RNA , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Vasopressinas/genética , Vasopressinas/metabolismoRESUMO
Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.
Assuntos
Transtorno Autístico , Predisposição Genética para Doença , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/metabolismo , Neuritos/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Córtex Pré-Frontal/metabolismo , Regulação para Cima/fisiologia , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Linhagem Celular Transformada , Células Cultivadas , Cromossomos Humanos Par 2 , Feto , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Técnicas In Vitro , Desequilíbrio de Ligação , Proteínas de Membrana Transportadoras/genética , Camundongos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Neurônios/citologia , Neurônios/metabolismo , Mudanças Depois da Morte , Córtex Pré-Frontal/embriologia , Córtex Pré-Frontal/patologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , RNA Mensageiro/metabolismo , Telencéfalo/citologia , TransfecçãoRESUMO
Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants (Ret-/-) did not survive after birth, our experiments were performed in wild-type (wt) and Ret-/- fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret-/- fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret-/- fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem 'en bloc' preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret-/- fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.
Assuntos
Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-ret/fisiologia , Mecânica Respiratória/fisiologia , Sistema Respiratório/crescimento & desenvolvimento , Sistema Respiratório/inervação , Animais , Monoaminas Biogênicas/metabolismo , Tronco Encefálico/citologia , Tronco Encefálico/crescimento & desenvolvimento , Feminino , Genótipo , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rede Nervosa/embriologia , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/fisiologia , Norepinefrina/fisiologia , Ponte/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Sistema Respiratório/embriologia , Rombencéfalo/enzimologia , Rombencéfalo/crescimento & desenvolvimentoRESUMO
INTRODUCTION: Studies into the contribution of genetic factors to respiratory control disorders are scarce, with impediments to their conduct including difficulties in characterizing these disorders, the large number of genes involved in respiratory control, and interactions between genetic and environmental factors. STATE OF THE ART: The rare congenital central hypoventilation syndrome (CCHS) has opened up the field of respiratory control genetics. Heterozygous mutations of genes involved in neural crest development were discovered recently. Studies in mutant mice have identified respiratory control disturbances related to loss of function of genes involved in neural crest development, genes encoding transcription factors, diffusible factors, and proteins contributing to neurotransmission. PERSPECTIVES: Future genetic epidemiological studies in humans and new models of mutant mice should describe genes involved in respiratory control. Better knowledge of CCHS genetics should provide guideposts for investigating the genetics of other respiratory control disorders. CONCLUSIONS: Respiratory control genetics is opening up new paths for research into respiratory physiology and pathophysiology.
Assuntos
Animais Geneticamente Modificados/genética , Transtornos Respiratórios/genética , Respiração/genética , Centro Respiratório/fisiologia , Apneia do Sono Tipo Central/genética , Adolescente , Adulto , Animais , Animais Recém-Nascidos , Células Quimiorreceptoras/fisiologia , Previsões , Predisposição Genética para Doença , Heterozigoto , Humanos , Hipóxia/fisiopatologia , Recém-Nascido , Mecanorreceptores/fisiologia , Camundongos/genética , Mutação , Crista Neural/crescimento & desenvolvimento , Fenótipo , Ratos/genética , Pesquisa , Apneia do Sono Tipo Central/congênito , Transmissão Sináptica/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
OBJECTIVE: To evaluate the understanding of written information contained in the information sheet for patients intended to receive an homologous transfusion and to know their opinion about this document. TYPE OF THE STUDY: A prospective cohort survey carried out by people unrelated to clinical units and transfusion services. METHODS: A document divided in two parts, the first one summarized, the second detailed, was distributed to transfused adult patients. The patients were hospitalized in the general surgery and orthopedic wards of two hospitals and in the hematology and oncology wards of two different hospitals. A questionnaire was filled out in the presence of the inquirer. RESULTS: Sixty one subjects have been enrolled, among them 53 considered the information as adequate; 53 as comforting and neutral. 53 patients considered a written information as essential and 52 estimated that both part of the information sheet (summarized and detailed) were mandatory. Conversely, a more in depth investigation revealed there was a gap between patients statements and their true understanding. CONCLUSION: The value of a written information for the patients is confirmed by the study. In addition, patients were not generally worried by this information. The partition of the document has been appreciated. It is noteworthy that a gap exist between the patient's perception of the information and their actual level of understanding.
Assuntos
Transfusão de Sangue , Educação de Pacientes como Assunto , Adulto , Estudos de Coortes , Coleta de Dados , Documentação , Feminino , Humanos , Consentimento Livre e Esclarecido , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Estudos Prospectivos , Inquéritos e QuestionáriosRESUMO
We identified new transcribed sequences, using a differential display paradigm to select genes expressed in proliferating neuroblasts from mouse telencephalon at 10 days of embryonic development. In this systematic search, we isolated a 361-bp partial 3' untranslated region (3' UTR) homologous to the 3' UTR of the human gene encoding a putative intracellular kinase regulator, glia maturation factor beta (GMFB). We cloned a full-length, 4,311-bp mouse cDNA containing a 270-bp 5' UTR, a 3,615-bp 3' UTR, and an open reading frame of 426 nucleotides encoding a putative 142 amino-acid protein, identical to human GMFB, with the exception of two amino acids. This 4.3-kb transcript is present in a variety of adult tissues and is developmentally regulated as shown by Northern blot analysis. Differential expression in telencephalon was demonstrated by quantification of radioactive relative RT-PCR and confirmed by in situ hybridization. The isolation of this full-length clone of mouse Gmfb should facilitate investigation of the intracellular mechanisms involved in the development of telencephalon.
Assuntos
Fator de Maturação da Glia/genética , Proteínas Quinases/metabolismo , Telencéfalo/metabolismo , Regiões 3' não Traduzidas/análise , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica no Desenvolvimento , Fator de Maturação da Glia/química , Fator de Maturação da Glia/farmacologia , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/enzimologiaRESUMO
PURPOSE: Wilms tumor or nephroblastoma is a developmental tumor of the kidney and one of the most frequent solid tumors in childhood. It derives from metanephrotic blastema and mimics nephrogenesis in a disorganized manner, offering an adequate model for study of human nephrogenesis. GDNF (glial cell line derived neurotrophic factor), a potent proliferation and survival factor of dopaminergic neurons, has recently been shown to have an early and major role in nephrogenesis. We studied the expression of GDNF in Wilms tumor. MATERIALS AND METHODS: The study included 20 patients with nephroblastoma whose age at surgery ranged from 2 months to 13 years. Expression of GDNF protein was analyzed by an immunohistochemical technique using anti-GDNF antibody. Presence of GDNF-messenger (m)RNA and receptors GFRalpha1 and GFRalpha2-mRNA was analyzed by reverse transcription polymerase chain reaction. Specimens were also studied to evaluate apoptosis, proliferation index and Bcl-2 expression. RESULTS: GDNF expression was mainly found in the epithelial cells of the most differentiated tubes, GDNF and co-receptors mRNA were found in specimens and proliferative activity was found on the same tubes as GDNF expression. Bcl-2 was expressed strongly in epithelial cells, in an intermediate fashion in the blastema and faintly in mesenchyma. Apoptosis was of low frequency in structures strongly expressing GDNF. CONCLUSIONS: We have shown that GDNF is expressed by nephroblastoma tissue of human kidneys. This expression is mainly in the differentiated epithelial component of the nephroblastoma. We have also shown that tissue strongly expressing GDNF is positively proliferative and has less apoptotic activity. We speculate that the role of GDNF may not be limited to normal nephrogenesis but may interact with other factors in the process of proliferation and apoptosis involved in nephroblastoma tumorigenesis.
Assuntos
Neoplasias Renais/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tumor de Wilms/metabolismo , Adolescente , Antígenos Nucleares , Apoptose , Criança , Pré-Escolar , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Imuno-Histoquímica , Lactente , Masculino , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Endothelin-converting-enzyme (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to mature endothelin-1. Most homozygous ECE-1-/- embryos die in utero and show severe craniofacial, enteric, and cardiac malformations precluding ventilatory function assessment. In contrast, heterozygous ECE-1+/- embryos develop normally. Their respiratory function at birth has not been studied. Taking into account previous respiratory investigations in mice with endothelin-1 gene disruption, we hypothesized that ECE-1-deficient mice may have impaired ventilatory control. We analyzed ventilatory responses to hypercapnia (8% CO(2)) and hypoxia (10% O(2)) in newborn and adult mice heterozygous for ECE-1 deficiency (ECE-1+/-) and in their wild-type littermates (ECE-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypoxia were significantly weaker in ECE-1+/- than in ECE-1+/+ newborn mice (percentage ventilation increase: 1 +/- 25% versus 33 +/- 29%, p = 0.010). Baseline breathing variables and ventilatory responses to hypercapnia were normal in the ECE-1+/- newborn mice. No differences were observed between adult ECE-1+/- and ECE-1+/+ mice. We conclude that ECE-1 is required for normal ventilatory response to hypoxia at birth.
Assuntos
Hipóxia/fisiopatologia , Metaloendopeptidases/fisiologia , Respiração , Animais , Animais Recém-Nascidos , Sequência de Bases , Primers do DNA , Feminino , Genótipo , Heterozigoto , Hipóxia/enzimologia , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Other studies have shown that caffeine accelerates telencephalic vesicle evagination in early post-implantation mouse embryos. The present study examines the effect of caffeine on gene modulation in post-implantation mouse embryos. Using mRNA differential display, we observed that caffeine increased gene expression of the regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA). RT--PCR analysis confirmed an increase in expression of this gene in caffeine-exposed embryos when compared with saline-treated controls. Using a fluorescent substrate of PKA, we found that PKA activity in the presence of cAMP was lower in caffeine-treated embryos than in controls. Treatment with H89 and PKI(12-24)amide, two inhibitors of PKA activity, mimicked the effects of caffeine on telencephalic vesicle formation. Together these data suggest that in early post-implantation mouse embryos caffeine modulates gene expression of the RI alpha subunit of PKA and that caffeine-induced inhibition of PKA activity plays a role in early telencephalic evagination.
Assuntos
Cafeína/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Inibidores de Fosfodiesterase/farmacologia , Telencéfalo/efeitos dos fármacos , Animais , Sequência de Bases , Proteínas de Transporte/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Expressão Gênica/fisiologia , Camundongos , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Telencéfalo/embriologia , Telencéfalo/metabolismoRESUMO
We took advantage of the combination of a rat heart transplantation model with a modified differential display RT-PCR method to identify transcriptome changes in the right atria from transplanted compared with native hearts. Based on sequence homology search, the 37 cDNAs differentially displayed both 2 and 7 days posttransplantation were categorized into 7 unknown transcripts, 16 expressed sequence tags (ESTs), and 14 partially or completely characterized genes. The last group cDNAs, validated by relative RT-PCR, belonged to diverse gene families involved in specific metabolisms, protein synthesis, cell signaling, and transcription. Furthermore, we identified differential transcripts corresponding to denervation and fetal gene reexpression. We found coordinate downregulation of genes involved in energy metabolism and protein synthesis regulation, similar to that reported for senescent skeletal muscle. From these transcriptome changes, we propose that heart transplants and senescent muscles share common molecular mechanisms.
Assuntos
Regulação da Expressão Gênica/fisiologia , Transplante de Coração/fisiologia , Família Multigênica/fisiologia , Animais , DNA Complementar/análise , DNA Complementar/genética , Denervação , Regulação para Baixo/fisiologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Átrios do Coração/inervação , Átrios do Coração/metabolismo , Masculino , Modelos Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transplante Heterotópico/fisiologia , Transplante Isogênico/fisiologia , Regulação para Cima/fisiologiaRESUMO
Respiratory abnormalities have been described in MASH-1 (mammalian achaete-scute homologous gene) and c-RET ("rearranged during transfection") mutant newborn mice. However, the neural mechanisms underlying these abnormalities have not been studied. We tested the hypothesis that the MASH-1 mutation may impair c-RET expression in brain stem neurons involved in the control of breathing. To do this, we analyzed brain stem c-RET expression and respiratory phenotype in MASH-1 +/+ wild-type, MASH-1 +/- heterozygous, and MASH-1 -/- knock-out newborn mice during the first 2 h of life. In MASH-1 -/- newborns, c-RET gene expression was absent in the noradrenergic nuclei (A2, A5, A6, A7) that contribute to modulate respiratory frequency and in scattered cells of the rostral ventrolateral medulla. The c-RET transcript levels measured by quantitative RT-PCR were lower in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ brain stems (P = 0.001 and P = 0.003, respectively). Breath durations were shorter in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ mice (P = 0.022) and were weakly correlated with c-RET transcript levels (P = 0.032). Taken together, these results provide evidence that MASH-1 is upstream of c-RET in noradrenergic brain stem neurons important for respiratory rhythm modulation.
Assuntos
Tronco Encefálico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Respiração , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Heterozigoto , Homozigoto , Hibridização In Situ , Camundongos , Camundongos Knockout , Rede Nervosa/fisiologia , Periodicidade , Fenótipo , Pletismografia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to susceptibility loci for autism. This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30-36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos. CAA17006 and CAB54261). Nevertheless, no homology to any known gene was found. RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate. Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism.
Assuntos
Transtorno Autístico/genética , Cromossomos Humanos Par 6/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Normal control of breathing is characterized by maintenance of CO2 and O2 arterial pressures at constant levels by appropriate ventilatory responses to changes in CO2 production and O2 consumption. Abnormal development of this regulatory system during embryogenesis may produce early impairments in chemosensitivity, as in congenital central hypoventilation syndrome. The present study addresses the role of the mammalian achaetescute homologous gene (Mash-1) in the development of respiratory control. We analyzed ventilatory responses to hypercapnia (8% CO2, 21% O2, 71% N2) and hypoxia (10% O2, 3% CO2, 87% N2) in newborn and adult Mash-1 heterozygous mice (Mash-1+/-) and their wild-type littermates (Mash-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypercapnia were significantly weaker in newborn male Mash-1+/- compared with Mash-1+/+ mice as a result of a weaker breath-duration response. No differences were observed between adult Mash-1+/- and Mash-1+/+ mice. Our data suggest that Mash-1 may be involved in respiratory control development via mechanisms linked to the X chromosome.
Assuntos
Proteínas de Ligação a DNA/genética , Heterozigoto , Hipóxia/fisiopatologia , Sistema Respiratório/fisiopatologia , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Feminino , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/crescimento & desenvolvimentoRESUMO
Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed in a restricted number of areas, and subject to numerous regulations during development and in adulthood. Two transcription factor binding sites present in the proximal region of the TH gene, the TPA-responsive element (TRE) and the c-AMP responsive element (CRE), have been shown to play important roles in TH gene regulation in vitro. In order to elucidate in vivo the role of these two sites, we produced transgenic mice bearing a 5.3-kb fragment from the 5' flanking sequence of the TH gene with mutations in either the CRE-or TRE-sites. Using the intact 5.3-kb fragment fused to two different reporter genes (HSV1-tk and lacZ), we show that this promoter fragment is able to specifically direct expression in catecholaminergic tissues both in adult mice and embryos. Interestingly, the CRE- and TRE-mutated transgenes were not expressed in adult mice, contrary to the situation in embryos where they were specifically expressed in catecholaminergic regions. These results demonstrate that the CRE and TRE play an essential role in basal TH expression in adult tissues in vivo. Moreover, they suggest that distinct transcription factors are involved in TH regulation in developing and adult tissues. In support of this, gel mobility shift experiments revealed a complex present only in embryonic tissues. Taken together, these data highlight the diversity of the mechanisms underlying the establishment and maintenance of the catecholaminergic phenotype.
Assuntos
Encéfalo/embriologia , Encéfalo/enzimologia , Proteínas de Ligação a DNA/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Repressoras , Tirosina 3-Mono-Oxigenase/biossíntese , Medula Suprarrenal/metabolismo , Envelhecimento/metabolismo , Animais , Química Encefálica/genética , Corantes , Modulador de Elemento de Resposta do AMP Cíclico , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/genética , Imuno-Histoquímica , Camundongos , Mutação/genética , Mutação/fisiologia , Plasmídeos/genética , Plasmídeos/imunologia , Gravidez , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Peripherin is a neuron-specific type III intermediate filament protein expressed in well-defined populations of neurons projecting towards peripheral targets. To investigate the molecular mechanisms by which a gene is expressed in a specific subset of neurons, we used a transgenic approach in order to define peripherin gene sequences that are necessary for cell-type specific expression. Transgenic mice carrying different various genomic regions of the mouse peripherin gene fused to the Escherichia coli lacZ reporter gene were generated. We used three different peripherin/lacZ constructs containing either 5.8 kb upstream sequences, or both 5.8 kb upstream and 1.1 kb intragenic sequences, or 1.1 kb intragenic sequences associated with an heterologous promoter. Analysis of lacZ gene expression in transgenic mouse embryos showed that cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences. Analysis of transgenic mouse lines expressing the construct containing both upstream and intragenic sequences showed that this transgene contains all regulatory elements essential for both spatial and temporal expression of the mouse peripherin gene during embryogenesis. Furthermore, lacZ+ positive cells isolated from these transgenic lines by fluorescence-activated cell sorting (FACS) can be stained with a peripherin antibody, demonstrating that the transgene containing both upstream and intragenic sequences is expressed in peripherin neurons. These mouse peripherin upstream and intragenic sequences can now be used to identify cis-acting regulatory elements and transcription factors involved in peripherin gene regulation.
Assuntos
Embrião de Mamíferos/fisiologia , Proteínas do Olho/genética , Expressão Gênica , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana , Camundongos Transgênicos/genética , Proteínas do Tecido Nervoso , Neurônios/fisiologia , Animais , Sequência de Bases , Separação Celular , Embrião de Mamíferos/citologia , Citometria de Fluxo , Óperon Lac , Camundongos/embriologia , Dados de Sequência Molecular , Neurônios/classificação , Neuropeptídeos/genética , PeriferinasRESUMO
Dominant megacolon (Dom) is one of four mutations in the mouse which can produce a phenotype similar to Hirschsprung disease in man. Here, we report that it is possible to take advantage of two microsatellite markers to genotype Dom embryos and to study enteric neuronal development in Dom embryos using whole-mount immunohistochemistry. Dom embryos present a variable defect in the ileo-caecal region, as do embryos of other murine models of Hirschsprung disease.
Assuntos
Doença de Hirschsprung/patologia , Megacolo/genética , Megacolo/patologia , Neurônios/fisiologia , Animais , Movimento Celular/fisiologia , DNA/análise , Sistema Digestório/metabolismo , Sistema Digestório/patologia , Modelos Animais de Doenças , Feminino , Genes Dominantes , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Repetições de Microssatélites , Neurônios/efeitos dos fármacos , Fenótipo , Reação em Cadeia da Polimerase , GravidezRESUMO
Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to the lacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression of lacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promote lacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression of lacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences contain cis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directs lacZ expression in embryonic neurons.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes , Proteínas de Neurofilamentos/genética , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , beta-Galactosidase/biossíntese , Animais , Animais Recém-Nascidos , Sequência de Bases , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/genética , Feminino , Genes Reporter , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , Regiões Promotoras GenéticasRESUMO
Cell lineage analysis in both the central and peripheral nervous system of vertebrates has revealed that many neural progenitor cells are multipotent. These observations have raised the general issue of when and how such multipotent progenitors generate their various differentiated progeny. The environment of these progenitors controls the cell lineage decisions in the neural crest. This review considers the roles of the environmental signals in the context of the development of several different neural crest-derived lineages.
Assuntos
Substâncias de Crescimento/fisiologia , Crista Neural/citologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Melanócitos/citologia , Neurônios Aferentes/citologia , Células-Tronco/citologiaRESUMO
We developed improved immobilization conditions which permitted (i) to immobilize neuroblastoma cells (N18) in calcium-alginate gel beads, (ii) to test the function of ionic channels using patch-clamp electrophysiological techniques and (iii) to quantitatively analyze ligand interactions with voltage-dependent sodium channels in neurons inside the beads. These results qualify this immobilization technique for the isolation and/or purification of ligands specific for neuronal cells.