RESUMO
The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6+/Hopx+ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.
Assuntos
Cérebro/crescimento & desenvolvimento , Cérebro/metabolismo , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Animais , Astrócitos/metabolismo , Cérebro/citologia , Cérebro/embriologia , Embrião de Mamíferos , Proteínas de Homeodomínio/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Macaca , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fator de Transcrição PAX6/metabolismo , Primatas , Proteínas Supressoras de Tumor/metabolismoRESUMO
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Assuntos
Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Microtúbulos/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/metabolismo , Interfase , Ventrículos Laterais/anatomia & histologia , Glândulas Mamárias Animais/citologia , Camundongos , Tamanho do Órgão , Organoides/citologiaRESUMO
Neural progenitors with distinct potential to generate progeny are associated with a spatially distinct microenvironment. Neocortical intermediate progenitors (IPs) in the subventricular zone (SVZ) of the developing brain generate neurons for all cortical layers and are essential for cortical expansion. Here, we show that spatial control of IP positioning is essential for neocortical development. We demonstrate that HDAC1 and HDAC2 regulate the spatial positioning of IPs to form the SVZ. Developmental stage-specific depletion of both HDAC1 and HDAC2 in radial glial progenitors results in mispositioning of IPs at the ventricular surface, where they divide and differentiate into neurons, thereby leading to the cortical malformation. We further identified the proneural gene Neurogenin2 as a key target of HDAC1 and HDAC2 for regulating IP positioning. Our results demonstrate the importance of the spatial positioning of neural progenitors in cortical development and reveal a mechanism underlying the establishment of the SVZ microenvironment.
Assuntos
Células Ependimogliais/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Ventrículos Laterais/embriologia , Malformações do Desenvolvimento Cortical/genética , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Malformações do Desenvolvimento Cortical/embriologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , NeurogêneseRESUMO
Abstract Objectives This study was done to evaluate the normal fetal cerebral lateral ventricle dimensions with transabdominal ultrasonography. The atrial width (AW), ventricle-tochoroid measurement (V-C), ventricle-to-hemisphere ratio (VHR), and combined anterior horn measurement (CAHM) were taken. Methods This was a cross-sectional study involving 400 normal singleton pregnant subjects whose gestational ages were between 14 and 40 weeks. Transabdominal sonography was performed to obtain the values of the fetal cerebral lateral ventricle (FCLV) parameters. Data were reported as mean standard deviation (SD) for continuous variables. The degrees of correlation between FCLV parameters and the estimated gestational age (EGA) were obtained using Pearson's correlation. Regression equations were used to generate the reference limits for the FCLV measurements. Results The values of AW, V-C measurements and CAHM increased with advancing gestation. The mean values of the AW, V-C and CAHM from 14 to 40 weeks increased from 6.60 0.94 mm to 9.75 0.07 mm (R2 = 0.114), 0.80 0.00 mm to 1.90 0.14 mm (R2= 0.266), and 6.95 0.06 mm to 23.07 4.02 mm (R2= 0.692) respectively, while the mean VHR decreased from 61.20 1.60% to 42.84 2.91% (R2 = 0.706) over the same period. Conclusion The AW, V-C, and CAHM increase, while VHR decreases with advancing gestation.
Resumo Objetivos O presente estudo objetiva avaliar as dimensões do ventrículo lateral de cérebros fetais por meio de ultrassonografia transabdominal. Foram medidos a largura do átrio (LA), a medida do ventrículo ao coroide (V-C), a razão ventrículo/ hemisfério (RVH), e a medida dos cornos anteriores combinados ( CAC ). Métodos Estudo transversal com 400 grávidas de único feto com idades gestacionais entre 14 e 40 semanas. Sonografias transabdominais foram realizadas para obter os valores dos parâmetros do ventrículo lateral de cérebros fetais (VLCF). Dados foram apresentados em média desvio padrão para variáveis contínuas. Os graus de correlação entre parâmetros de VLCF e idade gestacional estimada foram obtidos usando a correlação de Pearson. Equações de regressão foram usadas para gerar as referências-limite para medidas de VLCF. Resultados Os valores de LA, medida do V-C e CAC aumentaram com o avanço da gestação. Os valores médios de LA, V-C e CAC de 14 a 40 semanas aumentaram de 6,60 0,94 mm a 9,75 0,07 mm (R2 = 0,114), de 0,80 0,00 mm a 1,90 0,14 mm (R2 = 0,266), e de 6,95 0,06 mm a 23,07 4,02 mm (R2 = 0,692), respectivamente, enquanto a RVH média diminuiu de 61,20 1,60% para 42,84 2,91% (R2 = 0,706) no mesmo período. Conclusão A LA, V-C, e CAC aumentaram, enquanto a RVH diminuiu com o avanço da gestação.
Assuntos
Humanos , Feminino , Gravidez , Adolescente , Adulto , Adulto Jovem , Ventrículos Laterais/diagnóstico por imagem , Ventrículos Laterais/embriologia , Ultrassonografia Pré-Natal , Estudos Transversais , Estudos Prospectivos , Valores de ReferênciaRESUMO
Gas1 is a pleiotropic protein that inhibits cell growth when overexpressed in tumors but during development, it acts as a co-receptor for sonic hedgehog to promote the proliferation and survival of various growing organs and systems. This protein has been extensively studied during development in the cerebellum. However, in other structures of the central nervous system, information concerning Gas1 is limited to in situ hybridization studies. We investigate the pattern of Gas1 expression during various developmental stages of the cortex and dentate gyrus of the mouse brain. The levels of Gas1 decrease in the developing brain and the protein is mainly found in progenitor cells during the development of the cortex and dentate gyrus.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Giro Denteado/embriologia , Giro Denteado/metabolismo , Hipocampo/embriologia , Hipocampo/metabolismo , Células-Tronco/metabolismo , Animais , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Organogênese/fisiologiaRESUMO
The ERK/MAPK pathway is an important developmental signaling pathway. Mutations in upstream elements of this pathway result in neuro-cardio-facial cutaneous (NCFC) syndromes, which are typified by impaired neurocognitive abilities that are reliant upon hippocampal function. The role of ERK signaling during hippocampal development has not been examined and may provide critical insight into the cause of hippocampal dysfunction in NCFC syndromes. In this study, we have generated ERK1 and conditional ERK2 compound knock-out mice to determine the role of ERK signaling during development of the hippocampal dentate gyrus. We found that loss of both ERK1 and ERK2 resulted in 60% fewer granule cells and near complete absence of neural progenitor pools in the postnatal dentate gyrus. Loss of ERK1/2 impaired maintenance of neural progenitors as they migrate from the dentate ventricular zone to the dentate gyrus proper, resulting in premature depletion of neural progenitor cells beginning at E16.5, which prevented generation of granule cells later in development. Finally, loss of ERK2 alone does not impair development of the dentate gyrus as animals expressing only ERK1 developed a normal hippocampus. These findings establish that ERK signaling regulates maintenance of progenitor cells required for development of the dentate gyrus.
Assuntos
Giro Denteado , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Giro Denteado/embriologia , Giro Denteado/enzimologia , Giro Denteado/crescimento & desenvolvimento , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
Signaling via MET receptor tyrosine kinase (MET) has been implicated in a number of neurodevelopmental events, including cell migration, dendritic and axonal development and synaptogenesis. Related to its role in the development of forebrain circuitry, we recently identified a functional promoter variant of the MET gene that is associated with autism spectrum disorder (ASD). The association of the MET promoter variant rs1858830 C allele is significantly enriched in families with a child who has ASD and co-occurring gastrointestinal conditions. The expression of MET in the forebrain had been mapped in detail in the developing mouse and rhesus macaque. However, in mammals, its expression in the developing brainstem has not been studied extensively throughout developmental stages. Brainstem and autonomic circuitry are implicated in ASD pathophysiology and in gastrointestinal dysfunction. To advance our understanding of the neurodevelopmental influences of MET signaling in brainstem circuitry development, we employed in situ hybridization and immunohistochemistry to map the expression of Met and its ligand, Hgf, through prenatal development of the mouse midbrain and hindbrain. Our results reveal a highly selective expression pattern of Met in the brainstem, including a subpopulation of neurons in cranial motor nuclei (nVII, nA and nXII), B6 subgroup of the dorsal raphe, Barrington's nucleus, and a small subset of neurons in the nucleus of solitary tract. In contrast to Met, neither full-length nor known splice variants of Hgf were localized in the prenatal brainstem. RT-PCR revealed Hgf expression in target tissues of Met-expressing brainstem neurons, suggesting that MET in these neurons may be activated by HGF from peripheral sources. Together, these data suggest that MET signaling may influence the development of neurons that are involved in central regulation of gastrointestinal function, tongue movement, swallowing, speech, stress and mood.
Assuntos
Tronco Encefálico/embriologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Núcleos da Rafe/embriologia , Animais , Tronco Encefálico/metabolismo , Nervos Cranianos/citologia , Fator de Crescimento de Hepatócito/metabolismo , Ventrículos Laterais/embriologia , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Glândula Pineal/embriologia , Núcleos da Rafe/metabolismoRESUMO
Stroke is a leading cause of mortality and chronic disability. Therapies aimed at reducing stroke related morbidity are currently limited. Therefore it is very important to develop effective treatments that will maximize rehabilitation after stroke. Current efforts in the field of cellular therapy focus on stem cell transplantations. This approach involves biological and ethical complications and therefore, the use of endogenous neural stem cells (eNSC) for repairing damage in various neurological disorders has been suggested. eNSCs reside in specialized vascular niches in defined regions, such as the subventricular zone (SVZ) of the lateral ventricle. These cells have an unlimited potential to create newborn cells. Interrelations between newborn neural and endothelial cells have an important role in eNSC survival, maturation, migration and differentiation and neurogenesis occurs in close spatio-temporal association with vessel growth in these niches. Previous studies have shown that application of external factors can boost long-term endogenous repair mechanisms in the cerebral cortex. Activated platelets and their microparticles contain a variety of growth and trophic factors essential to angiogenesis and neurogenesis and may therefore serve as novel therapeutic agents for brain injury. Specifically, factors from platelets and their microparticles may promote neurogenesis by stimulating eNSC proliferation, migration and differentiation, and by stimulating niche angiogenesis and the release of neurogenic signals from endothelial cells and astrocytes. In this review we will show that combined augmentation of angiogenesis, neurogenesis and neuroprotection using platelets and their microparticles is feasible and results in improved functional gain after stroke.
Assuntos
Plaquetas/metabolismo , Isquemia Encefálica/terapia , Micropartículas Derivadas de Células/metabolismo , Córtex Cerebral/patologia , Neovascularização Fisiológica , Neurogênese , Reabilitação do Acidente Vascular Cerebral , Indutores da Angiogênese/metabolismo , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/uso terapêutico , Animais , Plaquetas/patologia , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Micropartículas Derivadas de Células/patologia , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Descoberta de Drogas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Humanos , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/transplante , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Ativação Plaquetária , Nicho de Células-Tronco , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Inactivation of FoxO proteins by phosphorylation is the result of a number of stimuli, including the insulin/IGF pathway. We were interested in the consequence of blunting this pathway by employing transgenic mice with tetracycline-controllable conditional expression of a constitutively active allele of FOXO3 under the control of the forebrain-specific CaMKIIα promoter. Although transgene-expressing mice were viable, brain weight was reduced by 30% in adult animals. Brains showed an isocortex compression with normal cortical layering, and a size reduction in regions known to depend on adult neurogenesis, i.e., the olfactory bulbs and the dentate gyrus. On postnatal activation of the transgene, adult neurogenesis was also severely affected. Investigating the molecular basis of this phenotype, we observed enhanced apoptosis starting from embryonic day E10.5 and a subsequent loss of progenitors in the ventricular/subventricular zones, but not in the isocortex or the striatum of adult mice. The enhanced apoptosis was accompanied by increased expression of PIK3IP1, which we identified as a direct transcriptional target of FOXO3. Transfection of Pik3ip1 into differentiating neural progenitors resulted in a significant reduction of viable cells. We therefore conclude that neural progenitors are particularly vulnerable to FOXO3-induced apoptosis, which is mediated by PIK3IP1, a negative PI3 kinase regulator.
Assuntos
Fatores de Transcrição Forkhead/genética , Células-Tronco Neurais/metabolismo , Prosencéfalo/metabolismo , Animais , Apoptose/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Análise por Conglomerados , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Interneurônios/fisiologia , Ventrículos Laterais/citologia , Células-Tronco Neurais/fisiologia , Actinas/metabolismo , Amidas/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular/genética , Desoxiuridina/análogos & derivados , Proteínas do Domínio Duplacortina , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Forminas , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Corpos Geniculados/citologia , Corpos Geniculados/embriologia , Corpos Geniculados/crescimento & desenvolvimento , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/metabolismo , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Técnicas de Cultura de Órgãos , Parvalbuminas/metabolismo , Transporte Proteico/genética , Piridinas/farmacologia , Proteína Reelina , Proteínas Repressoras/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo , Ácido gama-Aminobutírico/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Netrin1 (NTN1) deficiency in mouse brain causes defects in axon guidance and cell migration during embryonic development. Here we show that NTN1 is required for olfactory bulb (OB) development at late embryogenesis and at early postnatal stages to facilitate the accumulation of proper numbers of granular and glomerular neuron subtypes and oligodendrocytes into the OB. In addition to the analysis of Ntn1-/- mice we made tissue and neurosphere cultures to clarify the role of NTN1 in the anterior forebrain. We propose that a subset of neural progenitors/precursors requires NTN1 to efficiently enter the rostral migratory stream to migrate into the OB. The analysis of postnatal Ntn1-/- OBs revealed a reduction of specific types of interneurons which have been shown to originate from particular subregions of the lateral ventricle walls. Based on Ntn1 expression in ventral parts of the ventricle walls, we observed a decrease in the mainly ventrally derived type II interneurons that express calcium-binding proteins calretinin and calbindin. Instead, no change in the numbers of dorsally derived tyrosine hydroxylase expressing interneurons was detected. In addition to the specific reduction of type II interneurons, our results indicate that NTN1 is required for oligodendroglial migration into the OB. Furthermore, we characterised the Ntn1 expressing subpopulation of neurosphere-forming cells from embryonic and adult brain as multipotent and self-renewing. However, NTN1 is dispensable for the proliferation of neurosphere forming progenitor cells and for their differentiation.
Assuntos
Movimento Celular , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Bulbo Olfatório/crescimento & desenvolvimento , Proteínas Supressoras de Tumor/metabolismo , Animais , Calbindina 2 , Calbindinas , Diferenciação Celular , Células Cultivadas , Interneurônios , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Camundongos , Fatores de Crescimento Neural/genética , Netrina-1 , Células-Tronco Neurais/citologia , Neuroglia/citologia , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteínas Supressoras de Tumor/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Mental retardation in Down syndrome (DS) appears to be related to severe neurogenesis impairment during critical phases of brain development. Recent lines of evidence in the cerebellum of a mouse model for DS (the Ts65Dn mouse) have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. By using an in vitro model of NPCs obtained from the subventricular zone and hippocampus, we found that trisomic NPCs had an increased expression of the Shh receptor Patched1 (Ptch1), a membrane protein that suppresses the action of a second receptor, Smoothened (Smo), thereby maintaining the pathway in a repressed state. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. The overexpression of Ptch1 in trisomic NPCs resulted from increased levels of AICD [a transcription-promoting fragment of amyloid precursor protein (APP)] and increased AICD binding to the Ptch1 promoter. Our data provide novel evidence that Ptch1 overexpression underlies derangement of the Shh pathway in trisomic NPCs with consequent proliferation impairment. The demonstration that Ptch1 overexpression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Síndrome de Down/genética , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Receptores de Superfície Celular/biossíntese , Acetilação , Animais , Ciclo Celular/genética , Proliferação de Células , Cicloexilaminas/farmacologia , Metilação de DNA , Síndrome de Down/embriologia , Síndrome de Down/metabolismo , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacologia , Hipocampo/embriologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Ventrículos Laterais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Receptores Patched , Receptor Patched-1 , Complexo Repressor Polycomb 1 , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Proteínas Repressoras/genética , Receptor Smoothened , Tiofenos/farmacologia , Regulação para Cima , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
ABCB1 is a human ABC transporter originally characterized by its ability to cause resistance to chemotherapy drugs in cancer cells, and later found to be functionally expressed in human neural stem/progenitor cells (NSPCs) in vitro. Here, we performed a detailed examination of ABCB1's expression on human NSPCs in vitro and in human fetal brain tissues, and analyzed the cellular properties of the human NSPCs expressing ABCB1. We confirmed that ABCB1 was expressed on the surface of human NSPCs, and its level correlated well with those of Nestin and CD133. The population of fluorescence-activated cell sorter-sorted human NSPCs expressing high levels of ABCB1 showed enrichment of proliferating cells, higher expression of 246 genes (e.g., RGS6, IGFBP7, GFAP, TNC, Hes1), and lower expression of 71 genes (e.g., STMN2, DLX5, BASP1, DCX, CD24) compared with human NSPCs expressing low or no ABCB1. In situ, ABCB1 was selectively expressed in cells in the ventricular or subventricular regions of lateral ventricles that expressed Nestin in human development. These findings suggest that ABCB1 is predominantly expressed in immature human fetal NSPCs in vitro and at early developmental stages in vivo, and that it may be a useful marker for human NSPCs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Antígeno AC133 , Subfamília B de Transportador de Cassetes de Ligação de ATP , Idoso , Antígenos CD/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/citologia , Proliferação de Células , Células Cultivadas , Feto , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicoproteínas/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Peptídeos/metabolismo , Esferoides Celulares , Células-Tronco/citologia , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/metabolismoRESUMO
microRNAs are short RNA molecules that are often expressed in specific tissues and regulate a variety of developmental processes. We used locked nucleic acid probes in in situ hybridisation reactions to study the distribution of microRNA-449 (miR449) during mouse embryonic development in order to obtain clues about its function/s. Between E9.75 and E11.5, miR449 was found to be expressed specifically in the developing roof plate of the fourth ventricle within the domain of roof plate marker, Lmx1a. From E12.5 onwards, this expression became restricted to the epithelial cell layer of the fourth ventricle choroid plexus. MiR449 also became detectable specifically in the choroid plexuses of the lateral and 3rd ventricles at E13.5 and E15.5, respectively. Northern blot analysis of adult brain also showed a selective and enriched expression in the choroid plexus tissue. Potential target genes regulated by miR449 were selected for experimental validation in luciferase-reporter assays and the transcription factor E2f5, which regulates CSF production, was verified as a miR449 target gene. Taken together, these findings suggest that miR449 has a specific role in the development and functioning of choroid plexuses.
Assuntos
Plexo Corióideo/embriologia , Plexo Corióideo/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Northern Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Plexo Corióideo/crescimento & desenvolvimento , Plexo Corióideo/metabolismo , Fator de Transcrição E2F5/metabolismo , Embrião de Mamíferos/metabolismo , Células Epiteliais/metabolismo , Quarto Ventrículo/embriologia , Quarto Ventrículo/metabolismo , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas com Homeodomínio LIM , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Camundongos , Oligonucleotídeos/metabolismo , Terceiro Ventrículo/embriologia , Terceiro Ventrículo/metabolismo , Fatores de Transcrição , Proteína Wnt1/metabolismoRESUMO
Subventricular zone (SVZ)-derived adult neurospheres express two ectonucleotidases, nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and tissue non-specific alkaline phosphatase (TNAP). Agonists of the nucleotide receptors P2Y(1) and P2Y(2) as well as adenosine augment growth factor-mediated progenitor cell proliferation. NTPDase2 converts ATP and UTP to ADP and UDP, respectively, which are all P2Y receptor agonists. TNAP hydrolyzes nucleoside triphosphates and diphosphates and produces the P1 receptor agonist adenosine. In the SVZ, NTPDase2 is specifically expressed by type B cells. In order to further scrutinize the association of key molecules of the purinergic signaling pathway with neurogenic regions, we analyzed the expression of TNAP at the lateral ventricles of the adult and developing mouse brain. In the adult brain, TNAP was expressed by type B, type A and at least subsets of type C cells of the SVZ and throughout the rostral migratory stream. Almost 100% of the proliferating, Ki-67-positive cells of the adult SVZ stained for TNAP, supporting the notion of a ubiquitous association of TNAP with SVZ progenitors. In contrast, NTPDase2-positive progenitors of the dentate gyrus were TNAP-negative. Essentially all cells of the telencephalic vesicle at embryonic day (E) 14 revealed TNAP activity, including doublecortin-positive neuroblasts. During further embryonic development, enhanced TNAP activity became restricted to cells of the ventricular and SVZ. In contrast to TNAP, NTPDase2 was first expressed in the SVZ perinatally, in association with TNAP-positive SVZ border cells. During later development, NTPDase2-positive cells disappeared from the ventricular surface and began to form sheaths around clusters of subventricular doublecortin-positive cells, apparently transforming into type B cells. Our results identify TNAP and NTPDase2 as novel markers for subsets of progenitors in the adult and developing mouse brain. They further support the notion that signaling via extracellular nucleotides and nucleosides contributes to embryonic and adult neurogenesis.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Ventrículos Laterais/citologia , Nucleosídeo-Trifosfatase/metabolismo , Células-Tronco/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Proteínas do Domínio Duplacortina , Embrião de Mamíferos , Transportador 1 de Aminoácido Excitatório , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Antígeno Ki-67/metabolismo , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , GravidezRESUMO
Nicotine, as has been shown in animal studies, is a neuroteratogen, even in concentrations that do not cause growth retardation. In humans, there is only indirect evidence for negative influences of nicotine on brain development from studies on the association between maternal smoking in pregnancy and behavioural and cognitive development in the offspring. We investigated the associations of maternal smoking in pregnancy with foetal head growth characteristics in 7042 pregnant women. This study was embedded in the Generation R Study, a population-based prospective cohort study from foetal life until adulthood. Maternal smoking was assessed by questionnaires in early, mid- and late pregnancy. Head circumference, biparietal diameter, transcerebellar diameter and atrial width of lateral ventricles were repeatedly measured by ultrasound. When mothers continued to smoke during pregnancy, foetal head circumference showed a growth reduction of 0.13 mm [95% confidence interval (CI): -0.18, -0.09] per week compared to foetuses of mothers who never smoked during pregnancy. Biparietal diameter of foetuses with smoking mothers grew 0.04 mm (95% CI: -0.05, -0.02) less per week than that of foetuses of nonsmoking mothers. Atrial width of lateral ventricle was 0.12 mm (95% CI: -0.22, -0.02) smaller and transcerebellar diameter was 0.08 mm (95% CI: -0.15, -0.00) smaller if mothers smoked, but growth per week of these characteristics was not affected by maternal smoking in pregnancy. In conclusion, continuing to smoke during pregnancy leads to reduced growth of the foetal head. Further research should focus on the causal pathway from prenatal cigarette exposure via brain development to behavioural and cognitive functions.
Assuntos
Encéfalo/embriologia , Ecoencefalografia , Desenvolvimento Fetal , Fumar/efeitos adversos , Adulto , Cerebelo/diagnóstico por imagem , Cerebelo/embriologia , Estudos de Coortes , Feminino , Cabeça/diagnóstico por imagem , Cabeça/embriologia , Humanos , Ventrículos Laterais/diagnóstico por imagem , Ventrículos Laterais/embriologia , Lobo Parietal/diagnóstico por imagem , Lobo Parietal/embriologia , Gravidez , Estudos Prospectivos , Abandono do Hábito de FumarRESUMO
In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.
Assuntos
DNA/administração & dosagem , Eletroporação/métodos , Idade Gestacional , Injeções Intraventriculares/métodos , Ratos/embriologia , Animais , DNA/farmacocinética , Feminino , Laparotomia , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Gravidez , Células-Tronco/metabolismo , ÚteroRESUMO
In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however it is most commonly performed at E15 as shown in this video.
Assuntos
Eletroporação/métodos , Idade Gestacional , Injeções Intraventriculares/métodos , Camundongos/embriologia , Animais , DNA/administração & dosagem , DNA/farmacocinética , Feminino , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Neocórtex/embriologia , Neocórtex/metabolismo , Gravidez , Células-Tronco/metabolismo , ÚteroRESUMO
During development there is a clear correlation between position of dividing progenitor cells, mode of division and developmental potential, suggesting that the local environment of progenitor cells may influence their cell fate [ 17 (6), 639-647]. The contribution of these conditions was investigated here by transplantation of radial glial progenitor cells into isotopic, isochronic, heterotopic and heterochronic environment conditions. Neuronal cells were removed from E14 spinal cords using negative immunoselection. The remaining radial glia were transplanted into the ventricular system of host embryos and pups. Distance of migration as well as morphological and antigenic phenotype of transplanted radial glia was examined after various survival times post transplantation. Host age clearly influenced migration and differentiation of transplant cells, with transplant cells migrating further in younger hosts and differentiating earlier in older aged host environments. Evidence is presented showing that most transplanted spinal cord radial glia give rise to astrocytes. In addition some transplanted radial glia were shown to give rise to neurons in spinal cord regions. Radial glia did not appear to generate neurons in the brains of host animals until postnatal ages, perhaps because transplanted radial glia were isolated from spinal cord and thus may not have been influenced to behave as endogenous radial glia in the brain which commonly produce neurons.
Assuntos
Neuroglia/citologia , Neuroglia/transplante , Medula Espinal/embriologia , Medula Espinal/transplante , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Feminino , Sobrevivência de Enxerto/fisiologia , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/cirurgia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/fisiologia , Fenótipo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Ratos , Ratos Wistar , Células-Tronco/metabolismoRESUMO
Corticosteroids can influence brain function, and glucocorticoid hormone receptors (GRs) are present in brain tissue. We observed that GR and also mineralocorticoid receptor (MR) are expressed by embryonic rat neural stem cells (NSCs). NSCs in developing ventricular epithelium were positive for GR. Stimulation of cultured NSCs with the specific receptor ligands dexamethasone and corticosterone reduced cell proliferation, shown by 5'-bromo-2-deoxy-uridine labeling. The effect of the hormones was dose dependent and inhibited by the GR blocker mifepristone but not by spironolactone, blocking MR. Dexamethasone inhibited the cell cycle by decreasing the levels of cyclin D1 in NSCs. The hormone-induced decline was inhibited by MG132 (benzyloxycarbonyl-leucyl-leucyl-leucinal), showing an involvement of the ubiquitin proteasome system, In keeping with this, dexamethasone increased the ubiquitination of cyclin D1. In embryonic brain, dexamethasone inhibited cell proliferation of NSCs. This demonstrates that embryonic NSCs are critically influenced by glucocorticoids, which can have long-term effects in the brain.