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1.
Development ; 146(14)2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239243

RESUMO

Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.


Assuntos
Proteína Morfogenética Óssea 4/fisiologia , Diferenciação Celular/genética , Organoides/fisiologia , Proteínas Smad/metabolismo , Coluna Vertebral/citologia , Animais , Linhagem da Célula/genética , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Crista Neural/citologia , Crista Neural/fisiologia , Tubo Neural/citologia , Tubo Neural/embriologia , Neurônios/citologia , Neurônios/fisiologia , Organoides/citologia , Transdução de Sinais/genética , Proteínas Smad/genética
2.
Dev Biol ; 432(1): 24-33, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28625870

RESUMO

Transcription factors are key orchestrators of the emergence of neuronal diversity within the developing spinal cord. As such, the two paralogous proteins Pax3 and Pax7 regulate the specification of progenitor cells within the intermediate neural tube, by defining a neat segregation between those fated to form motor circuits and those involved in the integration of sensory inputs. To attain insights into the molecular means by which they control this process, we have performed detailed phenotypic analyses of the intermediate spinal interneurons (IN), namely the dI6, V0D, V0VCG and V1 populations in compound null mutants for Pax3 and Pax7. This has revealed that the levels of Pax3/7 proteins determine both the dorso-ventral extent and the number of cells produced in each subpopulation; with increasing levels leading to the dorsalisation of their fate. Furthermore, thanks to the examination of mutants in which Pax3 transcriptional activity is skewed either towards repression or activation, we demonstrate that this cell diversification process is mainly dictated by Pax3/7 ability to repress gene expression. Consistently, we show that Pax3 and Pax7 inhibit the expression of Dbx1 and of its repressor Prdm12, fate determinants of the V0 and V1 interneurons, respectively. Notably, we provide evidence for the activity of several cis-regulatory modules of Dbx1 to be sensitive to Pax3 and Pax7 transcriptional activity levels. Altogether, our study provides insights into how the redundancy within a TF family, together with discrete dynamics of expression profiles of each member, are exploited to generate cellular diversity. Furthermore, our data supports the model whereby cell fate choices in the neural tube do not rely on binary decisions but rather on inhibition of multiple alternative fates.


Assuntos
Proteínas de Homeodomínio/fisiologia , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fator de Transcrição PAX3/fisiologia , Fator de Transcrição PAX7/fisiologia , Medula Espinal/citologia , Animais , Diferenciação Celular/fisiologia , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/citologia , Camundongos , Tubo Neural/fisiologia , Medula Espinal/embriologia , Células-Tronco/citologia , Células-Tronco/fisiologia
3.
Biol Open ; 4(12): 1614-24, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26538636

RESUMO

Skeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120 kb upstream of the Mrf4-Myf5 locus. An enhancer, located at -57/-58 kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development.

4.
Development ; 141(8): 1726-36, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24715462

RESUMO

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Medula Espinal/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Interneurônios/citologia , Interneurônios/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Medula Espinal/embriologia , Células-Tronco/metabolismo
5.
Development ; 138(24): 5393-402, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22071108

RESUMO

The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. Here, we provide evidence for the involvement of homeobox Msx transcription factors in foetal meiosis initiation in mammalian germ cells. Upon meiosis initiation, Msx1 and Msx2 genes are strongly expressed in the foetal ovary, possibly stimulated by soluble factors found there: bone morphogenetic proteins Bmp2 and Bmp4, and retinoic acid. Analysis of Msx1/Msx2 double mutant embryos revealed a majority of undifferentiated germ cells remaining in the ovary and, importantly, a decrease in the number of meiotic cells. In vivo, the Msx1/Msx2 double-null mutation prevented full activation of Stra8, a gene required for meiosis. In F9 cells, Msx1 can bind to Stra8 regulatory sequences and Msx1 overexpression stimulates Stra8 transcription. Collectively, our data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line.


Assuntos
Proteínas de Homeodomínio/fisiologia , Fator de Transcrição MSX1/fisiologia , Meiose/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Mutação , Técnicas de Cultura de Órgãos , Ovário/fisiologia , Proteínas/metabolismo , Tretinoína/metabolismo , Tretinoína/fisiologia
6.
Development ; 132(8): 1807-18, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15772136

RESUMO

Targeted disruption of effectors molecules of the apoptotic pathway have demonstrated the occurrence and magnitude of early programmed cell death (EPCD), a form of apoptosis that affects proliferating and newly differentiated cells in vertebrates, and most dramatically cells of the central nervous system (CNS). Little is known about the molecular pathways controlling apoptosis at these early developmental stages, as the roles of EPCD during patterning of the developing nervous system. We describe a new function, in Xenopus neurodevelopment, for a highly conserved homeodomain protein Barhl2. Barhl2 promotes apoptosis in the Xenopus neuroectoderm and mesoderm, acting as a transcriptional repressor, through a mechanism that cannot be attributed to an unspecific cellular stress response. We show that the pro-apoptotic activity of Barhl2 is essential during normal neural plate formation as it limits the number of chordin- and Xshh-expressing cells in the prospective notochord and floorplate, which act as organizing centers. Our findings show that Barhl2 is part of a pathway regulating EPCD. They also provide evidence that apoptosis plays an important role in regulating the size of organizing centers.


Assuntos
Apoptose/genética , Padronização Corporal/genética , Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Sistema Nervoso Central/metabolismo , Análise por Conglomerados , Primers do DNA , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Ensaio de Imunoadsorção Enzimática , Galactosídeos , Glicoproteínas/metabolismo , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Indóis , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Filogenia , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Xenopus/genética , Proteínas de Xenopus/genética
7.
J Neurosci ; 23(36): 11444-52, 2003 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-14673009

RESUMO

Glial calcium signals play important roles during CNS development. Calcium transients induced by ATP, acting on purinergic receptors, stimulate DNA synthesis, increase astrocytic and neural stem cell proliferation, and are prominent during the differentiation of radial glia. We have shown previously that expression of P2Y receptors in astrocytes is altered when connexin43 (Cx43) is downregulated. To evaluate the consequences of Cx43 deletion on calcium signaling during neural progenitor development, studies were performed on neurospheres derived from embryonic striatum. After adhesion, cells migrating from wild-type (WT) and Cx43-null neurospheres displayed spontaneous calcium oscillations. Such activity was blunted by apyrase, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179), and suramin, suggesting that ATP released by neural cells acts on purinergic receptors to induce calcium oscillations. The amplitudes of Ca2+ transients induced by P2Y but not P2X receptor agonists were larger in WT than in Cx43-null progenitors, suggesting that these two cell populations express different P2 receptors. Suramin, a nonselective P2 receptor antagonist, and MRS-2179, a P2Y1 receptor-selective antagonist, reduced the proliferation rate and the migration of WT progenitor cells to levels similar to those of Cx43-null cells. Conversely, exogenous expression of P2Y1 receptors in Cx43-null cells restored their migration pattern to levels seen in WT progenitors. However, treatment with P2 receptor antagonists did not alter the ratio of nestin to GFAP expression in WT neural progenitors. These data show that altered autocrine-paracrine communication attributable to reduced levels of P2Y1 receptors in neural progenitor cells lacking Cx43 affects proliferation and migration but not cell differentiation during early CNS development.


Assuntos
Sinalização do Cálcio , Movimento Celular , Conexina 43/fisiologia , Neurônios/citologia , Receptores Purinérgicos P2/metabolismo , Células-Tronco/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Conexina 43/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y1 , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica
8.
J Cell Sci ; 115(Pt 16): 3241-51, 2002 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12140256

RESUMO

Embryonic neural progenitors isolated from the mouse striatal germinal zone grow in vitro as floating cell aggregates called neurospheres, which, upon adhesion, can be induced to differentiate into the three main cell types of the central nervous system (CNS), that is, astrocytes, neurons and oligodendrocytes. To study the possible role of connexins and junctional communication during differentiation of neural progenitors, we assessed cell-to-cell communication by microinjecting Lucifer Yellow into neurospheres at various times after adhesion. Cells located in neurospheres were strongly coupled, regardless of the differentiation time. Microinjections performed on the cell layers formed by differentiated cells migrating out of the neurosphere established that only astrocytes were coupled. These observations suggest the existence of at least three distinct communication compartments: coupled proliferating cells located in the sphere, uncoupled cells undergoing neuronal or oligodendrocytic differentiation and coupled differentiating astrocytes. A blockade of junctional communication by 18-beta-glycyrrhetinic acid (betaGA) reduced, in a concentration-dependent manner, the viability of undifferentiated neural progenitor cells. This effect appeared to be specific, inasmuch as it was reversible and that cell survival was not affected in the presence of the inactive analog glycyrrhyzic acid. Addition of betaGA to adherent neurospheres also decreased cell density and altered the morphology of differentiated cells. Cx43 was strongly expressed in either undifferentiated or differentiated neurospheres, where it was found both within the sphere and in astrocytes, the two cell populations that were dye coupled. Western blot analysis further showed that Cx43 phosphorylation was strongly increased in adherent neurospheres, suggesting a post-translational regulation during differentiation. These results point to a major role of cell-to-cell communication and Cx43 during the differentiation of neural progenitor cells in vitro.


Assuntos
Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Conexina 43/metabolismo , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Divisão Celular/fisiologia , Tamanho Celular , Sobrevivência Celular , Células Cultivadas , Conexina 43/genética , Feminino , Corantes Fluorescentes , Ácido Glicirretínico/química , Ácido Glicirretínico/farmacologia , Humanos , Junções Intercelulares/metabolismo , Isoquinolinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Neurônios/citologia , Neurônios/efeitos dos fármacos , Gravidez , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
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