RESUMO
Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2(+) ventricular zone progenitors into Nurr1(+) postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dopamina/metabolismo , Mesencéfalo/citologia , Mesencéfalo/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas com Homeodomínio LIM , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Fatores de Transcrição SOXB1 , Células-Tronco/citologia , Células-Tronco/fisiologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The dorsal spinal cord contains a diverse array of neurons that connect sensory input from the periphery to spinal cord motoneurons and brain. During development, six dorsal neuronal populations (dI1-dI6) have been defined by expression of homeodomain factors and position in the dorsoventral axis. The bHLH transcription factors Mash1 and Ngn2 have distinct roles in specification of these neurons. Mash1 is necessary and sufficient for generation of most dI3 and all dI5 neurons. Unexpectedly, dI4 neurons are derived from cells expressing low levels or no Mash1, and this population increases in the Mash1 mutant. Ngn2 is not required for any specific neuronal cell type but appears to modulate the composition of neurons that form. In the absence of Ngn2, there is an increase in the number of dI3 and dI5 neurons, in contrast to the effects produced by activity of Mash1. Mash1 is epistatic to Ngn2, and, unlike the relationship between other neural bHLH factors, cross-repression of expression is not detected. Thus, bHLH factors, particularly Mash1 and related family members Math1 and Ngn1, provide a code for generating neuronal diversity in the dorsal spinal cord with Ngn2 serving to modulate the number of neurons in each population formed.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interneurônios/citologia , Interneurônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Padronização Corporal/genética , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Epistasia Genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Medula Espinal/embriologia , Fatores de Transcrição/genéticaRESUMO
The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution.
Assuntos
Sequência Conservada/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Elementos Facilitadores Genéticos/genética , Proteínas do Olho , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Elementos de Resposta/genética , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Alinhamento de Sequência , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Transcrição Gênica/genética , Transgenes/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The transcriptional control of the differentiation of central serotonergic (5-HT) neurons in vertebrates has recently come under scrutiny and has been shown to involve the homeobox genes Nkx2-2 and Lmx1b, the Ets-domain gene Pet1 (also known as Fev) and the zinc-finger gene Gata3. The basic helix-loop-helix (bHLH) gene Ascl1 (also known as Mash1) is coexpressed with Nkx2-2 in the neuroepithelial domain of the hindbrain, which gives rise to 5-HT neurons. Here we show in the mouse that Ascl1 is essential for the birth of 5-HT neurons, both as a proneural gene for the production of postmitotic neuronal precursors and as a determinant of the serotonergic phenotype for the parallel activation of Gata3, Lmx1b and Pet1. Thus Ascl1, which is essential for noradrenergic differentiation, is also a determinant of the serotonergic phenotype.
