RESUMO
The striatum is a central regulator of behavior and motor function through the actions of D1 and D2 medium-sized spiny neurons (MSNs), which arise from a common lateral ganglionic eminence (LGE) progenitor. The molecular mechanisms of cell fate specification of these two neuronal subtypes are incompletely understood. Here, we found that deletion of murine Meis2, which is highly expressed in the LGE and derivatives, led to a large reduction in striatal MSNs due to a block in their differentiation. Meis2 directly binds to the Zfp503 and Six3 promoters and is required for their expression and specification of D1 and D2 MSNs, respectively. Finally, Meis2 expression is regulated by Dlx1/2 at least partially through the enhancer hs599 in the LGE subventricular zone. Overall, our findings define a pathway in the LGE whereby Dlx1/2 drives expression of Meis2, which subsequently promotes the fate determination of striatal D1 and D2 MSNs via Zfp503 and Six3.
Assuntos
Corpo Estriado/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ventrículos Laterais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteína Homeobox SIX3RESUMO
In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.
Assuntos
Proteínas de Homeodomínio , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Diferenciação Celular/genética , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.
Assuntos
Córtex Cerebral/embriologia , Redes Reguladoras de Genes , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Fator I de Transcrição COUP/metabolismo , Córtex Cerebral/metabolismo , Epigenoma , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Fatores de Transcrição/genéticaRESUMO
Dopamine receptor DRD1-expressing medium spiny neurons (D1 MSNs) and dopamine receptor DRD2-expressing medium spiny neurons (D2 MSNs) are the principal projection neurons in the striatum, which is divided into dorsal striatum (caudate nucleus and putamen) and ventral striatum (nucleus accumbens and olfactory tubercle). Progenitors of these neurons arise in the lateral ganglionic eminence (LGE). Using conditional deletion, we show that mice lacking the transcription factor genes Sp8 and Sp9 lose virtually all D2 MSNs as a result of reduced neurogenesis in the LGE, whereas D1 MSNs are largely unaffected. SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants. Finally, ChIP-Seq reveals that SP9 directly binds to the promoter and a putative enhancer of Six3 Thus, this study defines components of a transcription pathway in a regionally restricted LGE progenitor domain that selectively drives the generation of D2 MSNs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurônios/citologia , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Fatores de Transcrição/genética , Proteína Homeobox SIX3RESUMO
Distinct cortical interneuron (CIN) subtypes have unique circuit functions; dysfunction in specific subtypes is implicated in neuropsychiatric disorders. Somatostatin- and parvalbumin-expressing (SST+ and PV+) interneurons are the two major subtypes generated by medial ganglionic eminence (MGE) progenitors. Spatial and temporal mechanisms governing their cell-fate specification and differential integration into cortical layers are largely unknown. We provide evidence that Coup-TF1 and Coup-TF2 (Nr2f1 and Nr2f2) transcription factor expression in an arc-shaped progenitor domain within the MGE promotes time-dependent survival of this neuroepithelium and the time-dependent specification of layer V SST+ CINs. Coup-TF1 and Coup-TF2 autonomously repress PV+ fate in MGE progenitors, in part through directly driving Sox6 expression. These results have identified, in mouse, a transcriptional pathway that controls SST-PV fate.
Assuntos
Fator II de Transcrição COUP/metabolismo , Fator I de Transcrição COUP/metabolismo , Interneurônios/metabolismo , Neocórtex/citologia , Animais , Fator I de Transcrição COUP/genética , Fator II de Transcrição COUP/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Parvalbuminas/genética , Parvalbuminas/metabolismo , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismo , Somatostatina/genética , Somatostatina/metabolismoRESUMO
Immature neurons generated by the subpallial MGE tangentially migrate to the cortex where they become parvalbumin-expressing (PV+) and somatostatin (SST+) interneurons. Here, we show that the Sp9 transcription factor controls the development of MGE-derived cortical interneurons. SP9 is expressed in the MGE subventricular zone and in MGE-derived migrating interneurons. Sp9 null and conditional mutant mice have approximately 50% reduction of MGE-derived cortical interneurons, an ectopic aggregation of MGE-derived neurons in the embryonic ventral telencephalon, and an increased ratio of SST+/PV+ cortical interneurons. RNA-Seq and SP9 ChIP-Seq reveal that SP9 regulates MGE-derived cortical interneuron development through controlling the expression of key transcription factors Arx, Lhx6, Lhx8, Nkx2-1, and Zeb2 involved in interneuron development, as well as genes implicated in regulating interneuron migration Ackr3, Epha3, and St18. Thus, Sp9 has a central transcriptional role in MGE-derived cortical interneuron development.
Assuntos
Córtex Cerebral/citologia , Interneurônios/citologia , Eminência Mediana/embriologia , Neurogênese/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Interneurônios/metabolismo , Eminência Mediana/citologia , Camundongos , Fatores de Transcrição/metabolismoRESUMO
The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Neurônios GABAérgicos/fisiologia , Proteínas de Homeodomínio/fisiologia , Interneurônios/fisiologia , Sinapses/fisiologia , Fatores de Transcrição/fisiologia , Ácido gama-Aminobutírico/biossíntese , Animais , Córtex Cerebral/citologia , Feminino , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Glutamato Descarboxilase/metabolismo , Proteínas de Homeodomínio/genética , Interneurônios/citologia , Masculino , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura , Fatores de Transcrição/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Transcription factors (TFs) bind combinatorially to genomic cis-regulatory elements (cREs), orchestrating transcription programs. While studies of chromatin state and chromosomal interactions have revealed dynamic neurodevelopmental cRE landscapes, parallel understanding of the underlying TF binding lags. To elucidate the combinatorial TF-cRE interactions driving mouse basal ganglia development, we integrated ChIP-seq for twelve TFs, H3K4me3-associated enhancer-promoter interactions, chromatin and transcriptional state, and transgenic enhancer assays. We identified TF-cREs modules with distinct chromatin features and enhancer activity that have complementary roles driving GABAergic neurogenesis and suppressing other developmental fates. While the majority of distal cREs were bound by one or two TFs, a small proportion were extensively bound, and these enhancers also exhibited exceptional evolutionary conservation, motif density, and complex chromosomal interactions. Our results provide new insights into how modules of combinatorial TF-cRE interactions activate and repress developmental expression programs and demonstrate the value of TF binding data in modeling gene regulatory wiring.
RESUMO
The conserved mRNA export receptor NXF1 (Mex67 in yeast) assembles with messenger ribonucleoproteins (mRNP) in the nucleus and guides them through the nuclear pore complex into the cytoplasm. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage. However, the molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. Here we report that the human NXF1-binding protein RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. We found that RBM15 is targeted to the nuclear envelope, where it colocalizes extensively with DBP5 and NXF1. Gene silencing of RBM15 leads to cytoplasmic depletion and nuclear accumulation of general mRNA as well as individual endogenous transcripts, indicating that RBM15 is required for efficient mRNA export. We propose a model in which RBM15 acts locally at the nuclear pore complex, by facilitating the recognition of NXF1-mRNP complexes by DBP5 during translocation, thereby contributing to efficient mRNA export.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Humanos , Membrana Nuclear/metabolismo , Interferência de RNA , Splicing de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/metabolismoRESUMO
GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.
Assuntos
Diferenciação Celular , Neurônios GABAérgicos/fisiologia , Redes Reguladoras de Genes , Hipotálamo/citologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sobrevivência Celular , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Hipotálamo/metabolismo , Camundongos , Proteínas Nucleares , Sono/fisiologiaRESUMO
The human SPEN family proteins SHARP, RBM15/OTT1, and RBM15B/OTT3 share the structural domain architecture but show distinct functional properties. Here, we examined the function of OTT3 and compared it with its paralogues RBM15 and SHARP. We found that OTT3, like RBM15, has post-transcriptional regulatory activity, whereas SHARP does not, supporting a divergent role of RBM15 and OTT3. OTT3 shares with RBM15 the association with the splicing factor compartment and the nuclear envelope as well as the binding to mRNA export factors NXF1 and Aly/REF. Mutational analysis revealed direct interaction of OTT3 and RBM15 with NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knockdown of RBM15 in mouse is embryonically lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles.
Assuntos
Membrana Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Células HeLa , Humanos , Camundongos , Membrana Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a RNA/genética , Homologia Estrutural de ProteínaRESUMO
Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction.
Assuntos
Transtorno Autístico , Interneurônios , Elementos Reguladores de Transcrição , Esquizofrenia , Animais , Animais Geneticamente Modificados , Dependovirus , Vetores Genéticos , Camundongos , Fatores de TranscriçãoRESUMO
âMaf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.
Assuntos
Regulação da Expressão Gênica , Interneurônios/metabolismo , Fator de Transcrição MafB/fisiologia , Proteínas Proto-Oncogênicas c-maf/fisiologia , Animais , Feminino , Fatores de Transcrição MEF2/metabolismo , Camundongos , Doenças do Sistema Nervoso/etiologia , Gravidez , Precursores de Proteínas/genética , Receptores CXCR4/metabolismo , Receptores Opioides/genética , Análise de Célula Única , Proteína 25 Associada a Sinaptossoma/metabolismo , TranscriptomaRESUMO
DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.
Assuntos
Neurônios GABAérgicos/metabolismo , Redes Reguladoras de Genes , Genoma , Proteínas de Homeodomínio/metabolismo , Prosencéfalo/embriologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Camundongos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6â Tbr2â Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6â Tbr2â Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.
RESUMO
TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Grânulos Citoplasmáticos/química , Humanos , Camundongos , Dados de Sequência Molecular , Membrana Nuclear/química , Sinais de Localização Nuclear , Proteínas de Transporte Nucleocitoplasmático/análise , Proteínas de Transporte Nucleocitoplasmático/genética , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genéticaRESUMO
Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate.
Assuntos
Diferenciação Celular/genética , Elementos Facilitadores Genéticos/genética , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Gânglios da Base/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Otx2 homeodomain transcription factor is essential for gastrulation and early neural development. We generated Otx2 conditional knockout (cKO) mice to investigate its roles in telencephalon development after neurulation (approximately embryonic day 9.0). We conducted transcriptional profiling and in situ hybridization to identify genes de-regulated in Otx2 cKO ventral forebrain. In parallel, we used chromatin immunoprecipitation sequencing to identify enhancer elements, the OTX2 binding motif, and de-regulated genes that are likely direct targets of OTX2 transcriptional regulation. We found that Otx2 was essential in septum specification, regulation of Fgf signaling in the rostral telencephalon, and medial ganglionic eminence (MGE) patterning, neurogenesis, and oligodendrogenesis. Within the MGE, Otx2 was required for ventral, but not dorsal, identity, thus controlling the production of specific MGE derivatives.
Assuntos
Córtex Cerebral/embriologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Animais , Córtex Cerebral/citologia , Feminino , Expressão Gênica , CamundongosRESUMO
We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2(+/-) sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.
Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Homeodomínio/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Transgênicos , Mitose/fisiologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteína Reelina , Células-Tronco/fisiologiaRESUMO
Elucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreER(T2) and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define a comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6, and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions.