RESUMO
During meiosis, RNA polymerase II transcribes pachytene piRNA precursors with unusually long and unspliced transcripts from discrete autosomal loci in the mouse genome. Despite the importance of piRNA for male fertility and a well-defined maturation process, the transcriptional machinery remains poorly understood. Here, we document that D1PAS1, an ATP-dependent RNA helicase, is critical for pachytene piRNA expression from multiple genomic loci and subsequent translocation into the cytoplasm to ensure mature piRNA biogenesis. Depletion of D1PAS1 in gene-edited mice results in the accumulation of R-loops in pachytene spermatocytes, leading to DNA-damage-induced apoptosis, disruption of piRNA biogenesis, spermatogenic arrest, and male infertility. Transcriptome, genome-wide R-loop profiling, and proteomic analyses document that D1PAS1 regulates pachytene piRNA transcript elongation and termination. D1PAS1 subsequently forms a complex with nuclear export components to ensure pachytene piRNA precursor translocation from the nucleus to the cytoplasm for processing into small non-coding RNAs. Thus, our study defines D1PAS1 as a specific transcription activator that promotes R-loop unwinding and is a critical factor in pachytene piRNA biogenesis.
RESUMO
Hemogen is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells; however, the mechanism underlying its function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role for hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of corepressors nucleosome remodeling and histone deacetylase (NuRD) complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a coactivator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To determine whether hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen-knockout/knockin mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in embryonic days 12.5 to 16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. Chromatin immunoprecipitation sequencing in wild-type and hemogen-knockout animals revealed that BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, the hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.
Assuntos
DNA Helicases , Eritropoese , Proteínas Nucleares , Nucleossomos , Fatores de Transcrição , Animais , Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Eritropoese/genética , Hemoglobinas/genética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Germ-cell transcription factors control gene networks that regulate oocyte differentiation and primordial follicle formation during early, postnatal mouse oogenesis. Taking advantage of gene-edited mice lacking transcription factors expressed in female germ cells, we analyzed global gene expression profiles in perinatal ovaries from wildtype, FiglaNull, Lhx8Null and Sohlh1Null mice. Figla deficiency dysregulates expression of meiosis-related genes (e.g. Sycp3, Rad51, Ybx2) and a variety of genes (e.g. Nobox, Lhx8, Taf4b, Sohlh1, Sohlh2, Gdf9) associated with oocyte growth and differentiation. The absence of FIGLA significantly impedes meiotic progression, causes DNA damage and results in oocyte apoptosis. Moreover, we find that FIGLA and other transcriptional regulator proteins (e.g. NOBOX, LHX8, SOHLH1, SOHLH2) are co-expressed in the same subset of germ cells in perinatal ovaries and Figla ablation dramatically disrupts KIT, NOBOX, LHX8, SOHLH1 and SOHLH2 abundance. In addition, not only do FIGLA, LHX8 and SOHLH1 cross-regulate each other, they also cooperate by direct interaction with each during early oocyte development and share downstream gene targets. Thus, our findings substantiate a major role for FIGLA, LHX8 and SOHLH1 as multifunctional regulators of networks necessary for oocyte maintenance and differentiation during early folliculogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Redes Reguladoras de Genes , Proteínas com Homeodomínio LIM/metabolismo , Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células/genética , Dano ao DNA , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas com Homeodomínio LIM/genética , Meiose/genética , Camundongos , Oócitos/citologia , Ovário/metabolismo , Fatores de Transcrição/genéticaRESUMO
PIWI-interacting RNAs (piRNAs) are small non-coding RNAs essential for animal germ cell development. Despite intense investigation of post-transcriptional processing, chromatin regulators for piRNA biogenesis in mammals remain largely unexplored. Here we document that BTBD18 is a pachytene nuclear protein in mouse testes that occupies a subset of pachytene piRNA-producing loci. Ablation of Btbd18 in mice disrupts piRNA biogenesis, prevents spermiogenesis, and results in male sterility. Transcriptome profiling, chromatin accessibility, and RNA polymerase II occupancy demonstrate that BTBD18 facilitates expression of pachytene piRNA precursors by promoting transcription elongation. Thus, our study identifies BTBD18 as a specific controller for transcription activation through RNA polymerase II elongation at a subset of genomic piRNA loci.
Assuntos
Loci Gênicos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Elongação da Transcrição Genética , Animais , Apoptose/genética , Deleção de Genes , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Meiose/genética , Camundongos , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética , Testículo/citologiaRESUMO
Monospermic fertilization is mediated by the extracellular zona pellucida composed of ZP1, ZP2 and ZP3. Sperm bind to the N-terminus of ZP2 which is cleaved after fertilization by ovastacin (encoded by Astl) exocytosed from egg cortical granules to prevent sperm binding. AstlNull mice lack the post-fertilization block to sperm binding and the ability to rescue this phenotype with AstlmCherry transgenic mice confirms the role of ovastacin in providing a definitive block to polyspermy. During oogenesis, endogenous ovastacin traffics through the endomembrane system prior to storage in peripherally located cortical granules. Deletion mutants of ovastacinmCherry expressed in growing oocytes define a unique 7 amino acid motif near its N-terminus that is necessary and sufficient for cortical granule localization. Deletion of the 7 amino acids by CRISPR/Cas9 at the endogenous locus (AstlΔ) prevents cortical granule localization of ovastacin. The misdirected enzyme is present within the endomembrane system and ZP2 is prematurely cleaved. Sperm bind poorly to the zona pellucida of AstlΔ/Δ mice with partially cleaved ZP2 and female mice are sub-fertile.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Fertilização , Metaloproteases/metabolismo , Oócitos/metabolismo , Sinais Direcionadores de Proteínas , Glicoproteínas da Zona Pelúcida/metabolismo , Animais , Feminino , Metaloproteases/química , Metaloproteases/genética , Camundongos , Transporte Proteico , ProteóliseRESUMO
Although many genes that specify neocortical projection neuron subtypes have been identified, the downstream effectors that control differentiation of those subtypes remain largely unknown. Here, we demonstrate that the LIM domain-binding proteins Ldb1 and Ldb2 exhibit dynamic and inversely correlated expression patterns during cerebral cortical development. Ldb1-deficient brains display severe defects in proliferation and changes in regionalization, phenotypes resembling those of Lhx mutants. Ldb2-deficient brains, on the other hand, exhibit striking phenotypes affecting layer 5 pyramidal neurons: Immature neurons have an impaired capacity to segregate into mature callosal and subcerebral projection neurons. The analysis of Ldb2 single-mutant mice reveals a compensatory role of Ldb1 for Ldb2 during corticospinal motor neuron (CSMN) differentiation. Animals lacking both Ldb1 and Ldb2 uncover the requirement for Ldb2 during CSMN differentiation, manifested as incomplete CSMN differentiation, and ultimately leading to a failure of the corticospinal tract.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/deficiência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas com Domínio LIM/deficiência , Neurônios Motores/metabolismo , Tratos Piramidais/metabolismo , Fatores de Transcrição/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular/fisiologia , Camundongos Transgênicos , Neurogênese/fisiologia , Fatores de Transcrição/metabolismoRESUMO
LIM domain-binding protein 1 (Ldb1) is a nuclear cofactor that interacts with LIM homeodomain proteins to form multiprotein complexes that are important for transcription regulation. Ldb1 has been shown to play essential roles in various processes during mouse embryogenesis. To determine the role of Ldb1 in mid- and hindbrain development, we have generated a conditional mutant with a specific deletion of the Ldb1 in the Engrailed-1-expressing region of the developing mid- and hindbrain. Our study showed that the deletion impaired the expression of signaling molecules, such as fibroblast growth factor 8 (FGF8) and Wnt1, in the isthmic organizer and the expression of Shh in the ventral midbrain. The midbrain and the cerebellum were severely reduced in size, and the midbrain dopaminergic (mDA) neurons were missing in the mutant. These defects are identical to the phenotype that has been observed previously in mice with a deletion of the LIM homeodomain gene Lmx1b. Our results thus provide genetic evidence supporting that Ldb1 and Lmx1b function cooperatively to regulate mid- and hindbrain development. In addition, we found that mouse embryonic stem cells lacking Ldb1 failed to generate several types of differentiated neurons, including the mDA neurons, serotonergic neurons, cholinergic neurons, and olfactory bulb neurons, indicating an essential cell-autonomous role for Ldb1 in the development of these neurons.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Proteínas com Domínio LIM/metabolismo , Mesencéfalo/citologia , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Cerebelo/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/embriologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação/genéticaRESUMO
The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.
RESUMO
Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Proteínas do Tecido Nervoso/genética , Palato/embriologia , Palato/metabolismo , Fatores de Transcrição/genética , Animais , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Maxila/embriologia , Maxila/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Organogênese/genética , Palato/patologia , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
BACKGROUND: LIM domain binding protein 1 (LDB1) is a transcriptional co-factor, which interacts with multiple transcription factors and other proteins containing LIM domains. Complete inactivation of Ldb1 in mice resulted in early embryonic lethality with severe patterning defects during gastrulation. Tissue-specific deletions using a conditional knockout allele revealed additional roles of Ldb1 in the development of the central nervous system, hematopoietic system, and limbs. The goal of the current study was to determine the importance of Ldb1 function during craniofacial development in mouse embryos. RESULTS: We generated tissue-specific Ldb1 mutants using Wnt1-Cre, which causes deletion of a floxed allele in the neural crest; neural crest-derived cells contribute to most of the mesenchyme of the developing face. All examined Wnt1-Cre;Ldb1(fl/-) mutants suffered from cleft secondary palate. Therefore, we performed a series of experiments to investigate how Ldb1 regulated palate development. First, we examined the expression of Ldb1 during normal development, and found that Ldb1 was expressed broadly in the palatal mesenchyme during early stages of palate development. Second, we compared the morphology of the developing palate in control and Ldb1 mutant embryos using sections. We found that the mutant palatal shelves had abnormally blunt appearance, and failed to elevate above the tongue at the posterior domain. An in vitro head culture experiment indicated that the elevation defect was not due to interference by the tongue. Finally, in the Ldb1 mutant palatal shelves, cell proliferation was abnormal in the anterior, and the expression of Wnt5a, Pax9 and Osr2, which regulate palatal shelf elevation, was also altered. CONCLUSIONS: The function of Ldb1 in the neural crest-derived palatal mesenchyme is essential for normal morphogenesis of the secondary palate.
Assuntos
Fissura Palatina/genética , Proteínas de Ligação a DNA/genética , Proteínas com Domínio LIM/genética , Crista Neural/metabolismo , Palato/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Células Cultivadas , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Proteínas com Domínio LIM/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Crista Neural/embriologia , Crista Neural/patologia , Fator de Transcrição PAX9 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Palato/embriologia , Palato/patologia , Gravidez , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
The progenitor zones of the embryonic mouse ventral telencephalon give rise to GABAergic and cholinergic neurons. We have shown previously that two LIM-homeodomain (LIM-HD) transcription factors, Lhx6 and Lhx8, that are downstream of Nkx2.1, are critical for the development of telencephalic GABAergic and cholinergic neurons. Here we investigate the role of Ldb1, a nuclear protein that binds directly to all LIM-HD factors, in the development of these ventral telencephalon derived neurons. We show that Ldb1 is expressed in the Nkx2.1 cell lineage during embryonic development and in mature neurons. Conditional deletion of Ldb1 causes defects in the expression of a series of genes in the ventral telencephalon and severe impairment in the tangential migration of cortical interneurons from the ventral telencephalon. Similar to the phenotypes observed in Lhx6 or Lhx8 mutant mice, the Ldb1 conditional mutants show a reduction in the number of both GABAergic and cholinergic neurons in the telencephalon. Furthermore, our analysis reveals defects in the development of the parvalbumin-positive neurons in the globus pallidus and striatum of the Ldb1 mutants. These results provide evidence that Ldb1 plays an essential role as a transcription co-regulator of Lhx6 and Lhx8 in the control of mammalian telencephalon development.
Assuntos
Neurônios Colinérgicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Nucleares/metabolismo , Telencéfalo/embriologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Globo Pálido/embriologia , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Telencéfalo/crescimento & desenvolvimento , Fator Nuclear 1 de TireoideRESUMO
The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.
Assuntos
Vias Aferentes/fisiologia , Evolução Biológica , Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Bulbo Olfatório , Fatores Etários , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diencéfalo/citologia , Diencéfalo/fisiologia , Eletroporação/métodos , Embrião de Mamíferos , Feminino , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Microinjeções/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Oócitos , Técnicas de Cultura de Órgãos , Gravidez , Telencéfalo/citologia , Telencéfalo/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor , Órgão Vomeronasal/citologia , Órgão Vomeronasal/embriologia , Órgão Vomeronasal/crescimento & desenvolvimento , XenopusRESUMO
BACKGROUND: Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2-/-) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. RESULTS: Dlx1/2-/- mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down-regulated in the same region. Previous studies established that the epithelial-mesenchymal signaling loop involving Shh, Bmp4, and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2-/- palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2-/- mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. CONCLUSIONS: The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis.
Assuntos
Fissura Palatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Palato/embriologia , Palato/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
Lhx6 and Lhx8 transcription factor coexpression in early-born MGE neurons is required to induce neuronal Shh expression. We provide evidence that these transcription factors regulate expression of a Shh enhancer in MGE neurons. Lhx6 and Lhx8 are also required to prevent Nkx2-1 expression in a subset of pallial interneurons. Shh function in early-born MGE neurons was determined by genetically eliminating Shh expression in the MGE mantle zone (MZ). This mutant had reduced SHH signaling in the overlying progenitor zone, which led to reduced Lhx6, Lhx8, and Nkx2-1 expression in the rostrodorsal MGE and a preferential reduction of late-born somatostatin(+) and parvalbumin(+) cortical interneurons. Thus, Lhx6 and Lhx8 regulate MGE development through autonomous and nonautonomous mechanisms, the latter by promoting Shh expression in MGE neurons, which in turn feeds forward to promote the developmental program of the rostrodorsal MGE.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Corpos Geniculados/citologia , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Corpos Geniculados/embriologia , Corpos Geniculados/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismoRESUMO
The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Desenvolvimento Fetal , Células-Tronco Hematopoéticas/metabolismo , Transferência Adotiva , Células-Tronco Adultas/citologia , Células-Tronco Adultas/imunologia , Células-Tronco Adultas/transplante , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Feminino , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Ligação Proteica , Elementos Reguladores de Transcrição/genética , Elementos Reguladores de Transcrição/imunologia , Ativação Transcricional/genética , Ativação Transcricional/imunologiaRESUMO
Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Serina Endopeptidases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular , Técnicas de Cultura Embrionária , Proteínas da Matriz Extracelular/genética , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteína Reelina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Fatores de Transcrição/genéticaRESUMO
The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke's pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke's pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. In this study, we show that the LIM-homeobox gene Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary. Deletion of Lhx2 gene results in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke's pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe. Our study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland.
Assuntos
Proteínas de Homeodomínio/metabolismo , Hipófise/embriologia , Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Padronização Corporal/genética , Linhagem da Célula , Proliferação de Células , Diencéfalo/embriologia , Diencéfalo/metabolismo , Diencéfalo/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Modelos Biológicos , Mutação/genética , Hipófise/patologia , Neuro-Hipófise/embriologia , Neuro-Hipófise/metabolismo , Neuro-Hipófise/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Vertebrate limb development is controlled by three signaling centers that regulate limb patterning and growth along the proximodistal (PD), anteroposterior (AP) and dorsoventral (DV) limb axes. Coordination of limb development along these three axes is achieved by interactions and feedback loops involving the secreted signaling molecules that mediate the activities of these signaling centers. However, it is unknown how these signaling interactions are processed in the responding cells. We have found that distinct LIM homeodomain transcription factors, encoded by the LIM homeobox (LIM-HD) genes Lhx2, Lhx9 and Lmx1b integrate the signaling events that link limb patterning and outgrowth along all three axes. Simultaneous loss of Lhx2 and Lhx9 function resulted in patterning and growth defects along the AP and the PD limb axes. Similar, but more severe, phenotypes were observed when the activities of all three factors, Lmx1b, Lhx2 and Lhx9, were significantly reduced by removing their obligatory co-factor Ldb1. This reveals that the dorsal limb-specific factor Lmx1b can partially compensate for the function of Lhx2 and Lhx9 in regulating AP and PD limb patterning and outgrowth. We further showed that Lhx2 and Lhx9 can fully substitute for each other, and that Lmx1b is partially redundant, in controlling the production of output signals in mesenchymal cells in response to Fgf8 and Shh signaling. Our results indicate that several distinct LIM-HD transcription factors in conjunction with their Ldb1 co-factor serve as common central integrators of distinct signaling interactions and feedback loops to coordinate limb patterning and outgrowth along the PD, AP and DV axes after limb bud formation.
Assuntos
Padronização Corporal , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Fatores de Transcrição/genéticaRESUMO
Here we analyze the role of the Lhx6 lim-homeobox transcription factor in regulating the development of subsets of neocortical, hippocampal, and striatal interneurons. An Lhx6 loss-of-function allele, which expresses placental alkaline phosphatase (PLAP), allowed analysis of the development and fate of Lhx6-expressing interneurons in mice lacking this homeobox transcription factor. There are Lhx6+;Dlx+ and Lhx6-;Dlx+ subtypes of tangentially migrating interneurons. Most interneurons in Lhx6(PLAP/PLAP) mutants migrate to the cortex, although less efficiently, and exhibit defects in populating the marginal zone and superficial parts of the neocortical plate. By contrast, migration to superficial parts of the hippocampus is not seriously affected. Furthermore, whereas parvalbumin+ and somatostatin+ interneurons do not differentiate, NPY+ interneurons are present; we suggest that these NPY+ interneurons are derived from the Lhx6-;Dlx+ subtype. Striatal interneurons show deficits distinct from pallial interneurons, including a reduction in the NPY+ subtype. We provide evidence that Lhx6 mediates these effects through promoting expression of receptors that regulate interneuron migration (ErbB4, CXCR4, and CXCR7), and through promoting the expression of transcription factors either known (Arx) or implicated (bMaf, Cux2, and NPAS1) in controlling interneuron development.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interneurônios/citologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Telencéfalo/citologia , Telencéfalo/metabolismo , Animais , Animais Recém-Nascidos , Movimento Celular , Córtex Cerebral/citologia , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Embrião de Mamíferos , Hipocampo/citologia , Hipocampo/metabolismo , Interneurônios/metabolismo , Interneurônios/fisiologia , Proteínas com Homeodomínio LIM , Camundongos/embriologia , Camundongos/crescimento & desenvolvimento , Camundongos Endogâmicos , Neocórtex/citologia , Neocórtex/metabolismo , Fatores de TranscriçãoRESUMO
Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.