RESUMO
Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.
Assuntos
Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/fisiologia , Animais , Diferenciação Celular , Neurônios Colinérgicos/fisiologia , Análise por Conglomerados , Simulação por Computador , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Neurotransmissores/genética , Neurotransmissores/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl(-/-) embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl(-/-) hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl(fl/fl)Rosa26Cre-ER(T2) embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl(-/-) endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endotélio Vascular/embriologia , Coração/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Caderinas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Mesoderma/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Placenta/irrigação sanguínea , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/metabolismo , Saco Vitelino/irrigação sanguíneaRESUMO
Tissue-specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.
Assuntos
Degeneração Macular , Canais de Cátion TRPM , Humanos , Camundongos , Animais , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Estudo de Associação Genômica Ampla , Proteômica , Degeneração Macular/genética , Degeneração Macular/metabolismo , Diferenciação Celular , Epitélio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Canais de Cátion TRPM/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismoRESUMO
Primates and rodents, which descended from a common ancestor around 90 million years ago1, exhibit profound differences in behaviour and cognitive capacity; the cellular basis for these differences is unknown. Here we use single-nucleus RNA sequencing to profile RNA expression in 188,776 individual interneurons across homologous brain regions from three primates (human, macaque and marmoset), a rodent (mouse) and a weasel (ferret). Homologous interneuron types-which were readily identified by their RNA-expression patterns-varied in abundance and RNA expression among ferrets, mice and primates, but varied less among primates. Only a modest fraction of the genes identified as 'markers' of specific interneuron subtypes in any one species had this property in another species. In the primate neocortex, dozens of genes showed spatial expression gradients among interneurons of the same type, which suggests that regional variation in cortical contexts shapes the RNA expression patterns of adult neocortical interneurons. We found that an interneuron type that was previously associated with the mouse hippocampus-the 'ivy cell', which has neurogliaform characteristics-has become abundant across the neocortex of humans, macaques and marmosets but not mice or ferrets. We also found a notable subcortical innovation: an abundant striatal interneuron type in primates that had no molecularly homologous counterpart in mice or ferrets. These interneurons expressed a unique combination of genes that encode transcription factors, receptors and neuropeptides and constituted around 30% of striatal interneurons in marmosets and humans.
Assuntos
Interneurônios/citologia , Primatas , Animais , Callithrix , Córtex Cerebral/citologia , Feminino , Furões , Hipocampo/citologia , Humanos , Interneurônios/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Macaca , Masculino , Camundongos , Neostriado/citologia , Proteínas do Tecido Nervoso/metabolismo , RNA/genética , Especificidade da Espécie , Fatores de Transcrição/metabolismoRESUMO
In the mammalian cerebral cortex, the hippocampal primordium (Hcp) occupies a discrete position in the dorsal telencephalic neuroepithelium adjacent to the neocortical primordium (Ncp). We examined transcriptomic and chromatin-level features that distinguish the Hcp from the Ncp in the mouse during the early neurogenic period, embryonic day (E)12.5. ATAC-seq revealed that the Hcp was more accessible than the Ncp at this stage. Motif analysis of the differentially accessible loci in these tissues revealed LHX2 as a candidate transcription factor for modulating gene regulatory networks (GRNs). We analyzed LHX2 occupancy profiles and compared these with transcriptomic data from control and Lhx2 mutant Hcp and Ncp at E12.5. Our results revealed that LHX2 directly regulates distinct genes in the Hcp and Ncp within a set of common pathways that control fundamental aspects of development namely pluripotency, axon pathfinding, Wnt, and Hippo signaling. Loss of Lhx2 caused a decrease in accessibility, specifically in hippocampal chromatin, suggesting that this factor may play a unique role in hippocampal development. We identified 14 genes that were preferentially enriched in the Hcp, for which LHX2 regulates both chromatin accessibility and mRNA expression, which have not thus far been examined in hippocampal development. Together, these results provide mechanistic insight into how LHX2 function in the Hcp may contribute to the process by which the hippocampus acquires features distinct from the neocortex.
Assuntos
Cromatina , Neocórtex , Animais , Camundongos , Hipocampo , Proteínas com Homeodomínio LIM , Mamíferos , Fatores de Transcrição , TranscriptomaRESUMO
Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm and left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons, and, recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. These cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here, we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. We use RNA sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons as having a restricted spinal expression pattern with no Shox2 neuron overlap. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons leads to a decrease in the frequency of locomotor-like activity without change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the locomotor activity. Calcium activities of Lhx9-derived neurons show strong left-right out-of-phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.
Assuntos
Interneurônios , Proteínas com Homeodomínio LIM , Locomoção , Medula Espinal , Fatores de Transcrição , Animais , Interneurônios/fisiologia , Camundongos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Locomoção/fisiologia , Medula Espinal/fisiologia , Medula Espinal/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Masculino , Feminino , Ácido Glutâmico/metabolismo , Animais Recém-Nascidos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismoRESUMO
The inner ear sensory neurons play a pivotal role in auditory processing and balance control. Though significant progresses have been made, the underlying mechanisms controlling the differentiation and survival of the inner ear sensory neurons remain largely unknown. During development, ISL1 and POU4F transcription factors are co-expressed and are required for terminal differentiation, pathfinding, axon outgrowth and the survival of neurons in the central and peripheral nervous systems. However, little is understood about their functional relationship and regulatory mechanism in neural development. Here, we have knocked out Isl1 or Pou4f1 or both in mice of both sexes. In the absence of Isl1, the differentiation of cochleovestibular ganglion (CVG) neurons is disturbed and with that Isl1-deficient CVG neurons display defects in migration and axon pathfinding. Compound deletion of Isl1 and Pou4f1 causes a delay in CVG differentiation and results in a more severe CVG defect with a loss of nearly all of spiral ganglion neurons (SGNs). Moreover, ISL1 and POU4F1 interact directly in developing CVG neurons and act cooperatively as well as independently in regulating the expression of unique sets of CVG-specific genes crucial for CVG development and survival by binding to the cis-regulatory elements including the promoters of Fgf10, Pou4f2, and Epha5 and enhancers of Eya1 and Ntng2 These findings demonstrate that Isl1 and Pou4f1 are indispensable for CVG development and maintenance by acting epistatically to regulate genes essential for CVG development.
Assuntos
Orelha Interna , Regulação da Expressão Gênica no Desenvolvimento , Animais , Feminino , Masculino , Camundongos , Gânglios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5-1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5-1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.
Assuntos
Padronização Corporal , Planárias , Regeneração , Fatores de Transcrição , Animais , Planárias/genética , Planárias/fisiologia , Regeneração/genética , Regeneração/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Padronização Corporal/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.
Assuntos
Conectina , Proteínas com Domínio LIM , Proteínas Musculares , Miócitos Cardíacos , Proteínas do Tecido Nervoso , Proteínas Nucleares , Sarcômeros , Animais , Humanos , Camundongos , Ratos , Conectina/metabolismo , Conectina/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Sarcômeros/metabolismo , Fatores de TranscriçãoRESUMO
Non-syndromic sensorineural hearing loss (NSHL) is a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. There is, at present, no curative treatment for genetic hearing loss (HL). Early molecular diagnosis of progressive disorders and elucidation of the causes and pathomechanisms are essential for developing therapeutic strategies. Here, we identified a novel rare frameshift variant of LMX1A (c.915dup), which resulted in the C-terminal-altered and -truncated LMX1A (p.Val306Cysfs*32). This C-terminal frameshift mutation co-segregated with autosomal dominant (AD) NSHL in a four-generation Chinese family, suggesting that the LMX1A non-missense mutation is also contributed to ADNSHL. In this family, the affected individuals exhibited the variable auditory phenotypes ranging from profound congenital deafness at birth or to mild/moderate HL in adulthood. We also found that the embryonic cells carrying with the heterozygous variant significantly expressed several upregulated HL-associated genes at transcriptional level. In vitro splicing assay suggested that the LMX1A mRNA with c.915dup did not cause nonsense-mediated decay and was translated into a truncated LMX1A. In addition, electrophoresis mobility shift assay and luciferase assays have shown that the highly conserved C-terminal domain (amino acid 306-382) of the LMX1A was required for regulating the protein-DNA interaction and transactivation in vitro. Furthermore, apoptosis assays suggested that the C-terminal domain of the LMX1A was important for mediating apoptosis in the cochlear hair cells. Our work provided the multiline of the evidence to support that non-missense mutation of LMX1A leads to ADNSHL and the C-terminal domain of LMX1A is important for mediating transcriptional activity and associated with promoting apoptosis in the cells.
Assuntos
Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Humanos , Surdez/genética , Mutação da Fase de Leitura , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Proteínas com Homeodomínio LIM/genética , Linhagem , Fatores de Transcrição/genéticaRESUMO
The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/metabolismo , Telencéfalo/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Colina/metabolismo , Proteína Duplacortina/genética , Proteína Duplacortina/metabolismo , Feto , Ontologia Genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/classificação , Neurônios/citologia , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Transdução de Sinais , Estatmina/genética , Estatmina/metabolismo , Telencéfalo/citologia , Telencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Migration of myoblasts derived from the occipital somites is essential for tongue morphogenesis. However, the molecular mechanisms of myoblast migration remain elusive. In this study, we report that deletion of Isl1 in the mouse mandibular epithelium leads to aglossia due to myoblast migration defects. Isl1 regulates the expression pattern of chemokine ligand 12 (Cxcl12) in the first branchial arch through the Shh/Wnt5a cascade. Cxcl12+ mesenchymal cells in Isl1ShhCre embryos were unable to migrate to the distal region, but instead clustered in a relatively small proximal domain of the mandible. CXCL12 serves as a bidirectional cue for myoblasts expressing its receptor CXCR4 in a concentration-dependent manner, attracting Cxcr4+ myoblast invasion at low concentrations but repelling at high concentrations. The accumulation of Cxcl12+ mesenchymal cells resulted in high local concentrations of CXCL12, which prevented Cxcr4+ myoblast invasion. Furthermore, transgenic activation of Ihh alleviated defects in tongue development and rescued myoblast migration, confirming the functional involvement of Hedgehog signaling in tongue development. In summary, this study provides the first line of genetic evidence that the ISL1/SHH/CXCL12 axis regulates myoblast migration during tongue development.
Assuntos
Quimiocina CXCL12 , Proteínas Hedgehog , Proteínas com Homeodomínio LIM , Transdução de Sinais , Língua , Fatores de Transcrição , Animais , Camundongos , Movimento Celular/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligantes , Transdução de Sinais/genética , Língua/embriologia , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Quimiocina CXCL12/genéticaRESUMO
The genome is partitioned into topologically associated domains and genomic compartments with shared chromatin valence. This architecture is constrained by the DNA polymer, which precludes interactions between genes on different chromosomes. Here we report a marked divergence from this pattern of nuclear organization that occurs in mouse olfactory sensory neurons. Chromatin conformation capture using in situ Hi-C on fluorescence-activated cell-sorted olfactory sensory neurons and their progenitors shows that olfactory receptor gene clusters from 18 chromosomes make specific and robust interchromosomal contacts that increase with differentiation of the cells. These contacts are orchestrated by intergenic olfactory receptor enhancers, the 'Greek islands', which first contribute to the formation of olfactory receptor compartments and then form a multi-chromosomal super-enhancer that associates with the single active olfactory receptor gene. The Greek-island-bound transcription factor LHX2 and adaptor protein LDB1 regulate the assembly and maintenance of olfactory receptor compartments, Greek island hubs and olfactory receptor transcription, providing mechanistic insights into and functional support for the role of trans interactions in gene expression.
Assuntos
Cromossomos de Mamíferos/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Receptores Odorantes/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/genética , Cromossomos de Mamíferos/metabolismo , Feminino , Masculino , Camundongos , Família Multigênica/genética , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismoRESUMO
Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.
Assuntos
Diferenciação Celular , Interneurônios , Proteínas com Homeodomínio LIM , Medula Espinal , Fatores de Transcrição , Animais , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Interneurônios/metabolismo , Interneurônios/citologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Medula Espinal/embriologia , Embrião de Galinha , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/citologia , Humanos , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.
Assuntos
Injúria Renal Aguda , Túbulos Renais Proximais , Proteínas com Homeodomínio LIM , Proteínas Musculares , Traumatismo por Reperfusão , Fatores de Transcrição , beta Catenina , Animais , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/genética , Camundongos , beta Catenina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Masculino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transdução de Sinais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Proliferação de Células , ApoptoseRESUMO
Ischemia-reperfusion injury (IRI) is a major event in renal transplantation, leading to adverse outcomes. Bone marrow mesenchymal stem cells (BMSCs) are novel promising therapeutics for repairing kidney injuries. The therapeutic efficacy of BMSCs with ISL1 overexpression in renal IRI and its underlying mechanism need to be investigated. The unilateral renal IRI rat model was established to mimic clinical acute kidney injury. Rats were injected with PBS, BMSCs-Scrambled or BMSCs-ISL1 via the tail vein at the timepoint of reperfusion, and then sacrificed after 24 h of reperfusion. The administration of BMSCs-ISL1 significantly improved renal function, inhibited tubular cells apoptosis, inflammation, oxidative stress in rats. In vitro, HKC cells subjected to H2O2 stimulation were pretreated with the conditioned medium (CM) of BMSCs-Scrambled or BMSCs-ISL1. The pretreatment of ISL1-CM attenuated apoptosis and oxidative stress induced by H2O2 in HKC cells. Our proteomic data suggested that haptoglobin (Hp) was one of the secretory proteins in ISL1-CM. Subsequent experiments confirmed that Hp was the important paracrine factor from BMSCs-ISL1 that exerted anti-apoptotic and antioxidant functions. Mechanistically, Hp played a cytoprotective role via the inhibition of ERK signaling pathway, which could be abrogated by Ro 67-7476, the ERK phosphorylation agonist. The results suggested that paracrine action may be the main mechanism for BMSCs-ISL1 to exert protective effects. As an important anti-apoptotic and antioxidant factor in ISL1-CM, Hp may serve as a new therapeutic agent for treating IRI, providing new insights for overcoming the long-term adverse effects of stem cell therapy.
Assuntos
Apoptose , Proteínas com Homeodomínio LIM , Células-Tronco Mesenquimais , Estresse Oxidativo , Comunicação Parácrina , Traumatismo por Reperfusão , Fatores de Transcrição , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/terapia , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Ratos , Masculino , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ratos Sprague-Dawley , Rim/metabolismo , Rim/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/terapia , Meios de Cultivo Condicionados/farmacologia , Linhagem CelularRESUMO
A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.
Assuntos
Vias Auditivas , Núcleo Coclear , Células Ciliadas Auditivas , Proteínas com Homeodomínio LIM , Neurogênese , Gânglio Espiral da Cóclea , Fatores de Transcrição , Animais , Vias Auditivas/embriologia , Cóclea/embriologia , Cóclea/inervação , Núcleo Coclear/embriologia , Células Ciliadas Auditivas/fisiologia , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Camundongos , Neurogênese/genética , Gânglio Espiral da Cóclea/enzimologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFß, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.
Assuntos
Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Cromatina , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Guanilato Ciclase , Humanos , Proteínas com Homeodomínio LIM/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína-Arginina N-Metiltransferases , Ativação TranscricionalRESUMO
The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal ß-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.
Assuntos
Conectina , Mecanotransdução Celular , Camundongos Knockout , Animais , Camundongos , Conectina/metabolismo , Conectina/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Condicionamento Físico Animal , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Modelos Animais de Doenças , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Proteínas Quinases , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
The establishment of body pattern is a fundamental process in developmental biology. In Drosophila, the wing disc is subdivided into dorsal (D) and ventral (V) compartments by the D/V boundary. The dorsal fate is adopted by expressing the selector gene apterous (ap). ap expression is regulated by three combinational cis-regulatory modules which are activated by EGFR pathway, Ap-Vg auto-regulatory and epigenetic mechanisms. Here, we found that the Tbx family transcription factor Optomotor-blind (Omb) restricted ap expression in the ventral compartment. Loss of omb induced autonomous initiation of ap expression in the middle third instar larvae in the ventral compartment. Oppositely, over-activation of omb inhibited ap in the medial pouch. All three enhancers apE, apDV and apP were upregulated in omb null mutants, indicating a combinational regulation of ap modulators. However, Omb affected ap expression neither by directly regulating EGFR signaling, nor via Vg regulation. Therefore, a genetic screen of epigenetic regulators, including the Trithorax group (TrxG) and Polycomb group (PcG) genes was performed. We found that knocking down the TrxG gene kohtalo (kto), domino (dom) or expressing the PcG gene grainy head (grh), the ectopic ap in omb mutants was repressed. The inhibition of apDV by kto knockdown and grh activation could contribute to ap repression. Moreover, Omb and the EGFR pathway are genetically parallel in ap regulation in the ventral compartment. Collectively, Omb is a repressive signal for ap expression in the ventral compartment, which requires TrxG and PcG genes.