RESUMO
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
The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Proteínas com Domínio LIM/fisiologia , Animais , Citoesqueleto/química , Citoesqueleto/fisiologia , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/análise , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , alfa Catenina/metabolismoRESUMO
Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.
Assuntos
Proteínas de Peixes/genética , Linguado/genética , Proteínas com Homeodomínio LIM/genética , Animais , Evolução Molecular , Feminino , Proteínas de Peixes/fisiologia , Linguado/fisiologia , Técnicas de Silenciamento de Genes/veterinária , Células HeLa , Humanos , Proteínas com Homeodomínio LIM/fisiologia , Masculino , Oogênese/genética , SinteniaRESUMO
Abnormal expression of insulin gene enhancer-binding protein 1 (ISL1) has been demonstrated to be closely associated with cancer development and progression in several cancers. However, little is known about ISL1 expression in metastatic castration-resistant prostate cancer (CRPC). ISL1 has also been recognized as a positive modulator of epithelial-mesenchymal transition (EMT). In this study, we focused on ISL1 which showed maximum upregulation at the mRNA level in the enzalutamide-resistant cell line. Accordingly, we found that ISL1 was overexpressed in enzalutamide-resistant C4-2B cells and its expression was significantly related to EMT. Our findings reveal the important role of ISL1 in androgen receptor (AR)-dependent prostate cancer cell growth; ISL1 knockdown reduced the AR activity and cell growth. ISL1 knockdown using small-interfering RNA inhibited AR, PSA, and EMT-related protein expression in C4-2B ENZR cells. In addition, knock-down ISL1 reduced the levels of AKT and p65 phosphorylation in C4-2B ENZR cells and these suggest that knock-down ISL1 suppresses EMT in part by targeting the AKT/NF-κB pathway. Further, ISL1 downregulation could effectively inhibit tumor growth in a human CRPC xenograft model. Together, the present study shows that downregulation of ISL1 expression is necessary for overcoming enzalutamide resistance and improving the survival of CRPC patients.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas com Homeodomínio LIM/fisiologia , Nitrilas/uso terapêutico , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/fisiopatologia , Fatores de Transcrição/fisiologia , Antagonistas de Androgênios/farmacologia , Animais , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Masculino , Camundongos , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma (NB) is the most common extracranial solid malignancy in children and its mortality rate is relatively high. However, driver genes of NB are not clearly identified. Using bioinformatics analysis, we determined the top 8 differentially expressed genes (DEGs) in NB, including GFAP, PAX6, FOXG1, GAD1, PTPRC, ISL1, GRM5, and GATA3. Insulin gene enhancer binding protein 1 (ISL1) is a LIM homeodomain transcription factor which has been found to be highly expressed in a variety of malignant tumors, but the function of ISL1 in NB has not been fully elucidated. We identified ISL1 as an oncogene in NB. ISL1 is preferentially upregulated in NB tissues compared with normal tissues. High ISL1 expression is significantly associated with poor outcome of NB patients. Knockdown of ISL1 markedly represses proliferation and induces cell apoptosis in vitro, and suppresses tumorigenicity in vivo, while overexpression of ISL1 has the opposite effects. Mechanistically, we demonstrate that ISL1 promotes cell proliferation and EMT transformation through PI3K/AKT signaling pathway by upregulating Aurora kinase A (AURKA), a serine-threonine kinase that is essential for the survival of NB cells. The blockade of AURKA attenuates the function of ISL1 overexpression in the regulation of cell proliferation and migration, Conclusively, this study showed that ISL1 targeted AURKA to facilitate the development of NB, which provided new insights into the tumorigenesis of NB. Thus, ISL1 may be a promising therapeutic target in the future.
Assuntos
Carcinogênese/genética , Transição Epitelial-Mesenquimal/genética , Proteínas com Homeodomínio LIM/fisiologia , Neuroblastoma/genética , Fatores de Transcrição/fisiologia , Animais , Aurora Quinase A/metabolismo , Aurora Quinase A/fisiologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
Direct reprogramming has been widely explored to generate various types of neurons for neurobiological research and translational medicine applications, but there is still no efficient reprogramming method to generate retinal ganglion cell (RGC)-like neurons, which are the sole projection neurons in the retina. Here, we show that three transcription factors, Ascl1, Brn3b, and Isl1, efficiently convert fibroblasts into RGC-like neurons (iRGCs). Furthermore, we show that the competence of cells to enter iRGC reprogramming route is determined by the cell-cycle status at a very early stage of the process. The iRGC reprogramming route involves intermediate states that are characterized by a transient inflammatory-like response followed by active epigenomic and transcriptional modifications. Our study provides an efficient method to generate iRGCs, which would be a valuable cell source for potential glaucoma cell replacement therapy and drug screening studies, and reveals the key cellular events that govern successful neuronal fate reprogramming.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Reprogramação Celular , Fibroblastos/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Neurônios/fisiologia , Células Ganglionares da Retina/fisiologia , Fator de Transcrição Brn-3B/fisiologia , Fatores de Transcrição/fisiologia , Animais , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Neurogênese , Retina/citologiaRESUMO
The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57Kip2 is a member of the Cip/Kip family of CKIs and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and CDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2-regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity by HDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Proteínas Musculares/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Montagem e Desmontagem da Cromatina , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/metabolismo , Ligação Proteica , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Mesenchymal stem cells (MSCs) specifically differentiate into cardiomyocytes as a potential way to reverse myocardial injury diseases, and uncovering this differentiation mechanism is immensely important. We have previously shown that histone acetylation/methylation and DNA methylation are involved in MSC differentiation into cardiomyocytes induced by islet-1. These modifications regulate cardiac-specific genes by interacting with each other in the promoter regions of these genes, but the molecular mechanism of these interactions remains unknown. In this study, we found that the key enzymes that regulate GATA4/Nkx2.5 expression are Gcn5/HDAC1, G9A, and DNMT-1. When α-methylene-γ-butyrolactone 3 (MB-3) was used to inhibit Gcn5 expression, we observed that the interactions among these key enzymes in the GATA4/Nkx2.5 promoters were blocked, and MSCs could not be induced into cardiomyocytes. Our results indicated that islet-1 could induce Gcn5 binding to GATA4/Nkx2.5 promoter regions and induce the interactions among Gcn5, HDAC1, G9A and DNMT-1, which upregulated GATA4/Nkx2.5 expression and promoted MSC differentiation into cardiomyocytes.
Assuntos
Diferenciação Celular , Proteínas com Homeodomínio LIM/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos Cardíacos/fisiologia , Fatores de Transcrição/fisiologia , Fatores de Transcrição de p300-CBP/fisiologia , Acetilação , Animais , Western Blotting , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Metilação de DNA , Fator de Transcrição GATA4/metabolismo , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Imunoprecipitação , Proteínas com Homeodomínio LIM/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The four and a half LIM domain protein 2 (FHL2) is a member of the four and a half LIM domain (FHL) gene family, and it is associated with cholesterol-enriched diet-promoted atherosclerosis. However, the effect of FHL2 protein on vascular remodelling in response to hemodynamic alterations remains unclear. Here, we investigated the role of FHL2 in a model of restricted blood flow-induced atherosclerosis. To promote neointimal hyperplasia in vivo, we subjected FHL2+/+ and FHL2-/- mice to partial ligation of the left carotid artery (LCA). The expression of p-ERK and p-AKT was decreased in FHL2-/- mice. FHL2 bound to AKT regulated AKT phosphorylation and led to Rac1-GTP inactivation. FHL2 silencing in human aortic smooth muscle cells down-regulated the PDGF-induced phosphorylation of ERK and AKT. Furthermore, FHL2 silencing reduced cytoskeleton conformational changes and caused cell cycle arrest. We concluded that FHL2 is essential for the regulation of arterial smooth muscle cell function. FHL2 modulates proliferation and migration via mitogen-activated protein kinase (MAPK) and PI3K-AKT signalling, leading to arterial wall thickening and thus neointimal hyperplasia.
Assuntos
Aterosclerose/prevenção & controle , Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Deleção de Genes , Proteínas com Homeodomínio LIM/fisiologia , Proteínas Musculares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Artérias Carótidas/cirurgia , Movimento Celular , Proliferação de Células , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Transdução de SinaisRESUMO
Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.
Assuntos
Embrião de Mamíferos/citologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas com Homeodomínio LIM/fisiologia , Neurônios Motores/citologia , Nervo Oculomotor/citologia , Medula Espinal/citologia , Fatores de Transcrição/fisiologia , Nervo Troclear/citologia , Animais , Técnicas de Cultura de Células , Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Nervo Oculomotor/metabolismo , Medula Espinal/metabolismo , Nervo Troclear/metabolismoRESUMO
The 4-and-a-half LIM domain protein 2 (FHL2) is a multifunctional adaptor protein that can interact with cell surface receptors, cytosolic adaptor and structural proteins, kinases, and nuclear transcription factors. It is involved in numerous functional activities, including the epithelial-mesenchymal transition, cell proliferation, apoptosis, adhesion, migration, structural stability, and gene expression. Despite this, FHL2-knockout (KO) mice are viable and fertile with no obvious abnormalities, rather suggesting a high capacity for fine-tuning adjustment and functional redundancy of FHL2. Indeed, challenging FHL2-KO cells or mice provided numerous evidences for the great functional significance of FHL2. In recent years, several reviews have been published describing the high capacity of FHL2 to bind diverse proteins as well as the versatile functions of FHL2, emphasizing in particular its role in cardiovascular diseases and carcinogenesis. Here, we view the function of FHL2 from a different perspective. We summarize the published data demonstrating the impact of FHL2 on wound healing and inflammation. FHL2 seems to be involved in numerous steps of these extremely complex and multidirectional but tightly regulated tissue remodeling processes, supporting tissue repair and coordinating inflammation. Deficiency of FHL2 not only slows down ongoing wound healing but also often turns it into a chronic condition.-Wixler, V. The role of FHL2 in wound healing and inflammation.
Assuntos
Inflamação/fisiopatologia , Proteínas com Homeodomínio LIM/fisiologia , Proteínas Musculares/fisiologia , Fatores de Transcrição/fisiologia , Cicatrização/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Citocinas/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Inflamação/imunologia , Proteínas com Homeodomínio LIM/biossíntese , Proteínas com Homeodomínio LIM/deficiência , Proteínas com Homeodomínio LIM/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/biossíntese , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Miofibroblastos/fisiologia , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Regulação para CimaRESUMO
During neocortical development, neurons are produced by a diverse pool of neural progenitors. A subset of progenitors express the Cux2 gene and are fate restricted to produce certain neuronal subtypes; however, the upstream pathways that specify these progenitor fates remain unknown. To uncover the transcriptional networks that regulate Cux2 expression in the forebrain, we characterized a conserved Cux2 enhancer that recapitulates Cux2 expression specifically in the cortical hem. Using a bioinformatic approach, we identified putative transcription factor (TF)-binding sites for cortical hem-patterning TFs. We found that the homeobox TF Lmx1a can activate the Cux2 enhancer in vitro Furthermore, we showed that Lmx1a-binding sites were required for enhancer activity in the cortical hem in vivo Mis-expression of Lmx1a in hippocampal progenitors caused an increase in Cux2 enhancer activity outside the cortical hem. Finally, we compared several human enhancers with cortical hem-restricted activity and found that recurrent Lmx1a-binding sites are a top shared feature. Uncovering the network of TFs involved in regulating Cux2 expression will increase our understanding of the mechanisms pivotal in establishing Cux2 lineage fates in the developing forebrain.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Íntrons , Proteínas com Homeodomínio LIM/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação , Linhagem da Célula , Biologia Computacional , Feminino , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prosencéfalo/embriologia , Telencéfalo/embriologia , Fatores de Transcrição/genéticaRESUMO
A hundred years after Lhx2 ortholog apterous was identified as a critical regulator of wing development in Drosophila, LIM-HD gene family members have proved to be versatile and powerful components of the molecular machinery that executes the blueprint of embryogenesis across vertebrate and invertebrate species. Here, we focus on the spatio-temporally varied functions of LIM-homeodomain transcription factor LHX2 in the developing mouse forebrain. Right from its earliest known role in telencephalic and eye field patterning, to the control of the neuron-glia cell fate switch, and the regulation of axon pathfinding and dendritic arborization in late embryonic stages, LHX2 has been identified as a fundamental, temporally dynamic, always necessary, and often sufficient factor in a range of critical developmental phenomena. While Lhx2 mutant phenotypes have been characterized in detail in multiple brain structures, only recently have we advanced in our understanding of the molecular mechanisms by which this factor acts. Common themes emerge from how this multifunctional molecule controls a range of developmental steps in distinct forebrain structures. Examining these shared features, and noting unique aspects of LHX2 function is likely to inform our understanding of how a single factor can bring about a diversity of effects and play central and critical roles across systems and stages. The parallels in LHX2 and APTEROUS functions, and the protein complexes they participate in, offer insights into evolutionary strategies that conserve tool kits and deploy them to play new, yet familiar roles in species separated by hundreds of millions of years.
Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/fisiologia , Prosencéfalo/embriologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Evolução Biológica , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Camundongos , Neurogênese/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Análise Espaço-Temporal , Fatores de Transcrição/genéticaRESUMO
The delicate balance of excitation and inhibition is crucial for proper function of the cerebral cortex, relying on the accurate number and subtype composition of inhibitory gamma-aminobutyric (GABA)-expressing interneurons. Various intrinsic and extrinsic factors precisely orchestrate their multifaceted development including the long-range migration from the basal telencephalon to cortical targets as well as interneuron survival throughout the developmental period. Particularly expressed guidance receptors were described to channel the migration of cortical interneurons deriving from the medial ganglionic eminence (MGE) and the preoptic area (POA) along distinct routes. Hence, unveiling the regulatory genetic networks controlling subtype-specific gene expression profiles is key to understand interneuron-specific developmental programs and to reveal causes for associated disorders. In contrast to MGE-derived interneurons, little is known about the transcriptional networks in interneurons born in the POA. Here, we provide first evidence for the LIM-homeobox transcription factor LHX1 as a crucial key player in the post-mitotic development of POA-derived cortical interneurons. By transcriptional regulation of related genes, LHX1 modulates their survival as well as the subtype-specific expression of guidance receptors of the Eph/ephrin family, thereby affecting directional migration and layer distribution in the adult cortex.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Interneurônios/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Área Pré-Óptica/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Movimento Celular , Sobrevivência Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Efrina-B3/genética , Efrina-B3/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/citologia , Interneurônios/metabolismo , Proteínas com Homeodomínio LIM/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Área Pré-Óptica/citologia , Área Pré-Óptica/metabolismo , Receptor EphA4/genética , Receptor EphA4/fisiologia , Fatores de Transcrição/genéticaRESUMO
Metastatic clear cell renal cell carcinoma (CCC) remains incurable despite advances in the development of anti-angiogenic targeted therapies and the emergence of immune checkpoint inhibitors. We have previously shown that the sonic hedgehog-Gli signaling pathway is oncogenic in CCC allowing us to identify the developmental Lim1 transcription factor as a Gli target and as a new oncogene in CCC regulating cell proliferation and apoptosis, and promoting tumor growth. In this previous study, preliminary in vitro results also suggested that Lim1 may be implicated in metastatic spread. Here we investigated the potential pro-metastatic role of Lim1 in advanced CCC (1) in vitro using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene either naturally or by gene transfer and (2) ex vivo in 30 CCC metastatic tissues, including lymph nodes, lung, skin, bone, and adrenal metastases, and (3) in vivo, using a metastatic model by intravenous injection of siRNA-transfected cells into Balb/c nude. Our in vitro results reveal that Lim1 knockdown time-dependently decreased CCC cell motility, migration, invasion, and clonogenicity by up to 50% regardless of their VHL status. Investigating the molecular machinery involved in these processes, we identified a large panel of Lim1 targets known to be involved in cell adhesion (paxillin and fibronectin), epithelial-mesenchymal transition (Twist1/2 and snail), invasion (MMP1/2/3/8/9), and metastatic progression (CXCR4, SDF-1, and ANG-1). Importantly, Lim1 was found constitutively expressed in all metastatic tissues. The H-score in metastatic tissues being significantly superior to the score in the corresponding primary tumor tissues (P value = 0.009). Furthermore, we showed that Lim1 silencing decreases pulmonary metastasis development in terms of number and size in the in vivo metastatic model of human CCC. Taken together, these experiments strengthen the potential therapeutic value of Lim1 targeting as a promising novel approach for treating metastatic human CCC.
Assuntos
Carcinoma de Células Renais/secundário , Neoplasias Renais/terapia , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Oncogenes , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Renais/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologiaRESUMO
KEY POINTS: Classifying different subtypes of neurons in deep brain structures is a challenge and is crucial to better understand brain function. Understanding the diversity of neurons in the globus pallidus (GP), a brain region positioned to influence afferent and efferent information processing within basal ganglia, could help to explain a variety of brain functions. We present a classification of neurons from the GP using electrophysiological data from wild-type mice and confirmation using transgenic mice. This work will help researchers to identify specific neuronal subsets in the GP of wild-type mice when transgenic mice with labelled neurons are lacking. ABSTRACT: Classification of the extensive neuronal diversity in the brain is fundamental for neuroscience. The globus pallidus external segment (GPe), also referred to as the globus pallidus in rodents, is a large nucleus located in the core of the basal ganglia whose circuitry is implicated in action control, decision-making and reward. Although considerable progress has been made in characterizing different GPe neuronal subtypes, no work has directly attempted to characterize these neurons in non-transgenic mice. Here, we provide data showing the degree of overlap in expression of neuronal PAS domain protein (Npas1), LIM homeobox 6 (Lhx6), parvalbumin (PV) and transcription factor FoxP2 biomarkers in mouse GPe neurons. We used an unbiased statistical method to classify neurons based on electrophysiological properties from nearly 200 neurons from C57BL/6J mice. In addition, we examined the subregion distribution of the neuronal subtypes. Cluster analysis using firing rate and hyperpolarization-induced membrane potential sag variables revealed three distinct neuronal clusters: type 1, characterized by low firing rate and small sag potential; type 2, with low firing rate and larger sag potential; and type 3, with high firing rate and small sag potential. We used other electrophysiological variables and data from marker-expressing neurons to evaluate the clusters. We propose that the GPe GABAergic neurons should be classified into three subgroups: arkypallidal, low-firing prototypical and high-firing prototypical neurons. This work will help researchers identify GPe neuron subtypes when transgenic mice with labelled neurons cannot be used.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Biomarcadores/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Neurônios GABAérgicos/classificação , Neurônios GABAérgicos/metabolismo , Globo Pálido/metabolismo , Proteínas com Homeodomínio LIM/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Potenciais de Ação , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parvalbuminas/metabolismoRESUMO
The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches.
Assuntos
Dendritos/genética , Dendritos/metabolismo , Nociceptores/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/fisiologia , Proteínas de Membrana/metabolismo , Nociceptores/fisiologia , Elementos Reguladores de Transcrição/genética , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Dedos de ZincoRESUMO
Regulation of the neuron-glia cell-fate switch is a critical step in the development of the CNS. Previously, we demonstrated that Lhx2 is a necessary and sufficient regulator of this process in the mouse hippocampal primordium, such that Lhx2 overexpression promotes neurogenesis and suppresses gliogenesis, whereas loss of Lhx2 has the opposite effect. We tested a series of transcription factors for their ability to mimic Lhx2 overexpression and suppress baseline gliogenesis, and also to compensate for loss of Lhx2 and suppress the resulting enhanced level of gliogenesis in the hippocampus. Here, we demonstrate a novel function of Dmrt5/Dmrta2 as a neurogenic factor in the developing hippocampus. We show that Dmrt5, as well as known neurogenic factors Neurog2 and Pax6, can each not only mimic Lhx2 overexpression, but also can compensate for loss of Lhx2 to different extents. We further uncover a reciprocal regulatory relationship between Dmrt5 and Lhx2, such that each can compensate for loss of the other. Dmrt5 and Lhx2 also have opposing regulatory control on Pax6 and Neurog2, indicating a complex bidirectionally regulated network that controls the neuron-glia cell-fate switch.SIGNIFICANCE STATEMENT We identify Dmrt5 as a novel regulator of the neuron-glia cell-fate switch in the developing hippocampus. We demonstrate Dmrt5 to be neurogenic, and reciprocally regulated by Lhx2: loss of either factor promotes gliogenesis; overexpression of either factor suppresses gliogenesis and promotes neurogenesis; each can substitute for loss of the other. Furthermore, each factor has opposing effects on established neurogenic genes Neurog2 and Pax6 Dmrt5 is known to suppress their expression, and we show that Lhx2 is required to maintain it. Our study reveals a complex regulatory network with bidirectional control of a fundamental feature of CNS development, the control of the production of neurons versus astroglia in the developing hippocampus.Finally, we confirm that Lhx2 binds a highly conserved putative enhancer of Dmrt5, suggesting an evolutionarily conserved regulatory relationship between these factors. Our findings uncover a complex network that involves Lhx2, Dmrt5, Neurog2, and Pax6, and that ensures the appropriate amount and timing of neurogenesis and gliogenesis in the developing hippocampus.
Assuntos
Hipocampo/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Neurogênese/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Hipocampo/citologia , Hipocampo/embriologia , Masculino , Camundongos , Camundongos Transgênicos , GravidezRESUMO
The identification of distinct cell types in the basal ganglia has been critical to our understanding of basal ganglia function and the treatment of neurological disorders. The external globus pallidus (GPe) is a key contributor to motor suppressing pathways in the basal ganglia, yet its neuronal heterogeneity has remained an untapped resource for therapeutic interventions. Here we demonstrate that optogenetic interventions that dissociate the activity of two neuronal populations in the GPe, elevating the activity of parvalbumin (PV)-expressing GPe neurons over that of Lim homeobox 6 (Lhx6)-expressing GPe neurons, restores movement in dopamine-depleted mice and attenuates pathological activity of basal ganglia output neurons for hours beyond stimulation. These results establish the utility of cell-specific interventions in the GPe to target functionally distinct pathways, with the potential to induce long-lasting recovery of movement despite the continued absence of dopamine.
Assuntos
Dopamina/metabolismo , Globo Pálido/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Locomoção/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Parvalbuminas/fisiologia , Fatores de Transcrição/fisiologia , Potenciais de Ação/fisiologia , Animais , Feminino , Globo Pálido/efeitos dos fármacos , Proteínas com Homeodomínio LIM/biossíntese , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Oxidopamina , Parvalbuminas/biossíntese , Fatores de Transcrição/biossínteseRESUMO
Transforming growth factor ß1 (TGF-ß1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-ß1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-ß1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-ß1 promoter, whereas the upregulation of TGF-ß1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-ß1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-ß1 gene transcription. Enhanced expression of TGF-ß1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-ß1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-ß1 expression and contribute to the understanding of the TGF-ß-mediated fibrogenic response.