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1.
Cell ; 143(1): 35-45, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20887891

RESUMO

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.


Assuntos
Histona Desacetilases/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Miogenina/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Ubiquitina-Proteína Ligases/genética , Animais , Atrofia , Camundongos , Camundongos Knockout , Proteínas com Motivo Tripartido
2.
Nature ; 574(7778): 359-364, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31619788

RESUMO

The mechanisms that extend lifespan in humans are poorly understood. Here we show that extended longevity in humans is associated with a distinct transcriptome signature in the cerebral cortex that is characterized by downregulation of genes related to neural excitation and synaptic function. In Caenorhabditis elegans, neural excitation increases with age and inhibition of excitation globally, or in glutamatergic or cholinergic neurons, increases longevity. Furthermore, longevity is dynamically regulated by the excitatory-inhibitory balance of neural circuits. The transcription factor REST is upregulated in humans with extended longevity and represses excitation-related genes. Notably, REST-deficient mice exhibit increased cortical activity and neuronal excitability during ageing. Similarly, loss-of-function mutations in the C. elegans REST orthologue genes spr-3 and spr-4 elevate neural excitation and reduce the lifespan of long-lived daf-2 mutants. In wild-type worms, overexpression of spr-4 suppresses excitation and extends lifespan. REST, SPR-3, SPR-4 and reduced excitation activate the longevity-associated transcription factors FOXO1 and DAF-16 in mammals and worms, respectively. These findings reveal a conserved mechanism of ageing that is mediated by neural circuit activity and regulated by REST.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Longevidade , Neurônios/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo
3.
Development ; 148(9)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33914865

RESUMO

Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret-expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret. Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret-dependent cell behaviors play an important role in normal branching morphogenesis.


Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Transdução de Sinais , Ureter/metabolismo , Animais , Análise por Conglomerados , Epitélio/metabolismo , Feminino , Rim/crescimento & desenvolvimento , Rim/metabolismo , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Organoides , Proteínas Tirosina Quinases/metabolismo , Células-Tronco/metabolismo
4.
Genes Dev ; 27(9): 997-1002, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23651855

RESUMO

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.


Assuntos
Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/metabolismo , Animais , Padronização Corporal/genética , Linhagem Celular , Embrião de Mamíferos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Mutação , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Development ; 144(9): 1698-1711, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28356311

RESUMO

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.


Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Fatores de Transcrição/metabolismo , Vertebrados/metabolismo , Células Amácrinas/citologia , Células Amácrinas/metabolismo , Animais , Apoptose/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Contagem de Células , Divisão Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Células Cultivadas , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/metabolismo , Eletroporação , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Brn-3B/deficiência , Fatores de Transcrição/deficiência
6.
Genes Dev ; 24(16): 1816-26, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20713522

RESUMO

Little is known about how, during its formidable expansion in development and evolution, the cerebral cortex is able to maintain the correct balance between excitatory and inhibitory neurons. In fact, while the former are born within the cortical primordium, the latter originate outward in the ventral pallium. Therefore, it remains to be addressed how these two neuronal populations might coordinate their relative amounts in order to build a functional cortical network. Here, we show that Tbr2-positive cortical intermediate (basal) neuronal progenitors (INPs) dictate the migratory route and control the amount of subpallial GABAergic interneurons in the subventricular zone (SVZ) through a non-cell-autonomous mechanism. In fact, Tbr2 interneuron attractive activity is moderated by Cxcl12 chemokine signaling, whose forced expression in the Tbr2 mutants can rescue, to some extent, SVZ cell migration. We thus propose that INPs are able to control simultaneously the increase of glutamatergic and GABAergic neuronal pools, thereby creating a simple way to intrinsically balance their relative accumulation.


Assuntos
Córtex Cerebral , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Movimento Celular , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Interneurônios/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Proteínas com Domínio T/genética
7.
Development ; 140(3): 541-51, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23293286

RESUMO

The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element.


Assuntos
Células Amácrinas/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Reprogramação Celular , Proteínas do Tecido Nervoso/metabolismo , Células Ganglionares da Retina/citologia , Células Amácrinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Imunoprecipitação da Cromatina , Eletrorretinografia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Imuno-Histoquímica , Íntrons , Camundongos , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Ligação Proteica , Retina/citologia , Retina/embriologia , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
8.
Proc Biol Sci ; 283(1826): 20152978, 2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26962139

RESUMO

Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos/genética , Células Ganglionares da Retina/metabolismo , Strongylocentrotus purpuratus/genética , Fator de Transcrição Brn-3B/genética , Animais , Embrião de Mamíferos/embriologia , Embrião não Mamífero/embriologia , Proteínas de Homeodomínio/metabolismo , Camundongos/crescimento & desenvolvimento , Camundongos/metabolismo , Células Ganglionares da Retina/citologia , Strongylocentrotus purpuratus/metabolismo , Fator de Transcrição Brn-3B/metabolismo
9.
Proc Natl Acad Sci U S A ; 110(42): E4026-35, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24082127

RESUMO

The mammalian striatum controls the output of the basal ganglia via two distinct efferent pathways, the direct (i.e., striatonigral) and the indirect (i.e., striatopallidal) pathways. The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in a subpopulation of striatal progenitors; however, its specific role in striatal development remains unknown. Our genetic fate-mapping results show that Isl1-expressing progenitors give rise to striatal neurons belonging to the striatonigral pathway. Conditional inactivation of Isl1 in the telencephalon resulted in a smaller striatum with fewer striatonigral neurons and reduced projections to the substantia nigra. Additionally, conditional inactivation in the ventral forebrain (including both the telencephalon and diencephalon) revealed a unique role for Isl1 in diencephalic cells bordering the internal capsule for the normal development of the striatonigral pathway involving PlexinD1-Semaphorin 3e (Sema3e) signaling. Finally, Isl1 conditional mutants displayed a hyperlocomotion phenotype, and their locomotor response to psychostimulants was significantly blunted, indicating that the alterations in basal ganglia circuitry contribute to these mutant behaviors.


Assuntos
Corpo Estriado/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Substância Negra/embriologia , Fatores de Transcrição/metabolismo , Animais , Comportamento Animal/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Corpo Estriado/citologia , Proteínas do Citoesqueleto , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Homeodomínio LIM/genética , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Semaforinas , Substância Negra/citologia , Fatores de Transcrição/genética
10.
J Biol Chem ; 288(18): 12580-95, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23515314

RESUMO

Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and ß-catenin. Lef-1, ß-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Animais , Células CHO , Núcleo Celular/genética , Cricetinae , Cricetulus , Proteínas de Homeodomínio/genética , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , beta Catenina/genética , beta Catenina/metabolismo , Proteína Homeobox PITX2
11.
J Neurosci ; 32(26): 8831-44, 2012 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-22745484

RESUMO

The olfactory bulb (OB) is the first relay station in the brain where odor information from the olfactory epithelium is integrated, processed through its intrinsic neural circuitry, and conveyed to higher olfactory centers. Compared with profound mechanistic insights into olfactory axon wiring from the nose to the OB, little is known about the molecular mechanisms underlying the formation of functional neural circuitry among various types of neurons inside the OB. T-box transcription factor Tbr2 is expressed in various types of glutamatergic excitatory neurons in the brain including the OB projection neurons, mitral and tufted cells. Here we generated conditional knockout mice in which the Tbr2 gene is inactivated specifically in mitral and tufted cells from late embryonic stages. Tbr2 deficiency caused cell-autonomous changes in molecular expression including a compensatory increase of another T-box member, Tbr1, and a concomitant shift of vesicular glutamate transporter (VGluT) subtypes from VGluT1 to VGluT2. Tbr2-deficient mitral and tufted cells also exhibited anatomical abnormalities in their dendritic morphology and projection patterns. Additionally, several non-cell-autonomous phenotypes were observed in parvalbumin-, calbindin-, and 5T4-positive GABAergic interneurons. Furthermore, the number of dendrodendritic reciprocal synapses between mitral/tufted cells and GABAergic interneurons was significantly reduced. Upon stimulation with odorants, larger numbers of mitral and tufted cells were activated in Tbr2 conditional knockout mice. These results suggest that Tbr2 is required for not only the proper differentiation of mitral and tufted cells, but also for the establishment of functional neuronal circuitry in the OB and maintenance of excitatory-inhibitory balance crucial for odor information processing.


Assuntos
Neurônios/classificação , Neurônios/fisiologia , Bulbo Olfatório/citologia , Sinapses/fisiologia , Proteínas com Domínio T/deficiência , Animais , Caderinas/genética , Dendritos/metabolismo , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Inibição Neural/fisiologia , Neurônios/citologia , Odorantes , Receptores Odorantes/genética , Sinapses/genética , Fatores de Transcrição/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo
12.
Dev Biol ; 364(2): 114-27, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22326227

RESUMO

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA(+) afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC(+) proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB(+) and TrkC(+) neurons as well as TrkA(+)/TrkB(+) and TrkA(+)/TrkC(+) double positive cells at early embryonic stages. At later stages in the mutant, TrkB(+), TrkC(+) and parvalbumin(+) neurons diminish while there is a significant increase of CGRP(+) and c-ret(+) neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets.


Assuntos
Axônios/fisiologia , Gânglios Espinais/embriologia , Medula Espinal/embriologia , Fator de Transcrição Brn-3A/fisiologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/análise , Subunidade alfa 2 de Fator de Ligação ao Core/análise , Regulação para Baixo , Feminino , Gânglios Espinais/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Neurogênese/fisiologia , Proteínas Proto-Oncogênicas c-ret/análise , Receptor trkA/análise , Receptor trkB/análise , Receptor trkC/análise , Medula Espinal/crescimento & desenvolvimento , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/fisiologia
13.
Exp Eye Res ; 106: 24-33, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23142158

RESUMO

Mutations in the Ceramide kinase like (CERKL) gene are associated with retinitis pigmentosa (RP26) and cone-rod dystrophy. CERKL is homologous to Ceramide kinase (CERK), and its function is still unknown. The purpose of this study was to test the expression and distribution of this gene and its protein in rat and in mouse tissues, in light-stressed rat retinas and in the retinas of NeuroD1 knock-out mice to understand the role of CERKL in the retina. Expression of Cerkl and Cerk mRNA was determined by quantitative RT-PCR. Expression of the protein was determined by Western blotting with anti-CERKL antibody. Localization of the protein was determined by using immunofluorescence microscopy. With qRT-PCR, we revealed that the relative mRNA expression of Cerkl was the highest in the retina among all the rat tissue tested; it was >10-fold higher than in the brain. On the other hand, Cerk has ubiquitous expression and its relative abundance is >2 fold of Cerkl in the retina. Cerkl was expressed minimally in the developing mouse eyes and reached a peak at retinal maturity at 2 months. Western blots of retinal tissues revealed two major CERKL protein bands: 59 kDa (C1) and 37 kDa (C2). However, only C2 CERKL was found in the rat retinal rod outer segment (ROS) at level of that was not changed in light vs. dark adaptation. In the light-stressed retina, expression of Cerkl mRNA increased significantly, which was reflected in only on C2 CERKL protein. The CERKL protein localized prominently to the ganglion cells, inner nuclear layers (INL), retinal pigment epithelial (RPE) cells, and photoreceptor inner segments in the retinal sections. Nuclear localization of CERKL was not affected in RPE, INL and the ganglion cell layers in the light-stressed retina; however, the perinuclear and outer segment locations appear to be altered. In the NeuroD1 knock-out mouse retina, the expression of Cerkl mRNA and protein decreased and that decrease also pertains to C2 CERKL. In conclusion, the retina had the highest level of Cerkl mRNA and protein expression, which reached its maximum in the adult retina; CERKL localized to ROS and RPE cells and the light-adaptation did not change the level of CERKL in ROS; light-stress induced Cerkl expression in the retina; and its expression decreased in NeuroD1 knock-out retina. Thus, CERKL may be important for the stress responses and protection of photoreceptor cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica/fisiologia , Luz/efeitos adversos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Lesões Experimentais por Radiação/genética , Retina/efeitos da radiação , Degeneração Retiniana/genética , Animais , Western Blotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia de Fluorescência , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Retina/embriologia , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo
14.
Dev Biol ; 349(1): 90-9, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20969844

RESUMO

As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas Repressoras/genética , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismo
15.
Mol Vis ; 18: 2658-72, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23170059

RESUMO

PURPOSE: Retinal ganglion cell (RGC) death and optic nerve degeneration are complex processes whose underlying molecular mechanisms are only vaguely understood. Treatments commonly used for optic nerve degeneration have little long-term value and only prolong degeneration. Recent advances in stem cell replacement therapy offer new ways to overcome RGC loss by transferring healthy cells into eyes of afflicted individuals. However, studies on stem cell replacement for optic nerve degeneration are hampered by limitations of the available animal models, especially genetic models. We have developed a mouse model in which RGCs are genetically ablated in adult mice with subsequent degeneration of the optic nerve. In the study reported here, we used this model to determine whether embryonic retinal progenitor cells (RPCs) removed from donor retinas when RPCs are committing to an RGC fate could restore lost RGCs. METHODS: We used the RGC-depleted model as a host for transplanting donor green fluorescent protein (GFP)-labeled RPCs from embryonic retinas that are maximally expressing Atoh7, a basic helix-loop-helix gene essential for RGC specification. Dissociated GFP-labeled RPCs were characterized in situ by immunolabeling with antibodies against proteins known to be expressed in RPCs at embryonic day (E)14.5. Dissociated retinal cells were injected into the vitreous of one eye of RGC-depleted mice at two to six months of age. The injected and non-injected retinas were analyzed for gene expression using immunolabeling, and the morphology of optic nerves was assessed visually and with histological staining at different times up to four months after injection. RESULTS: We demonstrate the successful transfer of embryonic GFP-labeled RPCs into the eyes of RGC-depleted mice. Many transplanted RPCs invaded the ganglion cell layer, but the efficiency of the invasion was low. GFP-labeled cells within the ganglion cell layer expressed genes associated with early and late stages of RGC differentiation, including Pou4f1, Pou4f2, NFL, Map2, and syntaxin. Several GFP-labeled cells were detected within the injected optic nerves of RGC-depleted mice, and in most cases, we observed a significant increase in the thickness of the RPC-injected optic nerves compared with non-injected controls. We also observed more bundled axons emanating from RPC-injected retinas compared with RGC-depleted controls. CONCLUSIONS: The results offer a new approach for regenerating damaged optic nerves and indicate that a significant number of E14.5 RPCs are able to differentiate into RGCs in the foreign environment of the adult retina. However, the proportion of RPCs that populated the ganglion cell layer and contributed to the optic nerve was not sufficient to account for the increased thickness and higher number of axons. The results support the hypothesis that the injected E14.5 RPCs are contributing autonomously and non-autonomously to restoring damaged optic nerves.


Assuntos
Células-Tronco Embrionárias/transplante , Degeneração Neural/terapia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Embrião de Mamíferos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde , Injeções Intravítreas , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
16.
Proc Natl Acad Sci U S A ; 105(19): 6942-7, 2008 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-18460603

RESUMO

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Animais , Imunofluorescência , Deleção de Genes , Genes Controladores do Desenvolvimento , Proteínas de Homeodomínio/genética , Hibridização In Situ , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Nervo Óptico/metabolismo , Nervo Óptico/ultraestrutura , Retina/anormalidades , Retina/embriologia , Retina/ultraestrutura , Células Ganglionares da Retina/patologia , Fator de Transcrição Brn-3B/genética , Fatores de Transcrição
17.
Dev Dyn ; 238(9): 2309-17, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19459208

RESUMO

Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5(HA) and Pou4f2(HA) alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Epitopos/genética , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Southern Blotting , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismo
18.
Dev Biol ; 322(2): 406-14, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18721801

RESUMO

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.


Assuntos
Células-Tronco Adultas/metabolismo , Diferenciação Celular/fisiologia , Músculo Esquelético/metabolismo , Miogenina/fisiologia , Células-Tronco Adultas/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Fatores de Regulação Miogênica/fisiologia , Miogenina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez
19.
Exp Eye Res ; 88(3): 542-52, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19109949

RESUMO

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.


Assuntos
Degeneração Neural/patologia , Doenças do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Morte Celular/efeitos dos fármacos , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Deleção de Genes , Gliose/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Doenças do Nervo Óptico/genética , Fragmentos de Peptídeos/farmacologia , Retina/efeitos dos fármacos , Retina/embriologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/ultraestrutura , Tamoxifeno/farmacologia , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismo , Acuidade Visual
20.
Cell Rep ; 27(3): 900-915.e5, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995485

RESUMO

In the mouse retina, more than 30 retinal ganglion cell (RGC) subtypes have been classified based on a combined metric of morphological and functional characteristics. RGCs arise from a common pool of retinal progenitor cells during embryonic stages and differentiate into mature subtypes in adult retinas. However, the cellular and molecular mechanisms controlling formation and maturation of such remarkable cellular diversity remain unknown. Here, we demonstrate that T-box transcription factor T-brain 1 (Tbr1) is expressed in two groups of morphologically and functionally distinct RGCs: the orientation-selective J-RGCs and a group of OFF-sustained RGCs with symmetrical dendritic arbors. When Tbr1 is genetically ablated during retinal development, these two RGC groups cannot develop. Ectopically expressing Tbr1 in M4 ipRGCs during development alters dendritic branching and density but not the inner plexiform layer stratification level. Our data indicate that Tbr1 plays critical roles in regulating the formation and dendritic morphogenesis of specific RGC types.


Assuntos
Células Ganglionares da Retina/metabolismo , Proteínas com Domínio T/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Toxina da Cólera/toxicidade , Dendritos/fisiologia , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Potássio/farmacologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Proteínas com Domínio T/genética
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