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1.
Cell ; 187(2): 276-293.e23, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38171360

RESUMO

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.


Assuntos
Estruturas da Membrana Celular , Miosinas , Tubo Neural , Transdução de Sinais , Animais , Camundongos , Transporte Biológico , Estruturas da Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo
2.
Dev Biol ; 480: 39-49, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34419458

RESUMO

The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Ependimogliais/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células/genética , Epêndima/metabolismo , Células Ependimogliais/fisiologia , Via de Sinalização Hippo , Camundongos/embriologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese , Proteínas Serina-Treonina Quinases , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/genética
3.
Dev Cell ; 47(5): 576-591.e8, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30523785

RESUMO

The Hippo pathway controls the activity of YAP/TAZ transcriptional coactivators through a kinase cascade. Despite the critical role of this pathway in tissue growth and tumorigenesis, it remains unclear how YAP/TAZ-mediated transcription drives proliferation. By analyzing the effects of inactivating LATS1/2 kinases, the direct upstream inhibitors of YAP/TAZ, on mouse brain development and applying cell-number-normalized transcriptome analyses, we discovered that YAP/TAZ activation causes a global increase in transcription activity, known as hypertranscription, and upregulates many genes associated with cell growth and proliferation. In contrast, conventional read-depth-normalized RNA-sequencing analysis failed to detect the scope of the transcriptome shift and missed most relevant gene ontologies. Following a transient increase in proliferation, however, hypertranscription in neural progenitors triggers replication stress, DNA damage, and p53 activation, resulting in massive apoptosis. Our findings reveal a significant impact of YAP/TAZ activation on global transcription activity and have important implications for understanding YAP/TAZ function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Células Cultivadas , Via de Sinalização Hippo , Camundongos , Células-Tronco Neurais/citologia , Neurogênese , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição/genética , Ativação Transcricional , Transcriptoma , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP
4.
Autophagy ; 14(5): 796-811, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29099309

RESUMO

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)+ or ubiquitin+ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Orientação de Axônios , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteínas Relacionadas à Autofagia , Axônios/metabolismo , Axônios/ultraestrutura , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Córtex Somatossensorial/metabolismo , Proteínas Ubiquitinadas/metabolismo
5.
Cell Rep ; 21(6): 1534-1549, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29117559

RESUMO

Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.


Assuntos
Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/metabolismo , Trombospondinas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Placa Neural/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Retina/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Trombospondinas/genética , Fatores de Transcrição/genética , Proteínas Wnt , Proteína Homeobox SIX3
6.
J Neurosci ; 35(37): 12869-89, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26377473

RESUMO

Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT: Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking.Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons.


Assuntos
Córtex Cerebral/citologia , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Interneurônios/citologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Biomarcadores , Calbindina 2/análise , Moléculas de Adesão Celular Neuronais/análise , Linhagem da Célula , Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Proteínas da Matriz Extracelular/análise , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Interneurônios/classificação , Interneurônios/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteína Reelina , Serina Endopeptidases/análise , Proteínas Supressoras de Tumor/deficiência , Peptídeo Intestinal Vasoativo/análise
7.
Development ; 141(21): 4182-93, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25336744

RESUMO

The corpus callosum connects cerebral hemispheres and is the largest axon tract in the mammalian brain. Callosal malformations are among the most common congenital brain anomalies and are associated with a wide range of neuropsychological deficits. Crossing of the midline by callosal axons relies on a proper midline environment that harbors guidepost cells emitting guidance cues to instruct callosal axon navigation. Little is known about what controls the formation of the midline environment. We find that two components of the Hippo pathway, the tumor suppressor Nf2 (Merlin) and the transcriptional coactivator Yap (Yap1), regulate guidepost development and expression of the guidance cue Slit2 in mouse. During normal brain development, Nf2 suppresses Yap activity in neural progenitor cells to promote guidepost cell differentiation and prevent ectopic Slit2 expression. Loss of Nf2 causes malformation of midline guideposts and Slit2 upregulation, resulting in callosal agenesis. Slit2 heterozygosity and Yap deletion both restore callosal formation in Nf2 mutants. Furthermore, selectively elevating Yap activity in midline neural progenitors is sufficient to disrupt guidepost formation, upregulate Slit2 and prevent midline crossing. The Hippo pathway is known for its role in controlling organ growth and tumorigenesis. Our study identifies a novel role of this pathway in axon guidance. Moreover, by linking axon pathfinding and neural progenitor behaviors, our results provide an example of the intricate coordination between growth and wiring during brain development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Corpo Caloso/embriologia , Neurofibromatose 2/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Corpo Caloso/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Feminino , Lobo Límbico/embriologia , Lobo Límbico/metabolismo , Camundongos , Sistema Nervoso , Neurofibromatose 2/genética , Fosfoproteínas/genética , Gravidez , Fatores de Transcrição/genética , Proteínas de Sinalização YAP
8.
Dev Biol ; 388(1): 11-21, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24530424

RESUMO

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Giro Denteado/citologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Neurogênese/fisiologia , Receptor Notch1/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proliferação de Células , Giro Denteado/embriologia , Giro Denteado/patologia , Deleção de Genes , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Mutação , Células-Tronco Neurais/citologia , Receptor Notch1/genética , Proteínas Serrate-Jagged , Células-Tronco/citologia , Tamoxifeno/química
9.
Development ; 140(16): 3323-34, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23863479

RESUMO

Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Neurais/metabolismo , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Padronização Corporal , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Polaridade Celular , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/embriologia , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/patologia , Neurofibromina 2/genética , Fenótipo , Fosfoproteínas/genética , Gravidez , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas de Sinalização YAP
10.
Development ; 138(24): 5291-300, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22071110

RESUMO

Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.


Assuntos
Epêndima/crescimento & desenvolvimento , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Movimento Celular , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Hidrocefalia/etiologia , Ventrículos Laterais/crescimento & desenvolvimento , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Neuroglia/fisiologia , Proteína Homeobox SIX3
11.
PLoS Biol ; 8(8)2010 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-20808958

RESUMO

The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.


Assuntos
Diferenciação Celular , Giro Denteado/citologia , Proteínas de Homeodomínio/farmacologia , Células-Tronco Neurais/citologia , Neurogênese/efeitos dos fármacos , Proteínas Supressoras de Tumor/farmacologia , Células-Tronco Adultas/citologia , Animais , Encéfalo/embriologia , Giro Denteado/embriologia , Giro Denteado/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
12.
PLoS One ; 5(2): e9377, 2010 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-20186345

RESUMO

BACKGROUND: The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1(Flox/Flox); Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1(Flox/Flox); Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons. CONCLUSIONS/SIGNIFICANCE: These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.


Assuntos
Orelha Interna/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Cóclea/embriologia , Cóclea/metabolismo , Orelha Interna/embriologia , Orelha Interna/ultraestrutura , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação , Gravidez , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Vestíbulo do Labirinto/embriologia , Vestíbulo do Labirinto/metabolismo
13.
Development ; 135(3): 441-50, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18094027

RESUMO

The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.


Assuntos
Padronização Corporal , Diencéfalo/embriologia , Proteínas do Tecido Nervoso/deficiência , Animais , Diencéfalo/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Somitos/embriologia , Somitos/metabolismo , Telencéfalo/embriologia , Telencéfalo/metabolismo , Tálamo/embriologia , Tálamo/metabolismo , Proteínas Wnt/metabolismo , Proteína Homeobox SIX3
14.
Gene Expr Patterns ; 7(3): 252-7, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17084678

RESUMO

Recently, sequence analyses have identified a large number of opposite strand transcripts in the vertebrate genome. Although the transcripts appear to be spliced and polyadenylated, many of them are predicted to represent noncoding RNAs. High levels of noncoding transcripts of the Six3 Opposite Strand (Six3OS) were recently identified in the embryonic and postnatal retina of the mouse. In this study, we expanded those initial expression analyses, elucidated in detail the developmental expression profile of mouse Six3OS in the brain and visual system, and compared it with that of Six3. Our results show that Six3OS expression overlaps extensively with that of Six3 and is not altered in Six3-null embryos.


Assuntos
Desenvolvimento Embrionário/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , RNA Antissenso/genética , RNA não Traduzido/genética , Animais , Encéfalo/embriologia , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Camundongos , Mutação , Retina/embriologia , Retina/metabolismo , Proteína Homeobox SIX3
15.
Dev Dyn ; 236(2): 518-24, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17117441

RESUMO

Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood.


Assuntos
Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Encéfalo/metabolismo , Galactosídeos , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Indóis , Camundongos , Proteínas Supressoras de Tumor/genética
16.
J Neurochem ; 96(4): 1201-11, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16445854

RESUMO

Albino mammals have profound retinal abnormalities, including photoreceptor deficits and misrouted hemispheric pathways into the brain, demonstrating that melanin or its precursors are required for normal retinal development. Tyrosinase, the primary enzyme in melanin synthesis commonly mutated in albinism, oxidizes l-tyrosine to l-dopaquinone using l-3,4-dihydroxyphenylalanine (L-DOPA) as an intermediate product. L-DOPA is known to signal cell cycle exit during retinal development and plays an important role in the regulation of retinal development. Here, we have mimicked L-DOPA production by ectopically expressing tyrosine hydroxylase in mouse albino retinal pigment epithelium cells. Tyrosine hydroxylase can only oxidize l-tyrosine to L-DOPA without further progression towards melanin. The resulting transgenic animals remain phenotypically albino, but their visual abnormalities are corrected, with normal photoreceptor numbers and hemispheric pathways and improved visual function, assessed by an increase of spatial acuity. Our results demonstrate definitively that only early melanin precursors, L-DOPA or its metabolic derivatives, are vital in the appropriate development of mammalian retinae. They further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities.


Assuntos
Albinismo/enzimologia , Melaninas/deficiência , Retina/anormalidades , Tirosina 3-Mono-Oxigenase/genética , Visão Ocular/fisiologia , Animais , Células Cultivadas , Clonagem Molecular , Humanos , Camundongos , Proteínas Mutantes Quiméricas/metabolismo , Células PC12 , Feocromocitoma , Epitélio Pigmentado Ocular/enzimologia , Epitélio Pigmentado Ocular/patologia , Ratos , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Segmento Externo da Célula Bastonete/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
17.
Front Biosci ; 11: 743-52, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16146766

RESUMO

Albino animals display a hypopigmented phenotype associated with several visual abnormalities, including rod photoreceptor cell deficits, abnormal patterns of connections between the eye and the brain and a general underdevelopment of central retina. Oculocutaneous albinism type I, a common form of albinism, is caused by mutations in the tyrosinase gene. In mice, the albino phenotype can be corrected by functional tyrosinase transgenes. Tyrosinase transgenic animals not only show normal pigmentation but the correction of all visual abnormalities associated with albinism, confirming a role of tyrosinase, a key enzyme in melanin biosynthesis, in normal retinal development. Here, we will discuss recent work carried out with new tyrosinase transgenic mouse models, to further analyse the role of tyrosinase in retinal development. We will first report a transgenic model with inducible tyrosinase expression that has been used to address the regulated activation of this gene and its associated effects on the development of the visual system. Second, we will comment on an interesting yeast artificial chromosome (YAC)-tyrosinase transgene, lacking important regulatory elements, that has highlighted the significance of local interactions between the retinal pigment epithelium (RPE) and developing neural retina.


Assuntos
Regulação da Expressão Gênica , Monofenol Mono-Oxigenase/fisiologia , Retina/embriologia , Retina/patologia , Albinismo , Albinismo Oculocutâneo/genética , Animais , Cromossomos Artificiais Bacterianos/metabolismo , Cromossomos Artificiais de Levedura , Cruzamentos Genéticos , Modelos Animais de Doenças , Éxons , Levodopa/metabolismo , Melaninas/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Mutação , Fenótipo , Ativação Transcricional , Transgenes
18.
J Comp Neurol ; 485(4): 338-47, 2005 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-15803509

RESUMO

The retinal pigment epithelium (RPE) plays a key role in regulating retinal development. The critical enzyme in pigment production is tyrosinase. Transgenic mice with a tyrosinase construct where the locus control region was deleted (YRT4) display a variegated phenotype of tyrosinase expression. Their central retina is largely pigment free, whereas more peripheral regions are heavily pigmented. We have used this model to ask whether the influence of pigmented RPE over the retina during development is fundamentally governed by local interactions or is global. Our data show that YRT4 eyes have intermediate melanin content and relatively low tyrosinase activity compared with wild-type and albino animals. Rod counts are comparable to those in pigmented mice in peripheral regions but similar to those in albinos centrally. Anterograde labelling of retinal pathways demonstrates the presence of relatively normal ipsilateral chiasmatic projection in YRT4 mice, comparable with that in pigmented animals and consistent with the peripheral pigmented origin of this pathway. Examination of cellular proliferation levels during retinal development reveals that YRT4 mice display an extended period of mitosis, similar to that found in albinos. Hence, our results show that the regulatory influence of the RPE over the developing retina depends on localized interactions between these tissues.


Assuntos
Hipopigmentação/embriologia , Hipopigmentação/genética , Epitélio Pigmentado Ocular/anormalidades , Retina/anormalidades , Albinismo Ocular/embriologia , Albinismo Ocular/genética , Albinismo Ocular/metabolismo , Animais , Hipopigmentação/metabolismo , Melaninas/biossíntese , Melaninas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Epitélio Pigmentado Ocular/enzimologia , Epitélio Pigmentado Ocular/metabolismo , Retina/metabolismo
19.
BMC Cell Biol ; 6(1): 18, 2005 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-15826307

RESUMO

BACKGROUND: A number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalize melanocyte cell cultures from postnatal mouse skin. RESULTS: Here, we describe the efforts towards obtaining melanocyte cultures from our Tyr transgenic mice. We have bred our Tyr transgenic mice into Tyr c-32DSD mutant background, lacking the endogenous Tyr locus. In these conditions, we failed to obtain immortalized melanocytes. We decided to include the inactivation of the Ink4a-Arf locus to promote melanocyte immortalisation. For this purpose, we report the segregation of the Ink4a-Arf null allele from the brown (Tyrp1b) mutation in mice. Finally, we found that Ink4a-Arf +/- and Ink4a-Arf -/- melanocytes had undistinguishable tyrosine hydroxylase activities, although the latter showed reduced cellular pigmentation content. CONCLUSION: The simultaneous presence of precise genomic deletions that include the tyrosinase locus, such as the Tyr c-32DSD allele, the Tyr transgene itself and the inactivated Ink4a-Arf locus in Tyrp1B genetic background appear as the crucial combination to perform forthcoming experiments. We cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, subsequent experiments with melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.


Assuntos
Melanócitos/citologia , Melanócitos/enzimologia , Monofenol Mono-Oxigenase/genética , Transgenes , Alelos , Animais , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina , Métodos , Camundongos , Camundongos Transgênicos , Mutação , Proteína Supressora de Tumor p14ARF/genética
20.
J Biol Chem ; 280(6): 4817-24, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15572362

RESUMO

Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyrc-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyrc-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyrc-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyrc-m mice. Genomic analyses of Tyrc-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyrc-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.


Assuntos
Camundongos Mutantes , Monofenol Mono-Oxigenase/genética , Animais , Southern Blotting , Linhagem Celular , Cobre/metabolismo , Cruzamentos Genéticos , DNA/metabolismo , DNA Complementar/metabolismo , Retículo Endoplasmático/metabolismo , Éxons , Glicosilação , Complexo de Golgi/metabolismo , Heterozigoto , Humanos , Imuno-Histoquímica , Melaninas/biossíntese , Melaninas/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Modelos Genéticos , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/biossíntese , Mutação , Fenótipo , Pigmentação/genética , Tirosina/genética , Tirosina/metabolismo
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