RESUMO
As part of a high-throughput mutagenesis and phenotyping process designed to discover novel drug targets, we generated and characterized mice with a targeted mutation in Slc24a5, a gene encoding a putative cation exchanger. Upon macroscopic examination, Slc24a5-/- mice were viable, fertile, and indistinguishable by coat color from their heterozygous and wild-type litter mates. Ophthalmoscopic examination revealed diffuse retinal hypopigmentation, and a histologic examination of the eye confirmed the presence of moderate-to-marked hypopigmentation of the retinal pigmented epithelium (RPE), ciliary body, and iris pigment epithelium (IPE). Hypopigmentation was most severe in the anterior layer cells of the IPE, where melanosomes were smaller, paler, and more indistinct than those of the anterior stroma and posterior IPE. The pigment granules of the posterior IPE appeared to be nearly as dark as those in stromal melanocytes; however, both cell layers were thinner and paler than corresponding layers in wild-type mice. Ultrastructural analysis of the RPE, IPE, and ciliary body pigmented cells confirmed that mutation of Slc24a5 results in marked hypopigmentation of melanosomes in optic cup-derived pigmented neuroepithelium in the eyes. Milder reductions in melanosome size and pigmentation were noted in neural crest-derived melanocytes. The severe hypopigmentation of neuroepithelium-derived cells in the eyes resulted in a novel form of ocular albinism in Slc24a5-/- mice. Our findings suggest that SLC24A5 may be a candidate gene for some forms of ocular albinism and for the BEY1/EYCL2 locus previously associated with central brown eye color in humans.
Assuntos
Albinismo Ocular/genética , Antiporters/genética , Hipopigmentação/genética , Albinismo Ocular/ultraestrutura , Animais , Modelos Animais de Doenças , Feminino , Cor de Cabelo/genética , Histocitoquímica , Óperon Lac/genética , Masculino , Melanossomas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-DirigidaRESUMO
Disabled-1 (Dab1) is an intracellular adapter protein that mediates the effect of Reelin on neuronal migration and cell positioning during mammalian brain development. To identify components of the Reelin-Dab1 signaling pathway, we searched for proteins that interact with Dab1 using a yeast two-hybrid strategy. We found that the Dab1 phosphotyrosine binding (PTB) domain interacts with a novel protocadherin, orthologous to human protocadherin 18. Mouse Pcdh18 (mPcdh18), which consists of four exons similar to other protocadherin family members, maps to chromosome 3. The deduced amino acid sequence of mPcdh18 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. The site of Dab1 interaction was localized to the C-terminal 243 residues of mPcdh18. Expression analyses revealed that mPcdh18 is present in a variety of tissues in the embryo, but in adult mice it is primarily expressed in lung and kidney. In embryonic brain, mPcdh18 expression is temporally and spatially regulated. Our results indicate that mPcdh18 participates in signaling pathways involving PTB-containing proteins and suggest that it may play a role during brain development.
Assuntos
Caderinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Células Cultivadas , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/metabolismo , Embrião de Mamíferos/metabolismo , Éxons , Humanos , Hibridização In Situ , Rim/metabolismo , Pulmão/metabolismo , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteína Reelina , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Tempo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
The formation of synaptic connections requires the coordination of specific guidance molecules and spontaneous neuronal activity. The visual system has provided a useful model for understanding the role of these cues in shaping the precise connections from the neural retina to the brain. Here, we demonstrate that two essential genes in the Reelin signaling pathway function during the patterning of synaptic connectivity in the retina. Physiological studies of mice deficient in either reelin or disabled-1 reveal an attenuation of rod-driven retinal responses. This defect is associated with a decrease in rod bipolar cell density and an abnormal distribution of processes in the inner plexiform layer. These results imply that, in addition to its essential role during neuronal migration, the Reelin pathway contributes to the formation of neuronal circuits in the central nervous system.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Olho/fisiologia , Retina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fatores Etários , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Contagem de Células , Movimento Celular , Eletrorretinografia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/genética , Interneurônios/metabolismo , Proteínas Relacionadas a Receptor de LDL , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Receptores de LDL/fisiologia , Receptores de Lipoproteínas/fisiologia , Proteína Reelina , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/patologia , Serina Endopeptidases , Sinapses/fisiologia , Sinapses/ultraestrutura , Vias Visuais/fisiologiaRESUMO
The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.
Assuntos
Técnicas de Cultura de Células/métodos , Células Cultivadas/citologia , Citometria de Fluxo/métodos , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Camundongos Transgênicos/metabolismo , Células de Purkinje/citologia , Animais , Células Cultivadas/metabolismo , Proteínas de Fluorescência Verde , Camundongos , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Regiões Promotoras Genéticas/fisiologia , Células de Purkinje/metabolismo , Retina/citologia , Retina/metabolismo , Transcrição Gênica/fisiologiaRESUMO
The neurological mutant mouse reeler has played a critical role in the evolution of our understanding of normal brain development. From the earliest neuroanatomic studies of reeler, it was anticipated that the characterization of the gene responsible would elucidate important molecular and cellular principles governing cell positioning and the formation of synaptic circuits in the developing brain. Indeed, the identification of reelin has challenged many of our previous notions and has led to a new vision of the events involved in the migration of neurons. Several neuronal populations throughout the brain secrete Reelin, which binds to transmembrane receptors located on adjacent cells triggering a tyrosine kinase cascade. This allows neurons to complete migration and adopt their ultimate positions in laminar structures in the central nervous system. Recent studies have also suggested a role for the Reelin pathway in axonal branching, synaptogenesis, and pathology underlying neurodegeneration.
Assuntos
Encéfalo/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Neurônios/fisiologia , Animais , Axônios/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Proteína Reelina , Serina Endopeptidases , Transdução de Sinais , Sinapses/fisiologiaRESUMO
Drosophila Numb functions as a cell fate determinant during neurogenesis. We isolated a novel mammalian protein, Lnx2, which interacts with mammalian Numb and Numblike. Lnx2 and the related Lnx1 are multimodular proteins that bind to Numb via their NPXY motifs. In addition, Lnx proteins form oligomers either via their PDZ domains binding to PDZ-binding consensus motifs located in their C-termini or by homophilic oligomerization of their RING fingers. Therefore, Lnx proteins may form large networks by homomeric binding. In situ hybridization analysis revealed complementary patterns of Lnx1 and Lnx2 expression in developing and adult brain, although in several structures they are present in the same cell populations. Moreover, their expression patterns overlap with those of the Numb proteins. Oligomerization of Lnx2 and Numb binding occurs simultaneously. Therefore, our findings suggest that Lnx proteins may serve as molecular scaffolds that localize unrelated, interacting proteins, such as Numb, to specific subcellular sites.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/genética , Divisão Celular/genética , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologiaRESUMO
The organization of several laminated structures in the brain is controlled by a signaling pathway activated by Reelin, a large glycoprotein secreted by pioneer neurons in the developing brain. Reelin binds to transmembrane receptors, including VLDLR and ApoER2, and stimulates tyrosine phosphorylation of Disabled-1 (Dab1), which associates with an NPxY motif present in the cytoplasmic domain of the receptors. Disruption of reelin, dab1, or both the vldr and apoer2 genes results in similar cell positioning defects in laminated brain regions including the cerebellum, hippocampus, and cerebral cortex. Although retinal ganglion cells express reelin during development, there is no obvious disruption of cell positioning in the retina of reeler mice. Here, we examine the expression pattern of Dab1 as a first step toward understanding the function of the Reelin signaling pathway in neural retina. Immunohistochemical analysis of the adult retina revealed that Dab1 is expressed in a specific type of amacrine cell. These cells display a narrow dendritic field and they project to two distinct sublaminae within the inner plexiform layer. Dab1 co-localizes with the high-affinity glycine transporter, indicating that these amacrine cells are glycinergic. Cells that express Dab1 are surrounded by dopaminergic fibers originating from wide-field amacrine cells. These features are characteristic of type AII amacrine cells described in other mammalian species. Analysis of the retina at several stages of development revealed that Dab1 is expressed shortly after birth during the time at which AII amacrine cells extend neurites and form synaptic connections in the inner retina. This raises the possibility that the Reelin/Dab1 signaling pathway contributes to formation of intraretinal circuitry in the neural retina.
Assuntos
Camundongos/metabolismo , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Retina/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Contagem de Células , Dopamina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicina/metabolismo , Camundongos/anatomia & histologia , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteína Reelina , Retina/citologia , Retina/crescimento & desenvolvimento , Serina Endopeptidases , Transdução de Sinais/fisiologiaRESUMO
Tyrosinase is a key enzyme involved in the synthesis of melanin in the retinal pigment epithelium (RPE). Mice that are homozygous for the albino allele at the tyrosinase locus have fewer retinal ganglion cells with uncrossed projections at the optic chiasm. To determine the site of the albino gene action we studied the projections of retinal ganglion cells in two types of pigmentation mosaic mice. First, we generated mosaic mice that contain a translocated allele of the wild-type tyrosinase on one X chromosome but that also have the lacZ reporter transgene on the opposite X chromosome. In these lacZ/tyrosinase mice, which are homozygous for the albino allele on chromosome 7, X-inactivation ensures that tyrosinase cannot be functional within 50% of the retinal ganglion cells and that these individual cells can be identified by their expression of the lacZ reporter gene product, beta-galactosidase. The proportion of uncrossed retinal ganglion cells expressing beta-galactosidase was found to be identical to the proportion that did not express it, indicating that the albino mutation associated with axonal behavior at the optic chiasm must affect ganglion cells in a cell-extrinsic manner. Second, to determine whether the RPE is the source of the extrinsic signal, we generated aggregation chimeras between pigmented and albino mice. In these mosaic mice, the extent of the uncrossed projection corresponded with the amount of pigmented cells within the RPE, but did not correspond with the genotypes of neural retinal cells. These studies demonstrate that the albino mutation acts indirectly upon retinal ganglion cells, which in turn respond by making axonal guidance errors at the optic chiasm.
Assuntos
Albinismo/genética , Quimera/genética , Epitélio Pigmentado Ocular/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Genótipo , Histocitoquímica , Óperon Lac , Camundongos , Camundongos Endogâmicos , Monofenol Mono-Oxigenase/genética , Transgenes , Translocação Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Células COS , Cálcio/fisiologia , Linhagem Celular , Células/metabolismo , Humanos , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Proteínas do Tecido Nervoso , Receptores de LDL/metabolismo , Proteína Reelina , Serina EndopeptidasesAssuntos
Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Transdução de Sinais , Animais , Encéfalo/citologia , Encéfalo/embriologia , Moléculas de Adesão Celular Neuronais/genética , Sistema Nervoso Central/citologia , Proteínas da Matriz Extracelular/genética , Camundongos , Camundongos Mutantes Neurológicos/embriologia , Camundongos Mutantes Neurológicos/genética , Camundongos Mutantes Neurológicos/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo , Receptores de Lipoproteínas/fisiologia , Proteína Reelina , Serina EndopeptidasesRESUMO
Disruption of the disabled-1 gene (Dab1) results in aberrant migration of neurons during development and disorganization of laminar structures throughout the brain. Dab1 is thought to function as an adapter molecule in signal transduction processes. It contains a protein-interaction (PI) domain similar to the phosphotyrosine-binding domain of the Shc oncoprotein, it is phosphorylated by the Src protein tyrosine kinase, and it binds to SH2 domains in a phosphotyrosine-dependent manner. To investigate the function of Dab1, we searched for binding proteins using the yeast two-hybrid system. We found that the PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1). The association of Dab1 with APLP1 was confirmed in biochemical assays, and the site of interaction was localized to a cytoplasmic region of APLP1 containing the amino acid sequence motif Asn-Pro-x-Tyr (NPxY). NPxY motifs are involved in clathrin-mediated endocytosis, and they have been shown to bind to PI domains present in several proteins. This region of APLP1 is conserved among all members of the amyloid precursor family of proteins. Indeed, we found that Dab1 also interacts with amyloid precursor protein (APP) and APLP2 in biochemical association experiments. In transiently transfected cells, Dab1 and APLP1 colocalized in membrane ruffles and vesicular structures. Cotransfection assays in cultured cells indicated that APP family members increased serine phosphorylation of Dab1. Dab1 and APLP1 are expressed in similar cell populations in developing and adult brain tissue. These results suggest that Dab1 may function, at least in part, through association with APLP1 in the brain.
Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animais , Sítios de Ligação , Células COS , Córtex Cerebral/metabolismo , Sequência Consenso , Citoplasma/metabolismo , Expressão Gênica , Hipocampo/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Domínios de Homologia de srcRESUMO
The AML1/core binding factor beta (CBFbeta) transcription factor is essential for definitive hematopoiesis; however, the downstream pathways through which it functions remain incompletely defined. Using a differential cloning approach to define components of this pathway, we have identified a novel gene designated HERF1 (for hematopoietic RING finger 1), whose expression during development is dependent on the presence of functional AML1/CBFbeta. HERF1 contains a tripartite RING finger-B box-alpha-helical coiled-coil domain and a C-terminal region homologous to the ret proto-oncogene-encoded finger protein. Expression of HERF1 during embryogenesis coincides with the appearance of definitive erythropoiesis and in adult mice is restricted to erythroid cells, increasing 30-fold during terminal differentiation. Importantly, inhibition of HERF1 expression blocked terminal erythroid differentiation of the murine erythroleukemia cell line MEL, whereas its overexpression induced erythroid maturation. These results suggest an important role for this protein in erythropoiesis.
Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , Eritropoese/genética , Proteínas Proto-Oncogênicas , Dedos de Zinco/genética , Animais , Linhagem Celular , Clonagem Molecular , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA , Dimetil Sulfóxido , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Tetraciclina/farmacologia , Fatores de Transcrição/genética , Proteínas com Motivo TripartidoRESUMO
The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.
Assuntos
Oftalmopatias/genética , Olho/crescimento & desenvolvimento , Retina/crescimento & desenvolvimento , Animais , Ciclo Celular/genética , Morte Celular/genética , Diferenciação Celular/genética , Divisão Celular/genética , Desenvolvimento Embrionário e Fetal , Olho/embriologia , Histocitoquímica , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Mutantes , Morfogênese/genética , Mutação/genética , Fenótipo , Retina/embriologiaRESUMO
Mutation of either reelin (Reln) or disabled-1 (Dab1) results in widespread abnormalities in laminar structures throughout the brain and ataxia in reeler and scrambler mice. Both exhibit the same neuroanatomical defects, including cerebellar hypoplasia with Purkinje cell ectopia and disruption of neuronal layers in the cerebral cortex and hippocampus. Despite these phenotypic similarities, Reln and Dab1 have distinct molecular properties. Reln is a large extracellular protein secreted by Cajal-Retzius cells in the forebrain and by granule neurons in the cerebellum. In contrast, Dab1 is a cytoplasmic protein which has properties of an adapter protein that functions in phosphorylation-dependent intracellular signal transduction. Here, we show that Dab1 participates in the same developmental process as Reln. In scrambler mice, neuronal precursors are unable to invade the preplate of the cerebral cortex and consequently, they do not align within the cortical plate. During development, cells expressing Dab1 are located next to those secreting Reln at critical stages of formation of the cerebral cortex, cerebellum and hippocampus, before the first abnormalities in cell position become apparent in either reeler or scrambler. In reeler, the major populations of displaced neurons contain elevated levels of Dab1 protein, although they express normal levels of Dab1 mRNA. This suggests that Dab1 accumulates in the absence of a Reln-evoked signal. Taken together, these results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/biossíntese , Cerebelo/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Hipocampo/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/biossíntese , Fosfoproteínas/biossíntese , Proteína Reelina , Serina EndopeptidasesRESUMO
PURPOSE: The autosomal semidominant mutation Bst (belly spot and tail) is often associated with small and atrophic optic nerves in adult mice and shares several important attributes with heritable optic nerve atrophy in humans. In this article, the authors present adult and developmental studies on the retinal phenotype in Bst/+ mice. METHODS: Retinal ganglion cells in adult Bst/+ mice were labeled retrogradely with horseradish peroxidase injected into the right optic tract. Labeled ganglion cells were mapped in whole-mounted retinas ipsilateral and contralateral to the injection site. The number of axons in optic nerves of these and other cases were quantified using an electron microscopic method. Eyes of neonatal, embryonic day 15 (E15), and embryonic day 12 (E12) Bst/+ mutants were examined histologically to understand the etiology of the retinal phenotype. RESULTS: Approximately 60% of adult Bst/+ mice have deficient direct pupillary light responses. This neurologic phenotype is associated with a reduction in the number of retinal ganglion cells from the wild-type average of 67,000 to less than 20,000 in Bst/+ mutants. Ganglion cells with crossed projections are more severely affected than those with uncrossed projections. Histologic analysis of eyes from E12 mice reveals a delayed closure of the optic fissure. Despite this abnormality, other ocular structures appear relatively normal. However, some E15 mutants exhibit marked disorganization of the retinal neuroepithelium, and ganglion cell axons are found between pigmented and neural retina. At birth, optic nerves of affected mice are smaller than those of wild-type mice, ectopic axons are found within the eyes, and the ganglion cell layer contains many dying cells. CONCLUSIONS: The expression of the retinal phenotype in Bst/+ mutants is highly variable-ranging from a complete absence of ganglion cells to numbers comparable to that in wild-type mice. The reduction in ganglion cell number in affected adult Bst/+ mice is attributable to the failure of ganglion cell axons to reach the optic nerve head early in development. Delayed fusion of the fissure is consistently associated with the Bst/+ genotype and probably contributes to the failure of ganglion cell axons to grow out of the eye.
Assuntos
Axônios/patologia , Atrofias Ópticas Hereditárias/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Animais Recém-Nascidos , Contagem de Células , Feminino , Peroxidase do Rábano Silvestre , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Atrofias Ópticas Hereditárias/genética , Disco Óptico/embriologia , Disco Óptico/patologia , Nervo Óptico/anormalidades , Nervo Óptico/embriologia , Fenótipo , Gravidez , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/patologiaRESUMO
Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in reelin which cause widespread misplacement of neurons and associated ataxia in reeler mice. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment. Two new autosomal recessive mouse mutations, scramble and yotari have been described that exhibit a phenotype identical to reeler. Here we report that scrambler and yotari arise from mutations in mdab1, a mouse gene related to the Drosophila gene disabled (dab). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. Expression analysis indicates that mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signalling molecules that regulate cell positioning in the developing brain.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Mutação , Proteínas do Tecido Nervoso/genética , Animais , Axônios/fisiologia , Encéfalo/embriologia , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/fisiologia , Mapeamento Cromossômico , Cruzamentos Genéticos , Proteínas da Matriz Extracelular/fisiologia , Expressão Gênica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/fisiologia , Fenótipo , RNA Mensageiro/metabolismo , Proteína Reelina , Serina EndopeptidasesRESUMO
How much of the remarkable variation in neuron number within a species is generated by genetic differences, and how much is generated by environmental factors? We address this problem for a single population of neurons in the mouse CNS. Retinal ganglion cells of inbred and outbred strains, wild species and subspecies, and F1 hybrids were studied using an unbiased electron microscopic method with known technical reliability. Ganglion cell numbers among diverse types of mice are highly variable, ranging from 32,000 to 87,000. The distribution of all cases (n = 252) is close to normal, with a mean of 58,500 and an SD of 7800. Genetic factors are most important in controlling this variation; 76% of the variance is heritable and up to 90% is attributable to genetic factors in a broad sense. Strain averages have an unanticipated bimodal distribution, with distinct peaks at 55,500 and 63,500 cells. Three pairs of closely related strains have ganglion cell populations that differ by > 20% (10,000 cells). These findings indicate that different alleles at one or two genes have major effects on normal variation in ganglion cell number. Nongenetic factors are still appreciable and account for a coefficient of variation that averages approximately 3.6% within inbred strains and isogenic F1 hybrids. Age- and sex-related differences in neuron number are negligible. Variation within isogenic strains appears to be generated mainly by developmental noise.
Assuntos
Variação Genética , Células Ganglionares da Retina/citologia , Envelhecimento/fisiologia , Animais , Peso Corporal , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Contagem de Células , Meio Ambiente , Feminino , Genes Dominantes , Genética Populacional , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica , Tamanho do Órgão , Células Ganglionares da Retina/ultraestrutura , Caracteres Sexuais , Especificidade da EspécieRESUMO
The study of chimeric retinas has yielded insight on the early development of retina. The close match in chimerism ratios between right and left retinas is significant and supports the idea that both retinas originate from a common population of progenitors. We are able to estimate numbers of progenitor cells that contribute to the formation of the retina and the approximate time at which this small group is isolated from surrounding prosencephalic cell fields. These cells undergo at least five rounds of division before the first retinal neurons are generated. The mouse retina is not build from the center outward. There is simultaneous expansion and differentiation in all parts of the retina and as a result clones are not arranged in wedges. Instead the mouse retina is a patchwork of clones that do not differ greatly in size from center to periphery. The most consistent radial feature in mouse retina is a raphe left at the line of fusion of the margins of the ventral fissure. Processes that shape the clonal patchwork are both passive and active, intrinsic and extrinsic. Certain features of the clonal architecture of the retina, such as the size differences of clones are primarily passive responses to extrinsic forces on progenitor cells and their progeny. The fifteen-fold range in the size of cohorts is not due to intrinsic differences in the proliferative capacity of individual progenitor cells, but is due to the extent of cell movement and mixing at early stages of development. In contrast, active or intrinsic processes are illustrated by the partial (and still controversial) restriction of retinal progenitors, the possible clonal differences between ganglion cells with crossed and uncrossed projections, and the consistent differences in ratios of albino and pigmented genotypes in peripheral and central retina.
Assuntos
Retina/citologia , Animais , Linhagem da Célula , Quimera/fisiologia , Células Clonais , Camundongos , FenótipoRESUMO
Mutations in the tyrosinase gene are often associated with a misrouting of retinal ganglion cell axons at the optic chiasm. In albinos, tyrosinase activity is lost and some ganglion cell axons that would normally project into the ipsilateral optic tract instead cross midline and project into the contralateral tract. The developmental mechanisms that cause this modification in neuronal connectivity are unknown. In this study, we screened six diverse strains of albino mice (strains 129, A, AKR, BALB/c, C57BL/6-c/c, and CD-1) to discover genetically determined variations and possible gene loci that might affect the severity of the albino decussation abnormality. Ganglion cells were retrogradely labeled with horseradish peroxidase, and the ipsilaterally and contralaterally projecting cells were counted. The average number of ipsilaterally projecting ganglion cells in the six albino strains varies from 1,000 to 1,300. Despite this variation, 1.8-1.9% of the total population projects ipsilaterally in each strain. In comparison, 2.8% project ipsilaterally in the pigmented strain, C57BL/6(-)+/+. However, the percentage of displaced, ipsilaterally projecting cells varies substantially among albino strains--from a low of 4% in strain CD-1 to a high of nearly 10% in C57BL/6-c/c. We conclude that even with large differences in genetic background and in absolute numbers of ganglion cells, there is no appreciable variation in the magnitude of decussation error among albino mice. The consistent effect of null alleles at tyrosinase suggests a comparably tight linkage between the biochemical activity of this enzyme and the mechanisms that control decussation phenotype.