RESUMO
The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.
Assuntos
Carcinogênese , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Meduloblastoma/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , CamundongosRESUMO
Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Hedgehog/metabolismo , Animais , Astrócitos/citologia , Axônios/metabolismo , Células CHO , Células COS , Proliferação de Células , Chlorocebus aethiops , Cricetinae , Cricetulus , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Mutação/genética , Nervo Óptico/metabolismo , Células PC12 , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ratos , Células Ganglionares da Retina/metabolismo , Via SecretóriaRESUMO
Neurogenesis is regulated by the dynamic and coordinated activity of several extracellular signalling pathways, but the basis for crosstalk between these pathways remains poorly understood. Here we investigated regulatory interactions between two pathways that are each required for neural progenitor cell maintenance in the postnatal retina; Hedgehog (Hh) and Notch signalling. Both pathways are activated in progenitor cells in the postnatal retina based on the co-expression of fluorescent pathway reporter transgenes at the single cell level. Disrupting Notch signalling, genetically or pharmacologically, induces a rapid downregulation of all three Gli proteins and inhibits Hh-induced proliferation. Ectopic Notch activation, while not sufficient to promote Hh signalling or proliferation, increases Gli2 protein. We show that Notch regulation of Gli2 in Müller glia renders these cells competent to proliferate in response to Hh. These data suggest that Notch signalling converges on Gli2 to prime postnatal retinal progenitor cells and Müller glia to proliferate in response to Hh.
Assuntos
Células Ependimogliais/citologia , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Receptores Notch/metabolismo , Animais , Proliferação de Células/fisiologia , Feminino , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/genética , Receptores Notch/genética , Retina/citologia , Retina/embriologia , Transdução de Sinais , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.
Assuntos
Cegueira/congênito , Proteínas do Olho/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/genética , Retina/metabolismo , Espasmos Infantis/genética , Via de Sinalização Wnt , Animais , Cegueira/genética , Cegueira/metabolismo , Ciclo Celular/genética , Proliferação de Células , Epistasia Genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Doenças Genéticas Ligadas ao Cromossomo X , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/metabolismo , Ligação Proteica , Retina/crescimento & desenvolvimento , Degeneração Retiniana , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/metabolismo , Espasmos Infantis/metabolismo , Via de Sinalização Wnt/genética , Proteína Gli2 com Dedos de ZincoRESUMO
Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Intoxicação por MPTP/genética , Proteínas Oncogênicas/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Adenoviridae/genética , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Técnicas de Transferência de Genes , Genes Recessivos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/induzido quimicamente , Degeneração Neural/genética , Degeneração Neural/metabolismo , Neurotoxinas/farmacologia , Proteínas Oncogênicas/metabolismo , Proteína Desglicase DJ-1 , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Estresse Fisiológico/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Retina/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.
Assuntos
Células-Tronco Multipotentes/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/fisiologia , Retina/embriologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Regulação para Baixo/genética , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Distribuição Aleatória , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Retina/citologia , Retina/fisiologia , Transdução de Sinais/genéticaRESUMO
Sonic hedgehog (Shh) is an indispensable, extrinsic cue that regulates progenitor and stem cell behavior in the developing and adult mammalian central nervous system. Here, we investigate the link between the Shh signaling pathway and Hes1, a classical Notch target. We show that Shh-driven stabilization of Hes1 is independent of Notch signaling and requires the Shh effector Gli2. We identify Gli2 as a primary mediator of this response by showing that Gli2 is required for Hh (Hedgehog)-dependent up-regulation of Hes1. We also show using chromatin immunoprecipitation that Gli2 binds to the Hes1 promoter, which suggests that Hes1 is a Hh-dependent direct target of Gli2 signaling. Finally, we show that Shh stimulation of progenitor proliferation and cell diversification requires Gli2 and Hes1 activity. This paper is the first demonstration of the mechanistic and functional link between Shh, Gli, and Hes1 in the regulation of progenitor cell behavior.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas Hedgehog/fisiologia , Proteínas de Homeodomínio/fisiologia , Receptores Notch/fisiologia , Retina/citologia , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proliferação de Células , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptores Notch/química , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Retina/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição HES-1 , Proteína Gli2 com Dedos de ZincoRESUMO
ATRX is an SWI/SNF-like chromatin remodeling protein that is mutated in several X-linked mental retardation syndromes, including the ATR-X syndrome. In mice, Atrx expression is widespread and attempts to understand its function in brain development are hampered by the lethality associated with ubiquitous or forebrain-restricted ablation of this gene. One way to circumvent this problem is to study its function in a region of the brain that is dispensable for long-term survival of the organism. The retina is a well-characterized tractable model of CNS development and in our review of 202 ATR-X syndrome patients, we found ocular defects present in approximately 25% of the cases, suggesting that studying Atrx in this tissue will provide insight into function. We report that Atrx is expressed in the neuroprogenitor pool in embryonic retina and in all cell types of the mature retina with the exception of rod photoreceptors. Conditional inactivation of Atrx in the retina during embryogenesis ultimately results in a loss of only two types of neurons, amacrine and horizontal cells. We show that this defect does not arise from a failure to specify these cells but rather a defect in interneuron differentiation and survival post-natally. The timing of cell loss is concomitant with light-dependent changes in synaptic organization in the retina and with a change in Atrx subnuclear localization within these interneurons. Moreover, these interneuron defects are associated with functional deficits as demonstrated by reduced b-wave amplitudes upon electroretinogram analysis. These results implicate a role for Atrx in interneuron survival and differentiation.
Assuntos
DNA Helicases/metabolismo , Interneurônios/fisiologia , Deficiência Intelectual Ligada ao Cromossomo X/fisiopatologia , Proteínas Nucleares/metabolismo , Visão Ocular , Adulto , Células Amácrinas/fisiologia , Animais , Sobrevivência Celular , DNA Helicases/genética , Feminino , Expressão Gênica , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/embriologia , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteína Nuclear Ligada ao XRESUMO
The development of glial precursor cells in the mammalian optic nerve depends on retinal ganglion cell (RGC) axons, but the signals that mediate this neuron-to-glia interaction have not been fully characterized. Sonic hedgehog (Shh) is expressed by RGCs, and we showed previously that it is required for the specification of astrocyte lineage cells at the optic disc. To study the role of RGC-derived Shh on astrocyte development at later developmental stages, we generated mice with a conditional ablation of Shh in the peripheral retina and analyzed gene expression and glial cell development in the optic nerve. Astrocyte development was initiated in the optic nerves of these mutant mice; however, the expression of Hedgehog (Hh) target genes, Gli1 and Ptch1 and cell cycle genes, Ccnd1 and Cdc25b in the optic nerves were downregulated. Astrocyte proliferation was markedly reduced. Oligodendrocyte precursor cells were fewer in the optic nerves of mutant mice, possibly as a consequence of reduced secretion of growth factors by astrocytes. At a later developmental stage, optic nerve axons displayed signs of Wallerian degeneration, including reduction of astrocyte processes, degenerating glial cells and formation of distended axons. We also demonstrate that the Hh pathway can be activated in optic nerve-derived astrocytes in vitro, but fails to induce cell cycle gene expression and proliferation. RGC-derived Shh signalling isthus necessary in vivo for maintenance of astrocyte proliferation, affecting both axo-glial and normal glial cell development in the optic nerve.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Proteínas Hedgehog/fisiologia , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Ciclina D , Ciclinas/genética , Ciclinas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Neuroglia/citologia , Neuroglia/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/ultraestrutura , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína GLI1 em Dedos de Zinco , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.
Assuntos
Células Endoteliais/metabolismo , Proteínas Hedgehog/metabolismo , Epitélio Pigmentado Ocular/embriologia , Esclera/embriologia , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Corioide/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/metabolismo , Hipopigmentação/patologia , Fatores de Transcrição Kruppel-Like , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Órbita/metabolismo , Epitélio Pigmentado Ocular/anormalidades , Epitélio Pigmentado Ocular/ultraestrutura , Retina/embriologia , Retina/patologia , Esclera/anormalidades , Esclera/ultraestrutura , Transativadores/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.
Assuntos
Retina/embriologia , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Corpo Ciliar/embriologia , Corpo Ciliar/metabolismo , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Técnicas In Vitro , Óperon Lac , Camundongos , Camundongos Transgênicos , Microftalmia/embriologia , Microftalmia/genética , Microftalmia/metabolismo , Retina/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , beta Catenina/deficiência , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The morphogen Sonic hedgehog (Shh) is expressed by the projection neurons of the retina, retinal ganglion cells (RGCs) and promotes retinal precursor cell (RPC) proliferation. To distinguish between direct and indirect effects of Hedgehog (Hh) pathway activation in the perinatal mouse retina, we followed the fate of cells that expressed a constitutively active allele of Smoothened (SMO-M2), the signal transduction component of the Hh pathway. SMO-M2 expression promoted a cell-autonomous increase in CyclinD1 expression and RPC proliferation and promoted the development of cells with an inner nuclear layer identity. SMO-M2 expression also inhibited rhodopsin expression in uninfected cells, thus highlighting an unexpected non-cell autonomous effect of Hh pathway activation on photoreceptor development.
Assuntos
Retina/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Células Cultivadas , Proteínas Hedgehog , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Receptor Smoothened , Transativadores/genética , Transativadores/fisiologiaRESUMO
Hedgehog (Hh), Notch, and Wingless (Wnt) signaling control normal development of the cerebellum, and dysregulation of these signaling pathways are associated with medulloblastoma (MB). As an initial step in the study of the role of interacting signaling pathways in MB pathogenesis, we demonstrate the expression of several components of the Notch and Wnt signaling pathways, and activation of Notch signaling in MB from Ptch +/- mice that have elevated Hh signaling. We also show a marked downregulation in the expression of Notch2, Jagged1, Hes1, mSfrp1, and mFrz7 in cerebella of developing mice with reduced Hh signaling, suggesting that Hh signaling regulates the expression of these genes. Together with recent published data, these findings indicate that Hh signaling might synergize simultaneously with Notch and Wnt signaling in MB development by controlling Notch and Wnt pathway ligand, receptor and/or target gene expression.
Assuntos
Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Animais , Heterozigoto , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Receptores Patched , Receptor Patched-1 , RNA Mensageiro/análise , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
The timing of cell cycle exit and temporal changes in the developmental competence of precursor cells are key components for the establishment of the normal complement of cell types in the mammalian retina. The identity of cell extrinsic cues that control these processes is largely unknown. We showed previously in mouse retina that sonic hedgehog (Shh) signalling from retinal ganglion cells (RGCs) to retinal precursor cells (RPC) is required for the establishment of normal retinal organization. Here, we show that conditional ablation of Shh expression in the peripheral mouse results in a depletion of the RPC pool, owing to precocious cell-cycle exit and neuronal differentiation. These changes were correlated with the downregulation of cyclin D1 and Hes1 gene expression. Shh inactivation also results in an increase in RGC number owing to a bias of RPC towards RGC production. In contrast to zebrafish, where Shh signalling drives cell cycle exit and RGC development, our findings indicate that in the mouse retina Shh signalling is required to maintain RPC proliferation and to control the timing of RGC development.
Assuntos
Proliferação de Células , Retina/embriologia , Células Ganglionares da Retina/fisiologia , Transativadores/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Regulação para Baixo , Proteínas Hedgehog , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Ganglionares da Retina/citologia , Transdução de Sinais/fisiologia , Fatores de Transcrição HES-1RESUMO
Mutations in Patched (PTCH), encoding the Hedgehog (Hh) receptor, underlie Basal Cell Naevus syndrome (BCNS) and, in addition to tumor predisposition, are associated with a wide range of 'patterning' defects. The basis for the underlying patterning problems in Hh-dependent tissues in BCNS and their long-term consequences on tissue homeostasis are, however, not known. Hh signaling is required for normal growth and organization of the mammalian retina and we show that PtchlacZ+/- mice exhibit vitreoretinal abnormalities resembling those found in BCNS patients. The retinas of PtchlacZ+/- mice exhibit abnormal cell cycle regulation, which culminates in photoreceptor dysplasia and Müller cell-derived gliosis. In BCNS, the intraretinal glial response results in epiretinal membrane (ERM) formation, a proliferative and contractile response on the retinal surface. ERMs are a cause of significant visual loss in the general, especially elderly, population. We hypothesize that alteration of Müller cell Hh signaling may play a role in the pathogenesis of such age-related 'idiopathic' ERMs.
Assuntos
Proteínas de Membrana/genética , Retina/anormalidades , Transdução de Sinais , Transativadores/metabolismo , Animais , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/metabolismo , Síndrome do Nevo Basocelular/patologia , Regulação da Expressão Gênica , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular , Retina/crescimento & desenvolvimento , Retina/ultraestrutura , Displasia Retiniana/genética , Displasia Retiniana/patologia , Transativadores/genéticaRESUMO
The development of optic stalk neuroepithelial cells depends on Hedgehog (Hh) signaling, yet the source(s) of Hh protein in the optic stalk is unknown. We provide genetic evidence that sonic hedgehog (Shh) from retinal ganglion cells (RGCs) promotes the development of optic disc and stalk neuroepithelial cells. We demonstrate that RGCs express Shh soon after differentiation, and cells at the optic disc in close proximity to the Shh-expressing RGCs upregulate Hh target genes, which suggests they are responding to RGC-derived Shh signaling. Conditional ablation of Shh in RGCs caused a complete loss of optic disc astrocyte precursor cells, resulting in defective axon guidance in the retina, as well as conversion of the neuroepithelial cells in the optic stalk to pigmented cells. We further show that Shh signaling modulates the size of the Pax2(+) astrocyte precursor cell population at the optic disc in vitro. Together, these data provide a novel insight into the source of Hh that promotes neuroepithelial cell development in the mammalian optic disc and stalk.
Assuntos
Olho/embriologia , Morfogênese/fisiologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Biomarcadores , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Técnicas de Cultura , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Nervo Óptico/citologia , Nervo Óptico/embriologia , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/patologia , Receptores Patched , Fenótipo , Pigmentação/fisiologia , Receptores de Superfície Celular , Retina/citologia , Retina/fisiologia , Células Ganglionares da Retina/citologia , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The regulatory elements of the murine Thy1.2 gene were used to drive Cre recombinase expression in the nervous system (NS) of transgenic mice. Eleven Thy1-Cre lines exhibited transgene expression in several regions of the central and peripheral nervous systems, including the cerebral cortex, cerebellum, spinal cord, retina, and dorsal root ganglion. Thy1-Cre expression also resulted in region-specific activation of Cre reporter transgenes. Although Thy-1 expression is normally initiated in postmitotic neurons in the perinatal period, we show that the Thy-1.2 expression cassette drives Cre expression in immature proliferative zones in the NS as early as embryonic day 11 and in non-neural tissue. The Thy1.2 transgene cassette, therefore, does not impart transgene expression that is restricted to the NS or to postmitotic neurons within the NS. This panel of Thy1-Cre transgenic mice, however, will be useful reagents for the ablation of genes whose transcripts are spatially or temporally restricted in the developing NS.