RESUMO
Entosis, a form of cell cannibalism, is a newly discovered pathogenic mechanism leading to the development of small brains, termed microcephaly, in which P53 activation was found to play a major role. Microcephaly with entosis, found in Pals1 mutant mice, displays P53 activation that promotes entosis and apoptotic cell death. This previously unappreciated pathogenic mechanism represents a novel cellular dynamic in dividing cortical progenitors which is responsible for cell loss. To date, various recent models of microcephaly have bolstered the importance of P53 activation in cell death leading to microcephaly. P53 activation caused by mitotic delay or DNA damage manifests apoptotic cell death which can be suppressed by P53 removal in these animal models. Such genetic studies attest P53 activation as quality control meant to eliminate genomically unfit cells with minimal involvement in the actual function of microcephaly associated genes. In this review, we summarize the known role of P53 activation in a variety of microcephaly models and introduce a novel mechanism wherein entotic cell cannibalism in neural progenitors is triggered by P53 activation.
Assuntos
Apoptose , Entose , Microcefalia , Proteína Supressora de Tumor p53 , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Humanos , Camundongos , Modelos Animais de DoençasRESUMO
The multi-systemic genetic disorder tuberous sclerosis complex (TSC) impacts multiple neurodevelopmental processes including neuronal morphogenesis, neuronal migration, myelination and gliogenesis. These alterations contribute to the development of cerebral cortex abnormalities and malformations. Although TSC is caused by mTORC1 hyperactivation, cognitive and behavioral impairments are not improved through mTORC1 targeting, making the study of the downstream effectors of this complex important for understanding the mechanisms underlying TSC. As mTORC1 has been shown to promote the activity of the transcriptional co-activator Yap, we hypothesized that altered Yap/Taz signaling contributes to the pathogenesis of TSC. We first observed that the levels of Yap/Taz are increased in human cortical tuber samples and in embryonic cortices of Tsc2 conditional knockout (cKO) mice. Next, to determine how abnormal upregulation of Yap/Taz impacts the neuropathology of TSC, we deleted Yap/Taz in Tsc2 cKO mice. Importantly, Yap/Taz/Tsc2 triple conditional knockout (tcKO) animals show reduced cortical thickness and cortical neuron cell size, despite the persistence of high mTORC1 activity, suggesting that Yap/Taz play a downstream role in cytomegaly. Furthermore, Yap/Taz/Tsc2 tcKO significantly restored cortical and hippocampal lamination defects and reduced hippocampal heterotopia formation. Finally, the loss of Yap/Taz increased the distribution of myelin basic protein in Tsc2 cKO animals, consistent with an improvement in myelination. Overall, our results indicate that targeting Yap/Taz lessens the severity of neuropathology in a TSC animal model. This study is the first to implicate Yap/Taz as contributors to cortical pathogenesis in TSC and therefore as potential novel targets in the treatment of this disorder.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Esclerose Tuberosa , Proteínas de Sinalização YAP , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neurônios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteínas de Sinalização YAP/genéticaRESUMO
Mutations in the CRB1 (Crumbs homolog 1) cause rare retinal diseases like retinitis pigmentosa type 12 (RP12) and Leber congenital amaurosis type 8 (LCA8). RP12 results in progressively worsening peripheral vision, whereas LCA8 causes severe visual impairment at birth or in early life. While several mouse models have been proposed for RP12, few replicate the full spectrum of human LCA8 pathology, such as disorganized retinal layering, abnormal retinal thickening, pigmentary defects, hyperreflective lesions, and severely attenuated electroretinogram responses at birth. Six models have been proposed utilizing the Cre-loxP system to delete candidate genes in specific retinal cell types and developmental stages. The model ablating Crb1 and its homolog Crb2 (using mRx-Cre) from the beginning of the eye development is the most complete as it shows blindness during the eye-opening stage, pigmentary defects in the RPE, ganglion cell layer heterotopia, disruption of retinal lamination, and acellular patches. LCA8 represents a unique type of retinal dystrophy among LCA subtypes, driven by dysfunctional retinal progenitor cells during eye development. In contrast, other LCA types and RP12 are caused by photoreceptor defects. Therefore, the most accurate LCA8-like mouse model must target both alleles of the Crb1 and Crb2 genes in the optic vesicle or earlier.
Assuntos
Amaurose Congênita de Leber , Retinose Pigmentar , Animais , Modelos Animais de Doenças , Proteínas do Olho/genética , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Retina/metabolismo , Retinose Pigmentar/genéticaRESUMO
Membrane Protein Palmitoylated 5 (MPP5) is a highly conserved apical complex protein essential for cell polarity, fate and survival. Defects in cell polarity are associated with neurologic disorders including autism and microcephaly. MPP5 is essential for neurogenesis in animal models, but human variants leading to neurologic impairment have not been described. We identified three patients with heterozygous MPP5 de novo variants (DNV) and global developmental delay (GDD) and compared their phenotypes and magnetic resonance imaging (MRI) to ascertain how MPP5 DNV leads to GDD. All three patients with MPP5 DNV experienced GDD with language delay/regression and behavioral changes. MRI ranged from normal to decreased gyral folding and microcephaly. The effects of MPP5 depletion on the developing brain were assessed by creating a heterozygous conditional knock out (het CKO) murine model with central nervous system (CNS)-specific Nestin-Cre drivers. In the het CKO model, Mpp5 depletion led to microcephaly, decreased cerebellar volume and cortical thickness. Het CKO mice had decreased ependymal cells and Mpp5 at the apical surface of cortical ventricular zone compared with wild type. Het CKO mice also failed to maintain progenitor pools essential for neurogenesis. The proportion of cortical cells undergoing apoptotic cell death increased, suggesting that cell death reduces progenitor population and neuron number. Het CKO mice also showed behavioral changes, similar to our patients. To our knowledge, this is the first report to show that variants in MPP5 are associated with GDD, behavioral abnormalities and language regression/delay. Murine modeling shows that neurogenesis is likely altered in these individuals, with cell death and skewed cellular composition playing significant roles.
Assuntos
Deficiências do Desenvolvimento/etiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Doenças do Sistema Nervoso/etiologia , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/fisiologia , Adolescente , Adulto , Animais , Criança , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Adulto JovemRESUMO
Yap/Taz are well-established downstream effectors of the Hippo pathway, known to regulate organ size by directing proliferation and apoptosis. Although the functions of Yap/Taz have been extensively studied, little is known about their role in brain development. Here, through genetic ablation, we show that Yap/Taz are required for cerebellar morphogenesis. Yap/Taz deletion in neural progenitors causes defects in secondary fissure formation, leading to abnormal folia development. Although they seemed very likely to serve an important function in the development of cerebellar granule cell precursors, Yap/Taz are dispensable for their proliferation. Furthermore, Yap/Taz loss does not rescue the medulloblastoma phenotype caused by constitutively active Smoothened. Importantly, Yap/Taz are highly expressed in radial glia and play a crucial role in establishing the radial scaffold and cellular polarity of neural progenitors during embryogenesis. We found that Yap/Taz are necessary to establish and maintain junctional integrity of cerebellar neuroepithelium as prominent junction proteins are not maintained at the apical junction in the absence of Yap/Taz. Our study identifies a novel function of Yap/Taz in cerebellar foliation and finds that they are required to establish the radial glia scaffold and junctional stability.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cerebelo/embriologia , Organogênese , Transativadores/metabolismo , Animais , Proliferação de Células , Cerebelo/citologia , Cerebelo/metabolismo , Células Ependimogliais , Camundongos , Tamanho do Órgão , Células-Tronco/metabolismo , Proteínas de Sinalização YAPRESUMO
BUB-related 1 (BubR1) encoded by Budding Uninhibited by Benzimidazole 1B (BUB1B) is a crucial mitotic checkpoint protein ensuring proper segregation of chromosomes during mitosis. Mutations of BUB1B are responsible for mosaic variegated aneuploidy (MVA), a human congenital disorder characterized by extensive abnormalities in chromosome number. Although microcephaly is a prominent feature of MVA carrying the BUB1B mutation, how BubR1 deficiency disturbs neural progenitor proliferation and neuronal output and leads to microcephaly is unknown. Here we show that conditional loss of BubR1 in mouse cerebral cortex recapitulates microcephaly. BubR1-deficient cortex includes a strikingly reduced number of late-born, but not of early-born, neurons, although BubR1 expression is substantially reduced from an early stage. Importantly, absence of BubR1 decreases the proportion of neural progenitors in mitosis, specifically in metaphase, suggesting shortened mitosis owing to premature chromosome segregation. In the BubR1 mutant, massive apoptotic cell death, which is likely due to the compromised genomic integrity that results from aberrant mitosis, depletes progenitors and neurons during neurogenesis. There is no apparent alteration in centrosome number, spindle formation or primary cilia, suggesting that the major effect of BubR1 deficiency on neural progenitors is to impair the mitotic checkpoint. This finding highlights the importance of the mitotic checkpoint in the pathogenesis of microcephaly. Furthermore, the ependymal cell layer does not form in the conditional knockout, revealing an unrecognized role of BubR1 in assuring the integrity of the ventricular system, which may account for the presence of hydrocephalus in some patients.
Assuntos
Proteínas de Ciclo Celular/genética , Microcefalia/genética , Mitose/genética , Neurogênese/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Animais , Apoptose/genética , Proteínas de Ciclo Celular/deficiência , Proliferação de Células/genética , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/fisiopatologia , Segregação de Cromossomos/genética , Modelos Animais de Doenças , Humanos , Camundongos , Microcefalia/metabolismo , Microcefalia/fisiopatologia , Mosaicismo , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Proteínas Serina-Treonina Quinases/deficiência , Fuso Acromático/genética , Fuso Acromático/patologiaRESUMO
The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.
Assuntos
Olho/metabolismo , Olho/patologia , Deleção de Genes , Amaurose Congênita de Leber/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Adulto , Animais , Modelos Animais de Doenças , Eletrorretinografia , Humanos , Amaurose Congênita de Leber/patologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Células Fotorreceptoras de Vertebrados/metabolismo , Pigmentação , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.
Assuntos
Proliferação de Células/genética , Cerebelo/embriologia , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Núcleosídeo-Fosfato Quinase/metabolismo , Células-Tronco/citologia , Animais , Ciclo Celular/genética , Cerebelo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuroglia/citologia , Núcleosídeo-Fosfato Quinase/genética , Organogênese/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Receptor SmoothenedRESUMO
The Hippo-Yes associated protein (Yap) pathway plays an important role in organ size control by regulating cell proliferation, apoptosis, and stem cell renewal. Hippo-Yap signaling also functions at the level of cellular development in a variety of organs through its effects on cell cycle control, cell survival, cell polarity, and cell fate. Because of its important roles in normal development and homeostasis, abnormal regulation of this pathway has been shown to lead to pathological outcomes such as tissue overgrowth, tumor formation, and abnormal organogenesis, including ocular-specific disorders. In this review, we summarize how normal and perturbed control of Yap signaling is implicated in ocular development and disease Developmental Dynamics 247:794-806, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Oftalmopatias/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Polaridade Celular/genética , Polaridade Celular/fisiologia , Oftalmopatias/genética , Via de Sinalização Hippo , Humanos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Yap functions as a transcriptional regulator by acting together with sequence-specific DNA binding factors and transcription cofactors to mediate cell proliferation in developing epithelial tissues and tumors. An upstream kinase cascade controls nuclear localization and function in response to partially identified exogenous signals, including cell-to-cell contact. Nevertheless, its role in CNS development is poorly understood. In order to investigate Yap function in developing CNS, we characterized the cellular outcomes after selective Yap gene ablation in developing ocular tissues. When Yap was lost, presumptive retinal pigment epithelium acquired anatomical and molecular characteristics resembling those of the retinal epithelium rather than of RPE, including loss of pigmentation, pseudostratified epithelial morphology and ectopic induction of markers for retinal progenitor cells, like Chx10, and neurons, like ß-Tubulin III. In addition, developing retina showed signs of progressive degeneration, including laminar folding, thinning and cell loss, which resulted from multiple defects in cell proliferation and survival, and in junction integrity. Furthermore, Yap-deficient retinal progenitors displayed decreased S-phase cells and altered cell cycle progression. Altogether, our studies not only illustrate the canonical function of Yap in promoting the proliferation of progenitors, but also shed new light on its evolutionarily conserved, instructive role in regional specification, maintenance of junctional integrity and precise regulation of cell proliferation during neuroepithelial development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas do Olho/fisiologia , Olho/embriologia , Fosfoproteínas/fisiologia , Epitélio Pigmentado da Retina/citologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Adesão Celular , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Divisão Celular , Linhagem da Célula , Polaridade Celular , Transdiferenciação Celular , Olho/metabolismo , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/análise , Camundongos , Microscopia de Fluorescência , Placa Neural/citologia , Placa Neural/metabolismo , Organelas/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Retina/embriologia , Epitélio Pigmentado da Retina/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/análise , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Mutations of Crb1 gene cause irreversible and incurable visual impairment in humans. This study aims to use an LCA8-like mouse model to identify host-mediated responses that might interfere with survival, retinal integration and differentiation of grafted cells during neonatal cell therapy. METHODS: Mixed retinal donor cells (1 ~ 2 × 104) isolated from neural retinas of neonatal eGFP transgenic mice were injected into the subretinal space of LCA8-like model neonatal mice. Markers of specific cell types were used to analyze microglial attraction, CSPG induction and retinal cell differentiation. The positions of host retinal cells were traced according to their laminar location during disease progression to look for host cell rearrangements that might inhibit retinal integration of the transplanted cells. RESULTS: Transplanted retinal cells showed poor survival and attracted microglial cells, but CSPG was not greatly induced. Retinas of the LCA8 model hosts underwent significant cellular rearrangement, including rosette formation and apical displacement of inner retinal cells. CONCLUSIONS: Local disease environment, particularly host immune responses to injected cells and formation of a physical barrier caused by apical migration of host retinal cells upon disruption of outer limiting membrane, may impose two major barriers in LCAs cell transplantation therapy.
Assuntos
Transplante de Células/métodos , Retina/citologia , Degeneração Retiniana/cirurgia , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Retina/patologia , Degeneração Retiniana/patologiaRESUMO
Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, ß-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polaridade Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cristalino/embriologia , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Forma Celular/fisiologia , Primers do DNA/genética , Células Epiteliais/citologia , Imunofluorescência , Via de Sinalização Hippo , Hibridização In Situ , Cristalino/citologia , Camundongos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Proteínas de Sinalização YAP , beta-Cristalinas/metabolismoRESUMO
Mutation of the polarity gene Crumbs homolog 1 (CRB1) is responsible for >10% of Leber congenital amaurosis (LCA) cases worldwide; LCA is characterized by early-onset degenerative retinal dystrophy. The role of CRB1 in LCA8 pathogenesis remains elusive since Crb1 mouse mutants, including a null allele, have failed to mimic the early-onset of LCA, most likely due to functional compensation by closely related genes encoding Crb2 and Crb3. Crb proteins form an evolutionarily conserved, apical polarity complex with the scaffolding protein associated with lin-seven 1 (Pals1), also known as MAGUK p55 subfamily member 5 (MPP5). Pals1 and Crbs are functionally inter-dependent in establishing and maintaining epithelial polarity. Pals1 is a single gene in the mouse and human genomes; therefore, we ablated Pals1 to establish a mouse genetic model mimicking human LCA. In our study, the deletion of Pals1 leads to the disruption of the apical localization of Crb proteins in retinal progenitors and the adult retina, validating their mutual interaction. Remarkably, the Pals1 mutant mouse exhibits the critical features of LCA such as early visual impairment as assessed by electroretinogram, disorganization of lamination and apical junctions and retinal degeneration. Our data uncover the indispensible role of Pals1 in retinal development, likely involving the maintenance of retinal polarity and survival of retinal neurons, thus providing the basis for the pathologic mechanisms of LCA8.
Assuntos
Amaurose Congênita de Leber/metabolismo , Proteínas de Membrana/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Retina/metabolismo , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células , Eletrorretinografia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Hibridização In Situ , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Masculino , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas do Tecido Nervoso/metabolismo , Núcleosídeo-Fosfato Quinase/genética , Retina/embriologia , Retina/crescimento & desenvolvimento , Células-Tronco/patologia , Células-Tronco/ultraestrutura , Acuidade VisualRESUMO
Tuberous sclerosis complex (TSC) is a dominant tumor suppressor disorder caused by mutations in either TSC1 or TSC2. TSC causes substantial neuropathology, often leading to autism spectrum disorders (ASDs) in up to 60% of patients. The anatomic and neurophysiologic links between these two disorders are not well understood. We have generated and characterized a novel TSC mouse model with Purkinje cell specific Tsc2 loss. These Tsc2f/-;Cre mice exhibit progressive Purkinje cell degeneration. Since loss of Purkinje cells is a well reported postmortem finding in patients with ASD, we conducted a series of behavior tests to asses if Tsc2f/-;Cre mice displayed autistic-like deficits. Tsc2f/-;Cre mice demonstrated increased repetitive behavior as assessed with marble burying activity. Using the three chambered apparatus to asses social behavior, we found that Tsc2f/-;Cre mice showed behavioral deficits, exhibiting no preference between a stranger mouse and an inanimate object, or between a novel and a familiar mouse. We also detected social deficits in Tsc2f/f;Cre mice, suggesting that Purkinje cell pathology is sufficient to induce ASD-like behavior. Importantly, social behavior deficits were prevented with rapamycin treatment. Altogether, these results demonstrate that loss of Tsc2 in Purkinje cells in a Tsc2-haploinsufficient background leads to autistic-like behavioral deficits. These studies provide compelling evidence that Purkinje cell loss and/or dysfunction may be an important link between TSC and ASD as well as a general anatomic phenomenon that contributes to the ASD phenotype.
Assuntos
Comportamento Animal/fisiologia , Células de Purkinje/metabolismo , Esclerose Tuberosa/fisiopatologia , Proteínas Supressoras de Tumor/deficiência , Animais , Criança , Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Transtornos Globais do Desenvolvimento Infantil/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Células de Purkinje/patologia , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-ß levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-ß up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-ß-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.
Assuntos
Caderinas/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Fibrose Pulmonar/etiologia , Fator de Crescimento Transformador beta/biossíntese , Animais , Bleomicina/toxicidade , Caderinas/antagonistas & inibidores , Caderinas/deficiência , Caderinas/genética , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/fisiopatologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologiaRESUMO
Entosis is cell cannibalism utilized by tumor cells to engulf live neighboring cells for pro- or anti-tumorigenic purposes. It is unknown whether this extraordinary cellular event can be pathogenic in other diseases such as microcephaly, a condition characterized by a smaller than normal brain at birth. We find that mice mutant for the human microcephaly-causing gene Pals1, which exhibit diminished cortices due to massive cell death, also exhibit nuclei enveloped by plasma membranes inside of dividing cells. These cell-in-cell (CIC) structures represent a dynamic process accompanied by lengthened mitosis and cytokinesis abnormalities. As shown in tumor cells, ROCK inhibition completely abrogates CIC structures and restores the normal length of mitosis. Moreover, genetic elimination of Trp53 produces a remarkable rescue of cortical size along with substantial reductions of CIC structures and cell death. These results provide a novel pathogenic mechanism by which microcephaly is produced through entotic cell cannibalism.
Assuntos
Microcefalia , Humanos , Animais , Camundongos , Microcefalia/genética , Entose/fisiologia , Carcinogênese , Mitose/genética , Núcleo CelularRESUMO
Specification of the peripheral optic cup by Wnt signaling is critical for formation of the ciliary body/iris. Identification of marker genes for this region during development provides a starting point for functional analyses. During transcriptional profiling of single cells from the developing eye, two cells were identified that expressed genes not found in most other single cell profiles. In situ hybridizations demonstrated that many of these genes were expressed in the peripheral optic cup in both early mouse and chicken development, and in the ciliary body/iris at subsequent developmental stages. These analyses indicate that the two cells probably originated from the developing ciliary body/iris. Changes in expression of these genes were assayed in embryonic chicken retinas when canonical Wnt signaling was ectopically activated by CA-beta-catenin. Twelve ciliary body/iris genes were identified as upregulated following induction, suggesting they are excellent candidates for downstream effectors of Wnt signaling in the optic cup.
Assuntos
Corpo Ciliar/metabolismo , Iris/metabolismo , Animais , Embrião de Galinha , Corpo Ciliar/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Iris/embriologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Animais , Bromodesoxiuridina/farmacologia , Linhagem da Célula , Mapeamento Cromossômico , Análise por Conglomerados , Biologia Computacional , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Hibridização In Situ , Interneurônios/metabolismo , Camundongos , Mitose , Dados de Sequência Molecular , Neuroglia/citologia , Neuroglia/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Retina/metabolismo , Células-Tronco/citologia , Fatores de TempoRESUMO
Nuclear exclusion of the transcriptional regulators and potent oncoproteins, YAP/TAZ, is considered necessary for adult tissue homeostasis. Here we show that nuclear YAP/TAZ are essential regulators of peripheral nerve development and myelin maintenance. To proliferate, developing Schwann cells (SCs) require YAP/TAZ to enter S-phase and, without them, fail to generate sufficient SCs for timely axon sorting. To differentiate, SCs require YAP/TAZ to upregulate Krox20 and, without them, completely fail to myelinate, resulting in severe peripheral neuropathy. Remarkably, in adulthood, nuclear YAP/TAZ are selectively expressed by myelinating SCs, and conditional ablation results in severe peripheral demyelination and mouse death. YAP/TAZ regulate both developmental and adult myelination by driving TEAD1 to activate Krox20. Therefore, YAP/TAZ are crucial for SCs to myelinate developing nerve and to maintain myelinated nerve in adulthood. Our study also provides a new insight into the role of nuclear YAP/TAZ in homeostatic maintenance of an adult tissue.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bainha de Mielina/metabolismo , Fosfoproteínas/metabolismo , Células de Schwann/fisiologia , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Camundongos , Proteínas de Sinalização YAPRESUMO
Timely generation and normal maturation of ependymal cells along the aqueduct are critical for preventing physical blockage between the third and fourth ventricles and the development of fetal non-communicating hydrocephalus. Our study identifies Yap, the downstream effector of the evolutionarily conserved Hippo pathway, as a central regulator for generating developmentally controlled ependymal cells along the ventricular lining of the aqueduct. Yap function is necessary for proper proliferation of progenitors and apical attachment of ependymal precursor cells. Importantly, an injury signal initiated by lysophosphatidic acid (LPA), an upstream regulator of Yap that can cause fetal haemorrhagic hydrocephalus, deregulates Yap in the developing aqueduct. LPA exposure leads to the loss of N-cadherin concentrations at the apical endfeet, which can be partially restored by forced Yap expression and more efficiently by phosphomimetic Yap. These results reveal a novel function of Yap in retaining tissue junctions during normal development and after fetal brain injury.