RESUMEN
The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.
Asunto(s)
Centrosoma , Células-Madre Neurales , Neurogénesis , Neuronas , Heterotopia Nodular Periventricular , Mapas de Interacción de Proteínas , Empalme Alternativo , Animales , Encéfalo/anomalías , Centrosoma/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Microtúbulos/metabolismo , Neuronas/metabolismo , Heterotopia Nodular Periventricular/metabolismo , Proteoma/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Asunto(s)
Centrosoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Microtúbulos/metabolismo , Neurogénesis , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Uniones Intercelulares/metabolismo , Interfase , Ventrículos Laterales/anatomía & histología , Glándulas Mamarias Animales/citología , Ratones , Tamaño de los Órganos , Organoides/citologíaRESUMEN
Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Proteoma/análisis , Proteómica , Virus Zika/patogenicidad , Animales , Diferenciación Celular , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno/genética , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas , Proteoma/genética , Proteoma/metabolismo , ARN Interferente Pequeño/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virus Zika/genética , Virus Zika/metabolismoRESUMEN
BACKGROUND: Precise spatiotemporal control of gene expression is essential for the establishment of correct cell numbers and identities during brain development. This process involves epigenetic control mechanisms, such as those mediated by the polycomb group protein Ezh2, which catalyzes trimethylation of histone H3K27 (H3K27me3) and thereby represses gene expression. RESULTS: Herein, we show that Ezh2 plays a crucial role in the development and maintenance of the midbrain. Conditional deletion of Ezh2 in the developing midbrain resulted in decreased neural progenitor proliferation, which is associated with derepression of cell cycle inhibitors and negative regulation of Wnt/ß-catenin signaling. Of note, Ezh2 ablation also promoted ectopic expression of a forebrain transcriptional program involving derepression of the forebrain determinants Foxg1 and Pax6. This was accompanied by reduced expression of midbrain markers, including Pax3 and Pax7, as a consequence of decreased Wnt/ß-catenin signaling. CONCLUSION: Ezh2 is required for appropriate brain growth and maintenance of regional identity by H3K27me3-mediated gene repression and control of canonical Wnt signaling.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/crecimiento & desarrollo , Complejo Represivo Polycomb 2/genética , Prosencéfalo/crecimiento & desarrollo , Vía de Señalización Wnt , Animales , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Mesencéfalo/embriología , Ratones , Complejo Represivo Polycomb 2/metabolismo , Prosencéfalo/embriologíaRESUMEN
The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. ß-Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of ß-catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel ß-catenin mutant allele with conditional inactivation approaches. Wnt/ß-catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/ß-catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper-layer neurons and later, to precocious astrogenesis. Indeed, a genome-wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/ß-catenin signaling-ablated cortex. Thus, ß-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage-specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain.
Asunto(s)
Corteza Cerebral/citología , Células-Madre Neurales/citología , Neuronas/citología , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Corteza Cerebral/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Transducción de SeñalRESUMEN
The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is suppressed in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. Here we demonstrate that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. Our data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program.
Asunto(s)
Cartílago/embriología , Condrogénesis/fisiología , Epigénesis Genética/fisiología , Huesos Faciales/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Cresta Neural/fisiología , Osteogénesis/fisiología , Complejo Represivo Polycomb 2/metabolismo , Azul Alcián , Animales , Antraquinonas , Inmunoprecipitación de Cromatina , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Citometría de Flujo , Galactósidos , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/metabolismo , Inmunohistoquímica , Indoles , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Cresta Neural/metabolismo , Complejo Represivo Polycomb 2/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/ß-catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/ß-catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/ß-catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/ß-catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the ß-catenin-activated Wnt-reporter mouse and by pharmacological stimulation of Wnt/ß-catenin by infusion of the specific glycogen synthase kinase 3ß inhibitor, AR-A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/ß-catenin, respectively, increased and decreased the differentiation of dSVZ-NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/ß-catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/ß-catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/ß-catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs.
Asunto(s)
Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Proteína Wnt3/metabolismo , beta Catenina/metabolismo , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Ácido Glutámico/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ventrículos Laterales/citología , Ratones Transgénicos , Microscopía Confocal , Células-Madre Neurales/citología , Neuronas/citología , Oligodendroglía/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiazoles/farmacología , Urea/análogos & derivados , Urea/farmacología , Proteína Wnt3/genética , beta Catenina/genéticaRESUMEN
ß-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous ß-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant ß-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/ß-catenin signaling in dorsal neural tube development. While loss of ß-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/ß-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of ß-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , beta Catenina/genética , beta Catenina/metabolismo , Uniones Adherentes/genética , Animales , Células Epiteliales/citología , Células Epiteliales/patología , Gastrulación/genética , Ratones , Ratones Endogámicos , Mutación , Transducción de Señal/genética , Médula Espinal/citología , Médula Espinal/embriología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.