RESUMEN
Cell commitment to tumourigenesis and the onset of uncontrolled growth are critical determinants in cancer development but the early events directing tumour initiating cell (TIC) fate remain unclear. We reveal a single-cell transcriptome profile of brain TICs transitioning into tumour growth using the brain tumour (brat) neural stem cell-based Drosophila model. Prominent changes in metabolic and proteostasis-associated processes including ribogenesis are identified. Increased ribogenesis is a known cell adaptation in established tumours. Here we propose that brain TICs boost ribogenesis prior to tumour growth. In brat-deficient TICs, we show that this dramatic change is mediated by upregulated HEAT-Repeat Containing 1 (HEATR1) to promote ribosomal RNA generation, TIC enlargement and onset of overgrowth. High HEATR1 expression correlates with poor glioma patient survival and patient-derived glioblastoma stem cells rely on HEATR1 for enhanced ribogenesis and tumourigenic potential. Finally, we show that HEATR1 binds the master growth regulator MYC, promotes its nucleolar localisation and appears required for MYC-driven ribogenesis, suggesting a mechanism co-opted in ribogenesis reprogramming during early brain TIC development.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Antígenos de Histocompatibilidad Menor , Proteínas Proto-Oncogénicas c-myc , Proteínas de Unión al ARN , Animales , Humanos , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Carcinogénesis/patología , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glioblastoma/metabolismo , Glioma/patología , Antígenos de Histocompatibilidad Menor/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. beta1 integrins regulate the myelination of peripheral nerves, but their function during the myelination of axonal tracts in the CNS is unclear. Here we show that genetically modified mice lacking beta1 integrins in the CNS present a deficit in myelination but no defects in the development of the oligodendroglial lineage. Instead, in vitro data show that beta1 integrins regulate the outgrowth of myelin sheaths. Oligodendrocytes derived from mutant mice are unable to efficiently extend myelin sheets and fail to activate AKT (also known as AKT1), a kinase that is crucial for axonal ensheathment. The inhibition of PTEN, a negative regulator of AKT, or the expression of a constitutively active form of AKT restores myelin outgrowth in cultured beta1-deficient oligodendrocytes. Our data suggest that beta1 integrins play an instructive role in CNS myelination by promoting myelin wrapping in a process that depends on AKT.
Asunto(s)
Sistema Nervioso Central/metabolismo , Integrina beta1/metabolismo , Vaina de Mielina/metabolismo , Animales , Linaje de la Célula , Células Cultivadas , Integrina beta1/genética , Ratones , Ratones Noqueados , Vaina de Mielina/patología , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Oligodendroglía/citología , Oligodendroglía/fisiología , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Neuregulin-1 (NRG1) and its ErbB2/B4 receptors are encoded by candidate susceptibility genes for schizophrenia, yet the essential functions of NRG1 signaling in the CNS are still unclear. Using CRE/LOX technology, we have inactivated ErbB2/B4-mediated NRG1 signaling specifically in the CNS. In contrast to expectations, cell layers in the cerebral cortex, hippocampus, and cerebellum develop normally in the mutant mice. Instead, loss of ErbB2/B4 impairs dendritic spine maturation and perturbs interactions of postsynaptic scaffold proteins with glutamate receptors. Conversely, increased NRG1 levels promote spine maturation. ErbB2/B4-deficient mice show increased aggression and reduced prepulse inhibition. Treatment with the antipsychotic drug clozapine reverses the behavioral and spine defects. We conclude that ErbB2/B4-mediated NRG1 signaling modulates dendritic spine maturation, and that defects at glutamatergic synapses likely contribute to the behavioral abnormalities in ErbB2/B4-deficient mice.
Asunto(s)
Corteza Cerebral/citología , Espinas Dendríticas/patología , Proteínas del Tejido Nervioso/fisiología , Receptor ErbB-2/fisiología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Antipsicóticos/farmacología , Sistema Nervioso Central , Clozapina/farmacología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Neurregulina-1 , Proteínas Oncogénicas v-erbB/deficiencia , Proteínas Oncogénicas v-erbB/fisiología , Receptores de GlutamatoRESUMEN
Here, we describe a protocol to remove single identified cells directly from Drosophila living brains and analyze their transcriptome. We detail the steps to harvest fluorescent cells using a capillary under epifluorescence and transmitted light to avoid contamination. We then outline the procedure to obtain the transcriptome by reverse transcription and amplification. The process from cell harvesting to the initiation of reverse transcription only takes 2 min, thus avoiding transcriptional activation of cell damage response or cell death genes. For complete details on the use and execution of this protocol, please refer to Barros and Bossing (2021), Bossing et al. (2012), Gil-Ranedo et al. (2019), and Liu and Bossing (2016).
Asunto(s)
Drosophila melanogaster , Perfilación de la Expresión Génica , Animales , Drosophila , Encéfalo , TranscriptomaRESUMEN
Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-ß activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.
Asunto(s)
Sistema Nervioso Central/patología , Drosophila melanogaster/metabolismo , Microtúbulos/patología , Mitosis , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Muerte Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Desarrollo Embrionario , Humanos , Microtúbulos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas tau/metabolismoRESUMEN
Radial glial cells (RGCs) in the developing cerebral cortex are progenitors for neurons and glia, and their processes serve as guideposts for migrating neurons. So far, it has remained unclear whether RGC processes also control the function of RGCs more directly. Here, we show that RGC numbers and cortical size are reduced in mice lacking beta1 integrins in RGCs. TUNEL stainings and time-lapse video recordings demonstrate that beta1-deficient RGCs processes detach from the meningeal basement membrane (BM) followed by apoptotic death of RGCs. Apoptosis is also induced by surgical removal of the meninges. Finally, mice lacking the BM components laminin alpha2 and alpha4 show defects in the attachment of RGC processes at the meninges, a reduction in cortical size, and enhanced apoptosis of RGC cells. Our findings demonstrate that attachment of RGC processes at the meninges is important for RGC survival and the control of cortical size.
Asunto(s)
Corteza Cerebral/anomalías , Meninges/fisiología , Neuroglía/fisiología , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Corteza Cerebral/crecimiento & desarrollo , Electroporación/métodos , Embrión de Mamíferos , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/metabolismo , Etiquetado Corte-Fin in Situ/métodos , Técnicas In Vitro , Integrasas/genética , Integrina beta1/genética , Proteínas de Filamentos Intermediarios/deficiencia , Laminina/genética , Meninges/citología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Nestina , Neurogénesis/fisiología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Células Madre/metabolismoRESUMEN
Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.
Asunto(s)
Drosophila/embriología , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Neuronas/fisiología , Células Madre/fisiología , Animales , Animales Modificados Genéticamente , Transporte Biológico Activo/fisiología , Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula/fisiología , Polaridad Celular/fisiología , Proteínas de Drosophila/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Masculino , Neuronas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/fisiología , Células Madre/citología , Proteínas Supresoras de Tumor/metabolismo , Quinasas Asociadas a rhoRESUMEN
Adult stem cells reactivate from quiescence to maintain tissue homeostasis and in response to injury. How the underlying regulatory signals are integrated is largely unknown. Drosophila neural stem cells (NSCs) also leave quiescence to generate adult neurons and glia, a process that is dependent on Hippo signaling inhibition and activation of the insulin-like receptor (InR)/PI3K/Akt cascade. We performed a transcriptome analysis of individual quiescent and reactivating NSCs harvested directly from Drosophila brains and identified the conserved STRIPAK complex members mob4, cka, and PP2A (microtubule star, mts). We show that PP2A/Mts phosphatase, with its regulatory subunit Widerborst, maintains NSC quiescence, preventing premature activation of InR/PI3K/Akt signaling. Conversely, an increase in Mob4 and Cka levels promotes NSC reactivation. Mob4 and Cka are essential to recruit PP2A/Mts into a complex with Hippo kinase, resulting in Hippo pathway inhibition. We propose that Mob4/Cka/Mts functions as an intrinsic molecular switch coordinating Hippo and InR/PI3K/Akt pathways and enabling NSC reactivation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Insulina/metabolismo , Transcriptoma/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/metabolismo , Proliferación Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis/genética , Proteínas del Tejido Nervioso/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Análisis de la Célula IndividualRESUMEN
The analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species-most notably Drosophila-neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4-UASG. One limitation of Gal4-UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system's key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 ('Zal1') was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1-UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Red Nerviosa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Multimerización de Proteína , Neuronas Serotoninérgicas/metabolismoRESUMEN
Stem cells control their mitotic activity to decide whether to proliferate or to stay in quiescence. Drosophila neural stem cells (NSCs) are quiescent at early larval stages, when they are reactivated in response to metabolic changes. Here we report that cell-contact inhibition of growth through the canonical Hippo signalling pathway maintains NSC quiescence. Loss of the core kinases hippo or warts leads to premature nuclear localization of the transcriptional co-activator Yorkie and initiation of growth and proliferation in NSCs. Yorkie is necessary and sufficient for NSC reactivation, growth and proliferation. The Hippo pathway activity is modulated via inter-cellular transmembrane proteins Crumbs and Echinoid that are both expressed in a nutrient-dependent way in niche glial cells and NSCs. Loss of crumbs or echinoid in the niche only is sufficient to reactivate NSCs. Finally, we provide evidence that the Hippo pathway activity discriminates quiescent from non-quiescent NSCs in the Drosophila nervous system.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/citología , Drosophila/metabolismo , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas de Drosophila/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transporte de ProteínasRESUMEN
Mechanisms of CNS repair have vital medical implications. We show that traumatic injury to the ventral midline of the embryonic Drosophila CNS activates cell divisions to replace lost cells. A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra). Ectopic Miro expression can prevent midline divisions after damage, whereas Miro depletion destabilizes cortical ß-tubulin and increases divisions. Disruption of cortical microtubules, either by chemical depolymerization or by overexpression of monomeric tubulin, triggers ectopic mitosis in the midline and induces Jra expression. Conversely, loss of Jra renders midline cells unable to replace damaged siblings. Our data indicate that upon injury, the integrity of the microtubule cytoskeleton controls cell division in the CNS midline, triggering extra mitosis to replace lost cells. The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates.
Asunto(s)
Drosophila melanogaster/embriología , Microtúbulos , Mitosis , Animales , Diferenciación Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Activación Enzimática , Microtúbulos/química , Microtúbulos/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Tubulina (Proteína)/química , Proteínas de Unión al GTP rho/metabolismoRESUMEN
An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.