RESUMEN
Glioblastoma (GBM) is a lethal brain cancer with no effective treatment; understanding how GBM cells respond to tumor microenvironment remains challenging as conventional cell cultures lack proper cytoarchitecture while in vivo animal models present complexity all at once. Developing a culture system to bridge the gap is thus crucial. Here, we employed a multicellular approach using human glia and vascular cells to optimize a 3-dimensional (3D) brain vascular niche model that enabled not only long-term culture of patient derived GBM cells but also recapitulation of key features of GBM heterogeneity, in particular invasion behavior and vascular association. Comparative transcriptomics of identical patient derived GBM cells in 3D and in vivo xenotransplants models revealed that glia-vascular contact induced genes concerning neural/glia development, synaptic regulation, as well as immune suppression. This gene signature displayed region specific enrichment in the leading edge and microvascular proliferation zones in human GBM and predicted poor prognosis. Gene variance analysis also uncovered histone demethylation and xylosyltransferase activity as main themes for gene adaption of GBM cells in vivo . Furthermore, our 3D model also demonstrated the capacity to provide a quiescence and a protective niche against chemotherapy. In summary, an advanced 3D brain vascular model can bridge the gap between 2D cultures and in vivo models in capturing key features of GBM heterogeneity and unveil previously unrecognized influence of glia-vascular contact for transcriptional adaption in GBM cells featuring neural/synaptic interaction and immunosuppression.
RESUMEN
Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioma/patología , Glioblastoma/patología , Hipoxia/genética , Hipoxia/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Células Madre Neoplásicas/metabolismo , Hipoxia de la Célula/genéticaRESUMEN
During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.
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Actomiosina/metabolismo , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Madre/metabolismo , Actinas , Sistemas CRISPR-Cas , Diferenciación Celular , Uniones Célula-Matriz/metabolismo , Células Madre Embrionarias , Edición Génica , Expresión Génica , Humanos , Mecanotransducción Celular , Morfogénesis , Células-Madre Neurales , Semaforinas , Transducción de SeñalRESUMEN
The Olig genes encode members of the basic helix-loop-helix (bHLH) family of transcription factors. Olig1, Olig2, and Olig3 are expressed in both the developing and mature central nervous system (CNS) and strictly regulate cellular specification and differentiation. Extensive studies have established functional roles of Olig1 and Olig2 in directing neuronal and glial formation during different stages in development. Recently, Olig2 overexpression was implicated in neurodevelopmental disorders down syndrome (DS) and autism spectrum disorder (ASD) but its influence on cognitive and intellectual defects remains unknown. In this review, we summarize the biological functions of the Olig family and how it uniquely promotes cellular diversity in the CNS. This is followed up with a discussion on how abnormal Olig2 expression impacts brain development and function in DS and ASD. Collectively, the studies described here emphasize vital features of the Olig members and their distinctive potential roles in neurodevelopmental disease states.
RESUMEN
The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia (TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.
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Neoplasias Cerebelosas/prevención & control , Modelos Animales de Enfermedad , Proteínas Hedgehog/fisiología , Meduloblastoma/prevención & control , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Macrófagos Asociados a Tumores/inmunología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proliferación Celular , Neoplasias Cerebelosas/etiología , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Femenino , Humanos , Masculino , Meduloblastoma/etiología , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.
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Coroides/inmunología , Coroides/patología , Endotelio/inmunología , Inmunomodulación , Análisis de la Célula Individual , Animales , Proliferación Celular , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Hedgehog/metabolismo , Inflamación/genética , Mastocitos/metabolismo , Melanocitos/metabolismo , Melanocitos/patología , Ratones Endogámicos C57BL , Especificidad de Órganos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Transcripción Genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
PURPOSE: Genetically engineered mouse models of sporadic cancers are critical for studying tumor biology and for preclinical testing of therapeutics. We present an MRI-based pipeline designed to produce high resolution, quantitative information about tumor progression and response to novel therapies in mouse models of medulloblastoma (MB). METHODS: Sporadic MB was modeled in mice by inducing expression of an activated form of the Smoothened gene (aSmo) in a small number of cerebellar granule cell precursors. aSmo mice were imaged and analyzed at defined time-points using a 3D manganese-enhanced MRI-based pipeline optimized for high-throughput. RESULTS: A semi-automated segmentation protocol was established that estimates tumor volume in a time-frame compatible with a high-throughput pipeline. Both an empirical, volume-based classifier and a linear discriminant analysis-based classifier were tested to distinguish progressing from nonprogressing lesions at early stages of tumorigenesis. Tumor centroids measured at early stages revealed that there is a very specific location of the probable origin of the aSmo MB tumors. The efficacy of the manganese-enhanced MRI pipeline was demonstrated with a small-scale experimental drug trial designed to reduce the number of tumor associated macrophages and microglia. CONCLUSION: Our results revealed a high level of heterogeneity between tumors within and between aSmo MB models, indicating that meaningful studies of sporadic tumor progression and response to therapy could not be conducted without an imaging-based pipeline approach.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética , Meduloblastoma/diagnóstico por imagen , Algoritmos , Animales , Cerebelo/metabolismo , Análisis Discriminante , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Imagenología Tridimensional , Modelos Lineales , Ratones , Reconocimiento de Normas Patrones Automatizadas , Transducción de Señal , Receptor Smoothened/genéticaRESUMEN
For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.
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Proliferación Celular , Corteza Cerebelosa/crecimiento & desarrollo , Núcleos Cerebelosos/citología , Células de Purkinje/fisiología , Animales , Técnicas de Inactivación de Genes , Proteínas de Homeodominio/genética , Ratones , Proteínas del Tejido Nervioso/deficienciaRESUMEN
BACKGROUND: The cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known. METHODS: We used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) Our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atoh1-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion. RESULTS: Mosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved. CONCLUSION: We demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of Atoh1-Cre conditional mutants affecting GCPs.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cerebelo/fisiología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Células Precursoras de Granulocitos/metabolismo , Proteínas Hedgehog/metabolismo , Regeneración Nerviosa/fisiología , Nestina/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Transducción de Señal/fisiología , Proteína Gli2 con Dedos de Zinc/fisiología , Animales , Animales Recién Nacidos , Cerebelo/crecimiento & desarrollo , Embrión de Mamíferos , Ratones , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Proteína Gli2 con Dedos de Zinc/genéticaRESUMEN
Outside of the neurogenic niches of the brain, postmitotic neurons have not been found to undergo efficient regeneration. We demonstrate that mouse Purkinje cells (PCs), which are born at midgestation and are crucial for development and function of cerebellar circuits, are rapidly and fully regenerated following their ablation at birth. New PCs are produced from immature FOXP2+ Purkinje cell precursors (iPCs) that are able to enter the cell cycle and support normal cerebellum development. The number of iPCs and their regenerative capacity, however, diminish soon after birth and consequently PCs are poorly replenished when ablated at postnatal day five. Nevertheless, the PC-depleted cerebella reach a normal size by increasing cell size, but scaling of neuron types is disrupted and cerebellar function is impaired. Our findings provide a new paradigm in the field of neuron regeneration by identifying a population of immature neurons that buffers against perinatal brain injury in a stage-dependent process.
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Proliferación Celular , Cerebelo/crecimiento & desarrollo , Cerebelo/lesiones , Células de Purkinje/fisiología , Regeneración , Células Madre/fisiología , Factores de Edad , Animales , RatonesRESUMEN
The main cell of origin of the Sonic hedgehog (SHH) subgroup of medulloblastoma (MB) is granule cell precursors (GCPs), a SHH-dependent transient amplifying population in the developing cerebellum. SHH-MBs can be further subdivided based on molecular and clinical parameters, as well as location because SHH-MBs occur preferentially in the lateral cerebellum (hemispheres). Our analysis of adult patient data suggests that tumors with Smoothened (SMO) mutations form more specifically in the hemispheres than those with Patched 1 (PTCH1) mutations. Using sporadic mouse models of SHH-MB with the two mutations commonly seen in adult MB, constitutive activation of Smo (SmoM2) or loss-of-Ptch1, we found that regardless of timing of induction or type of mutation, tumors developed primarily in the hemispheres, with SmoM2-mutants indeed showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high-level SHH signaling compared with GCPs in the medial cerebellum (vermis), as more SmoM2 or Ptch1-mutant hemisphere cells remain undifferentiated and show increased tumorigenicity when transplanted. Finally, we identified location-specific GCP gene-expression profiles, and found that deletion of the genes most highly expressed in the hemispheres (Nr2f2) or vermis (Engrailed1) showed opposing effects on GCP differentiation. Our studies thus provide insights into intrinsic differences within GCPs that impact on SHH-MB progression.
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Neoplasias Cerebelosas/patología , Cerebelo/patología , Proteínas Hedgehog/metabolismo , Meduloblastoma/patología , Receptor Patched-1/metabolismo , Receptor Smoothened/metabolismo , Adulto , Animales , Diferenciación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Cerebelo/metabolismo , Proteínas Hedgehog/genética , Humanos , Lactante , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Receptor Patched-1/genética , Transducción de Señal , Receptor Smoothened/genética , TranscriptomaRESUMEN
Regeneration of several organs involves adaptive reprogramming of progenitors, but the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. Here we found that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors, specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog signaling in two Nestin-expressing progenitor populations is crucial not only for the compensatory replenishment of granule neurons but also for scaling interneuron and astrocyte numbers. Thus, we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration.
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Cerebelo/fisiología , Nestina/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Astrocitos/fisiología , Cerebelo/efectos de la radiación , Femenino , Proteínas Hedgehog/fisiología , Interneuronas/fisiología , Masculino , Ratones , Ratones Transgénicos , Nestina/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/efectos de la radiaciónRESUMEN
Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 µm in 2 hours) and high-throughput (150 µm in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm(3). Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.
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Neoplasias Cerebelosas/diagnóstico , Cloruros , Medios de Contraste , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética/métodos , Compuestos de Manganeso , Meduloblastoma/diagnóstico , Animales , Progresión de la Enfermedad , Imagenología Tridimensional , Ratones , Ratones Noqueados , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/genéticaRESUMEN
Sulfs are secreted sulfatases that catalyse removal of sulfate from Heparan Sulfate Proteoglycans (HSPGs) in the extracellular space. These enzymes are well known to regulate a number of crucial signalling pathways during development. In this study, we report that DSulfatase-1 (DSulf1), the unique Drosophila Sulf protein, is a regulator of Hedgehog (Hh) signalling during wing development. DSulf1 activity is required in both Hh source and Hh receiving cells for proper positioning of Hh target gene expression boundaries. As assessed by loss- and gain-of-function experiments in specific compartments, DSulf1 displays dual functions with respect to Hh signalling, acting as a positive regulator in Hh producing cells and a negative regulator in Hh receiving cells. In either domain, DSulf1 modulates Hh distribution by locally lowering the concentration of the morphogen at the apical pole of wing disc cells. Thus, we propose that DSulf1, by its desulfation catalytic activity, lowers Hh/HSPG interaction in both Hh source and target fields, thereby enhancing Hh release from its source of production and reducing Hh signalling activity in responding cells. Finally, we show that Dsulf1 pattern of expression is temporally regulated and depends on EGFR signalling, a Hh-dependent secondary signal in this tissue. Our data reveal a novel Hh regulatory feedback loop, involving DSulf1, which contributes to maintain and stabilise expression domains of Hh target genes during wing disc development.
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Tipificación del Cuerpo/fisiología , Proteínas de Drosophila/metabolismo , Retroalimentación Fisiológica/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Sulfatasas/metabolismo , Sulfotransferasas/metabolismo , Alas de Animales/crecimiento & desarrollo , Animales , Drosophila , Receptores ErbB/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Inmunohistoquímica , Hibridación in SituRESUMEN
Spatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation.