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Here, we have developed an automated image processing algorithm for segmenting lungs and individual lung tumors in in vivo micro-computed tomography (micro-CT) scans of mouse models of non-small cell lung cancer and lung fibrosis. Over 3000 scans acquired across multiple studies were used to train/validate a 3D U-net lung segmentation model and a Support Vector Machine (SVM) classifier to segment individual lung tumors. The U-net lung segmentation algorithm can be used to estimate changes in soft tissue volume within lungs (primarily tumors and blood vessels), whereas the trained SVM is able to discriminate between tumors and blood vessels and identify individual tumors. The trained segmentation algorithms (1) significantly reduce time required for lung and tumor segmentation, (2) reduce bias and error associated with manual image segmentation, and (3) facilitate identification of individual lung tumors and objective assessment of changes in lung and individual tumor volumes under different experimental conditions.
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KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
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Carcinogénesis/metabolismo , Dimerización , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas ras/genéticaRESUMEN
The utilization of suitable mouse models is a critical step in the drug discovery oncology workflow as their generation and use are important for target identification and validation as well as toxicity and efficacy assessments. Current murine models have been instrumental in furthering insights into the mode of action of drugs before transitioning into the clinic. Recent advancements in genome editing with the development of the CRISPR/Cas9 system and the possibility of applying such technology directly in vivo have expanded the toolkit of preclinical models available. In this review, a brief presentation of the current models used in drug discovery will be provided with a particular emphasis on the novel CRISPR/Cas9 models.
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PURPOSE: Lung adenocarcinomas comprise the largest fraction of non-small cell lung cancer, which is the leading cause of cancer-related deaths. Seventy-five percent of adenocarcinomas lack targeted therapies because of scarcity of druggable drivers. Here, we classified tumors on the basis of signaling similarities and discovered subgroups within this unmet patient population. EXPERIMENTAL DESIGN: We leveraged transcriptional data from >800 early- and advanced-stage patients. RESULTS: We identified three robust subtypes dubbed mucinous, proliferative, and mesenchymal with respective pathway phenotypes. These transcriptional states lack discrete and causative mutational etiology as evidenced by similarly distributed oncogenic drivers, including KRAS and EGFR. The subtypes capture heterogeneity even among tumors lacking known oncogenic drivers. Paired multi-regional intratumoral biopsies demonstrated unified subtypes despite divergently evolved prooncogenic mutations, indicating subtype stability during selective pressure. Heterogeneity among in vitro and in vivo preclinical models is expounded by the human lung adenocarcinoma subtypes and can be leveraged to discover subtype-specific vulnerabilities. As proof of concept, we identified differential subtype response to MEK pathway inhibition in a chemical library screen of 89 lung cancer cell lines, which reproduces across model systems and a clinical trial. CONCLUSIONS: Our findings support forward translational relevance of transcriptional subtypes, where further exploration therein may improve lung adenocarcinoma treatment.See related commentary by Skoulidis, p. 913.
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Adenocarcinoma del Pulmón/tratamiento farmacológico , Biomarcadores de Tumor/genética , Neoplasias Pulmonares/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenocarcinoma del Pulmón/diagnóstico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Conjuntos de Datos como Asunto , Femenino , Heterogeneidad Genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Estadificación de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , RNA-Seq , Transcriptoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings-while capturing dynamics within and crosstalk between all relevant tumor compartments-provides a unique opportunity to better design and sequence appropriate clinical interventions.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Matriz Extracelular/patología , Melanoma/tratamiento farmacológico , Melanoma/patología , Animales , Azetidinas/farmacología , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , Resistencia a Antineoplásicos/fisiología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Melanoma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Piperidinas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Vemurafenib/farmacología , Secuenciación del ExomaRESUMEN
Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness.Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images.Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy-based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment.Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455-67. ©2018 AACR.
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Recuento de Células , Diagnóstico por Imagen de Elasticidad , Matriz Extracelular/patología , Melanoma Experimental/diagnóstico por imagen , Animales , Línea Celular Tumoral , Colágeno/metabolismo , Colágeno/ultraestructura , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Humanos , Ácido Hialurónico/ultraestructura , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Microambiente Tumoral/genéticaRESUMEN
KRAS mutant tumors are largely recalcitrant to targeted therapies. Genetically engineered mouse models (GEMMs) of Kras mutant cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through comprehensive profiling of exomes and matched transcriptomes of >200 KrasG12D-initiated GEMM tumors from one lung and two pancreatic cancer models, we discover that significant intratumoral and intertumoral genomic heterogeneity evolves during tumorigenesis. Known oncogenes and tumor suppressor genes, beyond those engineered, are mutated, amplified, and deleted. Unlike human tumors, the GEMM genomic landscapes are dominated by copy number alterations, while protein-altering mutations are rare. However, interspecies comparative analyses of the genomic landscapes demonstrate fidelity between genes altered in KRAS mutant human and murine tumors. Genes that are spontaneously altered during murine tumorigenesis are also among the most prevalent found in human indications. Using targeted therapies, we also demonstrate that this inherent tumor heterogeneity can be exploited preclinically to discover cancer-specific and genotype-specific therapeutic vulnerabilities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness advantage of Kras-mutant cells. These data reveal previously unknown genomic diversity among KrasG12D-initiated GEMM tumors, places them in context of human patients, and demonstrates how to exploit this inherent tumor heterogeneity to discover therapeutic vulnerabilities.
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Genes ras , Heterogeneidad Genética , Neoplasias/genética , Neoplasias/patología , Alelos , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genómica/métodos , Humanos , Neoplasias Pulmonares/genética , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Neoplasias/metabolismo , Neoplasias/mortalidad , Pronóstico , Selección Genética , TranscriptomaRESUMEN
The adult prostate possesses a significant regenerative capacity that is of great interest for understanding adult stem cell biology. We demonstrate that leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is expressed in a rare population of prostate epithelial progenitor cells, and a castration-resistant Lgr5(+) population exists in regressed prostate tissue. Genetic lineage tracing revealed that Lgr5(+) cells and their progeny are primarily luminal. Lgr5(+) castration-resistant cells are long lived and upon regeneration, both luminal Lgr5(+) cells and basal Lgr5(+) cells expand. Moreover, single Lgr5(+) cells can generate multilineage prostatic structures in renal transplantation assays. Additionally, Lgr5(+) cell depletion revealed that the regenerative potential of the castrated adult prostate depends on Lgr5(+) cells. Together, these data reveal insights into the cellular hierarchy of castration-resistant Lgr5+ cells, indicate a requirement for Lgr5(+) cells during prostatic regeneration, and identify an Lgr5(+) adult stem cell population in the prostate.
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Próstata/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Andrógenos/farmacología , Animales , Linaje de la Célula , Células Cultivadas , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Orquiectomía , Próstata/citología , Próstata/patología , Ratas , Ratas Sprague-Dawley , Regeneración/efectos de los fármacos , Células Madre/citología , Testosterona/farmacologíaRESUMEN
Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non-small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner.
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Mice lacking the Dlx1 and Dlx2 homeobox genes (Dlx1/2 mutants) have severe deficits in subpallial differentiation, including overexpression of the Gsx1 and Gsx2 homeobox genes. To investigate whether Gsx overexpression contributes to the Dlx1/2 mutant phenotypes, we made compound loss-of-function mutants. Eliminating Gsx2 function from the Dlx1/2 mutants rescued the increased expression of Ascl1 and Hes5 (Notch signaling mediators) and Olig2 (oligodendrogenesis mediator). In addition, Dlx1/2;Gsx2 mutants, like Dlx1/2;Ascl1 mutants, exacerbated the Gsx2 and Dlx1/2 patterning and differentiation phenotypes, particularly in the lateral ganglionic eminence (LGE) caudal ganglionic eminence (CGE), and septum, including loss of GAD1 expression. On the other hand, eliminating Gsx1 function from the Dlx1/2 mutants (Dlx1/2;Gsx1 mutants) did not severely exacerbate their phenotype; on the contrary, it resulted in a partial rescue of medial ganglionic eminence (MGE) properties, including interneuron migration to the cortex. Thus, despite their redundant properties, Gsx1 and -2 have distinct interactions with Dlx1 and -2. Gsx2 interaction is strongest in the LGE, CGE, and septum, whereas the Gsx1 interaction is strongest in the MGE. From these studies, and earlier studies, we present a model of the transcriptional network that regulates early steps of subcortical development.
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Encéfalo/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Neurogénesis/fisiología , Factores de Transcripción/genética , Animales , Encéfalo/embriología , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Ratones , Ratones Mutantes , FenotipoRESUMEN
Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.
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Adenocarcinoma/tratamiento farmacológico , Anticuerpos Antiidiotipos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Tumores Neuroendocrinos/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/inmunología , Adenocarcinoma/genética , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Modelos Animales de Enfermedad , Quimioterapia Combinada , Ingeniería Genética , Indoles/uso terapéutico , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Ratones , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirroles/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/genética , Sunitinib , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
CXCL12/CXCR4 signaling is critical for cortical interneuron migration and their final laminar distribution. No information is yet available on CXCR7, a newly defined CXCL12 receptor. Here we demonstrated that CXCR7 regulated interneuron migration autonomously, as well as nonautonomously through its expression in immature projection neurons. Migrating cortical interneurons coexpressed Cxcr4 and Cxcr7, and Cxcr7(-/-) and Cxcr4(-/-) mutants had similar defects in interneuron positioning. Ectopic CXCL12 expression and pharmacological blockade of CXCR4 in Cxcr7(-/-) mutants showed that both receptors were essential for responding to CXCL12 during interneuron migration. Furthermore, live imaging revealed that Cxcr4(-/-) and Cxcr7(-/-) mutants had opposite defects in interneuron motility and leading process morphology. In vivo inhibition of Gα(i/o) signaling in migrating interneurons phenocopied the interneuron lamination defects of Cxcr4(-/-) mutants. On the other hand, CXCL12 stimulation of CXCR7, but not CXCR4, promoted MAP kinase signaling. Thus, we suggest that CXCR4 and CXCR7 have distinct roles and signal transduction in regulating interneuron movement and laminar positioning.
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Movimiento Celular/fisiología , Interneuronas/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Quimiocina CXCL12/metabolismo , Inmunohistoquímica , Interneuronas/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Receptores CXCR/deficiencia , Receptores CXCR/genética , Receptores CXCR4/deficiencia , Receptores CXCR4/genéticaRESUMEN
Dlx5 and Dlx6 homeobox genes are expressed in developing and mature cortical interneurons. Simultaneous deletion of Dlx5 and 6 results in exencephaly of the anterior brain; despite this defect, prenatal basal ganglia differentiation appeared largely intact, while tangential migration of Lhx6(+) and Mafb(+) interneurons to the cortex was reduced and disordered. The migration deficits were associated with reduced CXCR4 expression. Transplantation of mutant immature interneurons into a wild-type brain demonstrated that loss of either Dlx5 or Dlx5&6 preferentially reduced the number of mature parvalbumin(+) interneurons; those parvalbumin(+) interneurons that were present had increased dendritic branching. Dlx5/6(+/-) mice, which appear normal histologically, show spontaneous electrographic seizures and reduced power of gamma oscillations. Thus, Dlx5&6 appeared to be required for development and function of somal innervating (parvalbumin(+)) neocortical interneurons. This contrasts with Dlx1, whose function is required for dendrite innervating (calretinin(+), somatostatin(+), and neuropeptide Y(+)) interneurons (Cobos et al., 2005).
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Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Proteínas de Homeodominio/metabolismo , Interneuronas/fisiología , Parvalbúminas/metabolismo , Animales , Ganglios Basales/crecimiento & desarrollo , Ganglios Basales/fisiología , Ganglios Basales/fisiopatología , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Corteza Cerebral/fisiopatología , Dendritas/patología , Dendritas/fisiología , Proteínas de Homeodominio/genética , Interneuronas/citología , Interneuronas/patología , Proteínas con Homeodominio LIM , Factor de Transcripción MafB/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Periodicidad , Receptores CXCR4/metabolismo , Convulsiones/patología , Convulsiones/fisiopatología , Telencéfalo/crecimiento & desarrollo , Telencéfalo/fisiología , Telencéfalo/fisiopatología , Factores de TranscripciónRESUMEN
Signaling mechanisms mediated by the Transforming Growth Factor-beta (TGF-beta) superfamily regulate a variety of developmental processes. Here we show that components of both bone morphogenetic protein/growth differentiation factor and TGF-beta/activin/Nodal branches of TGF-beta superfamily signaling are expressed in the developing subpallium. Furthermore, Smad proteins, transcriptional effectors of TGF-beta signaling, are co-expressed and physically interact in the basal ganglia with Dlx homeodomain transcription factors, which are critical regulators of the differentiation, migration and survival of telencephalic GABAergic neurons. We also show that Dlx and Smad proteins localize to promoters/enhancers of a number of common telencephalic genes in vivo and that Smad proteins co-activate transcription with Dlx family members, except with certain mutated human DLX proteins identified in autistic individuals. In agreement with these observations, expression of dominant-negative Smads in the developing basal ganglia phenocopies the cell migration defects observed in Dlx1/2-deficient mice. Together, these results suggest that TGF-beta superfamily signaling plays a role in telencephalic GABAergic neuron development through functional interactions with Dlx transcription factors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11689-009-9035-6) contains supplementary material, which is available to authorized users.
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Precise control of neuronal migration is essential for proper function of the brain. Taking a forward genetic screen, we isolated a mutant mouse with defects in interneuron migration. By genetic mapping, we identified a frame shift mutation in the pericentrin (Pcnt) gene. The Pcnt gene encodes a large centrosomal coiled-coil protein that has been implicated in schizophrenia. Recently, frame shift and premature termination mutations in the pericentrin (PCNT) gene were identified in individuals with Seckel syndrome and microcephalic osteodysplastic primordial dwarfism (MOPD II), both of which are characterized by greatly reduced body and brain sizes. The mouse Pcnt mutant shares features with the human syndromes in its overall growth retardation and reduced brain size. We found that dorsal lateral ganglionic eminence (dLGE)-derived olfactory bulb interneurons are severely affected and distributed abnormally in the rostral forebrain in the mutant. Furthermore, mutant interneurons exhibit abnormal migration behavior and RNA interference knockdown of Pcnt impairs cell migration along the rostal migratory stream (RMS) into the olfactory bulb. These findings indicate that pericentrin is required for proper migration of olfactory bulb interneurons and provide a developmental basis for association of pericentrin function with interneuron defects in human schizophrenia.
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Antígenos/genética , Movimiento Celular/fisiología , Interneuronas/citología , Mutación , Bulbo Olfatorio/metabolismo , Animales , Centrosoma/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
During central nervous system development, several transcription factors regulate the differentiation of progenitor cells to postmitotic neurons. Here we describe a novel role for Ikaros-1 in the generation of late-born striatal neurons. Our results show that Ikaros-1 is expressed in the boundary of the striatal germinal zone (GZ)/mantle zone (MZ), where it induces cell cycle arrest of neural progenitors by up-regulation of the cyclin-dependent kinase inhibitor (CDKi) p21(Cip1/Waf1). This effect is coupled with the neuronal differentiation of late precursors, which in turn is critical for the second wave of striatal neurogenesis that gives rise to matrix neurons. Consistently, Ikaros(-/-) mice had fewer striatal projecting neurons and, in particular, enkephalin (ENK)-positive neurons. In addition, overexpression of Ikaros-1 in primary striatal cultures increases the number of calbindin- and ENK-positive neurons. Our results also show that Ikaros-1 acts downstream of the Dlx family of transcription factors, insofar as its expression is lost in Dlx1/2 double knockout mice. However, we demonstrate that Ikaros-1 and Ebf-1 independently regulate the final determination of the two populations of striatal projection neurons of the matrix compartment, ENK- and substance P-positive neurons. In conclusion, our findings identify Ikaros-1 as a modulator of cell cycle exit of neural progenitors that gives rise to the neurogenesis of ENK-positive striatal neurons.
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Proteínas de Ciclo Celular/metabolismo , Cuerpo Estriado/embriología , Encefalinas/metabolismo , Factor de Transcripción Ikaros/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Animales , Calbindinas , Proteínas de Ciclo Celular/genética , Diferenciación Celular/fisiología , Cuerpo Estriado/citología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Vías Eferentes/citología , Vías Eferentes/embriología , Genes cdc/fisiología , Proteínas de Homeodominio/genética , Factor de Transcripción Ikaros/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Proteína G de Unión al Calcio S100/metabolismo , Células Madre/citología , Células Madre/metabolismo , Sustancia P/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Here we define the expression of approximately 100 transcription factors (TFs) in progenitors and neurons of the developing mouse medial and caudal ganglionic eminences, anlage of the basal ganglia and pallial interneurons. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most gamma-aminobutyric acidergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 basic helix-loop-helix (b-HLH) TF. These analyses define core transcriptional components that differentially specify the identity and differentiation of the globus pallidus, basal telencephalon, and pallial interneurons.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/fisiología , Encéfalo/citología , Encéfalo/metabolismo , Proteínas de Homeodominio/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Animales , Encéfalo/embriología , Diferenciación Celular , Ratones , Ratones Endogámicos C57BLRESUMEN
Here we define the expression of approximately 100 transcription factors in progenitors and neurons of the developing basal ganglia. We have begun to elucidate the transcriptional hierarchy of these genes with respect to the Dlx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Dlx-dependent and Dlx-independent pathways. The Dlx-independent pathway depends in part on the function of the Mash1 b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Cuerpo Estriado , Proteínas de Homeodominio/metabolismo , Morfogénesis , Vías Nerviosas , Factores de Transcripción/metabolismo , Animales , Ganglios Basales/anatomía & histología , Ganglios Basales/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Corteza Cerebral/anatomía & histología , Corteza Cerebral/fisiología , Cuerpo Estriado/anatomía & histología , Cuerpo Estriado/fisiología , Epistasis Genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Vías Nerviosas/anatomía & histología , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/fisiología , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Growth, differentiation and regional specification of telencephalic domains, such as the cerebral cortex, are regulated by the interplay of secreted proteins produced by patterning centers and signal transduction systems deployed in the surrounding neuroepithelium. Among other signaling molecules, members of the fibroblast growth factor (FGF) family have a prominent role in regulating growth, differentiation and regional specification. In the mouse telencephalon the rostral patterning center expresses members of the Fgf family (Fgf8, Fgf15, Fgf17, Fgf18). FGF8 and FGF17 signaling have major roles in specification and morphogenesis of the rostroventral telencephalon, whereas the functions of FGF15 and FGF18 in the rostral patterning center have not been established. RESULTS: Using Fgf15-/- mutant mice, we provide evidence that FGF15 suppresses proliferation, and that it promotes differentiation, expression of CoupTF1 and caudoventral fate; thus, reducing Fgf15 and Fgf8 dosage have opposite effects. Furthermore, we show that FGF15 and FGF8 differentially phosphorylate ERK (p42/44), AKT and S6 in cultures of embryonic cortex. Finally, we show that FGF15 inhibits proliferation in cortical cultures. CONCLUSION: FGF15 and FGF8 have distinct signaling properties, and opposite effects on neocortical patterning and differentiation; FGF15 promotes CoupTF1 expression, represses proliferation and promotes neural differentiation.