RESUMO
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Assuntos
Neoplasias Encefálicas/genética , Modelos Animais de Doenças , Marcação de Genes/métodos , Loci Gênicos/genética , Glioma/genética , Mutagênese Insercional/métodos , Transgenes/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Recombinases/metabolismo , TransfecçãoRESUMO
During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Córtex Cerebral , Receptores ErbB , Proteínas Hedgehog , Proteínas do Tecido Nervoso , Células-Tronco Neurais , Neurogênese , Fator de Transcrição 2 de Oligodendrócitos , Fator de Transcrição PAX6 , Animais , Neurogênese/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Camundongos , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição PAX6/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Neuroglia/metabolismo , Neuroglia/citologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Bulbo Olfatório/metabolismo , Bulbo Olfatório/citologia , Linhagem da Célula , HumanosRESUMO
Both the canonical Wnt and androgen receptor (AR) signaling pathways are important for prostate organogenesis and homeostasis. How they crosstalk to regulate prostate stem cell behaviors remains unclear. Here, we show in lineage-tracing mouse models that although Wnt is essential for basal stem cell multipotency, ectopic Wnt activity promotes basal cell over-proliferation and squamous phenotypes, which are counteracted by elevated levels of androgen. In prostate basal cell organoids, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated growth in a concentration-dependent manner. DHT down-regulates the expressions of a Wnt reporter and target genes, and RNA sequencing (RNA-seq) analyses identify Wnt signaling as a key altered pathway. Mechanistically, DHT enhances AR and ß-catenin protein binding, and CUT&RUN analyses reveal that ectopic AR sequesters ß-catenin away from its Wnt-related cistrome. Our results suggest that an intermediate level of Wnt activity in prostate basal stem cells, achieved via AR-ß-catenin interaction, is essential for normal prostate homeostasis.
Assuntos
Androgênios , Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Androgênios/farmacologia , Próstata/metabolismo , beta Catenina/metabolismo , Receptores Androgênicos/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Via de Sinalização WntRESUMO
Projection neuron subtype identities in the cerebral cortex are established by expressing pan-cortical and subtype-specific effector genes that execute terminal differentiation programs bestowing neurons with a glutamatergic neuron phenotype and subtype-specific morphology, physiology, and axonal projections. Whether pan-cortical glutamatergic and subtype-specific characteristics are regulated by the same genes or controlled by distinct programs remains largely unknown. Here, we show that FEZF2 functions as a transcriptional repressor, and it regulates subtype-specific identities of both corticothalamic and subcerebral neurons by selectively repressing expression of genes inappropriate for each neuronal subtype. We report that TLE4, specifically expressed in layer 6 corticothalamic neurons, is recruited by FEZF2 to inhibit layer 5 subcerebral neuronal genes. Together with previous studies, our results indicate that a cortical glutamatergic identity is specified by multiple parallel pathways active in progenitor cells, whereas projection neuron subtype-specific identity is achieved through selectively repressing genes associated with alternate identities in differentiating neurons.
Assuntos
Córtex Cerebral/citologia , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transcrição Gênica , Alelos , Animais , Diferenciação Celular/genética , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Camundongos Knockout , Mitose/genética , Neurônios/citologia , Ligação Proteica , Proteínas Repressoras/metabolismoRESUMO
Neural stem cells (NSCs) in the prenatal neocortex progressively generate different subtypes of glutamatergic projection neurons. Following that, NSCs have a major switch in their progenitor properties and produce γ-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, we provide evidence for the molecular mechanism that underlies this switch in the state of neocortical NSCs. We show that, at around E16.5, mouse neocortical NSCs start to generate GSX2-expressing (GSX2+) intermediate progenitor cells (IPCs). In vivo lineage-tracing study revealed that GSX2+ IPC population gives rise not only to OB interneurons but also to cortical oligodendrocytes and astrocytes, suggesting that they are a tri-potential population. We demonstrated that Sonic hedgehog signaling is both necessary and sufficient for the generation of GSX2+ IPCs by reducing GLI3R protein levels. Using single-cell RNA sequencing, we identify the transcriptional profile of GSX2+ IPCs and the process of the lineage switch of cortical NSCs.