RESUMO
Exome sequencing studies have identified multiple genes harboring de novo loss-of-function (LoF) variants in individuals with autism spectrum disorders (ASD), including TBR1, a master regulator of cortical development. We performed ChIP-seq for TBR1 during mouse cortical neurogenesis and show that TBR1-bound regions are enriched adjacent to ASD genes. ASD genes were also enriched among genes that are differentially expressed in Tbr1 knockouts, which together with the ChIP-seq data, suggests direct transcriptional regulation. Of the nine ASD genes examined, seven were misexpressed in the cortices of Tbr1 knockout mice, including six with increased expression in the deep cortical layers. ASD genes with adjacent cortical TBR1 ChIP-seq peaks also showed unusually low levels of LoF mutations in a reference human population and among Icelanders. We then leveraged TBR1 binding to identify an appealing subset of candidate ASD genes. Our findings highlight a TBR1-regulated network of ASD genes in the developing neocortex that are relatively intolerant to LoF mutations, indicating that these genes may play critical roles in normal cortical development.
Assuntos
Transtorno do Espectro Autista/genética , Proteínas de Ligação a DNA/genética , Neocórtex/fisiopatologia , Neurogênese/genética , Animais , Transtorno do Espectro Autista/fisiopatologia , Modelos Animais de Doenças , Exoma/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutação , Neocórtex/crescimento & desenvolvimento , Neurônios/metabolismo , Neurônios/patologia , Fatores de Risco , Proteínas com Domínio TRESUMO
Generation of distinct cortical projection neuron subtypes during development relies in part on repression of alternative neuron identities. It was reported that the special AT-rich sequence-binding protein 2 (Satb2) is required for proper development of callosal neuron identity and represses expression of genes that are essential for subcerebral axon development. Surprisingly, Satb2 has recently been shown to be necessary for subcerebral axon development. Here, we unravel a previously unidentified mechanism underlying this paradox. We show that SATB2 directly activates transcription of forebrain embryonic zinc finger 2 (Fezf2) and SRY-box 5 (Sox5), genes essential for subcerebral neuron development. We find that the mutual regulation between Satb2 and Fezf2 enables Satb2 to promote subcerebral neuron identity in layer 5 neurons, and to repress subcerebral characters in callosal neurons. Thus, Satb2 promotes the development of callosal and subcerebral neurons in a cell context-dependent manner.
Assuntos
Córtex Cerebral/embriologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Axônios/metabolismo , Sítios de Ligação , Mapeamento Encefálico , Linhagem da Célula , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Prosencéfalo/embriologia , RNA Mensageiro/metabolismoRESUMO
The molecular mechanisms regulating fate divergence of closely related, but distinct, layer 6 corticothalamic and layer 5 subcerebral projection neurons are largely unknown. We present evidence for central transcriptional mechanisms that regulate fate specification of corticothalamic (layer 6) and subcerebral (layer 5) projection neurons. We found that TBR1 promotes the identity of corticothalamic neurons and represses subcerebral fates through reducing expression of Fezf2 and CTIP2. These conclusions are based on the following: (1) In Tbr1(-/-) mice, the number of cells expressing layer 6 markers was reduced, and the number of cells expressing layer 5 markers was increased. Early-born (birthdated on E11.5) neurons ectopically expressed subcerebral neuronal markers, and extended their axons into subcerebral targets. (2) Ectopic Tbr1 expression in layer 5 neurons prevented them from extending axons into the brainstem and the spinal cord. (3) Chromatin immunoprecipitation analysis using TBR1 antibodies showed that TBR1 bound to a conserved region in the Fezf2 gene. (4) Analysis of Fezf2 mutants and Tbr1(-/-); Fezf2(-/-) compound mutants provided evidence that Fezf2 blocks corticothalamic fate in layer 5 by reducing Tbr1 expression in subcerebral neurons. All neocortical regions appear to use this core transcriptional program to specify corticothalamic (layer 6) and subcerebral (layer 5) projection neurons.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neocórtex/citologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Animais , Axônios/fisiologia , Tronco Encefálico/fisiologia , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação , Neocórtex/embriologia , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurogênese , Neurônios/metabolismo , Ligação Proteica , Proteínas Repressoras/biossíntese , Medula Espinal/fisiologia , Proteínas com Domínio T , Tálamo/citologia , Tálamo/embriologia , Tálamo/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/biossínteseRESUMO
In addition to their role as chemorepellent netrin-1 receptors, UNC5 proteins may mediate cell death because they induce apoptosis in cultured cells. To test this in vivo, we generated Unc5a (formerly Unc5h1) knockout mice and found that this deletion decreased apoptosis and increased the number of neurons in the spinal cord. In contrast, loss of netrin-1 (Ntn1) did not affect the amount of apoptosis, suggesting that NTN1 is not required for neuronal apoptosis in vivo.
Assuntos
Apoptose/fisiologia , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Receptores de Netrina , Netrina-1 , Neurônios/patologia , Receptores de Superfície Celular/genética , Medula Espinal/anormalidades , Medula Espinal/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Many modern crop varieties contain patented biotechnology traits, and an increasing number of these crops have multiple (stacked) traits. Fast and accurate determination of transgene levels is advantageous for a variety of use cases across the food, feed and fuel value chain. With the growing number of new transgenic crops, any technology used to quantify them should have robust assays that are simple to design and optimize, thereby facilitating the addition of new traits to an assay. Here we describe a PCR-based method that is simple to design, starts from whole seeds, and can be run to end-point in less than 5 minutes. Subsequent relative quantification (trait vs. non-trait) using capillary electrophoresis performed in 5% increments across the 0-100% range showed a mean absolute error of 1.9% (s.d. = 1.1%). We also show that the PCR assay can be coupled to non-optical solid-state nanopore sensors to give seed-to-trait quantification results with a mean absolute error of 2.3% (s.d. = 1.6%). In concert, the fast PCR and nanopore sensing stages demonstrated here can be fully integrated to produce seed-to-trait quantification results in less than 10 minutes, with high accuracy across the full dynamic range.
Assuntos
Produtos Agrícolas/genética , Glycine max/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/métodos , Transgenes , DNA de Plantas/genética , Eletroforese Capilar/economia , Eletroforese Capilar/métodos , Nanoporos , Reação em Cadeia da Polimerase/economia , Característica Quantitativa Herdável , Sementes/genética , Fatores de TempoRESUMO
Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCalpha) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCalpha and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCalpha-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCalpha-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.
Assuntos
Axônios/metabolismo , Membrana Celular/metabolismo , Fatores de Crescimento Neural/fisiologia , Receptor A2B de Adenosina/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Agonistas do Receptor A2 de Adenosina , Animais , Axônios/efeitos dos fármacos , Células COS , Células Cultivadas , Galinhas , Chlorocebus aethiops , Receptores de Netrina , Netrina-1 , Ligação Proteica/fisiologia , Ratos , Receptor A2B de Adenosina/fisiologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismoRESUMO
Accessible point-of-care technologies that can provide immunoassay and molecular modalities could dramatically enhance diagnostics, particularly for infectious disease control in low-resource settings. Solid-state nanopores are simple and durable sensors with low-energy instrumentation requirements. While nanopore sensors have demonstrated efficacy for nucleic acid targets, selective detection and quantification of target proteins from sample background has not been demonstrated. We present a simple approach for electronic detection and quantification of target proteins that combines novel biomolecular engineering methods, a portable reader device and disposable nanopore test strips. The target of interest can be varied by swapping the binding domain on our engineered detection reagent, which eficiently binds in the bulk-phase to the target and subsequently generates a unique signature when passing through the pore. We show modularity of the detection reagent for two HIV antibodies, TNFα and tetanus toxin as targets. A saliva swab-to-result is demonstrated for clinically relevant HIV antibody levels (0.4-20 mg/liter) in under 60 seconds. While other strip-like assays are qualitative, the presented method is quantitative and sets the stage for simultaneous immunoassay and molecular diagnostic functionality within a single portable platform.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Equipamentos Descartáveis , Nanoporos , Anticorpos Monoclonais/análise , Anticorpos Anti-HIV/análise , Humanos , Indicadores e Reagentes , Modelos Teóricos , Imagem Individual de Molécula , Toxina Tetânica/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.
Assuntos
Proteínas de Transporte/metabolismo , Sistema Nervoso Central/embriologia , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Córtex Cerebelar/citologia , Córtex Cerebelar/embriologia , Córtex Cerebelar/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia/fisiologia , Sinais (Psicologia) , Proteínas do Citoesqueleto , Endocitose/fisiologia , Ativação Enzimática/fisiologia , Cones de Crescimento/ultraestrutura , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Receptores de Netrina , Netrina-1 , Fosforilação , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
Netrin-1 is a bifunctional guidance cue that directs migrating neurons and axons based on specific receptors expressed on the cell surface. Attraction occurs through the receptor Deleted in Colorectal Cancer (DCC) and repulsion occurs through a receptor complex of DCC and UNC5H, the vertebrate homolog to Caenorhabditis elegans UNC-5, but how the specific surface expression of these receptors is achieved remains unknown. Here, we demonstrate that surface expression of UNC5H1 is regulated in neurons by protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC), and show that one mechanism by which cells control their response to netrin-1 is by changing the surface availability of receptors. We identified PICK1 as a binding partner for UNC5H1 using the yeast two-hybrid system and found that the extreme three C-terminal amino acids of UNC5H1 interact with the PSD-95/Dlg/ZO-1 (PDZ) domain of PICK1. Coexpression of UNC5H1 and PICK1 in heterologous cells results in the recruitment of PICK1 to UNC5H1 clusters. Endogenous UNC5H1 and PICK1 coimmunoprecipitate from extracts of cultured hippocampal neurons and P4 cortices, and immunohistochemistry shows that UNC5H1, PICK1, and PKC are all present in growth cones. PKC activation induces the formation of UNC5H1/PICK1/PKC complexes and leads to the specific removal of UNC5H1, but not DCC, from the surface of neurons and growth cones via a PICK1/PKC-dependent mechanism. Lastly, we demonstrate that activating PKC, which decreases surface expression of UNC5H1, inhibits netrin-1-dependent collapse of hippocampal growth cones. Together, our results suggest that by regulating the surface expression of UNC5Hs, an axon can modulate its repellent response to netrin-1.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Neurônios/metabolismo , Proteínas Nucleares , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sítios de Ligação , Células COS , Proteínas de Transporte/análise , Linhagem Celular , Membrana Celular/enzimologia , Células Cultivadas , Cricetinae , Proteínas do Citoesqueleto , Cones de Crescimento/química , Cones de Crescimento/enzimologia , Cones de Crescimento/metabolismo , Hipocampo/citologia , Receptores de Netrina , Neurônios/enzimologia , Proteína Quinase C/análise , Proteína Quinase C-alfa , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores de Superfície Celular/análise , Proteínas Recombinantes de Fusão/análiseRESUMO
We recently published genetic lineage-tracing experiments using the Fezf2 and Cux2 loci. These experiments demonstrated that at both the clonal and population levels Fezf2(+) RGCs are multipotent and that at the population level Cux2(+) RGCs are multipotent. Here, we extend our work on the lineages of Fezf2(+) and Cux2(+) RGCs. Clonal analysis of E10.5 neocortical progenitors suggests that most, if not all, Cux2(+) and Fezf2(+) RGCs generate diverse projection neuron subtypes located throughout layers 2-6. These results support our previous conclusion that both Fezf2(+) and Cux2(+) RGCs are multipotent neocortical progenitors. This Matters Arising Response paper addresses the Gil-Sanz et al. (2015) Matters Arising paper, published concurrently in Neuron.
Assuntos
Linhagem da Célula/fisiologia , Proteínas de Homeodomínio/metabolismo , Neocórtex/metabolismo , Neurônios/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Multipotentes/citologia , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , OligodendrogliaRESUMO
BACKGROUND: The genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons. RESULTS: Using a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons. CONCLUSIONS: These results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.
Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Elementos Reguladores de Transcrição , Animais , Córtex Cerebral/citologia , Elementos Facilitadores Genéticos , Camundongos , Camundongos TransgênicosRESUMO
Progenitor cells in the cerebral cortex sequentially generate distinct classes of projection neurons. Recent work suggests the cortex may contain intrinsically fate-restricted progenitors marked by expression of Cux2. However, the heterogeneity of the neocortical ventricular zone as well as the contribution of lineage-restricted progenitors to the overall cortical neurogenic program remains unclear. Here, we utilize in vivo genetic fate mapping to demonstrate that Fezf2-expressing radial glial cells (RGCs) exist throughout cortical development and sequentially generate all major projection neuron subtypes and glia. Moreover, we show that the vast majority of CUX2⺠cells in the VZ and SVZ are migrating interneurons derived from the subcortical telencephalon. Examination of the embryonic cortical progenitor population demonstrates that Cux2⺠RGCs generate both deep- and upper-layer projection neurons. These results identify Fezf2⺠radial glial cells as a multipotent neocortical progenitor and suggest that the existence, and molecular identity, of laminar-fate-restricted RGCs awaits further investigation.
Assuntos
Astrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Multipotentes/fisiologia , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Diferenciação Celular , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Endopeptidases/genética , Endopeptidases/metabolismo , Lateralidade Funcional , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/metabolismoRESUMO
The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion.
Assuntos
Movimento Celular , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Forkhead/genética , Redes Reguladoras de Genes , Genoma , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neocórtex/embriologia , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/classificação , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The murine olfactory system consists of main and accessory systems that perform distinct and overlapping functions. The main olfactory epithelium (MOE) is primarily involved in the detection of volatile odorants, while neurons in the vomeronasal organ (VNO), part of the accessory olfactory system, are important for pheromone detection. During development, the MOE and VNO both originate from the olfactory pit; however, the mechanisms regulating development of these anatomically distinct organs from a common olfactory primordium are unknown. Here we report that two closely related zinc-finger transcription factors, FEZF1 and FEZF2, regulate the identity of MOE sensory neurons and are essential for the survival of VNO neurons respectively. Fezf1 is predominantly expressed in the MOE while Fezf2 expression is restricted to the VNO. In Fezf1-deficient mice, olfactory neurons fail to mature and also express markers of functional VNO neurons. In Fezf2-deficient mice, VNO neurons degenerate prior to birth. These results identify Fezf1 and Fezf2 as important regulators of olfactory system development and sensory neuron identity.