RESUMEN
Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.
Asunto(s)
Trastorno Autístico , Trastorno Autístico/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Organoides/metabolismo , Protocadherinas , TelencéfaloRESUMEN
Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Axones/metabolismo , Roturas del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Expresión Génica/fisiología , Ratones Endogámicos C57BL , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Proyección Neuronal/fisiología , Nervio Ciático/metabolismoRESUMEN
We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR-Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed. The procedure begins with genetic editing of mouse ESCs via CRISPR-Cas9, which can be accomplished in ~4-8 weeks. We provide protocols to achieve fluorescent labeling of ESCs in ~2-3 weeks, which allows tracing of the injected, ESC-derived donor cells in chimeras generated via NBC. Once modified ESCs are ready, NBC chimeras are generated in ~3 weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudo-pregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as 3 months, in the context of a whole organism.
Asunto(s)
Mapeo Encefálico/métodos , Encéfalo/embriología , Células Madre Embrionarias de Ratones/fisiología , Animales , Blastocisto , Diferenciación Celular , Quimera , Femenino , Masculino , Ratones , Organogénesis , FenotipoRESUMEN
Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.
Asunto(s)
Blastocisto/citología , Blastocisto/metabolismo , Organogénesis , Prosencéfalo/citología , Prosencéfalo/embriología , Animales , Quimera/embriología , Quimera/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Dominio Doblecortina , Femenino , Prueba de Complementación Genética , Células Germinativas/metabolismo , Hipocampo/anatomía & histología , Hipocampo/citología , Hipocampo/embriología , Hipocampo/fisiología , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/deficiencia , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Neocórtex/anatomía & histología , Neocórtex/citología , Neocórtex/embriología , Neocórtex/fisiología , Neuronas/citología , Neuronas/metabolismo , Neuropéptidos/deficiencia , Fenotipo , Prosencéfalo/anatomía & histología , Prosencéfalo/fisiologíaRESUMEN
Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; â¼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Carácter Cuantitativo Heredable , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Amiloide/metabolismo , Evolución Molecular , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Sistemas de Lectura Abierta , Priones/química , Priones/metabolismo , Proteoma , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.
Asunto(s)
Roturas del ADN de Doble Cadena , Células-Madre Neurales/metabolismo , Transcripción Genética , Translocación Genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/metabolismo , Células Cultivadas , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes myc , Genes p53 , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-myc/deficiencia , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Sitio de Iniciación de la Transcripción , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions.
Asunto(s)
Roturas del ADN , Células-Madre Neurales/metabolismo , Animales , Afidicolina/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/citología , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Roturas del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteínas Ligadas a GPI/metabolismo , Genoma , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Sinapsis , Factores de Transcripción/metabolismo , Translocación GenéticaRESUMEN
In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR(+)], a protein-based epigenetic element, allows yeast to circumvent this "glucose repression" and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR(+)]. [GAR(+)] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR(+)] presents a unique example of Lamarckian inheritance.
Asunto(s)
Epigénesis Genética , Priones/metabolismo , Saccharomyces cerevisiae/metabolismo , Staphylococcus hominis/metabolismo , Fermentación , Glucosa/metabolismo , Saccharomyces cerevisiae/genética , Staphylococcus hominis/genética , Vino/microbiología , Levaduras/genética , Levaduras/metabolismoRESUMEN
Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sµ and in a downstream acceptor S region, with a DSB in Sµ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sµ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.
Asunto(s)
Linfocitos B/inmunología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Neoplasias/genética , Recombinación Genética/genética , Cartilla de ADN/genética , Reparación del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Neoplasias/inmunología , Reacción en Cadena de la Polimerasa , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI(+)] or [MOT3(+)], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities.
Asunto(s)
Evolución Biológica , Fenotipo , Priones/metabolismo , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/genética , Pared Celular/metabolismo , Citoplasma/metabolismo , Epigénesis Genética , Estudios de Asociación Genética , Variación Genética/genética , Genotipo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Laboratorios , Meiosis , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Distant metastases, rather than the primary tumors from which these lesions arise, are responsible for >90% of carcinoma-associated mortality. Many patients already harbor disseminated tumor cells in their bloodstream, bone marrow, and distant organs when they initially present with cancer. Hence, truly effective anti-metastatic therapeutics must impair the proliferation and survival of already-established metastases. Here, we assess the therapeutic potential of acutely expressing the microRNA miR-31 in already-formed breast cancer metastases. Activation of miR-31 in established metastases elicits metastatic regression and prolongs survival. Remarkably, even brief induction of miR-31 in macroscopic pulmonary metastases diminishes metastatic burden. In contrast, acute miR-31 expression fails to affect primary mammary tumor growth. miR-31 triggers metastatic regression in the lungs by eliciting cell cycle arrest and apoptosis; these responses occur specifically in metastases and can be explained by miR-31-mediated suppression of integrin-α5, radixin, and RhoA. Indeed, concomitant re-expression of these three proteins renders already-seeded pulmonary metastases refractory to miR-31-conferred regression. Upon miR-31 activation, Akt-dependent signaling is attenuated and the proapoptotic molecule Bim is induced; these effects occur in a metastasis-specific manner in pulmonary lesions and are abrogated by concurrent re-expression of integrin-α5, radixin, and RhoA. Collectively, these findings raise the possibility that intervention strategies centered on restoring miR-31 function may prove clinically useful for combating metastatic disease.
Asunto(s)
Neoplasias de la Mama/fisiopatología , MicroARNs/metabolismo , Metástasis de la Neoplasia/fisiopatología , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Ciclo Celular/fisiología , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Neoplasias Pulmonares/secundario , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia/terapia , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Tumorales Cultivadas , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
miR-31 inhibits breast cancer metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin alpha(5) (ITGA5), radixin (RDX), and RhoA. We previously showed that the concomitant overexpression of ITGA5, RDX, and RhoA was capable of overriding the antimetastatic effects of ectopically expressed miR-31 in vivo. However, these prior studies failed to investigate whether the combined suppression of the endogenous mRNAs encoding these three proteins recapitulated the in vivo consequences of miR-31 expression on metastasis. We show here that short hairpin RNA-mediated concurrent downregulation of ITGA5, RDX, and RhoA is sufficient to phenocopy the full spectrum of described influences of miR-31 on metastasis in vivo, including the effects of this microRNA (miRNA) on local invasion, early post-intravasation events, and metastatic colonization. These findings provide mechanistic insights into the metastatic process and have implications about the importance of pleiotropy for the biological actions of miRNAs.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas del Citoesqueleto/metabolismo , Integrina alfa5/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular , Movimiento Celular , Proliferación Celular , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Integrina alfa5/química , Integrina alfa5/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
It remains unclear whether a microRNA (miRNA) affects a given phenotype via concomitant down-regulation of its entire repertoire of targets or instead by suppression of only a modest subset of effectors. We demonstrate that inhibition of breast cancer metastasis by miR-31-a miRNA predicted to modulate >200 mRNAs-can be entirely explained by miR-31's pleiotropic regulation of three targets. Thus, concurrent re-expression of integrin-alpha5, radixin, and RhoA abrogates miR-31-imposed metastasis suppression. These effectors influence distinct steps of the metastatic process. Our findings have implications concerning the importance of pleiotropy for the biological actions of miRNAs and provide mechanistic insights into metastasis.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Carcinoma Ductal de Mama/fisiopatología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , MicroARNs/metabolismo , Metástasis de la Neoplasia/fisiopatología , Animales , Línea Celular Tumoral , Femenino , Humanos , RatonesRESUMEN
The identification of reliable peripheral biomarkers for clinical diagnosis, patient prognosis, and biological functional studies would allow for access to biological information currently available only through invasive methods. Traditional approaches have so far considered aspects of tissues and biofluid markers independently. Here we introduce an information theoretic framework for biomarker discovery, integrating biofluid and tissue information. This allows us to identify tissue information in peripheral biofluids. We treat tissue-biofluid interactions as an information channel through functional space using 26 proteomes from 45 different sources to determine quantitatively the correspondence of each biofluid for specific tissues via relative entropy calculation of proteomes mapped onto phenotype, function, and drug space. Next, we identify candidate biofluids and biomarkers responsible for functional information transfer (p < 0.01). A total of 851 unique candidate biomarkers proxies were identified. The biomarkers were found to be significant functional tissue proxies compared to random proteins (p < 0.001). This proxy link is found to be further enhanced by filtering the biofluid proteins to include only significant tissue-biofluid information channels and is further validated by gene expression. Furthermore, many of the candidate biomarkers are novel and have yet to be explored. In addition to characterizing proteins and their interactions with a systemic perspective, our work can be used as a roadmap to guide biomedical investigation, from suggesting biofluids for study to constraining the search for biomarkers. This work has applications in disease screening, diagnosis, and protein function studies.