RESUMEN
Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Femenino , Embarazo , Placenta , Diferenciación Celular/genética , Trofoblastos/metabolismo , Proteína Morfogenética Ósea 5/metabolismoRESUMEN
OBJECTIVE: Schizophrenia is a brain disorder that originates during neurodevelopment and has complex genetic and environmental etiologies. Despite decades of clinical evidence of altered striatal function in affected patients, studies examining its cellular and molecular mechanisms in humans are limited. To explore neurodevelopmental alterations in the striatum associated with schizophrenia, the authors established a method for the differentiation of induced pluripotent stem cells (iPSCs) into ventral forebrain organoids (VFOs). METHODS: VFOs were generated from postmortem dural fibroblast-derived iPSCs of four individuals with schizophrenia and four neurotypical control individuals for whom postmortem caudate genotypes and transcriptomic data were profiled in the BrainSeq neurogenomics consortium. Individuals were selected such that the two groups had nonoverlapping schizophrenia polygenic risk scores (PRSs). RESULTS: Single-cell RNA sequencing analyses of VFOs revealed differences in developmental trajectory between schizophrenia and control individuals in which inhibitory neuronal cells from the patients exhibited accelerated maturation. Furthermore, upregulated genes in inhibitory neurons in schizophrenia VFOs showed a significant overlap with upregulated genes in postmortem caudate tissue of individuals with schizophrenia compared with control individuals, including the donors of the iPSC cohort. CONCLUSIONS: The findings suggest that striatal neurons derived from high-PRS individuals with schizophrenia carry abnormalities that originated during early brain development and that the VFO model can recapitulate disease-relevant cell type-specific neurodevelopmental phenotypes in a dish.
RESUMEN
Yeast replicative aging is a process resembling replicative aging in mammalian cells. During aging, wild-type haploid yeast cells enlarge, become sterile, and undergo nucleolar enlargement and fragmentation; we sought gene expression changes during the time of these phenotypic changes. Gene expression studied via microarrays and quantitative real-time reverse-transcription polymerase chain reaction (qPCR) has shown reproducible, statistically significant changes in messenger RNA (mRNA) of genes at 12 and 18-20 generations. Our findings support previously described changes towards aerobic metabolism, decreased ribosome gene expression, and a partial environmental stress response. Our findings include a pseudostationary phase, downregulation of methylation-related metabolism, increased nucleotide excision repair-related mRNA, and a strong upregulation of many of the regulatory subunits of protein phosphatase I (Glc7). These findings are correlated with aging changes in higher organisms as well as with the known involvement of protein phosphorylation states during yeast aging.
Asunto(s)
Envejecimiento/genética , Regulación Fúngica de la Expresión Génica , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Regulación hacia Abajo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Neuronal development is characterized by a period of exuberant synaptic growth that is well studied. However, the mechanisms that restrict this process are less clear. Here we demonstrate that glycosylphosphatidylinositol-anchored cell-surface receptors of the Nogo Receptor family (NgR1, NgR2, and NgR3) restrict excitatory synapse formation. Loss of any one of the NgRs results in an increase in synapse number in vitro, whereas loss of all three is necessary for abnormally elevated synaptogenesis in vivo. We show that NgR1 inhibits the formation of new synapses in the postsynaptic neuron by signaling through the coreceptor TROY and RhoA. The NgR family is downregulated by neuronal activity, a response that may limit NgR function and facilitate activity-dependent synapse development. These findings suggest that NgR1, a receptor previously shown to restrict axon growth in the adult, also functions in the dendrite as a barrier that limits excitatory synapse number during brain development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Proteínas de la Mielina/metabolismo , Neuronas/fisiología , Receptores de Superficie Celular/metabolismo , Sinapsis/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Células Cultivadas , Dendritas/genética , Dendritas/ultraestructura , Homólogo 4 de la Proteína Discs Large , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas de la Mielina/deficiencia , Neuronas/citología , Receptor Nogo 1 , Técnicas de Cultivo de Órganos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/genética , Sinapsis/ultraestructura , Transfección/métodos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Although transcription factors that repress gene expression play critical roles in nervous system development, their mechanism of action remains to be understood. Here, we report that the Olig-related transcription factor Bhlhb5 (also known as Bhlhe22) forms a repressor complex with the PR/SET domain protein, Prdm8. We find that Bhlhb5 binds to sequence-specific DNA elements and then recruits Prdm8, which mediates the repression of target genes. This interaction is critical for repressor function since mice lacking either Bhlhb5 or Prdm8 have strikingly similar cellular and behavioral phenotypes, including axonal mistargeting by neurons of the dorsal telencephalon and abnormal itch-like behavior. We provide evidence that Cadherin-11 functions as target of the Prdm8/Bhlhb5 repressor complex that must be repressed for proper neural circuit formation to occur. These findings suggest that Prdm8 is an obligate partner of Bhlhb5, forming a repressor complex that directs neural circuit assembly in part through the precise regulation of Cadherin-11.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Red Nerviosa/metabolismo , Neuronas/metabolismo , Animales , Axones/metabolismo , Cadherinas/metabolismo , Proteínas de Unión al ADN , Histona Metiltransferasas , Ratones , Ratones Transgénicos , Tractos Piramidales/metabolismoRESUMEN
Itch is the least well understood of all the somatic senses, and the neural circuits that underlie this sensation are poorly defined. Here we show that the atonal-related transcription factor Bhlhb5 is transiently expressed in the dorsal horn of the developing spinal cord and appears to play a role in the formation and regulation of pruritic (itch) circuits. Mice lacking Bhlhb5 develop self-inflicted skin lesions and show significantly enhanced scratching responses to pruritic agents. Through genetic fate-mapping and conditional ablation, we provide evidence that the pruritic phenotype in Bhlhb5 mutants is due to selective loss of a subset of inhibitory interneurons in the dorsal horn. Our findings suggest that Bhlhb5 is required for the survival of a specific population of inhibitory interneurons that regulate pruritus, and provide evidence that the loss of inhibitory synaptic input results in abnormal itch.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Interneuronas/patología , Células del Asta Posterior/patología , Prurito/genética , Prurito/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Supervivencia Celular/fisiología , Técnicas de Sustitución del Gen/métodos , Interneuronas/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Inhibición Neural/fisiología , Células del Asta Posterior/metabolismo , Prurito/fisiopatología , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Neuronal activity-regulated gene expression has been suggested to be an important mediator of long-lasting, experience-dependent changes in the nervous system, but the activity-dependent component of gene transcription has never been selectively isolated and tested for its functional significance. Here, we demonstrate that introduction of a subtle knockin mutation into the mouse Bdnf gene that blocks the ability of the activity-regulated factor CREB to bind Bdnf promoter IV results in an animal in which the sensory experience-dependent induction of Bdnf expression is disrupted in the cortex. Neurons from these animals form fewer inhibitory synapses, have fewer spontaneous inhibitory quantal events, and exhibit reduced expression of inhibitory presynaptic markers in the cortex. These results indicate a specific requirement for activity-dependent Bdnf expression in the development of inhibition in the cortex and demonstrate that the activation of gene expression in response to experience-driven neuronal activity has important biological consequences in the nervous system.