RESUMEN
Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.2 CNVs lead to disease are unclear. Relevant models, such as human cortical organoids (hCOs), are needed to understand the human-specific mechanisms of neurodevelopmental disease. We generated hCOs from 17 patients and controls, profiling 167,958 cells with single-cell RNA-sequencing analysis, which revealed neuronal-specific differential expression of genes outside the 16p11.2 region that are related to cell-cell adhesion, neuronal projection growth, and neurodevelopmental disorders. Furthermore, 16p11.2 deletion syndrome organoids exhibited reduced mRNA and protein levels of RBFOX1, a gene that can also harbor CNVs linked to neurodevelopmental phenotypes. We found that the genes previously shown to be regulated by RBFOX1 are also perturbed in organoids from patients with the 16p11.2 deletion syndrome and thus identified a novel link between independent CNVs associated with neuronal development and autism. Overall, this work suggests convergent signaling, which indicates the possibility of a common therapeutic mechanism across multiple rare neuronal diseases.
Asunto(s)
Deleción Cromosómica , Variaciones en el Número de Copia de ADN , Humanos , Variaciones en el Número de Copia de ADN/genética , Encéfalo , Fenotipo , Organoides , Factores de Empalme de ARN/genéticaRESUMEN
Animals use olfactory receptors to navigate mates, food, and danger. However, for complex olfactory systems, it is unknown what proportion of primary olfactory sensory neurons can individually drive avoidance or attraction. Similarly, the rules that govern behavioral responses to receptor combinations are unclear. We used optogenetic analysis in Drosophila to map the behavior elicited by olfactory-receptor neuron (ORN) classes: just one-fifth of ORN-types drove either avoidance or attraction. Although wind and hunger are closely linked to olfaction, neither had much effect on single-class responses. Several pooling rules have been invoked to explain how ORN types combine their behavioral influences; we activated two-way combinations and compared patterns of single- and double-ORN responses: these comparisons were inconsistent with simple pooling. We infer that the majority of primary olfactory sensory neurons have neutral behavioral effects individually, but participate in broad, odor-elicited ensembles with potent behavioral effects arising from complex interactions.
Asunto(s)
Quimiotaxis , Drosophila melanogaster/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Animales Modificados Genéticamente , Reacción de Prevención , Femenino , Masculino , OptogenéticaRESUMEN
Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.
Asunto(s)
Descubrimiento de Drogas , Transcriptoma , Descubrimiento de Drogas/métodos , Humanos , ARN , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodosRESUMEN
Geneticists use olfactory conditioning in Drosophila to identify learning genes; however, little is known about how these genes are integrated into short-term memory (STM) pathways. Here, we investigated the hypothesis that the STM evidence base is weak. We performed systematic review and meta-analysis of the field. Using metrics to quantify variation between discovery articles and follow-up studies, we found that seven genes were both highly replicated, and highly reproducible. However, â¼80% of STM genes have never been replicated. While only a few studies investigated interactions, the reviewed genes could account for >1000% memory. This large summed effect size could indicate irreproducibility, many shared pathways, or that current assay protocols lack the specificity needed to identify core plasticity genes. Mechanistic theories of memory will require the convergence of evidence from system, circuit, cellular, molecular, and genetic experiments; systematic data synthesis is an essential tool for integrated neuroscience.
Asunto(s)
Drosophila/genética , Memoria a Corto Plazo , Animales , Drosophila/fisiología , Memoria a Corto Plazo/fisiologíaRESUMEN
In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal's survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved.