RESUMEN
Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in Caenorhabditis elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single-molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene.
Asunto(s)
Proteínas de Caenorhabditis elegans , Factores de Transcripción , Animales , Factores de Transcripción/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Retroalimentación , Caenorhabditis elegans/metabolismo , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Polyploid cells contain more than 2 copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either 1 nucleus (mononucleation) or 2 or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy is largely unknown. Here, we assess the function of multinucleation in Caenorhabditis elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single-worm RNA sequencing, we find that binucleation is important for tissue-specific gene expression, most prominently for genes that show a rapid up-regulation at the transition from larval development to adulthood. Regulated genes include vitellogenins, which encode yolk proteins that facilitate nutrient transport to the germline. We find that reduced expression of vitellogenins in mononucleated intestinal cells leads to progeny with developmental delays and reduced fitness. Together, our results show that binucleation facilitates rapid up-regulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function.
Asunto(s)
Poliploidía , Vitelogeninas , Animales , Caenorhabditis elegans/genética , División Celular , Núcleo Celular/genética , Expresión Génica , Vitelogeninas/genéticaRESUMEN
RNA tomography or tomo-seq combines mRNA sequencing and cryo-sectioning to spatially resolve gene expression. We have adapted this method for the nematode Caenorhabditis elegans to generate anteroposterior gene expression maps at near-cellular resolution. Here, we provide a detailed overview of the method and present two approaches: one that includes RNA isolation for maximum sensitivity and one that is suitable for partial automatization and is therefore less time-consuming. For complete details on the use and execution of this protocol, please refer to Ebbing et al. (2018).