RESUMEN
The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.
Asunto(s)
Elementos de Facilitación Genéticos , Notocorda , Ratones , Animales , Elementos de Facilitación Genéticos/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genéticaRESUMEN
Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized "trunk-like structures" (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.
