Project description:Human stem cell technologies including self-assembling 3D tissue models provide unprecedented access to early neurodevelopment and are enabling fundamental insights into neuropathologies. Gastruloid models have yet to be used to investigate developing neuronal systems. Here we generate elongating multi-lineage-organized (EMLO) gastruloids with trunk identity that co-develop central and peripheral nervous system (CNS, PNS) correlates. We identify neural crest cells that differentiate to form peripheral neurons integrated with an upstream spinal cord region. This follows initial EMLO polarization events and is coordinated with primitive gut tube elongation and multicellular spatial reorganization. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and use them to investigate the impact of mu opioid receptor modulation on neuronal activity. This comprehensive study demonstrates the first combined human CNS-PNS model of early organogenesis in the trunk to benefit biomedical research.
Project description:Stem cell technologies including self-assembling 3D tissue models provide access to early human neurodevelopment and fundamental insights into neuropathologies. Gastruloid models have not been used to investigate co-developing central and peripheral neuronal systems with trunk mesendoderm which we achieve here in elongating multi-lineage organized (EMLO) gastruloids. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and evaluation of ectodermal and non-ectodermal neural crest cells (NCCs). We identify NCCs that differentiate to form peripheral neurons integrated with an upstream spinal cord region after day 8. This follows initial EMLO polarization events that coordinate with endoderm differentiation and primitive gut tube formation during multicellular spatial reorganization. This combined human central-peripheral nervous system model of early organogenesis highlights developmental events of mesendoderm and neuromuscular trunk regions and enables systemic studies of tissue interactions and innervation of neuromuscular, enteric and cardiac relevance.
Project description:Procedure: three independent experiments were done to generate three 96-well plates of E14-Tg2A gastruloids made with either homemade or commercial N2B27. 48 gastruloids per sample were used for RNA extraction using the Qiagen RNeasy Micro Kit. Library preparation and sequencing were done by the CRG Genomics Facility (Spain). Sequencing was done in a NextSeq 2000 and generated around 30M paired-end reads per sample.
Project description:Single cell transcriptomic study (using 10x Genomics v3 kits) of gastruloids, aggregates of mESCs, at different developmental timepoints (24h, 48h and 72h post-aggregation). mESCs, corresponding to the 0h timepoint, were also sequenced. The aim of this study was to delineate the cell state transition dynamics underlying anteroposterior symmetry breaking and germ layer formation in gastruloids. Gastruloids used in this study were generated from Bra::GFP reporter mESCs (Fehling et al., 2003).
Project description:Gastruloids are three-dimensional aggregates of embryonic stem cells (ESCs) that display key features of mammalian post-implantation development, including germ layer specification and axial organization. Gastruloids have mostly been characterized with microscopy-based approaches, limiting the number of genes that can be explored. It is therefore unclear to what extent gene expression in gastruloids reflects in vivo embryonic expression. Using both single-cell RNA-seq (scRNA-seq) and spatial transcriptomics we systematically compared cell types and spatial expression patterns between mouse gastruloids and mouse embryos.
Project description:Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites, and diverse cell types including neural crest, neural progenitors, renal progenitors, and myocytes. Through in silico staging based on single-cell RNA-seq (scRNA-seq), we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.
Project description:This study evaluated transcriptomic responses to submergence in elongating and non-elongating leaves of rice near-isogenic lines with and without SUB1A using RNA-Seq. SUB1A is an ERF transcription factor gene and the key regulator of submergence tolerance in rice, restricting underwater elongation and avoiding starvation under the stress. Submergence induces mRNA accumulation of SUB1A similarly in elongating and non-elongating leaves. This study uncovered SUB1A-dependent and independent regulation of adaptive responses to submergence in the two functionally distinct leaves at the global level.
Project description:Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Indeed, there is a lack of an in vitro model replicating the differentiation of both heart and head muscles to study these mechanisms. Such models are required to allow live-imaging and high throughput genetic and drug screening. Here, we show that the formation of self-organizing or pseudo-embryos from mouse embryonic stem cells (mESCs), also called gastruloids, reproduces cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm establishment and differentiation in gastruloids and comparing it to mouse embryos, we present the first evidence for skeletal myogenesis in gastruloids. By inferring lineage trajectories from the gastruloids single-cell transcriptomic data, we further suggest that heart and head muscles formed in gastruloids derive from cardiopharyngeal mesoderm progenitors. We identify different subpopulations of cardiomyocytes and skeletal muscles, which most likely correspond to different states of myogenesis with “head-like” and “trunk-like” skeletal myoblasts. These findings unveil the potential of mESC-derived gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases.