Supplementary MaterialsSupplementary information develop-146-173740-s1
December 15, 2020
Supplementary MaterialsSupplementary information develop-146-173740-s1. Remarkably, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates. cells show one of the clearest examples of self-organisation during development. Upon starvation, cells initiate a programme of differentiation resulting in the generation of the two major cell fates: stalk and spore. After 6?h JI-101 of starvation, single cells chemotax together to form a multicellular mound. Cells entering this mound are initially equivalent, before deciding over the next few hours whether to become stalk or spore progenitors (prestalk and prespore, respectively). The final developed structure, formed 24?h after the induction of differentiation, consists of a JI-101 spore head suspended over the substrate by a thin cellular stalk. Prestalk and prespore markers have been identified (Brown and Firtel, 1999; Maeda et al., 2003; Maruo et al., 2004; Mehdy et al., 1983; Williams, 2006), and specific perturbations and intrinsic cell states can favour specific developmental choices. In particular, the choice between stalk and spore fates is influenced by a cell’s position in the cell cycle at the onset of starvation (Gomer and Firtel, 1987; Gruenheit et al., 2018; Weijer et al., 1984), with cells dividing around the onset of starvation favouring the stalk fate. These intrinsic destiny tendencies could be modulated by a number of different extracellular indicators additional, such as for example cAMP and DIF (Dark brown and Firtel, 1999; Kay et al., 1999; Loomis, 2014) as well as the dietary background of the cell (Thompson and Kay, 2000). The entire developmental program of requires a complex group of gene manifestation changes related to different stages of differentiation (Rosengarten et al., 2015). Nevertheless, the original gene manifestation transitions occuring in JI-101 specific cells during cell destiny separation never have been described. To characterise the adjustments in gene manifestation accompanying the development from an equal human population of cells through a bifurcation to two distinct fates, we completed solitary cell transcriptomics for the mound stage of advancement. Our data reveal that cells getting into the mound changeover through distinct intermediate cell areas with spore or stalk tendencies. Pursuing these intermediates, cells completely communicate the classical prestalk markers or even more stimulate the prespore program strongly. Transitions between cell areas are cell and fast areas show up separated, with small spillover in the manifestation of cell type-specific markers between fates. Remarkably, most changes in transcript abundance occurring during fate separation were common to both spore and stalk, with almost step-like progression in global expression profiles occurring alongside the initiation of cell type-specific programmes. RESULTS To characterise the gene expression decisions occurring during cell fate choice in aggregates. (A) Schematic of development. Single cell transcriptomics was carried out on 116 cells, over three replicates, at the mound stage (outlined). (B) Patterns of correlation within lineage-specific genes. JI-101 Correlation heatmaps, split into prespore and prestalk genes, are shown for 0, 3, 6 and 14?h timepoints. We selected cell type-specific genes from the data of Parikh et al. (2010) with |log2FC| 1, FDR 0.1 and an expression level of at least 100 normalised read counts, in at least one cell. (C) PCA of individual cell transcriptomes reveals distinct subpopulations at the mound stage. Shown here are the first two principal components. Each dot is a cell, colour-coded by developmental time. The single cell transcriptomes show clear indications of cell fate divergence. Fig.?1B and Fig.?S1A show correlation heatmaps for markers of stalk and spore fate, at different stages of development. The fate markers were extracted from transcriptomic data of prespore and prestalk cells separated by gradient centrifugation (Parikh et al., 2010). In data from the unicellular phase of development (0-6?h) (Antolovic et al., 2017) there was no clear segregation of heatmaps into Itga2 stalk and spore clusters. In contrast, the 14?h heatmap shows widespread single cell correlations between spore genes, clear correlations between stalk genes and anti-correlations between genes of the two fates. Divergence between cells at 14?h is also observed using principal component analysis (PCA) (Fig.?1C). Cells from 0-6?h of development projected as single populations in PCA. In contrast, 14?h data were even more dispersed, showing in least two distinct clusters. Evaluation of known cell destiny.