Fig 1.
inDrop analysis of cultured bloodstream form and procyclic trypanosomes.
A. inDrop performed in a microfluidic format, using a single primer to replace the BHMs (no single-cell resolution). The plots show the reads per kilobase per million mapped (RPKM) for annotated T. brucei transcripts aligned from their 5´splice site to the 3´polyadenylation sequence. Profile plots (above) show the average RPKM across the ~7,500 non-redundant gene set for trypanosomes. Heatmaps (below) show the RPKM for individual transcripts. C. UMAP projection of single-cell barcoding data for a population of cells containing calculated proportions of 47.5% for each BSF and PCF, and 5% Ramos cells. Measured cell proportions are 51% BSF, 41% PCF and 8% Ramos. D. Violin plots depict the total number of UMIs and genes captured per cell within BSF, PCF and Ramos cell clusters. E. UMAP projections from C overlaid with colour scale of gene expression values for EP1 procyclin (Tb927.10.10260), VSG-2 (Tb427.BES40.22) and Immunoglobulin lambda constant 3 (IGLC3).
Fig 2.
InDrop barcoding of salivary gland T. brucei.
A. UMAP projection of two technical replicates (Lib1 and Lib2, red and blue, respectively). B. Merged UMAP projection of technical replicates with clustering information as indicated. C. Gene expression levels for marker genes in the salivary glands, EP1 procyclin (midgut stages) [51]: Tb927.10.10260, BARP (epimastigote stage): Tb927.9.15640 [52], VSG-393: (Genbank) KC612418.1 [48], RBP6: Tb927.3.2930 [12], Calflagin: Tb927.8.5440 [6], HAP2: Tb927.10.10770 [53].
Fig 3.
Cluster-based analysis of main energy metabolism transcripts.
A. The schematic shows the glycolytic and TCA pathways [82]. Compounds (black text) and enzymes (red text for glycolysis, blue text for TCA cycle) are shown and reactions are represented by arrows. B. Plot shows the expression Z-scores for each glycolysis or TCA cycle enzyme transcript retained after SCT normalization [43]. Thicker lines show the average across each cohort. AE: attached epimastigote cells; G: Gamete cells; Pre-M: pre-metacyclic cells: M: metacyclic cells. C. Single cell level analysis of metabolic transcripts. Cells are arranged by cluster (left to right) and relative transcript expression shown per cell. Colours represent Z-transformed expression values. Cluster abbreviations as for B. Abbreviations of metabolites: G-6-P: glucose-6-phosphate; F-6-P: fructose-6-phosphate; F-1,6-BP: fructose-1,6-bisphosphate; DHA-P: dihydroxyacetone phosphate; GA-3-P: glyceraldehyde-3-phosphate; 1,3-BP-GA: 1,3-bisphosphoglycerate; 3-P-GA: 3-phosphoglycerate; 2-P-GA: 2-phosphoglycerate; P-EP: phospho-enol pyruvate. Abbreviations of enzymes: HK1: hexokinase 1; PGI phosphoglucose isomerase; PFK: phosphofructokinase; ALD: aldolase; TIM: triose-phosphate isomerase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PGK(C/A): phosphoglycerate kinase, PGAM: phosphoglycerate mutase; ENO: enolase; PYK1: pyruvate kinase; CS: citrate synthase; ACO: aconitase; IDH: isocitrate dehydrogenase; SCSα: succinyl coenzyme A synthetase; FHc, fumarate hydratase, cytosolic; gMDH: glycosomal malate dehydrogenase; mMDH: mitochondrial malate dehydrogenase.
Fig 4.
A. Normalised expression values for BARP (Tb927.9.15640) and HAP2 (Tb927.10.10770) overlaid on UMAP projections of attached epimastigote and gamete cell clusters. B. Normalised expression values for canonical histones: H1 Tb927.11.1800, H2A Tb927.7.2820, H2B Tb927.10.10460, H3 Tb927.1.2430 and H4 Tb927.5.4170. C. Sub-clustering of cells in S-Phase. Clusters were manually re-annotated using the Seurat function CellSelector. Two new clusters of cells expressing both core histones and either BARP or HAP2 were created and are indicated in brown and red, respectively.
Fig 5.
Developmental progression of VSG expression.
A. VSG expression data for a random subset of 50 pre-metacyclic cells, with a total VSG UMI count > 10. Data are raw (unscaled) UMI counts. Each column represents a cell and each colour a different VSG. B. Histogram showing the number of VSG expressed for all pre-metacyclic cells (per VSG UMI count > 2, all cells in cluster). C. As for A except that the data are a subset of 50 metacyclic cells with total VSG UMI counts >10. D. Histogram as for B except showing metacyclic VSG transcript diversity (per VSG UMI count >2, all cells in cluster). E. UMAP projection from Fig 2B coloured according to the number of mVSG genes detected per cell.
Fig 6.
Single molecule RNA-FISH of in vitro metacyclogenesis.
A. Panel of representative images of VSG+ and VSG++ cells containing both VSG-397 and VSG-531 transcripts (i-iii) and VSG+++ cells where a single VSG type is present (iv and v). B. Above, proportions of VSG-397 and VSG-531 transcripts for VSG+, VSG++ and VSG+++ cells. Each cell is plotted as a single column on the x-axis. An estimate of transcript abundance for VSG++ and VSG+++ cells was used based on published estimates of VSG transcript abundance in BSF [46]. Below, transcript abundance for VSG+ cells, and estimated transcript abundance for VSG+ and VSG++ cells.
Fig 7.
Model of the initiation of VSG monoallelic expression during metacyclogenesis.
A. Pre-metacyclic cells initiate a transcriptional race for monoallelic expression where a single VSG gene must reach a threshold of transcriptional activity. B. In metacyclic cells, this threshold has been reached for a single VSG that is active, while the transcription of the other mVSGs is silenced.