Fig 1.
scRNAseq atlas of the adult schistosome reveals two tegument related lineages (TRLs).
(A) Uniform Manifold Approximation and Projection (UMAP) showing a scRNAseq atlas of the adult schistosome revealing two lineages predicted to produce tegumental cells. One lineage is characterized by the expression of tsp-2 and sm13, while the other lineage expresses tsp-2 along with egc, meg-1 and zfp-1-1. Different highlighted colors represent distinct TRL+ cell populations. (B-F) UMAP expression of tsp-2 (B), sm13 (C), egc (D), meg-1 (E) and zfp-1-1 (F) in TRLs.
Fig 2.
klf4 is expressed in the egc+/meg-1+/zfp-1-1+ TRL.
(A) UMAP showing klf4 expression in egc+/meg-1+/zfp-1-1+ TRL. (B) Colorimetric whole in situ hybridization (WISH) showing expression pattern of klf4. Scale bar, 100 µm; anterior towards the top. (C) Double fluorescence in situ hybridization (FISH) showing expression of klf4 relative to the egc+, meg-1+, zfp-1-1+ and tsp-2+ cells (co-expression indicated by arrows). 65%, 35%, 19% and 32% of klf4+ cells are egc+ (81/125 cells, n=3 parasites), meg-1+ (88/255 cells, n=4 parasites), zfp-1-1+ (59/319 cells, n=6 parasites) and tsp-2+ (70/217 cells, n=6 parasites), respectively, as indicated in the Venn diagram in the upper right. The approximate selected region for cell quantification is indicated by the dotted rectangle in panel (B). Scale bar, 10 µm.
Fig 3.
klf4 is required for the maintenance of the egc+/meg-1+/zfp-1-1+ TRL.
(A) Graphic depicting the workflow for exploring function of klf4 by scRNAseq and bulk RNAseq transcriptional profiling analysis. Adult male schistosome worms were treated with dsRNA targeting klf4 in vitro at Days 0, 1, 2, 5, 8 and 11. After 17 days, both control (RNAi) and klf4 (RNAi) worms were collected for scRNAseq and bulk RNAseq analyses. (B) scRNAseq analysis shows klf4 RNAi resulted in a loss of the egc+/meg-1+/zfp-1-1+ TRL. (C) Volcano plot depicting bulk RNAseq analysis. This analysis identified 67 differentially expressed genes (Padj<0.05, 55 down- and 12 up-regulated genes) following knockdown of klf4. Grey dots represent genes with a log2 fold change (log2FC) between -1 and 1. Blue dots represent log2FC<-1 or >1. Green dots indicate marker genes expressed in the egc+/meg-1+/zfp-1-1+ TRL. Magenta dots indicate down-regulated genes validated to be expressed in the tegument and zfp-1-1+ cells. Red dots indicate up-regulated genes validated to be expressed in sm13+ cells. (D) A dot-plot summarizing the expression of the bulk RNAseq down-regulated differentially expressed genes (DEGs, log2 fold change<-2, Padj<0.05) in clusters from the klf4 RNAi scRNAseq profile in panel 3B. Cluster populations are on the vertical axis and gene IDs are on the horizontal axis. Expression levels are colored by gene expression (blue = low, red = high). Percentage of cells in the cluster expressing the gene is indicated by the size of the circle. (E) FISH results validating the loss of klf4+, egc+, meg-1+ and a substantial reduction in zfp-1-1+ cells following klf4 RNAi. We noted no changes in the number of EdU+ proliferative cells. Scale bar, 100 µm. Numbers at bottom left represent the fraction of parasites displaying the observed phenotype. (F) FISH depicting a modest decrease in tsp-2+ cell number following klf4 RNAi. Scale bar, 100 µm. (G) Quantification of the number of tsp-2+ cells per mm of worm. Control (RNAi) n= 33, klf4 (RNAi) n=29. (H) qPCR quantification of expression of tsp-2 following klf4 RNAi. n=12 experiments. Data are presented as mean ± standard deviation (mean ± SD). An unpaired t-test and a paired t-test were performed in Panel G and H, respectively.
Fig 4.
+/meg-1+/zfp-1-1+ TRL is required for producing a specific tegumental subpopulation. (A) FISH monitoring the expression for a cocktail of tegumental markers (calpain Smp_214190, npp-5 Smp_153390, annexin Smp_077720 and gtp-4 Smp_105410) in control (RNAi) and klf4 (RNAi) worms. Scale bar: 50 µm. (B) Quantification of tegumental cell density. Control (RNAi) n= 12, klf4 (RNAi) n=14. (C) qPCR detection of expression of the tegumental marker calpain [11]. n=10 experiments. (D) FISH results confirm loss of egf-1+ and meg-10+ (highlighted with magenta dots in (Fig 3C)) following klf4 RNAi. Numbers at bottom left represent the fraction of parasites displaying the observed phenotype. (E) Double FISH showing expression of egf-1 and meg-10 relative to the calpain+ and zfp-1-1+ cells, respectively (indicated in arrows). The Venn diagram in the upper right shows the percentage of egf-1+ cells that are calpain+ cells (201/235 cells, n=17 parasites) or zfp-1-1+ (12/60 cells n=10 parasites) and the percentage of meg-10+ cells that are calpain+ or zfp-1-1+ (127/221 cells are calpain+, n=13 parasites; 66/233 cells are zfp-1-1+, n=10 parasites). Scale bars, 100 µm. Data are presented as mean ± SD. An unpaired t-test and a paired t-test were performed in Panel B and C, respectively.
Fig 5.
Knockdown of klf4 alters the heterogeneity of the sm13
+ TRL. (A) FISH result showing klf4 RNAi causes no significant effect on the number of cells expressing sm13 (left), quantification of the number of sm13+ cells per mm of worm (right). Control (RNAi) n= 28, klf4 (RNAi) n=26. (B) qPCR detection of the expression of sm13 (n=10 experiments) and up-regulated DEGs including ludp (Smp_319910) (n=10 experiments), ly6e (Smp_334920) (n=6 experiments) and litaf (Smp_333330) (n=10 experiments) following klf4 RNAi. (C) FISH results showing a significant increase in the number of ludp+ cells following klf4 RNAi (left); Double FISH (inset region indicated by the dotted rectangle from the left panel) showing expression of sm13 relative to the ludp+ cells (right); the Venn diagram in upper right shows the percentage of sm13+ cells expressing ludp. White arrows indicate the sm13+ludp+ cells, red arrows indicate the sm13-ludp+ cells. Scale bar, 100 µm. (D) Quantification of the number of ludp+ cells per mm of worm. Control (RNAi) n=34, klf4 (RNAi) n=38. (E) Quantification of percentage of sm13+ cells expressing ludp. Control (RNAi) n=20, klf4 (RNAi) n=21. (F) Double FISH showing expression of calpain relative to the ludp+ cells, and the Venn diagram in upper right shows the percentage of calpain+ cells expressing ludp. Control (RNAi) n=19, klf4 (RNAi) n=13. Scale bar, 10 µm. (G) Quantification of percentage of calpain+ cells expressing ludp. Scale bar, 10 µm. Data are presented as mean ± SD. Welch’s t-tests were performed in Panel A, D, E and G, and multiple paired t-tests were performed in Panel B.
Fig 6.
Knockdown of klf4 increases the production of ludp
+sm13+ cells. (A) FISH for tegumental markers with EdU detection in control (RNAi) and klf4 (RNAi) worms at day seven (D7) following an EdU pulse. Arrows represent EdU+ tegumental cells. (B) Quantification of the percentage of tegumental cells that are EdU+ following a 7-day chase period. Control (RNAi) n=28, klf4 (RNAi) n=29. (C) Double FISH for sm13 and ludp with EdU detection in control (RNAi) and klf4 (RNAi) worms at D7 following an EdU pulse. Red arrows represent EdU+sm13+ cells, white arrows represent EdU+ludp+ cells and red arrows with white outline represent EdU+ludp+sm13 cells. (D) Quantification of percentage of ludp+sm13+ cells that are EdU+ following a 7-day chase period following klf4 RNAi. Control (RNAi) n=16, klf4 (RNAi) n=16. Scale bar, 10 µm. Data are presented as mean ± SD. An unpaired t-test and a Welch’s t-test were performed in Panel B and D, respectively.
Fig 7.
Model for the role of klf4 in tegumental maintenance.
Left: The tegument depends on pool of tsp-2+ tegument progenitor cells. Cells of the egc+/meg-1+/zfp-1-1+ TRL commit to transition state egf-1+ cells that fuse with the tegument, while sm13+ TRL cells commit to transition state ludp+ cells that likewise fuse with the tegument. Each TRL delivers a unique set of protein cargos to the tegumental syncytium (green or yellow circles). Right: In klf4 (RNAi) worms, loss of klf4 blunts the commitment of neoblasts to the egc+/meg-1+/zfp-1-1+ TRL resulting in an increase in the rate at which sm13+ lineage seeds new tegumental cell birth, thereby leading to alternation in the molecular composition of the tegumental syncytium.