Fig 1.
F. hepatica neoblast-like cells are inherently linked to juvenile growth.
(A) Confocal images of juvenile F. hepatica cultured for nine days in either growth inducing media (+CS) or non-growth inducing media (-CS) with labelling of proliferative cells (EdU, green) and all nuclei (DAPI, magenta), scale bars = 50 μm. (B) Comparison of # EdU+ nuclei in juveniles maintained in +CS and -CS shows that growth inducing +CS leads to a significant increase in the number of EdU+ nuclei (unpaired t test). (C) Plot of # EdU+ cells against worm area (each point represents an individual worm grown in +CS) shows a positive correlation (linear regression analysis, p<0.0001), symbols represent different worms of different ages. ****, p<0.0001.
Fig 2.
Evidence of differentiation by F. hepatica neoblast-like cells.
(A) Confocal images with EdU labelling (green) show pulse-chase trial where EdU+ cells migrate to worm midline and lateral margins over a one-week chase period (blue = # of days post-pulse). (B) The dissociation of juveniles just after EdU incubation illustrates how EdU+ cells (images on left) have ovoid morphology with scant cytoplasm, in contrast to EdU- cells (image on right). (C) The dissociation of juveniles 72 hours after EdU incubation illustrates how EdU+ cells (images on left) have a similar morphology to EdU- cells (image on right), suggesting cell differentiation. Differential interference contrast microscopy and DAPI counterstain (magenta) used to provide context of images. Scale bars; A = 50 μm; B/C = 10 μm. Schematic figure created with BioRender.com.
Fig 3.
hepatica neoblast-like cells are capable of self-renewal.
F. (A) Timeline of dual labelling with the thymidine analogues EdU and BrdU where colocalisation would be indicative of self-renewal. (B) Confocal images of EdU (green), BrdU (red) and merged channels showing colocalisation of EdU/BrdU (yellow/orange) in worms treated according to above timeline, supporting the presence of self-renewing cells. Scale bar = 100 μm. (C) Confocal image of the mid-region of a juvenile labelled with EdU/BrdU according to the above timeline shows colocalisation of EdU/BrdU (orange arrowheads) as well as the presence of doublets (white arrowheads). Scale bar = 10 μm. (D) Asymmetric pairing of EdU+/BrdU+ and EdU+/BrdU- cells (white arrowhead). Scale bar = 10 μm. (E) Based on the percentage of proliferating cells that were EdU+, BrdU+ or EdU/BrdU+ (n = 6), it was evident that ~50% of these cells had self-replicated within the experimental timeframe. Schematic figure created with BioRender.com.
Fig 4.
The neoblast-like cells of F. hepatica are sensitive to ionising radiation.
(A) Confocal images of three-day old juvenile F. hepatica irradiated with 200 Gy before 39 further days of culture shows that irradiated worms do not recover EdU+ nuclei (blue text = age; black text = # days post irradiation). EdU (green), DAPI (magenta); Scale bars = 50 μm. (B) # EdU+ nuclei in 200 Gy irradiated and non-irradiated worms tracked over 39 days following irradiation (blue text = age; black text = #days post irradiation) shows significant reduction in # EdU+ at all time points (Kruskal-Wallis and Dunn’s posthoc test). (C/D) Dark-field images of control (C) and 200 Gy irradiated (D) worms 39 days post irradiation shows reduced growth in irradiated worms; scale bars = 200 μm. (E) Confocal images of two-day-old juvenile F. hepatica treated with 0–200 Gy radiation as metacercariae prior to excystment demonstrates dose dependent effects of irradiation on EdU+ nuclei (green), DAPI (magenta); scale bars = 50 μm. (F) # EdU+ nuclei in two-day-old juvenile F. hepatica treated with 10, 100 or 200 Gy radiation as metacercariae (Kruskal-Wallis and Dunn’s posthoc test). **, p<0.01; ****, p<0.0001.
Fig 5.
Irradiation of juvenile F. hepatica downregulates stem cell associated transcripts.
(A) Timeline of experiment to generate irradiated transcriptomes where juvenile F. hepatica were grown for 20 days in vitro before treatment group was irradiated with 200 Gys to ablate stem cells. (B) Heatmap of significantly differentially expressed genes, as identified by DESeq2, highlights 124 downregulated genes following irradiation. (C) TOPGO analysis shows genes associated with cell replication, and localised to nucleus, overrepresented among downregulated genes following irradiation. (D) Significantly downregulated KEGG pathways following irradiation. (E) KEGG cell cycle pathway shows that many pathway components are downregulated (upregulated, red; no change, grey; downregulated, blue; unassigned KEGG ID, white; differentially expressed, orange box). (F) TOPGO analysis shows genes associated with microtubules and TOR signalling overrepresented among upregulated genes following irradiation. Schematic figure created with BioRender.com.
Fig 6.
RNAi mediated knockdown of fhh2b significantly reduces stem cell associated transcripts and neoblast-like nuclei in juvenile F. hepatica in vitro.
(A) Expression of fhh2b (white), fhh2a (green), fhfgfrA (purple) and fhzfp-1 (pink) relative to fhgapdh in in vitro juvenile F. hepatica following three weeks of repeated exposures to fhh2b dsRNA shows significant knockdown of stem cell associated transcripts fhh2b, fhh2a and fhfgfrA relative to control dsRNA treated worms (unpaired t tests, n = 3). (B) The in vitro growth (area in μm2) of juvenile F. hepatica was significantly reduced after three weeks of repeated exposure to fhh2b dsRNA (Kruskal-Wallis and Dunn’s posthoc tests; week 1, n = 173; week 2, n = 171; week 3, n = 161). (C) Darkfield images of three-week old in vitro juvenile F. hepatica show how juveniles treated repeatedly with fhh2b dsRNA are significantly less developed than those treated with control dsRNA; scale bars = 1 mm. (D) Confocal images of EdU staining (green) in three-week-old in vitro F. hepatica treated with fhhh2b dsRNA show ablation of EdU+ nuclei compared to control dsRNA treated worms; scale bars = 100 μm, DAPI counterstain (magenta). (E) # EdU+ nuclei are significantly reduced in three-week-old F. hepatica juveniles following repeated treatment with fhh2b dsRNA (Mann-Whitney U test, n = 48). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.