Fig 1.
cbp1(RNAi) results in an increase in neoblast proliferation.
(A, B) Whole mount in situ hybridization showing cbp1 mRNA expression in (A) male and (B) female parasites. cbp1 is expressed broadly in the worm including in the testes and ovaries. (C, D) FISH showing expression of cbp1 with (C) Histone H2B and (D) tsp-2. Nuclei are in blue. (E, F) EdU labeling showing cell proliferation in (E) control and (F) cbp1(RNAi) male parasites. Images are tiled from multiple confocal stacks acquired from parasites at D14 of RNAi. Parasites were fixed after an overnight EdU pulse. (G,H) Quantification of EdU+ neoblasts per μm3 from (G) heads and (H) tails. Each dot represents counts from a confocal stack taken from a single male parasite and error bars represent 95% confidence intervals. Differences are statistically significant (p < 0.002, t-test). Quantification was performed on male parasites at D14 of RNAi. (I,J) WISH for neoblast markers (I) Histone H2B and (J) fgfrA in control and cbp1(RNAi) animals. Images are from D14 of RNAi and are representative of n > 10 male parasites. (K) FISH for Histone H2B in control and cbp1(RNAi) animals. Nuclei are in gray. Images are from D11 of RNAi. (L) FISH for tsp-2 in control and cbp1(RNAi) animals. D13 of RNAi. Anterior faces left in A-B, E-F, I, J, and L. Scale Bars: A,B 100 μm; C,D, and K 10 μm; E,F 500 μm; I,J, and L 250 μm.
Fig 2.
cbp1(RNAi) results in degeneration of the esophageal glands.
(A) Cartoon depicting the position of the schistosome esophageal gland relative to other tissues. (B) FISH for Histone H2B showing a mass of proliferative neoblasts in proximity to the esophageal glands at D11 of RNAi treatment. (C) Double FISH for the esophageal gland marker meg-4 and the neoblast marker Histone H2B following 11 days of control or cbp1 RNAi treatments. The number of esophageal gland cells is reduced and the number of proliferative neoblasts in increased. In extreme cases only a few meg-4+ cells remain in cbp1(RNAi) animals. Top and bottom panels for cbp1(RNAi) depict animals with differing phenotypic severities. Nuclei are in blue. (D) WISH for meg-4 at D10 of RNAi treatment. At this time point, by WISH three distinct phenotypic severities are observed: normal, intermediate, and severe. Normal animals possess relatively normal gland structure and meg-4+ labeling. Intermediate animals have clearly abnormal or degenerated gland structure and often possess ectopic meg-4+ cells (arrowhead) outside of the region were the gland resides in control animals. Severe animals possess few, if any, meg-4+ cells. (E) Plots depicting the relative fraction of animals that display normal, intermediate, or severe phenotypes with respect to the esophageal glands as observed by WISH for meg-4 at D2–D18 of RNAi. >12 animals from two separate experiments were observed for each time point. (F) Double FISH for Histone H2B and meg-4 at D11 in irradiated (+IRR) and unirradiated worms. Degeneration of esophageal glands in cbp1(RNAi) worms is not abrogated following irradiation. However, notably more isolated meg-4+ cells (Arrowheads) remain in unirradiated cbp1(RNAi) worms. Numbers indicate fraction of worms displaying phenotypes similar to those pictured. Nuclei are in blue. Anterior faces up in all images. Scale Bars: B, F 50 μm; C 20 μm; D 100 μm.
Fig 3.
cbp1(RNAi) leads to an elevation of cell death in degenerating esophageal glands and throughout the bodies of male parasites.
(A) Cartoon showing approximate positions of parasites examined for panels B-F. (B, C) TUNEL in the esophageal glands of (B) control(RNAi) or (C) cbp1(RNAi) parasites at D10 of treatment. The lectin PNA (green) is used as a marker of the esophageal gland. At this time point 28% of cbp1(RNAi) parasites displayed large number of TUNEL+ cells in the region around the esophageal gland. Numbers indicate fraction of animals observed with large numbers of TUNEL+ cells within the region of the esophageal gland. Insets are magnified views of the boxed regions. Nuclei are in blue. (D, E) Quantification of TUNEL+ cells per μm3 from (D) trunks and (E) tails. Each dot represents counts from a confocal stack taken from a single male parasite and error bars represent 95% confidence intervals. Differences are statistically significant at D10 and D14: D10 trunks (p < 0.02, t-test); D10 Tails (p < 0.001, t-test), D14 trunks (p < 0.005, t-test), and D14 tails (p < 0.0002, t-test). At least ten male parasites were examined at each time point. (F) TUNEL staining in control and cbp1(RNAi) parasites at D10 of RNAi. cbp1(RNAi) results in elevations in cell death relative to controls. Insets are magnified views of the boxed regions. Anterior faces up in all images. Scale Bars: B, C 20 μm; F, 50 μm.
Fig 4.
Physical injury initially induces cell death and increases in neoblast proliferation.
(A) Cartoon showing strategy to injure worms. (B) 4 hours post-injury the number of TUNEL+ cells is increased at the site of injury. Dashed line approximates the site of injury. Images representative of n = 21/23 male parasites. (C) EdU-incorporating neoblasts accumulate near wound site at 48 hours post-injury (n = 18/19 parasites). Parasites were fixed after a 4 hour EdU pulse. Dashed line in inset approximates the site of injury. (D) Histone H2B+ neoblasts at 72 hours post-injury are enriched around wound sites (n = 11/11 male parasites). Parasites were labeled with EdU for 4 hours at 48 hours post-injury and were fixed 24 hours later (72 hours post-injury). Arrowhead indicates approximate site of injury. (E) TUNEL staining for cell death and Phospho-Histone H3 (pH3) labeling for neoblasts in mitosis in animals at 48 hours post-injury. Mitotic neoblasts 48 hours post-injury are clustered at wound sites (n = 28/29 parasites), whereas the number of TUNEL+ cells are reduced in tissues adjacent to wound sites (n = 24/26 male parasites). Arrowhead indicates approximate site of injury. Scale Bars: 100 μm. D and E are titled images from multiple confocal stacks.
Fig 5.
cbp1 is essential for schistosome survival in vivo.
(A) Images of control and cbp1 RNAi-treated worms during in vitro culture. Until at least D19 of in vitro culture, control(RNAi) animals remain as male and female worm pairs, are firmly attached to the substrate, and maintain vigorous movement. By D8 of in vitro culture, cbp1(RNAi) animals become unpaired and fail to firmly attach to the substrate. Over time, movements of cbp1(RNAi) worms become less vigorous and oftentimes their heads curled ventrally (Cyan box, D15 time point). Cartoon depicting surgical procedure to examine the requirement for cbp1 function in vivo. (C) Images of three mice that were transplanted with control or cbp1 RNAi-treated parasites following hepatic vein perfusion. Unlike mice that received control(RNAi) worms, livers from mice receiving cbp1(RNAi) worms were normal-sized and the mice had few signs of infection. Plot showing quantification of the percent recovery of control and cbp1 RNAi-treated parasites from mice. Each dot represents percent recovery from an individual mouse. Two separate sets of transplantations were performed with n = 5 mice for controls and n = 8 mice for cbp1(RNAi). p < 0.0001, t-test. Representative livers from mice transplanted with control or cbp1 RNAi-treated parasites. Livers from mice that received control(RNAi) parasites were large and contained large numbers of granulomas. Livers from mice receiving cbp1(RNAi) parasites were normal sized and contained very few granulomas. Few large granuloma-like structures were often found at the periphery of livers from mice that received cbp1(RNAi) worms (arrowhead in inset). Plot depicting number of schistosome eggs per liver section from mice transplanted with control or cbp1 RNAi-treated parasites. Each dot represents the mean number of eggs counted from two liver sections from an individual mouse. n = 4 livers for both control and cbp1 RNAi treatment groups. H&E staining of liver tissue from mice transplanted with control or cbp1 RNAi-treated worms. Arrowheads point to eggs inside granulomas. Large granuloma-like masses in livers of mice from cbp1(RNAi) treatment group correspond to worms at various stages of degeneration. Left panel shows a male worm with a clearly identifiable tegument and intestine (labeled teg and gut in inset, respectively) surrounded by neutrophils and lymphocytes. As panels move to the right, worms appear to become structurally compromised and lesions contain more host fibroblasts, suggesting these lesions possess a more mature immune response to the parasites. Scale Bars: A,E 1 mm; G-H 100 μm.
Fig 6.
Model for observed consequences of loss of cbp1.
Reduced cbp1 function induces cell death and tissue degeneration. This triggers neoblast proliferation and an increased rate of neoblast differentiation to repair damaged tissues. Since cbp1 levels remain reduced, tissues continue to degenerate, triggering more neoblast proliferation. Parasites eventually die due to defects in tissue function.