Genome-wide transcriptome profiling and spatial expression analyses identify signals and switches of development in tapeworms

Abstract

Background: Tapeworms are agents of neglected tropical diseases responsible for significant health problems and economic loss. They also exhibit adaptations to a parasitic lifestyle that confound comparisons of their development with other animals. Identifying the genetic factors regulating their complex ontogeny is essential to understanding unique aspects of their biology and for advancing novel therapeutics. Here we use RNA sequencing to identify up-regulated signalling components, transcription factors and post-transcriptional/translational regulators (genes of interest, GOI) in the transcriptomes of Larvae and different regions of segmented worms in the tapeworm Hymenolepis microstoma and combine this with spatial gene expression analyses of a selection of genes.

Results: RNA-seq reads collectively mapped to 90% of the > 12,000 gene models in the H. microstoma v.2 genome assembly, demonstrating that the transcriptome profiles captured a high percentage of predicted genes. Contrasts made between the transcriptomes of Larvae and whole, adult worms, and between the Scolex-Neck, mature strobila and gravid strobila, resulted in 4.5-30% of the genes determined to be differentially expressed. Among these, we identified 190 unique GOI up-regulated in one or more contrasts, including a large range of zinc finger, homeobox and other transcription factors, components of Wnt, Notch, Hedgehog and TGF-β/BMP signalling, and post-transcriptional regulators (e.g. Boule, Pumilio). Heatmap clusterings based on overall expression and on select groups of genes representing 'signals' and 'switches' showed that expression in the Scolex-Neck region is more similar to that of Larvae than to the mature or gravid regions of the adult worm, which was further reflected in large overlap of up-regulated GOI.

Conclusions: Spatial expression analyses in Larvae and adult worms corroborated inferences made from quantitative RNA-seq data and in most cases indicated consistency with canonical roles of the genes in other animals, including free-living flatworms. Recapitulation of developmental factors up-regulated during larval metamorphosis suggests that strobilar growth involves many of the same underlying gene regulatory networks despite the significant disparity in developmental outcomes. The majority of genes identified were investigated in tapeworms for the first time, setting the stage for advancing our understanding of developmental genetics in an important group of flatworm parasites.

Keywords: Differential gene expression; Hymenolepis; Post-transcriptional regulators; RNA-seq; Signalling factors; Tapeworms; Transcription factors; Transcriptomics.

Fig. 1

RNA-seq samples. a Hymenolepis microstoma life cycle illustrating the stages and regions sampled for transcriptome profiling (dashed boxes) described in Table 1 (whole worm illustration modified from [42]). b Principal component analysis showing clustering of sample replicates and relative variance among samples across PC1 and PC2. c Heatmap clustering of sample replicates showing hierarchical relationships based on overall expression similarity (n.b. each of the three larval samples was split and sequenced as two technical replicates). Note clustering of the Scolex-Neck samples with the Larvae samples rather than with the other adult samples

Fig. 2

Heatmaps based on mean, normalised expression of selected factors representing ‘signals’ and ‘switches’. a All homeobox transcription factors [35]. b Wnt, Notch and Hedgehog signalling components [35, 63, 178]. c Differentially expressed genes of interest (Table 2, Additional file 4: Table S4; note that some of the factors in a, b were differentially expressed in one or more of the contrasts and are therefore also represented in c). All three suites of genes produce the same hierarchical clustering of the samples, congruent with that based on overall gene expression (Fig. 1c)

Fig. 3

Comparison of Gene Ontology biological process annotations (slimmed) for the gene models across the samples. GO hits were counted only for models with mean, normalised expression levels ≥ 10. Note a generally smaller number of GO annotations in Larvae compared to the samples representing the adult worm

Fig. 4

Spatial gene expression during larval metamorphosis. Larvae are staged as shown in Fig. 1a. Asterisks in d the nascent suckers and rostellar bulb, and arrows indicate the regions of the Larvae that give rise to the juvenile worm, cyst tissues and tail. Dotted line in e the boundary between the encysted juvenile worm and surrounding tissues. Arrows show oncospheral hooks where visible, indicating the larval posterior [63]. Gene models: aris-like (HmN_000064700), foxQ2 (HmN_000125600), myoD (HmN_000553800), otp (HmN_000845400), pou4 (HmN_000747700), pou-like (HmN_000074300), soxPF-1 (HmN_000208300), zf581200 (HmN_000581200), zf798800 (HmN_000798800). All scale bars 50 μm

Fig. 5

Spatial gene expression in the Scolex-Neck. a–d Expression in the scolex, neck and immature strobila. f Expression of snail in the genital primordia. g Expression of bZIP137200 in the nascent seminal receptacle. h, i Expression of tgfb-like in immature segments. Arrows in h segmental, dorsoventrally paired foci, and dotted box in i punctate stripe of expression across the segments, both patterns consistent with the positions of segmentally repeated elements of the nervous system [39]. j Expression of zf631300 showing punctate expression in the cortex. Gene models: bZIP137200 (HmN_000137200), six3/6 (HmN_000022100), snail (HmN_000348000), tgfb-like (HmN_000204000), zf631300 (HmN_000631300). Scale bars 200 μm (a–e), 50 μm (f–j)

Fig. 6

Spatial gene expression in the mature strobila. Arrows in f–h indicate expression foci around the genital pore. esc external seminal vesicle, gp genital pore, esv external seminal vesicle, isv internal seminal vesicle, oc osmoregulatory canal, ov ovary, sr seminal receptacle, t testes, ut uterus, v vagina, vt vitellarium. Gene models: boule (HmN_000762300), extra-like (HmN_000610200), foxC-like (HmN_000142300), msxlx (HmN_000016500), nk1 (HmN_000601100), otx (HmN_000666600), pax-like (HmN_000199100), prox2 (HmN_000961900), zf621400 (HmN_000621400), zyg11-like (HmN_000995400). All scale bars 100 μm