Gonad RNA-specific qRT-PCR analyses identify genes with potential functions in schistosome reproduction such as SmFz1 and SmFGFRs

Abstract

In the search for new strategies to fight schistosomiasis, the unique reproductive biology of Schistosoma mansoni has come into the focus of research. The development of the gonads and the ability of egg production are fundamental not only for continuing the life cycle but also for pathogenicity. Previous studies of schistosome biology demonstrated an influence of pairing on gonad development of the female and on gene expression profiles in both genders. Due to the limited access to specific tissues, however, most of these studies were done at the level of whole worms neglecting individual tissues that may be targets of pairing-dependent processes. Recently, we established a protocol allowing the isolation of testes and ovaries from adult S. mansoni. Here, we describe tissue-specific qRT-PCR analyses comparing transcript levels of selected genes on the basis of RNA from gonads and whole worms. Gene expression in ovary and testes was in some cases found to be significantly influenced by pairing, which was not traceable in whole worms. Among the candidate genes identified as regulated by pairing in gonads were the frizzled homolog SmFz1 and the two fibroblast growth factor receptor homologs SmFGFR-A and SmFGFR-B. First functional characterizations were done, including comparative qRT-PCR analyses, in situ-localization experiments, heterologous expression in Xenopus oocytes (SmFGFR-A/B), and inhibitor studies using the Fz/Dvl-pathway inhibitor 3289-8625, or BIBF1120 blocking FGFR-signaling. Besides confirming gonad localization and receptor functions, inhibitor-induced phenotypes were observed in vitro such as decreased egg production as well as drastic effects on gonad differentiation, morphology, embryogenesis, and survival of adult worms. In summary, these results emphasise the usefulness of tissue-specific qRT-PCRs for selection of candidate genes with important roles in reproduction, allowing subsequent studies to determine their suitability as drug targets.

The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction

“…We therefore carried out additional BLASTP searches on the E. multilocularis genome using human VEGF- and PDGF-receptors as queries, but only obtained significant hits with the above mentioned TKDs of EmFR1, EmFR2, and EmFR3 (data not shown). These data indicated that members of the VEGF- and PDGF-receptor families are absent in Echinococcus , as has already been described for the closely related schistosomes [24].…”

“…Furthermore, similar FGF receptors were also detected in stem cells of the planarian Schmidtea mediterranea [23]. In the genome of the flatworm parasite species Schistosoma mansoni , two FGFR-encoding genes were identified of which fgfr A codes for a predicted protein with two extracellular immunoglobulin domains and a split tyrosine kinase domain whereas the fgfr B gene product only comprises one immunoglobulin domain in the extracellular region [24]. Expression of fgfr A and fgfr B in neoblast-like somatic stem cells has been shown and evidence was obtained for an important role of both receptors in schistosome stem cell maintenance [25-27].…”

Serine/threonine protein phosphatase 1 (PP1) controls growth and reproduction in Schistosoma japonicum

“…In addition to down-regulation of eggshell synthesis-associated genes in females, such as putative eggshell protein, trematode eggshell synthesis protein, and tyrosinases after Sj-pp1cs knockdown ( Table 1), stem cell and neoblast-associated genes such as fibroblast growth factor receptor (Sjp_ 0112660), argonaute-2 (Sjp_0045200), and nanos (Sjp_ 0060520) were also down-regulated (genes known to participate in gonadal stem cell activities) (24,54). Among these, fibroblast growth factor receptors are not only involved in regulation and maintenance of proliferating somatic cells but are also required for gonad differentiation and reproduction in S. mansoni (24,55). Argonaute and nanos proteins are indispensable for germinal stem cell maintenance in planarians (56,57); their gonad-specific transcription in S. mansoni (54,58,59) also indicates that both genes could function in germline development and maintenance of schistosomes.…”

Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

“…Recently, two Venus Kinase Receptors (VKR), that belong to a novel family of RTKs (discovered for the first time in schistosomes) ( Vicogne et al, 2003; Vanderstraete et al, 2013a; Dissous et al, 2014b ) and that possess a TK domain similar to that of IR ( Ahier et al, 2009; Gouignard et al, 2012 ), were demonstrated to control parasite reproductive activities ( Vanderstraete et al, 2014 ). Also, two proteins of the FGFR (Fibroblast Growth Factor Receptor) family could participate with these RTKs in the development of reproductive organs ( Hahnel et al, 2014; Morel et al, 2014 ). All these schistosome RTKs can be inhibited by commercially available kinase inhibitors as well as the non-receptor CTKs that participate to their downstream signaling.…”