Hahnel S et al. (2014), Gonad RNA-specific qRT-PCR analyses identify ge...

XB-ART-49737

Front Genet 2014 Jun 10;5:170. doi: 10.3389/fgene.2014.00170.

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

Hahnel S , Quack T , Parker-Manuel SJ , Lu Z , Vanderstraete M , Morel M , Dissous C , Cailliau K , Grevelding CG .

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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.

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Species referenced: Xenopus
Genes referenced: acta4 dvl2 msi1 notch1 tbx2

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	Figure 1. Influence of pairing on gene transcription in adult worms and gonads. Analyses of the influence of pairing on the transcription rates of candidate genes in whole worms and gonads. Transcription of SmFGFR-A, SmFGFR-B, SmPMRC1, and the musashi homolog were up-regulated in em compared to um, whereas SmFz1 and Notch were not differentially transcribed at the whole worm level. In contrast, focusing on the testes, all analyzed genes were transcribed more abundantly in the testes of em compared to those of um (A). Comparably, in female worms, most of the candidate genes were up-regulated following pairing with the exception of Notch and SmFz1, which appeared not differentially and less transcribed in ef compared to uf, respectively. Focusing on ovary-specific gene expression, transcription of SmPMRC1, Musashi as well as SmFz1 were strongly up-regulated by pairing. Compared to this, pairing led to a less remarkable effect on the expression the two FGFR-homologs and Notch in the female gonad (B). In both diagrams the statistical evaluation of three technical replicates is shown (error bars indicated).
	Figure 2. Domain structure analyses of SmFz1, SmFGFR-A, and SmFGFR-B. Schematic structures of the frizzled homolog SmFz1, and the two FGFR homologs SmFGFR-A and SmFGFR-B from S. mansoni. SmFz1 comprises an N-terminal signal peptide sequence (red) and an extracellular CRD domain (yellow) sufficient for Wnt ligand binding. The frizzled transmembrane domain (gray box) contains seven TMHs (I–VII). Following TMH VII, a Dvl-binding motif (KTLVSW) is located at the beginning of the intracellular C-terminus. Both FGFRs possess TK domains within their intracellular C-termini but differ in the structures of their extracellular parts (TMHs in blue). SmFGFR-A consists of an N-terminal signal peptide sequence (red) followed by two IG-like domains (green) sufficient for ligand binding (A). In contrast the N-terminus of SmFGFR-B is smaller in size and contains only one putative IG-like domain (dashed line), which was not rated as significant (B).
	Figure 3. Localization of transcripts of SmFz1, SmFGFR-A, and SmFGFR-B in adult S. mansoni. Results of in situ-hybridization experiments to localize transcripts of SmFz1 (A–C), SmFGFR-A (D–F), and SmFGFR-B (G–I) using DIG-labeled antisense-RNA probes on 5 μm sections of S. mansoni couples. Transcripts of all genes were detected in the testes of the male and the ovary of the female. SmFGFR-B transcripts were also observed in the vitellarium of the female and the parenchyma as well as the gastrodermis of both genders. Negative controls using sense-RNA probes showed no color reaction (J,K). [te, testes; ov, ovary; v, vitellarium; p, parenchyma, g, gastrodermis; ♂, male; ♀, female].
	Figure 4. Morphology of S. mansoni couples after treatment with the Frizzled-Dvl inhibitor 3289-8625 in vitro. S. mansoni couples were cultured in vitro for 96 h with different concentrations of the Frizzled-Dvl inhibitor 3289-8625 and subsequently studied by bright field microscopy and CLSM for morphological changes. Whereas no morphological alterations were seen in control worms (A–C), inhibitor treatment (500 μM) led to a reduced vitality accompanied by a separation of couples (D). Furthermore, CLSM-analyses revealed severe effects of the inhibitor on the morphology of the gonads. The testes of treated males contained large pore-like structures (E, arrows) whereas ovaries of females were mostly affected in the anterior part containing several damaged cells and cell fragments (F, arrows). [te, testes; sv, seminal vesicle; ov, ovary; io, immature oocytes; mo, mature oocytes; ♂, male; ♀, female].
	Figure 5. Influence of the inhibitor 3289-8625 on the development of S. mansoni eggs. S. mansoni couples were treated with different inhibitor concentrations in vitro. After 24 h worms were removed from the dishes, and the deposited eggs were cultured for an additional 3-4 day period. Subsequent analyses revealed a concentration-dependent effect of 3289-8625 on egg development compared to the DMSO control (A). The statistical evaluation of three independent experiments is shown (error bars indicated). Untreated eggs showed continued development and were categorized into the stages II and III of embryogenesis as defined by Jurberg et al. (2009) (B). Upon increasing inhibitor concentrations embryogenesis was remarkably affected resulting in a larger proportion of undeveloped eggs (C). [em: embryo; ma: macromeres; oc: oocyte; vc: vitelline cells].
	Figure 6. Influence of BIBF1120 on pairing stability and egg production on S. mansoni couples in vitro. Treatment of S. mansoni couples with BIBF1120 for 96 h in vitro revealed a concentration-dependent effect of the FGFR inhibitor on pairing stability (A) and egg production (B) compared to the control (DMSO). Whereas 1 μM BIBF1120 had no influence on pairing, couples treated with 5 μM separated during the experiment period. The highest inhibitor concentration (10 μM) supplied, led to a separation of all couples within the first 24 h (A). An obvious effect of BIBF1120 on egg production was also detected after 24 h. At this time point the inhibitor treatment led to a decrease of eggs down to 50% (1 μM) and to 20% (5 μM) compared to the control. At higher concentrations egg production was nearly completely disrupted (B). In both diagrams the statistical evaluation of three independent experiments is shown (error bars indicated).
	Figure 7. Morphological analyses of S. mansoni couples after in vitro treatment with the inhibitor BIBF1120. Couples of S. mansoni were treated with different concentrations of the FGFR inhibitor BIBF1120 for 96 h in vitro (D–I) und subsequently analyzed for morphological alterations. Worms supplied with 1 μM inhibitor showed no obvious changes in vitality and morphology compared to those of the DMSO control using bright field microscopy (A,D). A more detailed examination by CLSM revealed an effect of BIBF1120 treatment on the gonads of both genders. Testicular lobes of treated males were reduced in their diameter and the seminal vesicle contained fewer mature sperms (E, asterix). In females 1 μM of inhibitor led to a partial degradation of immature oocytes in the anterior part of the ovary (F, asterix) (DMSO control: B,C). In contrast 5 μM BIBF11120 had severe effects on worm vitality and pairing stability as well as morphology in general (G–I). Those worms showed a dramatically swollen intestinal tract and cells of the gonads were severely damaged. [te, testes; sv, seminal vesicle; ov, ovary; io, immature oocytes; mo, mature oocytes v, vitellarium; ga, gastrodermis; ♂, male; ♀, female].
	Figure 8. EdU-incorporation of BIBF1120-treated S. mansoni couples. S. mansoni couples were co-cultured in vitro for 48 h with BIBF1120, with the addition of EdU after 24 h, to investigate the influence of the inhibitor on mitotically active cells. In worms of the control group EdU+-cells were detected in the parenchyma and the gonads of both genders as well as in the vitellarium of the female (A–D). Application of 5 μM BIBF1120 led to a drastic decline of EdU+-cells in all tissues, although the effect on the ovary was the weakest (E–H). [te, testes; ov, ovary; io, immature oocytes; v, vitellarium; p, parenchyma; ♂, male; ♀, female].

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