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Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization

Published online by Cambridge University Press:  09 August 2007

G. P. DILLON ,
J. C. ILLES ,
H. V. ISAACS  and
R. A. WILSON
G. P. DILLON*
Affiliation:
Biology Department, University of York, Heslington, York YO10 5DD, UK
J. C. ILLES
Affiliation:
Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK
H. V. ISAACS
Affiliation:
Biology Department, University of York, Heslington, York YO10 5DD, UK
R. A. WILSON
Affiliation:
Biology Department, University of York, Heslington, York YO10 5DD, UK
*
*Corresponding author: Biology Department, University of York, Heslington, York YO10 5DD, UK. Tel: +44 (0) 1904 328592. Fax: +44 (0) 1904 328505. E-mail: [email protected]
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Summary

As a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

Keywords

Schistosoma mansoniRNA probeconfocal microscopy
Type
Research Article
Information
Parasitology , Volume 134 , Issue 11 , October 2007 , pp. 1589 - 1597
Copyright
Copyright © Cambridge University Press 2007

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