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

TY - JOUR

T1 - Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization

AU - Dillon, G.P.

AU - Illesh, J.C.

AU - Isaacs, H.V.

AU - Wilson, R.A.

N1 - Copyright © Cambridge University Press 2007. Reproduced in accordance with the publisher's self archiving policy. Available from August 2008.

PY - 2007/10

Y1 - 2007/10

N2 - rom 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.

AB - rom 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.

KW - Schistosoma mansoni

KW - RNA probe

KW - confocal microscopy

U2 - 10.1017/S0031182007002995

DO - 10.1017/S0031182007002995

M3 - Article

SN - 0031-1820

VL - 134

SP - 1589

EP - 1597

JO - Parasitology

JF - Parasitology

IS - 11

ER -