Localization of gender-regulated gene expression in the filarial nematode Brugia malayi.

We used in situ hybridization (ISH) to localize expression of gender-biased genes in the filarial parasite Brugia malayi that were previously identified by microarray analysis and quantitative reverse transcriptase PCR (qRT-PCR). We studied seven genes with male-biased expression, 11 genes with female-biased expression, and one control gene with equal expression in males and females. RNA probes were hybridized to frozen sections of adult worms. ISH confirmed gender-biased expression for all 18 of the differentially expressed genes and non-biased expression for the control. We identified six patterns of expression for these genes. As expected, most of the gender-biased genes were expressed in reproductive organs, developing gametes and embryos. Hybridization signal intensities correlated with relative mRNA levels as assessed by qRT-PCR. Some of the differentially expressed genes had tightly regulated expression patterns. For example, a high mobility group protein gene (Bm-hmg) was exclusively expressed in developing larvae in females. Expression was first detected in late stage oocytes, peaked in morula stage embryos and no signal was detected in late pretzel stage or in stretched microfilariae. Another female up-regulated gene (microfilarial sheath protein Bm-shp-1) was exclusively expressed in the epithelium of uterine sections that contained morulae or early pretzel embryos. No signal was detected in other female structures, in late embryos or in male worms. This result suggests that microfilarial sheath proteins are produced by the uterus epithelium and not by embryos. Transcripts of the male-upregulated major sperm protein-1 (Bm-msp-1) were detected in spermatocytes in the early spermatogenesis zone and in spermatids but not in spermatozoa in the vas deferens. Thus, ISH provides a means to independently confirm differential expression of genes identified by other methods. In addition, localization patterns provide insight regarding the function of known or novel genes in the parasite.