Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

Abstract

Background: The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells.

Method: The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction.

Results: Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0-2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1-3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function.

Conclusions: These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

Figure 1

A. Expression of KIT gene using semi-quantitative RT-PCR, in gastric fundic tissues from wild type and W/W^V mutant mice maintained under fasted conditions. GAPDH levels were measured as a control. B. Representative image of DNA microarray hybridization. Alterations in gene expression that were represented by the ratio of intensity of fluorescence measured using Cy3 and Cy5 fluorochromes. Expression profiles were evaluated by reverse transcription of poly (A)+ RNAs from gastric fundic muscles from wild type and W/W^V mice in the presence of Cy3 or Cy5 fluorescent labeling dyes followed by hybridization to mouse GEM 1 microarrays. The image shown was produced by superimposing the Cy5 fluorescence image (pseudo-coloured green that represents expression level of RNA from the tunica muscularis of W/W^V fundus) and the Cy3 fluorescence image (pseudo-coloured red that represents RNA expression from wild type tissues). Arrays were counterstained with DAPI (blue). C. Confirmation of the reliability of the microarray data by semi-quantitative RT-PCR. Representative examples of RT-PCR analysis, in fundic tissues from six wild type and six W/W^V mutant mice maintained under starved conditions. Expression of the CPO gene was considerably reduced in the fundus of W/W^V mice compared to age matched wild type mice, whereas the MCM7 gene showed an increase in expression in W/W^V mice. D. Relative expression level of CPO (CPO/GAPDH) and MCM7 (MCM7/GAPDH) in fundic tissues from six wild type and six W/W^V mutant mice. The data represent the mean ( ± SD) of six different RT-PCR experiments using six wild type or W/W^V fundus RNA as templates.