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A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract
Published on: August 19, 2011
In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs
1Department of Pharmacology, Faculty of Medicine, Kyoto University, Japan.
Abstract:
1. Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co-localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2. IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3. IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4. IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small- and large-sized neurones were labelled but no labelling was found in the glia. Expression of PPTA mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor-positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor-positive neurons co-expressed the EP1, EP3 and EP4 receptor, respectively. 5. These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.
Insights
This study reveals prostacyclin receptor (IP receptor) mRNA expression in mouse thymus, spleen, vascular smooth muscle cells, and dorsal root ganglion neurons. IP receptor actions in these tissues warrant further investigation.
Area of Science:
- Immunology
- Neuroscience
- Cardiovascular Biology
Background:
- Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation.
- The prostacyclin receptor (IP receptor) mediates these effects.
- Understanding IP receptor distribution is crucial for elucidating prostacyclin's physiological roles.
Purpose of the Study:
- To investigate the expression patterns of IP receptor mRNA in various mouse organs.
- To identify specific cell types expressing IP receptor mRNA.
- To examine co-localization with other key molecular markers.
Main Methods:
- In situ hybridization to detect IP receptor mRNA expression.
- Double in situ hybridization to analyze co-localization with preprotachykinin A (PPTA), prostaglandin E receptor subtypes (EP1, EP3, EP4), and renin mRNA.
- Analysis in thymus, spleen, vascular tissues, dorsal root ganglion, and kidney.
Main Results:
- IP receptor mRNA was detected in thymus medulla (mature thymocytes), spleen (lymphocytes, megakaryocytes), vascular smooth muscle cells (aorta, coronary, pulmonary, cerebral arteries), and dorsal root ganglion neurons.
- IP receptor mRNA was absent in veins and juxtaglomerular (JG) cells of the kidney.
- Significant co-expression of IP receptor mRNA with PPTA mRNA in dorsal root ganglion neurons was observed, alongside co-expression with EP1, EP3, and EP4 receptors.
Conclusions:
- IP receptor expression is confirmed in tissues sensitive to prostacyclin and identified in novel locations like the thymus, spleen, and neurons.
- Prostacyclin's actions on blood vessels and platelets are mediated by the same receptor type.
- Prostacyclin's effect on renin release may be indirect due to the absence of IP receptors in JG cells.

