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Comparative Study
. 2009 Feb 11:9:10.
doi: 10.1186/1471-213X-9-10.

Expression analysis of the CLCA gene family in mouse and human with emphasis on the nervous system

Marko Piirsoo  1 , Dies Meijer, Tõnis Timmusk
Affiliations
Comparative Study

Expression analysis of the CLCA gene family in mouse and human with emphasis on the nervous system

Marko Piirsoo et al. BMC Dev Biol. .

Abstract

Background: Members of the calcium-activated chloride channel (CLCA) gene family have been suggested to possess a variety of functions including cell adhesion and tumor suppression. Expression of CLCA family members has mostly been analyzed in non-neural tissues. Here we describe the expression of mouse and human CLCA genes in the nervous system.

Results: We show that from the six mouse CLCA family members only Clca1, Clca2 and Clca4 mRNAs are expressed in the adult brain, predominantly in olfactory ensheathing cells. During mouse nervous system development Clca1/2 is more widely expressed, particularly in cranial nerves, the diencephalon and in the cerebral cortex. While human CLCA2 and CLCA4 genes are widely expressed in brain, and at particularly high levels in the optic nerve, human CLCA3, the closest homologue of mouse Clca1, Clca2 and Clca4, is not expressed in the brain. Furthermore, we characterize the expression pattern of mouse Clca1/2 genes during embryonic development by in situ hybridization.

Conclusion: The data published in this article indicate that within the nervous system mouse Clca1/2 genes are highly expressed in the cells ensheathing cranial nerves. Human CLCA2 and CLCA4 mRNAs are expressed at high level in optic nerve. High level expression of CLCA family members in mouse and human glial cells ensheathing nerves suggests a specific role for CLCA proteins in the development and homeostasis of these cells.

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Figures

Figure 1
Figure 1
A) Semiquantitative RT-PCR analysis of mClca1-mClca6 and control mRNA gapdh expression in mouse tissues. B) Real-time PCR analysis of mClca1, mClca2 and mClca4 expression in the brain at indicated developmental timepoints. Expression level is shown relative to the expression level of the respective mRNA at embryonic day 13. C) Real-time PCR analysis of mClca1, mClca2 and mClca4 expression in mouse brain regions. Expression levels are shown relative to the expression level in the cerebral cortex.
Figure 2
Figure 2
In situ hybridization analysis of mClca1/2 and mClca4 expression in the mouse brain. Dark-field emulsion autoradiographs of whole P9 and adult brain mid saggital sections are shown in A-C. Hematoxylin-eosin stained bright-field images (D, E and F) and corresponding dark-field emulsion autoradiographs (G, H and I) are shown at 40× magnification. Lack of in situ hybridization signal using corresponding sense probes is shown in J-O. Cellular distribution of Clca1/2 expression in P9 mouse cerebral cortex and olfactory bulb and adult mouse olfactory bulb are shown in J-L at 600× magnification. Abbreviations: CBL – cerebellum; CTX – cortex; GL – glomerular cell layer; LI – cerebral cortex layer I; LII-III – cerebral cortex layers II-III; OB – olfactory bulb; ONL – olfactory nerve layer.
Figure 3
Figure 3
In situ hybridization analysis of mClca1/2 expression in E13 mouse embryo. Dark-field emulsion autoradiograph depicting whole embryo is shown in A. Hematoxylin-eosin stained bright-field images (B, E, H, K, N, R at 100× magnification and D, G, J, M, P, T at 600× magnification) and corresponding dark-field emulsion autoradiographs (C, F, I, L, O and S) are shown corresponding to various parts of the embryo. Abbreviations: ao-aorta; he-heart; in-intestine; on-olfactory nerve nc-nasal cavity; ne-spinal nerve; ur-urethra; ve-vertebrae.
Figure 4
Figure 4
In situ hybridization analysis of mClca1/2 expression in E17 mouse embryo. Dark-field emulsion autoradiograph depicting whole embryo is shown in A. Hematoxylin-eosin stained bright-field images (B, D, F, H, J, L, N, P, S, U, W) and corresponding dark-field emulsion autoradiographs (C, E, G, I, K, M, O, R, T, V, X) taken at 100× magnification, corresponding to various parts of the embryo are shown. Abbreviations: di-diencephalon; fv-follicles of vibrissae; he-heart; in-intestine; li-liver; lu-lung; oe-olfactory epithelium; on-optic nerve; sg-submandibular gland; sk-skin; tr-trachea; tri-trigeminal nerve; ur-urethra; ve-vertebrae.
Figure 5
Figure 5
High magnification images of mClca1/2 expression in the nervous system at E17. Images of diencephalon (A), optic nerve (B), olfactory nerve (C) and trigeminal nerve (D) are shown at 600× magnification. Abbreviations: di-diencephalon; oln-olfactory nerve; on-optic nerve; tri-trigeminal nerve.
Figure 6
Figure 6
Structure of human, mouse and rat CLCA locus and homology between CLCA family members. Schematic depiction of order of succession of genes within human, mouse and rat CLCA locus is shown in A (the figure is not drawn in scale). Homology tree showing relationship between human, mouse and rat CLCA proteins (B). The dendrogram was generated using DNAMAN software (Lynnon Biosoft) with complete amino acid sequences of the CLCA proteins.
Figure 7
Figure 7
A) Semiquantitative RT-PCR analysis of human CLCA1-4 mRNA expression and control mRNA hprt in adult human tissues. B) Real-time PCR analysis of CLCA2 and CLCA4 expression in adult human brain regions. Expression levels are shown relative to the expression level in the cerebral cortex.
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