ARTICLE |
Correspondence to: Richard E. Tashian, Dept. of Human Genetics, Med Sci II, M4708, U. of Michigan Medical School, Ann Arbor, MI 48109-0618.
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Summary |
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The spatial expression patterns of the two -carbonic anhydrase genes, CA VII and CA-RP VIII (called Car7 and Car8 in the mouse) were examined in the mouse brain by in situ hybridization. These two genes are the most highly conserved evolutionarily among the mammalian
-CAs. Both genes showed a similarly wide expression pattern in the brain. In the cerebrum, mRNA expression was detected in the pia, choroid plexus, and neurons of the cortical layer, thalamus, and medial habenulae. A high level of expression appeared in the pyramidal and granular cells of the hippocampus. In the cerebellum, both Car7 and Car8 were transcribed to different degrees in the Purkinje cells, and a lower expression level occured in the molecular and granular cell layers. Transcription signals for both genes were excluded from the white matter regions. (J Histochem Cytochem 45:657-662, 1997)
Key Words: CA VII (Car7), CA-RP VIII (Car8), in situ hybridization, mouse brain, gene expression
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Introduction |
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Expression of the -carbonic anhydrases (
-CAs) in amniotes is under the control of 10 autosomal genes (
CA activity was demonstrated in neural tissues as early as the1940s. This was further confirmed by many subsequent studies carried out by many different investigators. Most of these studies used enzymatic cytochemical approaches that detect CA activity without identifying the specific isozyme. These studies localized the CA activity extraneuronally to the oligodendroglial and choroid plexus cells (
In this study, we examined by in situ hybridization the mRNA expression of one catalytic CA (CA VII) and one acatalytic CA (CA-RP VIII) in the mouse brain. The amino acid sequences of their protein products are the most highly conserved of the -CAs between the human and mouse homologues, the percent sequence identity being
95% for the CA VIIs and
98% for the CA VIIIs (
The CA VII gene was isolated and characterized from a human genomic library (
Throughout this report, the mouse CA VII and CA-RP VIII genes will be given the designations Car7 and Car8, respectively, in keeping with nomenclature policy for the naming of murine CA genes.
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Materials and Methods |
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Tissue Preparation
Healthy 18-day-old female mice (C57 Bl/6J) were sacrificed according to the guidelines of the University of Michigan Commitee on the Use and Care of Animals (UCUCA), and their brains were rapidly isolated. The cerebrum and the cerebellum were separated, frozen in isopentane cooled to -30C and stored frozen at -80C. They were then equilibrated to cryostat temperature (-18C), mounted in OCT medium (Lab-Tek Division; Miles, Elkhart, IN), and frozen as solid blocks, which were later cryosectioned at 12-µm thickness. Sections were thaw-mounted on poly-L-lysine-coated slides and stored at -80C until processed.
Riboprobe Preparation
Specific probes for each gene were prepared by reverse transcription-polymerase chain reaction (RT-PCR) of mRNA isolated from the mouse brain. This mRNA was first reverse-transcribed into cDNA by superscript II (Life Technologies; Bethesda, MD). The cDNA probes for both Car7 and Car8 genes were made by PCR of this cDNA template using Car7- and Car8-specific sets of primers. The amplified cDNAs for Car7 (446 BP) and Car8 (500 BP) were independently cloned in pAMP1 vector (Life Technologies) such that the anti-sense transcript was under control of the T7 promoter, whereas the sense transcript was under control of the SP6 promoter. Transcription reaction was performed according to standard procedures (
In Situ Hybridization and Autoradiography
The in situ hybridization procedure was an adaptation of previously described methods (
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Results and Discussion |
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In this study, we used in situ hybridization of 35S-labeled anti-sense riboprobes, specific for Car7 and Car8, to examine the regional distribution in the mouse brain of mRNAs encoded by these two functionally different CA genes. The enzyme histochemical studies used earlier for detecting CA expression in animal tissues did not demonstrate the specificity of the isozyme. These methods are unable to detect the acatalytic isozymes, such as CA-RP VIII (Car8).
The two riboprobes specific for each of these two genes were transcribed from two cDNA clones isolated from mouse brain by RT-PCR. The specificity of the hybridization signal was verified by using sense riboprobes for both genes that produced (in both cases) only a weak background random scatter of grains (Figure 1A, Figure 1B, and Figure 2A-2C).
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The transcripts of the two genes displayed slightly different anatomic distributions in the cerebellum (Figure 1). Both were expressed at a relatively high level in the Purkinje cells, although the Car8 transcription signal (Figure 1E and Figure 1F) was slightly stronger. A transcription signal for both genes appeared in the granular layer, but in this case the signal for Car7 (Figure 1C and Figure 1D) appeared to be stronger than that of Car8. In addition, a low level of mRNA expression for both genes was present in the molecular layer of the cerebellum. It is not clear whether this positive signal is due to late-migrating granular cells or to scattered basket and stellate cells present in the molecular layer. There was no detectable signal for either gene in the white matter.
The mRNA from both genes appeared to be distributed throughout the cerebrum in a fairly similar pattern (Figure 2). An mRNA expression signal appeared at the pial surface and in the large neurons throughout the cortical layers. A very strong transcription signal was present in the hippocampal formation, specifically in the pyramidal cells of Ammon's horn and in the granular cells of the dentate gyrus. In addition, a moderately strong signal for both genes was present in the medial habenulae and neurons of the thalamus. The signal appeared to be absent in the glial end-feet (glial limitans) at the pial surface and in the subventricular zone. In general, signal was also absent in the white matter layer. No expression was seen in blood vessels and capillaries. However, signal appeared in the choroid plexus and cerebrospinal fluid-containing channels (not shown).
The significance of the expression pattern of these two genes is not clear. Even though their expression appeared to be overlapping in the cerebrum, there was a slightly different pattern of expression in the cerebellum. Cross-hybridization is very unlikely under the experimental conditions used because the two sequences shared no more than 52% identity in their nucleo-tides, in addition to the insertion or deletion of 10 nucleotides between the two sequences. These results demonstrate a wide distribution of mRNA for both genes, suggesting a nonspecific but rather generalized function for the products of these genes in the brain. Their expression is characteristically strong in neurons. Clearly, because CA-RP VIII lacks CO2-hydrase activity, the two gene products are expected to have very different functions.
One can speculate on the possible function(s) of CA VII on the basis of what is known about other active CAs. CA activity is known to play a role in cerebrospinal fluid production and in regulation of its ionic constituents and pH (
It is more difficult to speculate on the possible function(s) of CA-RP VIII. The fact that 19 acidic amino acids (i.e., 15 Glu, 4 Asp) are found in the first 35 N-terminal residues of human and mouse CA-RP VIII (
In the longer term, it would be of interest to determine if alterations in expression of these genes can be correlated with different physiological conditions or activities of the animal. The nonspecific expression of these genes is not a characteristic only of the brain, because they are also expressed in a variety of other tissues during embryonic development (Lakkis et al., in press) as well as in adults, implying an important general role in the various tissues or perhaps very different roles.
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Acknowledgments |
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Supported by NIH grant GM24681.
We thank the Morphology Core Facility for assistance in tissue sectioning and the Cell Biology Laboratory, particularly Bruce Donohoe and Thomas Komorowsky, at the University of Michigan, for assistance in microscopy and computer scanning.
Received for publication July 25, 1996; accepted December 6, 1996.
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