Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse

Biochimica et Biophysica Acta 1428 (1999) 397^405 www.elsevier.com/locate/bba

Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse David A. Zacharias 1 , Claudia Kappen * Department of Biochemistry and Molecular Biology, Samuel C. Johnson Medical Research Center, Mayo Clinic Scottsdale, 13400 E. Shea Boulevard, Scottsdale, AZ 85259, USA Received 10 February 1999; accepted 13 April 1999

Abstract The plasma membrane calcium ATPases are critical components in the regulation of cellular calcium homeostasis and signaling. In mammals, there are 4 Pmca genes, and information on the cellular and tissue distribution of their expression during development will provide insight into the regulation and possible function of each Pmca isoform. Using specific probes and in situ hybridization, we found that the four Pmca genes are expressed in spatially overlapping but distinct patterns in the mouse embryo. The dynamic temporal patterns of expression indicate that the individual isoforms are subject to both positive and negative regulation. The differential and restricted expression of Pmca genes supports the notion that they play unique functional roles in mammalian development. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Calcium pump; Development; Embryo; Plasma membrane ATPase ; In situ hybridization; mRNA expression

1. Introduction Calcium is arguably the most ubiquitous second messenger molecule in eukaryotic cells. The proper regulation of Ca2‡ is critical for general cellular metabolism, neuronal signaling and formation of the skeleton [1^3]. All eukaryotic cells maintain a very low cytosolic level of free Ca2‡ . In neurons, for example, the resting level of free Ca2‡ ranges from

* Corresponding author. Fax: +1-602-301-7017; E-mail: [email protected] 1 Present address: Howard Hughes Medical Institute, Cellular and Molecular Medicine Q. 310, University of California School of Medicine, San Diego, La Jolla, CA 92093-0647, USA.

50 to 200 nM, 10 000 times lower than what is found in the extracellular milieu. Extrusion of Ca2‡ to the extracellular side and maintenance of the low level of free cytosolic Ca2‡ is accomplished primarily by the high-a¤nity, plasma membrane calcium ATPase (Pmca). In mammals, these proteins are encoded by four genes, and to date, there is only limited information available on their patterns of expression during development [4]. The Pmca1 and Pmca4 genes and corresponding proteins are expressed ubiquitously in the adult, and isoforms 2 and 3 are expressed almost exclusively in the nervous system (for a complete description see Stau¡er et al. [5,6]). Additionally, the mRNA transcript for each gene can be alternatively spliced, generating a variety of pumps with unique functional characteristics. Given the high degree of corresponding homology between species and the lower degree of similarity

0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 5 8 - 6

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between isoforms within a species, strong selective pressure appears to have maintained all four isoforms throughout evolution [7]. Conserved between isoforms are domains that are essential to the catalytic and transport functions, whereas regions of higher diversity are likely to re£ect isoform-speci¢c regulatory and functional specializations of each pump [7]. On the basis of their structural similarities, it is possible that some Pmca isoforms could be functionally redundant, so that the malfunction or absence of one isoform may be compensated for by another expressed in the same cell. To gain a clearer understanding of the functional signi¢cance for the presence of four distinct genes in the Pmca family and of the di¡erential regulation of Pmca expression, we have undertaken a study of Pmca gene expression in the developing mouse embryo. Information about the temporal and spatial pattern of expression during development is important to understanding the possible functional relevance of each Pmca gene for the tissues in which it is expressed, as well as for the survival of the organism. 2. Materials and methods 2.1. Nomenclature Pmca1 is encoded by the Atp2b1 gene (Human and Mouse Genome nomenclatures), Pmca2 by Atp2b2, Pmca3 by Atp2b3, Pmca4 by Atp2b4, respectively. Because of the prevalence of use of the Pmca designation in the literature, we chose this more popular designation throughout for the mRNAs, cDNAs and corresponding genes. 2.2. Generation of cRNA probes Complementary DNAs of the 3P-untranslated region (UTR) of each mouse Pmca gene were generated by reverse transcriptase polymerase chain reaction (RT-PCR) [8] using primers speci¢c for the 3PUTR of the corresponding rat messages (each primer is listed in the 5P to 3P orientation, and in brackets are given the ¢rst and last corresponding nucleotide positions in the rat cDNA sequence as in the respective GenBank entries for Pmca cDNAs):

rPmca1-3P-UTR.up

(3842; J03753)

rPmca1-3P-UTR.dn

(4177; J03753)

rPmca2-3P-UTR.up

(6139; J03754)

rPmca2-3P-UTR.dn

(6928; J03754)

rPmca3-3P-UTR.up

(4260; J05087)

rPmca3-3P-UTR.dn

(5013; J05087)

rPmca4-5P-UTR.up

(71; U15408)

rPmca4-5P-UTR.dn

(439; U15408)

5P-CACAACTTTATGACACACCCCGAG-3P 5P-TGTGTCTTCTGTTGAAGTCCGGAG-3P 5P-GGAAAAGAGCCATAGTCCGCTG-3P 5P-GGGGTGAACTGCCAAACGTATC-3P 5P-CTACACCCACAATATTCCGCTCA-3P 5P-GTATAGCAAAGCTCCAAATAGGCTC-3P 5P-GTGGGAAGAACGAAGAAGAG-3P 5P-TGGGCTTTTTCGGAGGTATC-3P.

Sequence comparisons were performed using McMolly (SoftGene, Berlin, Germany) under conditions of minimal match length of 8 bp, gap penalty 3 and mismatch penalty 1. Total RNA was isolated from whole mouse brain (strain 129Sv/J) using TRI Reagent (Molecular Research Center, Cincinnati, OH) and reverse transcription was performed using SuperScriptII (GibcoBRL) as described previously [9]. Each of the PCR products was cloned into pBluescriptKS (Stratagene) which had been linearized at the EcoRV site and T-tailed [10]. Sequence analysis con¢rmed the authenticity of each 3P-UTR cDNA clone. The plasmids were linearized with either XhoI or XbaI and radiolabeled antisense or sense RNA probes were generated using either T3 or T7 RNA polymerase (Promega), and 35 S-UTP (NEN, Dupont). 2.3. Embryonic stem cells Embryonic stem cells (GK-1 at passage 13; [11]) were a kind gift from Dr. Sandra Gendler (Mayo Clinic Scottsdale) with the permission of Graham Kay (London, UK). 2.4. Staging and harvesting of mouse embryos Embryos resulting from mating superovulated female FVB to male FVB mice were harvested at 9.5, 10.5, 12.5, 13.5, 15.5 and 18.5 days post coitum (dpc). Midday on the day of vaginal plug was considered 0.5 dpc. Embryos were ¢xed overnight in 4% paraformaldehyde at 4³C, dehydrated in an ethanol

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series, cleared in xylene and embedded in para¤n as described elsewhere [12]. 2.5. Tissue sectioning and in situ hybridization The tissue blocks were sectioned at 10 Wm onto glass slides subbed with silane (Sigma). In situ hybridization was performed as described by Toth et al. [13] with modi¢cations according to Lee et al. [14]. Contact autoradiograms for all experiments were generated by exposing the slides to BioMax MR ¢lm (Kodak) overnight at 370³C. Emulsion autoradiographs were generated using NTB-2 emulsion (Kodak) and exposing them at 4³C for 7 or 8 days. Sections were then counterstained with methyl green and examined and photographed on a Leica microscope. 3. Results 3.1. Pmca mRNAs are expressed in embryonic stem cells ES cells are totipotent cell lines derived from the inner cell mass of mouse blastocysts. In these cells, we found each of the four Pmca mRNAs expressed (Fig. 1). Non-quantitative RT-PCR was performed using primers speci¢c for the 3P-UTR of Pmca1, Pmca2 and Pmca3, and for the 5P-UTR of Pmca4

Fig. 1. Expression of Pmca mRNA in embryonic stem cells. All four Pmca gene transcripts were expressed in RNA from ES cells, as determined by RT-PCR (35 cycles). Identical amounts of cDNA were used in all reactions. Sequence analysis con¢rmed the authenticity of each PCR product. M, molecular weight marker (1 kb ladder, Gibco-BRL). Lanes 1^4: reactions with primers speci¢c for Pmca1, Pmca2, Pmca3, and Pmca4, respectively.

399

(see Section 2). Expression of all four Pmca genes seems to be a common occurrence in many cell lines ([8], and unpublished observations), despite more restricted patterns of expression in di¡erentiated tissues. The reasons for this are unclear, but suggest that expression becomes more restricted as cells differentiate. 3.2. Identity of the mouse cDNA fragments In order to con¢rm the speci¢city of our PCR reactions and to correctly identify the respective ampli¢ed fragment, we cloned and sequenced the PCR products. The respective primer pairs would, from rat cDNA, produce hypothetical amplicons of 359 bp for Pmca1, of 789 bp for Pmca2, 705 bp for Pmca3 and 369 pb for Pmca4 (including the lengths of the primers). The rat sequences for these hypothetical fragments did not detect any similarities with non-Pmca sequences in GenBank and did not `cross-react' by BLAST search with a cDNA sequence of any Pmca cDNA other than their respective isoform (data not shown). The PCR products we ampli¢ed from adult mouse brain RNA (not shown) and embryonic stem cells (Fig. 1) were of sizes corresponding to those predicted from the rat sequences, except for the Pmca3 product which was slightly smaller. DNA sequence analysis of the cloned mouse cDNA fragments revealed a high degree of similarity with the published cDNA sequences from rat and human but not with any other non-Pmca sequence (BLAST results not shown). Fig. 2 shows the comparisons for each of the mouse sequences with the respective rat cDNA sequence. The nearly continuous diagonal lines indicate regions of high degrees of sequence identity. As predicted, the Pmca1, Pmca2, and Pmca3 products were derived from the 3P-UTR of their corresponding mRNAs, and the Pmca4 product was derived from the 5P-UTR. The alignment for Pmca3 also revealed the likely reason for the size di¡erence of the mouse amplicon from the size predicted from the rat cDNA sequence. The rat sequence contains several shorter AC repeats between bases 4506 and 4646 that do not appear to be conserved in length in the mouse. However, there was high similarity in the immediate surrounding regions. In addition, the absence of interruptions with concomitant major parallel shifts in the diagonals

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Fig. 2. Sequence comparisons of mouse and rat Pmca cDNA sequences. Sequences from the cloned mouse PCR products were ¢rst compared to GenBank where they identi¢ed matches with the corresponding Pmca sequences from other species. Shown here are comparisons between each mouse-derived product and its corresponding rat cDNA sequence. The diagonal lines indicate the alignment and high similarity of the mouse with the rat sequences.

indicated that the murine PMCA amplicons were corresponding to the UTRs of the respective rat cDNAs. These similarity plots therefore do not provide evidence for altered splice forms in the mouse. Taken together, these data demonstrate that we have cloned cDNA fragments corresponding to the 3PUTR for Pmca1, Pmca2, and Pmca3, respectively, and to the 5P-UTR for Pmca4, and thus con¢rm the identity of the probes used in our in situ hybridization experiments. 3.3. Pmca1 gene expression sets on early in the developing mouse embryo The Pmca1 gene was expressed throughout the

embryo from the earliest time point examined, 9.5 dpc (Fig. 3). By day 15.5 dpc, expression was highest in the lung, the spinal cord and the dorsal root ganglia (DRG; Fig. 4A), and by day 18.5, in the brain and spinal cord, the lung and skeletal muscle (Figs. 3 and 4B^D and Table 1). Expression within the spinal cord was not restricted to any particular region or cell type (not shown), and expression in the brain was comparatively lower. In adult mammals, expression of this isoform is highest in brain, intestine, liver, lung, kidney and stomach [5,15^17]. Although Pmca1 expression commences early and is widespread, stronger expression in the lung and spinal cord may imply a special functional role in these tissues.

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Fig. 3. Temporal and spatial distribution of Pmca gene expression during development. Contact autoradiograms of in situ hybridizations to sections of embryos isolated from 9.5 to 18.5 dpc. All sections were sagittal/parasagittal, sections on the left were stained with Alcian blue (AB) and Nuclear fast red (NFR) to illustrate overall anatomy and morphology for each stage (scale bar: 0.5 cm). Pmca1 was expressed from at least 9.5 dpc on. Signal is distributed throughout the embryo, but is greatest in the nervous system, heart, skeletal muscle and intestine. Pmca2 was ¢rst detected at 12.5 dpc. Its expression was con¢ned to the nervous system (CNS, DRG and pituitary) throughout development. Pmca3 was expressed ¢rst at 12.5 dpc. At the early stages, expression was greatest in the nervous system, but it was also found transiently in skeletal muscle. The onset of Pmca4 expression was at 12.5 dpc. Its expression was greatest in the liver, but decreased by 18.5 dpc.

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D.A. Zacharias, C. Kappen / Biochimica et Biophysica Acta 1428 (1999) 397^405 Fig. 4. Tissues with abundant Pmca gene expression. In situ hybridization analysis: (A^D) Pmca 1; (E^H) Pmca 2; (I,J) Pmca 3; and (K,L) Pmca 4. Dark ¢eld photomicrographs of sagittal sections, dorsal is top and rostral is to the left. (A) The dorsal root ganglia, DRG, in the thoracic region of a 15.5 dpc embryo. TRP, trapezius muscle. (B) Hippocampal formation at 18.5 dpc. CA1 and CA3 are regions of Ammon's horn; DG, dentate gyrus. (C) Spinal cord in the thoracic region at 18.5 dpc. (D) Lung at 18.5 dpc. (E) Partial brain at 13.5 dpc. Signal intensity was greatest in the neopallidal layer of forebrain, FB, olfactory bulb, OB, and throughout the septal area, SA. (F) The cerebellum, at 18.5 dpc, contained the most signal in the external granular layer, EGL, the site of early Purkinje cell somata, and the interpositus nucleus, I. (G) The developing eye at 18.5 dpc. Signal intensity was strongest in the retinoblast layer, R. (H) Expression of Pmca2 was greatest in the adult cerebellum within the Purkinje cells, PC. (I) Pmca 3 expression was found in the DRG ; however, the choroid plexus (J) has the greatest Pmca3 abundance in the late embryo. (K) Pmca4 expression at 18.5 dpc in the DRG and (L) in the wall of the intestine.

6

3.4. The Pmca2 gene is speci¢cally expressed in the developing nervous system Pmca2 gene expression was detectable from approximately 12.5 dpc on (Fig. 3) and was restricted to the nervous system. By 13.5 dpc, the brain contained the highest signal intensity (Fig. 4E). By 18.5 dpc, the central nervous system was strongly labeled, as were the DRG (Fig. 3), and the retinoblast cell layer of the developing eye (Fig. 4G). Within the brain, the greatest signal emanated from the external granular layer of the cerebellum (the site of developing Purkinje neurons) as well as the interpeduncular nucleus (Fig. 4F). In the adult, cerebellar Purkinje neurons have the highest level of Pmca2 mRNA (Fig. 4H) and protein expression [6,18,19]. Expression of this isoform has been observed outside of the nervous system by RT-PCR [5,16,20]; however, the level of expression in non-neuronal tissues is below the level of detection by in situ hybridization (this study), or by Western blot or immunohistochemistry [6,21]. 3.5. Pmca3 gene expression becomes restricted to speci¢c tissues Onset of Pmca3 gene expression was around 12.5

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dpc (Fig. 3), and was initially widespread. By day 16.5, Pmca3 message was found more localized in the nervous system, the lung, and the developing limb. At 18.5 dpc, Pmca3 mRNA was detected in the spinal cord, the DRG, the retinoblast cell layer of the eye, the lung and in skeletal muscle (Fig. 3). Transient Pmca3 expression in fetal muscle has also been observed in human tissue [5]; whether Pmca3 continues to be expressed in adult mouse skeletal muscle is not known. Examination of the emulsion autoradiographs revealed that, in addition to hybridization in the DRG (Fig. 4I), the strongest signal intensity was in the choroid plexus (Fig. 4J). This is consistent with reports that the choroid plexus and the medial habenula are the regions of highest expression within the adult rat brain [15,22], suggesting a unique role for Pmca3 in choroid plexus functions, such as, for example, the regulation of CSF production.

with exception of the liver. Interestingly, the expression in liver decreased by 18.5 days, but increased in the bladder and spinal cord. Pmca4 message was also present in the brain, the heart, the DRG and the intestine (Fig. 4K,L and Table 1). Notably, compared to Pmca1, Pmca4 is expressed at much lower levels. In contrast, Pmca4 mRNA and protein were reported to be expressed at relatively high levels throughout most tissues in the adult rat, especially the brain and the heart [5,6]. According to Stau¡er and colleagues, Pmca4 comprised 25% or more of the total Pmca mRNA expressed in each tissue; 51% in the heart. Within the adult rat brain, expression was shown to be highest within lamina 2 of the piriform cortex and the neocortex [23]. The weak expression of Pmca4 in mouse embryos suggests that expression levels of this isoform rise around birth or during postnatal development.

3.6. The Pmca4 gene is expressed in spinal cord and bladder, and transiently, in the liver

4. Discussion

The expression of Pmca4 mRNA was ¢rst detected at about 12.5 dpc (Fig. 3) at a generally low level, Table 1 Summary of the pattern of strongest Pmca mRNA expression at 18.5 dpc Nervous System Eye Heart Lung Kidney Bladder Skeletal muscle Liver Intestine

Pmca1

Pmca2

Pmca3

Pmca4

X

X X

X X X X

X

X X X X X X X

X

X

Xa X

The X indicates tissues in which the individual Pmca mRNAs were expressed abundantly. Expression within the nervous system proper varied for subregions, with Pmca2 and Pmca3 mRNA found most concentrated in the cerebellum and choroid plexus, respectively. Outside of the central nervous system, Pmca1, Pmca2 and Pmca3 were also detected within the dorsal root ganglia. Within the eye, Pmca2 and Pmca3 were expressed in the retinoblast layer. This overall pattern of expression in mice was consistent with what is found in adult mammals (see Sections 3 and 4). a Abundance decreased by 18.5 dpc.

4.1. Unique and overlapping sites of expression of the four Pmca genes Our results demonstrate distinct temporal and spatial distribution patterns for the four Pmca genes in the developing mouse embryo. The distinct expression patterns add support to previous biochemical data indicating that the members of the Pmca gene family possess unique functional characteristics [24^ 27]. There was some overlap of the four mRNAs in di¡erent embryonic organs and tissues, albeit at different levels of expression for each gene. However, the expression of multiple mRNA species in the same tissue or cell type does not necessarily imply functional redundancy as the Pmca proteins are likely to have a di¡erential distribution in regions of the plasma membrane and may interact with di¡erent cytoplasmic proteins [28]. It is also important to note here that the onset of expression is quite di¡erent for the four Pmca genes: Pmca1 is detected from day 9.5 on while the other three genes are expressed only from day 12.5 on. In short, our data demonstrate the unique nature of the individual Pmca family members: (1) the distinct and restricted spatial patterns of expression suggest specialized functions for each Pmca isoform; and (2) the variable and

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dynamic temporal pattern of expression indicate differential mechanisms of gene regulation. 4.2. Temporal and spatial regulation Regulation of the expression of the Pmca proteins occurs at least at two levels, the ¢rst concerns the particular cell type in which the pumps are expressed, the second relates to the membrane sub-domain in which the protein resides. The promoter of each gene will direct the ¢rst aspect, and alternative splicing has been implicated in the second aspect [28]. Virtually nothing is known about the promoters that regulate the expression of the four Pmca gene family members. Only the Pmca1 gene promoter has been examined to date [29] and contains some classic characteristics of a housekeeping-gene promoter, such as CpG islands, no TATA box and many SP1 binding sites [30]. However, the expression pattern of Pmca1 in the embryo is spatially restricted, suggesting that tissue-speci¢c factors contribute to its regulation. The same applies to Pmca2, which has the most restricted pattern of expression only in neuronal tissues. Pmca3 has been referred to previously as brain-speci¢c [6]. However, in the embryo, it was clearly expressed in the nervous system as well as in other tissues, including the developing limb, the liver, heart and skeletal muscle. While the Pmca4 gene was reported to be highly expressed in the adult mammal, its expression is relatively weak in the developing mouse embryo. Its decline in the liver and increase in the spinal cord and bladder suggest that Pmca4 is subject to both positive and negative regulation. This may apply to the regulation of the other Pmca genes and their alternative splice variants as well [5,31]. 4.3. Conclusion The dynamic spatial and temporal expression patterns of the murine Pmca genes suggest that each of these genes has evolved to a special function in the regulation of Ca2‡ . Recently, the ¢rst targeted disruption of a mouse Pmca gene revealed that the absence of Pmca2 causes hearing loss in heterozygotes and hearing and balance de¢cits in homozygous mutant mice [32]. Correspondingly, the Pmca2 gene was found to be mutated in deafwaddler mutant mice which are deaf and have di¤culty maintaining bal-

ance [33]. These results demonstrate a crucial role for Pmca2 in development and function of the inner ear and the cerebellum. Development of better pharmacological tools and further gene knockout experiments will aid in de¢ning the biological relevance of each of the genes in this family. Acknowledgements We thank Anita Jennings for sectioning, Teresa Tinder for excellent support with the in situ hybridization experiments, Drs. J. Michael Salbaum, Emanuel E. Strehler, and Paul J. Yaworsky for discussions and critical reading of the manuscript, Carol Williams for help with its preparation and Marv Ruona and Julie Jensen for graphics. We wish to express special thanks to Dr. Gary Sieck for his support. This work was funded by NIH Training Grant 5-T32GM08288-08 and Mayo Foundation for Medical Education and Research.

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