ARTICLE |
Correspondence to: Guy Boileau, Département de Biochimie, Université de Montréal, CP 6128, Succ. Centre-Ville, Montréal, Qc, Canada H3C 3J7.
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Summary |
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Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptasenested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-BP Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-KB Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues. (J Histochem Cytochem 46:459468, 1998)
Key Words: bone, teeth, extracellular matrix, ontogenesis, X-linked hypophosphatemia, in situ hybridization, Northern blot analysis
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Introduction |
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A POSITIONAL CLONING approach was recently used to identify PEX (Phosphate regulating gene with homologies to Endopeptidases on the X chromosome) as the candidate gene for X-linked hypophosphatemia (XLH) (
Human and mouse PEX/Pex cDNAs have now been cloned and sequenced (
The mechanism by which loss of PEX function elicits the bone and renal abnormalities observed in XLH patients is not clear. There are no data suggesting the presence of PEX/Pex mRNA in the kidney (
To identify a specific role for PEX/Pex, we were interested first in its tissue and cell distribution. For this reason, using ISH we examined Pex mRNA temporal and spatial patterns of expression on sagittal sections of embryonic mice from Day 13 to 19 and in newborn mice. At this period of development, most tissues are already formed and many are functional. For example, the ossification process starts on Day 14.5 post coitum (
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Materials and Methods |
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Animals
For ISH, we used unfixed, frozen tissues from fetuses of CD1 time-pregnant female mice. The fetuses were grouped according to embryonic age10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 intrauterine days of life (e10, e11, etc.) and postnatal Day 3 and 7 (p3 and p7) and were prepared as described (
DNA Manipulations
All DNA manipulations, including the reverse transcription from RNA, PCR, and cloning, were done according to standard protocols (
Cloning of a Mouse Partial Pex cDNA
To obtain a mouse Pex probe for ISH and Northern blot analysis, degenerate oligonucleotides corresponding to conserved regions among members of the neutral endopeptidase family were designed using the published human PEX gene sequence (
Preparation of cRNA Probes
The pCRII plasmid containing the Pex cDNA fragment was linearized with Xho1 and used as a template in an in vitro transcription assay to synthesize a single-stranded anti-sense RNA probe with SP6 RNA polymerase. For control, sense RNA probe was synthesized with T7 RNA polymerase after linearization of the plasmid with Kpn1. For ISH, Pex riboprobes were labeled with both [35S]-UTP and [35S]-CTP (1250 Ci/mmol; Amersham, Arlington Heights, IL) because very low mRNA levels were reported previously (
In Situ Hybridization
ISH was undertaken using RNase-free solutions, starting with frozen cryostat tissue sections that were slowly immersed in cold formaldehyde in 0.1 M phosphate buffer (pH 7.2) and maintained in this solution for 4560 min, then washed extensively with PBS. The tissues were treated for 10 min with acetic anhydride in 0.1 M TEA. After dehydration with alcohol, the tissue sections were dried and then incubated overnight at 55C with a hybridization solution consisting of 75% formamide, 10% polyethylene glycol, 3 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M Na citrate, pH 7.2), 50 mM phosphate buffer, pH 7.2, 1 x Denhardt's (made from 50 x stock solution: 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin in water), 0.5 mg/ml yeast tRNA, and 0.1 mg/ml sonicated denatured salmon sperm DNA. To increase the signal/noise ratio, the dithiothreitol (DTT) concentration was set at 200 mM (
Alkaline Phosphatase Activity
Localization of alkaline phosphatase activity (
RNA Extraction and Northern Blot Analysis
Northern blot analysis was performed using total RNA from newborn and adult calvariae and teeth, and from newborn brain, lung, and liver. For this purpose, the frozen tissues were mixed with TRIzol Reagent (Life Technologies/GibcoBRL; Burlington, Ontario, Canada) and disrupted with a Polytron. Total RNA was extracted according to
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Results |
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Pex ISH at Anatomic Resolution in Mouse Embryo
The Pex expression pattern was analyzed by ISH using anti-sense riboprobes on histological sections obtained from embryonic and postnatal mice (Figure 1 Figure 2 Figure 3 Figure 4 Figure 5). ISH at anatomic resolution was examined after emulsion autoradiography on embryonic Day 16 (e16) (Figure 1). Pex mRNA was readily detectable in regions of calvaria, mandible, and ribs. Although not apparent in Figure 1, the vertebrae and long bones also contained Pex mRNA on e16, whereas later, around birth, Pex mRNA could also be detected in developing teeth. Overall, Pex mRNA was localized within alkaline phosphatase territory (not shown). Controls were performed with sense riboprobes, which produced nonspecific background elevated in skin (see also Figure 3d and Figure 4d) and skeletal muscles (not shown). Other tissues, including kidney, lung, liver, and brain, were negative.
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Pex mRNA in Osteoblasts
To identify cells synthesizing Pex mRNA, representative tissues were examined under higher microscopic magnification. Figure 2a and Figure 2a', which are higher magnifications of the area shown in Figure 4c, demonstrate the presence of Pex mRNA on e16 in mandibular intramembraneous ossification centers. This topographically heterogeneous region is composed of an ossification zone (OZ) characterized by the presence of (a) a bone extracellular matrix with a dense cell population on the border zone and (b) an undifferentiated mesenchyme (M). ISH revealed the presence of Pex mRNA within cells bordering bone extracellular matrix. This topography suggests that Pex-expressing cells are osteoblasts. The majority of osteoblasts display Pex ISH labeling (Figure 2a').
Hybridization sites were also identified in vertebral en- dochondral ossification centers (Figure 2b). Pex mRNA was seen within an ossification zone (OZ) adjacent to an unlabeled calcified zone (CZ) and the cartilage (Ca). Bone extracellular matrix was well delineated within OZ, with a significant concentration of Pex-labeled cells on a border zone. There was no apparent hybridization labeling outside of OZ.
Pex mRNA in Odontoblasts
Within tooth, Pex mRNA was concentrated in the layer of odontoblasts (Od) (Figure 2c, which represents a higher magnification of the area shown in Figure 5b and Figure 2c'). In contrast, the layer of ameloblasts (Am) and the stellate reticulum of the enamel organ (SR) were unlabeled. Because the dentinoenamel matrix separates the odontoblast epithelium from the ameloblast epithelial layer, the cell population expressing Pex mRNA is particularly well-defined in this tissue.
Pex mRNA Ontogeny
To determine temporal and tissue-specific patterns of Pex gene expression, the calvaria, mandible, and teeth were examined at the stages preceding and following the onset of chondrification (e11), ossification (e14.5), and odontogenesis (e14). ISH results are shown in Figure 3 Figure 4 Figure 5.
Figure 3 shows the presence of Pex mRNA in the calvaria from e15 to p3. Although rudimental calvaria is already visible on e14 (Figure 3a and Figure 3a'), Pex mRNA is not detectable at this time but is evident thereafter. It is worth noting the presence of bone extracellular matrix within rudimentory calvaria at e15 but not e14. This matrix was seen as a deposition of acellular eosinophilic material along and within a space delineated by a front of osteoblasts (data not shown). Pex mRNA remains present in the calvaria during later gestation and early postnatal development.
Figure 4 depicts Pex mRNA distribution in the mandible from e15 to p3. Despite the absence of the bone extracellular matrix, which is not present early on, the rudiments of the mandible bone are evident by e14. Similar to calvaria, a dramatic elevation of Pex mRNA is evident on e15 (Figure 4b). Pex mRNA remains present until p3.
In addition to bones, Pex mRNA labeling can be observed in both incisors and molars, although the onset of expression varies with time of tooth development (Figure 4 and Figure 5). All teeth shown in Figure 5 are from the same section of e19 mouse and include one molar (Figure 5a and Figure 5a'), one inferior incisor (Figure 5b and Figure 5b'), and one superior incisor (Figure 5c and Figure 5c'). Incisors are strongly labeled, whereas rudimental molars not. The presence of dental extracellular matrix was noted within the two incisors but not in the molar. In addition to incisors, first molars showed PEX mRNA labeling on p3, whereas second molars were positive on p7 (results not shown).
Characterization of Pex mRNA in Adult Bone and Teeth
To verify the presence of Pex mRNA in adult mouse, Northern analysis was performed with total RNA obtained from p3 and adult tissues. A Pex transcript of approximately 7 KB was detected in calvariae and teeth (Figure 6). With 18S RNA as an internal control for mRNA loading, it is clear that PEX mRNA levels in calvariae are higher on p3 than in adulthood. Pex mRNA was observed in p3 and adult teeth. Knowing that incisors, but not molars, present continuous growth in rodents, we isolated total RNA from adult incisors and molars and compared their levels of Pex mRNA. This comparison demonstrated that adult incisors express higher mRNA levels of Pex than adult molars. Pex mRNA was not detectable in newborn lungs, liver, and brain by Northern analysis.
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Discussion |
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This report provides histochemical evidence for Pex mRNA expression in murine embryonic and postnatal bones and teeth. In these tissues, Pex mRNA was detected in osteoblasts and odontoblasts, respectively. With both ISH and Northern analysis, bones and teeth were the only tissues in which the presence of the Pex mRNA could be detected, suggesting that these two tissues are privileged sites for Pex expression in the developing mouse. A significant concentration of Pex mRNA was also detected in adult incisors, which grow continuously in rodents, whereas in adult calvariae and in nongrowing molars Pex mRNA expression appeared to be considerably lower. Taken together, these data suggest a role for Pex in the development of bones and teeth.
Osteoblasts and chondroblasts/chondrocytes are important cell components of developing bone. Major products of osteoblasts and chondrocytes are the extracellular matrix of bone and cartilage, respectively. The bone extracellular matrix is composed of 90% collagen and 10% noncollagenous proteins (
Our results are in concordance with the clinical features of Hyp and Gy mice, which harbor large deletions in the Pex gene (
Like patients with XLH, both Hyp and Gy mutant mice develop hypophosphatemia secondary to impaired renal phosphate reabsorption (
In conclusion, we demonstrate the presence of Pex mRNA in both embryonic and adult bones and teeth. Cells expressing Pex have been identified as osteoblasts and odontoblasts. Therefore, Pex could be a useful marker for these two cell types. In adult bones, the levels of Pex mRNA were lower than those in embryos. In adult teeth, Pex mRNA concentration remained elevated in incisors and was lower in molars. These results suggest that Pex is involved in the development of bone and tooth tissues.
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Acknowledgments |
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Supported by grants from FCAR (to LDG, PC, and GB) and the Medical Research Council of Canada (MT-12686, to MM). AFR is supported by the Canadian International Development Agency (CIDA), Canada and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
We are grateful to Ms J. Marcinkiewicz for skillful technical help and Mr C. Charboneau for photographic work.
Received for publication July 15, 1997; accepted December 10, 1997.
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