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Endocrinology Vol. 142, No. 12 5303-5310
Copyright © 2001 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Partial Rescue of PTH/PTHrP Receptor Knockout Mice by Targeted Expression of the Jansen Transgene

D. W. Soegiarto, S. Kiachopoulos, E. Schipani, H. Jüppner, R. G. Erben and B. Lanske

Molecular Endocrinology, Max-Planck-Institute for Biochemistry, Martinsried 85152, Germany; Endocrine Unit, Massachusetts General Hospital and Harvard Medical School (E.S., H.J.), Boston, Massachusetts 02114; and Institute of Animal Physiology, Ludwig-Maximilian University of Munich (R.G.E.), Munich 80539, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homozygous ablation of the gene encoding the PTH/PTHrP receptor (PPR-/-) leads to early lethality and limited developmental defects, including an acceleration of chondrocyte differentiation. In contrast to the findings in homozygous PTHrP-ablated (PTHrP-/-) animals, these PPR-/- mice show an increase in cortical bone, a decrease in trabecular bone, and a defect in bone mineralization. Opposite observations are made in Jansen’s metaphyseal chondrodysplasia, a disorder caused by constitutively active PPR mutants, and in transgenic animals expressing one of these receptor mutants (HKrk-H223R) under control of the type {alpha}1(I) collagen promoter. Expression of the Jansen transgene under the control of the type {alpha}1(II) collagen promoter was, furthermore, shown to delay chondrocyte differentiation and to prevent the dramatic acceleration of chondrocyte differentiation in PTHrP-/- mice, thus rescuing the early lethality of these animals. In the present study we demonstrated that the type {alpha}1(II) collagen promoter Jansen transgene restored most of the bone abnormalities in PPR-/- mice, but did not prevent their perinatal lethality. These findings suggested that factors other than impaired gas exchange due to an abnormal rib cage contribute to the early death of PPR-/- mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SKELETOGENESIS TAKES place by two different mechanisms (2). Craniofacial bones are formed by intramembranous ossification, in which precursor cells directly differentiate into osteoblasts. In contrast, the axial as well as the appendicular skeleton develops through endochondral ossification, in which a cartilaginous bone template is formed first. Chondrocytes in the center of this anlage then differentiate into hypertrophic cells, mineralize, and are eventually replaced by bone. In the peripheral layers of the developing bones, chondrocytic cells form the perichondrium, and the differentiation of mesenchymal cells into osteoblasts within this portion of the skeletal structure leads to the formation of the bone collar.

Many signaling molecules and their receptors are known to influence endochondral ossification, including PTHrP and the PTH/PTHrP receptor (PPR) (3). The PPR, a G protein-coupled receptor with seven membrane-spanning helices, mediates the actions of PTH and PTHrP, two distinct, but related, ligands. PTH is, besides 1,25-hydroxyvitamin D3, the most important endocrine regulator of calcium homeostasis that acts primarily on kidney and bone (4). PTHrP was first discovered as the most frequent cause of the humoral hypercalcemia of malignancy syndrome (5, 6). However, PTHrP is expressed in a large variety of different tissues and usually acts as an autocrine/paracrine regulator rather than as an endocrine hormone (7). Important insights into these roles were obtained through the generation of mice in which both copies of the PTHrP gene were ablated or PTHrP was overexpressed under control of the type II collagen promoter, which targets expression to growth plate chondrocytes (8, 9, 10). These and additional studies (11, 12, 13) showed that the actions of PTHrP on growth plate cartilage are mediated via the PPR, which is most abundantly expressed in chondrocytes residing in the zone between proliferation and hypertrophy. It was, therefore, not unexpected that PPR-ablated mice (PPR-/-) show skeletal abnormalities that are similar to but more severe than those observed in PTHrP-/- animals (11, 12, 13).

Consistent with these findings in mice, activating PPR mutations are the most likely cause of Jansen’s metaphyseal chondrodysplasia, a rare autosomal dominant disorder characterized by hypercalcemia, hypophosphatemia, and short-limbed dwarfism (14, 15, 16, 17, 18). When introduced into the wild-type human PPR (19) and expressed in mammalian cells, each of the three Jansen mutations identified to date leads to ligand-independent constitutive cAMP accumulation (14, 15, 16, 17, 18), thus providing a reasonable explanation for abnormal regulation of mineral homeostasis and chondrocyte differentiation. Furthermore, to prove that the constitutively active PPR is indeed responsible for the growth plate abnormalities observed in Jansen’s disease (20), one of the PPR mutants, HKrK-H223R, was expressed in mice under the control of the {alpha}1(II) collagen promoter to target expression to chondrocytes. The resulting transgenic mice were viable and showed growth plate abnormalities mimicking those observed in the Jansen’s metaphyseal chondrodysplasia (18), i.e. a delay in chondrocyte differentiation, a delay in vascular invasion, and a reduction or absence of mineralization of bone elements that are formed through the endochondral process. Because of this delay in endochondral bone formation, these mice were used to prevent the acceleration of chondrocyte differentiation and thus the bone abnormalities during fetal development and the early lethality of PTHrP-/- animals (18).

The goal of the present study was to determine whether the targeted expression of HKrk-H223R in the growth plate can also normalize the skeletal phenotype of PPR-/- mice, and whether expression of the transgene can rescue, at least partially, the early lethality of these animals. For this purpose, we generated and analyzed mice that lack the endogenous PPR, but express a constitutively active PPR in growth plate chondrocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Riboprobes
Complementary 35S-labeled riboprobes (cRNAs) were transcribed from plasmids encoding type I collagen (21), type II collagen (22), type X collagen (23), osteocalcin (provided by G. Karsenty) (24), osteopontin (25), collagenase 3 (provided by Y. Eeckhout) (26), Indian hedgehog (provided by A. McMahon) (27), and patched (the receptor for Indian hedgehog; provided by M. Scott) (28). Sense and antisense probes were synthesized from the linearized plasmids using [35S]UTP (800 Ci/mmol; Amersham Pharmacia Biotech, Braunschweig, Germany) and Sp6, T3, and T7 RNA polymerases (Promega Corp., Mannheim, Germany). The following reagents were purchased: paraformaldehyde, silver nitrate, sodium thiosulfate, Alizarin Red S (Sigma-Aldrich Corp., Taufkirchen, Germany), methylmethacrylate (Merck Eurolab, Bruchsal, Germany), NTB-2 photoemulsion, Kodak Dektol developer and Kodak fixer (Integra Biosciences, Fernwald, Germany), and x-ray film (BioMax MR-1, Amersham Pharmacia Biotech, Freiburg, Germany).

Animals
The generation of PPR-ablated mice (PPR-/-) (11) and transgenic lines (Tg-A or Tg-B) expressing a constitutively active form of the human PPR (HKrk-H223R mutant) under the control of the rat {alpha}1(II) collagen promoter was described previously (18). PPR+/- mice were intercrossed with each of the Jansen transgenic lines to obtain mice that lack one copy of the PPR gene and contain one copy of the transgene (Tg-A+ or Tg-B+). Matings of those double heterozygous mice resulted in offspring with different genotypes, i.e. wild-type, heterozygous, or homozygous for the receptor knockout (PPR+/-, PPR-/-), and heterozygous or homozygous for the Jansen transgene (Tg-A/B+ or Tg-A/B++). All mice were genotyped by Southern blot analysis of tail clip DNA. Animals were maintained in the facilities operated by the Max-Planck-Institute for Biochemistry (Martinsried, Germany) in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were employed using protocols approved by the institution’s subcommittee on animal care.

Skeletal analysis
The mineralization pattern of the skeleton was analyzed on embryonic day 18.5 (E18.5) as described previously by McLeod (29). Briefly, on E18.5 embryos were harvested by cesarean section. Fetuses were skinned, eviscerated, and fixed in 95% ethanol. Subsequently, acetone was used to remove fat. Then skeletons were stained by Alizarin Red S and sequentially cleared in 1% potassium hydroxide. Mineralized bones were visualized by the staining.

Histology and tissue preparation
For histological analyses, either paraffin or methylmethacrylate sections of bones were produced on E18.5. For paraffin sections, fetuses were fixed in 4% paraformaldehyde (PFA)/PBS, pH 7.4, at 4 C, rinsed in PBS, dehydrated at room temperature through an ethanol series (70% for 6 h, 80% for 1 h, 96% for 1 h, and 100% for 3 h), cleared twice in xylene for 1 h/step, embedded in paraffin, sectioned at 6 µm with a Microm HM 355 microtome (Microm International GmbH, Walldorf, Germany), and mounted on SuperFrost Plus slides (Carl Roth GmbH & Co., Karlsruhe, Germany).

To obtain methylmethacrylate sections, samples were fixed in 4% PFA for 24 h at 4 C and washed overnight in PBS containing 10% sucrose at 4 C. Subsequently, the left and right hindlimbs of each embryo were dehydrated and embedded undecalcified in conventional methylmethacrylate (30) or using a modified methylmethacrylate embedding method suitable for histochemistry and immunohistochemistry, respectively (31). Three-micron-thick sections were prepared in the midsagittal plane of the knee joint with an HM 360 microtome and stained with von Kossa/toluidine blue (30).

Riboprobes and in situ hybridizations
Complementary [35S]UTP-labeled riboprobes (cRNAs) were used for in situ hybridizations. Plasmids encoding the cDNA were linearized with appropriate restriction enzymes to transcribe either antisense or sense riboprobes in vitro using the appropriate RNA polymerase. In situ hybridization was carried out as described previously (12), only antisense riboprobes showed specific signals.

Briefly, bone sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series (100%, 90%, and 70%). After proteinase K treatment and postfixation in 4% PFA, sections were incubated in 0.2 N HCl. Sections were then acetylated with 0.25% acetic anhydride in triethanolamine buffer. Before hybridization was performed, sections were dehydrated in 70% and 95% ethanol and air-dried. Sections were hybridized with 35S-labeled antisense or sense riboprobes in a humidified chamber at 50 C for 16 h. After hybridization, unspecifically bound riboprobes were removed by washing the slides with 2 x SSC and 2 x SSC/50% formamide at 50 C and treating them with ribonuclease at 37 C for 20 min. The final wash steps were performed once in 2 x SSC and twice in 0.2 x SSC at 50 C for 20 min. To detect the hybridization of riboprobes on tissues, sections were dehydrated in 70% and 95% ethanol and air-dried. To estimate the intensity of bound riboprobes, slides were exposed to x-ray film (Kodak Biomax MR-1) overnight at room temperature. Sections were then coated with Kodak NTB2 emulsion diluted 1:1 with H2O, exposed for the time needed (determined by autoradiography), developed with Kodak Dektol developer, and fixed with Kodak fixer. After counterstaining with hematoxylin-eosin, tissue sections were analyzed with a Carl Zeiss microscope (New York, NY) using bright- and darkfield optics.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of homozygous mutant PPR animals expressing the Jansen transgene in chondrocytes
The goal of this study was to determine whether targeted expression of a constitutively active human PPR in chondrocytes could prevent the acceleration of endochondral bone formation and thus rescue the early lethality of PPR-/- mice. Mice lacking both copies of the PPR gene (11) and two Jansen transgenic lines (Tg-A and Tg-B) expressing a constitutively active form of the human PPR under the control of the rat {alpha}1(II) collagen promoter (18) were previously generated. To ensure that the transgene is specifically expressed in the growth plate and not in bone, we performed in situ hybridizations using a riboprobe specific for a vector-derived portion of the transgene. Although growth plate chondrocytes revealed a clearly detectable signal, no expression was observed in osteoblasts of calvariae (data not shown); the presence of osteoblasts in calvaria was, however, confirmed by hybridization with a probe encoding collagen type I (data not shown). These studies confirmed earlier findings (18), making it possible to determine whether expression of the transgene in chondrocytes alone could rescue the early lethality of PPR-/- embryos. To generate animals of the genotypes PPR+/-/Tg-A+ or PPR+/-/Tg-B+, we first mated PPR-/- animals with one of the two Jansen transgenic mice. These were then intercrossed to generate animals that express the transgene and are homozygous for ablation of the PPR (genotypes: PPR-/-/Tg-A+, PPR-/-/Tg-A++, or PPR-/-/Tg-B+, PPR-/-/Tg-B++). Genotypes were established by Southern blot analysis (data not shown).

Survival rate
Genotyping by Southern blot analysis of embryos on E18.5 verified the expected Mendelian ratio; i.e. the genotypes wild-type and PPR-/- were present in 25% of the offspring, and 50% had the genotype PPR+/-. However, all PPR-/- animals in the absence or presence of the Jansen transgene died perinatally, indicating that targeted expression of the constitutively active PPR failed to rescue the early lethality of PPR-/- animals (data not shown). This result was different from that in PTHrP-/-/Tg+ mice that survived for up to 3 wk (18). Due to the persistent perinatal lethality of PPR-/-/Tg+ animals, histological analyses were performed on E18.5.

Gross and histological phenotype of PPR-/-/Tg+ mice expressing a constitutively active Jansen receptor in the growth plate
PPR-/- embryos died at birth or earlier and were at each developmental stage smaller than their normal littermates (11). Heterozygous Tg-A+ and Tg-B+ mice were macroscopically indistinguishable from their normal littermates, and only homozygous Tg-B++ animals showed foreshortening of limbs and tail (18). With the exception of their smaller size, PPR-/-/Tg-A+ and PPR-/-/Tg-B+ mice were indistinguishable from the wild-type animals. In particular, the former mice lacked abnormalities of the skulls and extremities, suggesting that the Jansen transgene was able to prevent the skeletal phenotype of the PPR-/- embryos (Fig. 1Go).



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  		Figure 1. Lateral view of a wild-type (WT), Jansen transgenic (Tg-A+), PPR-/-, and PPR-/-/Tg-A+ embryo on E18.5. Tg-A+ embryos were largely indistinguishable from their normal littermates (WT). The expression of the Jansen transgene in chondrocytes partially corrected the macroscopic appearance of PPR-/- embryos. With the exception of their smaller size, PPR-/-/Tg-A+ embryos were similar to WT embryos and showed normally shaped skull and extremities.

 
To further evaluate the impact of the transgene in the PPR-/- background, the intact skeleton of wild-type, Tg-A+, Tg-B+, PPR-/-, PPR-/-/Tg-A+, and PPR-/-/Tg-B+ animals was stained with Alizarin Red S. Skeletons of Tg-A+ and Tg-B+ animals showed the expected delay in endochondral bone formation, with reduced or absent mineralization of some bone elements (18). In contrast, mineralization of the skeletons of PPR-/- embryos was accelerated, as shown in bones formed through replacement of a cartilaginous mold (11). Similar to the findings in PTHrP-/- mice, the Jansen transgene in either Tg-A (Fig. 2Go) or Tg-B mice (data not shown) prevented, at least partially, the skeletal abnormalities observed in PPR-/- animals. Figure 2AGo shows the skull of a wild-type (left), PPR-/- (middle), and PPR-/-/Tg-A+ (right) embryo on E18.5. Excessive mineralization was present in the bones forming the base of the skull of PPR-/- animals. These abnormalities were not observed in PPR-/-/Tg-A+ animals, which were indistinguishable from their normal littermates. Correction of the advanced mineralization was also achieved in forelimbs (Fig. 2BGo) and vertebrae (Fig. 2DGo). The ribs, which were completely mineralized in PPR-/- mice (Fig. 2CGo), in the presence of the Jansen transgene showed no evidence for calcium deposition; in fact, there appeared to be a delay in mineralization, as indicated by the nonmineralized sternum (Fig. 2CGo). Taken together, these data suggested that the expression of a constitutively active PPR prevented the accelerated formation of mineralized cartilage in PPR-/- mice.



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  		Figure 2. Alizarin Red S staining of wild-type (A–D, left panel), PPR-/- (A–D; middle panel), and PPR-/-/Tg-A+ (A–D; right panel) skeletons on E18.5. Representative examples of more than six litters are shown: base of skull (A), forelimb (B), ribs and sternum (C), and vertebrae (D). An abnormal mineralization pattern, depicted by the arrows, was apparent throughout the skeleton of PPR-/- mice, but not in those bones from wild-type littermates. In every case the expression of the Jansen transgene in chondrocytes was thus able to correct or even overcorrect (see sternum) the defect in mineralization.

 
To further assess the growth plate chondrocytes, we performed histological analyses of hematoxylin/eosin-stained sections and in situ hybridizations with type X collagen, which is most abundantly expressed in hypertrophic chondrocytes. Paraffin sections of the sternebrae of a wild-type embryo on E18.5 revealed hypertrophic chondrocytes, blood vessel invasion, primary spongiosa, and bone marrow cavity formation (Fig. 3Go, A and E). In contrast, sternebrae of Jansen transgenic animals exhibited a delay in chondrocyte differentiation and, therefore, no evidence for blood vessel invasion (Fig. 3Go, B and F). The sternebrae of PPR-/- mutants were completely occupied by hypertrophic chondrocytes (Fig. 3CGo), resulting in sternebrae that consisted mainly of type X collagen-positive cells (Fig. 3GGo). In contrast, PPR-/- mutants expressing the constitutively active PPR in chondrocytes (PPR-/-/Tg-A+ or PPR-/-/Tg-B+) exhibited relatively normal sternebrae (Fig. 3Go, D and H), that is hypertrophic cells were only present in the center of bones, where one would expect chondrocyte differentiation to occur. Interestingly, blood vessel invasion was delayed to a similar extent in sternebrae of Jansen transgenic animals (Tg-A or Tg-B; Fig. 3Go, B and F) with or without the endogenous PPR gene, i.e. Tg-A+ or PPR-/-/Tg-A+.



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  		Figure 3. Histological sections of sternebrae of wild-type (A and E), Jansen transgenic Tg-A+ (B and F), PPR-/- (C and G), and PPR-/-/Tg-A+ (D and H) embryos on E18.5. Upper panels (A–D), Hematoxylin/eosin staining; lower panels (E–H), in situ hybridization using a 35S-labeled riboprobe encoding type X collagen. Darkfield views are shown.

 
The tibiae of wild-type embryos on E18.5 contained two growth plates located at either end of the bone, followed by metaphyseal regions with adjacent trabecular bone and diaphyseal areas comprising most of the bone marrow cavity (Fig. 4AGo). Tg-A+ or Tg-B+ animals showed a slight reduction in the length of their bones, possibly caused by the delayed replacement of cartilage by bone (Fig. 4BGo). In contrast, limbs of PPR-/- mice were disproportionally shorter, and their long bones were misshapen, with a diminution in the size of the growth plates and lack of trabecular bone (Fig. 4CGo). The disturbed chondrocyte differentiation process was also accompanied by changes in osteoblast development. Intramembranous bones adjacent to the hypertrophic chondrocytes of the PPR-/- embryo showed a dramatic accumulation of matrix-producing osteoblasts. Osteoid failed to mineralize, as indicated by the lack of von Kossa staining (Fig. 4GGo). The tibia of PPR-/-/Tg-A+ embryos (Fig. 4DGo) were almost normal in appearance, although the bones remained shorter, but proportionate to the smaller size of the animals. The shape of the bones was restored, the growth plates were normal in size, and the organization of the chondrocytes within the growth plates was similar to that of wild-type animals. In addition, the mineralization defect in hypertrophic chondrocytes was restored. In contrast, trabecular bone remained absent (Fig. 4DGo), but this may have been related to the delay in cartilage replacement, as observed in Tg-A+ or Tg-B+ animals (Fig. 4BGo). Surprisingly, the thickening of the bone collar and the diaphyseal cortex of the long bones of PPR-/- mutants (Fig. 4Go, E and G) had been normalized by the presence of the transgene in the growth plate (Fig. 4Go, F and H).



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  		Figure 4. Tibiae of wild-type (A), Jansen transgenic Tg-A+ (B), PPR-/- (C), and PPR-/-/Tg-A+ (D) embryos on E18.5. E and F, Enlargements of the cortical bone area (see arrows). Three-micron methylmethacrylate sections stained with toluidine blue and von Kossa are shown. The bone from a Tg-A+ mouse (B) is somewhat shorter and shows some hypertrophic cells in the diaphysis compared with the same bone from a wild-type mouse (A). Shortening and misshaping of the tibiae of PPR-/- mice as well as thickening of the cortical bone (C, arrow) were prevented by the presence of the Jansen transgene, as demonstrated in D. The size of the growth plate (bars) and the shape of the bone were corrected. The rescued bone resembles that of Tg-A+ mice, in that it is somewhat smaller compared with the same bone from wild-type littermates and shows persisting chondrocytes in the middle of the bone. Enlargements at the level of the metaphyseal region illustrate the abnormal formation of a mineralized bone collar and the abnormal width of the osteoblast layers in PPR-/- mice (asterisk; G). PPR-/-/Tg-A+ embryos exhibit a correctly mineralized bone collar, the additional layers of osteoblasts layers are no longer present, and the osteoid has undergone normal mineralization as revealed by von Kossa staining (black, asterisk; H).

 
In addition to these histological changes, in situ hybridizations were performed to assess possible changes in the expression of genes specific for osteoblasts and chondrocytes. When using a riboprobe encoding collagenase 3 (Fig. 5Go, A–D), high levels of the mRNA encoding this metalloproteinase were detected in the diaphyseal region of PPR-/-/Tg-A+ or PPR-/-/Tg-B+ animals, which is consistent with the continuous presence of hypertrophic chondrocytes. Collagen type I gene expression was strongest in the cortical bones of PPR-/- mice. These changes, which were probably due to the excessive intramembranous bone formation, were normalized in PPR-/-/Tg-A+ or PPR-/-/Tg-B+ mice (Fig. 5Go, E–J). Animals homozygous for the Jansen transgene exhibited no detectable collagen type I expression, consistent with the observation that their bones were still cartilaginous. All other probes, including osteopontin, osteocalcin, Indian hedgehog, collagen type II, collagen type X, and patched, revealed only minor changes in mRNA expression (data not shown), and these alterations appeared to be related to chondrocyte differentiation rather than altered expression by the same cell population within equivalent areas of the bones.



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  		Figure 5. In situ hybridizations of tibia on E18.5 using probes encoding collagenase 3 (A–D) and collagen type I (E–J). Brightfield images are shown in the upper panels, and darkfield exposures in the lower panels. The diaphysis of PPR-/-/Tg-A+ embryos (C and D) is still occupied by hypertrophic chondrocytes and, therefore, is strongly positive for collagenase 3 gene expression. PPR-/- embryos (A and B) exhibit normal gene expression when compared with wild-type animals (data not shown). The increased number of osteoblasts in the cortical region of PPR-/- mice (E and F) is strongly positive for collagen type I. In contrast, PPR-/-/Tg-A+ animals (G and H) exhibit normal levels of expression, whereas tibiae of PPR-/-/Tg-A++ animals (homozygous for the Jansen transgene; I and J) do not show a detectable signal. Note that these bones are mainly comprised of cartilaginous tissue.

 
Taken together, we determined that expression of a constitutively active Jansen receptor in growth plate chondrocytes prevented most of the skeletal abnormalities of PPR-/- animals, but the transgene failed to normalize body size and rescue them from perinatal lethality.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During endochondral bone formation, skeletal elements are formed by replacing a cartilaginous mold with bone-forming osteoblasts. Among the multiple factors that are involved in this complex process, the PPR and its ligands, PTHrP and PTH, were shown to play important roles in chondrocyte and osteoblast differentiation and bone turnover (11, 12, 13, 32). Consequently, mutations in the PPR lead to severe abnormalities in growth plate development and bone formation, i.e. activating PPR mutations lead to Jansen’s disease and homozygous or compound heterozygous inactivating mutations lead to Blomstrand’s disease (33, 34, 35); note that the findings in this latter disease are similar to those in PPR-ablated mice.

Transgenic mice expressing a constitutively active human PPR mutant under the control of the rat {alpha}1(II) collagen promoter display phenotypic changes similar to those observed in mice overexpressing PTHrP under control of the mouse {alpha}1(II) collagen promoter (9), yet the opposite of those observed in PPR-/- animals (11). Based on these findings, expression of the Jansen transgene in PPR-/- mutants was predicted to prevent the skeletal abnormalities and the early lethality of PPR-/- mice.

Our studies have now shown that the targeted expression of a constitutively active PPR in chondrocytes is able to almost completely prevent the skeletal abnormalities of homozygous PPR-ablated fetuses by slowing the abnormal acceleration of chondrocyte differentiation and by correcting osteoblast development. With the exception of the smaller body size, the macroscopic appearance of PPR-/-/Tg+ mice was, on E18.5, identical to that of their normal littermates. In addition to the correction of growth plates, the rescued animals (PPR-/-/Tg+) showed normalization of long bones and decreased thickening of the cortical bone. Furthermore, the presence of the Jansen transgene restored the abnormal bone collar in PPR-/- mice. Given that the transgene is not expressed in osteoblasts (see above), these findings indicate that through paracrine actions a cross-talk occurs between cartilage and bone (36).

On E18.5 PPR-/- mice were present at the expected Mendelian ratio (see Results). However, all of the rescued PPR-/-/Tg+ mice died postnatally despite their almost normal skeletal phenotype. In contrast to the transgene-induced survival of PTHrP-ablated mice, expression of the constitutively active PPR was unable to prevent the perinatal death of PPR-/- animals. These data are consistent with previous findings indicating that PPR-ablated mice exhibit a more severe phenotype than PTHrP-/- mice (11).

The early lethality could be related to the lack of functional PPRs leading in utero to PTH resistance in bone (and kidney) and an abnormal regulation of mineral ion homeostasis. However, in preliminary studies on E18.5, PPR-/- mice showed blood ionized calcium concentrations approximately 25% lower than those in wild-type animals, consistent with our previous findings (37); PPR-/-/Tg-A+ mice showed calcium levels approximately 11% below normal. These findings suggested that the intrauterine regulation of calcium homeostasis is not severely impaired, although postnatal calcium concentrations would be predicted to be as low as those in Gmc-2-ablated mice that had undergone thymectomy to remove extraparathyroidal PTH production (38). Thus, although fetal hypocalcemia may have contributed to the perinatal lethality of PPR-/-/Tg+ mice, it appears more likely that additional factors contribute to the perinatal death of PPR-/-/Tg+ mice. In fact, recent studies have demonstrated that the early lethality of PPR-/- mice that occurs around E10.5 in the C57BL/6 background may be due to cardiac dysmorphogenesis and subsequent vascular collapse or circulatory insufficiency (39, 40). However, a lack of functional PPR may also affect PTHrP-dependent functions in the central nervous system (41, 42).

In conclusion, we have demonstrated that the targeted expression of a constitutively active PPR in chondrocytes is able to almost completely rescue the bone phenotype of homozygous PPR-ablated fetuses by preventing the abnormalities in chondrocyte differentiation; this finding is similar to the observations in rescued PTHrP-/- mice. Furthermore, there was a correction of the anomalies in osteoblast development, but underlying mechanisms leading to this effect remain uncertain. In contrast to prolonged postnatal survival of rescued PTHrP-/- mice, the presence of the Jansen transgene did not improve the early lethality of PPR-/- animals, indicating that persisting abnormalities in mineral ion homeostasis and other PTH- and PTHrP-dependent actions, not only respiratory insufficiency due to the rib cage abnormalities, contribute to the perinatal lethality. Further investigations are needed to determine the cause of the lethality and to fully rescue the PPR-/- embryos.


   Acknowledgments
 
The authors thank the following for their technical support: Wenke Barkey, Cornelia Wölfle, El Mokhtar Bousadik, and Dr. Kerstin Stahr.


   Footnotes
 
Current address for all correspondence and requests for reprints: Beate Lanske, Ph.D., Harvard-Forsyth Department of Oral Biology, The Forsyth Institute, 140 Fenway, Boston, Massachusetts 02115.

This work was supported by Roche Diagnostics and the Bavarian Ministry for Economic Affairs, Transport, and Technology, and by the NIH (DK 50708) (to H.J.).

Abbreviations: E18.5, Embryonic d 18.5; JMC, Jansen’s metaphyseal chondrodysplasia; PFA, paraformaldehyde; PPR, PTH/PTHrP receptor; Tg-A+ or Tg-B+, transgenic line A heterozygous or transgenic line B heterozygous; Tg-A++ or Tg-B++, transgenic line A homozygous or transgenic line B homozygous.

Received April 16, 2001.

Accepted for publication August 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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