Article |
Address correspondence to Gerard Karsenty or Lawrence Chan, Baylor College of Medicine, One Baylor Plaza, Rm. S921, Houston, TX 77030. Tel.: (713) 798-4490. Fax: (713) 798-1530. E-mail: karsenty{at}bcm.tmc.edu or lchan{at}bcm.tmc.edu
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Abstract |
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Key Words: low bone mass; blindness; Wnt; osteoblast function; vascular regression
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Introduction |
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In Drosophila, arrow encodes a single-pass transmembrane protein thought to act as a coreceptor for wingless, as inactivation of arrow results in a wingless-like phenotype (Wehrli, 1999). Arrow is an LRP strikingly homologous to vertebrate LRP5 and LRP6 (Wehrli, 1999; Pinson et al., 2000; Tamai et al., 2000). This sequence homology is consistent with the multiple Wnt deficiencylike phenotypes of Lrp6-deficient mice (Pinson et al., 2000). Lrp5, the other homologue of arrow and a close relative of Lrp6, is involved in Wnt signaling in vitro. Indeed, in NIH3T3 fibroblasts, the interaction of Lrp5 with Axin is initiated by Wnt treatment of these cells (Mao et al., 2001). The observation that overexpression of Lrp6, but not of Lrp5, induced dorsal axis duplication in Xenopus (Tamai et al., 2000) indicates that these two receptors control different functions, presumably by interacting with distinct ligands of the Wnt family.
The Wnt proteins are secreted proteins that control multiple developmental processes including mesoderm induction, cell fate determination, limb patterning, and organogenesis (Parr and McMahon, 1998; Wodarz and Nusse, 1998; Vainio et al., 1999; Hartmann and Tabin, 2001). Wingless in Drosophila and Wnt proteins in vertebrates initiate these events by binding to seven transmembrane domain receptors of the Frizzled family (Bhanot et al., 1996; Wodarz and Nusse, 1998). Wnt binding to Frizzled results in stabilization of ß-catenin, which then interacts with transcription factors of the Lef/Tcf family to activate specific gene expression programs (Huelsken and Birchmeier, 2001). Distinct Wnt proteins control early events during skeletal development such as limb patterning (Perrimon and McMahon, 1998) and joint formation (Hartmann and Tabin, 2001). However, the recent findings that LRP5 is inactivated in osteoporosis-pseudoglioma syndrome patients (Gong et al., 2001) and is mutated in patients with the high bone mass syndrome (Little et al., 2002) strongly suggest that Wnt proteins may control other aspects of skeletal biology later during development and postnatally.
Through the analysis of mice lacking most of the Lrp5 protein we present evidence that the Lrp5 signaling pathway is required for osteoblast proliferation as well as for bone matrix deposition by differentiated osteoblasts. Surprisingly, these phenotypic abnormalities occur in the context of normal Cbfa1/Runx2 (Cbfa1) expression, a gene usually viewed as controlling osteogenesis (Karsenty, 1999). Thus, these results suggest a role for Cbfa1-independent pathways in the control of osteoblast proliferation and function. Biochemical and expression evidence indicates that Lrp5 binds directly to Wnt proteins and is required for optimal signaling of a distinct subset of Wnt proteins belonging to the Wnt1 subfamily in osteoblasts. Lrp5 is also necessary for the normal regression of embryonic vasculature in the eye. Together, these results demonstrate that the Lrp5 signal transduction pathway, and thereby Wnt proteins, regulate osteoblast proliferation, function, and eye vascularization during late development and after birth.
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Results |
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Immunolocalization of Lrp5
To identify the cell types producing Lrp5, we used an antibody directed against the amino terminal part of the molecule which is still transcribed in Lrp5-/- mice (Figueroa et al., 2000). By immunohistochemistry we observed the presence of the protein in osteoblasts lining the endosteal and trabecular bone surfaces (Fig. 2, AG). We could not detect any expression in osteoclasts (unpublished data). Lrp5 protein was detected only in F4/80-positive macrophages found closely associated with the vitreous microvasculature of the eye (Fig. 2, HM). Consistent with its broad pattern of RNA expression, Lrp5 was also detected in other tissues including liver, pancreas, skin, and brain (Figueroa et al., 2000; unpublished data).
Low bone mass phenotype in Lrp5-/- mice
A small number of mutant animals were limping while walking, suggesting the existence of a skeleton-related abnormality. Radiographic analysis of one of these mutant mice at 2 mo of age revealed the presence of a fracture in the tibia betraying the existence of a low bone mass phenotype (Fig. S2) that was present in all Lrp5-/- mice at that age (Fig. 3 A; unpublished data).
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Decreased bone formation postnatally in Lrp5-/- mice
The cellular basis of this low bone mass phenotype was studied using histomorphometric analyses and biochemical and cell-based assays. The bone formation aspect of bone remodeling was analyzed by measuring the bone formation rate (BFR), an indicator of osteoblast activity, after in vivo double labeling with calcein, a marker of newly formed bone. In 6-mo-old Lrp5-/- mice there was a twofold decrease in the BFR compared with wild-type littermates (Fig. 4 A). A similar difference was observed in 2- and 4-mo-old Lrp5-/- mice (unpublished data). The decreased BFR was solely due to a decreased matrix apposition rate (MAR) (Fig. 4 A, arrow), an index of the amount of bone matrix deposited per osteoblast cluster (0.75 ± 0.05 vs. 0.45 ± 0.06 µm/d; P < 0.05) (Aaron et al., 1984). This decreased MAR directly demonstrates a functional defect of osteoblasts in vivo in the absence of Lrp5. Consistent with the dominant nature of the osteoporosis-like phenotype, the BFR was also significantly decreased in Lrp5+/- mice (88.6 ± 4.8 vs. 60.5 ± 3.9 µm3/µm2/y; P < 0.05). We also performed osteoblast counts in the primary and secondary spongiosa. The results obtained were identical in both cases: total osteoblast number per bone area was significantly decreased in Lrp5-/- mice (Fig. 4 B). To further study osteoblast function, we assayed the ability of primary osteoblast cultures to mineralize an extracellular matrix (ECM). As shown in Fig. 4 C, mineralization of the ECM surrounding Lrp5-/- osteoblasts was delayed compared with what we observed in wild-type osteoblasts at day 10 of culture. This latter finding supports the notion that Lrp5-/- osteoblasts have a functional defect. However, it may alternatively be explained by the abnormal proliferation of Lrp5-/- osteoblasts (see below). The growth plate of Lrp5-/- mice appeared normal, indicating that chondrogenesis was not overtly affected (Fig. 4 D).
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Abnormal osteoblast proliferation and normal Cbfa1 expression in Lrp5-/- mice
The perinatal onset of the osteogenesis defect, along with the decreased number of osteoblasts in Lrp5-/- mice, suggested that Lrp5 could be required for osteoblast proliferation and/or differentiation. To determine if osteoblast proliferation was affected, we measured the number of cells actively synthesizing DNA by 5-bromo-2'-deoxy-uridine (BrdU) incorporation in vivo. Fig. 5 E shows sections of calvaria from 4-d-old wild-type and Lrp5-/- mice. Significantly fewer labeled nuclei were detected in sections from Lrp5-/- mice compared to those derived from wild-type mice (Fig. 5 E). The total number of osteoblasts was also decreased in Lrp5-/- calvaria but even after correction for this decreased cell number there was still a significantly lower mitotic index for mutant osteoblasts in vivo (Fig. 5 F). To assess whether this proliferation defect extended to progenitors of the osteoblast lineage, we determined the number of stromal cell progenitors (CFU-F) in wild-type and Lrp5-/- bone marrow. The numbers of nucleated bone marrow cells obtained from wild-type and Lrp5-/- bone marrow were similar (unpublished data). However, as shown in Fig. 5 G, Lrp5-/- bone marrow cells formed almost twofold fewer alkaline phosphatasepositive colonies than wild-type bone marrow cells, suggesting the existence of an early proliferation defect in the osteoblast lineage. There was no increased apoptosis in the calvaria of Lrp5-/- mice (Fig. 5 H), indicating that the low mitotic index of mutant osteoblasts was solely due to a proliferation defect.
To determine if the osteoblast phenotype affected the classical Cbfa1 pathway controlling osteoblast differentiation we examined the expression of Cbfa1 in calvaria, long bones, and primary osteoblasts. As shown in Fig. 5 I, Cbfa1 expression was not decreased in the absence of Lrp5, indicating that this bone phenotype develops in a Cbfa1-independent manner. Because there are fewer osteoblasts in Lrp5-/- calvaria and long bones, it is possible that Cbfa1 may be upregulated in these mice. Moreover, the normal expression of Osteocalcin in Lrp5-/- (Fig. 5 J) mice suggests also that osteoblast differentiation is not affected in the absence of Lrp5.
In summary, in vivo and cell-based assays support the hypothesis that the low bone mass phenotype observed in Lrp5-/- mice is due to two main defects, both occurring in a Cbfa1-independent manner: a decrease in osteoblast proliferation evidenced by the BrdU in vivo labeling experiments, and a decrease in bone matrix deposition shown by the decreased BFR.
Lrp5 acts as a Wnt receptor in osteoblasts
The homology between arrow, a coreceptor for wingless in Drosophila, and Lrp5, as well as the requirement of Lrp5 for Wnt signaling in NIH3T3 fibroblasts (Wehrli, 1999; Mao et al., 2001), suggested that the bone phenotype of Lrp5-/- mice may be secondary to a defect in Wnt signaling in osteoblasts.
To test this hypothesis, we first performed DNA cotransfection experiments in primary calvarial osteoblasts obtained from wild-type or Lrp5-/- 4-d-old mice. Cells were transfected with a luciferase reporter construct containing multiple copies of an oligonucleotide containing the Lef1 binding site (TOPtkluc) (Korinek et al., 1997), as well as a Lef1 expression vector. Using this assay, cotransfection of a Wnt1 expression vector consistently enhanced Lef1-dependent transcription to a higher degree in wild-type than in Lrp5-/- primary osteoblasts (Fig. 6 A). Importantly, cotransfection of an LRP5 expression vector into Lrp5-/- osteoblasts restored the ability of Wnt1 to induce Lef1-dependent gene expression to wild-type levels (Fig. 6 B).
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Expression of Wnts, and of genes encoding Wnt signaling molecules in bone
Lrp5 is expressed in osteoblasts at early and late stages of differentiation (Fig. 1 B). To determine if the low bone mass phenotype in the Lrp5-/- mice was consistent with the expression pattern of other molecules involved in Wnt signaling, we analyzed their expression in bone and in osteoblasts. Wnt1 was expressed at high levels in calvaria in both wild-type and Lrp5-/- mice (Fig. 7 A). Wnt1 expression was detected in primary osteoblasts at higher amplification levels, but was absent from day 0 Lrp5-/- osteoblasts (Fig. 7 A), suggesting that Wnt1 expression by proliferating osteoblasts requires the presence of Lrp5. The normal level of Wnt1 expression in day 10 Lrp5-/- osteoblasts suggests that Lrp5 is not necessary for Wnt1 expression at later stages of differentiation when proliferation ceases. Wnt4 and 14 were also expressed in calvaria and in osteoblasts in wild-type and Lrp5-/- samples (Fig. 7 B). Wnt3a expression was not detected in calvaria, long bones, or primary osteoblasts, regardless of the level of amplification indicating that it may not be the ligand for Lrp5 in bone (unpublished data).
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Abnormal macrophage-mediated apoptosis in the eyes of Lrp5-/- mice
Postnatal development of the rodent eye involves the regression of three related vascular networks called the pupillary membrane (PM), tunica vasculosa lentis (TVL), and the hyaloid vessels (Fig. 8 A). In wild-type mice, regression of these networks is normally complete by P12 for the PM and by P16 for the TVL and hyaloid vessels (Ito and Yoshioka, 1999). Histological analysis of eyes at 6 mo of age revealed the presence of hyaloid vessels in 70% (28/40) of Lrp5-/- mice and in none of the wild-type controls. To define the onset of this phenotype, we performed a series of dissections of the capillary networks in order to compare their rates of regression. Although both the PM and TVL showed mild delays in regression (unpublished data), this response was dramatic in the hyaloid vessels; quantification of the number of capillary segments over a P3P8 time course (Fig. 8 B) indicated that loss of segments was much slower in Lrp5-/- mice. The presence of equal numbers of capillary segments in wild-type and Lrp5-/- mice at P3 indicated that the persistence was not a trivial consequence of early vessel overgrowth in Lrp5-/- mice. The persistence of the hyaloid vessels is readily visualized when comparing histological preparations from wild-type (Fig. 8, DF) and Lrp5-/- mice (Fig. 8, GI) over the P3P8 time course. Higher magnifications of hyaloid vessel preparations (Fig. 2, GL) indicate that the number of macrophages associated with wild-type and Lrp5-/- hyaloid vessels is not different. No delay in hyaloid vessel regression was detected in Lrp+/- mice (unpublished data).
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Discussion |
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Lrp5 as a determinant of peak bone mass in vertebrates
Three aspects of osteoblast biology could be controlled by the Lrp5 signaling pathway: differentiation, proliferation, and function. The decreased number of bone marrow stromal cell progenitors may be viewed as a differentiation defect. However, the normal expression of Cbfa1, the earliest marker of osteoblast differentiation, and of Osteocalcin, the latest marker of osteoblast differentiation, together with the absence of any detectable phenotypic abnormalities in Lrp5-/- mice before birth, strongly suggest that the Lrp5 signaling pathway does not play a major role in the process of Cbfa1-dependent osteoblast differentiation. Several lines of evidence suggest that the Lrp5 signaling pathway plays an important role in osteoblast proliferation. First, the total number of osteoblasts was decreased in the skeleton of Lrp5-/- mice compared with wild-type controls. Second, and more importantly, by in vivo BrdU labeling we observed a lower proportion of proliferating osteoblasts in Lrp5-/- mice than in wild-type controls. Third, the number of stromal cell progenitors was markedly reduced in the bone marrow of Lrp5-/- mice. These developmental defects are not the only ones observed in osteoblasts in the absence of Lrp5. Indeed, the reduction in the MAR in Lrp5-/- mice and the delay in mineralization observed in primary cultures of Lrp5-/- osteoblasts suggest that the function of differentiated osteoblasts, i.e., bone matrix deposition, is decreased in Lrp5-/- mice.
Together, these two abnormalities lead to the inability of Lrp5-/- mice to reach a normal bone mass early during life. The bone mass normally achieved early on in vertebrate life, also called peak bone mass, has been shown to be genetically controlled (Johnston and Slemenda, 1993). Our data are consistent with the hypothesis that the Lrp5 signal transduction pathway is one of the genetic determinants of peak bone mass in vertebrates.
Uncovering a Cbfa1-independent control of osteoblast biology
Our findings, together with those of Gong et al. (2001) and Little et al. (2002), have two implications. First, the observation that Lrp5 signaling is important for osteoblast proliferation and bone formation suggests an additional, postnatal function for the Wnt proteins in skeletal biology. However, we are aware that despite the interaction between Wnt1 and Lrp5 reported here, the Wnt proteins may not be the only class of secreted molecules transducing their signals through Lrp5. This is an important concern, as other Lrps have been shown to have multiple ligands (Herz and Strickland, 2001).
Second, Lrp5 disruption reveals the existence of an additional, evolutionarily conserved, pathway besides Cbfa1-dependent gene expression, for controlling osteoblast proliferation and bone formation. The normal expression of Cbfa1 in Lrp5-/- mice implicates other transcriptional mediators in this alternate pathway. Because Lef/Tcf proteins are well characterized transcriptional mediators of Wnt actions in other organ systems (Merrill et al., 2001) and are expressed in osteoblasts, one or more members of this family are likely to control some aspects of osteoblast biology. An important question in bone biology will now be to define which aspects of bone biology are affected by Wnt and Lef/Tcf proteins and how these functions relate to Lrp5 function.
Wnt proteins and postnatal bone formation
Cell culture analyses have shown that LRP5 acts as a coreceptor for Wnt proteins (Mao et al., 2001). Our analysis defines this interaction as being one of direct binding and demonstrates its biological importance in vivo. We also observed expression of Wnt, Frizzled, ß-catenin, and Tcf genes in osteoblasts. The decreased expression of Lef1 in Lrp5-/- osteoblasts suggests that Lef1 expression is controlled directly or indirectly by the Lrp5 signaling pathway, and that Lef1 may be the preferred transcriptional effector of the various functions of this pathway in osteoblasts.
What may be the Wnt protein(s) that control osteoblast proliferation and function? The absence of Wnt3a expression in any bone sample analyzed implies that it is not the ligand of Lrp5 in bone. The accelerated ossification caused by Wnt4 overexpression in chick limb buds (Hartmann and Tabin, 2000) is the mirror image of the bone phenotype of Lrp5-/- mice. To our surprise, DNA cotransfection experiments showed that Wnt1, but not Wnt4, induces Lef1-dependent gene expression. Wnt proteins have been separated into two categories based on their ability to induce dorsal axis duplication in Xenopus embryos. Members of the Xwnt8 family induce axis duplication, whereas members of the Xwnt5a do not. In that respect, it is interesting to note that Wnt1 belongs to the first family, whereas Wnt4 belongs to the second family (Wodarz and Nusse, 1998). The function of Wnt1 itself during skeletal development and during bone formation has not been reported, in part because Wnt1-deficient mice die shortly after birth (McMahon and Bradley, 1990). This question can now be addressed by a conditional gene inactivation strategy. The identification of the ligand(s) for Lrp5 is an urgent need in the field of bone biology.
Lrp5 requirement during eye development
The mammalian eye is vascularized during embryonic development with three connected but anatomically distinct networks: the PM, TVL, and hyaloid vessels (Fig. 8 A). In the mouse, all three networks regress postnatally due to apoptosis of capillary cells (Lang et al., 1994; Meeson et al., 1996; Lang, 1997; Ito and Yoshioka, 1999). Lrp5-/- mice have a reduced level of capillary cell apoptosis and a defect in hyaloid vessel regression resulting in the retention of the hyaloid vasculature throughout life. It has been shown previously that ocular macrophages found closely associated with these vascular networks are necessary and sufficient for the cell death that drives the regression process (Lang and Bishop, 1993; Diez-Roux and Lang, 1997; Lang, 1997). Lrp5 is produced by macrophages but is not detectable in endothelial cells, suggesting that Lrp5 has a crucial role to play in macrophage-induced capillary cell death.
The presence of normal numbers of ocular macrophages in Lrp5-/- mice indicates that Lrp5 is not critical for macrophage survival or migration. This raises the hypothesis that an Lrp5 ligand, perhaps a member of the Wnt family, is critical for macrophage function, and more specifically, for the killing action of macrophages. Although a precise understanding of the role of Lrp5 in programmed capillary regression will require further experimentation, this phenotype is of great interest because it indicates for the first time a signaling pathway that is required for macrophages to kill other cells in a developmental context. Even more intriguing is the possibility that this mouse model will lead us to an understanding of the means by which macrophage-mediated endothelial cell killing might be harnessed for therapeutic benefit in the treatment of diseases characterized by inappropriate angiogenesis.
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Materials and methods |
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Immunohistochemistry
Frozen sections were cut at 10 µm, and Lrp5 was detected with the AS884 murine-specific N-terminal antibody as previously described (Figueroa et al., 2000). Primary antibody was detected with an Alexa Fluor-594 chicken antirabbit secondary antibody (Molecular Probes). Anti-Cbfa1 antibody #770 was used to detect osteoblasts on alternate sections and detected with an antirabbit HRP secondary antibody (Santa Cruz Biotechnology), followed by detection with DAB (Vector Laboratories). Macrophages were identified using an F4/80 antibody (Serotec) followed by detection with an Alexa Fluor-647 chicken antirat secondary antibody (Molecular Probes). Sections were counterstained with Hoechst stain to visualize nuclei.
Morphological and histological analyses
Skeletons from pups were prepared as described (Kochhar, 1973) and stained with Alcian blue and Alizarin red. All mice were injected with calcein, 10 and 2 d prior to sacrifice (Vignery and Baron, 1980). Undecalcified bones were fixed, dehydrated through an ethanol series, embedded in methylmethacrylate at 4°C, and 7-µm sections were prepared. Sections were stained with von Kossa reagent and counterstained by van Gieson reagent. Histomorphometric analysis was performed according to standard protocols (Parfitt et al., 1987) using the OsteoMeasure Analysis System (Osteometrics). Two sections from the third lumbar vertebra (L3) and two sections from L4 were measured for bone volume and tissue volume. All sections were >25 µm, i.e., more than one trabecular width apart. The mean distance between the calcein double labels was measured from 12-µm sections and used to calculate the MAR. These sections were also used to determine the mineralizing surface. Bone formation rate was calculated as the product of mineralizing surface per bone surface (Parfitt et al., 1987). Osteoblasts were counted separately in the primary and secondary spongiosa according to morphological criteria; cuboidal cells attached to bone occurring in clusters and having a large, asymmetrically positioned nucleus. The primary spongiosa was defined as the metaphyseal bone within one high power field (40x objective) of the growth plate. Statistical differences between groups were assessed by Student's t test.
Proliferation studies
Stromal cell progenitors were obtained by flushing bone marrow from 13-mo-old wild-type and Lrp5-/- mouse femora and tibiae. 0.5, 1.0, 2.0, and 4.0 x 107 nucleated cells were plated in 75-cm2 flasks in MEM/15% FBS, which was supplemented with 50 µg/ml ascorbic acid and fresh medium after 3 d of culture. The medium was then changed every 2 d with fresh ascorbic acid added until harvest at day 1014. Colonies were stained for alkaline phosphatase activity as previously described (Ducy et al., 1999), and the experiment repeated three times (n = 58 mice per genotype per experiment). For in vivo BrdU labeling, 4-d-old mice were injected intraperitoneally with 100 µl of 100 µM BrdU in PBS, sacrificed 4 h later, calvaria were isolated, fixed for 6 h in 10% formalin in PBS and processed in paraffin. Sections of 5 µm were prepared and stained with the BrdU staining kit (Zymed). Apoptotic cells were detected in calvarial sections by measuring terminal transferase labeling of cells (DeadEnd; Promega). Eight animals per genotype were used and three sections per animal were studied. Statistical differences between groups were assessed by Student's t test.
Cell culture, DNA transfection, Western blotting, and immunoprecipitation
Primary osteoblast cultures from calvaria of newborn, wild-type, and Lrp5-/- mice were established and mineralized in vitro as previously described (Ducy et al., 1999). Transfections were performed in 24-well plates (20,000 cells/well; Fugene6; Roche) in triplicate and repeated at least three times; cells were harvested 40 h after transfection. Empty vector was added to keep the total amount of DNA constant at 225 ng/well. The FOPtkluc reporter, containing mutated Lef1 binding sites, was used in control transfections. All luciferase values were corrected for ß-galactosidase activity as a control for transfection efficiency. For Western blot analysis, primary osteoblast lysates were collected in physiological buffer with protease inhibitor cocktail (Roche), homogenized, fractionated into cytosolic and membrane fractions by ultracentrifugation at 100,000 g for 90 min, and separated by SDS-PAGE. The epitope tags were detected by the anti-FLAG M2 antibody (Sigma-Aldrich) and anti-HA antibody (Santa Cruz Biotechnology). The antiß-catenin antibody was obtained from Transduction Laboratories. Coimmunoprecipiation assay was performed using transfected COS-7 cells that were lysed with 400 µl of RIPA buffer (50 mM Tris/Cl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS) with protease inhibitor cocktail. After removing cell debris by brief centrifugation, 200 µl of cell lysate were incubated with 10 µg of anti-HA or anti-Flag antibody for 90 min. 25 µl of protein G Sepharose (Amersham Pharmacia Biotech) was added and incubated for 90 min. The Sepharose was washed three times, and precipitated proteins were separated by SDS-PAGE followed by Western blot analysis using the anti-Flag or anti-HA antibodies.
Gene expression analyses
Total RNA was isolated from calvaria, long bones stripped of bone marrow and primary osteoblasts from P4 wild-type and mutant mice using Trizol (Invitrogen), and treated with DNaseI (Invitrogen). Reverse transcription was performed on 4 µg of RNA using SuperscriptII (Invitrogen) according to the manufacturer's protocol. Reverse transcription reactions were amplified by PCR using gene-specific primers in standard reaction conditions with a 2-min initial denaturation step followed by 24 or 30 cycles of 94°C20 s, 58°C20 s, and 72°C60 s. Reverse transcribed cDNA from day 11 embryos (E11) was used as a positive control for all PCR reactions. All products were resolved on a 1.6% agarose gel and analyzed by Southern blot hybridization using specific probes. PCR primers used for amplification are available upon request. RNase protection assays were performed on 5 µg total RNA (pretreated with DNaseI) using gel eluted probes prepared with the MAXIscript kit (Ambion). The RPAIII kit (Ambion) was used, products were separated on a 7.5% acrylamide gel and detected by autoradiography. The sizes of the full-length probes and protected fragments were confirmed using the RNA Century ladder (Ambion). The Lef1 and Tcf4 templates were those used for in situ hybridization analysis in Hartmann and Tabin (2000).
Preparation of hyaloid vessels, TUNEL labeling, and quantification
Animals were anesthetized, perfused with 4% PFA in PBS, eyeballs enucleated, and injected with a 1.5% solution of low melting point agarose in PBS. After 30 min at room temperature, eyeballs were incised around the equator. The hyaloid vessels embedded in agarose were removed from the retinal cup, heated on a glass slide, washed with PBS, and air dried; hyaloid vessel preparations were permeabilized with 0.05% Triton X-100 in PBS and processed for TUNEL labeling. Apoptotic cells were labeled using the DeadEnd kit (Promega) and visualized with streptavidin-Alexa Fluor 568 conjugate (Molecular Probes). Images were taken using Zeiss Axioplan microscope and a Sony DKC5000 digital camera. The total number of blood vessels and the number of TUNEL-labeled segments were quantified using established methods (Ito and Yoshioka, 1999) where we counted the number of vessels crossing a circle drawn at 50% of the maximum radial spread of the preparations. The apoptotic index was calculated as the ratio of the number of TUNEL-labeled vessels to the total number of blood vessels. At least three hyaloid vessel preparations were quantified for each time-point. Error bars represent standard errors.
Chemistry and hormone measurements
Calcium and phosphate were measured by their respective kits (Sigma-Aldrich). Deoxypyridinoline crosslinks were measured in morning urines using the Pyrilinks-D immunoassay kit (Metra Biosystems). Creatinine values were used for standardization between urine samples (Creatinine kit; Metra Biosystems) (n = 8 animals per genotype).
Online supplemental material
Online supplemental materials are available at http://www/jcb.org/cgi/content/full/200201089/DC1. Fig. S1 depicts the targeting strategy used for Lrp5, the Southern blot confirming correct targeting and genotyping data showing a normal Mendelian distribution at birth but slightly fewer animals (22.1%) alive at 1 mo of age. Fig. S2 shows a radiograph of a tibial fracture that occurred in a 2-mo-old Lrp52/2 mouse. Fig. S3 shows Western blots for the Wnt3a and Wnt4 transfection experiments to confirm expression and show cytoplasmic accumulation of ß-catenin.
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Footnotes |
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M. Kato, M.S. Patel, R. Levasseur, Ivan Lobov, and B.H.-J. Chang contributed equally to this work.
* Abbreviations used in this paper: BFR, bone formation rate; BrdU; 5-bromo-2'-deoxy-uridine; dpc, days postcoitum; ECM, extracellular matrix; ES, embryonic stem; LDLR, low-density lipoprotein receptor; Lrp, LDLR-related protein; MAR, matrix apposition rate; PM, pupillary membrane; TRAP, tartrate-resistant acid phosphatase; TVL, tunica vasculosa lentis.
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Acknowledgments |
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This work was supported by the following: March of Dimes (FY99-489); National Institutes of Health (DK58882, AR42919, and DE11290 to G. Karsenty, and HL51586 and HL16512 to L. Chan); a grant from the Societe Française de Rhumatologie; a grant from the Philippe Fondation (R. Levasseur); and a Canadian Institutes of Health Research Fellowship (M.S. Patel).
Submitted: 22 January 2002
Revised: 19 February 2002
Accepted: 5 March 2002
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