Autopodial development is selectively impaired by misexpression of chordin-like 1 in the chick limb

AbstractChordin-like 1 (CHRDL1) is a secreted bone morphogenetic protein (BMP) antagonist expressed in mesenchymal tissues whose function in development of the skeleton has not been examined in detail. Here we show Chrdl1 is dynamically expressed in the early distal limb bud mesenchyme, with expression becoming downregulated as development proceeds. Chrdl1 expression is largely excluded from the critical signaling center of the posterior limb bud, the Zone of Polarizing Activity (ZPA), as has been described for the BMP antagonist Gremlin (GREM1) (Scherz et al., 2004, Science, 305, 396–399). Unlike Grem1, Chrdl1 is expressed in the hindlimb by a small subset of ZPA cells and their descendants suggesting divergent regulation and function between the various BMP antagonists. Ectopic expression of Chrdl1 throughout the avian limb bud using viral misexpression resulted in an oligodactyly phenotype with loss of digits from the anterior limb, although the development of more proximal elements of the zeugopod and stylopod were unaffected. Overgrowths of soft tissue and syndactyly were also observed, resulting from impaired apoptosis and failure of the anterior mesenchyme to undergo SOX9-dependent chondrogenesis, instead persisting as an interdigital-like soft tissue phenotype. Sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling were upregulated and persisted later in development, however these changes were only detected late in limb development at timepoints when endogenous Grem1 would normally be downregulated and increasing BMP signaling would cause termination of Shh and Fgf expression. Our results suggest that the early stages of the GREM1–SHH–FGF signaling network are resistant to Chrdl1-overexpression, leading to normal formation of proximal limb structures, but that later Bmp expression, impaired by ectopic CHRDL1, is essential for formation of the correct complement of digits

A Wisp3 Cre-knockin Allele Produces Efficient Recombination in Spermatocytes during Early Prophase of Meiosis I

Individuals with the autosomal recessive skeletal disorder Progressive Pseudorheumatoid Dysplasia have loss-of-function mutations in WISP3, and aberrant WISP3 expression has been detected in tumors from patients with colon and breast cancer. In mice however, neither absence nor over-expression of WISP3 was found to cause a phenotype, and endogenous Wisp3 expression has been difficult to detect. To confirm that Wisp3 knockout mice have no phenotype and to identify potential sites of endogenous Wisp3 expression, we generated mice with a knockin allele (Wisp3GFP-Cre) designed to express Green Fluorescent Protein (GFP) and Cre-recombinase instead of WISP3. Heterozygous and homozygous knockin mice were fertile and indistinguishable from their wild-type littermates, confirming that mice lacking Wisp3 have no phenotype. We could not detect GFP-expression from the knockin allele, but we could detect Cre-expression after crossing mice with the knockin allele to Cre-reporter mice; the double heterozygous offspring had evidence of Cre-mediated recombination in several tissues. The only tissue that had high levels of Cre-mediated recombination was the testis, where recombination in spermatocytes occurred by early prophase of meiosis I. As a consequence, males that were double heterozygous for a Wisp3GFP-Cre and a floxed allele only contributed a recombined allele to their offspring. We detected no evidence of Cre-mediated recombination in the female ovary, although when double heterozygous females contributed the reporter allele to their offspring it had recombined ~7% of the time. Wisp3GFP-Cre expression therefore occurs less frequently and most likely at a later stage of oocyte development in female mice compared to male mice. We conclude that although WISP3 is dispensable in mice, male mice with a Wisp3GFP-Cre allele (Jackson Laboratory stock # 017685) will be useful for studying early prophase of meiosis I and for efficiently recombining floxed alleles that are passed to offspring

Presphenoidal synchondrosis fusion in DBA/2J mice

Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice. Electronic supplementary material The online version of this article (doi:10.1007/s00335-012-9437-8) contains supplementary material, which is available to authorized users

Protective Effects of C-Type Natriuretic Peptide on Linear Growth and Articular Cartilage Integrity in a Mouse Model of Inflammatory Arthritis

Objective: The C-type natriuretic peptide (CNP) signaling pathway is a major contributor to postnatal skeletal growth in humans. This study was undertaken to investigate whether CNP signaling could prevent growth delay and cartilage damage in an animal model of inflammatory arthritis. Methods: We generated transgenic mice that overexpress CNP (B6.SJL-Col2a1-NPPC) in chondrocytes. We introduced the CNP transgene into mice with experimental systemic inflammatory arthritis (K/BxN T cell receptor [TCR]) and determined the effect of CNP overexpression in chondrocytes on the severity of arthritis, cartilage damage, and linear growth. We also examined primary chondrocyte cultures for changes in gene and protein expression resulting from CNP overexpression. Results: K/BxN TCR mice exhibited linear growth delay (P < 0.01) compared to controls, and this growth delay was correlated with the severity of arthritis. Diminished chondrocyte proliferation and matrix production was also seen in K/BxN TCR mice. Compared to non–CNP-transgenic mice, K/BxN TCR mice with overexpressed CNP had milder arthritis, no growth delay, and less cartilage damage. Primary chondrocytes from mice overexpressing CNP were less sensitive to inflammatory cytokines than wild-type mouse chondrocytes. Conclusion: CNP overexpression in chondrocytes can prevent endochondral growth delay and protect against cartilage damage in a mouse model of inflammatory arthritis. Pharmacologic or biologic modulation of the CNP signaling pathway may prevent growth retardation and protect cartilage in patients with inflammatory joint diseases, such as juvenile idiopathic arthritis

High-bone-mass causing mutant LRP5 receptors are resistant to endogenous inhibitors in vivo

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). We previously reported the creation of two Lrp5 HBM knock-in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock-in mice are resistant to SOST- or DKK1-induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes ((8kb) Dmp1-SOST) or mouse DKK1 in osteoblasts and osteocytes ((2.3kb) Col1a1-Dkk1). We observed that the (8kb) Dmp1-SOST transgene significantly lowered whole-body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone-formation rate (BFR) in wild-type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The (2.3kb) Col1a1-Dkk1 transgene significantly lowered whole-body BMD, BMC, and vertebral BV/TV in wild-type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM-causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1

Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues

In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3caH1047R) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3caH1047R/+ offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3caH1047R/+ offspring survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3caH1047R/+ and Tg(DMP1-Cre);Pik3caH1047R/+ mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude that differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation are due to Cre-recombination being induced at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination

Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, μCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8–17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population

Expression of a Degradation‐Resistant β‐Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation‐Induced Bone Wasting

Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β‐catenin—an intracellular signaling node in the canonical Wnt pathway—in disuse mechanotransduction is not defined. Using the β‐catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen‐inducible, osteocyte‐selective Cre driver, we evaluated the effects of degradation‐resistant β‐catenin on bone properties during disuse. We hypothesized that if β‐catenin plays an important role in Wnt‐mediated osteoprotection, then artificial stabilization of β‐catenin in osteocytes would protect the limbs from disuse‐induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum‐toxin (botox)‐induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail‐suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox‐treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail‐suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail‐suspended β‐catenin stabilized mice and tail‐suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β‐catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease

Co-deletion of Lrp5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration

The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of Lrp5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when Lrp5 is suppressed), or through other yet undiscovered mechanisms independent of Lrp5/6. To distinguish between these two possibilities, we generated mice with compound deletion of Lrp5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both Lrp5 and Lrp6 (Lrp5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre−) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre− mice, as assessed by DXA, μCT, pQCT, and histology, indicating that Lrp5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre− mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of Lrp5 alone, the bone-selective late-stage co-deletion of Lrp5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both Lrp5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody