Discovery and characterization of spontaneous mouse models of craniofacial dysmorphology.

Craniofacial abnormalities are among the most common features of human genetic syndromes and disorders. The etiology of these conditions is often complex, influenced by both genetic context and the environment. Frequently, craniofacial abnormalities present as part of a syndrome with clear comorbid phenotypes, providing additional insight into mechanisms of the causative gene or pathway. The mouse has been a key tool in our understanding of the genetic mechanisms of craniofacial development and disease, and can provide excellent models for human craniofacial abnormalities. While powerful genetic engineering tools in the mouse have contributed significantly our understanding of craniofacial development and dysmorphology, forward genetic approaches provide an unbiased means to identify new genes and pathways. Moreover, spontaneous mutations can occur on any number of genetic backgrounds, potentially revealing critical genes that require a specific genetic context. Here we report discovery and phenotyping of 43 craniofacial mouse models, derived primarily from a screen for spontaneous mutations in production colonies at the Jackson Laboratory. We identify the causative gene for 33 lines, including novel genes in pathways not previously connected to craniofacial development, and novel alleles of known genes that present with unique phenotypes. Together with our detailed characterization, this work provides a valuable gene discovery resource for the craniofacial community, and a rich source of mouse models for further investigation.

Synthetic Peptide CK2.3 Enhances Bone Mineral Density in Senile Mice.

BACKGROUND: Osteoporosis is a silent disease caused by low bone mineral density that results in bone fractures in 1 out of 2 women and 1 in 4 men over the age of 50. Although several treatments for osteopenia and osteoporosis are available, they have severe side effects and new treatments are desperately needed. Current treatments usually target osteoclasts and inhibit their activity or differentiation. Treatments that decrease osteoclast differentiation and activity but enhance osteogenesis and osteoblast activity are not available. We recently developed a peptide, CK2.3, that induces bone formation and increases bone mineral density as demonstrated by injection over the calvaria of 6 to 9-day-old mice and tail vein injection of 8-week-old mice. CK2.3 also decreased osteoclast formation and activity. However, these studies raise questions: does CK2.3 induce similar results in old mice and if so, what is the effective CK2.3 concentration and, is the bone mineral density of vertebrae of the spinal column increased as well? METHODS: CK2.3 was systematically injected into the tail vein of female 6-month old mice with various concentrations of CK2.3: 0.76 µg/kg, 2.3 µg/kg, or 6.9 µg/kg per mice. Mice were sacrificed one week, two weeks, and four weeks after the first injection. Their spines and femurs were collected and analyzed for bone formation. RESULTS: Femur and lumbar spine analyses found increased bone mineral density (BMD) and mineral apposition rate, with greater stiffness observed in femoral samples four weeks after the first injection. Histochemistry showed that osteoclastogenesis was suppressed in CK2.3 treated senile mice. CONCLUSIONS: For the first time, this study showed the increase of lumbar spine BMD by CK2.3. Moreover, it showed that enhancement of femur BMD was accompanied by increased femur stiffness only at medium concentration of CK2.3 four weeks after the first injection indicating the maintenance of bone's structural integrity by CK2.3.

Genetic dissection of mouse distal chromosome 1 reveals three linked BMD QTLs with sex-dependent regulation of bone phenotypes.

UNLABELLED: Genetic analyses with mouse congenic strains for distal Chr1 have identified three closely linked QTLs regulating femoral vBMD, mid-diaphyseal cortical thickness, and trabecular microstructure in a sex-dependent fashion. The homologous relationship between distal mouse Chr 1 and human 1q21-24 offers the possibility of finding common regulatory genes for cortical and trabecular bone. INTRODUCTION: The distal third of mouse chromosome 1 (Chr 1) has been shown to carry a major quantitative trait locus (QTL) for BMD from several inbred mouse strain crosses. Genetic and functional analyses are essential to identify genes and cellular mechanisms for acquisition of peak bone mass. MATERIALS AND METHODS: Nested congenic sublines of mice were developed with a C57BL/6J (B6) background carrying <1- to 9-Mbp-sized segments donated from C3H/HeJ (C3H). Isolated femurs from 16-wk-old female and male mice were measured by pQCT and microCT40 for volumetric (v)BMD, mid-diaphyseal cortical thickness, and distal trabecular phenotypes. Static and dynamic histomorphologic data were obtained on selected females and males at 16 wk. RESULTS AND CONCLUSIONS: We found that the original BMD QTL, Bmd5, mapped to distal Chr 1 consists of three QTLs with different effects on vBMD and trabecular bone in both sexes. Compared with B6 controls, femoral vBMD, BMD, and cortical thickness (p < 0.0001) were significantly increased in congenic subline females, but not in males, carrying C3H alleles at QTL-1. Both females and males carrying C3H alleles at QTL-1 showed marked increases in BV/TV by microCT compared with B6 mice (p < 0.0001). Females increased BV/TV by increasing trabecular thickness, whereas males increased trabecular number. In addition, the microCT40 data showed two unique QTLs for male trabecular bone, QTL-2 and QTL-3, which may interact to regulate trabecular thickness and number. These QTLs are closely linked with and proximal to QTL-1. The histomorphometric data revealed sex-specific differences in cellular and bone formation parameters. Mice and humans share genetic homology between distal mouse Chr 1 and human Chr 1q20-24 that is associated with adult human skeletal regulation. Sex- and compartment-specific regulatory QTLs in the mouse suggest the need to partition human data by sex to improve accuracy of mapping and genetic loci identification.

Systemic injection of CK2.3, a novel peptide acting downstream of bone morphogenetic protein receptor BMPRIa, leads to increased trabecular bone mass.

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal-related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.

Multiple quantitative trait loci for cortical and trabecular bone regulation map to mid-distal mouse chromosome 4 that shares linkage homology to human chromosome 1p36.

The mid-distal region of mouse chromosome 4 (Chr 4) is homologous with human Chr 1p36. Previously, we reported that mouse Chr 4 carries a quantitative trait locus (QTL) with strong regulatory effect on volumetric bone mineral density (vBMD). The intent of this study is to utilize nested congenic strains to decompose the genetic complexity of this gene-rich region. Adult females and males from 18 nested congenic strains carrying discrete C3H sequences were phenotyped for femoral mineral and volume by pQCT and for trabecular bone volume (BV), tissue volume (TV), trabecular number (Trab.no), and trabecular thickness (Trab.thk) by MicroCT 40. Our data show that the mouse Chr 4 region consists of at least 10 regulatory QTL regions that affected either or both pQCT and MicroCT 40 phenotypes. The pQCT phenotypes were typically similar between sexes, whereas the MicroCT 40 phenotypes were divergent. Individual congenic strains contained one to seven QTL regions. These regions conferred large positive or negative effects in some congenic strains, depending on the particular bone phenotype. The QTL regions II to X are syntenic with human 1p36, containing from 1 to 102 known genes. We identified 13 candidate genes that can be linked to bone within these regions. Six of these genes were linked to osteoblasts, three linked to osteoclasts, and two linked to skeletal development. Three of these genes have been identified in Genome Wide Association Studies (GWAS) linked to 1p36. In region III, there is only one gene, Lck, which conferred negative pQCT and MicroCT 40 phenotypes in both sexes. This gene is important to development and functioning of T cells, has been associated with osteoclast activity, and represents a novel bone regulatory gene that merits further experimental evaluation. In summary, congenic strains are powerful tools for identifying regulatory regions that influence bone biology and offer models for testing hypotheses about gene-gene and gene-environment interactions that are not available to experimental work in humans.

BMD regulation on mouse distal chromosome 1, candidate genes, and response to ovariectomy or dietary fat.

The distal end of mouse chromosome 1 (Chr 1) harbors quantitative trait loci (QTLs) that regulate bone mineral density (BMD) and share conserved synteny with human chromosome 1q. The objective of this article was to map this mouse distal Chr 1 region and identify gene(s) responsible for BMD regulation in females. We used X-ray densitometry [ie, dual-energy X-ray Absorptiometry (DXA), micro-computed tomography (µCT), and peripheral quantitative computed tomography (pQCT)] to phenotype a set of nested congenic strains constructed from C57BL/6BmJ (B6/Bm) and C3H/HeJ (C3H) mice to map the region associated with the BMD QTL. The critical region has been reduced to an interval of 0.152 Mb that contributes to increased BMD when C3H alleles are present. Histomorphometry and osteoblast cultures indicated that increased osteoblast activity was associated with increased BMD in mouse strains with C3H alleles in this critical region. This region contains two genes, Aim2, which binds with cytoplasmic dsDNA and results in apoptosis, and AC084073.22, a predicted gene of unknown function. Ovariectomy induced bone loss in the B6/Bm progenitor and the three congenic strains regardless of the alleles present in the critical BMD region. High dietary fat treatment (thought to suppress distal Chr 1 QTL for BMD in mice) did not induce bone loss in the congenics carrying C3H alleles in the critical 0.152 Mb carrying the AIM2 and AC084073.22 genes. Gene expression studies in whole bone of key congenics showed differential expression of AC084073.22 for strains carrying B6/Bm versus C3H alleles but not for Aim2. In conclusion, our data suggest that osteoblasts are the cellular target of gene action and that AC084073.22 is the best candidate for female BMD regulation in the distal region of mouse Chr 1.