Differential cutaneous wound healing in thermally injured MRL/MPJ mice

Adult wound repair occurs with an initial inflammatory response, reepithelialization, and the formation of a permanent scar. MRL/MpJ mice following ear‐hole punch biopsies display accelerated healing and tissue regeneration. In this study, we characterized the healing responses in both MRL/MpJ and BALB/c mice following a 15% total body surface area full‐thickness cutaneous burn injury. Macroscopic and histological observations show that delayed wound closure in MRL/MpJ mice is accompanied by an increase in edema, reduced neutrophil infiltration, and more prominent eschar. In vivo bromodeoxyuridine labeling showed no defect in keratinocyte proliferation and migration (reepithelialization). In comparison with BALB/c mice, MRL/MpJ wounds had greater collagen deposition, less granulation tissue formation, and contained fewer α‐smooth muscle actin‐positive myofibroblasts. An observed reduction in dermal neutrophil infiltration and myofibroblast development correlated with enhanced angiogenesis. Overall, BALB/c wounds contracted sooner and to a larger degree, resulting in a significant decrease in scar formation. Interestingly, MRL/MpJ mice showed overt abnormalities in hair follicle proliferation, morphogenesis, and subsequent hair regrowth postburn injury. No substantial evidence of tissue regeneration was observed in either BALB/c or MRL/MpJ wounds. Our results convincingly demonstrate that MRL/MpJ skin burn wounds heal with scar formation with delays in two critical wound healing events: wound closure, and myofibroblast development.     

Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice

The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Furthermore, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F=16.49, P=0.0002), while no significant effect was found on total femur BMD (F=2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168-172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice.     

Tissue profiling MALDI mass spectrometry reveals prominent calcium-binding proteins in the proteome of regenerative MRL mouse wounds

MRL/MpJ‐Fas^lpr mice exhibit the ability to regenerate ear tissue excised by dermal punches. This is an exceptional model to identify candidate proteins that may regulate regeneration in typically nonregenerative tissues. Identification of key molecules involved in regeneration can broaden our understanding of the wound‐healing process and generate novel therapeutic approaches. Tissue profiling by matrix‐assisted laser desorption ionization mass spectrometry is a rapid, powerful proteomic tool that allows hundreds of proteins to be detected from specific regions of intact tissue specimens. To identify these candidate molecules, protein expression in ear punches was examined after 4 and 7 days using tissue profiling of MRL/MpJ‐Fas^lpr mice and the nonregenerative mouse strain C57BL/6J. Spectral analysis revealed distinct proteomic differences between the regenerative and nonregenerative phenotypes, including the calcium‐binding proteins calgranulin A and B, calgizzarin, and calmodulin. Spatial distributions for these differentially expressed proteins within the injured regions were confirmed by immunohistochemistry.     

Detecting Novel Bone Density and Bone Size Quantitative Trait Loci Using a Cross of MRL/MpJ and CAST/EiJ Inbred Mice

Most previous studies to identify loci involved in bone mineral density (BMD) regulation have used inbred strains with high and low BMD in generating F₂ mice. However, differences in BMD may not be a requirement in selecting parental strains for BMD quantitative trait loci (QTL) studies. In this study, we intended to identify novel QTL using a cross of two strains, MRL/MpJ (MRL) and CAST/EiJ (CAST), both of which exhibit relatively high BMD when compared to previously used strains. In addition, CAST was genetically distinct. We generated 328 MRL × CAST F₂ mice of both sexes and measured femur BMD and periosteal circumference (PC) using peripheral quantitative computed tomography. Whole-genome genotyping was performed with 86 microsatellite markers. A new BMD QTL on chromosome 10 and another suggestive one on chromosome 15 were identified. A significant femur PC QTL identified on chromosome 9 and a suggestive one on chromosome 2 were similar to those detected in MRL × SJL. QTL were also identified for other femur and forearm bone density and bone size phenotypes, some of which were colocalized within the same chromosomal positions as those for femur BMD and femur PC. This study demonstrates the utility of crosses involving inbred strains of mice which exhibit a similar phenotype in QTL identification.     

Regeneration in MRL mice: further genetic loci controlling the ear hole closure trait using MRL and M.m. Castaneus mice

The MRL mouse has been shown to display an epimorphic regenerative response after ear hole punching leading to complete closure within 30 days and cartilage regrowth. The regenerative capacity of the MRL has also been seen after a severe cryoinjury to the heart leads to complete healing without scarring and functional myocardium. The wound healing ear hole closure response that occurs in MRL mice has been shown to be genetically controlled. We have previously identified 11 quantitative trait loci (QTL) that govern healing in an intercross of (MRL x C57BL/6 J) mice. However, it is desirable to use another poorly healing mouse strain to elucidate the full range of genetic factors that affect this important process. In the current study, we have used an inbred subspecies of the mouse, M. castaneus, and have confirmed a number of loci identified previously. In addition, we report three new healing QTL. Furthermore, in this strain combination, we note a strong sexual dimorphism also observed in the MRL x C57BL/6 cross, both in the healing trait and in the QTL that control it.     

Multiple genetic loci from CAST/EiJ chromosome 1 affect vBMD either positively or negatively in a C57BL/6J background.

Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively. INTRODUCTION: BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1). MATERIALS AND METHODS: After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines. RESULTS: Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (-6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = -0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb. CONCLUSIONS: Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.     

Congenic Mice Reveal Sex-Specific Genetic Regulation of Femoral Structure and Strength

Genetic linkage studies in C3H/HeJ (C3H) and C57BL/6J (B6) mice identified several chromosomal locations or quantitative trait loci (QTL) linked to femoral volumetric bone mineral density (vBMD). From QTL identified on chromosomes (chr) 1, 4, 6, 13, and 18, five congenic mouse strains were developed. In each of these mice, genomic DNA from the QTL region of the donor C3H strain was transferred into the recipient B6 strain. Here we report the effects of donated C3H QTL on femoral structure, cortical vBMD and bending strength. Femoral structure was quantified by the polar moment of inertia (I_p) at the mid-diaphysis, which reflects the bending or torsional rigidity of the femur. Although the C3H progenitor mice have a smaller I_p than B6 progenitor mice, the congenic mice carrying the C3H segment at Chr 4 had significantly increased I_p in both males and females, giving these mice stronger femora. In female mice from the congenic Chr 1 strain, I_p was increased whereas male mice from the Chr 1 strain had smaller femoral cross-sections and significantly reduced I_p. This sex-specific effect on femoral structure was seen to a lesser extent in Chr 18 congenic mice. In addition, cortical vBMD was measured using peripheral quantitative computed tomography. Cortical vBMD was similar among most congenic strains except in Chr 6 congenic mice, where cortical vBMD was significantly less in females, but not in males. We conclude that (1) chromosomal QTL from C3H mice, which are genetically linked to total femoral vBMD, also regulate femoral structure; (2) the QTL on Chr 4 improves femoral structure and strength; (3) QTL on Chr 1 and 18 impart sex-specific effects on femoral structure; and (4) the QTL on Chr 6 imparts a sex-specific effect on cortical vBMD and femoral strength.     

Mapping quantitative trait loci that influence blood levels of alkaline phosphatase in MRL/MpJ and SJL/J mice

To examine the hypothesis that serum alkaline phosphatase (ALP) levels have a heritable component, we analyzed blood from two inbred strains of mice, MRL/MpJ and SJL, which exhibit 90% difference in total serum ALP activity (268+/-26 vs. 140+/-15 U/l, respectively, P<0.001). A genome-wide scan was carried out using 137 polymorphic markers in 518 F2 female mice. Serum ALP activity in the F2 progeny showed a normal distribution with an estimated heritability of 56%. Genome-wide scan for cosegregation of genetic marker data with serum ALP activity revealed three major quantitative trait loci (QTL), one each on chromosomes 2 (LOD score 3.8), chromosome 6 (LOD score 12.0), and chromosome 14 (LOD score 3.7). In addition, there was one suggestive QTL on chromosome 2 (LOD score of 3.3). In aggregate, these QTLs explain 22.5% of variance in serum ALP between these two strains. Serum ALP showed a moderate but significant correlation with body weight adjusted total body bone mineral density (r=0.12, P=0.0108) and periosteal circumference at midshaft tibia (r=0.15, P=0.0006) in F2 mice. The chromosome 6 locus harboring the major serum ALP QTL also contains a major BMD and bone size QTL, identified earlier, between these two strains of mice; in addition, this QTL is also close to the locus that regulates IGF-I levels (LOD score 8-9) in C3HB6 F2 mice. These common QTLs indicate that the observed difference in ALP and BMD or bone size may be regulated by same loci (or genes). Accordingly, the osteoblast cells isolated from femur and tibia of MRL mice showed a significantly higher number of ALP +ve cells/colony and two- to threefold higher ALP activity (P<0.001) as compared to the cells isolated from SJL mice, thus suggesting that differences in serum ALP between MRL and SJL reflect difference in ALP expression from osteoblasts from these strains of mice. These data suggest that serum ALP levels are genetically determined and correlate with cellular mechanisms that differentiate BMD accrual in these two strains of mice. The findings that ALP and BMD traits share the same loci on chromosome 6 suggest a role for genetic determinants of bone formation in overall BMD accretion.     

Skin wounds in the MRL/MPJ mouse heal with scar

Adult MRL/MpJ mice regenerate cartilage during repair of through-and-through ear punch wounds. However, the ability of this mouse strain to heal isolated cutaneous wounds by regeneration or with scar is unknown. The purpose of this study was to characterize the rate of reepithelialization and collagen architecture in dermal wounds from MRL/MpJ mice compared with C57bl/6 and Balb/c strains. Full-thickness incisional (5 mm) and excisional (2 mm diameter) skin wounds were made on the dorsum of 7-week-old MRL/MpJ, C57bl/6, and Balb/c mice. Ear punch wounds were made simultaneously on each animal. Reepithelialization was complete by 48 hours for incisional skin wounds in each strain. All excisional wounds showed incomplete reepithelialization at 24, 48, and 72 hours. At 14 days, all skin wounds had grossly healed. In contrast to the ear wounds made in C57bl/6 and Balb/c mice, MRL/MpJ ear wounds were completely healed by day 28. Dorsal skin wound sections at 14 and 28 days revealed dense collagen deposition and similar degrees of fibrosis between the three strains of mice. In conclusion, in contrast to wound healing in the ear, MRL/MpJ mouse dorsal cutaneous wounds heal similarly to C57bl/6 and Balb/c mice with dermal collagen deposition and scar formation.     

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. © 2011 American Society for Bone and Mineral Research.     

Congenic strains of mice for verification and genetic decomposition of quantitative trait loci for femoral bone mineral density.

Peak femoral volumetric bone mineral density (femoral bone mineral density) in C57BL/6J (B6) 4-month-old female mice is 50% lower than in C3H/HeJ (C3H) and 34% lower than in CAST/EiJ (CAST) females. Genome-wide analyses of (B6 x C3H)F2 and (B6 x CAST)F2 4-month-old female progeny demonstrated that peak femoral bone mineral density is a complex quantitative trait associated with genetic loci (QTL) on numerous chromosomes (Chrs) and with trait heritabilities of 83% (C3H) and 57% (CAST). To test the effect of each QTL on femoral bone mineral density, two sets of loci (six each from C3H and CAST) were selected to make congenic strains by repeated backcrossing of donor mice carrying a given QTL-containing chromosomal region to recipient mice of the B6 progenitor strain. At the N6F1 generation, each B6.C3H and B6.CAST congenic strain (statistically 98% B6-like in genomic composition) was intercrossed to obtain N6F2 progeny for testing the effect of each QTL on femoral bone mineral density. In addition, the femoral bone mineral density QTL region on Chr 1 of C3H was selected for congenic subline development to facilitate fine mapping of this strong femoral bone mineral density locus. In 11 of 12 congenic strains, 6 B6.C3H and 5 B6.CAST, femoral bone mineral density in mice carrying c3h or cast alleles in the QTL regions was significantly different from that of littermates carrying b6 alleles. Differences also were observed in body weight, femoral length, and mid-diaphyseal periosteal circumference among these 11 congenic strains when compared with control littermates; however, these latter three phenotypes were not consistently correlated with femoral bone mineral density. Analyses of eight sublines derived from the B6.C3H-1T congenic region revealed two QTLs: one located between 36.9 and 49.7 centiMorgans (cM) and the other located between 73.2 and 100.0 cM distal to the centromere. In conclusion, these congenic strains provide proof of principle that many QTLs identified in the F2 analyses for femoral bone mineral density exert independent effects when transferred and expressed in a common genetic background. Furthermore, significant differences in femoral bone mineral density among the congenic strains were not consistently accompanied by changes in body weight, femur length, or periosteal circumference. Finally, decomposition of QTL regions by congenic sublines can reveal additional loci for phenotypes assigned to a QTL region and can markedly refine genomic locations of quantitative trait loci, providing the opportunity for candidate gene testing.     

Quantitative trait loci that determine BMD in C57BL/6J and 129S1/SvImJ inbred mice.

BMD is highly heritable; however, little is known about the genes. To identify loci controlling BMD, we conducted a QTL analysis in a (B6 x 129) F2 population of mice. We report on additional QTLs and also narrow one QTL by combining the data from multiple crosses and through haplotype analysis. INTRODUCTION: Previous studies have identified quantitative trait loci (QTL) that determine BMD in mice; however, identification of genes underlying QTLs is impeded by the large size of QTL regions. MATERIALS AND METHODS: To identify loci controlling BMD, we performed a QTL analysis of 291 (B6 x 129) F2 females. Total body and vertebral areal BMD (aBMD) were determined by peripheral DXA when mice were 20 weeks old and had consumed a high-fat diet for 14 weeks. RESULTS AND CONCLUSIONS: Two QTLs were common for both total body and vertebral aBMD: Bmd20 on chromosome (Chr) 6 (total aBMD; peak cM 26, logarithm of odds [LOD] 3.8, and vertebral aBMD; cM 32, LOD 3.6) and Bmd22 on Chr 1 (total aBMD; cM 104, LOD 2.5, and vertebral aBMD; cM 98, LOD 2.6). A QTL on Chr 10 (Bmd21, cM 68, LOD 3.0) affected total body aBMD and a QTL on Chr 7 (Bmd9, cM 44, LOD 2.7) affected vertebral aBMD. A pairwise genome-wide search did not reveal significant gene-gene interactions. Collectively, the QTLs accounted for 21.6% of total aBMD and 17.3% of vertebral aBMD of the F(2) population variances. Bmd9 was previously identified in a cross between C57BL/6J and C3H/HeJ mice, and we narrowed this QTL from 34 to 22 cM by combining the data from these crosses. By examining the Bmd9 region for conservation of ancestral alleles among the low allele strains (129S1/SvImJ and C3H/HeJ) that differed from the high allele strain (C57BL/6J), we further narrowed the region to approximately 9.9 cM, where the low allele strains share a common haplotype. Identifying the genes for these QTLs will enhance our understanding of skeletal biology.     

Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice

Serum insulin-like growth factor-1 (IGF-1) and femoral bone mineral density (BMD) differ between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), by approximately 30% and 50%, respectively. Similarly, skeletal IGF-1 content, bone formation, mineral apposition, and marrow stromal cell numbers are higher in C3H than in B6 mice. Because IGF-1 and several bone parameters cosegregate, we hypothesize that the serum IGF-1 phenotype has a strong heritable component and that genetic determinants for serum IGF-1 are involved in the regulation of bone mass. We intercrossed (B6 x C3H)F1 hybrids and analyzed 682 F2 female offspring at 4 months of age for serum IGF-1 by radioimmunoassay and femoral BMD by peripheral quantitative computerized tomography (pQCT). Genomic DNA was assayed by polymerase chain reaction (PCR) to determine alleles for 114 Mit markers inherited in F2 mice at average distances of 14 centimorgans (cM) along each chromosome (Chr). Serum IGF-1 levels in the F2 progeny were relatively normal in distribution, but showed a greater range than either progenitor, indicating that serum IGF-1 level is a polygenic trait with an estimated heritability of 52%. Serum IGF-1 correlated with femoral length (r = 0.266, p < 0.0001) and femoral BMD (r = 0.267, p < 0.0001). Whole genome scans for main effects associated with serum IGF-1 levels revealed three significant QTLs (in order of significance) on mouse Chrs 6, 15, and 10. The QTL on Chr 6 showed a significant reduction in IGF-1 associated with increasing C3H allele number, whereas the Chr 15 and Chr 10 loci showed additive effects with increasing C3H allele number. A genome-wide search for interacting marker pairs identified a significant interaction between the Chr 6 QTL and a locus on Chr 11. This interactive effect suggested that when the Chr 11 locus was homozygous for C3H, there was no effect of the Chr 6 locus on serum IGF-1; however, the combination of C3H alleles on Chr 6 with B6 alleles on Chr 11 was associated with reduced serum IGF-1 concentrations. To test this in vivo, we tested congenic mice carrying the Chr 6 QTL region from C3H on a B6 background (B6.C3H-6). Both serum IGF-1 and femoral BMD were significantly lower in female congenic than progenitor B6 mice. In summary, we identified three major QTLs on mouse Chrs 6, 10, and 15, and noted a major locus-locus interaction between Chrs 6 and 11. We named these QTLs IGF-1 serum levels (Igf1sl1 to Igf1sl4). Functional isolation of the Igf1sl1 QTL on Chr 6 for IGF-1 in B6.C3H-6 congenic mice demonstrated effects on both the IGF-1 and BMD phenotypes. The genetic determinants of these Igf1sl QTLs will provide much insight into the regulation of IGF-1 and the subsequent acquisition of peak bone mass.     

Generation of a new congenic mouse strain to test the relationships among serum insulin-like growth factor I, bone mineral density, and skeletal morphology in vivo.

Insulin-like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF-I were related to strain-specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF-I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF-I) and C57BL/6J (B6; low total femoral BMD and low IGF-I) strains. The strongest QTL, IGF-I serum level 1 (Igflsl-1; log10 of the odds ratio [LOD] score, approximately 9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF-I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H-6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF-I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11-21% lower serum IGF-I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF-I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF-I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (-4.8%, p < 0.01) and 6T female (-2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8-week-old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (-29% and -19%, respectively), whereas Tb.Sp was higher (+29%) in 16-week-old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF-I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.     

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

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.     

Identification of quantitative trait loci influencing skeletal architecture in mice: Emergence of Cdh11 as a primary candidate gene regulating femoral morphology

Bone strength is influenced by many properties intrinsic to bone, including its mass, geometry, and mineralization. To further advance our understanding of the genetic basis of bone‐strength‐related traits, we used a large (n = 815), moderately (G₄) advanced intercross line (AIL) of mice derived from a high‐runner selection line (HR) and the C57BL/6J inbred strain. In total, 16 quantitative trait loci (QTLs) were identified that affected areal bone mineral density (aBMD) and femoral length and width. Four significant (p < .05) and one suggestive (p < .10) QTLs were identified for three aBMD measurements: total body, vertebral, and femoral. A QTL on chromosome (Chr.) 3 influenced all three aBMD measures, whereas the other four QTLs were unique to a single measure. A total of 10 significant and one suggestive QTLs were identified for femoral length (FL) and two measures of femoral width, anteroposterior (AP) and mediolateral (ML). FL QTLs were distinct from loci affecting AP and ML width, and of the 7 AP QTLs, only three affected ML. A QTL on Chr. 8 that explained 7.1% and 4.0% of the variance in AP and ML, respectively, was mapped to a 6‐Mb region harboring 12 protein‐coding genes. The pattern of haplotype diversity across the QTL region and expression profiles of QTL genes suggested that of the 12, cadherin 11 (Cdh11) was most likely the causal gene. These findings, when combined with existing data from gene knockouts, identify Cdh11 as a strong candidate gene within which genetic variation may affect bone morphology. © 2011 American Society for Bone and Mineral Research     

Quantitative trait loci for bone density in mice: the genes determining total skeletal density and femur density show little overlap in F2 mice

Bone mineral density variation is a highly heritable trait and is the best predictor of skeletal fragility. Total skeletal density was determined by PIXIMUS, and femur density was determined by pQCT. The data were analyzed for quantitative trait loci (QTL) to determine if bone density at a specific skeletal site (femur) would identify new gene loci or the same gene loci as total body (PIXIMUS). In order to show concordance and differences in QTL for total body bone density versus femur bone density, we performed a genome-wide scan from 633 (MRL x SJL) F2 mice. The bone mineral density (BMD) data from pQCT were used to identify nine QTL on chromosomes 1, 3, 4, 9, 12, 17, and 18, while nine QTL on chromosomes 1, 2, 4, 9, 11, 14, and 15 were identified by PIXIMUSdata, accounting for 32.5% and 30.4% variation in F2 mice, respectively. QTL on chromosomes 1, 2, 3, 9, 11, 12, 14, 15, 17, and 18 are unique to our study, as they have never been described before. Chromosome 1 (D1Mit33 and D1Mit362) had similar QTL between pQCT and PIXIMUS. Several QTL were identified for both femur and total body BMD but only two QTL were common for both of these phenotypes. This suggests that genes regulating bone density differ depending on the skeletal site analyzed.     

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. © 2012 American Society for Bone and Mineral Research