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, ∼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.
INSULIN-LIKE GROWTH factor (IGF) I is a ubiquitous growth factor that, in a tissue-specific manner, regulates cell proliferation, differentiation, and programmed cell death.(1–3) Growth hormone is the principal modulator of IGF-I expression in several tissues, especially in the liver, and hepatic IGF-I accounts for ∼75% of the total circulating IGF-I level.(3,4) Bone is both a source and a target for IGF-I action. In the skeleton, IGF-I is synthesized by osteoblasts and acts on other cells (i.e., lining cells, osteoclasts, and marrow stromal cells) in both a paracrine and autocrine fashion.(2,3,5) Within the skeletal matrix, IGF-I is bound to several IGF binding proteins (IGFBPs) and is released during osteoclast-mediated bone resorption.(2,3,6–9) Serum IGF-I also contributes to the overall skeletal pool, although the role of circulating IGF-I in the bone milieu is not well understood. IGF-I directly stimulates collagen synthesis in osteoblasts and at least partially mediates the anabolic effects of parathyroid hormone (PTH) on trabecular bone.(9–11)
During puberty, linear expansion and consolidation of new bone is associated with a rise in serum and skeletal IGF-I expression whereas during aging, local and circulating IGF-I concentrations decline.(2,3) In some, but not all studies, serum IGF-I levels correlate with total hip bone mineral density (BMD) and the lowest quartile of IGF-I is associated with a greater fracture risk in elderly women.(12–17) These studies imply that components of the IGF-I regulatory system are important in determining this growth factor's effects on adult bone mass and bone strength, although the precise mechanism of this regulation has not been clarified.
For several years, our laboratory has used inbred strains of mice to study the relationships among serum IGF-I, bone density, and skeletal morphology.(18) We previously reported that C3H/HeJ (C3H) mice have 50% greater total femoral bone mineral density BMD and 30% higher serum (and skeletal) IGF-I concentrations than C57BL/6J (B6) mice.(18,19) Moreover, in both C3H and B6, serum IGF-I levels during pubertal growth (4-8 weeks) are correlated closely with total femoral BMD and cross-sectional geometry at the femoral diaphysis.(20) In addition, using a B6C3F1 intercross strategy to produce F2 progeny for genetic analyses, we identified four quantitative trait loci (QTLs) with major effects on the serum IGF-I phenotype.(21) Together with several minor QTLs, which individually contribute 1-2% of the variability in the phenotype (i.e., chromosomes [Chr's] 1, 3, 4, 7, 8, 17, and 18), all these loci account for nearly 23% of the variance in serum IGF-I between the B6 and C3H progenitor strains.(21) For the four major QTLs (designated Igf1sl-1 to Igf1sl-4), three are highly significant as individual loci and one is epistatic or interactive. The QTL accounting for the greatest variance in IGF-I (i.e., Igf1sl-1, log10 of the odds ratio [LOD] ∼9.0) is located on the middle third of mouse Chr 6 in a 25-centimorgan (cM) region containing hundreds of genes.(21) Paradoxically, this Chr 6 QTL with C3H alleles has a significant negative effect on the serum IGF-I phenotype. More specifically, serum IGF-I is ∼15% lower in F2 mice that are homozygous for the C3H alleles (c3/c3) and carry at least one B6 allele (b6/b6 or b6/c3) at the interactive locus on Chr 11.(21) For the other two IGF-I QTLs (located on Chr's 10 and 15), homozygosity for the C3H alleles in the F2 mice is associated with an ∼15% increase in serum IGF-I.(21)
QTL analysis for an F2 population is a critical first step in decomposition of the genetic determinants underlying a polygenic trait such as serum IGF-I. However, then it is essential to verify both the existence and the phenotypic effects of a QTL in the context of a constant genetic background. To accomplish this for the Igf1sl QTL on Chr 6, we developed a congenic strain of mice in which the Chr 6 QTL from C3H is carried in the B6 strain of mice. In this study, we report the morphological and histomorphometric characteristics of female and male Chr 6 congenic mice. The results of these studies provide compelling evidence that congenic mice are excellent models for defining, in vivo, the effects of genetic determinants of IGF-I on skeletal morphology and density.
MATERIALS AND METHODS
Generation of congenic mice for
Igf1sl-1 on Chr 6: This congenic strain was developed by transfer of a Chr 6 region from the C3H strain to the B6 strain background for up to nine generations (Fig. 1). The C3H donor region was defined by the simple sequence length polymorphic (SSLP) markers D6Mit93 located at 25 recombination units (cM) and D6Mit150 located at ∼51 cM from the centromere, respectively.(22) Although exact ends of the donated chromosomal region have not been established yet by fine mapping, both total femoral BMD (Bmd8) and the IGF-I QTL (Igf1sl-1) map within this region.(23) Transfer of the C3H donor region was accomplished by first producing (B6 × C3H) N1F1 offspring and then backcrossing a N1F1 mouse to a recipient B6 mouse to obtain N2F1 progeny. Tail tip DNAs made from N2F1 offspring by standard NaOH digestion method were genotyped to find heterozygous carriers of the desired C3H Chr 6 segment (D6Mit93 to D6Mit150). These carriers were backcrossed to new B6 mice to generate N3F1 progeny for genotyping. This backcross/genotype system for identifying carriers was repeated for nine cycles (Fig. 1). At the N9 generation, the genome of the congenic mouse is estimated to be 99.8% B6 in composition. These mice are designated formally as B6.C3H-6T (D6Mit93 − Igf1sl1/Bmd8 − D6Mit150) and hereafter as 6T. When the N9F1 backcross male and female progeny were identified as carriers of the donated C3H Chr 6 region, these mice were intercrossed to produce N9F2 6T congenic offspring segregating the donated region. Studies for defining QTL effects used N9 female and male mice that were genotypically homozygous for C3H (c3/c3) or B6 (b6/b6, i.e., B6 controls) alleles at the D6Mit93 to D6Mit150 region. Heterozygous mice b6/c3 for the Chr 6 region were not retained.
Measurement of serum IGF-I
To measure serum IGF-I we used a radioimmunoassay (RIA) using a polyclonal antibody to IGF-I after separation of the IGFBPs. The extraction was done by acid-ethanol cryoprecipitation as reported previously.(15,17,21,24) For inbred strains of mice, the mean serum levels of IGF-I at 16 weeks of age ranged between 200 and 500 ng/ml. The sensitivity of the assay was 12 ng/ml and there was virtually no cross-reactivity with IGF-II. The intra-assay CV is 5% and the interassay CV is 11% using C3H and B6 pooled mouse serum as standards. Serum IGF-I was assessed in 6T congenic and B6 control female and male mice at 6, 8, and 16 weeks of age.
Isolation of bone cells and measurement of IGF-1 in conditioned media
Calvarial osteoblast isolation
The isolation and culture of neonatal mouse calvarial cells (NMCs) from 6T and B6 mice were performed using previously published methods.(25–27) Calvariae from 3-day-old neonatal mice were stripped of the periosteum and incubated in a mixture of collagenase and trypsin for five successive 20-minute periods at 37°C in a 5% CO2 incubator. Digestions 1 and 2 were discarded and NMCs were harvested from digestions 3-5. Cells were plated in α-modified essential medium (α-MEM) with 10% fetal bovine serum (FBS), 100 μg/ml of ascorbic acid, 100 U/ml of penicillin, and 100 μg/ml of streptomycin in a 100-mm plate and grown to confluence. After reaching confluence, the cells were trypsinized and half were added to a new 100-mm plate (passage 1) and the other half were frozen. The passage 1 cells were grown to confluence in complete medium and then washed and placed in medium in which 0.1% bovine serum albumin (BSA) was substituted for 10% FBS (serum-free medium). After 24 h, conditioned medium (CM) and cells were collected and processed for determination of cell number and viability.
IGF-I in culture media from calvarial osteoblasts
We examined osteoblast production of IGF-I using previously described methods.(19) Briefly, CMs from 6T and B6 calvarial cells isolated at passage 1 were measured for IGF-I using an ultrasensitive RIA (ALPCO, Windham, NH, USA) that incorporates the addition of recombinant IGF-II to block IGF-I binding to residual IGFBPs left after acid-ethanol extraction and before addition of a polyclonal antibody to IGF-I. For this assay, the sensitivity was 0.01 ng/ml, and there was no cross-reactivity with IGF-II. Interassay CVs using the ultrasensitive IGF-I method were ∼10%. IGF-I in CMs was measured by the ultrasensitive RIA and reported as IGF-I ng/106 cells for each of the strains (i.e., 6T congenic mice and B6 control mice).
Bone density and morphology measurements
Peripheral dual-energy X-ray absorptiometry
We used peripheral dual-energy X-ray absorptiometry (pDXA; PIXImus, GE-Lunar, Madison, WI, USA) to assess areal whole body BMD and body composition in female 6T congenic and B6 control mice at 8, 12, and 16 weeks of age. This methodology has been validated recently in small animals.(28) PIXImus measurements of total and site-specific areal BMD correlate well with volumetric density data from our pQCT. Moreover, PIXImus measurements of mineral content are very highly correlated with mineral content of hydroxyapatite standard of known density (r2 = 0.997).
Peripheral quantitative computed tomography
Isolated right femora from N9 6T and B6 controls were assessed using peripheral quantitative computed tomography (pQCT; Stratec XCT 960M; Norland Medical Systems, Ft. Atkinson, WI, USA) as described in our previous publications.(18,23,29–31) Briefly, thresholds of 1.300 attenuation units differentiated mouse bones from water, adipose tissue, muscle, and tendon; a threshold of 2.000 differentiated high-density cortical bone from low-density bone. Calibration of the densitometer was done with a set of hydroxyapatite standards (0.050-1.0 mg/mm3), yielding a correlation of 0.997 between standards and pQCT estimation of density. Precision of the XCT 960M for repeated measurements of the same femur and same vertebrae were 1.2% and 1.4%, respectively. The CVs for density measurements were obtained by measuring different sets such that for the progenitor B6 strain the CV was 3% for BMD, 9% for bone mineral content, 7% for bone volume (BV), 3% for middiaphyseal periosteal circumference, and 4% for cortical thickness. Femoral periosteal circumferences and cortical thickness were calculated at the midpoint of the diaphysis. Femoral length of excised specimens was measured with digital calipers to the nearest 0.01 mm.
We used high-resolution, desktop microtomography imaging systems (μCT20 and μCT40; Scanco Medical AG, Bassersdorf, Switzerland) to evaluate the bone density and morphology of the entire femur, the femoral midshaft, distal femoral metaphysis, and the cancellous bone of the fifth lumbar vertebral body at 8 weeks and 16 weeks of age.(32) For evaluation of the femur, the bone was scanned using a 34-μm slice increment, requiring ∼100-150 micro-CT (μCT) slices per specimen. For evaluation of the cancellous bone in the vertebral body, the vertebrae were scanned using a 12-μm slice increment, requiring ∼200-250 μCT slices. Images were reconstructed, filtered (δ = 0.8 and width = 1.0), and thresholded (22% of maximum possible gray scale value) as previously described.(33) The images were stored in three-dimensional 3D arrays with an isotropic voxel size of either 34 μm (for the femur) or 12 μm (for the vertebra). Morphometric parameters were computed using a direct 3D approach that does not rely on any assumptions about the underlying structure.(34) For the whole femur, we computed the total BV (mm3) and the apparent volume density (AVD, %), defined as the percent of mineralized tissue volume divided by the total volume of the external bone envelope.(28) For the cortical region, the BV (mm3), percent cortical volume (cBV, %), and cortical thickness (μm) were computed in a 1-mm-thick region at the middiaphysis. The final femur region included an evaluation of the secondary spongiosa in the distal metaphysis. In this region of cancellous bone, we assessed BV (mm3) and bone volume fraction (BV/TV, %), as well as trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm), trabecular number (Tb.N, 1/mm). For the cancellous bone region in the vertebral body, we assessed BV/TV (%), Tb.Th. (μm), Tb.Sp. (μm), Tb.N. (1/mm), and connectivity density (mm−3). In addition, we computed the structure model index (SMI), which indicates the platelike (SMI = 0) or rodlike (SMI = 3) nature of the underlying cancellous architecture.(35)
Histomorphometric procedures were performed using a semiautomatic image analysis system (OsteoMetrics, Inc., Atlanta, GA, USA) consisting of a Compaq computer with OsteoMeasure software interfaced with a microscope and image analysis system. Tibias were harvested from 7 female 6T and 7 B6 control mice at 8 weeks of age. Bones were dual-labeled with tetracycline using a 6-day interlabel interval as previously described.(36) Twenty-four hours after the second fluorochrome label was injected, mice were necropsied and tibias were dissected free of muscle and fixed in 10% neutral buffered formalin (NBF) on ice for 4 h and then changed to 70% ethyl alcohol (EtOH). The proximal tibia was isolated and dehydrated in a series of increasing concentrations of ethanol, embedded without demineralization in a mixture of methymathacrylate/2-hydroxyethylmethacrylate (12.5:1) to retain the fluorochrome labels, sectioned at a thickness of 5 μm, and viewed with a fluorescence microscope (Olympus BH-2; Olympus, New Hyde Park, NY, USA) to detect the fluorochrome labeling. Cortical and cancellous bone measurements were performed as previously described.(36)
Statistical analyses were performed using StatView 4.5 (SAS Institute, Cary, NC, USA) software. Unpaired Student's t-tests were used to compare IGF-I, body weight, and other skeletal phenotypes in 6T congenic and B6 control mice at each age. Gender comparisons were performed using a two-way analysis of variance (ANOVA) with sex and genotype as independent variables. Bivariate regression analysis was used to assess the relationship of IGF-I to total femur BMD. All values are reported as the mean ± SEM and statistical significance was accepted at p < 0.05.
Serum IGF-I was 11-21% lower in female 6T congenic mice compared with B6 control mice at 6, 8, and 16 weeks of age (p < 0.05 for all; Table 1; Fig. 2). In contrast to females, male 6T and B6 mice had nearly identical serum IGF-I levels at 6 weeks and 8 weeks of age (Fig. 2). However, consistent with females, at 16 weeks of age, serum IGF-I was 13% lower in male 6T congenic mice than in B6 control mice (Table 1, Fig. 2, p < 0.001). Thus, in female congenic 6T mice, serum IGF-I was lower than B6 at all three time points, whereas in the male congenic mice, the difference in serum IGF-I between 6T congenic mice and B6 control mice was first evident at 16 weeks of age.
Table Table 1.. Serum IGF-I, Body Weight, Femoral Length, and Femoral Bone Density and Geometry in 16-Week-Old 6T Congenic Mice and B6 Control Mice (Mean ± SEM)
Production of IGF-I by calvarial osteoblasts
IGF-I in CM from 6T congenic osteoblasts was significantly lower than B6 osteoblasts when corrected for the number of cells (0.883 ± 0.056 ng of IGF-I/106 cells for 6T vs. 1.443 ± 0.130 ng of IGF-I/106 cells for B6; p = 0.01). Strain-related differences in secreted IGF-I from the primary calvarial osteoblast cultures were similar (i.e., congenic 6T osteoblasts secreted ∼40% less IGF-I than B6) when the cells were cultured either in serum-free or serum-supplemented media (data not shown).
Body composition, femoral length, and whole body BMD
Body weight was similar in female 6T congenic mice and B6 control mice at 8, 12, and 16 weeks of age (Fig. 3). Similarly, there was no difference in body weight between male 6T congenic mice and B6 control mice at 16 weeks of age (Table 1). At 16 weeks, femoral length was slightly lower in female congenic mice compared with control mice but was similar in male congenic mice and control mice (Table 1). Total body areal BMD was similar in female congenic mice and control mice at 8 weeks and 12 weeks of age but was lower in 6T congenic mice at 16 weeks of age (Fig. 3). Body fat percentage was similar in female 6T congenic mice and B6 control mice at 8 and 16 weeks of age, but was decreased in 6T congenic mice at 12 weeks of age (Fig. 3).
Femoral bone density and geometry
At 16 weeks of age, total femoral BMD, measured by pQCT, was slightly lower in both female and male 6T congenic mice compared with their gender-matched B6 controls (females, −2.3%, p = 0.06; males, −4.8%, p < 0.01; Table 1). In addition, geometric properties of 6T femurs were reduced compared with controls, because femurs from 6T congenic mice had lower midshaft periosteal circumference and cross-sectional moments of inertia (Table 1). Serum IGF-I correlated with total femoral BMD, as measured by pQCT, in both female 6T and female B6 congenic mice (r = 0.70; p < 0.001). μCT measurements revealed that female 6T congenic femurs were slightly smaller than B6 controls, as evidenced by lower BV of the entire femur and midshaft regions, and had reduced cortical thickness at both 8 weeks and 16 weeks of age (Table 2). In addition to their slightly smaller size, AVD of the whole femur was 4.8% lower in 6T congenics compared with B6 controls at 8 weeks (p < 0.001) and 1.9% lower at 16 weeks (p < 0.05; Table 2). Moreover, in the distal metaphysis, cancellous BV density and Tb.N were ∼50% lower, whereas Tb.Sp was 90% higher in 8-week-old female 6T congenic mice compared with B6 controls (Table 2; p < 0.01 for all). These strain-related differences in cancellous bone density and architecture were smaller (and not significant) at 16 weeks.
Table Table 2.. μCT Evaluation of the Femur (Total, Cortical, and Cancellous Regions) and Vertebral Body in 8-Week and 16-Week-Old Female 6T Congenic and B6 Control Mice (Mean ± SEM)
Vertebral cancellous bone density and morphometry
μCT was used to evaluate the BV fraction and microarchitecture of cancellous bone in the vertebral body of 8-week- and 16-week-old female 6T congenic and B6 control mice (Table 2). At 16 weeks, BV/TV was 29% lower in the 6T congenics compared with B6 controls (p < 0.001; Fig. 4). This decreased bone density was associated with a 19% decrease in Tb.N and a 29% increase in Tb.Sp in the 6T congenic mice. Interestingly, Tb.Th was similar in 6T congenic and B6 control mice. The net effect of these changes in trabecular bone morphology was reflected by a 25% decrease in connectivity density in the 6T mice compared with B6 mice (p < 0.001; Table 2). In addition, the cancellous bone in the 6T congenics had a more platelike structure than that of the B6 controls (SMI = 0.24 vs. 1.03, respectively; Table 2; Fig. 4). Similar trends were observed at 8 weeks, although only the 17% decrease in BV/TV in the 6T congenics achieved statistical significance (Table 2).
Histomorphometry of the proximal tibia
To assess the cellular mechanism(s) responsible for these alterations in bone, we examined histomorphometric parameters in the proximal tibia from 8-week-old female 6T congenic and B6 control mice. Cortical cross-sectional area (CsAr) and periosteal mineral apposition rate (PsMAR) were similar in both strains (Table 3). However, trabecular BV/TV was 40% lower in the 6T congenic mice compared with B6 mice. Although the MAR was identical in both strains, there was a trend toward a decrease in labeled surface and bone formation rate per bone surface or per BV (Table 3), suggesting a potential reduction in the recruitment and/or differentiation of osteoblast precursors in the congenics.
Table Table 3.. Histomorphometric Analyses of Proximal Tibiae from 8-Week-Old Female 6T Congenic and B6 Control Mice (Mean ± SEM)
The goals of this study were to assess the effect of a single QTL on serum IGF-I in a new congenic mouse strain and to determine whether this genetic factor had a significant role in regulating bone morphology and density. Although we previously reported that in B6C3F2 mice, homozygosity for C3H alleles at the QTL region on Chr 6 (IGF1sI) altered serum IGF-I, its true biological effect could not be ascertained directly, in part because several QTLs for IGF-I also were segregating within the F2 population.(21) Indeed, phenotypic and genotypic analyses of F2 mice produce only a relative estimate of allele effects for a statistically significant QTL. This genetic heterogeneity complicates interpretation, even for QTLs with high LOD scores. Therefore, for several reasons, congenic mouse strains are a useful tool to test the significance of individual QTLs.(37) First, congenic mice establish “proof of concept” that a QTL, when placed on another genetic background, has a meaningful effect in vivo on a particular phenotype. Second, congenic mice can be used to test epistatic interactions between two chromosomal loci for a specific phenotype, if alleles for those genetic determinants are different in respect to progenitor origin. Third, generation of nested congenic sublines, with appropriate phenotyping, are useful for fine mapping and positional cloning of single QTLs. Last, congenic mice can provide important information regarding the potential biological mechanisms associated with genetic regulation of a particular phenotype.
The generation of our congenic 6T strain exhibiting reduced serum IGF-1 levels accompanied by alterations in both cortical and cancellous bone at different skeletal sites provides important insight into the role of IGF-I during bone acquisition. First, changes in circulating IGF-I in N9 mice reinforce our earlier findings in F2 mice, in which the QTL on Chr 6 has a powerful effect on levels of IGF-I.(21) Second, differences in serum IGF-I between congenic and B6 mice were reflected also in neonatal bone cell production of IGF-I. These data support our previous work showing that skeletal IGF-I production in C3H and B6 mice parallel changes in circulating levels.(19–21) Third, this Chr 6 QTL, which regulates serum IGF-I levels, also dramatically affects femoral cortical, vertebral cancellous, and tibial cancellous bone density in both genders, as well as femur length and cross-sectional geometry in females. The gender effect on bone size, which we noted, suggests there may be an important interaction between sex steroids, IGF-I expression, and bone modeling.
Our rationale for constructing a congenic for the QTL on Chr 6 was based on the very high LOD scores shown in our F2 population for this region (Igf1sl-1).(21) Moreover, this single locus accounted for >6% of the variance in serum IGF-I in the F2 population. From published databases, we determined that none of the four major QTLs for serum IGF-I contained genes related to growth hormone, its releasing factors, or somatostatin.(21) This was important because we were interested in assessing changes in skeletal morphology caused by alterations of IGF-I that were at least partially independent of growth hormone. We subsequently posited that if the Chr 6 IGF-I QTL had an effect on skeletal acquisition in the congenic mice, there would be more noticeable effects on trabecular rather than cortical bone parameters. The current data show that 6T congenic mice exhibit multiple skeletal phenotypes, although the major effect of this QTL is clearly on trabecular bone density and microarchitecture. These skeletal changes could be a result of the actions of a single pleiotropic gene within the QTL, multiple genes in that one locus, or, indirectly, from impaired expression of skeletal and/or serum IGF-I.
With regard to the possibility that other genes may be contributing to this skeletal phenotype, it is important to note that the observed phenotype of low cancellous bone density is consistent with our previous observations of reduced trabecular bone density in C3H mice.(29) Hence, it is very possible that the transfer of a relatively large Chr 6 region from C3H to the B6 genetic background (i.e., 25 cM) may introduce not-yet-identified C3H genes that lead to decreased trabecular bone density. As such, the final phenotype of the 6T congenic mice may reflect reduced serum IGF-I levels as well as other genetic contributions from the C3H background resulting in low cancellous bone density. New congenic sublines with a smaller and more refined Chr 6 locus will help to distinguish between these two possibilities.
Irrespective of the genetic mechanism, the phenotypic presentation of the 6T congenic mice suggests that the IGF regulatory system (either skeletal and/or circulatory), in concert with hormonal variables, exert a profound influence on bone development and acquisition. Conceptually, reduced local or systemic synthesis of IGF-I could produce significant changes in skeletal morphology, especially during periods of rapid growth (i.e., 8-12 weeks of age). In support of this idea, the absence of differences in MARs between the 6T congenic and B6 control mice, coupled with alterations in trabecular BV fraction, and labeled surface/total volume, suggest that skeletal IGF-I may affect bone acquisition through enhanced recruitment of osteoblast precursors or maintenance of greater numbers of mature osteoblasts. Indirect evidence for this tenet also comes from our recent study with genetically altered mice in which IGF-I was targeted to bone using an osteocalcin promoter. In this model, trabecular bone density was increased markedly at 6 weeks of age in mice overexpressing IGF-I in bone, whereas cortical bone density was only modestly increased.(38) Similarly, recently, it has been shown that targeted deletion of the type I IGF-I receptor in bone results in markedly reduced trabecular bone density, with smaller effects on cortical bone parameters.(39) Other studies have indicated that IGF-I can increase osteoblast number, either through enhanced recruitment of stromal cells or by a reduction in programmed cell death. The latter remains a distinct possibility in this study, because IGF-I is strongly antiapoptotic and the IGF-I type I receptor, when activated, inhibits programmed cell death in bone cells.
It is conceivable that the combination of reduced circulating levels of IGF-I and impaired skeletal expression of the peptide, together, contribute to the severity of the skeletal phenotype we have observed. Evidence to support this thesis is contradictory, although some studies suggest that circulating IGF-I is essential for optimal bone growth and consolidation. For example, growth hormone-deficient lit/lit and IGF-I null (IGF-I −/−) mice have shortened femurs and less cortical bone density than wild-type mice.(40,41) Moreover, Yakar et al. reported that mice null for both the acid labile subunit (ALS) and the hepatic IGF-I expression have reduced femoral length, lower cortical BMD, and shortened growth plates than wild-type control mice.(42) Taken together, these data imply that the circulating levels of IGF-I may be an important factor in the growth and development of the skeleton. However, further studies are needed to delineate the precise mechanism(s) by which low levels of circulating IGF influence bone density and morphology.
We previously reported that B6C3F2 progeny carrying two c3 alleles for the Chr 6 QTL had lower serum IGF-I concentrations than mice with two b6 alleles at the same locus.(21) At first glance, this would seem paradoxical, because progenitor C3H mice have 30% higher serum IGF-I levels than B6 mice. However, the IGF-I levels in C3H mice or any other strain (i.e., B6) are a net result of all relevant regulatory genes plus environmental stimuli and, therefore, are not simply the effect of a single QTL. In addition, it is not uncommon for progenitor mice to carry alleles that could act to either increase or decrease the phenotype, as we have reported for total femoral and vertebral BMD.(23,30) This does not diminish the importance of the allele source or the size of the effect for a QTL on that phenotype. Furthermore, we recognized that in the F2 progeny, significant epistatic interactions occurred between the Chr 6 QTL and a locus on mouse Chr 11. As such, the presence of c3/c3 alleles at Chr 6 and at least one b6 allele at the Chr 11 QTL resulted in 15% lower serum IGF-I level compared with B6 controls. If, on the other hand, the Chr 6 and Chr 11 QTLs were both c3/c3 in the F2 mice, serum IGF-I concentrations were not different from C3H progenitor mice.(21) Thus, we would have predicted a priori that our 6T congenic strain, which is c3/c3 at Chr 6 but b6 at Chr 11, would have lower serum IGF-I concentration than B6 controls. Indeed, this is exactly what we observed. More importantly, the epistatic relationship between the loci on Chr 6 and Chr 11 will provide a unique opportunity to study gene-gene interaction and its relationship to skeletal growth and development.
Probably the most important findings from this study are the altered skeletal phenotype of the 6T congenic mice and the possibility of gender-specific effects of this QTL on bone size. In addition to having reduced total femoral BMD, femurs from female congenic mice were slightly shorter and had reduced cortical cross-sectional geometric properties compared with B6 mice. In comparison, male 6T congenic mice had lower total femoral BMD and cortical cross-sectional geometric properties than B6 controls but did not have shorter femurs. Thus, like other reports suggesting gender specificity in respect to QTLs for BMD in mice, this heritable determinant of IGF-I and its effect on bone morphometry also may be influenced by gender.(43)
There are several limitations to this study. First, we recognize that there may be more than one gene in this QTL, resulting in variable phenotypes. Fine mapping of this Chr 6 QTL region, which has been facilitated by producing the N9 congenics, will allow us to test for closely linked genes in this region. The second limitation is that the complete developmental sequence of bone acquisition in the 6T congenic mice is still not complete. Our preliminary data suggest that the 6T mouse has altered expression of IGF-I, at least in some tissues, from birth. However, it is not clear precisely when changes in IGF-I have the greatest impact on bone acquisition and modeling, because the timing of the acquisition of peak bone mass varies with skeletal site. Thus, it was not until 16 weeks that total body areal BMD was lower in the congenic mice than the control mice. Similarly, strain-related differences in trabecular bone density in the vertebral body were most pronounced at 16 weeks, and in the femur, many of the changes were evident as early as 8 weeks of age. As such, it is possible that the impact of the Chr 6 QTL on skeletal morphology might be related to the timing of peak and/or sustained high levels of IGF-I expression in bone and/or serum.
An additional limitation of our study relates to the fact that B6 and C3H inbred strains are only ∼50% polymorphic at most MIT marker sites in their genomes. Thus, it is conceivable that there are other and possibly stronger genetic determinants for IGF-I that are not detectable in a cross between B6 and C3H progenitors because these strains carry the same alleles. The use of various recombinant inbred strains that exhibit differences in serum IGF-I may provide new QTLs that are important for regulating this phenotype.
In summary, we have produced a new mouse strain from a QTL for serum IGF-I, the congenic 6T mouse. These 6T congenic mice, with a donated segment from C3H on a B6 background, exhibit alterations in serum IGF-I concentrations as well as reduced vertebral, femoral, and tibial bone density. In addition, female 6T congenic mice tend to have shorter femurs with a reduced periosteal circumference. This newly engineered strain will become a useful model for understanding the complex relationship between serum IGF-I and bone acquisition, as well as a means for studying, in vivo, changes related to genetic manipulation of the IGF regulatory system.
The authors thank Dr. E.H. Leiter and Dr. T.P. O'Brien for critical review of this study. Research support for this work was provided by the National Institutes of Health (NIH) AR45433 (to C.J.R.), AR43618 and U.S. Army DAMD 17-96-1-6306 (to W.G.B.), NIR AR 45233 (to R.T.T.), and NIH AR46530 (to C.H.T.).