Fine map of the Gct1 spontaneous ovarian granulosa cell tumor locus.

The spontaneous development of juvenile-onset, ovarian granulosa cell (GC) tumors in the SWR/Bm (SWR) inbred mouse strain is a model for juvenile-type GC tumors that appear in infants and young girls. GC tumor susceptibility is supported by multiple Granulosa cell tumor (Gct) loci, but the Gct1 locus on Chr 4 derived from SWR strain background is fundamental for GC tumor development and uniquely responsive to the androgenic precursor dehydroepiandrosterone (DHEA). To resolve the location of Gct1 independently from other susceptibility loci, Gct1 was isolated in a congenic strain that replaces the distal segment of Chr 4 in SWR mice with a 47 × 10(6)-bp genomic segment from the Castaneus/Ei (CAST) strain. SWR females homozygous for the CAST donor segment were confirmed to be resistant to DHEA- and testosterone-induced GC tumorigenesis, indicating successful exchange of CAST alleles (Gct1 ( CA )) for SWR alleles (Gct1 ( SW )) at this tumor susceptibility locus. A series of nested, overlapping, congenic sublines was created to fine-map Gct1 based on GC tumor susceptibility under the influence of pubertal DHEA treatment. Twelve informative lines have resolved the Gct1 locus to a 1.31 × 10(6)-bp interval on mouse Chr 4, a region orthologous to human Chr 1p36.22.

The heparin-binding domain of IGFBP-2 has insulin-like growth factor binding-independent biologic activity in the growing skeleton.

Insulin-like growth factor-binding protein 2 (IGFBP-2) is a member of a family of six highly conserved IGFBPs that are carriers for the insulin-like growth factors (IGFs). IGFBP-2 levels rise during rapid neonatal growth and at the time of peak bone acquisition. In contrast, Igfbp2(-/-) mice have low bone mass accompanied by reduced osteoblast numbers, low bone formation rates, and increased PTEN expression. In the current study, we postulated that IGFBP-2 increased bone mass partly through the activity of its heparin-binding domain (HBD). We synthesized a HBD peptide specific for IGFBP-2 and demonstrated in vitro that it rescued the mineralization phenotype of Igfbp2(-/-) bone marrow stromal cells and calvarial osteoblasts. Consistent with its cellular actions, the HBD peptide ex vivo stimulated metacarpal periosteal expansion. Furthermore, administration of HBD peptide to Igfbp2(-/-) mice increased osteoblast number, suppressed marrow adipogenesis, restored trabecular bone mass, and reduced bone resorption. Skeletal rescue in the Igfbp2(-/-) mice was characterized by reduced PTEN expression followed by enhanced Akt phosphorylation in response to IGF-I and increased ß-catenin signaling through two mechanisms: 1) stimulation of its cytosolic accumulation and 2) increased phosphorylation of serine 552. We conclude that the HBD peptide of IGFBP-2 has anabolic activity by activating IGF-I/Akt and ß-catenin signaling pathways. These data support a growing body of evidence that IGFBP-2 is not just a transport protein but rather that it functions coordinately with IGF-I to stimulate growth and skeletal acquisition.

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype.

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin-1 alpha and Caveolin-1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H-1-12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re-localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H-1-12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H-1-12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2-induced Smad signaling in BMSCs isolated from B6.C3H-1-12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H-1-12 congenic and (ii) contributes to increase BMD of the B6.C3H-1-12 compared to the C57BL/6J control mice.

Insulin-like growth factor-1 increases bone calcium accumulation only during rapid growth in female rats.

Calcium retention varies with developmental state, which may be partially under the control of insulin-like growth factor 1 (IGF-1). IGF-1 levels can be manipulated through dietary and therapeutic interventions. We investigated the relationship between IGF-1 endogenous production and calcium utilization and bone accretion during growth as well as the effects of IGF-1 treatment on calcium utilization during rapid and slowed growth in intact female Sprague-Dawley rats. In 33 rats killed at 11 time points (n = 3 each) from age 4 to 24 wk, femoral and vertebral bone mass were paralleled by plasma IGF-1 up to 9 wk. Fractional calcium absorption was maximal at 9 wk, reduced by one-half at 12 wk, and there was no further change at 20 wk. From this study, we selected 2 stages of growth, rapid and slow, for a subsequent intervention study. A 4-wk intervention was initiated at 6 or 8 wk when rats (n = 15/group) received either continuous rhIGF-1/IGF binding protein 3 (IGFBP3) infusion (0.3 mg/d) or vehicle (control) by osmotic mini-pumps. In rapidly growing IGF-1/IGFBP3-treated rats compared to controls, but not in slowly growing treated compared to control rats, IGF-1 treatment increased (P < 0.05) calcium absorption (35 vs. 21%), bone calcium balance (0.55 vs. 0.3 mmol/d), and femoral calcium content (31 vs. 24% of dry weight). Exogenous IGF-1/IGFBP3 treatment increased calcium accretion during rapid growth, but rats past rapid growth were no longer as sensitive to this dose of IGF-1/IGFBP3. Thus, interventions designed to improve bone mass through increased IGF-1 will have the greatest impact during rapid growth.

Quantitative trait loci, genes, and polymorphisms that regulate bone mineral density in mouse.

This is an in silico analysis of data available from genome-wide scans. Through analysis of QTL, genes and polymorphisms that regulate BMD, we identified 82 BMD QTL, 191 BMD-associated (BMDA) genes, and 83 genes containing known BMD-associated polymorphisms (BMDAP). The catalogue of all BMDA/BMDAP genes and relevant literatures are provided. In total, there are substantially more BMDA/BMDAP genes in regions of the genome where QTL have been identified than in non-QTL regions. Among 191 BMDA genes and 83 BMDAP genes, 133 and 58 are localized in QTL regions, respectively. The difference was still noticeable for the chromosome distribution of these genes between QTL and non-QTL regions. These results have allowed us to generate an integrative profile of QTL, genes, polymorphisms that determine BMD. These data could facilitate more rapid and comprehensive identification of causal genes underlying the determination of BMD in mouse and provide new insights into how BMD is regulated in humans.

A close examination of genes within quantitative trait loci of bone mineral density in whole mouse genome.

Bone mineral density (BMD) is one of the strongest determinants of osteoporotic fracture risk. Over the last decade, a large number of quantitative trait loci (QTL) that regulate BMD have been identified using the mouse model. In an attempt to examine the relationship between those QTL and gene distribution in the mouse genome, we searched PubMed with keywords bone and QTL for every publication up to January 2007; we obtained a total of 75 QTL of BMD. We next obtained genes within a QTL for measurements of BMD from the Ensembl database. We then evaluated the potential connection of every gene with bone biology with Online Mendelian Inheritance in Man (OMIM) and PubMed by using eight key words: bone mineral density, BMD, bone strength, bone size, osteoporosis, osteoblast, osteoclast, and fracture. We obtained a total of 15,084 genes for 75 BMD QTL covering 1,211,376,097 base pairs of genomic sequence. Although this very large number of genes exists within QTL regions, only 291 were identified as candidate genes according to our bioinformatics search. Importantly, the association between polymorphism of many candidate genes and BMD has been reported in human studies. Thus, updated genome information and resources should provide new insight for gene identification of QTL. Accordingly, the comprehensive search of candidate genes in the genome for known QTL may provide unexpected benefits for QTL studies.

Genetic effects on bone mechanotransduction in congenic mice harboring bone size and strength quantitative trait loci.

UNLABELLED: The degree to which bone tissue responds to mechanical loading events is partially under genetic control. We assess the contribution of three genetic loci (QTLs linked to bone geometry and strength)--located on mouse Chrs. 1, 8, and 13--to mechanically stimulated bone formation, through in vivo skeletal loading of congenic strains. Bone size was not consistently associated with mechano-responsiveness, indicating that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific. INTRODUCTION: We showed previously that C57BL/6J (B6) mice are more responsive to mechanical stimulation than C3H/HeJ (C3H) mice and that B6 mice harboring a 40-Mb region of distal C3H Chromosome (Chr.) 4 are more responsive to mechanical stimulation than are fully B6 mice. Here, we assess the contribution of three more genetic loci--located on mouse Chrs. 1, 8, and 1--to mechanically stimulated bone formation. MATERIALS AND METHODS: Three congenic mouse strains were created in which a region of mouse Chr. 1 (approximately 64 cM; 150 Mb), Chr. 8 (approximately 45 cM; 86 Mb), or Chr. 13 (approximately 24 cM; 42 Mb) was moved from C3H stock to a B6 background through selective breeding over nine generations. The regions moved to the B6 background correspond to three of several quantitative trait loci (QTLs) identified for bone size and strength. The resulting congenic mice were 99% B6, with the remaining genomic DNA comprised of the Chr. 1, 8, or 13 QTLs of interest. Male and female congenic (1T, 8T, and 13B) and B6 control mice were subjected to in vivo loading of the right ulna at one of three different load magnitudes. A separate set of animals from each group had strain gauges applied at the ulnar midshaft to estimate strain at each loading level. Loading was conducted once per day for 3 days (60 cycles/d; 2 Hz). Fluorochrome labels were injected intraperitoneally 4 and 11 days after loading began. Using quantitative histomorphometry, bone formation rates were measured in loaded (right) and control (left) ulnas. RESULTS: All male congenic mice exhibited significantly reduced mechano-responsiveness compared with male B6 controls, but the same comparison among females yielded no difference from controls, with the exception of the 1T congenics, which showed increased responsiveness to loading. Among the congenic strains, smaller bone size was not consistently associated with reduced mechano-responsiveness. CONCLUSIONS: Our results indicate that the genetic regulation of mechanotransduction is a complex process that involves a number of genes and is sex-specific. Our data might explain why different individuals can engage in similar exercise protocols yet experience different results in terms of bone mass accrual.

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.

LH analog and dietary isoflavones support ovarian granulosa cell tumor development in a spontaneous mouse model.

The reproductive hormone environment is an important influence upon spontaneous ovarian granulosa cell (GC) tumor development in genetically susceptible (SWR x SWXJ-9) F1 female mice: androgenic support during puberty stimulates tumorigenesis, while exposure to 17beta-estradiol (E(2)) suppresses tumor initiation. We sought to determine whether gonadotropic stimulation was sufficient to initiate GC tumors in a grafted model system, and to determine the potential for dietary isoflavones (genistein and daidzein) as alternatives to E(2) for tumor chemoprevention in vivo. Isolated ovaries from pre-pubertal (SWR x SWXJ-9) F1 females were transferred to the kidney capsule of host mice homozygous for the hypogonadal (hpg/hpg) and severe combined immunodeficiency (scid/scid) mutations. CB17; HPG-Prkdc(scid) Gnrh1(hpg)/Bm host mice received either follicle-stimulating hormone (FSH), or a functional analog for LH human chorionic gonadotropin for 2 consecutive weeks, at which time the ovary grafts were examined for evidence of tumor initiation. LH analog administration, but not FSH, initiated GC tumorigenesis in the graft system, suggesting that the LH surge at puberty initiates GC tumor development in genetically susceptible female mice. To assess the chemopreventive potential of phytoestrogens, GC tumor frequency was compared between (SWR x SWXJ-9) F1 females reared on an isoflavone-free diet versus a diet supplemented with 125 mug/g each of the isoflavones daidzein and genistein. It was observed that (SWR x SWXJ-9) F1 females reared on isoflavone-supplemented diet maintained significantly higher GC tumor frequency (22%) than females reared on isoflavone-free diet (11%), and that non-tumor-bearing siblings reared on the isoflavones had significantly increased ovarian weight, indicative of an overall stimulation of the reproductive hormone axis. The stimulation of GC tumorigenesis by isoflavones, which contrasts with the chemopreventive action of E(2) (2.5 mg/kg) administration during pubertal maturation, may result from general stimulation of ovarian growth, and the inability of the genistein and daidzein supplements to suppress LH secretion.

Circulating levels of IGF-1 directly regulate bone growth and density.

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1-deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis.

Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the wdl mouse.

Recently, the waddles (wdl) mouse was identified as a carbonic anhydrase VIII (Car8) mutant. The mutation is associated with marked deficiency of Car8, an inositol triphosphate receptor 1-binding protein expressed at high levels in cerebellar Purkinje cells. To help unravel the molecular aberrations contributing to motor dysfunction in wdl mice, cerebellar gene expression profiles were examined in the mutants and their wild-type littermates. Genes involved in signaling, cell division, zinc ion-binding, synapse integrity and plasticity were downregulated in wdl mice. Several of the upregulated genes encode proteins that function in the Golgi apparatus which suggests that Car8 deficiency has important effects on synaptic vesicle formation and transport.

Evaluation of gene expression profiling in a mouse model of L-gulonolactone oxidase gene deficiency.

Humans and guinea pigs are species which are unable to synthesize ascorbic acid (vitamin C) because, unlike rodents, they lack the enzyme L-gulonolactone oxidase (Gulo). Although the phenotype of lacking vitamin C in humans, named scurvy, has long been well known, information on the impact of lacking Gulo on the gene expression profiles of different tissues is still missing. This knowledge could improve our understanding of molecular pathways in which Gulo may be involved. Recently, we discovered a deletion that includes all 12 exons in the gene for Gulo in the sfx mouse, characterized by spontaneous bone fractures. We report here the initial analysis of the impact of the Gulo gene deletion on the murine gene expression profiles in the liver, femur and kidney.

Distal Chr 4 harbors a genetic locus (Gct1) fundamental for spontaneous ovarian granulosa cell tumorigenesis in a mouse model.

The spontaneous development of juvenile-onset ovarian granulosa cell tumors in mice of the SWXJ-9 recombinant inbred strain is a model for juvenile-type granulosa cell tumors that appear in very young girls. To expedite gene discovery in this mouse model of childhood cancer, we did a gene mapping study with the SWXJ-9 recombinant inbred strain and the evolutionarily divergent Mus musculus castaneus (CAST/Ei) strain as a mapping partner. Our mapping strategy focused on autosomal determinants of susceptibility with a backcross scheme that exploited a paternal, parent-of-origin effect for a X-linked gene (Gct4) that strongly supports granulosa cell tumor development. Of 1,968 backcross females examined, we detected 81 granulosa cell tumor-bearing animals and compared their allelic inheritance patterns to non-tumor-bearing siblings in a case-control analysis. The results of our study have confirmed an important locus on mouse chromosome (Chr) 4 (Gct1) and have revealed new loci for granulosa cell tumor susceptibility (Gct7-Gct9) on Chrs 1, 2, and 13 with susceptibility alleles contributed by the SWXJ-9 progenitor. Two novel gene-gene interactions supportive for granulosa cell tumor development were also observed between loci on Chrs 17 and 18 and loci on Chrs 2 and 10. Our data substantiate the evidence that Gct1 on Chr 4 is a fundamental oncogene for granulosa cell tumorigenesis in mice and has identified additional interacting autosomal loci that support tumor development.

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

UNLABELLED: 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.

Femur mechanical properties in the F2 progeny of an NZB/B1NJ x RF/J cross are regulated predominantly by genetic loci that regulate bone geometry.

UNLABELLED: Genetic analysis of an NZB/B1NJ x RF/J cross has identified QTLs for femur mechanical, geometric, and densitometric phenotypes. Most mechanical QTLs were associated with geometric QTLs, strongly suggesting common genetic regulation. INTRODUCTION: Previous studies have shown that bone architecture and BMD are important factors affecting bone strength, and both are genetically regulated. We conducted genetic analyses for loci regulating femur mechanical properties, geometric properties, and BMD in a cohort of F2 mice derived from intercross matings of (NZB/B1NJ x RF/J)F1 parents. MATERIALS AND METHODS: Femurs were isolated from 662 10-week-old females. Mechanical properties were determined for a femur from each animal by three-point bending. Geometric properties and volumetric BMD (vBMD) were determined by pQCT. Genotype data were obtained by PCR assays for polymorphic markers carried in the genomic DNA of each mouse. Genome-wide scans were carried out for co-segregation of genetic marker data with values from 23 different phenotypes. Quantitative trait loci (QTLs) were identified for mechanical, geometric, and mineral density phenotypes. RESULTS: QTLs for many phenotypes were significantly refined by covariate analyses using body weight and femur length. Major QTLs for mechanical and geometric phenotypes were found on chromosomes 5, 7, 9, 11, and 12. Nine chromosomal locations were identified with mechanical QTLs and 17 locations with one or more geometric QTLs. The significance of five mechanical and nine geometric QTLs was affected by the inclusion of covariates. These changes included both decreases and increases in significance. The QTLs on chromosomes 5 and 12 were decreased by inclusion of the covariates in the analysis, but QTLs on 7 and 11 were unaffected. Mechanical QTLs were almost always associated with geometric QTLs and less commonly (two of six) with vBMD QTLs. CONCLUSIONS: Genetic regulation of mechanical properties in the F(2) mice of this NZB/B1NJ x RF/J cross seems to be caused by genes regulating femur geometry.

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

UNLABELLED: 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.

Genetic increase in serum insulin-like growth factor-I (IGF-I) in C3H/HeJ compared with C57BL/6J mice is associated with increased transcription from the IGF-I exon 2 promoter.

C3H/HeJ (C3H) mice exhibit 30-40% higher serum IGF-I than do C57BL/6J (B6) mice, in association with increased bone mineral density and strength. These differences are inherited and thus provide a model for determining molecular mechanisms for genetic variation of serum IGF-I and downstream actions. We now report that increased serum IGF-I in C3H mice is associated with increased transcription from the minor exon 2 promoter in liver from female and male mice. The increase in hepatic IGF-I gene expression caused by increased abundance of IGF-I mRNA transcribed from the exon 2 promoter can quantitatively account for the increased serum IGF-I in C3H mice. Also, levels of both Ea and Eb IGF-I mRNAs are increased in livers of male C3H mice. Fasting lowered serum IGF-I and liver IGF-I mRNA levels in female mice of both strains. However, serum IGF-I and liver IGF-I mRNA levels remained higher in fasted C3H mice compared with fasted B6 mice. Levels of IGF-I transcripts initiated from exon 2 are also significantly increased in skeletal muscle, fat, ovaries, and kidneys of C3H mice. IGF binding protein (IGFBP)-5 mRNA levels are significantly higher in muscle and fat of C3H mice than in B6 mice. Levels of exon 1-containing transcripts are increased in whole femurs of male and female C3H mice. We conclude that increased transcription of the IGF-I gene occurs in a promoter- and tissue-specific manner in C3H mice. The increased IGF binding protein-5 mRNA levels in fat and muscle suggest that IGF-I signaling is increased in these tissues in C3H mice.

Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice.

The waddles (wdl) mouse is a unique animal model that exhibits ataxia and appendicular dystonia without pathological abnormalities of either the central or the peripheral nervous systems. A 19-bp deletion in exon 8 of the carbonic anhydrase-related protein VIII gene (Car8) was detected by high-throughput temperature-gradient capillary electrophoresis heteroduplex analysis of PCR amplicons of genes and ESTs within the wdl locus on mouse chromosome 4. Although regarded as a member of the carbonic anhydrase gene family, the encoded protein (CAR8) has no reported enzymatic activity. In normal mice, CAR8 is abundantly expressed in cerebellar Purkinje cells as well as in several other cell groups. Compatible with nonsense-mediated decay of mutant transcripts, CAR8 is virtually absent in mice homozygous for the wdl mutation. These data indicate that the wdl mouse is a Car8 null mutant and that CAR8 plays a central role in motor control.

Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy.

BACKGROUND: Male transgenic mice expressing the human RAS gene on an FVB strain background develop adenocarcinoma of the breast between 7 and 8 weeks of age. We have utilized this mammary tumour model to investigate apoptotic responses following photodynamic therapy (PDT) with a chlorin-based, water-soluble photosensitizer. METHODS: Detection of apoptosis was accomplished by use of the antibody M30 against a neo-epitope of caspase-cleaved cytokeratin 18 that becomes available at an early stage of the apoptotic cascade. Mice bearing multiple tumours were injected with the photosensitizer intraperitoneally, and following a drug-light interval of 96h, 40J/cm(2) of 652nm laser light was applied to one tumour per animal, while the other tumours were protected from light to serve as host controls. The M30 antibody was used for standard immunohistochemistry of tumour sections and flow cytometric detection of epitope expression coupled to cell cycle analysis in tumour cell populations retrieved from paraffin blocks. RESULTS: M30 staining was significantly increased within 2h following light treatment and persisted until 96h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2h post PDT, and recovery of SPF within 96h. CONCLUSIONS: Cytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2h and 96h suggested distinct cellular dynamics at early and late time points, and we propose the M30/SPF ratio as a tumour dynamic index (TDI) to monitor events post PDT.

High-resolution genetic map of X-linked juvenile-type granulosa cell tumor susceptibility genes in mouse.

SWR/Bm (SWR) female mice spontaneously develop early-onset ovarian granulosa cell (GC) tumors that can progress to metastatic carcinoma and thus provide a model system for human, juvenile-type GC tumors. In SWR mice, GC tumor susceptibility is an inherited, polygenic trait that appears at a low frequency. A dramatic increase in tumor frequency occurs when the autosomal SWR genetic complement is combined with the X-linked Gct4 allele of the mouse strain SJL/Bm (SJL). The modifier effect of the SJL Gct4 allele (Gct4(J)) also shows a strong parent-of-origin effect, occurring only when the Gct4(J) allele is paternally inherited. To genetically localize Gct4, we generated seven congenic mouse strains (SWR.SJL-X1 through -X7) that contained a defined segment of the SJL X chromosome (Chr) on the SWR autosomal strain background and mapped Gct4 to a 3 cM region. To better define the location of Gct4, we created an additional congenic strain (SWR.CAST-X) that contains most of the genetically polymorphic Chr X from the strain CAST/Ei. From crosses of the SWR.CAST-X and SWR.SJL-X congenic strains, we derived males carrying unique combinations of SJL-X and CAST-X segments. Progeny testing subsequently revealed a second SJL-derived, GC tumor frequency modifier gene, Gct6, located 6.5 cM distal to Gct4 on Chr X. In summary, we have mapped two modifier genes on the mouse Chr X that cause high-frequency, juvenile-type GC tumor development in female mice. The identity of these genes will provide a solid foundation for determination of tumor susceptibility genes in human cases of juvenile-type GC tumors.