Abstract
Dedicator of Cytokinesis 7 (DOCK7) has recently emerged as a regulator of skeletal homeostasis, but existing Dock7 mutant models harbor only global mutations and are incompatible with tissue-specific deletion studies. We previously generated a Dock7-floxed allele in which exons 3–4 are flanked by LoxP sites. To validate the utility of this allele for future conditional strategies, we globally deleted exons 3–4 to generate Dock7em2/em2 mice and characterized their skeletal phenotype. Dock7em2/em2 mice exhibited a diluted coat color and white belly spot, consistent with spontaneous Dock7 mutations. Bone microarchitecture was assessed in 21-week-old males and females. Global deletion of Dock7 exons 3–4 resulted in a 30–37% reduction in trabecular bone volume in the distal femur and L5 vertebrae. Cortical bone thickness was unchanged in both sexes; however, male Dock7em2/em2 mice displayed reduced total femoral area, whereas females showed increased medullary area. These data suggest altered appositional bone growth with mutation of Dock7. To assess osteoblast function, bone marrow stromal cells (BMSCs) were differentiated in vitro. Dock7em2/em2 BMSCs exhibited reduced mineralization and decreased Bglap expression, indicating attenuated osteoblast differentiation. These findings demonstrate that Dock7 exons 3–4 are required for normal trabecular bone acquisition and osteoblast function. Loss of these exons disrupts DOCK7 activity, supporting the Dock7em2/em2 line as a valid loss-of-function model. The Dock7em2/em2 mouse provides a foundation for future tissue-specific deletion studies to define the cellular roles of DOCK7 in regulating bone formation and trabecular architecture.
Keywords: Dock7, osteoblast, bone, trabecular bone, Bglap
1. Introduction
Osteoporosis is a chronic condition characterized by skeletal fragility and an increased risk of bone fractures. Low bone mass affects approximately 54 million individuals in the United States (1). This disease contributes significantly to morbidity and mortality, emphasizing the need for a deeper understanding of its underlying mechanisms. Bone health depends on a finely tuned balance between bone formation by osteoblasts and bone resorption by osteoclasts. Disruption of this equilibrium leads to low bone mass and heightened fracture susceptibility. This balance is influenced by systemic factors, including nervous system activity and muscle mass. Recent studies have identified Dedicator of Cytokinesis 7 (DOCK7) as a critical regulator of bone homeostasis (2, 3).
Loss of DOCK7 in mice has been shown to result in low bone mass, impaired trabecular microarchitecture, and reduced periosteal expansion with age (2, 3). These phenotypes are accompanied by a decrease in osteoblast numbers, an increase in osteoclast numbers, and systemic alterations such as reduced brown adipose tissue thermogenesis and lean mass (2, 3). These findings suggest that DOCK7 influences bone metabolism through both direct and systemic mechanisms. While these studies have established the importance of DOCK7 in vivo, further exploration of its role in regulating bone metabolism is necessary to uncover novel pathways involved in bone health. However, current models to investigate the function of DOCK7 in bone are limited.
DOCK7 belongs to the Class C group of irregular guanine nucleotide exchange factors (GEFs) within the DOCK180 protein superfamily (4). It contains two conserved domains: Dock homology region 2 (DHR-2), which mediates GDP-to-GTP exchange in small GTPases such as Rac1 and Cdc42, and DHR-1, whose functions are less well characterized (5, 6). Emerging evidence suggests that the DHR-1 domain may regulate signaling pathways, including phosphatidylinositol (3,5)-bisphosphate signaling and AKT/mTORC regulation, through TSC (7–9). Beyond its GTPase-dependent functions, DOCK7 also modulates cellular processes like migration, polarity, and differentiation in diverse cell types, including osteoblasts, through interactions with proteins such as TACC3 and ErbB4 (2, 10–14). Its role in neuronal polarity and Schwann cell function further highlights its broad biological significance (10, 15–18).
Reports of individuals with compound heterozygous DOCK7 mutations describe phenotypes including seizures, blindness, and craniofacial abnormalities with mutations within the N-terminus, TACC3 binding domain, and DHR2 domain, among other regions (19–21). However, models for studying DOCK7 loss-of-function remain limited. RNAi-based approaches have been widely used for transient DOCK7 ablation. However, these approaches are time-intensive and limited by efficiency, off-target effects, and lack of tissue specificity (10, 15). Spontaneous Dock7 mutant mouse models, such as Misty and Moonlight, have provided insights into DOCK7 function (2, 3, 12). The Misty model, which carries a premature stop codon in Dock7 exon 18, exhibits a diluted coat color and white belly spot (12). DOCK7 was undetectable by western blotting in Misty mice, but in-depth analysis of DOCK7 protein products was not performed (3). While these constitutive mutant models offer insights into the physiological role of DOCK7, they lack the flexibility for tissue-specific studies necessary to understand the more nuanced role of DOCK7 in disease and its potential therapeutic opportunity.
To address these limitations, a new Dock7 floxed model (Dock7fl/fl) was recently developed using CRISPR/Cas9 to flank exons 3 and 4 with LoxP sites (22). Deletion of these exons is predicted to generate a premature stop codon and mimic features of spontaneous Dock7 mutants, including the diluted coat color phenotype. To better understand the implications of this deletion model for future conditional deletion studies, we then generated a mouse with global deletion of Dock7 exons 3–4 (Dock7em2/em2) and explored this impact on bone. We characterized bone microarchitecture, body composition, and in vitro osteoblastogenesis in Dock7em2/em2 mice and compared these findings with those of wild-type controls. Our results reveal that Dock7em2/em2 mice exhibit reduced trabecular bone mass and impaired mineralization in vitro, consistent with findings in other Dock7 mutant models. These data underscore the utility of this model for future tissue-specific studies on the role of DOCK7 in bone metabolism.
2. Methods
2.1. Mice and regulatory approval
The Dock7-em1 allele (Dock7 floxed allele) was generated in the Mouse Genomic Modification Core at MaineHealth Institute for Research (MHIR), as previously described (22). It was used to generate the Dock7-em2Cjr (Dock7-em2) allele, which was used in these studies. The C57BL/6J-Dock7em2/em2 mice (Dock7em2/em2) have global homozygous deletion of Dock7 exons 3 and 4. Briefly, the Dock7-em2Cjr (Dock7-em2) allele was generated by crossing the C57BL/6J-Dock7em1/em1 mice with the B6.Cg-Edil3Tg(Sox2-cre)1Amc/J (Sox2-cre, The Jackson Laboratory, 8454) line in the laboratory of Clifford Rosen at MHIR. To eliminate the Sox2-cre allele, the Dock7-em2 allele was isolated through breeding, thereby eliminating the Sox2-cre allele from the model. The Dock7-em2 allele was then bred to homozygosity (Dock7em2/em2) as confirmed by genotyping (S. Figure 1). Genotypes of the Dock7 em2/em2 mice were confirmed upon arrival at the University of New England.
All animal studies followed National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees (IACUC) of the University of New England or MaineHealth Institute for Research. Bone phenotyping cohorts were housed and bred in the MaineHealth Institute for Research barrier animal facility, an AALAC-accredited facility. Mice were on a 14:10-hour light/dark cycle and fed standard laboratory chow (Teklad irradiated global 18% protein diet, Envigo, 2918) and water ad libitum. Mice for in vitro differentiation of bone marrow stromal cells were housed and bred at the University of New England, kept on a 12:12-hour light/dark cycle, and fed standard laboratory chow (Teklad global 18% protein diet, Envigo, 2018).
2.2. Genotyping
Genotyping of the Dock7-em2 and Dock7+ alleles were performed on tail or toe clips incubated in 50 mM NaOH at 95°C for 1 hr and neutralized with 10% volume of 1M Tris, pH 8.0. DNA from tissue lysates was amplified using primers listed in S. Table 1, a Terra PCR Direct Polymerase Mix (Takara, 639270), and cycling conditions: 1. 98°C, 2 min; 2. 98°C, 10 sec; 3. 60°C, 15 sec; 4. 68°C, 1 min; 5. Steps 2–4, 26 cycles. The Dock7+ (271 bp) and Dock7-em2 (362 bp) PCR products were detected on a 3% agarose gel, using a 100 bp DNA ladder (New England Biolabs, N0467) as shown in S. Figure 1.
2.3. Bone and body composition phenotyping cohort analyses
2.3.1. General cohort information and tissue collection
Male and female Dock7+/+ control mice and Dock7em2/em2 experimental mice (n=14–15/sex/genotype) were aged to 21 weeks and euthanized with carbon dioxide asphyxiation. Femora, tibiae, lumbar vertebrae, and adipose depots were fixed in 10% neutral buffered formalin (10% NBF) for 72 hrs and transferred to 70% EtOH for long-term storage. Mice with oral malocclusions were excluded from experiments.
2.3.2. Body composition and bone mineral density analysis by dual-energy x-ray absorptiometry (DXA)
DXA was performed on male and female 20-week-old Dock7+/+ and Dock7em2/em2 mice (n=14–15/sex/genotype) using the GE-Lunar PIXImus system. All mice underwent DXA analysis under isoflurane anesthesia to assess whole-body composition, excluding the head. Measurements included fat mass, total tissue mass, total areal bone mineral density (aBMD), total areal bone mineral content (aBMC), femoral areal bone mineral density (fBMD) and femoral areal bone mineral content (fBMC). Total tissue and fat mass were used to determine fat-free mass, which approximates lean mass. The central femoral diaphysis was further analyzed as a region of interest (ROI).
2.3.3. Micro-computed tomography (μCT) analysis
Bone microarchitecture analysis was performed by μCT according to established guidelines for μCT analysis of rodent bones (23). Intact femora (n=10/group) and L5 vertebrae (n=10/group/sex) from Dock7+/+ and Dock7em2/em2 mice were randomly selected prior to analysis by μCT. Any bones found to have damage that impaired microarchitecture analysis were excluded. A benchtop μCT imaging system (μCT40, Scanco Medical AG, Brüttisellen, Switzerland) was used to scan bones at a 10μm3 isotropic voxel size, 70 kVP energy level, 114 μA intensity, and 200 ms integration time.
In femora, the trabecular bone microarchitecture was evaluated in a 1.5 mm (150 transverse slices) long region of the distal metaphysis beginning 200 μm superior to the peak of the growth plate and extending proximally. The trabecular region of the L5 vertebral body included the endocortical region beginning 100 μm inferior to the cranial endplate and extended to 100 μm superior to the caudal growth plate. Mineral density thresholds of 385 mgHA/cm3 and 370 mgHA/cm3 were used to segment bone from soft-tissue in the femur and L5 vertebrae, respectively. Trabecular microarchitecture was analyzed using the standard trabecular bone morphology script in the Scanco Evaluation Program. Cortical bone was assessed in 50 transverse μCT slices (500 μm long region) at the femoral mid-diaphysis, and the region included the entire outermost edge of the cortex. Cortical bone was segmented using a fixed threshold of 700 mgHA/cm3 and was analyzed using the Scanco Midshaft Evaluation script.
2.3.4. Adipose depot weights
Whole gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), and interscapular brown adipose tissue (iBAT) depot weights from 21-week-old Dock7+/+ and Dock7em2/em2 mice (n=14–15/sex/genotype) were recorded following fixation in 10% NBF and then transferred to 70% EtOH. No sample weights were excluded from the analysis.
2.4. Bone marrow stromal cell (BMSC) culture
2.4.1. BMSC isolation and general cell culture
Tibiae, femora, and iliac crests were isolated from age-matched male and female Dock7+/+ and Dock7em2/em2 mice at 6–9 weeks of age. Bone marrow was combined from 2 to 5 mice per genotype in each experiment. Total bone marrow was plated in MEMα (Thermo Fisher, 12571) supplemented with 10% fetal bovine serum (FBS, VWR, 97068–085, lot #164K18) and 1% penicillin/streptomycin (P/S) (Thermo Fisher, 15140122). After 48–72 hours, the non-adherent cell population was removed, and the adherent BMSCs were lifted using 0.25% trypsin EDTA (Sigma, T4049). BMSCs were plated at 1–2 million cells/well in 6-well plates (Corning, 353046). All cultures were incubated at 37°C and 5% CO2 throughout the experiment.
2.4.2. BMSC osteogenic differentiation
Osteoblast differentiation was induced at 40–90% BMSC confluence. BMSCs were incubated with MEMα supplemented with 5% FBS, 1% P/S, 8.0 mM β-glycerol phosphate (Sigma, G9422), and 50 μg/mL ascorbic acid to induce osteogenic differentiation. Media was changed every 2–3 days.
2.5. Analysis of osteogenic differentiation by alkaline phosphatase and Von Kossa staining
Staining for alkaline phosphatase activity and mineralization was analyzed in BMSCs isolated from Dock7+/+ and Dock7em2/em2 mice at day 7 of osteogenic differentiation. Individual experiments included 3 wells/genotype and were repeated 3 times/sex. BMSCs were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710). Alkaline phosphatase expression was detected using the Leukocyte Alkaline Phosphatase kit (Sigma, 86R-1KT). Mineral deposition was tested using Von Kossa staining. No wells were excluded from the analysis.
2.6. Analysis of gene expression by real time reverse transcription quantitative PCR (RT-qPCR)
2.6.1. RNA isolation and reverse transcription
Gene expression was analyzed in BMSCs isolated from Dock7+/+ and Dock7em2/em2 mice. Total RNA was isolated via standard extraction with Tri Reagent (Molecular Research Center, TR118) and resuspended in water. Samples were stored at −80°C for RNA analysis. cDNA was generated from 1 μg total RNA. RNA was treated with DNaseI and reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher, 4368814) according to the manufacturer’s instructions. cDNA was diluted 1:5 prior to quantitative analysis.
2.6.2. Quantitative analysis of Dock7 exons 3–4 deletion in BMSCs by RT-qPCR
Transcript expression of Dock7 exons 3–4 was analyzed in BMSCs differentiated in osteogenic media for 7 days. cDNA was generated as described in 2.6.1. Experiments were repeated 3 times/sex with 3 wells/genotype. Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556). Samples were run on the Applied Biosystems QuantStudio3 System V.1.5.3 using cycle conditions: 1. 95°C, 20 sec; 2. 95°C, 1 sec; 3. 60°C, 20 sec; 4. Steps 2–3, 40 cycles. All target transcripts were normalized to the Hprt transcript reference assay (Thermo Fisher, Mm03024075_m1, 4331182). Replicates from all 3 experiments were combined for analysis, and no samples were excluded from analysis.
2.6.3. Quantitative analysis of osteoblast-related genes and additional Dock7 exons by RT-qPCR
The mRNA expression of Runx2, Bglap, Alpl, Dock7 exons 11–12, and Dock7 exons 34–35 were analyzed in BMSCs differentiated in osteogenic media for 7 days. cDNA was generated as described in 2.6.1. Experiments were repeated 3 times/sex with 3 wells/genotype. Transcript expression was analyzed using AzuraQuant Green Fast qPCR HiRox Mix (Azura Genomics, AZ-2020) on the Applied Biosystems StepOnePlus System using cycle conditions: 1. 95°C, 3 min; 2. 95°C, 15 sec; 3. 60°C, 30 sec; 4. Steps 2–3, 40 cycles; 5. 55°C→95°C, 1°/sec; 6. 95°C, 15 sec. Sequences of target primers are provided in Table S1. All target transcripts were normalized to Hprt transcript levels. Replicates from all 3 experiments were combined for statistical analysis. Samples with indications of genomic DNA contamination or extreme deviations in Hprt expression were excluded from the analysis.
2.8. Mass spectrometry
BMSCs were isolated from individual male and female Dock7+/+ and Dock7em2/em2 mice (n=2 mice/group). BMSCs plated according to section 2.4 and harvested at 15–65% confluence. Briefly, cells were lifted with 0.25% trypsin EDTA, washed twice with PBS, snap frozen, and stored at −80°C prior to protein isolation. Samples from each mouse were separated into 1–3 replicates prior to proteomic analysis. As previously reported, tryptic peptides were separated by reverse phase liquid chromatography using a hand-fabricated C18 column (24). Protein identification and quantification was accomplished using a SWATH mass spectrometry workflow on a SCIEX 5600 TripleTOF mass spectrometer (25). Data were analyzed using the Peakview SWATH microapp and MarkerView software packages, as described previously (24). Technical replicates (n=3) from all mice were combined prior to statistical analysis.
2.9. Statistical analysis
Graphs are plotted as box-and-whisker plots, displaying median and sample range. Individual wells are displayed as separate data points. All statistical analyses were performed using Prism 10.1.3. Statistical significance (set at p<0.05) was determined using 2-way ANOVA with Šidák’s multiple comparisons post-hoc test. Statistical significance is indicated: *, p<0.05; **,p<0.01; ***, p<0.001; ****, p<0.0001.
2.10. Graphic representation
Graphic figures were generated using GraphPad Prism 10.1.3 and modified in Adobe Illustrator 2025. Diagrams were created in BioRender.
3. Results
3.1. Generation of the Dock7-em2 allele
A Dock7 gene allele with a deletion of exons 3–4 (Dock7-em2) was generated from the Dock7em1/em1 mouse (Dock7fl/fl, Figure 1A). This deletion is predicted to create a premature stop codon in the Dock7 mRNA transcript (Figure 1B). Homozygous Dock7em2/em2 mice exhibited a diluted coat color and white belly spot, consistent with previously reported phenotypes in Misty, Moonlight, and CRISPR-mediated Dock7 exon 3–4 deletion models (12, 22, 26). Heterozygous Dock7+/em2 mice did not display coat color changes (Figure 1C).
Figure 1. Deletion of Dock7 exons 3–4 in the Dock7-em2 allele results in a diluted coat color and white belly spot in homozygous mice.
(A) Schematic of the Dock7 wild type (+) and Dock7 exons 3–4 deletion (Dock7-em2) transgenic allele. Image created with BioRender.com. (B) Predicted translation of the Dock7+ and Dock7-em2 alleles. Image created with BioRender.com. (C) Representative images of Dock7+/+, Dock7+/em2, and Dock7em2/em2 mice.
3.2. Molecular analysis of the Dock7-em2 allele in bone marrow stromal cells (BMSCs)
To demonstrate the deletion of Dock7 exons 3–4 at the Dock7 transcript levels, the expression of Dock7 mRNA was assessed in BMSCs isolated from Dock7em2/em2 and Dock7+/+ mice. Dock7 exons 3–4 were undetectable at the transcript level, demonstrating germline deletion of these exons (Figure 2A). The expression of Dock7 exons 11–12 and 34–35, 2 loci following the deletion, were reduced by 63–70% (Figure 2A).
Figure 2. Dock7em2/em2 mice had similar levels of DOCK7 protein to those of Dock7+/+ mice.
(A) RNA expression of Dock7 exons 3–4, exons 11–12, and exons 34–35 was analyzed in BMSCs differentiated for 7 days in osteogenic media. Points represent individual replicates for all 3 experiments. (B) Quantitative protein analysis of DOCK7 peptides was performed by mass spectrometry in BMSC isolated from Dock7+/+ and Dock7em2/em2 mice. N=2 mice/group. Points represent replicates from all mice. (C) DOCK7 peptides were detected by mass spectrometry and used to quantify total DOCK7 levels. High confidence (green), medium confidence (yellow), and low confidence (red) DOCK7 peptides are indicated. The protein sequence corresponding to DOCK7 exons 3–4, the DHR1 domain, and the DHR2 domain is underlined and labeled.
Protein expression for Dock7 was quantified using tandem mass spectrometry in BMSCs isolated from Dock7+/+ and Dock7em2/em2 mice. High-confidence peptides associated with the DOCK7 protein were identified by sequential window acquisition of all theoretical mass spectra (SWATH) workflows in protein samples from both genotypes and used to quantify DOCK7 protein levels (Figure 2C). These data indicate the presence of DOCK7 protein in BMSCs isolated from both Dock7+/+ and Dock7em2/em2 mice. No significant differences in DOCK7 protein abundance was detected following deletion of exons 3–4 (Figure 2B). However, DOCK7 protein expression may be lower in BMSCs from male mice than in those from female mice. High confidence DOCK7 peptides were not detected in Dock7 exons 1–4, and thus, the loss of the N-terminal peptides prior to the exon 3–4 deletion or peptides within exons 3–4 could not be evaluated. A low to medium confidence peptide was detected in Dock7 exon 4 from both genotypes. Because the transcript analysis demonstrated that this region was not detected and that it shows sequence homology to other mouse proteins, the evidence does not support that this peptide is associated with the DOCK7 protein. Spectra and sequencing data are shown for representative high-confidence DOCK7 peptides (S. Figure 2–3). Confirmation of protein expression by western blot analysis was not completed as antibodies to mouse DOCK7 were not commercially available.
3.3. Bone microarchitecture analysis
To evaluate the effect of the Dock7 exon 3–4 deletion on bone microarchitecture, femora and L5 vertebrae from male and female Dock7em2/em2 mice at 21 weeks of age were analyzed and compared to age- and sex-matched Dock7+/+ control mice. Trabecular bone volume fraction (Tb.BV/TV) was reduced in Dock7em2/em2 mice by 37% in femora from both sexes and in L5 vertebrae from female mice, while L5 vertebrae from male mice displayed a 30% reduction (Table 1). Although the percentage reduction in trabecular parameters was similar between femora and vertebrae, trabecular bone reductions in the femora of female mice were only trending, likely due to the inherently low cancellous bone present in the distal femur at this age.
Table 1.
Deletion of Dock7 exons 3–4 results in reduced trabecular microarchitecture at 21 weeks of age.
| Female | Male | Two-way ANOVA p-values | Šidák’s Test post-hoc p -values | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
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| Dock7+/+ (n=9–10) | Dock7em2/em2 (n=10) | Dock7+/+ (n=9–10) | Dock7em2/em2 (n=9–10) | Interaction | Sex | Genotype | Females | Males | ||
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| Distal Femur | Femur length (mm) | 15.2±0.1 | 14.9±0.1 | 15.2±0.1 | 15.0±0.1 | 0.9280 | 0.5805 | 0.0184 | 0.1509 | 0.1922 |
| Tb.BV/TV (%) | 5.12±0.26 | 3.24±0.25 | 19.40±1.42 | 12.19±1.63**** | 0.0197 | <0.0001 | 0.0002 | 0.4126 | <0.0001 | |
| Tb.BMD (mgHA/cm3) | 101.0±2.6 | 80.0±2.4 | 227.0±13.9 | 162.9±14.4*** | 0.0408 | <0.0001 | 0.0002 | 0.2801 | 0.0002 | |
| Conn.D (1/mm3) | 31.8±3.9 | 19.1±2.5 | 119.2±7.1 | 73.1±8.0**** | 0.0069 | <0.0001 | <0.0001 | 0.2489 | <0.0001 | |
| Tb.N (1/mm) | 3.01±0.05 | 2.48±0.04*** | 4.47±0.13 | 3.75±0.10**** | 0.2776 | <0.0001 | <0.0001 | 0.0004 | <0.0001 | |
| Tb.Th (μm) | 47.3±0.7 | 45.2±1.5 | 58.6±2.0 | 53.9±2.2 | 0.4604 | <0.0001 | 0.0518 | 0.6116 | 0.1144 | |
| Tb.Sp (μm) | 332±6 | 404±8**** | 210±8 | 258±7*** | 0.1149 | <0.0001 | <0.0001 | <0.0001 | 0.0001 | |
| SMI | 3.35±0.08 | 3.39±0.08 | 1.62±0.13 | 2.30±0.17*** | 0.0110 | <0.0001 | 0.0048 | 0.9671 | 0.0006 | |
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| Femur Mid-shaft | Ct. Th (mm) | 0.193±0.003 | 0.192±0.003 | 0.197±0.004 | 0.193±0.006 | 0.7741 | 0.4990 | 0.5641 | 0.9735 | 0.7899 |
| Ct. TMD (mgHA/cm3) | 1238±3 | 1240±5 | 1190±4 | 1215±4*** | 0.0095 | <0.0001 | 0.0024 | 0.9156 | 0.0003 | |
| Ct.Ar (mm2) | 0.81±0.02 | 0.83±0.02 | 1.01±0.03 | 0.90±0.03** | 0.0127 | <0.0001 | 0.0848 | 0.7991 | 0.0073 | |
| Ma.Ar (mm2) | 0.88±0.02 | 1.00±0.02* | 1.28±0.04 | 1.11±0.04*** | <0.0001 | <0.0001 | 0.4209 | 0.0175 | 0.0008 | |
| Tt.Ar (mm2) | 1.70±0.03 | 1.84±0.03 | 2.29±0.06 | 2.01±0.06*** | 0.0001 | <0.0001 | 0.1743 | 0.0990 | 0.0006 | |
| Ct.Ar/Tt.Ar (%) | 48.0±0.6 | 45.4±0.3** | 44.2±0.7 | 45.0±0.8 | 0.0087 | 0.0014 | 0.1297 | 0.0084 | 0.6317 | |
| Ct. Por. (%) | 0.705±0.033 | 0.745±0.034 | 0.879±0.071 | 0.790±0.051 | 0.2025 | 0.0341 | 0.6345 | 0.8119 | 0.3867 | |
| pMOI(mm4) | 0.356±0.015 | 0.392±0.014 | 0.620±0.033 | 0.474±0.031*** | 0.0008 | <0.0001 | 0.0335 | 0.5164 | 0.0004 | |
| Imax(mm4) | 0.240±0.011 | 0.253±0.009 | 0.430±0.024 | 0.313±0.019**** | 0.0004 | <0.0001 | 0.0039 | 0.8259 | <0.0001 | |
| Imin(mm4) | 0.116±0.004 | 0.139±0.006 | 0.190±0.010 | 0.161±0.013 | 0.0057 | <0.0001 | 0.7447 | 0.1407 | 0.0527 | |
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| Vertebrae (L5) | BV/TV (%) | 21.40±0.50 | 13.44±0.85**** | 25.64±0.88 | 17.83±0.74**** | 0.9285 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| Conn.D (1/mm3) | 92.1±5.1 | 69.2±6.6* | 156.6±6.4 | 96.6±7.0**** | 0.0062 | <0.0001 | <0.0001 | 0.0282 | <0.0001 | |
| Tb.N (1/mm) | 3.60±0.10 | 2.95±0.12*** | 4.77±0.10 | 3.72±0.14**** | 0.1021 | <0.0001 | <0.0001 | 0.0008 | <0.0001 | |
| Tb.Th (μm) | 58.5±0.7 | 53.8±0.8** | 55.4±1.8 | 52.5±0.8 | 0.4050 | 0.0549 | 0.0013 | 0.0076 | 0.1391 | |
| Tb.Sp (μm) | 277±9 | 339±13** | 201 ±5 | 267±16*** | 0.8637 | <0.0001 | <0.0001 | 0.0011 | 0.0007 | |
| SMI | 0.71±0.04 | 1.47±0.08**** | 0.61±0.08 | 1.15±0.05**** | 0.0917 | 0.0025 | <0.0001 | <0.0001 | <0.0001 | |
Trabecular bone volume fraction (Tb.BV/TV), trabecular bone mineral density (Tb.BMD), specific bone surface (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), connectivity density (Conn.D), structural model index (SMI), total cross-sectional area (Tt.Ar), cortical bone area (Ct.Ar), medullary area (Ma.Ar), bone area fraction (Ct.Ar/Tt.Ar), cortical tissue mineral density (Ct.TMD), cortical thickness (Ct.Th), and maximum (Imax), minimum (Imin) and polar moments of inertia.
Error represented as SEM. Bold Values represent stastical significant with p<0.05.
No significant changes in cortical thickness (Ct.Th) were observed in femora from either sex. However, analysis of cortical bone parameters demonstrated sex-specific differences. Male Dock7em2/em2 mice showed decreases in total area (Tt.Ar), medullary area (Ma.Ar), and cortical area (Ct.Ar) (Table 1). In female mice, an increase in Ma.Ar and a trending increase in Tt.Ar area suggested a potential acceleration of appositional bone growth (Table 1). These findings indicate that deletion of Dock7 exons 3–4 may result in altered appositional bone growth, with distinct effects between sexes.
3.4. Body composition and bone mineral density
Body composition was analyzed by DXA analysis at 20 weeks of age in male and female Dock7em2/em2 mice and compared to age- and sex-matched Dock7+/+ control mice. Male Dock7em2/em2 mice exhibited reduced total body mass and fat-free mass, with no changes in fat mass, compared to Dock7+/+ control mice (Figure 3). No change in body composition was detected in female Dock7em2/em2 mice. Ex vivo analysis of fat pad weights confirmed no differences in adipose depot size in either sex (S. Figure 4). Reduced total and femoral areal bone mineral density (aBMD) and areal bone mineral content (aBMC) were observed exclusively in male Dock7em2/em2 mice, consistent with the decreased cortical area (Ct.Ar) identified in these mice by μCT analysis (S. Figure 5).
Figure 3. Body composition in Dock7em2/em2 mice at 20 weeks of age.
Body composition was measured by DXA analysis in male and female Dock7em2/em2 mice and compared to Dock7+/+ control mice. Data was analyzed by 2-way ANOVA and Holm-Šídák’s test for post-hoc analysis. N=14–15 mice/group. Points represent each mouse.
3.5. In vitro osteogenic differentiation
The role of DOCK7 in osteogenic differentiation was explored using bone marrow stromal cells (BMSCs) isolated from Dock7em2/em2 and Dock7+/+ mice. Osteogenic differentiation was stimulated in vitro and analyzed at Day 7 for mineral deposition, alkaline phosphatase activity, and osteoblast-related gene expression. BMSCs isolated from Dock7em2/em2 mice showed a decrease in mineralization by von Kossa staining (Figure 4A). No overt change in alkaline phosphatase staining was observed at this time point. Expression of Bglap was decreased in BMSCs from Dock7em2/em2 mice, while AlpI expression was only reduced in BMSCs from male mice. Runx2 gene expression was unaffected with mutation of Dock7 (Figure 4B). These results demonstrate that deletion of Dock7 exons 3–4 results in impaired in vitro mineralization.
Figure 4. Deletion of Dock7 exons 3–4 impaired in vitro mineralization and reduced Bglap gene expression in BMSCs.
Osteogenic differentiation was assessed in BMSCs that were isolated from Dock7+/+ and Dock7em2/em2 mice after 7 days of osteogenic differentiation. (A) Representative image of von Kossa and alkaline phosphatase staining. (B) Expression of the osteoblast-related genes Runx2, AlpI, and Bglap. Points represent individual replicates for all 3 experiments.
4. Discussion
These studies characterize a bone phenotype in a new model of Dock7 mutation. Genomic deletion of exons 3–4 resulted in a reduction in Dock7 mRNA levels in BMSCs with similar levels of DOCK7 protein in the wild-type and mutant mice as assessed by mass spectrometry. The deletion of Dock7 exons 3–4 resulted in a 37% reduction in trabecular bone volume fraction (Tb.BV/TV) in the femur across both sexes and in L5 vertebrae in female mice, with male mice showing a 30% decrease in trabecular bone in L5 vertebrae. The low trabecular bone volume observed in Dock7em2/em2 mice was accompanied by impaired in vitro mineralization with reduced Bglap gene expression during osteoblast differentiation, highlighting the functional consequences of deletion of Dock7 exons 3–4 in the Dock7-em2 allele. Our data suggest that Dock7 exon 3–4 may be necessary for Dock7 transcript stability and protein function. The genomic deletion of Dock7 exons 3–4 may serve as a valuable tool for investigating the cell-specific roles of DOCK7 when used in the conditional deletion format available with the Dock7-em1 allele.
The observed trabecular bone phenotype in Dock7em2/em2 mice aligns with findings from previous studies using Misty mice, a model with no detectable Dock7 protein (3). In Misty mice, reductions in Tb.BV/TV ranged from 22–60% at 16 weeks of age (2, 3). Consistent with this, Dock7em2/em2 mice exhibited trabecular bone loss within the narrower range of 30–37%. The similar degree of cancellous bone loss between these models supports the conclusion that DOCK7 is critical for maintaining approximately one-third of the trabecular bone mass in the femur and vertebrae. Together, these results validate Dock7em2/em2 mice as a robust and reproducible model for studying the role of DOCK7 in cancellous bone.
Cortical bone phenotypes in Dock7em2/em2 mice revealed sex-specific differences, consistent with prior studies of Misty mice (2, 3). At 21 weeks, Dock7em2/em2 mice exhibited no change in cortical thickness (Ct.Th) but showed distinct changes in medullary area (Ma.Ar): higher in females and lower in males compared to controls. This sex-dimorphic pattern aligns with findings from Misty mice, where female mice displayed early increases in periosteal and endosteal circumference followed by slow appositional bone growth. Over time, female Misty mice also developed reductions in Ct.Th, a phenotype not observed in males (2, 3). However, the absence of changes to Ct.Th in the Dock7em2/em2 mice suggest that the Dock7 exons 3–4 deletion may display a more subtle cortical bone phenotype than the Misty mice. While a direct comparison is complicated by differences in age between this study and prior reports, these data suggest that DOCK7 plays a sexually dimorphic role in regulating cortical bone growth and maintenance.
Deletion of Dock7 exons 3–4 also impaired in vitro osteoblast differentiation, as evidenced by reduced mineralization and diminished Bglap expression in differentiated BMSCs from Dock7em2/em2 mice. These results parallel observations in female Misty mice, where Dock7 mutations reduced AlpI and Bglap expression during calvarial osteoblast differentiation (3). However, this study expands upon prior work by demonstrating that in vitro mineralization defects are present in both male and female Dock7em2/em2 mice. This further underscores the critical role of DOCK7 in osteoblast function and mineralization.
In addition to sexually dimorphic changes in cortical bone, both total body mass and fat-free mass were reduced only in male Dock7em2/em2 mice. Furthermore, AlpI expression was only reduced in BMSCs isolated from male Dock7em2/em2 mice, indicating that deletion of Dock7 exons 3–4 may present with modified low bone mass phenotypes between male and female mice. While previous studies have reported similarities in the loss of fat-free mass in Misty mice, characterization of bone in Dock7-mutant mice has primarily been explored in female mice (2, 3). As our initial observations demonstrate that BMSCs from male mice express lower levels of DOCK7 protein than those from female mice, this differential expression of DOCK7 may be one mechanism underlying the observed sex differences in bone. However, we cannot eliminate the possibility that the sex differences observed in this study may also be linked to the Dock7 exons 3–4 deletion model itself.
One limitation of this study was that only bone microarchitecture was analyzed at a single time point, 21 weeks-of-age, where changes in bone microarchitecture between Dock7+/+ and Dock7em2/em2 mice may reflect a combination of bone acquisition and age-related bone loss. The current study cannot differentiate whether the observed skeletal phenotype was due to deficits in bone acquisition, enhanced bone loss, or a combination of both. Previous studies suggest that loss of DOCK7 in Misty mice primarily impacts bone acquisition with little impact on age-related bone loss (2, 3). Therefore, the bone loss in Dock7em2/em2 mice likely reflects a deficiency in bone acquisition. Future experiments should examine different ages to differentiate between these mechanisms.
No studies have indicated that Dock7 exons 3–4 serve an essential role in protein function. The Dock7em2/em2 mouse model exhibits a coat color change and a low bone mass phenotype similar to those reported for the Misty mouse model, a model with no detectable protein by western blot (3). Interestingly, the absence of changes in DOCK7 protein levels with the deletion of exons 3–4 suggests that the deletion of Dock7 exons 3–4 may result in a truncated protein product via an alternative translational start site. However, the size of the mutant protein could not be confirmed by western blot due to the lack of commercially available antibodies that reliably recognize the mouse DOCK7 protein. Furthermore, as SWATH analysis did not detect an N-terminal peptide prior to the Dock7 exons 3–4 deletion in the wild type mice, these data do not exclude the presence of a short N-terminal peptide prior to the deletion site. Therefore, results from these studies support the hypothesis that Dock7 exons 3–4 are essential for normal DOCK7 protein function, making the Dock7 exons 3–4 deletion model a novel tool to study the role of DOCK7 in physiology and cell signaling. Further studies are necessary to confirm the direct role of the deletion of Dock7 exons 3–4 on protein function.
Another key strength of the Dock7 exon 3–4 deletion model is that it can be used globally in mice with the Dock7-em2 allele, as well as in conditional deletion studies using cell-type-specific Cre alleles with the Dock7-em1 allele. However, the presence of a truncated DOCK7 protein product and possible N-terminal peptide is a limitation of the model. The detection of high confidence peptide fragment in the SWATH analysis does suggest that the DOCK7 DHR2 domain is still present in BMSCs from Dock7em2/em2 mice. This raises the possibility that these domains retain activity and that the Dock7-em2 allele encodes a nonfunctional, hypomorphic, or dominant-negative protein. However, the absence of the white belly spot in the Dock7+/em2 indicates that the presence of a dominant-negative DOCK7 protein is unlikely. Additional studies are needed to characterize the DOCK7 protein products and confirm their functional status. Because of the similarities in the bone and coat color phenotype between the Dock7 em2/em2 mice and the Misty phenotype, these data suggest that Dock7 exon 3–4 may be critical for protein function, indicating that the floxed Dock7-em1 allele will be a valuable tool to study the tissue-specific role of DOCK7 in future studies.
5. Conclusions
In summary, the deletion of Dock7 exons 3–4 replicates key phenotypes observed in Misty mice, including trabecular bone deficits and coat color changes, supporting its validity as a model for studying the role of DOCK7 in bone physiology. Although the functional status of the truncated DOCK7 protein remains unclear, the phenotypic similarities strongly suggest disrupted normal DOCK7 function. Importantly, the floxed allele design of the Dock7-em1 model enables future conditional deletion studies to explore cell type-specific roles of DOCK7 in bone development and disease. Further investigation into the functionality of the protein product from the Dock7-em2 allele will be necessary to understand the molecular mechanisms underlying these phenotypes.
Supplementary Material
Acknowledgements
The authors thank Clifford Rosen for providing the Dock7em2/em2 mice to the University of New England. Support from the Center for Cell Signaling and the Center for Pain Research in research development and dissemination of these studies is also gratefully acknowledged.
Funding
This work was supported by the National Institutes of Health (P20GM152330, F32AR067071, and P30GM145497) and, in part, by a Minigrant from the University of New England Office of Research and Scholarship. C.L. was supported by a Carmen Pettapiece Student Research Fellowship from the University of New England, College of Osteopathic Medicine, Student Government Association.
Footnotes
Declaration of competing interest
The authors have nothing to disclose.
Data availability
Raw data will be deposited in Dryad upon peer-reviewed manuscript acceptance. Mass spectrometry (proteomic) data will be deposited into PRoteomics IDEntifications Database, unless otherwise specified.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Raw data will be deposited in Dryad upon peer-reviewed manuscript acceptance. Mass spectrometry (proteomic) data will be deposited into PRoteomics IDEntifications Database, unless otherwise specified.