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. Author manuscript; available in PMC: 2025 Apr 26.
Published in final edited form as: Matrix Biol. 2024 Aug 6;133:1–13. doi: 10.1016/j.matbio.2024.07.005

Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

Cassandre Labelle-Dumais a, Courtney Mazur b,c, Serra Kaya b, Yoshihiro Obata e, Bryson Lee a, Claire Acevedo b,d,e, Tamara Alliston b,c, Douglas B Gould a,f,*
PMCID: PMC12032920  NIHMSID: NIHMS2069910  PMID: 39097038

Abstract

Skeletal defects are hallmark features of many extracellular matrix (ECM) and collagen-related disorders. However, a biological function in bone has never been defined for the highly evolutionarily conserved type IV collagen. Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form α1α1α2 (IV) heterotrimers that represent a fundamental basement membrane constituent present in every organ of the body, including the skeleton. COL4A1 and COL4A2 mutations cause Gould syndrome, a variable and clinically heterogenous multisystem disorder generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular manifestations. We have previously identified elevated TGFβ signaling as a pathological insult resulting from Col4a1 mutations and demonstrated that reducing TGFβ signaling ameliorate ocular and cerebrovascular phenotypes in Col4a1 mutant mouse models of Gould syndrome. In this study, we describe the first characterization of skeletal defects in Col4a1 mutant mice that include a developmental delay in osteogenesis and structural, biomechanical and vascular alterations of mature bones. Using distinct mouse models, we show that allelic heterogeneity influences the presentation of skeletal pathology resulting from Col4a1 mutations. Importantly, we found that TGFβ target gene expression is elevated in developing bones from Col4a1 mutant mice and show that genetically reducing TGFβ signaling partially ameliorates skeletal manifestations. Collectively, these findings identify a novel and unsuspected role for type IV collagen in bone biology, expand the spectrum of manifestations associated with Gould syndrome to include skeletal abnormalities, and implicate elevated TGFβ signaling in skeletal pathogenesis in Col4a1 mutant mice.

Keywords: COL4A1, COL4A2, Basement membrane, Gould syndrome, Osteogenesis imperfecta, Ehlers–Danlos syndrome, Skeletal defects, bone

Introduction

Skeletal defects are hallmark features of many extracellular matrix (ECM) disorders, including osteogenesis imperfecta (OI) and Ehlers Danlos syndrome (EDS) that are caused by mutations in genes encoding collagens or collagen-related proteins [1,2]. Collagens are major ECM components that comprise 28 different members in mammals [3]. Type I and type II collagens are the major collagens in skeletal tissues and their role in bone development and disease are well established. However, many other types of collagens are present in bone and while a subset of them have been implicated in skeletal development and disease, a role for type IV collagen remain to be defined [4,5]. Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form collagen α1α1α2(IV) heterotrimers that constitute a fundamental basement membrane component present throughout the animal kingdom [6] and in every organ of the body, including the skeleton [5,7–11]. Consistent with the broad distribution of collagen α1α1α2 (IV) heterotrimers, COL4A1 and COL4A2 mutations lead to Gould syndrome – a rare and clinically heterogenous multisystem disorder that includes variable cerebrovascular, ocular, renal, brain and neuromuscular manifestations [12–14]. While cerebrovascular, ocular, renal, and muscular defects have been the focus of multiple studies [15–28], there are many aspects of Gould syndrome that are still emerging and remain poorly characterized, including skeletal abnormalities. In addition, despite the increasing recognition of Gould syndrome, the underlying pathogenic mechanisms are poorly understood and there are currently no effective treatment options for individuals with COL4A1 and COL4A2 mutations.

Col4a1 and Col4a2 mutant mice recapitulate the spectrum of clinical manifestations observed in individuals with COL4A1 and COL4A2 mutations and represent a valuable resource to study pathologies associated with Gould syndrome and provide insight into the mechanisms contributing to disease [16,19,20,29–35]. Notably, the use of Col4a1 mutant mice was instrumental in describing novel pathological features of Gould syndrome not previously reported in humans, including optic nerve hypoplasia, neuronal migration defects associated with congenital cortical malformation, and peripheral nerve hypomyelination [19,33, 34], and demonstrating that genetic context and allelic heterogeneity influence disease outcome and contribute to the variable presentation of the syndrome [32,34–37].

Collagen α1α1α2 (IV) heterotrimers assemble within the endoplasmic reticulum before being secreted into the extracellular space, where they form an intricate network essential for the structural and functional integrity of the basement membrane and act as a multifunctional signaling platform regulating various developmental and homeostatic processes [13,38]. Although frameshifts, deletions, and duplications have all been reported, missense mutations of highly conserved glycine residues constitute the most common class of COL4A1 and COL4A2 mutations for which the primary consequence is impaired heterotrimer secretion [17,18,29,33,36,39–41]. Importantly, distinct mutations have different biosynthetic consequences that influence disease presentation in an allelic series of Col4a1 and Col4a2 mutant mice. Notably, cerebrovascular disease severity inversely correlates with collagen α1α1α2 (IV) secretion efficiency [35,36]. Consistent with this observation, pharmacologically promoting collagen α1α1α2 (IV) secretion using 4-phenylbutyrate (4PBA) reduces the severity of cerebrovascular manifestations in Col4a1 mutant mice [15,35,42,43]. However, the presence of mutant collagen α1α1α2 (IV) in the basement membrane can also contribute to pathology and we showed that while 4PBA had beneficial effects on cerebrovascular and ocular outcomes in most Col4a1 mutant strains examined, it ameliorates or exacerbates myopathy in a mutation-dependent manner [19,22,35,42]. Collectively, these findings revealed allelic-dependent and tissue-specific mechanistic heterogeneity in Gould syndrome pathogenesis and implicate a role for functional collagen α1α1α2 (IV) subdomains in mediating tissue-specific pathology. Therefore, depending on the mutation and pathological manifestations, targeting downstream signaling pathways may be a better therapeutic strategy than pharmacologically promoting collagen α1α1α2 (IV) secretion. Importantly, we have recently demonstrated that elevated TGFβ signaling contributes to ocular pathology and cerebrovascular disease in Col4a1 mutant mice and established the TGFβ pathway as a potential therapeutic target for individuals with Gould syndrome [15,21,22]. Notably, abnormal TGFβ signaling has also been reported in OI and EDS, raising the possibility of a convergent pathogenic mechanism shared between these three collagen-related ECM disorders [1,2,44,45–47].

In this study, we provide the first characterization of skeletal pathology in Col4a1 mutant mice. We identified delayed osteogenesis and changes in bone marker gene expression during development, as well as altered architecture, mechanical and material properties of mature bones. Using distinct Col4a1 mutant mouse models, we show that the severity of skeletal manifestations is modulated by allelic heterogeneity. Importantly, while 4PBA treatment had mixed effects on skeletal defects, genetically reducing TGFβ signaling partially ameliorates the skeletal pathology in Col4a1 mutant mice. Our findings expand the spectrum of manifestations associated with Gould syndrome to include bone abnormalities and suggest that elevated TGFβ signaling contributes, at least in part, to skeletal pathogenesis, highlighting phenotypic and potentially mechanistic overlap between Gould syndrome, EDS, and OI.

Results

Col4a1+/Δex41 mice exhibit skeletal defects

Col4a1 mutant mice typically have reduced body size and weight [17,19] (Fig. 1A). To determine if alterations of skeletal development could contribute to the smaller size of Col4a1 mutant mice, we performed Alcian blue and Alizarin red staining on whole mount skeletal preparations from Col4a1+/Δex41 mice to identify cartilage and bone, respectively. We selected this strain because Col4a1+/Δex41 mice exhibit significantly reduced body weight compared to their Col4a1+/+ littermates [19]. In addition to the smaller body size, we observed reduced ossification in Col4a1+/Δex41 mice during embryogenesis and early postnatal life (Figs. S1 and 1), where a decrease in Alizarin red staining was predominantly detected in the calvaria, ribs and vertebrae. Consistent with the developmental skeletal defects observed in Col4a1+/Δex41 mice, qPCR analysis revealed altered expression of bone marker genes in postnatal day (P)7 calvaria and femurs, which form through intramembranous and endochondral ossification, respectively (Fig. 2). In calvaria (Fig. 2A), the expression of genes associated with bone formation (Col1a1 and Bglap) was significantly reduced, while the expression of genes associated with inhibition of bone formation (Sost) and bone resorption (Acp5) was significantly increased in Col4a1+/Δex41 mice compared to their Col4a1+/+ littermates. Furthermore, although the expression of Tnfsf11 and Tnfrsf11b, encoding two bone remodeling markers with antagonistic activity, was increased in P7 Col4a1+/Δex41 calvaria, the Tnfsf11/Tnfrst11b ratio was reduced. In contrast, a general reduction in bone marker gene expression was observed in P7 Col4a1+/Δex41 femurs compared to their Col4a1+/+ counterparts (Fig. 2B). These findings are consistent with reduced bone turnover and delayed bone formation, a phenotype that aligns with the impaired developmental osteogenesis in Col4a1+/Δex41 mice.

Fig. 1.

Fig. 1.

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Altered skeletal development in Col4a1+/Δex41 mice. (A) Col4a1+/Δex41 mice exhibit reduced body size compared to their Col4a1+/+ littermates throughout life (shown are postnatal day (P) 7 mice). (B-H) Representative images of skeletal preparations from P7 mice stained with Alcian blue and Alizarin red showing reduced ossification in (C-D) calvaria (black arrow indicate reduced Alizarin red staining), (E-F) ribs (black asterisks demarcate regions where alizarin red staining is absent in E and F), and (E-H) vertebrae (white asterisks mark the vertebral column in E and F) from Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates. n = 3 mice per genotype. Shown are skeletal preparations from male mice, similar results were observed in female mice.

Fig. 2.

Fig. 2.

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Altered bone marker gene expression in P7 Col4a1+/Δex41 mice. (A-B) qPCR analyses showing altered expression of bone marker genes in calvaria (A) and femurs (B) from P7 Col4a1+/Δex41 mice compared to their Col4a1+/+ littermates. Expression of bone marker genes was normalized to the expression of β2-microglobulin. Data are presented as fold expression relative to Col4a1+/+ levels, mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, unpaired two-tailed Student t-test. Since no difference in bone marker gene expression were observed between male and female mice, both sexes were combined with similar numbers of males and females included.

While Col4a1+/Δex41 mice remained smaller than their Col4a1+/+ littermates at P21 and P90, no other gross skeletal defects were detected at these ages (Fig. S2A and B). Consistent with the smaller body size, femur length was also significantly reduced in 3 to 4-month-old (mo) Col4a1+/Δex41 mice (Fig. S2C). To investigate the integrity of mature bones in Col4a1+/Δex41 mice, we performed 3D morphometric analyses using ex vivo microcomputed tomography (μCT) to evaluate cortical bone morphology and trabecular microarchitecture of femurs from 3 to 4mo mice (Fig. 3, Table S1). μCT analysis revealed that although Col4a1+/Δex41 mice had significantly smaller total cross-sectional area (TtAr) and cortical area (CtAr) (Table S1), cortical bone area fraction (CtAr/TtAr) and cortical bone mineral density (BMD) were similar between Col4a1+/Δex41 mice and their sex-matched Col4a1+/+ littermates (Fig. 3C and E). However, cortical bone thickness was significantly decreased in Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates (Fig. 3D). In addition, significant reductions in femoral trabecular bone volume fraction (BV/TV) and trabecular number, and a significant increase in trabecular spacing were detected in Col4a1+/Δex41 mice of both sexes (Fig. 3F–H). Trabecular thickness was reduced in male, but not female Col4a1+/Δex41 mice (Fig. 3I). A small but significant reduction in femoral trabecular BMD was also observed in Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates (Fig. 3J, Table S1). We next assessed bone mechanical properties using 3-point bending flexural test on 3–4mo femurs and detected decreased stiffness and maximal load in Col4a1+/Δex41 mice that are consistent with reduced cortical thickness (Fig. 4, Table S2). Significant sex-specific reductions in yield force and work were also observed in Col4a1+/Δex41 female and male mice, respectively (Fig. 4B and D, Table S2). Together, these findings show that in addition to the developmental delay in osteogenesis, Col4a1+/Δex41 mice exhibit altered structural and mechanical properties of mature cortical bone, as well as altered trabecular microarchitecture and composition.

Fig. 3.

Fig. 3.

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Morphometric analyses of femoral cortical and trabecular bone in Col4a1+/Δex41 mice. (A-B) Representative 3D images generated from microcomputed tomography (μCT) analysis of cortical and trabecular parameters of femurs from 3 to 4 months old female (A) and male (B) Col4a1+/+ and Col4a1+/Δex41 mice. Scale bar=200μm. (C-J) Bone parameters of femurs analyzed by μCT. (C-E) Cortical bone parameters of the middle diaphysis. While cortical bone area fraction (Ct.Ar/T.Ar) (C) and cortical bone mineral density (BMD) (E) were not significantly different between genotypes, cortical bone thickness was significantly reduced in Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates (D). (F-J) Femoral trabecular bone parameters. Trabecular bone volume fraction (BV/TV) (F) and trabecular number (G) were significantly reduced and spacing (H) was significantly increased both in female and male Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates, while trabecular thickness was significantly reduced in male, but not female Col4a1+/Δex41 mice (I). Trabecular BMD was also reduced in Col4a1+/Δex41 mice compared to their sex-matched Col4a1+/+ littermates (J). n = 8 for Col4a1+/+ and Col4a1+/Δex41 female mice and n = 9 and 7 for Col4a1+/+ and Col4a1+/Δex41 male mice, respectively. Data are presented as mean ± SD. * p ≤ 0.05, ** p < 0.01, *** p < 0.001, unpaired two-tailed Student t-test. Additional parameters are shown in Table S1.

Fig. 4.

Fig. 4.

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Altered bone mechanical properties in Col4a1+/Δex41 mice.

(A-D) Assessment of femoral bone mechanical properties by 3-point bending flexural test revealed a trend toward reduced femoral stiffness in Col4a1+/Δex41 mice compared to sex-matched Col4a1+/+ littermates (A), a significant reduction in yield force in female but not male Col4a1+/Δex41 mice (B), a decrease in ultimate force that reached statistical significance in male but not female Col4a1+/Δex41 mice (C), and a significant decrease in work in male but not female Col4a1+/Δex41 mice (D). n = 4/group. Data are presented as mean ± SD. *p < 0.05; **p < 0.01, unpaired two-tailed Student t-test. Additional details are shown in Table S2.

Allelic heterogeneity modulates skeletal manifestations in Col4a1 mutant mice

We have previously demonstrated that allelic heterogeneity modulates the severity and penetrance of various phenotypes associated with Gould syndrome [35,36]. To evaluate if allelic heterogeneity also modulates skeletal pathology in Col4a1 mutant mice, we tested bone parameters in two additional Col4a1 mutant strains (Col4a1G394V and Col4a1G1344D) that were selected based on the differential phenotypic expression and distinct biosynthetic consequences of the mutations [19, 35,36]. Alcian blue and Alizarin red staining of P7 whole skeletal preparations revealed no gross abnormalities in Col4a1+/G394V mice and a mild reduction in ossification in Col4a1+/G1344D mice compared to their respective Col4a1+/+ littermates (Fig. S3). μCT analyses of 3–4mo femurs revealed that while cortical bone morphology was similar between Col4a1+/G394V, Col4a1+/G1344D, and Col4a1+/+ mice (Fig. 5A–E, Table S3), alterations in trabecular bone microarchitecture were present in both Col4a1 mutant strains (Fig. 5F–J, Table S3). The trabecular bone volume fraction was reduced in Col4a1 mutant mice compared to their Col4a1+/+ counterparts, although statistical significance was only observed for males (Fig. 5F). Trabecular number was reduced in Col4a1+/G394V mice of both sexes and in male, but not female Col4a1+/G1344D mice (Fig. 5G) and trabecular separation was increased in both Col4a1 mutant strains compared to sex-matched Col4a1+/+ mice (Fig. 5H). Trabecular thickness was also slightly elevated in Col4a1+/G394V females compared to sex-matched Col4a1+/+and Col4a1+/G1344D mice (Fig. 5I) and a trend towards decreased trabecular BMD was detected in Col4a1+/G394V but not Col4a1+/G1344D mice (Fig. 5J). Consistent with the absence of cortical bone alterations, no major impairment in bone mechanical properties were observed in Col4a1+/G394V mice and Col4a1+/G1344D female mice, however a decrease in femoral stiffness was detected in Col4a1+/G1344D male mice (Table S4). Overall, these findings highlight the influence of allelic heterogeneity in the manifestations of the skeletal defects in Col4a1 mutant mice and demonstrate that mild skeletal developmental defects were observed in Col4a1+/G1344D, but not Col4a1+/G394V mice, while structural alterations of mature trabecular bones occurred in both Col4a1 mutant strains and were generally more prominent in males than females.

Fig. 5.

Fig. 5.

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Morphometric analyses of femoral cortical and trabecular bones in Col4a1+/G394V and Col4a1+/G1344D mice. (A-B) Representative 3D images generated from μCT analysis of cortical and trabecular bone parameter of femurs from 3 to 4mo (A) female and (B) male Col4a1+/+, Col4a1+/G394V, and Col4a1+/G1344D mice. Scale bar=200μm. (C-J) Bone parameters of femurs analyzed by μCT. (C-E) Cortical bone parameters of the middle diaphysis, including cortical bone area fraction (Ct.Ar/T.Ar) (C), cortical bone thickness (D) and cortical BMD (E), were similar between Col4a1+/mut mice and their Col4a1+/+ littermates. (F-J) Femoral trabecular bone parameters. Trabecular bone density (BV/TV) (F) and trabecular numbers (G) are reduced, and trabecular separation (H) increased in Col4a1+/mut mice compared to Col4a1+/+ littermates. Femoral trabecular thickness (I) was slightly increased in Col4a1+/G394V females compared to sex-matched Col4a1+/+ and Col4a1+/G1344D mice and a trend towards reduced BMD was observed in Col4a1+/G394V mice compared to Col4a1+/+ mice (J). n = 11, 5, and 10 for Col4a1+/+, Col4a1+/G394V, and Col4a1+/G1344D female mice, and n = 11, 6, and 8 for Col4a1+/+, Col4a1+/G394V, and Col4a1+/G1344D male mice, respectively. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, one-way ANOVA. Additional parameters are shown in Table S3.

Tgfb1 heterozygosity reduces the severity of skeletal defects in Col4a1+/Δex41 mice

The pathogenicity of Col4a1 mutations is generally attributed to impaired collagen α1α1α2(IV) secretion. We recently demonstrated that elevated TGFβ signaling contributes to ocular and cerebrovascular pathologies in Col4a1 mutant mice and that promoting collagen α1α1α2 (IV) secretion using 4PBA or reducing TGFβ signaling ameliorates cerebrovascular and ocular manifestations [15,19,21,22,35,42]. qPCR analysis revealed increased expression of two bona fide TGFβ target genes in calvaria and femurs from P7 Col4a1+/Δex41 mice compared to Col4a1+/+ littermates (Fig. S4), raising the possibility that elevated TGFβ signaling might also contribute to skeletal manifestations in Col4a1 mutant mice. To test if promoting collagen α1α1α2(IV) secretion or genetically reducing TGFβ signaling can also ameliorate skeletal defects in Col4a1 mutant mice, we evaluated femoral bone parameters in Col4a1+/+ and Col4a1 mutant mice provided with 4PBA in drinking water from embryonic day (E) 9.5 or carrying a null allele of Tgfb1 (Tgfb1+/−). For these experiments we focused on Col4a1+/Δex41 male mice as the Col4a1Δex41 mutation had the most severe skeletal pathology and overall, skeletal defects tended to be more pronounced in Col4a1 mutant males than females. μCT analysis confirmed the presence of alterations in femoral cortical bone morphology and trabecular bone microarchitecture in an independent cohort of 3–4 mo untreated Col4a1+/Δex41 mice obtained from crosses between Col4a1+/Δex41 and Tgfb1+/− mice (Fig. 6, Table S5). Surprisingly, μCT analysis revealed that 4PBA treatment unexpectedly affected bone parameters in Col4a1+/+ mice and had mixed effects on skeletal manifestations in Col4a1+/Δex41 mice (Fig. 6, Table S5). 4PBA significantly increased trabecular thickness and tended to increase cortical thickness and trabecular bone volume fraction in Col4a1+/+ mice (Fig. 6I, D, and F) and although it improved trabecular number and separation in Col4a1+/Δex41 mice (Fig. 6G and H), it significantly decreased trabecular thickness and BMD (Fig. 6I and J). Interestingly, while Tgfb1 heterozygosity negatively impacted some bone parameters in Col4a1+/+ mice (Fig. 6, Table S5), it improved trabecular bone microarchitecture in Col4a1+/Δex41 mice. Tgfb1 heterozygosity significantly reduced cortical and trabecular bone thicknesses (Fig. 6D and I) and tended to reduce cortical bone area fraction, trabecular bone volume fraction and cortical BMD in Col4a1+/+ mice (Fig. 6C, F, and E). In contrast, Tgfb1 heterozygosity significantly improved trabecular number and separation and tended to ameliorate trabecular bone density in Col4a1+/Δex41 mice (Fig. 6G, H, and F). To further investigate the effect of 4PBA treatment and Tgfb1 heterozygosity on bone parameters in Col4a1+/Δex41 mice, we performed 3-point bending flexural tests. Significant reductions in femoral bone stiffness and maximal load were observed in untreated Col4a1+/Δex41 mice compared to their Col4a1+/+ counterparts, validating the presence of altered bone mechanical properties in an independent cohort of Col4a1+/Δex41 mice (Fig. 7A–C, Table S6). In addition, we found that Col4a1+/Δex41 mice also exhibit changes in bone material properties characterized by a significant increase in elastic modulus (Fig. 7D–F). While 4PBA treatment had no effect on bone mechanical and material properties in Col4a1+/+ mice, it exacerbated the reductions in femoral bone mechanical properties in Col4a1+/Δex41 mice without affecting material bone parameters. Genetically reducing TGFβ signaling led to a significant reduction in femoral maximal load and significant increase in elastic modulus in Col4a1+/+ mice (Fig. 7C and D) but had no effect on bone mechanical and material properties in Col4a1+/Δex41 mice. Collectively, these findings indicate that 4PBA treatment can have a detrimental effect on bone parameters in Col4a1 mutant mice and suggest that elevated TGFβ signaling contributes, at least partially, to the skeletal pathology in Col4a1 mutant mice.

Fig. 6.

Fig. 6.

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Genetically reducing Tgfb1 levels partially improves structural bone parameters in Col4a1+/Δex41 mice. Morphometric analyses of femurs from 3 to 4 mo male mice. (A-B) Representative 3D images generated from μCT analysis of cortical and trabecular femoral bones from 3 to 4mo untreated (NT), 4PBA-treated, and Tgfb1 heterozygous Col4a1+/+ (A) and Col4a1+/Δex41 (B) mice. Scale bar=200μm. (C-E) Cortical bone parameters of the middle diaphysis and (F-J) trabecular bone parameters analyzed by μCT showing significant reductions in cortical thickness, trabecular bone density and number and significant increase in trabecular separation in untreated Col4a1+/Δex41 mice compared their Col4a1+/+ counterparts (D, F, G, and H, respectively). 4PBA treatment increased trabecular bone volume fraction and cortical and trabecular thicknesses in Col4a1+/+ mice (F, D, and I). Although 4PBA improved trabecular number and separation (G, H) in Col4a1+/Δex41 mice, it significantly decreased trabecular bone thickness and BMD (I and J). While Tgfb1 heterozygosity significantly reduced cortical and trabecular thickness and tended to reduce cortical BMD and trabecular bone volume fraction in Col4a1+/+ mice (D, I, E, and F), it significantly improved trabecular bone number and separation and tended to increase trabecular bone volume fraction in Col4a1+/Δex41 mice (G, H, and F, respectively). n = 9, 10, 8 for untreated, 4PBA-treated and Tgfb1+/− Col4a1+/+ mice and n = 12, 8, and 7 for untreated, 4PBA-treated and Tgfb1+/− Col4a1+/Δex41 mice, respectively. Data are presented as mean ± SD. **P < 0.01; ****P < 0.0001, unpaired two-tailed Student t-test for comparison between untreated Col4a1+/+ and Col4a1+/Δex41mice. ‡p < 0.05; ‡‡p < 0.001, one-way ANOVA (or Kruskal-Wallis and Dunnett pairwise tests for non-Gaussian distribution in F and H) to test the effect of 4PBA treatment and Tgfb1 heterozygosity in mice of a given genotype. Additional parameters are shown in Table S5.

Fig. 7.

Fig. 7.

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4PBA and Tgfb1 heterozygosity do not ameliorate alterations in bone mechanical and material properties in Col4a1+/Δex41 mice. (A-F) 3-point bending flexural analysis of femurs from 3 to 4mo male mice showing altered femoral bone mechanical (A-C) and material (D-F) properties characterized by significant reductions in stiffness (A) and maximal load (C) and significant increase in elastic modulus (D) in Col4a1+/Δex41 mice compared to Col4a1+/+ mice. Neither 4PBA treatment nor Tgfb1 heterozygosity improved bone mechanical and material properties in Col4a1+/Δex41 mice. 4PBA treatment did not affect bone mechanical and material properties in Col4a1+/+ mice (A-F) but it exacerbated the reduction in femoral stiffness in Col4a1+/Δex41 mice (A). While Tgfb1 heterozygosity had deleterious effects in Col4a1+/+ mice, including significant reduction in maximal load (C) and increase in elastic modulus (D), it did not affect mechanical and material bone parameters in Col4a1+/Δex41 mice. n = 9, 10, 8 for untreated, 4PBA-treated and Tgfb1+/− Col4a1+/+ mice, and n = 12, 8, and 7 for untreated, 4PBA-treated and Tgfb1+/− Col4a1+/Δex41 mice, respectively. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ****p < 0.0001, unpaired two-tailed Student t-test for comparison between untreated Col4a1+/+ and Col4a1+/Δex41mice. ‡p < 0.05; ‡‡p < 0.001, one-way ANOVA (or Kruskal-Wallis and Dunnett pairwise tests for non-Gaussian distribution in A and F) to test the effect of 4PBA treatment and Tgfb1 heterozygosity in mice of a given genotype. Additional details are shown in Table S6.

Reduced bone vascular density in Col4a1+/Δex41 mice

The vasculature plays a central role in bone formation and homeostasis, and Col4a1 mutations are well-established to cause vascular defects in multiple organs [15,17,19,20,29,35]. Using synchrotron radiation μCT (SRμCT), we identified a reduction in vascular density and a trend towards decreased cortical bone porosity in femurs from 3mo Col4a1+/Δex41 mice, suggesting that vascular abnormalities might contribute to the skeletal phenotype in Col4a1 mutant mice (Fig. 8). To directly address this possibility, we selectively expressed mutant COL4A1Δex41 in vascular endothelial cells by crossing conditional Col4a1 mutant mice (Col4a1Flex41) [19,20] to the Tek-Cre transgenic line [48]. μCT analysis identified reductions in femoral total area and trabecular bone surface and total volume (TtAr, TbBS, and TbTV, Table S7) but no other defects in cortical bone morphology and trabecular microarchitecture in TekCre;Col4a1Flex41/Flex41 mice (Fig. S5, Table S7), indicating that selective expression of the Col4a1Δex41 mutation in vascular endothelial cells is not sufficient to recapitulate the skeletal defects observed in Col4a1+/Δex41 mice. However, 3-point bending flexural testing revealed significant changes in bone mechanical and material properties in TekCre;Col4a1Flex41/Flex41 mice compared to Col4a1Flex41/Flex41 littermates (Fig. S6, Table S8), including an increase in elastic modulus which was also observed in Col4a1+/Δex41 mice (Figs. S6D and 7D), suggesting a potential vascular contribution to some of the skeletal manifestations observed in Col4a1 mutant mice.

Fig. 8.

Fig. 8.

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Reduced vascular density in femora from Col4a1+/Δex41 mice. (A-B) Representative images and (C-E) quantification of synchrotron radiation micro-computed tomography (SRμCT) analysis of femoral longitudinal canals from 3 to 4 months old Col4a1+/+ and Col4a1+/Δex41 male mice. While vascular canal diameter was similar between Col4a1+/+ and Col4a1+/Δex41 mice (C), vascular canal density was significantly reduced in Col4a1+/Δex41 mice (D) which was reflected in a trend towards decrease cortical porosity (E). Data are presented as mean ± SD. *p < 0.05.

Discussion

COL4A1 and COL4A2 mutations are well established causes of cerebrovascular and ocular diseases and there is increased recognition for their roles in other multisystemic manifestations associated with Gould syndrome including neuromuscular and renal pathologies [12–14]. However, many aspects of the disease are still emerging or remain poorly characterized, and the underlying pathogenic mechanisms are poorly understood. Col4a1 and Col4a2 mutant mice recapitulate the spectrum of manifestations associated with Gould syndrome and have served as powerful tools to better define pathological features of the syndrome and provide insight into the mechanisms contributing to disease [16,19,20,29–35]. In this study we show that Col4a1 mutant mice exhibit skeletal defects, identifying a novel phenotype associated with Col4a1/Col4a2 mutations. Notably, our findings demonstrate that allelic heterogeneity modulates the severity of skeletal defects in Col4a1 mutant mice and suggest that elevated TGFβ signaling partially contributes to skeletal pathology.

Here we used a combination of whole-mount skeletal staining, molecular and μCT analyses, and flexural biomechanical tests to characterize skeletal pathology in Col4a1 mutant mice. Alcian blue and Alizarin red whole-mount skeletal staining revealed a mutation-dependent developmental delay in osteogenesis in Col4a1 mutant mice that mostly resolved by adulthood. Expression of bone marker genes was also altered in developing calvaria and femurs from Col4a1+/Δex41 mice. Because RNA yield from P7 bones was limiting, we did not evaluate Col4a1 or Col4a2 expression; however, our previous studies did not detect changes in Col4a1 and Col4a2 expression in multiple contexts [22,36]. The observed changes in bone marker gene expression in calvaria reflects a suppression of bone formation and resorption, with a decrease in anabolic markers (Col1a1, Bglap), increased levels of the inhibitor of osteogenesis Sost, and reduction in the ratio of pro/anti-osteoclastogenic factors (Tnfsf11/Tnfrsf11b ratio). Although Sost mRNA levels were reduced in femurs, rather than increased as it was in calvaria, Col1a1 levels remain suppressed. Further analyses are needed to determine why Sost expression differs between calvaria and femurs. Two interesting possibilities include 1) differential effects of the Col4a1 mutation on intramembranous vs endochondral ossification, and 2) differential effects of the Col4a1 mutation on mechanosensitive regulation of Sost, since femurs undergo dynamic loading in P7 mice but calvaria do not. Overall, the pattern of bone marker gene expression is consistent with the impaired osteogenesis observed in Col4a1 mutant mice, as well as with the eventual resolution of this delay in ossification before they reach 3 months of age.

Interestingly, while ossification appears grossly normal in whole-mount skeletal preparations from adult Col4a1+/Δex41 mice, body size and femur length remain smaller, with structural alterations of trabecular and cortical bone. Changes in trabecular bone microarchitecture were also observed in the two additional Col4a1 mutant mouse stains that we examined (Col4a1+/G394V, Col4a1+/G1344D), and overall skeletal pathology tended to be more pronounced in males than females. Estrogen is a well-established regulator of bone formation and homeostasis that could influence skeletal manifestations in a sex-specific manner in Col4a1 mutant mice via its interaction with the TGFβ signaling pathway, regulation of collagen α1α1α2(IV) turnover, and/or its role in angiogenesis [49–54]. While a small but significant decrease in trabecular BMD was detected in the initial cohort of Col4a1+/Δex41 mice (Fig. 3, Table S1), there was no alteration in trabecular BMD in a second cohort of Col4a1+/Δex41 male mice (Fig. 6, Table S5). The discrepancy in trabecular bone mineral BMD between the two cohorts could have stemmed from differences in general mouse colony health/maintenance between the Col4a1+/Δex41 mouse cohort used for our pilot study and subsequent cohorts to further investigate skeletal pathology in Col4a1 mutant mice.

Notably, the reduction in cortical thickness in Col4a1 mutant mice was mutation-dependent and three-point bending flexural tests revealed compromised femoral bone mechanical properties that aligned with the observed decrease in cortical thickness. Assessment of bone material properties revealed a significant increase in elastic modulus in Col4a1−/Δex41 male mice (Fig. 7D), even though cortical bone mineral density was unaffected (Figs. 3E and 6E). Although elastic modulus correlates well with bone mineralization [55], other factors, including those that are regulated by TGFβ signaling, impact elastic modulus independently of mineralization [56]. Among these, TGFβ signaling regulates bone porosity, which in turn can affect elastic modulus [57]. Prior observations of increased porosity in bone from mice with suppressed osteocytic TGFβ signaling [57] are consistent with the trend towards reduced porosity of femoral cortical bone and elevated TGFβ signaling in Col4a1+/Δex41 mice. Nonetheless, additional studies would be needed to identify the cellular and molecular mechanisms responsible for differences in the material properties of Col4a1 mutant bone.

The primary consequence of COL4A1 and COL4A2 mutations is impaired collagen α1α1α2 (IV) secretion. We have previously shown that pharmacologically promoting secretion using 4PBA is generally beneficial in preventing or reducing the severity of pathological manifestations in Col4a1 mutant mice [19,22,35,42,15,35,42,43]. However, promoting collagen α1α1α2(IV) secretion can also have detrimental effects when a mutation occurs in a functional subdomain necessary for COL4A1-mediated tissue-specific function [19]. Based on these studies, we hypothesized that 4PBA would reduce the severity of the skeletal phenotype in Col4a1+/Δex41 mice. Unexpectedly, we found that 4PBA treatment influenced trabecular bone thickness and volume fraction in Col4a1+/+ mice and had mixed effects on skeletal manifestations in Col4a1+/Δex41 mice. Notably, while 4PBA treatment improved trabecular bone microarchitecture (trabecular number and separation, and trend towards increase connectivity) in Col4a1+/Δex4 mice, it significantly reduced trabecular bone thickness and BMD. Furthermore, 4PBA tended to reduce cortical thickness and failed to rescue mechanical deficits in Col4a1+/Δex41 bones. Taken together, these data suggest that 4PBA may exert distinct effects on specific bone cell types, such that increased secretion shifts osteoblast/osteoclast activity to a more anabolic state but may have a different effect on osteocytic perilacunar/canalicular remodeling, which can shift bone matrix mineralization [58,59]. 4PBA has additional properties beside its general chemical chaperone activity and it is possible the detrimental effects of 4PBA on bone parameters could be independent from its effect on collagen α1α1α2(IV) secretion. Interestingly, 4PBA treatment improved bone longitudinal growth and trabecular parameters but not bone strength in a mouse model of OI [60]. Although additional studies will be needed to clarify the mechanisms by which Col4a1 mutations affect each bone cell type, these findings suggest that increasing secretion using 4PBA may not be the best therapeutic approach to target COL4A1-related skeletal defects.

Elevated TGFβ signaling is a pathogenic consequence of impaired collagen α1α1α2 (IV) secretion contributing to cerebrovascular and ocular manifestations in Col4a1 mutant mice [15,21,22]. Gene expression signatures suggest that TGFβ signaling is elevated in bones from Col4a1 mutant mice and morphometric analysis demonstrates that genetically reducing TGFβ signaling using heterozygosity for a null allele of Tgfb1 improved skeletal pathology in Col4a1+/Δex41 mice. These findings support a role for elevated TGFβ signaling in COL4A1-related skeletal pathogenesis. TGFβ signaling pathway plays critical roles in bone development, homeostasis, and disease [61,62]. Notably, excessive TGFβ signaling has been linked to several clinical conditions associated with bone loss or low bone mass and was identified as a common mechanism involved in the pathogenesis of OI, an inherited connective tissue disorder resulting from defects in type I collagen that is primarily characterized by extremely fragile bones that are prone to fractures [63, 64]. Interestingly, of the many known actions of TGFβ in the skeleton, including on osteoclasts, osteoblasts, osteocytes, and their progenitors, alterations in trabecular bone appears to be the most TGFβ-sensitive skeletal manifestation of Col4a1 mutations. Indeed, hyperactive TGFβ signaling uncouples the balance of osteoblast and osteoclast activity, escalates bone remodeling, and decreases trabecular bone volume [65], which is similar to the trabecular bone phenotype observed in Col4a1 mutant mice. In addition, genetically reducing TGFβ signaling selectively improved trabecular bone microarchitecture in Col4a1+/Δex41 mice. It is interesting to note that 4PBA treatment also had beneficial effects on trabecular bone parameters in Col4a1+/Δex41 mice, which is consistent with our previous work showing that 4PBA reduced TGFβ signaling in Col4a1 mutant mice [15,22]. The observation that Tgfb1 heterozygosity did not completely rescue skeletal pathology in Col4a1 mutant mice suggests that additional pathways might be involved.

Bone abnormality is a pathological hallmark of other ECM disorders including OI and EDS. Notably, Gould syndrome, OI and EDS are all caused by defects in collagens and share overlapping clinical manifestations. OI is caused by defects in type I collagen and is primarily characterized by bone abnormalities with variable involvement of other tissues including skin and joint [66]. EDS is associated with alterations in type I, type III, type V and type XII collagens, and is characterized by hyperextensible skin, joint laxity, and fragility of various tissues including blood vessels [1]. Similar to Gould syndrome, the clinical presentation of OI and EDS is highly variable, and genotype-phenotype correlations are well documented. Although the predominant pathological features are distinct between Gould syndrome, OI and EDS – with vascular/ocular, skeletal, and skin/joint abnormalities, respectively - there is a significant overlap in the multisystemic clinical presentation of these ECM disorders. Furthermore, vascular and ocular pathologies, which are cardinal features of Gould syndrome, are also well documented in EDS and OI [1,67–69]. In addition, skeletal muscle abnormalities have been described in Gould syndrome, EDS, and OI [70,71]. Here we expand further the spectrum of phenotypic similarities between Gould syndrome, EDS and OI by showing that Col4a1 mutant mice exhibit bone abnormalities including reductions in cortical thickness, reduced trabecular bone density and BMD, and bone fragility [72–74]. Since aberrant TGFβ signaling has also been implicated in OI and EDS pathogenesis, there might also be an overlap in the pathogenic mechanisms contributing to skeletal disease in Gould syndrome.

Despite being highly evolutionary conserved, the biological functions of collagen α1α1α2(IV) are poorly defined. This is particularly true in bone. While dominated by type I collagen, proteomic mass spectrometry analyses detect a small fraction of collagen IV in bone [5]. Type IV collagen is primarily found in blood vessels in bone tissue [75–77], and since the vasculature is essential for bone development and function [78], we were intrigued by the reduced vascular density in bones from Col4a1 mutant mice. Furthermore, collagen α1α1α2(IV) plays a critical role in vascular development, maintenance, and disease in other tissues, raising the possibility that primary vascular abnormalities could contribute to the skeletal manifestations described here. Determining if skeletal defects arise from vascular abnormalities in Col4a1 mutant mice is important because we previously reported that pharmacologically promoting collagen α1α1α2(IV) secretion and genetically reducing TGFβ signaling partially prevents developmental vascular defects and significantly reduces cerebral hemorrhages in Col4a1 mutant mice [15]. Conditional expression of the Col4a1Δex41 mutation in the vascular endothelium in TekCre;Col4a1Flex41/Flex41 mice does not phenocopy the bone phenotype observed in Col4a1+/Δex41 mice. We have previously shown that conditional expression of mutant COL4A1 in individual cell types of the neurovascular unit or developing eye does not result in robust phenotypes [20,35,79] and we speculate that other cell types, such as mural cells, may compensate for impaired collagen α1α1α2(IV) secretion from vascular endothelial cells. Additional studies will be needed to determine the cellular mechanisms responsible for the skeletal manifestations caused by Col4a1 mutations.

Skeletal abnormalities have been reported in individuals with Gould syndrome [80] but not as frequently as other manifestations. This might be explained by ascertainment bias as patients with skeletal pathology are not typically screened for COL4A1 or COL4A2 mutations. Interestingly mutations in genes encoding key enzymes involved in type IV collagen biosynthesis – including PLOD3, P4HA2, and GLT25D1 – cause manifestations overlapping with those observed in Gould syndrome including skeletal abnormalities [81–89]. In addition, conditional ablation of integrin beta 1– a cell surface receptor for collagen and other matrix proteins – from the vascular endothelium leads to bone structural abnormalities resembling those observed in Col4a1 mutant mice [90]. Together, these findings provide support for a role for collagen α1α1α2 (IV) in bone biology and leave open the possibility that primary vascular defects might be contributing to this phenotype.

Collectively, our findings expand the spectrum of manifestations associated with Gould syndrome to include skeletal pathology and suggest that vascular defects and elevated TGFβ signaling contribute to skeletal pathogenesis in Col4a1 mutant mice and highlight the clinical and mechanistic overlap between ECM disorders caused by defects in collagens.

Experimental procedures

Animals

All experiments were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee at University of California San Francisco (Protocols AN159737 and AN182181). The three Col4a1 mutant mouse lines (Col4a1Δex41, Col4a1G394V, andCol4a1G1344D) were described previously [29,32,36]. (Note that the Col4a1+/Δex41 mutation was originally reported as Col4a1+/Δex40 and later updated on subsequent genome annotations [12]). All Col4a1 mutant mice used in this study were heterozygous for a given Col4a1 mutation that was iteratively crossed to C57BL/6 J (B6) mice for a minimum of 16 generations. The conditional Col4a1Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [19,20]. The Tek-Cre line [48] was used to induce endothelial cell specific expression of the conditional Col4a1Flex41 allele. Tgfb1 mutant mice (Tgfb1tm1N) [91] were backcrossed to B6 mice for a minimum of 5 generations and bred to Col4a1mut/+mice to generate Col4a1mut/+and Col4a1+/+ littermates heterozygous for the Tgfb1 null mutation. Both male and female mice were used in all experiments and no samples were excluded from the study.

4PBA treatment

Sodium 4-phenylbutyrate (4PBA; Scandinavian Formulas) was provided in drinking water (50 mM) from embryonic day (E) 9.5 and refreshed weekly as described previously [19,42]. The day of fertilization (E0.5) was assessed by the presence of vaginal plug.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Calvaria and femurs were harvested from postnatal day (P)7 mice, snap frozen in liquid nitrogen and pulverized using a mortar and pestle in a liquid nitrogen bath. Pulverized bone sample were homogenized in TRizol Reagent. Total RNA was extracted using the RNeasy Mini Kit (74134, Qiagen) with on-column DNase digestion according to the manufacturer’s instructions. 1 ug of total RNA was reverse transcribed using the iScript cDNA synthesis kit (1708841, BioRad). qPCR was performed on a C1000 Touch Thermal cycler with CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) using SsoAdvanced SYBR Green Supermix (1725270, BioRad) and primers listed in Table S10. Briefly, 15 ng of cDNA and 25 μM primers were used per reaction in a final volume of 10 μl of Supermix. Each cycle consisted of denaturation at 95 °C for 5 s, followed by annealing and extension at 60 °C for 25 s. Each reaction was run as technical duplicates and a minimum of 6 biological replicates was used per group. The relative expression of each gene was normalized to the expression of beta-2 microglobulin (B2m) and calculated using the 2−ΔΔCT method [92].

Bone phenotyping

Whole-mount skeletal staining

Whole-mount skeletal staining was performed as previously described [93]. Briefly, pre- and postnatal mouse skeletons were dehydrated and fixed in 99 % ethanol or 95 % ethanol, respectively. Then, skeletal preparations were permeabilized in acetone and stained with Alcian blue and Alizarin red to identify cartilage and bone, respectively, cleared in 1 % potassium hydroxide and stored in glycerol.

Micro-computed tomography (μCT)

Femurs from 3 to 4 months old mice were scanned using a Scanco μCT50 specimen scanner (Scanco Medical, Wangen-Brüttisellen, Switzerland) at room temperature with an X-ray potential of 55 kVp, current of 109 μA, and voxel size of 10 μm. Cortical analysis was conducted in a 1-mm region equidistant from the proximal and distal ends of the bone. Trabecular analysis was conducted in a 2-mm region immediately proximal to the distal growth plate. Thresholding and quantification to determine cortical and trabecular parameters were obtained using Scanco analysis methods as described [94].

Mechanical testing

Whole-bone mechanical properties were assessed in intact femurs obtained from 3 to 4 months old male and female mice. Briefly, femurs were isolated, carefully cleaned of soft tissue, wrapped with gauze, soaked in Hanks’ balanced salt solution (HBSS), and stored at −20 °C. On the day of the experiment, the femurs were thawed at room temperature and subjected to a three-point bending mechanical test using a Bose ElectroForce 3200 test instrument. Throughout the test, the samples were kept hydrated in order to preserve mechanical properties. Each femur’s length was measured with a hand caliper to mark the midpoint, and then the pins were carefully placed, such that anterior side is in tension to replicate physiological bending. Prior to the test, a 0.5 N pre-load was applied to securely keep the samples in place. The three-point bending test was performed at a 0.01 mm/sec displacement rate until fracture occurred. During the test, load and displacement values were continuously recorded. Using a custom-written Matlab script, structural properties such as stiffness, ultimate load, post yield displacement were calculated. Additionally, by employing the moment of inertia measurements from microCT of the same bones, when available, the tissue-level material properties, such as elastic modulus, ultimate stress, and post yield strain, were determined.

Synchrotron radiation X-ray computed micro-tomography (SRμCT)

SRμCT of femurs from 3 to 4 months old mice was performed at the microtomography beamline 8.3.2 at the Advanced Light Source in Berkeley, CA [95]. In total, 1025 projections were acquired over 180° of rotation with a 400 ms exposure time at a beam energy of 20 keV. Following the acquisition of SRμCT images, 3D image volumes were reconstructed in the open-source python code TomoPy [96], with a final image pixel spacing of 1.25 μm/pixel. Image processing was performed in ImageJ (Fiji) [97] using BoneJ [98] and Dragonfly (Object Research Systems (ORS) Inc, Montreal, Canada).

Microscopy

Images of skeletal preparations were captured using SteREO Discovery. V8 microscope equipped with an AxioCam ICc3 digital camera and AxioVision software 4.6 (Carl Zeiss Microscopy, LLC, Germany).

Statistical analyses

Statistical analyses were performed using GraphPad Prism. Two group comparisons were performed using unpaired Student-test. Multiple group comparisons to test for difference between genotypes or test the effect of 4PBA treatment or Tgfb1 heterozygosity on a given genotype were performed using one-way ANOVA or using the Kruskal-Wallis and Dunnett pairwise tests for Gaussian and non-Gaussian data distribution, respectively. P values <0.05 were considered statistically significant.

Supplementary Material

Supplemental material

Acknowledgements

This work was supported by the UCSF Core Center for Musculoskeletal Biology and Medicine (P30 AR075055), and by the National Institutes of Health (NS096173). The UCSF Department of Ophthalmology is supported by a Core Grant from the National Eye Institute (P30 EY002162) and an unrestricted grant from Research to Prevent Blindness.

Footnotes

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.matbio.2024.07.005.

Data availability

Data will be made available on request.

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Supplementary Materials

Supplemental material
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