ABSTRACT
Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.
INTRODUCTION
The skull vault, one of the most important craniofacial bones, is formed through intramembranous ossification (Chai and Maxson, 2006; Szabo-Rogers et al., 2010; Wilkie and Morriss-Kay, 2001). In humans, intelligence and mental capacities highly depend on long-term expansion of the skull, which allows brain growth (Morriss-Kay and Wilkie, 2005). If the skull does not develop appropriately owing to variable ossification defects and/or premature cranial suture fusions, individuals will present diverse medical conditions such as hearing and vision loss, which require long-term care. Therefore, it is crucial to understand how craniofacial bones develop.
In mice, skull development (e.g. frontal bone) is initiated at embryonic day (E) 12.5 when osteogenic precursors derived from cranial neural crest (CNC) cells spread upward to the top of the skull (Ishii et al., 2005; Noden and Trainor, 2005). Experimental evidence shows that growth factor signaling, such as fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling, controls osteogenic proliferation and differentiation (Iseki et al., 1997, 1999; Kim et al., 1998; Komatsu and Mishina, 2016; Komatsu et al., 2013; Rice et al., 2000). These developmental processes occur at the unique niche between the cranial sutures and the edge of skull bones. Several studies reveal that aberrant osteoblast growth and differentiation frequently result in skull defects (Rice et al., 1999; Yu et al., 2005). These studies suggest that the initial step of osteogenesis is essential. However, it is unknown how collagen secretion, another essential step in osteogenesis, contributes to intramembranous ossification during skull formation.
We have previously reported that intraflagellar transport 20 (IFT20), known to be required for assembling the primary cilium, is important for intramembranous ossification in the skull (Kaku and Komatsu, 2017; Noda et al., 2016). Ciliary-mediated signaling regulates osteogenic cell proliferation and survival, but ciliary-independent IFT20 function is also crucial to control collagen secretion (Noda et al., 2016; Reiter and Leroux, 2017). Because craniofacial bone defects in Ift20 mutant mice cannot be simply explained by modest cell survival defects, we hypothesized that ciliary-independent IFT20 function may have a primary role in the regulation of craniofacial osteogenesis. However, it is unclear how IFT20 is involved in collagen trafficking. Interestingly, previous studies have shown that Golgi microtubule-associated protein 210 (GMAP210) anchors to IFT20, and that both proteins function together in the trafficking of proteins destined for the ciliary membrane (Finetti et al., 2009; Follit et al., 2008). Although IFT20 mutation(s) in humans have not yet been identified, an absent GMAP210 (encoded by the TRIP11 gene) results in lethal skeletal dysplasia (Smits et al., 2010). In addition, null mutations of Trip11 cause mineralization defects of craniofacial bones in zebrafish (Daane et al., 2019). Therefore, it may be reasonable to speculate that both IFT20 and GMAP210 function together to regulate skeletal formation. Importantly, a recent phenotypic analysis of GMAP210 in mice revealed that it is essential for trafficking-specific cargo in chondrocytes, but dispensable in other secretory cells such as osteoblasts (Bird et al., 2018). However, human fetuses with lethal skeletal dysplasia because of the GMAP210 mutation display a severe mineralization defect in the skull, in which chondrocytes are not present (Ornitz and Marie, 2002; Smits et al., 2010). These observations raise an important question: Although GMAP210 appears to function as a standalone for the regulation of chondrogenesis during endochondral ossification, does GMAP210 interact with other molecule(s) to participate in skull development?
In this study, we demonstrate the role of the molecular complex of IFT20 and GMAP210, which is crucial for orchestrating collagen trafficking in CNC-derived osteoblasts. Our study highlights that molecular regulation of IFT20 and GMAP210 is required for intramembranous ossification, which has not been previously characterized in skull formation.
RESULTS
Osteoblasts near the osteogenic front are crucial for intramembranous ossification
During skull development, growth at the osteogenic fronts (OFs) occurs via intramembranous ossification, in which mesenchymal osteoprogenitors proliferate and differentiate directly into osteoblasts (Iseki et al., 1997, 1999; Rice et al., 2000). A recent elegant study using single cell RNA-seq analysis demonstrated that one of the most significant OF-associated genes is Col22a1, which encodes cross-linking extracellular matrix (ECM) collagen (Holmes et al., 2020). This prompted us to hypothesize that osteoblasts residing on OFs may be actively involved in production of type I collagen (Fig. 1A), thus directly contributing to intramembranous ossification during skull development (Fig. 1B). To test this possibility, we examined the cellular localization of procollagen in CNC-derived osteoblasts in the mouse skull. Interestingly, only osteoblasts near the OFs of wild-type skulls were enriched for procollagen in the Golgi (Fig. 1C,D). At embryonic day (E) 18.5, nearly 80% of CNC-derived osteoblasts in the OF contained procollagen in the Golgi (Fig. 1E). A detailed quantitative imaging analysis revealed that procollagen was abundantly present in cis-Golgi labeled with GM130 (Fig. 1F,G). We also quantified Golgi size from the OF zone to non-OF zones. Although there was a tendency of decreased Golgi size toward non-OF zones, no statistical differences in Golgi size were found in the wild-type skull (Fig. S1). These results demonstrate that osteoblasts near the OFs are key in the regulation of spatiotemporal collagen secretion required for the intramembranous ossification in skull development.
Osteoblasts residing on the osteogenic front are crucial for skull development. (A) Skeletal staining analysis in skull of newborn wild-type mouse. Black lines indicate the osteogenic front (OF). fb, frontal bone; pb, parietal bone; sm, suture mesenchyme. (B) Picrosirius Red staining analysis in wild-type mouse skull tissues from E14.5 to E17.5. Asterisks indicate OF. (C) Immunohistological analysis using anti-GM130 (magenta) and anti-Runx2 (cyan) antibodies in wild-type skull tissues at E18.5. Yellow line indicates the osteoid nearby the OF. (D) Immunohistological analysis using anti-GM130 (magenta) and anti-type I collagen (labeling collagen I; green) antibodies in wild-type skull tissues at E18.5. Arrows show the localization of procollagen in the cis-Golgi. Lower panels show the high magnification images from the boxes in upper panels. Dashed lines indicate skull tissue. (E) Quantification of the percentage of cells co-localizing with procollagen in the cis-Golgi. Note that the osteoblasts near the OF have multiple cells with procollagen in the cis-Golgi (d), compared with other osteoblasts (Non-OF; a,b,c) in the skull. (F,G) Co-localization of type I collagen in the cis-Golgi was analyzed (F) and quantified (G) using IMARIS software. Note that the abundant procollagen in the cis-Golgi was specifically detected in the osteoblasts near the OF, but not in other osteoblasts (Non-OF) in the skull. Three individual wild-type skulls were harvested and immunohistochemistry was performed three times. Data are mean±s.d., n=3 in each group. *P<0.05 (t-test or ANOVA with Tukey's HSD post-hoc test). N.S., not significant.
Osteoblasts residing on the osteogenic front are crucial for skull development. (A) Skeletal staining analysis in skull of newborn wild-type mouse. Black lines indicate the osteogenic front (OF). fb, frontal bone; pb, parietal bone; sm, suture mesenchyme. (B) Picrosirius Red staining analysis in wild-type mouse skull tissues from E14.5 to E17.5. Asterisks indicate OF. (C) Immunohistological analysis using anti-GM130 (magenta) and anti-Runx2 (cyan) antibodies in wild-type skull tissues at E18.5. Yellow line indicates the osteoid nearby the OF. (D) Immunohistological analysis using anti-GM130 (magenta) and anti-type I collagen (labeling collagen I; green) antibodies in wild-type skull tissues at E18.5. Arrows show the localization of procollagen in the cis-Golgi. Lower panels show the high magnification images from the boxes in upper panels. Dashed lines indicate skull tissue. (E) Quantification of the percentage of cells co-localizing with procollagen in the cis-Golgi. Note that the osteoblasts near the OF have multiple cells with procollagen in the cis-Golgi (d), compared with other osteoblasts (Non-OF; a,b,c) in the skull. (F,G) Co-localization of type I collagen in the cis-Golgi was analyzed (F) and quantified (G) using IMARIS software. Note that the abundant procollagen in the cis-Golgi was specifically detected in the osteoblasts near the OF, but not in other osteoblasts (Non-OF) in the skull. Three individual wild-type skulls were harvested and immunohistochemistry was performed three times. Data are mean±s.d., n=3 in each group. *P<0.05 (t-test or ANOVA with Tukey's HSD post-hoc test). N.S., not significant.
IFT20 interacts with GMAP210 during collagen secretion in CNC-derived osteoblasts
To characterize the function of intraflagellar transport proteins (IFT) in craniofacial development, we previously deleted IFT20, known as the smallest IFT, using Wnt1-Cre mice (hereafter Ift20 cKO). Although deletion of Ift20 moderately affects proliferation and cell survival in CNC-derived osteoblasts (Noda et al., 2016), Ift20 cKO mice displayed severe skull defects (Fig. 2A). Thus, there must be a reason that explains loss of skull mineralization in Ift20 cKO mice. Also, it was not clear how loss of IFT20 leads to defective collagen structure. To address these points, collagen structure was examined by scanning electron microscopy (SEM) in vitro. Compared with controls, the collagen fibrils were thin and disorganized in Ift20 cKO osteoblasts (Fig. 2B). To characterize the collagen structure at the nanoscale level, atomic force microscopy (AFM) analysis was also performed. Consistent with the SEM analysis, the collagen fibril network in Ift20 cKO osteoblasts was severely damaged, with thinly formed collagen layers (Fig. 2C). To further examine ultrastructure of collagen in vivo, super-resolution microscopy analysis was performed. Compared with controls, the number of collagen fibrils was reduced in skull tissue from Ift20 cKO mice (Fig. 2D). These results suggest that IFT20 is essential for collagen biosynthesis, which is crucial for intramembranous ossification in the skull. Although the primary role of the IFT complex is to assemble primary cilia, recent studies have highlighted the importance of the extraciliary function of IFTs (Reiter and Leroux, 2017; Singla and Reiter, 2006). IFT20 participates in the regulation of intracellular membrane trafficking, Cbl-mediated ubiquitination and nucleation of Golgi-derived microtubules (Finetti et al., 2009; Nishita et al., 2017; Schmid et al., 2018). However, little is known about the cellular function of IFT20 during collagen biosynthesis. To determine the molecules that interact with IFT20 during intramembranous ossification, cell lysates from control and Ift20 cKO osteoblasts were immunoprecipitated using an IFT20 antibody and subjected to non-biased mass spectrometry (MS) and proteomic analysis (Fig. S2). As a result, GMAP210 was identified as a potential molecule binding with IFT20 (Fig. 2E). A co-immunoprecipitation assay confirmed the MS results by showing molecular binding between IFT20 and GMAP210 in CNC-derived osteoblasts (Fig. 2F). To examine whether IFT20 interacts with GMAP210 during skull development, immunocytochemical analysis was performed using wild-type CNC-derived osteoblasts. Consistent with results from the MS analysis (Tables S1-S5), IFT20 and GMAP210 colocalized in the cis-Golgi (Fig. 2G). To further define the molecular interaction between IFT20 and GMAP210 at the cellular level, a proximity ligation assay (PLA), which allows the in situ immunodetection of protein-protein complexes under endogenous circumstances, was performed. PLA determined the functional binding activity between IFT20 and GMAP210, but its localization is distinct from the presence of primary cilia (Fig. 2H), demonstrating the intracellular binding activity of IFT20 and GMAP210 as single proteins. These findings are consistent with a previous study showing that GMAP210 anchors IFT20 to the Golgi complex (Follit et al., 2008). Together, these results suggest that, as an extraciliary function, IFT20 interacts with GMAP210 to control collagen trafficking during skull development.
IFT20 interacts with GMAP210 during skull development. (A) Skeletal staining of skulls from wild-type (WT) and Ift20 cKO mice at E18.5. Yellow dashed lines indicate the osteogenic fronts (OF). fb, frontal bone; ib, interparietal bone; jb, jugal bone; nb, nasal bone; pb, parietal bone. (B) Scanning electron microscope analysis of type I collagen matrix in cultured wild-type (WT) and Ift20 cKO CNC-derived osteoblasts. Lower panels show high magnification images from upper panels. (C) Atomic force microscopy analysis in cultured WT and Ift20 cKO CNC-derived osteoblasts. The roughness of the surface and volume of collagen matrix were analyzed and quantified. Rq, root-square roughness. (D) Super-resolution microscopy analysis of type I collagen (green) in WT and Ift20 cKO skull tissues at E18.5. Lower panels show the high magnification images from the boxes in the upper panels. Dashed lines indicate skull tissue. Asterisks indicate OF. (E) Identification of molecular binding partners of IFT20. Mass spectrometry (MS) was performed using the cell lysates from three individual primary osteoblasts from WT and Ift20 cKO. (F) Immunoprecipitation analysis (IP) to determine the molecular binding activity between IFT20 and GMAP210. We used CNC-derived osteoblasts established from three individual control and Ift20 mutant skulls (#1, #2, #3). (G) Confocal microscopy analysis to examine the cellular localization of GMAP210 and IFT20 using WT CNC-derived osteoblasts. Intensity of fluorescent signal was quantified with Olympus imaging software, following the direction of the arrow. (H) Proximity ligation assay (PLA) to examine the in situ molecular binding of IFT20 and GMAP210 (red) in WT CNC-derived osteoblasts with the presence of primary cilia (green). Data are mean±s.d., n=3 in each group. *P<0.05 (t-test).
IFT20 interacts with GMAP210 during skull development. (A) Skeletal staining of skulls from wild-type (WT) and Ift20 cKO mice at E18.5. Yellow dashed lines indicate the osteogenic fronts (OF). fb, frontal bone; ib, interparietal bone; jb, jugal bone; nb, nasal bone; pb, parietal bone. (B) Scanning electron microscope analysis of type I collagen matrix in cultured wild-type (WT) and Ift20 cKO CNC-derived osteoblasts. Lower panels show high magnification images from upper panels. (C) Atomic force microscopy analysis in cultured WT and Ift20 cKO CNC-derived osteoblasts. The roughness of the surface and volume of collagen matrix were analyzed and quantified. Rq, root-square roughness. (D) Super-resolution microscopy analysis of type I collagen (green) in WT and Ift20 cKO skull tissues at E18.5. Lower panels show the high magnification images from the boxes in the upper panels. Dashed lines indicate skull tissue. Asterisks indicate OF. (E) Identification of molecular binding partners of IFT20. Mass spectrometry (MS) was performed using the cell lysates from three individual primary osteoblasts from WT and Ift20 cKO. (F) Immunoprecipitation analysis (IP) to determine the molecular binding activity between IFT20 and GMAP210. We used CNC-derived osteoblasts established from three individual control and Ift20 mutant skulls (#1, #2, #3). (G) Confocal microscopy analysis to examine the cellular localization of GMAP210 and IFT20 using WT CNC-derived osteoblasts. Intensity of fluorescent signal was quantified with Olympus imaging software, following the direction of the arrow. (H) Proximity ligation assay (PLA) to examine the in situ molecular binding of IFT20 and GMAP210 (red) in WT CNC-derived osteoblasts with the presence of primary cilia (green). Data are mean±s.d., n=3 in each group. *P<0.05 (t-test).
GMAP210 is an essential molecular partner of IFT20 during craniofacial bone development
Loss-of-function mutations in GMAP210 result in lethal skeletal dysplasia in humans and mice (Bird et al., 2018; Smits et al., 2010). Although these studies do not focus on addressing the molecular function of GMAP210 in intramembranous bones, patients with GMAP210 mutations display skull defects (Smits et al., 2010). The function of GMAP210 has been well studied in chondrocytes during endochondral ossification (Bird et al., 2018), but its molecular function during intramembranous ossification is unclear. To address this question, we disrupted Trip11 using Wnt1-Cre mice (hereafter Trip11 cKO). Interestingly, Trip11 cKO mice displayed craniofacial defects (Fig. 3A). The disruption was confirmed by absence of GMAP210 proteins in the skull (Fig. 3B). Although the majority of Trip11 cKO mice died after birth (n=9, >66.7%; Fig. S3A), this was not because of cardiac defects, because the structure of the cardiac outflow tract effectively labeled by Wnt1-Cre was normal in Trip11 cKO mice (Fig. S3B). Skeletal staining and histological analysis revealed that CNC-derived bones (e.g. nasal and frontal bones) were less mineralized in Trip11 cKO mice (Fig. 3C,D). These observations highlight the newly discovered function of GMAP210 in intramembranous bone formation.
CNC-specific disruption of GMAP210 results in skull defects. (A) Lateral view of craniofacial morphology in newborn wild-type (WT) and Trip11 cKO mice. (B) Immunohistological analysis to examine the production of GMAP210 in skulls from WT and Trip11 cKO mice at E18.5. Asterisks indicate osteogenic front (OF). (C) Skeletal staining analysis of craniofacial bone in WT and Trip11 cKO mice. fb, frontal bone; ib, interparietal bone; jb, jugal bone; md, mandible; mx, maxilla; nb, nasal bone; pb, parietal bone; tr, tympanic ring. Bottom panels show magnification of boxed area in middle panels. Dashed lines indicate OFs. (D) Von Kossa staining of WT and Trip11 cKO skull tissues at E17.5. Asterisks indicate OF. Lower panels indicate high magnification images around OF (boxed areas above). Arrows show dotted calcium deposition.
CNC-specific disruption of GMAP210 results in skull defects. (A) Lateral view of craniofacial morphology in newborn wild-type (WT) and Trip11 cKO mice. (B) Immunohistological analysis to examine the production of GMAP210 in skulls from WT and Trip11 cKO mice at E18.5. Asterisks indicate osteogenic front (OF). (C) Skeletal staining analysis of craniofacial bone in WT and Trip11 cKO mice. fb, frontal bone; ib, interparietal bone; jb, jugal bone; md, mandible; mx, maxilla; nb, nasal bone; pb, parietal bone; tr, tympanic ring. Bottom panels show magnification of boxed area in middle panels. Dashed lines indicate OFs. (D) Von Kossa staining of WT and Trip11 cKO skull tissues at E17.5. Asterisks indicate OF. Lower panels indicate high magnification images around OF (boxed areas above). Arrows show dotted calcium deposition.
Disruption of GMAP210 results in absence of bone mineralization in the skull
Because GMAP210 may play a pivotal role in the onset of craniofacial osteogenesis, we first determined proliferation activity in CNC-derived skulls using a BrdU assay and found that the ratio of BrdU incorporation was comparable between control and Trip11 cKO mice (Fig. 4A). Next, Col1a1, RUNX2 and OSX (also known as SP7), major markers of osteoblast differentiation, were examined by in situ hybridization and immunohistochemistry. Surprisingly, the examined osteogenic differentiation genes were still normally expressed in both control and Trip11 cKO skulls (Fig. 4B-D), suggesting that GMAP210 is unnecessary for regulating osteogenic proliferation and differentiation activities, but may be required for controlling the process of collagen biosynthesis in the skull. To examine this possibility, the process of mineralization was examined using CNC-derived osteoblasts in vitro. Although the wild-type osteoblasts formed solid nodules, which positively stained with Alizarin Red, no cell clusters developed and few osteogenic nodules stained in Trip11 cKO cells (Fig. 4E). Because collagen fibrils are required for maturation of the skull, Picrosirius Red staining was performed and revealed discontinuous collagen fibrils in the skull of Trip11 cKO mice (Fig. 4F). Because skull defects in Trip11 cKO mice are very similar to those observed in Ift20 cKO mice (Fig. 2), levels of IFT20 were examined by immunohistochemical staining and western blot analysis. Compared with controls, IFT20 production was reduced in Trip11 mice (Fig. S4), suggesting that stability of IFT20 may depend on the presence of GMAP210. Together, these results demonstrate that GMAP210 is important for regulating collagen biosynthesis by controlling collagen trafficking during skull development.
GMAP210 is dispensable for osteogenic proliferation and differentiation, but required for bone mineralization in the skull. (A) BrdU analysis of skulls from wild-type (WT) and Trip11 cKO mice at E18.5. Auto-fluorescence signals from blood cells (white color) were excluded for quantification analysis. Dashed lines indicate skull tissue. (B) Expression analysis of Col1a1 by section in situ hybridization in WT and Trip11 cKO skull tissues at E18.5. (C) Osteogenic marker analysis using anti-RUNX2 antibody in WT and Trip11 cKO skull tissues at E18.5. (D) Osteogenic marker analysis using anti-OSX antibody in WT and Trip11 cKO skull tissues at E18.5. In B-D, right panels show magnification of boxed areas in left panels. (E) Osteogenic mineralization was induced by ascorbic acid treatment for 2 weeks. Three individual CNC-derived primary osteoblast cultures derived from WT and Trip11 cKO (#1, #2, #3) were then examined following Alizarin Red staining. (F) Picrosirius Red staining of WT and Trip11 cKO skull tissues at E17.5. Asterisks indicate osteogenic front (OF). Data are mean±s.d., n=3 in each group (t-test). N.S., not significant.
GMAP210 is dispensable for osteogenic proliferation and differentiation, but required for bone mineralization in the skull. (A) BrdU analysis of skulls from wild-type (WT) and Trip11 cKO mice at E18.5. Auto-fluorescence signals from blood cells (white color) were excluded for quantification analysis. Dashed lines indicate skull tissue. (B) Expression analysis of Col1a1 by section in situ hybridization in WT and Trip11 cKO skull tissues at E18.5. (C) Osteogenic marker analysis using anti-RUNX2 antibody in WT and Trip11 cKO skull tissues at E18.5. (D) Osteogenic marker analysis using anti-OSX antibody in WT and Trip11 cKO skull tissues at E18.5. In B-D, right panels show magnification of boxed areas in left panels. (E) Osteogenic mineralization was induced by ascorbic acid treatment for 2 weeks. Three individual CNC-derived primary osteoblast cultures derived from WT and Trip11 cKO (#1, #2, #3) were then examined following Alizarin Red staining. (F) Picrosirius Red staining of WT and Trip11 cKO skull tissues at E17.5. Asterisks indicate osteogenic front (OF). Data are mean±s.d., n=3 in each group (t-test). N.S., not significant.
GMAP210 regulates collagen trafficking in CNC-derived osteoblasts
Although Col1a1 was normally expressed in Trip11 cKO mice (Fig. 4B), the amount of type I collagen was decreased in skull tissues (Fig. 4F). These results suggest that, although procollagen was produced, it was not secreted extracellularly, resulting in the attenuation of collagen fibril formation. This raised an important question about how GMAP210 functions in Golgi to control collagen trafficking. To examine intracellular protein transport from endoplasmic reticulum (ER) to Golgi, we employed a plasmid encoding vesicular stomatitis virus G protein tagged with EGFP (VSVG-GFP) (Presley et al., 1997). VSVG-GFP has been used to study membrane transport because of its reversible misfolding and retention in the ER at 40°C, and its ability to move out of ER and into Golgi upon reducing the temperature to 32°C. VSVG-GFP plasmid was transfected into control and Trip11 cKO osteoblasts, and GFP movement was examined by immunocytochemical co-staining with GM130 (GOLGA2), a marker of cis-Golgi. After temperature reduction, similar to the controls, GFP in Trip11 cKO osteoblasts was well co-localized with GM130 (Fig. 5A,B). Although a decreased Golgi size was found in Trip11 cKO osteoblasts, this did not prevent the transport of VSVG-GFP into Golgi (Fig. 5B). Time-lapse live imaging analysis confirmed these observations in vitro (Movies 1 and 2). To examine the process of collagen trafficking, control and CNC-derived Trip11 cKO cells were treated with ascorbic acid (AA) (Noda et al., 2016). After 2 h of AA stimulation, ∼80% of control and Trip11 cKO cells showed smooth collagen transport into the Golgi (Fig. 5C, second panels from the left, Fig. 5D). Starting from 4 h of AA stimulation, many of the control osteoblasts actively secreted collagen from the Golgi, suggesting that dynamic collagen trafficking occurs (Fig. 5C, third panels from the left, Fig. 5D). On the other hand, over 80% of Trip11 cKO osteoblasts still showed an insufficient collagen trafficking from the Golgi, suggesting that collagen trafficking is significantly slower in Trip11 cKO osteoblasts compared with controls (Fig. 5C, third and fourth left lower panels, Fig. 5D). Western blot analysis confirmed that intracellular collagen in Trip11 cKO osteoblasts remained much longer than in controls (Fig. 5E,F), indicating that collagen trafficking is hampered by GMAP210 dysfunction. To examine whether collagen trafficking was attenuated in Trip11 cKO skulls, the levels of collagen were examined by immunohistochemistry (Fig. 5G). Although Col1a1 was expressed normally (Fig. 4B), compared with controls, type I collagen was less presented in Trip11 cKO skull (Fig. 5G). Quantification analysis further confirmed that type I collagen in cis-Golgi was less present in Trip11 cKO compared with controls (Fig. S5). With these data, one might think that there is a discrepancy observed in cellular localization of collagen in vitro (Fig. 5C-F) and in vivo (Fig. 5G). However, this can be explained by the following reasons. Recently, we have reported that IFT20 plays a pivotal role in regulation of collagen quality (Yamaguchi et al., 2020). Because IFT20 directly interacted with GMAP210 (Fig. 2E-H), it is reasonable to infer that GMAP210 may also be involved in regulation of collagen quality. Therefore, in addition to a lesser amount of extracellular collagen secretion, intracellular collagen could not be detected due to the low quality of collagen in Trip11 cKO skulls. On the other hand, although the pattern of collagen kinetics in the Golgi was distinct compared with control cells, intracellular collagen was still detectable in Trip11 mutant cells (Fig. 5C-F). This is probably because cell culture experiments enabled us to examine collagen trafficking without being affected by abnormal collagen post-translational modifications, which may occur in Trip11 cKO skulls. Together, these results suggest that GMAP210 is required for the transport of collagen through the Golgi, thereby directly contributing to the intracellular trafficking of collagen during skull development.
GMAP210 controls collagen trafficking during skull development. (A) The VSVG-GFP plasmid was transfected into wild-type (WT) and Trip11 cKO osteoblasts. After incubation at 40°C for 24 h, the temperature was shifted to 32°C in order to allow VSVG-GFP transport from the ER into the Golgi. After a temperature shift to 32°C, cells were fixed (0, 2.5, 5, 7.5, 10, and 12.5 min), and localization of GFP in the cis-Golgi (labeled by GM130 antibody) was examined. (B) Quantification of the percentage of VSVG-GFP-positive cells in the Golgi. (C) WT and Trip11 cKO osteoblasts were treated with ascorbic acid (AA) for the indicated time, then the cellular localization of procollagen was examined by immunocytochemistry. Arrows show the localization of procollagen in the cis-Golgi. (D) Quantification of the percentage of cells co-localizing with procollagen in the cis-Golgi. (E) Western blot analysis for measuring the amount of intracellular type I collagen after AA stimulation for the indicated time course. (F) Quantification of relative levels of intracellular collagen in WT and Trip11 cKO osteoblasts. Protein levels were normalized to α-tubulin. (G) Immunohistochemical analysis to examine the production of type I collagen in skull tissues from WT and Trip11 cKO mice at E18.5. Dashed lines indicate skull tissue. Asterisks indicate the osteogenic front. Data are mean±s.d., n=3 in each group. ***P<0.001, **P<0.01, *P<0.05 (t-test). N.S., not significant.
GMAP210 controls collagen trafficking during skull development. (A) The VSVG-GFP plasmid was transfected into wild-type (WT) and Trip11 cKO osteoblasts. After incubation at 40°C for 24 h, the temperature was shifted to 32°C in order to allow VSVG-GFP transport from the ER into the Golgi. After a temperature shift to 32°C, cells were fixed (0, 2.5, 5, 7.5, 10, and 12.5 min), and localization of GFP in the cis-Golgi (labeled by GM130 antibody) was examined. (B) Quantification of the percentage of VSVG-GFP-positive cells in the Golgi. (C) WT and Trip11 cKO osteoblasts were treated with ascorbic acid (AA) for the indicated time, then the cellular localization of procollagen was examined by immunocytochemistry. Arrows show the localization of procollagen in the cis-Golgi. (D) Quantification of the percentage of cells co-localizing with procollagen in the cis-Golgi. (E) Western blot analysis for measuring the amount of intracellular type I collagen after AA stimulation for the indicated time course. (F) Quantification of relative levels of intracellular collagen in WT and Trip11 cKO osteoblasts. Protein levels were normalized to α-tubulin. (G) Immunohistochemical analysis to examine the production of type I collagen in skull tissues from WT and Trip11 cKO mice at E18.5. Dashed lines indicate skull tissue. Asterisks indicate the osteogenic front. Data are mean±s.d., n=3 in each group. ***P<0.001, **P<0.01, *P<0.05 (t-test). N.S., not significant.
IFT20 and GMAP210 regulate collagen trafficking in skull development
Although the absence of GMAP210 in mesoderm-derived osteoblasts does not result in overt skeletal defects, including craniofacial bone abnormalities (Bird et al., 2018), CNC-specific GMAP210 mutants showed skull defects (Fig. 3). These results suggest that GMAP210 may be associated with a specific molecular partner to regulate collagen trafficking in a cell-origin-specific manner, i.e. CNC-derived osteoblasts. Given the similar skull defects observed in both Ift20 cKO and Trip11 cKO mice and the functional molecular binding between IFT20 and GMAP210 (Figs 2 and 3), it is crucial to investigate whether IFT20 and GMAP210 function together during skull development. To address this question, CNC-specific Ift20 and Trip11 double-knockout mice (Ift20:Trip11 dKO) were generated. Similar to Ift20 cKO and Trip11 cKO mice, deletion of both Ift20 and Trip11 in CNC cells resulted in neonatal death (Fig. S6). Importantly, Ift20:Trip11 dKO mutants displayed more severe skull defects than either Ift20 cKO or Trip11 cKO mutants (Fig. 6A). Consistent with this observation, the size of Golgi in Ift20:Trip11 dKO skull was significantly decreased (Fig. S7). The defective area of nasal-frontal bones confirmed that the malformation in Ift20:Trip11 dKO mice was very severe (Fig. 6B). We also examined the amount of type I collagen among mutants using Picrosirius Red staining (Fig. 6C). As a result, the patency of skull ratio corresponds with decreasing amounts of collagen (Fig. S8A). Lastly, to examine whether the disruption of both Ift20 and Trip11 in CNC-derived osteoblasts causes abnormal collagen trafficking, transmission electron microscopy (TEM) analysis was performed. Golgi stacks in Ift20 cKO osteoblasts were moderately affected, with some showing increased cisternae size (Fig. 6D, second panel from the left). In Trip11 cKO osteoblasts, Golgi stack structures were severely affected in the Trip11 cKO mice (Fig. 6D, third panel from the left). Consistent with these observations, osteoblasts from Ift20:Trip11 dKO mice developed an even more severe Golgi phenotype with a complete disruption of the stacked cisternae structure (Fig. 6D, far right panel). Taken together, these results suggest that the IFT20-GMAP210 complex is required for appropriate collagen trafficking during skull development (Fig. S8B).
IFT20 and GMAP210 regulate collagen trafficking in skull development. (A,B) Skeletal staining (A) and schema (B) of skulls from wild-type (WT), Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO mice at E18.5. fb, frontal bone; ib, interparietal bone; jb, jugal bone; nb, nasal bone; pb, parietal bone. (C) Picrosirius Red staining of WT, Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO skull tissues at E18.5. Lower panels show the high magnification images from the boxed areas in the middle panels. Asterisks indicate osteogenic front (OF). Dashed lines indicate skull tissue. (D) Transmission electron microscopy analysis of skull tissues close to the OF, from WT, Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO mice at E18.5. Orange arrowheads indicate normal Golgi cisternae, red arrowheads indicate the swollen Golgi cisternae, and yellow arrowheads indicate packaged proteins coated with cell membrane proteins. Orange dashed line indicates Golgi shape.
IFT20 and GMAP210 regulate collagen trafficking in skull development. (A,B) Skeletal staining (A) and schema (B) of skulls from wild-type (WT), Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO mice at E18.5. fb, frontal bone; ib, interparietal bone; jb, jugal bone; nb, nasal bone; pb, parietal bone. (C) Picrosirius Red staining of WT, Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO skull tissues at E18.5. Lower panels show the high magnification images from the boxed areas in the middle panels. Asterisks indicate osteogenic front (OF). Dashed lines indicate skull tissue. (D) Transmission electron microscopy analysis of skull tissues close to the OF, from WT, Ift20 cKO, Trip11 cKO and Ift20:Trip11 dKO mice at E18.5. Orange arrowheads indicate normal Golgi cisternae, red arrowheads indicate the swollen Golgi cisternae, and yellow arrowheads indicate packaged proteins coated with cell membrane proteins. Orange dashed line indicates Golgi shape.
To summarize our results, the IFT20-GMAP210 complex in wild-type cis-Golgi plays a pivotal role in type I collagen trafficking in osteoblasts residing on the OF, which may function as a ‘center of collagen secretion’. This cellular mechanism contributes to smooth collagen trafficking and produces adequate amounts of collagen matrix required for normal skull development. On the other hand, lacking IFT20-GMAP210 complex diminishes the process of collagen trafficking via the Golgi, resulting in less collagen matrix formation and thus, defective skull formation (Fig. 7).
IFT20-GMAP210 complex is required for appropriate collagen trafficking during skull development. In wild-type mice, CNC-derived osteoblasts residing on osteogenic fronts serve as a ‘center of collagen secretion’, which regulates the intramembranous ossification in the skull (upper panel). The molecular complex of IFT20 and GMAP210 functions to control collagen trafficking in the Golgi. If IFT20 and GMAP210 do not appropriately function, that results in hampered collagen trafficking, and craniofacial bone mineralization is thus severely attenuated, which is frequently observed in patients with GMAP210 mutations (lower panel).
IFT20-GMAP210 complex is required for appropriate collagen trafficking during skull development. In wild-type mice, CNC-derived osteoblasts residing on osteogenic fronts serve as a ‘center of collagen secretion’, which regulates the intramembranous ossification in the skull (upper panel). The molecular complex of IFT20 and GMAP210 functions to control collagen trafficking in the Golgi. If IFT20 and GMAP210 do not appropriately function, that results in hampered collagen trafficking, and craniofacial bone mineralization is thus severely attenuated, which is frequently observed in patients with GMAP210 mutations (lower panel).
DISCUSSION
Intramembranous ossification gives rise to many craniofacial bones, including the skull. One of the important roles of osteoblasts is to secrete type I collagen. However, it remains unclear how collagen trafficking contributes to form complex skull structures. Here, we show that ciliary protein IFT20 and golgin protein GMAP210 interact together to regulate collagen trafficking and control the process of intramembranous ossification in mice. This is the first study to demonstrate that both ciliary and golgin proteins participate in the regulation of collagen biosynthesis during skull development.
Between adjacent skull bones, OFs are present within the developing suture consisting of fibrous joints (Morriss-Kay and Wilkie, 2005; Rice et al., 2000; Wilkie and Morriss-Kay, 2001). Growth at the OFs occurs through intramembranous ossification, by which osteoprogenitors proliferate and differentiate directly into osteoblasts (Iseki et al., 1997, 1999). Subsequently, osteoblasts secrete type I collagen required for bone mineralization. It has been well studied that the balance of transcriptional regulations and growth factor signaling is essential for regulating the proliferation and differentiation of these osteoblasts (Chai and Maxson, 2006; Noden and Trainor, 2005). However, it remains unknown how exactly osteoblasts participate in the collagen secretion process during skull development. We found that CNC-derived osteoblasts near the OF of wild-type skulls are enriched for procollagen in the cis-Golgi (Fig. 1). Because the Golgi apparatus in skeletogenic cells expands when collagen matrix is secreted in skeletogenic cells (Cameron, 1968; Kitami et al., 2019), we hypothesized that the process of collagen secretion in these osteoblasts is highly activated when they need to build bone ECM rapidly. Although the cellular function of osteoblasts residing on OFs has not been well characterized, our study may define the OF as a ‘center of collagen secretion’ required for the intramembranous ossification in the skull (Fig. 7).
The fundamental role of IFTs is to assemble primary cilia (Kozminski et al., 1993; Pazour and Rosenbaum, 2002). Once overlooked as an evolutionary vestige, primary cilia are now considered to be one of the crucial organelles required for many developmental aspects and tissue homeostasis (Eggenschwiler and Anderson, 2007; Goetz and Anderson, 2010). Therefore, understanding the complete role of IFTs will be very helpful in dissecting the complex etiology of human diseases linked to defective primary cilia formation and function, termed ‘ciliopathies’ (Chang et al., 2015; Reiter and Leroux, 2017). To date, ciliopathies have emerged as broad and serious clinical conditions, and considerable efforts have been made to understand how their pathogeneses attribute to alteration of Hedgehog signaling. However, skull defects vary in skeletal ciliopathies and their etiology cannot be solely explained by ciliary Hedgehog signaling mechanisms. Therefore, it is essential to understand how IFT functions in the regulation of skeletal development.
In humans, mutations in ciliary genes often affect the skeletal system. For example, skeletal ciliopathies are frequently observed in Verma-Naumoff syndrome, Majewski syndrome, Jeune syndrome and Ellis-van Creveld syndrome (Ashe et al., 2012; Duran et al., 2017; Miller et al., 2013). Consistent with the phenotypic features of these conditions, loss-of-function of IFT in animal models has clearly demonstrated that IFTs are crucial for ciliogenesis and that they orchestrate growth factor signaling to control skeletogenic cell proliferation, survival and differentiation (Serra, 2008; Yuan et al., 2015). However, at present, little is known about how IFTs function when organizing bone ECM. In this regard, we have recently reported that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in the craniofacial bone (Yamaguchi et al., 2020). This demonstrates that IFT20 participates in collagen post-translational modifications to control the ‘quality’ of collagen. In addition, we have also reported that IFT20 functions in collagen trafficking to regulate the ‘quantity’ of collagen (Noda et al., 2016). These findings are significant because elucidating the role of IFT20 will provide a novel insight in the understanding of the etiology of skeletal ciliopathies. However, it remains unclear what the primary role of IFT20 during collagen biosynthesis is. To address this, we performed a non-bias MS analysis to identify the binding partner of IFT20. Utilizing both genetic and proteomic approaches, we determined that both IFT20 and GMAP210 function together in CNC-derived osteoblasts. Our finding is consistent with those from a previous study showing that GMAP210 anchors IFT20 to the Golgi complex (Follit et al., 2008). In that study, the authors generated stable mouse kidney cell lines overexpressing both FLAG-tagged IFT20 and GMAP210 and found that GMAP210 functions as an IFT20 binding protein. Our study shows, for the first time, the importance of the IFT20-GMAP210 molecular complex in type I collagen secretion in vivo. Through our study, we are aware that disruption of IFT20 and GMAP210 causes craniofacial bone defects including micrognathia. Although the mandible is also formed by intramembranous ossification, its developmental mechanism is very different compared with the skull. For example, mandible formation is required for Meckel's cartilage, but skull is formed by the direct conversion process of mesenchymal cells to osteoblasts. We will examine the etiology of mandible defects in both Ift20 and Trip11 mutants in the future.
Our results support recent elegant studies that showed how hypomorphic mutations in TRIP11 cause odontochondrodysplasia, highlighting the important role of the IFT20-GMAP210 complex in the development of a clinical condition (Medina et al., 2020; Wehrle et al., 2019). Although IFT20 mutations have not been identified in humans, one might expect to observe phenotypic resemblance, at least in part, between IFT20 and TRIP11 mutations. Supporting this prediction, deleting either Ift20 and Trip11 using Wnt1-Cre leads to similar osteopenia-like skull defects, and the double knockout mice developed much more severe skull defects compared with either IFT20 or GMAP210 mutants (Fig. 6). Because cellular trafficking from the ER to the Golgi was comparable in both control and GMAP210 mutant osteoblasts (Fig. 5), the primary cause of bone mineralization defects was most likely attributed to collagen trafficking abnormalities through the Golgi onwards to the plasma membrane. This is well supported by the cellular localization of GMAP210 in the cis-Golgi (Follit et al., 2008, 2006), further reinforcing our idea that the molecular association between IFT20 and GMAP210 in the Golgi is essential for collagen trafficking. However, this cellular machinery may not be generally conserved; rather, it may function in cell origin- and/or developmental stage-specific manners. For example, deletion of GMAP210 in CNC-derived osteoblasts resulted in craniofacial bone defects (Fig. 3), but disruption of GMAP210 in mesoderm-derived osteoblasts did not (Bird et al., 2018). This suggests that the IFT20-GMAP210 complex may predominantly function in CNC-derived skeletogenic precursors. In fact, null Trip11 mutations in mice and loss-of-function of TRIP11 in humans results in lack of mineralization in the frontal area of skull where CNC-derived osteoblasts contribute to bone formation (Smits et al., 2010). It would be interesting to further examine the function of both Ift20 and Trip11 using a mesodermal Cre-driver such as Mesp1-Cre to determine whether these genes control skull development.
Chondrocytes in mice lacking Trip11 show ER swelling and stress, which is also the case in patients with achondrogenesis type 1A caused by mutations in GMAP210 (Bird et al., 2018; Smits et al., 2010). Interestingly, chondrogenesis in these mutant mice is severely affected; however, the secretion of major chondrocyte-related ECM proteins, including type II collagen or aggrecan, is normal. This suggests that GMAP210 may not function in all of the cellular trafficking processes. If this is the case, it may be reasonable to predict that GMAP210 specifically interacts with IFT20 to regulate type I collagen trafficking in CNC-derived osteoblasts. At this time, it is unclear how the molecular mechanism of the IFT20-GMAP210 complex participates in collagen trafficking. It is noteworthy though that GMAP210 possibly functions when collagen molecules tether in the Golgi (Roboti et al., 2015; Sato et al., 2015), suggesting that the IFT20-GMAP210 complex may play a role in the tethering process of type I collagen.
Collagen trafficking is an essential cellular process during craniofacial bone development (Lang et al., 2006). In humans, mutations in the SEC23A and SEC24D genes, which encode components of the coat protein complex II (COPII) required for anterograde transport from the ER to the Golgi, cause cranio-lenticulo-sutural dysplasia (CLSD), characterized by open cranial sutures and defects in craniofacial bones (Boyadjiev et al., 2006, 2003). Coat protein complex I (COPI) vesicles are required for the retrograde transport from the Golgi to the ER, and mutations in ARCN1, which encodes a component of the COPI, also cause craniofacial abnormalities in humans (Izumi et al., 2016). Our study further demonstrates the importance of collagen trafficking regulated by IFT20 and GMAP210, which is crucial for skull development.
MATERIALS AND METHODS
Animals
Trip11 floxed mice were generated as previously described (Bird et al., 2018). Ift20 floxed mice (Jonassen et al., 2008) and Wnt1-Cre mice (Danielian et al., 1998) were obtained from The Jackson Laboratory. All mice were maintained in the animal facility of The University of Texas Medical School at Houston, USA. The experimental protocol (AWC-18-0137) was reviewed and approved by the Animal Welfare Committee and the Institutional Animal Care and Use Committee of The University of Texas Medical School at Houston.
Skeletal preparations, histological analysis, and fluorescence imaging
Staining of craniofacial tissues with Alizarin Red and Alcian Blue was carried out as described in the supplementary Materials and Methods (Skeletal staining). Immunofluorescent staining of paraffin sections was performed as described in supplementary Materials and Methods (Histological analysis). Primary antibodies used in immunofluorescence staining were as follows: collagen type I (1:200, Cedarlane, CL50151AP), GM130 (1:200, BD Biosciences, 610822), GMAP210 (1:200, LS Biosciences, LS-C20059), BrdU (1:100, Abcam, ab6326), Runx2 (1:200, Cell Signaling Technology, 12556), Osx (1:100, Abcam, ab22552). Slides were viewed under an Olympus FluoView FV1000 laser scanning confocal microscope using the software FV10-ASW Viewer (version 3.1). Super-resolution images were obtained using the Nikon SIM super resolution microscope system. More detailed information is described in supplementary Materials and Methods (Super-resolution microscope analysis). Quantification of fluorescence images was performed using IMARIS software (version 9.6). More detailed information is described in supplementary Materials and Methods (3D reconstruction and quantification by IMARIS software).
Isolation and culture of primary osteoblast
We collected calvarial osteoblasts from the embryos at E18.5. Nasal and frontal bones were subjected to five sequential digestions with an enzyme mixture containing 1 mg/ml collagenase type I (Sigma-Aldrich, C0130) and 1 mg/ml collagenase type II (Sigma-Aldrich, C6885). Cell fractions (from two to five of the sequential digestions) were collected and cultured in growth medium [α-MEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin] and in osteogenic medium (growth medium supplemented with 50 μg/ml ascorbic acid and 2 mM β-glycerol phosphate).
SEM analysis
CNC-derived osteoblasts were fixed in 2.5% glutaraldehyde for 15 min at room temperature. After a series of ethanol dehydrations, samples were treated with 50% t-butanol/ethanol solution and placed on a stub (Ted Pella). SEM analysis was performed using an FEI Nova NanoSEM 230. SEM accelerating voltage was set at 5 kV, spot was set at 3 (the diameter of the e-beam was at 3 nm), and SEM work distance was 5 mm. More detailed information is described in supplementary Materials and Methods (Scanning electron microscopy (SEM) analysis).
AFM analysis
CNC-derived osteoblasts were fixed in 2.5% glutaraldehyde for 15 min at room temperature. After a series of ethanol dehydrations, samples were treated with 50% t-butanol/ethanol solution and placed on a stub (Ted Pella). AFM analysis was performed using the Bio-Catalyst AFM system (Bruker), with a scanning rate set at 1 Hz. 3D images and surface roughness were reconstructed and analyzed using NanoScope Analysis software 1.40 (Bruker). More detailed information is described in supplementary Materials and Methods (Atomic force microscope (AFM) analysis).
TEM analysis
Skull tissues were fixed for at least 24 h in Karnovsky's fixative, decalcified in 10% EDTA for one week, then refixed for at least 24 h in Karnovsky's fixative. Samples were then secondarily fixed in 1% osmium tetroxide for 1 h, dehydrated in increasing concentrations of ethanol, embedded in epoxy resin, sectioned at 100 nm thickness using an ultramicrotome (Leica EM UC7), stained with uranyl acetate and lead citrate, and imaged at 80 kV using a JEOL JEM-1230 TEM equipped with a Gatan digital camera.
Section in situ hybridization
A digoxygenin-labeled RNA probe was generated by in vitro transcription (Sigma-Aldrich). Section in situ hybridization was performed according to standard procedures (Komatsu et al., 2014).
Western blot analysis
Osteoblast lysates or tissue lysates were prepared in radioimmunoprecipitation assay buffer. After centrifugation at 15,000 g, the supernatants were separated by SDS/PAGE, blotted onto a PVDF membrane, analyzed with specific antibodies, and visualized with enhanced chemiluminescence. The antibodies used were as follows: GMAP210 (1:500, LS Biosciences, LS-C20059), IFT20 (1:500, Proteintech, 13615-1-AP), collagen type I (1:1000, Cedarlane, CL50151AP), α-tubulin (1:5000, Abcam, ab7291) and GAPDH (1:5000, Cell Signaling Technology, 2118). The Clarity Max ECL Substrate (Bio-Rad) was used for chemiluminescent detection, and signals were quantified with Image Lab Version 5.0 (Bio-Rad).
LC/MS/MS analysis
The gel band samples were subjected to in-gel digestion using a previously described procedure (Shevchenko et al., 2001). The tryptic digested samples were analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) using an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific™) interfaced with a Dionex UltiMate 3000 Binary RSLCnano HPLC System. We loaded 1 μg of each sample for the analysis. Peptides were separated with an AcclaimTM PepMap TM C18 column (75 µm ID×15 cm) at a flow rate of 300 nl/min. Gradient conditions were: 3%-22% of buffer B (0.1% formic acid in acetonitrile) versus buffer A (0.1% formic acid in water) for 12 min, 22%-35% buffer B for 10 min, 35%-90% buffer B for 10 min, followed by 90% buffer B for 10 min. The peptides were analyzed using the data-dependent acquisition (DDA) method. The survey scan was carried out at 120 K resolution at 400 m/z from 350 to 1500 m/z, with AGC target of 2e5 and maximum injection time of 50 msec. Monoisotopic masses were then selected for further fragmentation for ions with 2+ to 4+ charges within a dynamic exclusion range of 35 s. Fragmentation priority was given to the most intense ions. Precursor ions were isolated using the quadrupole with an isolation window of 1.6 m/z. The rapid scan speed in the ion trap was used for CID fragmentation (NCE 35), and an AGC target of 1e4 and maximum injection time of 35 ms were applied. The DDA cycle was limited to 3 s.
MS data processing and analysis
Raw data files were processed using Thermo Fisher Scientific™ Proteome Discoverer™ software version 1.4. The spectra were searched against the Uniprot Mus musculus database using both the Mascot (v2.3.02, Matrix Science) and Sequest HT search engine. The database search was restricted to the following parameters: allowing up to two missed cleavages, 10 ppm mass tolerance for MS1, 0.8 Da mass tolerance for MS/MS, carbamidomethylation on cysteine residues as fixed modification, oxidation of methionine and phosphorylation of serine, threonine and tyrosine as variable modifications. The search results were validated and trimmed to a 1% false discovery rate (FDR) using Percolator.
Proximity ligation assay
CNC-derived osteoblast from wild type at E18.5 were cultured and used to perform PLA using the Duolink PLA Kit (Millipore Sigma, DUO92101). More detailed information is described in supplementary Materials and Methods [Proximity ligation assay (PLA)].
VSVG-GFP time-lapse imaging and protein transport assays and immunocytochemistry
CNC-derived osteoblasts were transfected with the plasmid containing VSVG-GFP using Lipofectamine 3000 (Thermo Fisher Scientific). To shift the temperature from 40°C to 32°C, we used a Thermo Plate (Tokai Hit) for immunocytochemistry and a Stage Top Incubator (Tokai Hit) for time-lapse imaging. For quantification, ten images were captured from one coverslip with at least 33 GFP-positive cells. More detailed information is described in supplementary Materials and Methods (VSVG-GFP time-lapse imaging). To analyze intracellular COL1A1, cells were cultured for 12 h after seeding and then treated with 50 µg/ml ascorbic acid or vehicle. The primary antibodies used were as follows: IFT20 (1:500, Proteintech, 13615-1-AP), collagen type I (1:500, Cedarlane, CL50151AP), GM130 (1:500, BD Biosciences, 610822), GFP (1:2000, Abcam, ab13970) and GMAP210 (1:500, LS Biosciences, LS-C20059). More information is described in supplementary Materials and Methods (Protein transport assays and immunocytochemistry).
Statistical analysis
An unpaired t-test (two-tailed) or one-way ANOVA with Tukey's HSD post-hoc test was performed in the statistical analysis. A P-value of less than 0.05 was considered statistically significant.
Acknowledgements
We deeply thank Dr Patrick Smits for fruitful and helpful comments on the manuscript. We acknowledge Dr Patrick Smits and Dr Matthew Warman for the Trip11 floxed mice; Dr Gregory Pazour for the Ift20 floxed mice; Dr Andrew P. McMahon for the Wnt1-Cre mice; Dr Wei Hsu for in situ plasmids; Dr Jianhua Gu for SEM and AFM analysis; Dr Kandice Levental and Dr Olga Chumakova for the super-resolution microscope analysis; Dr Amanda Howard for the IMARIS image analysis; Dr Jennifer Lippincott-Schwartz for sharing the pEGFP-VSVG plasmid; and Dr Masayuki Ebina for the generous guidance during the mass spectrometry analysis. We also thank Patricia Fonseca for editorial assistance.
Footnotes
Author contributions
Conceptualization: Y.K.; Methodology: H.Y., M.D.M., L.H., L.S., S.P.; Software: H.Y.; Validation: H.Y., Y.K.; Investigation: H.Y., M.D.M., L.S., S.P., Y.K.; Data curation: H.Y., M.D.M., L.H., L.S., S.P.; Writing - original draft: Y.K.; Writing - review & editing: Y.K.; Supervision: Y.K.; Project administration: Y.K.; Funding acquisition: H.Y., Y.K.
Funding
This study was supported by a research grant from the National Institute of Dental and Craniofacial Research/National Institutes of Health (R01DE025897 to Y.K.) and by a fellowship from the Uehara Memorial Foundation (to H.Y.). This work was also supported in part by the Clinical and Translational Proteomics Service Center at The University of Texas Health Science Center at Houston. Deposited in PMC for release after 12 months.
Peer review history
The peer review history is available online at https://journals.biologists.com/dev/article-lookup/doi/10.1242/dev.199559
References
Competing interests
The authors declare no competing or financial interests.