Nuclear factor I-C regulates intramembranous bone formation via control of FGF signalling
Introduction The skull consists of the calvarial bone, craniofacial bone, and cranial base . The calvarial bone primarily includes a significant portion of the frontal bone, most of the two parietal bones, and smaller sections of the temporal and occipital bones . Cranial sutures are essential in the calvarial bone's development, growth, and maintenance. The suture also plays an essential function in preventing premature bone separation of the calvarial bone during the growth process . Craniosynostosis can occur if the cranial suture contains the skeletal stem and progenitor cells and does not maintain patency, and premature fusion occurs . Therefore, the osteoblast progenitor cells within the suture need to undergo appropriate proliferation and maintain good differentiation potential to support normal skull development and formation. Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) are crucial in bone development and related diseases. Numerous studies have demonstrated that FGFRs, particularly FGFR1, FGFR2, and FGFR3, are essential for regulating cranial suture development. FGFR1 is primarily found in osteoblasts and the mesenchymal tissue of the skull, where it is associated with the differentiation of osteoprogenitor cells. In contrast, FGFR2 is mainly expressed in proliferating osteogenic stem cells and regulates cell proliferation. Notably, before the differentiation of osteoprogenitor cells in the coronal sutures of mice, the expression of FGFR2 is downregulated while FGFR1 expression is upregulated [ , , , , , ]. These studies indicate that FGF signaling may be essential in cranial bone development. NFI-C was first reported as a critical regulator in tooth development, particularly the formation of the tooth root, at a specific time during postnatal development [ , , , , , , ]. Later, Nfic -deficient mice ( Nfic −/− mice) had abnormalities in odontoblast proliferation and differentiation, resulting in abnormal incisors and short molar root formation [ , , ]. It was reported that NFI-C acts downstream of the Runt-related transcription factor (Runx2), a transcription factor playing a central role in the development process of the teeth as well as bones, and upstream of another transcription factor, Osterix (Osx), and controls the formation of bones through the regulation of Osx . Moreover, NFI-C was a critical factor in the proliferation of epiphyseal chondrocytes, a growth plate for long bones . Additionally, we previously indicated that NFI-C is vital for maintaining the stem cell niche in mineralized tissues and may serve as an additional factor to improve reprogramming efficiency. We showed that Nfic regulates epithelial cell proliferation through epithelial-mesenchymal interactions in dental epithelial stem cells (DESCs), controlling the Fgf8-Nfic-Sox2 pathway in the epithelium while the Nfic-Fgf10 pathway in the mesenchyme . However, the correlation between Nfic and FGF signaling in calvarial bone development is unclear. Based on these previous findings, this study aims to determine the effects of Nfic on the proliferation of calvarial osteogenic progenitor cells and osteoblast differentiation in calvarial bone formation using Nfic -deficient mice and reveal the underlying molecular mechanism in this process. These findings suggest that Nfic plays an essential role in the proliferation and differentiation of calvarial osteogenic progenitors through regulating Fgfr1 during calvarial bone development. 

Materials and methods 2.1 Reagents and antibodies Rabbit cyclin D1 (2922), Fgfr1 (9740), Erk1/2 (9102), and p-Erk1/2 (4370) were obtained from Cell Signaling Technology. Antibodies against PCNA (sc-400), p21 (sc-6246), caspase-8 (sc-7890), and caspase-3 (sc-7148) were obtained from Santa Cruz Biotechnology, Inc. 2.2 Mice Experiments involving mice were conducted following the guidelines of the Dental Research Institute and the Institutional Animal Care and Use Committee at Seoul National University (SNU-181127-13-2). Nfic −/− mice were generated by deleting the second exon of the Nfic gene. Nfic −/− mice were obtained by breeding heterozygous Nfic +/- mice. The genotypes of the mice were determined as previously described . Due to the brittle teeth characteristic of Nfic −/− mice, a ground standard rodent chow was provided to all animals three times a week, starting three days before weaning and continuing for up to six weeks . 2.3 Micro-computed tomography (micro-CT) analysis, histology, and immunohistochemistry The skulls of two and six weeks wild-type (WT) and Nfic -deficient mice were removed, fixed in 4 % paraformaldehyde at 4 °C overnight, and subjected to micro-CT using a SkyScan scanner and associated software (Skyscan 1172, Kontich, Belgium). The instrument's isotopic resolution is 10 μm. After reconstruction, we selected a round region of interest (ROI) around the midline suture (4 mm in diameter) for further quantitative analysis. BMD and BV/TV were measured using the CTan program. Decalcified skulls were embedded in paraffin and cut into sections 5-μm thick. They were stained using hematoxylin-eosin (H&E) and then analyzed using immunohistochemistry (IHC) as previously described . 2.4 Skeletal analyses The skulls of two- and four weeks were prepared and stained using Alcian blue for cartilage and alizarin red S for bone. First, the skin and internal organs were removed, and the skulls were fixed for five days in 95 % ethanol, followed by two days in acetone. The samples were stained for three days with a solution containing 0.015 % Alcian blue 8GX (Sigma) and 0.005 % alizarin red S (Sigma) in a mixture of 5 % glacial acetic acid and 70 % ethanol. After staining, the samples were treated with 80 % glycerol and ultimately stored in 100 %. 2.5 MTT assays The proliferation rates of primary calvarial osteoprogenitor cells were assessed using MTT assays. The cells were seeded and cultured in 96-well plates at a density of 3000 cells per well. After washing with PBS, 50 μl of MTT solution (5 mg/ml) was added to each well, followed by an incubation period of 4 h at 37 °C. Once the incubation was complete, the MTT solution was removed, and the resulting dye was dissolved in DMSO. The absorbance at 540 nm was then measured using a microplate reader (Multiskan EX). Triplicate samples from two independent experiments were analyzed. 2.6 Cell culture and transfection Primary calvarial osteoprogenitor cells were isolated from the calvaria (parietal and frontal bones) of 7-day-old wild-type (WT) and Nfic −/− mice. In brief, the calvaria were harvested and dissected into fragments. These fragments were washed in phosphate-buffered saline (PBS), minced into several pieces, and digested for 1 h with 1 % Collagenase Type I (Gibco BRL) and 1.6 % Dispase II (Gibco BRL) in the alpha-MEM medium under an atmosphere containing 5 % CO 2 . The cells were then cultured in alpha-MEM supplemented with 1x antibiotic antimycotic solution and 10 % fetal bovine serum (FBS). Human bone marrow stem cells (hBMSCs) were obtained from Lonza Pte. Ltd. (Cat. No. PT-2501) and cultured in low glucose DMEM supplemented with 1x antibiotic antimycotic solution and 10 % FBS. For plasmid construction, a 0.56-kb genomic fragment was amplified from human genomic DNA to obtain the FGFR1 promoter region (−555 to +145). One microliter of human genomic DNA was subjected to PCR using the following cycling conditions: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for 35 cycles. The sense and anti-sense primers used were as follows: FGFR1 Sense: 5′-TCT CCG GTC CGA GCT CGG GGC GCC C-3′, FGFR1 Anti-sense: 5′-ACG AGC GCA GGG AGG GGG CGC AGG A-3′, FGFR1 Mutant Sense: 5′-GGG CGC GTC GTC CCG AGC GG-3′, and FGFR1 Mutant Anti-sense: 5′-CCG CTC GGG ACG ACG CGC CC-3'. The amplified fragment was then subcloned into the PCR®2.1 T vector and ligated into the Xho I and Hin dIII sites of the pGL3 luciferase (LUC) basic expression vector (Promega). 2.7 Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses 2 μg of total RNA was reverse transcribed using 0.5 μg of Oligo d(T) and one μL (50 IU) of Superscript III (Invitrogen) in a 20 μl reaction mixture at 50 °C for 1 h. The resulting cDNAs were amplified using PCR. Primers specific to mNfic, mRunx2, mOsx, mAlp, mBsp, mOc, mDmp1, mFgfr1, mFgfr2, hFGFR1, hFGFR2, hNFI-C , and hGAPDH were synthesized for real-time PCR (see ). 2.8 Western blot analysis Western blot analyses were executed precisely, following established protocols . In brief, we separated 30 μg of total proteins using 10 % SDS-PAGE. Proteins were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell BioScience) and incubated with specific antibodies. Labeled proteins were then detected using an enhanced chemiluminescence system from GE Healthcare, ensuring the reliability of the results. 2.9 ChIP assays ChIP assays were conducted as previously described . hBMSCs were transiently transfected with an NFI-C expression plasmid for 48 h. The samples were then sonicated, followed by chromatin immunoprecipitation using 30 μl of anti-NFIC antibodies and 10 μl of anti-IgG antibodies (Santa Cruz). The final DNA pellets were recovered and analyzed by polymerase chain reaction (PCR) using specific primers, as listed in . The PCR conditions were set as follows: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 35 cycles. The PCR products were analyzed using electrophoresis on a 2 % agarose gel. 2.10 Statistical analysis All quantitative data are presented as means ± standard deviation (SD) using SPSS 26. The significance of differences was assessed with a Student's t-test (∗P < .05, ∗∗P < .01, ∗∗∗P < .001). 

Reagents and antibodies Rabbit cyclin D1 (2922), Fgfr1 (9740), Erk1/2 (9102), and p-Erk1/2 (4370) were obtained from Cell Signaling Technology. Antibodies against PCNA (sc-400), p21 (sc-6246), caspase-8 (sc-7890), and caspase-3 (sc-7148) were obtained from Santa Cruz Biotechnology, Inc. 

Mice Experiments involving mice were conducted following the guidelines of the Dental Research Institute and the Institutional Animal Care and Use Committee at Seoul National University (SNU-181127-13-2). Nfic −/− mice were generated by deleting the second exon of the Nfic gene. Nfic −/− mice were obtained by breeding heterozygous Nfic +/- mice. The genotypes of the mice were determined as previously described . Due to the brittle teeth characteristic of Nfic −/− mice, a ground standard rodent chow was provided to all animals three times a week, starting three days before weaning and continuing for up to six weeks . 

Micro-computed tomography (micro-CT) analysis, histology, and immunohistochemistry The skulls of two and six weeks wild-type (WT) and Nfic -deficient mice were removed, fixed in 4 % paraformaldehyde at 4 °C overnight, and subjected to micro-CT using a SkyScan scanner and associated software (Skyscan 1172, Kontich, Belgium). The instrument's isotopic resolution is 10 μm. After reconstruction, we selected a round region of interest (ROI) around the midline suture (4 mm in diameter) for further quantitative analysis. BMD and BV/TV were measured using the CTan program. Decalcified skulls were embedded in paraffin and cut into sections 5-μm thick. They were stained using hematoxylin-eosin (H&E) and then analyzed using immunohistochemistry (IHC) as previously described . 

Skeletal analyses The skulls of two- and four weeks were prepared and stained using Alcian blue for cartilage and alizarin red S for bone. First, the skin and internal organs were removed, and the skulls were fixed for five days in 95 % ethanol, followed by two days in acetone. The samples were stained for three days with a solution containing 0.015 % Alcian blue 8GX (Sigma) and 0.005 % alizarin red S (Sigma) in a mixture of 5 % glacial acetic acid and 70 % ethanol. After staining, the samples were treated with 80 % glycerol and ultimately stored in 100 %. 

MTT assays The proliferation rates of primary calvarial osteoprogenitor cells were assessed using MTT assays. The cells were seeded and cultured in 96-well plates at a density of 3000 cells per well. After washing with PBS, 50 μl of MTT solution (5 mg/ml) was added to each well, followed by an incubation period of 4 h at 37 °C. Once the incubation was complete, the MTT solution was removed, and the resulting dye was dissolved in DMSO. The absorbance at 540 nm was then measured using a microplate reader (Multiskan EX). Triplicate samples from two independent experiments were analyzed. 

Cell culture and transfection Primary calvarial osteoprogenitor cells were isolated from the calvaria (parietal and frontal bones) of 7-day-old wild-type (WT) and Nfic −/− mice. In brief, the calvaria were harvested and dissected into fragments. These fragments were washed in phosphate-buffered saline (PBS), minced into several pieces, and digested for 1 h with 1 % Collagenase Type I (Gibco BRL) and 1.6 % Dispase II (Gibco BRL) in the alpha-MEM medium under an atmosphere containing 5 % CO 2 . The cells were then cultured in alpha-MEM supplemented with 1x antibiotic antimycotic solution and 10 % fetal bovine serum (FBS). Human bone marrow stem cells (hBMSCs) were obtained from Lonza Pte. Ltd. (Cat. No. PT-2501) and cultured in low glucose DMEM supplemented with 1x antibiotic antimycotic solution and 10 % FBS. For plasmid construction, a 0.56-kb genomic fragment was amplified from human genomic DNA to obtain the FGFR1 promoter region (−555 to +145). One microliter of human genomic DNA was subjected to PCR using the following cycling conditions: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for 35 cycles. The sense and anti-sense primers used were as follows: FGFR1 Sense: 5′-TCT CCG GTC CGA GCT CGG GGC GCC C-3′, FGFR1 Anti-sense: 5′-ACG AGC GCA GGG AGG GGG CGC AGG A-3′, FGFR1 Mutant Sense: 5′-GGG CGC GTC GTC CCG AGC GG-3′, and FGFR1 Mutant Anti-sense: 5′-CCG CTC GGG ACG ACG CGC CC-3'. The amplified fragment was then subcloned into the PCR®2.1 T vector and ligated into the Xho I and Hin dIII sites of the pGL3 luciferase (LUC) basic expression vector (Promega). 

Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses 2 μg of total RNA was reverse transcribed using 0.5 μg of Oligo d(T) and one μL (50 IU) of Superscript III (Invitrogen) in a 20 μl reaction mixture at 50 °C for 1 h. The resulting cDNAs were amplified using PCR. Primers specific to mNfic, mRunx2, mOsx, mAlp, mBsp, mOc, mDmp1, mFgfr1, mFgfr2, hFGFR1, hFGFR2, hNFI-C , and hGAPDH were synthesized for real-time PCR (see ). 

Western blot analysis Western blot analyses were executed precisely, following established protocols . In brief, we separated 30 μg of total proteins using 10 % SDS-PAGE. Proteins were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell BioScience) and incubated with specific antibodies. Labeled proteins were then detected using an enhanced chemiluminescence system from GE Healthcare, ensuring the reliability of the results. 

ChIP assays ChIP assays were conducted as previously described . hBMSCs were transiently transfected with an NFI-C expression plasmid for 48 h. The samples were then sonicated, followed by chromatin immunoprecipitation using 30 μl of anti-NFIC antibodies and 10 μl of anti-IgG antibodies (Santa Cruz). The final DNA pellets were recovered and analyzed by polymerase chain reaction (PCR) using specific primers, as listed in . The PCR conditions were set as follows: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 35 cycles. The PCR products were analyzed using electrophoresis on a 2 % agarose gel. 

Statistical analysis All quantitative data are presented as means ± standard deviation (SD) using SPSS 26. The significance of differences was assessed with a Student's t-test (∗P < .05, ∗∗P < .01, ∗∗∗P < .001). 

Results 3.1 Evaluation of mice calvaria formation by Nfic gene targeting The calvaria of two-week-old (P14) and four-week-old (P28) mice, both WT and Nfic -deficient, were removed, and calvaria formation was analyzed using micro-CT and Alizarin Red S and Alcian Blue staining to evaluate the effect of the absence of Nfic on the calvaria formation. The micro-CT analysis of 2-week-old mice showed that the nasal bone of mice deficient in the Nfic gene was observed to be different from that of wild-type mice, and the density of the nasal bone was also reduced ( A). Comparing the calvaria of WT and Nfic -deficient mice using Alizarin Red S and Alcian Blue staining, the calvaria of both 2-week-old and 4-week-old mice of the Nfic -deficient mice showed a delay in the fusion of the interfrontal suture. In contrast, the calvaria of 4-week-old mice of the Nfic -deficient mice was abnormal, with deformation pointing toward the right compared to WT mice ( B). In micro-CT analyses, Nfic -deficient mice exhibited significant decreases in bone volume (BV/TV, 36 %) and bone mineral density (BMD, 14 %) but no change in bone thickness (Tb. Th) ( C). We also identified males and females by histology and found no sex differences regarding reduced trabecular bone density and increased trabecular bone separation. However, we used only male littermates for all the experiments to reduce variation. 3.2 Evaluation of bone formation in mice with Nfic gene targeting The calvaria of six-week-old mice, both WT and deficient in Nfic , were removed, and calvaria formation was compared using micro-CT analysis to confirm the effects of Nfic 's absence on skull formation. The micro-CT analysis of 6-week-old mice showed that the basisphenoid bone of the skull of Nfic -deficient mice was diagnosed with osteoporosis. In contrast, the sagittal suture was diagnosed with interrupted suture closure compared to WT mice ( A). Using 3D micro-CT analysis, Nfic -deficient mice had reduced calvaria bone volume (BV/TV, 33 %), bone thickness (Tb. Th, 16 %), and bone mineral density (BMD, 23.5 %) compared to WT mice at a statistically significant level ( B). Through histological analysis, it was observed that the calvaria of Nfic -deficient mice had a delay in the sagittal suture closure and a reduction in thickness compared to those of WT mice ( C). 3.3 Effects of Nfic gene absence on osteoblast differentiation Osteoprogenitor cells were isolated from WT and Nfic -deficient mice's calvaria and investigated for their ability to differentiate into osteoblasts. ALP staining on day 7 of differentiation and Alizarin Red S (ARS) on day 14 showed that Nfic -deficient osteoprogenitor cells had lower ALP activation and reduced calcified nodules. ALP activity and quantity of ARS staining in Nfic deficiency calvarial osteoprogenitor cells decreased approximately five-fold compared to WT cells ( A). These results show that the NFI-C plays an essential role in the differentiation process of osteoblasts. Real-time polymerase chain reaction (PCR) was used on day 7 of differentiation to examine the WT and Nfic -deficient osteoprogenitor cells for osteoblast-specific gene expression. The expression of transcription factor Runx2 , which regulates osteoblast differentiation, showed no difference in WT and Nfic -deficient osteoprogenitor cells, but Osx expression was reduced by about 5-fold in osteoprogenitor cells deficient in the Nfic gene ( B and C). The gene expressions of Alp , osteocalcin ( Oc ), Bsp , and Dmp1 , all osteoblasts marker genes, were also significantly reduced in osteoprogenitor cells deficient in the Nfic gene ( D–G). These results showed that the Nfic acting downstream of Runx2 regulated Osx expression, which also regulated osteoblast differentiation. 3.4 Effects of Nfic on osteoprogenitor cell proliferation and apoptosis Cell proliferation in WT mice and Nfic -deficient mice sutures was observed to investigate the cellular mechanism underlying defective suture closure in mice deficient in the Nfic gene. The cell proliferation marker gene PCNA from wild-type and Nfic -deficient mice was immunohistochemically stained, and these results showed that while cells stained with PCNA antibodies were observed in mice deficient in the gene, the cells stained with the PCNA antibodies in the sutures of Nfic -deficient mice were decreased about fivefold compared to WT mice ( A). Osteoprogenitor cells were isolated from the calvaria of WT and Nfic -deficient mice, and an MTT assay was performed to confirm cell proliferation in vitro. The results of the MTT assay revealed that Nfic deficiency significantly reduced cell proliferation of osteoprogenitor cells from day 3 of cell culture compared to WT ( B). Furthermore, in osteoprogenitor cells deficient in Nfic , the protein level of Cyclin D, which promotes the cell cycle process, was reduced. In contrast, the protein level of p21, which is a factor that suppresses the cell cycle process, increased ( C). Next, the protein levels of Caspase-8 and Caspase-3, which play a central role in cell apoptosis, were compared between the osteoprogenitor cells of WT mice and Nfic -deficient mice using Western blot to investigate the effects of Nfic absence on osteoprogenitor cells in their apoptosis. Generally, the level of caspase-8 in its cleaved form, known as the initiator caspase, was higher in osteoprogenitor cells deficient in Nfic than in WT ( D). Active caspase-8 cleaves procaspase-3 and promotes its activity, leading to apoptosis. Procasepase-3 and cleaved caspase-3 levels were higher in Nfic -deficient osteoprogenitor cells ( E). 3.5 Fibroblast growth factor receptor 1 ( Fgfr1 ) and Fgfr2 gene expressions in osteoprogenitor cells of Nfic gene-targeted mice Fgfr1 and Fgfr2 expressions in osteoprogenitor cells were observed to investigate the relevance of the Fgf gene family in abnormal calvaria formation of Nfic -deficient mice. Fgfr1 and Fgfr2 gene expressions in osteoprogenitor cells deficient in the Nfic gene were observed to be reduced by 2.5-fold and 2-fold, respectively, compared to WT ( A and B). Furthermore, the Fgfr1 protein expression was also reduced in osteoprogenitor cells deficient in the Nfic gene ( C). The ERK/MAPK cascade has been demonstrated to be an important intracellular mediator of FGF signaling . Therefore, we investigated the activity of ERK in calvarial osteoprogenitor cells from WT and nfic deficient mice. Activated ERK1/2 was significantly reduced in Nfic deficient calvarial osteoprogenitor cells compared with WT ( C). 3.6 Effects of the NFI-C gene on the expression of FGFR1 The NFI-C gene was overexpressed in human-derived mesenchymal stem cells, following which FGFR1 and FGFR2 mRNA expressions were observed using real-time PCR to verify the effects of NFI-C on regulating Fgfr1 expression. NFI-C gene expression was increased by around 550-fold ( A), while the FGFR1 and FGFR2 expressions increased by around 5 times and 3.5 times, respectively, due to NFI-C overexpression ( B and C). Western blot was used to assess the FGFR1 protein expression, and the results showed that the FGFR1 protein expression was also increased in mesenchymal stem cells with overexpressed NFI-C, while phosphorylation of ERK acting downstream of the FGF signaling pathway was also found to increase ( D). Next, the NFI binding motif of FGFR1's promoter region was investigated to determine whether NFI-C directly regulated FGFR1 expression. The NFI binding motif was identified within positions −198 and −50 of the FGFR1 promoter region sequence, and the human-derived mesenchymal stem cells were overexpressed with the NFI-C gene, followed by a ChIP assay for verification. From the ChIP assay, NFI-C protein binding was not observed in the FGFR1 promoter region that does not contain the NFI binding motif (P2), but NFI-C protein binding was observed in the FGFR1 promoter region with the NFI binding motif (P1) ( E). In addition, NFI-C binds to its specific DNA binding site in the FGFR1 promoter ( E). When the NFI-C binding motif in the FGFR1 promoter was mutated, NFI-C could not enhance the activity of the FGFR1 promoter ( F). These data indicate that NFI-C is positioned upstream in the NFIC-FGFR1 signaling pathway. 

Evaluation of mice calvaria formation by Nfic gene targeting The calvaria of two-week-old (P14) and four-week-old (P28) mice, both WT and Nfic -deficient, were removed, and calvaria formation was analyzed using micro-CT and Alizarin Red S and Alcian Blue staining to evaluate the effect of the absence of Nfic on the calvaria formation. The micro-CT analysis of 2-week-old mice showed that the nasal bone of mice deficient in the Nfic gene was observed to be different from that of wild-type mice, and the density of the nasal bone was also reduced ( A). Comparing the calvaria of WT and Nfic -deficient mice using Alizarin Red S and Alcian Blue staining, the calvaria of both 2-week-old and 4-week-old mice of the Nfic -deficient mice showed a delay in the fusion of the interfrontal suture. In contrast, the calvaria of 4-week-old mice of the Nfic -deficient mice was abnormal, with deformation pointing toward the right compared to WT mice ( B). In micro-CT analyses, Nfic -deficient mice exhibited significant decreases in bone volume (BV/TV, 36 %) and bone mineral density (BMD, 14 %) but no change in bone thickness (Tb. Th) ( C). We also identified males and females by histology and found no sex differences regarding reduced trabecular bone density and increased trabecular bone separation. However, we used only male littermates for all the experiments to reduce variation. 

Evaluation of bone formation in mice with Nfic gene targeting The calvaria of six-week-old mice, both WT and deficient in Nfic , were removed, and calvaria formation was compared using micro-CT analysis to confirm the effects of Nfic 's absence on skull formation. The micro-CT analysis of 6-week-old mice showed that the basisphenoid bone of the skull of Nfic -deficient mice was diagnosed with osteoporosis. In contrast, the sagittal suture was diagnosed with interrupted suture closure compared to WT mice ( A). Using 3D micro-CT analysis, Nfic -deficient mice had reduced calvaria bone volume (BV/TV, 33 %), bone thickness (Tb. Th, 16 %), and bone mineral density (BMD, 23.5 %) compared to WT mice at a statistically significant level ( B). Through histological analysis, it was observed that the calvaria of Nfic -deficient mice had a delay in the sagittal suture closure and a reduction in thickness compared to those of WT mice ( C). 

Effects of Nfic gene absence on osteoblast differentiation Osteoprogenitor cells were isolated from WT and Nfic -deficient mice's calvaria and investigated for their ability to differentiate into osteoblasts. ALP staining on day 7 of differentiation and Alizarin Red S (ARS) on day 14 showed that Nfic -deficient osteoprogenitor cells had lower ALP activation and reduced calcified nodules. ALP activity and quantity of ARS staining in Nfic deficiency calvarial osteoprogenitor cells decreased approximately five-fold compared to WT cells ( A). These results show that the NFI-C plays an essential role in the differentiation process of osteoblasts. Real-time polymerase chain reaction (PCR) was used on day 7 of differentiation to examine the WT and Nfic -deficient osteoprogenitor cells for osteoblast-specific gene expression. The expression of transcription factor Runx2 , which regulates osteoblast differentiation, showed no difference in WT and Nfic -deficient osteoprogenitor cells, but Osx expression was reduced by about 5-fold in osteoprogenitor cells deficient in the Nfic gene ( B and C). The gene expressions of Alp , osteocalcin ( Oc ), Bsp , and Dmp1 , all osteoblasts marker genes, were also significantly reduced in osteoprogenitor cells deficient in the Nfic gene ( D–G). These results showed that the Nfic acting downstream of Runx2 regulated Osx expression, which also regulated osteoblast differentiation. 

Effects of Nfic on osteoprogenitor cell proliferation and apoptosis Cell proliferation in WT mice and Nfic -deficient mice sutures was observed to investigate the cellular mechanism underlying defective suture closure in mice deficient in the Nfic gene. The cell proliferation marker gene PCNA from wild-type and Nfic -deficient mice was immunohistochemically stained, and these results showed that while cells stained with PCNA antibodies were observed in mice deficient in the gene, the cells stained with the PCNA antibodies in the sutures of Nfic -deficient mice were decreased about fivefold compared to WT mice ( A). Osteoprogenitor cells were isolated from the calvaria of WT and Nfic -deficient mice, and an MTT assay was performed to confirm cell proliferation in vitro. The results of the MTT assay revealed that Nfic deficiency significantly reduced cell proliferation of osteoprogenitor cells from day 3 of cell culture compared to WT ( B). Furthermore, in osteoprogenitor cells deficient in Nfic , the protein level of Cyclin D, which promotes the cell cycle process, was reduced. In contrast, the protein level of p21, which is a factor that suppresses the cell cycle process, increased ( C). Next, the protein levels of Caspase-8 and Caspase-3, which play a central role in cell apoptosis, were compared between the osteoprogenitor cells of WT mice and Nfic -deficient mice using Western blot to investigate the effects of Nfic absence on osteoprogenitor cells in their apoptosis. Generally, the level of caspase-8 in its cleaved form, known as the initiator caspase, was higher in osteoprogenitor cells deficient in Nfic than in WT ( D). Active caspase-8 cleaves procaspase-3 and promotes its activity, leading to apoptosis. Procasepase-3 and cleaved caspase-3 levels were higher in Nfic -deficient osteoprogenitor cells ( E). 

Fibroblast growth factor receptor 1 ( Fgfr1 ) and Fgfr2 gene expressions in osteoprogenitor cells of Nfic gene-targeted mice Fgfr1 and Fgfr2 expressions in osteoprogenitor cells were observed to investigate the relevance of the Fgf gene family in abnormal calvaria formation of Nfic -deficient mice. Fgfr1 and Fgfr2 gene expressions in osteoprogenitor cells deficient in the Nfic gene were observed to be reduced by 2.5-fold and 2-fold, respectively, compared to WT ( A and B). Furthermore, the Fgfr1 protein expression was also reduced in osteoprogenitor cells deficient in the Nfic gene ( C). The ERK/MAPK cascade has been demonstrated to be an important intracellular mediator of FGF signaling . Therefore, we investigated the activity of ERK in calvarial osteoprogenitor cells from WT and nfic deficient mice. Activated ERK1/2 was significantly reduced in Nfic deficient calvarial osteoprogenitor cells compared with WT ( C). 

Effects of the NFI-C gene on the expression of FGFR1 The NFI-C gene was overexpressed in human-derived mesenchymal stem cells, following which FGFR1 and FGFR2 mRNA expressions were observed using real-time PCR to verify the effects of NFI-C on regulating Fgfr1 expression. NFI-C gene expression was increased by around 550-fold ( A), while the FGFR1 and FGFR2 expressions increased by around 5 times and 3.5 times, respectively, due to NFI-C overexpression ( B and C). Western blot was used to assess the FGFR1 protein expression, and the results showed that the FGFR1 protein expression was also increased in mesenchymal stem cells with overexpressed NFI-C, while phosphorylation of ERK acting downstream of the FGF signaling pathway was also found to increase ( D). Next, the NFI binding motif of FGFR1's promoter region was investigated to determine whether NFI-C directly regulated FGFR1 expression. The NFI binding motif was identified within positions −198 and −50 of the FGFR1 promoter region sequence, and the human-derived mesenchymal stem cells were overexpressed with the NFI-C gene, followed by a ChIP assay for verification. From the ChIP assay, NFI-C protein binding was not observed in the FGFR1 promoter region that does not contain the NFI binding motif (P2), but NFI-C protein binding was observed in the FGFR1 promoter region with the NFI binding motif (P1) ( E). In addition, NFI-C binds to its specific DNA binding site in the FGFR1 promoter ( E). When the NFI-C binding motif in the FGFR1 promoter was mutated, NFI-C could not enhance the activity of the FGFR1 promoter ( F). These data indicate that NFI-C is positioned upstream in the NFIC-FGFR1 signaling pathway. 

Discussion This study investigated the effects of the transcription factor Nfic on calvarial osteogenic progenitor cell proliferation and differentiation from the standpoint of the Runx2 - Nfic - Osx and Runx2 - Nfic - Fgf signaling pathways. Runx2 is part of the Runx family and the essential transcription factor in skeletal development. Runx2 is necessary for the proliferation of osteoprogenitor cells and osteoblast differentiation, and bone formation cannot proceed without Runx2. Furthermore, Runx2 induces Osx expression in preosteoblasts, leading to these cells' differentiation into osteoblasts . According to a study by Lee et al. , if NFI-C acting downstream from Runx2 is disrupted in the bone marrow, osteogenesis in the bone marrow is decreased, and adipogenesis increases and this is considered a result of the inability of Osx to act downstream of Nfic to be expressed resulting in relatively higher adipocytes within the bone marrow. In this study, the Nfic disruption did not affect Runx2 expression, but Osx expression was suppressed, and osteoblast differentiation was disrupted, leading to reduced calvarial bone length and density abnormalities. The study results revealed that Nfic regulates bone formation via the Runx2-Nfic-Osx signaling pathway, which acts on the intermediary stage of Runx2 and Osx in calvaria in the same manner as in the bone marrow. These results also suggest that if Nfic is targeted in calvaria, the upstream factor Runx2 is unaffected. In contrast, through regulation of the downstream factor Osx , the later stages of osteoblast differentiation could be selectively regulated, and Nfic could thus be effectively utilized in the prevention and treatment of calvaria formation diseases. Since the Fgf gene family plays an essential role in the proliferation and differentiation of osteoblasts in the development of bone, numerous studies have reported that the defective Fgf gene expression in calvaria leads to deformities in calvarial bone development and formation [ , , ]. The premature fusion of the cranial suture, an essential factor in defective cranial bone development, is modulated by basic Fgf and Fgfr . Fgfr1 and Fgfr2 are expressed in osteoprogenitor cells and regulate cranial osteoblast phenotype, osteoblast function, osteoblast, and osteocyte death and growth . More recently, it was revealed that Runx2 regulated Fgfr2 and Fgfr3 expressions and modulated osteoprogenitor cell proliferation [ , , ]. Moreover, FGF signaling plays an essential role during tooth development. We previously demonstrated that Nfic regulates FGF10 expression in the dental mesenchyme in mouse incisors. In addition, Nfic is activated through FGF8 signaling in dental epithelial tissues to regulate stem cell capacity and cell proliferation of these tissues . Here, we showed that Nfic regulates the proliferation of calvarial osteoprogenitor cells through modulation of FGFR1 expression. In addition, we identified that the expression of FGF1 and FGF2 was reduced in calvarial osteoprogenitor cells of Nfic -deficient mice (data not shown). Interestingly, the ChIP assay revealed that FGFR1 functioned by binding to NFI-C. Additionally, when Nfic is disrupted, Fgfr1 and Fgfr2 expressions are suppressed, while conversely, NFI-C overexpression in hBMSCs results in increased FGFR1 and FGFR2 expressions. In addition, when evaluating the effect of NFI-C on FGFR1 promoter activity, NFI-C increased FGFR1 promoter activity, but when the NFI binding motif was mutated in the FGFR1 prompter, it did not increase FGFR1 promoter activity. These results revealed that the NFI-C regulated FGFR1 and FGFR2 expressions via NFIC-FGF signaling pathway irrespective of Osx , resulting in calvarial osteoprogenitor cell proliferation modulation. Nonetheless, further research is needed to investigate the direct relevance between NFI-C and FGFR2 and whether NFI-C binds to the FGFR2 gene and regulates transcription and translation. Xingwen Wang et al. recently reported that miR-34a regulates osteoblast proliferation and apoptosis through FGFR1 . These results indicate the importance of FGFR1 in osteoblast proliferation and apoptosis. In addition, several studies have shown that the ERK MAPK pathway, a downstream factor of FGFR, increases osteoblast differentiation and bone formation in vitro and in vivo [ , , ]. Furthermore, mice lacking Erk1 and Erk2 in the limb and head mesenchyme (Erk1 −/− Erk2 Prx1) exhibited severe limb malformations and impaired skeletal mineralization . Therefore, these reports support our findings that the absence of Nfic causes defects in the FGFR1-Erk signaling pathway, leading to decreased cell proliferation and increased apoptosis in calvarial osteoprogenitor cells . p21 suppresses the activity of cyclin E/CDK2, cyclin D1/CDK4/6, cyclin A/CDK2, and so on, allowing cell growth to remain at the G1/S checkpoint, resulting in suppression of cell proliferation through inhibition of the cell cycle . This study showed that Nfic disruption increased the expression of p21 in calvarial osteoprogenitor cells. In turn, this reduced the expression of Cyclin D1, disrupting cell proliferation and relatively increased expression of cleaved-Caspase8 and cleaved-Caspase3 overall, leading to cell apoptosis. This suggests that, as opposed to Nfic disruption, calvarial osteoprogenitor cell proliferation could be induced by Nfic overexpression, thus regulating osteoblast differentiation and calvarial bone formation. In conclusion, this study revealed that the transcription factor Nfic regulates the cell proliferation of calvarial osteogenic progenitor cells via the Nfic - Fgf signaling pathway and the differentiation of calvarial osteogenic progenitor cells via the Nfic-Osx signaling pathway. The study results point toward further research on utilizing Nfic to prevent calvaria diseases such as craniosynostosis due to defective cell proliferation and differentiation of osteogenic progenitor cells. 

Jieun Lee: Writing – original draft, Investigation, Conceptualization. Joo-Cheol Park: Writing – review & editing, Data curation, Conceptualization. Heung‐Joong Kim: Data curation, Conceptualization. Hyun Sook Bae: Writing – review & editing, Data curation, Conceptualization. Dong-Seol Lee: Writing – review & editing, Data curation, Conceptualization. 

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Seoul National University with the approval number [SNU-181127-13-2] 

All data generated and analyzed during this study are included in this article. Requests for this information can be made to the corresponding author. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

