Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

Liao, Junguang; Huang, Yuping; Sun, Fuju; Zheng, Chenggong; Yao, Yifeng; Zhang, Cui; Zhou, Chenhe; Zhang, Xingen; Wu, Mengrui; Chen, Guiqian

doi:10.1038/s41467-025-57808-4

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Published: 12 March 2025

Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration

Nature Communications volume 16, Article number: 2478 (2025) Cite this article

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Abstract

Skeletal mesenchymal stem cells (MSCs) possess self-renewal capacities and play a leading role in the craniofacial system. However, their engagement in controlling cranial bone development and regeneration remains largely unidentified. Herein, we discovered the neurofibromin 2 (Nf2)-encoded regulator Merlin, demonstrating indispensableness in the craniofacial system. Mice lacking Nf2 in MSCs exhibit malformed cranial bones, diminished proliferation, increased apoptosis, and more severe osteogenesis impairment. Mechanically, we substantiate that Nf2 physically interacts with focal adhesion kinase (FAK) to preferentially mediate Erk1/2 and PI3K catalytic p110 subunit/Akt signaling. Meanwhile, Nf2-FAK disturbance in MSCs results in deficient migration, cytoskeletal organization and focal adhesion dynamics, and develops retarded regeneration of cranial bone defects. Collectively, our findings underscore an unrecognized scaffolding role for Nf2-FAK as upstream element in regulating PI3K/Akt and Erk1/2 action in osteoblasts, and illuminate its essentialness in coordinating cell migration, osteogenic lineage development, cranial bone ossification and regeneration.

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Introduction

The skull is a requisite bone that plays a central role in regulating stable brain development and growth. The morphogenesis of cranial bones is exquisitely organized during the embryonic and postnatal stage. The skeletal mesenchymal stem cells (MSCs) are vital for the development, construction, and remodeling of cranial bones. The commitment and recruitment of MSCs to differentiate into osteoblast lineage is critical for the calvaria formation^1,2,3,4,5,6. The disturbance in this process gives rise to serious birth defects or skeletal deformities^7,8. Moreover, cranial MSCs, harboring in the postnatal sutures, are competent to be invigorated and emigrated towards injury sites to restore bones defects^9,10,11. Manipulating MSCs was found sufficiently repairing craniofacial birth defects¹². Therefore, the concerted migration and differentiation of MSCs exerts a significant influence for osteoblast lineage development, cranial bone formation and the repair of skull defects.

Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase and has been documented essentially to guide mesenchymal stem cells differentiate to osteoblasts^13,14,15. FAK governs osteoblast proliferation, differentiation¹⁶, osteoblast-adipose commitment shift¹⁷, collagen synthesis¹⁸ and bone regeneration¹⁹. FAK and its mediated signaling were indicated through MAPK²⁰, PI3K/Akt^21,22 or mTOR¹⁶ in osteoblasts. Moreover, FAK plays essential role in cell migration, and FAK triggers focal adhesion positioning²³, establishes sub-areas within focal adhesions²⁴ and modulates cell protruding²⁵. Focal adhesion (FA) dynamic is a continued process connecting the adjustment between FA and actin cytoskeleton^26,27. FAK accumulates at the emerging FAs earlier at cell front²⁷. However, it is still largely unknown how FAK activity is determined to direct MSC migration and osteoblast differentiation.

Neurofibromin 2 (Nf2) encodes a protein called Merlin and firstly named in patients with Neurofibromatosis Type 2 disease^28,29,30. Merlin is a 70-kDa member of the Ezrin-Radixin-Moesin branch of the band 4.1 protein superfamily. Merlin consists of an N-terminal FERM domain, a central α-helical region, and a C-terminal domain (CTD). The intermolecular FERM-CTD interaction change allows Merlin to transit into an open (proliferation-permissiveness) or a closed (proliferation-inhibition) transformation. Nf2/Merlin was widely known as a tumor suppressor, but mechanism varied in various tumor cells^31,32,33. Remarkably, Nf2 extensively expressed at E6.5 and in migrating neural crest cells at E8.5³⁴, which was a critical cell lineage for skull development^3,4. Nf2 was found expressed in frontal and parietal bone³⁵. Nf2 mutant mice failed to launch the gastrulation³⁶. Nf2 in hematopoietic stem cells maintains the homeostasis of hematopoietic stem cell niche and confine bone and vascular components³⁷. However, how Nf2 performs in bone cells is still principally undetermined.

To characterize molecular function of Nf2/merlin in cranial bones, we generate a skeletal tissue-specific knockout mice model of Nf2 using Prx1-Cre, a generally used transgenic mouse line to label skeletal mesenchymal stem cell^38,39, and inducible Prx1-CreER, which exclusively mark suture stem cells that important for bone regeneration of cranial defects^40,41. Our data demonstrate that Nf2 acts as an upstream regulator to mediate FAK activity and Nf2-FAK reciprocity is indispensable in regulating MSC migration, osteoblast lineage differentiation, cranial bone formation and regeneration.

Results

Nf2 knockout in skeletal mesenchymal cells impaired the morphogenesis of cranial bones

In our study, Nf2 gene product, Merlin, was found expressed in brain, liver, spinal cord, skull and limb tissues at 18.5 embryonic stage (Fig. 1a, upper panel). In skull tissues, Merlin also maintained higher expressions at early postnatal cranial tissues (Fig. 1a, lower panel). To characterize the function of Nf2 in bone tissues, Nf2 floxed allele (f/f) mice from Inserm⁴² was introduced and its conditional knockout was generated using Prx1-Cre, which essentially mark skeletal mesenchymal cells^38,43, including frontal and parietal bone but not dura mater⁴⁴. Nf2 knockout mice (Nf2-cKO) mice presented severe skeletal deficiencies, such as smaller body size and irregular cranial bones (Fig. 1b–d). This phenotype was uniform to skeletal deformities observed using inducible Prx1-CreER mediated Nf2 deletion (Supplemental Fig. S1a–c), and Nf2 mutant mice died post-delivery, probably from asphyxia due to non-fusion sternum during the development (Fig. S1d, red arrow).

**Fig. 1: *Nf2* knockout in skeletal MSC impaired cranial bones development.**

At the craniofacial level, Nf2-cKO mice displayed deformed appearance, the angles from the body axis to the head were significantly enlarged compared to littermate controls (E15.5 p < 0.0001; P1 p < 0.0001, n = 3) (Fig. 1b, Fig. S1e). Using skeletal staining (E16.5-P1) and micro-CT (P1, Fig. S1f) to visualize the skeletal integrity, we found that Nf2-cKO mice exhibited unossified empty frontal and parietal bone, and partially the intraparietal bones (Fig. 1c, d, red arrow). At coronal level, the width distance between two parietal bones (Fig. 1c, red arrow) was significantly expanded in Nf2-cKO compared to the litter controls (Fig. S1g, E16.5 p < 0.0001; E18.5 p < 0.0001; P1 p < 0.0001, n = 3). The distances between nasal bone and coronal suture (Fig. 1c, black arrow) were a significantly elongated in Nf2-cKO compared to the controls (Fig. S1h, E16.5 p < 0.0001; E18.5 p < 0.0001; P1 p = 0.011, n = 3) (Fig. 1g), suggesting a malformed skull size in Nf2 mutant mice.

Due to the severe affected frontal bone and parietal bone in Nf2-cKO mice, we scanned E18.5 Nf2-cKO and littermate control mice using micro-CT and reconstructed the 3D images⁴⁵ (Fig. 1d–e, n = 8). We found that the volume of defective frontal and parietal bone (Fig. 1f1, p = 0.0005; Fig. 1g1, p = 0.0072), the ratio of the defective volume relative to its respective bone volume (Fig. 1f2, p < 0.0001; Fig. 1g2, p = 0.0384) were both significantly diminished in Nf2-cKO mice. The ratio of surface defect area relative to the whole frontal bone (Fig. 1f3, p < 0.0001) and parietal bone (Fig. 1g3, p < 0.0001) were significantly increased in Nf2 mutant mice. Next, we isolated frontal bone (Fig. 1h, n = 8) and parietal bone (Fig. 1j, n = 8) from E18.5 control and Nf2-cKO mice and located the anatomical landmarks (Fig. 1h1, j1, triangle indicated) and compared the size especially. In frontal bones, Nf2 mutant mice displayed significant reduction on lateral bone size between points 2–3 (Fig. 1i1, p = 0.00054) and points 1–3 (Fig. 1i1, p = 0.0025) but not significant difference in points 1-2 (Fig. 1i1, p = 0.467), significant increase on defect width between points 1–1 (Fig. 1i2, p < 0.0001), 2–2 (Fig. 1i2, p = 0.001) and 3–3 (Fig. 1i2, p = 0.0002), and the ratios of width defect ratio relative to points 3-3 (1-1/3-3, 2-2/3-3) were significantly increased (Fig. 1i3, p < 0.0001). In parietal bones, we found that length distances between points 1–2 (Fig. 1k1, p < 0.0001), 3–4 (Fig. 1k1, p < 0.0001), 1–3 (Fig. 1k1, p = 0.012), and 2–4 (Fig. 1k1, p = 0.001) were significantly reduced, the width defect distances between points 1-1 (Fig. 1k2, p = 0.007), 2–2 (Fig. 1k2, p = 0.0005), 3-3 (Fig. 1k2, p = 0.001) and 4–4 (Fig. 1k2, p < 0.0001) were significantly disorganized, the ratios on 1-1/2-2 (Fig. 1k3, p = 0.0012) and 3-3/4-4 (Fig. 1k3, p < 0.0001) were significantly increased in Nf2 mutant mice, thus presenting severely affected bone volume, surface area, size and shape in frontal and parietal bones in Nf2 mutant mice. Collectively, these data have demonstrated that Nf2 in skeletal mesenchymal cells plays a leading role in shaping the morphogenesis of cranial bones during the development.

Nf2 deficiency in MSCs resulted in a disrupted osteogenic lineage development

Cranial primordium is initially structured to advance cranial bones formation. Firstly, we showed that Nf2 expression in Runx2⁺ labeling osteoprogenitor was effectively reduced at the section of cranial primordium of Nf2 mutants at E12.5 (Fig S2a). Next, coronal cranial primordium sections at E12.5 were co-stained with Runx2 and EdU (Fig. 2a) to evaluate the cell proliferation. In Nf2-cKO mutants, the number of Runx2⁺ labeling osteoprogenitors was significantly reduced compared to the control (Fig. 2b, p = 0.026, n = 3), and EdU⁺/Runx2⁺ ratio was declined with a mild significance in Nf2-cKO mutants compared to the control (Fig. 2c, p = 0.034, n = 3), and EdU⁺/Runx2^- ratio was not altered in Nf2-cKO mutants compared to the control (Fig. 2d, p = 0.616, n = 3), suggesting that Nf2 deletion in skeletal MSCs leads to attenuated proliferation in EdU⁺ osteoprogenitors. Besides, TUNEL⁺ immunosignals in Nf2-cKO were found increased in Runx2⁺ osteoprogenitors (Fig S2b, arrow, p < 0.0001, n = 6), suggesting that apoptosis was increased in Runx2⁺ osteoprogenitors. Alkaline Phosphatase (ALP) staining was used to evaluate the activities of pre-osteoblasts and osteoblasts at histological level, we found that Nf2 mutant mice exhibited remarkably reduced activities of osteoprogenitors at E12.5, and the extents and distributions of osteoprogenitors, pre-osteoblasts and mature osteoblasts were dramatically deficient from E12.5 to E17.5 (Fig. 2e, f, arrow). von Kossa staining was used to evaluate ossification process, and we found that Nf2 mutant calvaria was minimally ossified for most cranial regions at E18.5 sagittal section of cranial bone (Fig. 2g, arrow). Consequently, the immunosignals of osteoblasts-special markers osteopontin (OPN)⁺ (Fig. 2h, arrow), Runx2⁺(Fig. 2i, arrow), and Osterix/Sp7⁺ (Fig. 2j, arrow) were hardly detected at E18.5 sagittal section of cranial bone in Nf2 mutant mice, suggesting that osteogenic differentiation was more severely impaired in Nf2 mutants.

**Fig. 2: *Nf2* deficiency in skeletal MSC disrupted osteoblast lineage development.**

Using calvaria primary osteoblast culture system, the same number of osteoblasts were seeded and induced for osteoblast differentiation at D7 and D14. ALP (D7) (Fig. 2k) and Alizarin S Red staining (D14) (Fig. 2l) showed that osteoblast differentiation and ossification was seriously reduced in Nf2 mutants. Nf2 mutant osteoblasts were found easy to detach from the plate. In primary pre-osteoblasts, the level of PCNA (proliferating cell nuclear antigen) in Nf2 mutants was significantly reduced (Fig. 2m, S3a, p = 0.0166, n = 3), and the levels of cyclin D1 and PCNA were significantly declined (Fig. 2o, p < 0.0001, n = 3). The osteoblast marker genes (ALP, Runx2, Sp7, Osteocalcin (OCN), Type I collagen (Col1), Fibronectin (FN), Bone sialoprotein (BSP)) were significantly decreased at D7 (Fig S3b, n = 3) and D14 (Fig. 2p, n = 3). The expressions of osteoblastic transcriptional factors (Runx2, Osterix/Sp7) and the secretion of type I collagen by mature osteoblasts (Fig. 2n, n = 3) were substantially diminished (Fig S3a, n = 3). Besides, Dlx5 was an essential transcription factor for the differentiation of calvarial osteoblasts. Dlx5 mutant mice was shown delayed skull ossification⁴⁶. Msx1/2 double knockout mice displayed rigorous defects in skull formation⁴⁷, and the quantity and proliferation of skeletogenic mesenchyme were decreased in Msx2-Twist double mutants⁴⁸. We found that Dlx5, Msx2, Twist1/2 were dramatically declined in Nf2 mutant osteoblasts at D7 (Fig S3b, n = 3) and D14 stage (Fig. 2p, n = 3). These data unveiled that Nf2 in mesenchymal stem cell in vivo is indispensable to orchestrate a standard osteogenic lineage development.

Nf2 was required for cell migration, adhesion, and cytoskeletal organization

Due to the defects that massive unossified cranial tissues and loosen primary osteoblasts in Nf2 mutants, we wondered whether it was due to the flaws in pre-osteoblasts migration and adhesion. We performed a scratch-wound assay to evaluate cell migrations and found decreased migration but with no notable difference in osteoblast migration in Nf2 mutants compared to the control (Fig. S4a, b, dotted red line). Then, we tested if cranial MSCs migration was deficient, to lessen the consequence of proliferation in Nf2 mutants, the collected primary suture MSCs^49,50 were firstly used to be transfected with lentivirus mediated knockdown of Nf2 in culture medium with no FBS supplement, and the healing distances at different time interval after the scratch-wound were calculated and analyzed, and we found that the healing distance was significantly reduced in Nf2 knockdown MSC compared to that in the controls at 12 h (p = 0.032, n = 3) and 24 h (p = 0.017, n = 3) (Fig. S4c, d). Meanwhile, the same migration assay was performed using Nf2 control and Nf2-cKO MSC (Fig. 3a, dotted line), and we observed that the healing distance was significantly declined in Nf2-cKO MSC compared to that in the controls at 12 h (p = 0.016, n = 8), 24 h (p < 0.001, n = 8), 36 h (p < 0.001, n = 8) and 48 h (p < 0.001, n = 8) (Fig. 3a, b).

**Fig. 3: *Nf2* deletion resulted in defective cell migration, adhesion, and attachment.**

Next, cell adhesion and detachment in osteoblasts were evaluated. After plating the same number of primary osteoblasts for different times, the non-attached cells were washed off with caution, the remnant osteoblasts were stained with crystal violet solution and counted. We found that Nf2 mutant osteoblasts showed significantly lower capacity for the cell adhesion at post-plating 30 min (Fig. 3c, p < 0.001, n = 3). Similarly, the detachment assay was performed, and 80–90% confluent primary osteoblasts were incubated using 0.2% EDTA for 10 min, and the remnant adherent osteoblasts were numbered. We found that the number of adherent osteoblasts after 0.2% EDTA treatment in Nf2 mutants was notably diminished compared to the control (Fig. 3d, p = 0.0045, n = 3), suggesting that adherent Nf2 mutant osteoblasts were easier to be detached compared to the controls. Cytoskeletal arrangement was requisite for cell migration, F-actin and β-Tubulin were primary cytoskeleton proteins, and the immunosignals showed a chaotic cytoskeletal assembly in Nf2 mutant osteoblasts (Fig. 3e). GTPase activity was one influential adjustor in cytoskeleton, and we found that the activity of RhoA enzyme was specially decreased in Nf2 mutant MSCs using rhotekin-RBD beads pulling down the RhoA in lysate (Fig. 3f, p = 0.0010, n = 3). These data illuminated that Nf2 is principally required for normal cell migration, adhesion and cytoskeleton organization in MSCs and osteoblasts.

Nf2 deficiency led to defective focal adhesion formation

To explore the potential cause of defective migration and adhesion in Nf2 mutants, we tested how focal adhesion was altered. Focal adhesion (FA) dynamics were a constant manner engaging in adjusting between FA and actin cytoskeleton^26,27. The spatial-temporal localization of Paxillin played essential roles in FA formation and is necessary for FA turnover during cell migration^51,52. Paxillin mutations affected focal adhesions⁵³. We found that phospho-Paxillin-labeled focal adhesion was extensively distributed in protrusion of cell fronts in control after plating cells at 3 h, and the phospho-Paxillin-labeled focal adhesion was attached to numerous actin stress fibers in FA (Fig. 4a, b), but Nf2 mutant MSC showed markedly reduced formation of focal adhesion (Fig. 4b, arrow) and actin stress fibers in the mutant cells (Fig. 4a, b). The area of FA size (Fig. 4b, arrow) was significantly declined in Nf2 mutants (Fig. 4c, n = 12, p = 0.0387). Due to the difficulty to transfect the Paxillin-GFP plasmid into MSC, we then transfected Paxillin-GFP plasmid directly into primary osteoblasts. After 24 h, the primary osteoblasts were digested and plated in fibronectin-precoated plates for live cell imaging to estimate the proceeding of cell adhesion. We found that the distributions of Paxillin-GFP labeled FA were properly grown and diffused at the fronts in primary osteoblasts of the control (Fig. 4d, d’ arrow). However, in Nf2 mutant osteoblasts, we found that Paxillin-GFP labeled FAs were mostly situated in cytoplasm and less at the fronts (Fig. 4d, d” arrow). The ratio between the cells with properly formed Paxillin-GFP-labeled FA at the fronts and total cells showed a notable decrease in Nf2 mutants compared to the controls (p < 0.001, n = 3) (Fig. 4e), suggesting that the focal adhesion formation at the fronts was considerably inactive in Nf2 mutant osteoblasts, which may be the cause for the phenotype that Nf2 mutant osteoblasts disclosed deficient adhesion and delayed migration. These data presented that Nf2 is essentially important for the formation of focal adhesion dynamics in part through the regulation of Paxillin.

**Fig. 4: *Nf2* disturbance led to deformed focal adhesion dynamics.**

Nf2 acted as an upstream regulator to mediate FAK activity with a physical interaction

Mechanically, we found that the level of phospho-397 FAK, a momentous site for cellular FAK action, was declined in Nf2 mutants (Fig. 5a), implying that FAK may function as a downstream factor of Nf2. At sections of cranial primordium at E12.5, we demonstrated that the immunosignals of p-FAK (397) were predominantly reduced in Runx2⁺ osteoprogenitor in Nf2 mutant mice (Fig. 5h, arrow). At late stage of cranial bone development at E18.5, we confirmed that immunosignals of p-FAK (397) were substantially diminished in Nf2 mutants (Fig. 5i, arrow) on sagittal sections of the skull. While the overexpression of Nf2 or FAK in Nf2 mutant osteoblasts was effectively rescued deficient phenotype of Nf2 mutant osteoblasts for 5 days using ALP staining (Fig. 5b). Additionally, overexpression of Nf2 in Nf2 knockout osteoblasts can sufficiently rescue the levels of phospho-397FAK, collagen type 1, Runx2 and SP7 in Nf2 mutant osteoblasts (Fig. 5c), thus presenting that Nf2 works as an upstream regulator to modify FAK activity in osteoblasts.

**Fig. 5: *Nf2*-FAK interaction alteration impaired osteoblast differentiation.**

In osteoblasts, how FAK is regulated is unidentified. To evaluate the interactive role between Nf2 and FAK in osteoblasts, we generated the transient plasmids for FAK and Nf2 with a tag protein FLAG and HA using the HEK293T cell. In vitro co-immunoprecipitation assay (co-IP) showed that FLAG-FAK and HA-Nf2 formed a complex (Fig. 5d), and single transfection of either FLAG-FAK or HA-Nf2 as a control (Fig S5). In primary cultured osteoblasts, we certified that FAK interacts with Nf2 protein (Fig. 5e). Thereafter, we applied the NanoBiT protein, a protein interaction system, which has been demonstrated to be an effective tool in discerning protein interaction in live cells based on structural complementation^54,55. We made all the plasmids for FAK and Nf2 fuse proteins blended with large BiT and small BiT domains based on individual amino acid directions (N2015, Promega) (Fig. 5f). Then the plasmids containing with large or small BiT domains were transfected into osteoblast cell line MC3T3-E1 (3T3) to examine the bioluminescence that produced based on structural complementation. Our results showed that C-terminal-Nf2 domain in small subunit structurally and physically interacts with N-terminal-FAK domain in large subunit, as a result, a physical Nf2-FAK reciprocity can develop the bioluminescence. The bioluminescence ratio between Nf2-FAK interaction and negative control was about 37.6-fold, which was noticeably greater than the definition of active protein: protein interaction threshold (about 10 folds) (p = 0.0001, n = 3) (Fig. 5g). Our data demonstrated that Nf2-FAK interaction is physical in structural domain in osteoblasts.

Nf2-FAK interaction preferably mediated PI3K/Akt and Erk1/2 signaling in osteoblasts

FAK mediated signaling is an established intracellular signaling pathway that organizes cell behavior¹⁵. FAK promoted phospholipase C-gamma activity⁵⁶, FAK also functioned as the upstream of PI3K/Akt in fibroblasts⁵⁷. In skeletal cells, FAK was shown mediated intracellular signaling through either MAPK²⁰, PI3K/Akt^21,22 or mTOR¹⁶. How Nf2-FAK mediates downstream signaling in osteoblasts is unclear. Our data demonstrated that the activity of phospho-PLC-γ decreased in Nf2 mutant osteoblasts (Fig. 6a). Akt was regarded as the principal output of PI3K signaling⁵⁷, our data confirmed that the activity of phospho-Akt significantly reduced (Fig S3) in Nf2 mutant osteoblasts, suggesting that PI3K/Akt signaling is altered in Nf2 mutant osteoblasts (Fig. 6a). Additionally, MAPK signaling pathways, including Erk1/2⁵⁸, p38 MAPK⁸, and JNK pathways⁵⁹, are important signaling for the osteoblast differentiation. Our data exhibited that the activities of phoshpo-p38 and phospho-JNK were not altered in Nf2 mutants, but the level of phospho-Erk1/2 was significantly declined (Fig. S3a) (Fig. 6b), suggesting that Erk1/2 signaling was particularly disturbed in Nf2 knockout osteoblasts. At the section of skull tissue at E18.5, the immunosignals of phospho-AKT (Fig. 6f, arrow) and phospho-Erk1/2 (Fig. 6g, arrow) in Nf2 mutant mice were significantly diminished. At the section of cranial primordium at E12.5, we also authenticated that the immunosignals of phospho-Akt (Fig. 6h, arrow) and phospho-Erk1/2 (Fig. 6i, arrow) were significantly declined in Runx2⁺ osteoprogenitors in Nf2 mutant mice compared to the controls. These data exhibited that Nf2-FAK interaction preferentially mediated PI3K/Akt and Erk1/2 signaling in osteoblasts at cellular and histological level.

**Fig. 6: Nf2-FAK interaction mediated PI3K/AKT and ERK1/2 signaling in osteoblasts.**

PI3K is a heterodimer including a p85 regulatory subunit and a p110 catalytic subunit⁶⁰, and plays an essential function in osteoblasts differentiation^61,62. In fibroblasts, FAK was reported to bind PI3K-p85 regulatory subunit to regulate cell behavior⁶³. In osteoblasts, how FAK and PI3K/Akt is associated remains unknown. We performed co-IP assay to assess their association in primary osteoblasts, our data revealed that FAK interacts with the catalytic subunit p110 of PI3K complex, and Nf2 mutant osteoblasts attenuated the connection between FAK and subunit p110/PI3K (Fig. 6c–e), suggesting a direct cellular interplay of Nf2/FAK/p110-PI3K/Akt in osteoblasts. These results uncover a new frame for Nf2-FAK as upstream component in regulating PI3K action in osteoblasts.

FAK signaling activation sufficiently restored aberrant differentiation in Nf2 mutant osteoblasts

To investigate the efficacy of FAK and its downstream AKT and ERK1/2 actions in osteoblasts, pharmaceutical FAK inhibitor (PF573228)⁶⁴ was used to inhibit the activity of FAK in primary osteoblasts for 12 h at D3, we found that FAK inhibition can induce decreased activities of phospho-FAK (397) at low concentration (Fig. 7a) starting from 5 μm of PF573228. The inhibitory FAK action resulted in markedly decreased activities of phospho-Erk1/2, phospho-Akt, and reduced activities of Runx2 and Sp7 in primary osteoblasts (Fig. 7a), and the concentration of FAK inhibitor displayed dose-dependent impediment to both osteoblast differentiation and activities of phospho-Erk1/2 and phospho-Akt signaling.

**Fig. 7: Activation of FAK mediated ERK and AKT signaling rescued the deficiency in *Nf2* mutant osteoblasts.**

Next, we applied Adhesamine, a dumbbell-shaped molecule to trigger FAK actions⁶⁵ to treat primary osteoblasts for 24 h at the concentration of 20 μm, and we found that the level of phospho-FAK (397) was significantly enhanced in Nf2 mutant osteoblasts compared to the Nf2 mutant control and negative control (Fig. 7b). Besides, the activities of phospho-Erk1/2 and phospho-Akt and osteogenic transcriptional factor Runx2 were significantly elevated in Nf2 mutant primary osteoblasts compared to the Nf2 mutant control and negative control (Fig. 7b), suggesting that pharmaceutical activation of FAK on phospho-FAK(397) can sufficiently rescue the activities of downstream signaling phospho-Erk1/2 and phospho-Akt in Nf2 mutant osteoblasts, and the flawed differentiation of osteoblasts in Nf2 mutants can be sufficiently recovered (Fig. 7b). Furthermore, SC79 (cat # HY-18749, MCE) was a specific AKT catalyst in the cytoplasma⁶⁶. SC79 was used to treat Nf2 mutant osteoblasts at 20 μm. Pamoic acid disodium could (cat # HY-W010907, MCE) stimulate ERK1/2 activation⁶⁷, which was utilized to treat Nf2 mutant osteoblasts at 50 μm. Alizarin S Red staining was used to estimate mineralization matrix in osteoblasts. We found that FAK activator can sufficiently rescue the defective calcium deposition in Nf2 mutant osteoblasts compared to the controls for 14 days (Fig. 7c). Treating Nf2 mutant osteoblasts for 14 days using AKT or Erk1/2 activator can sufficiently rescue the inferior ossification in Nf2 mutant osteoblasts (Fig. 7c), suggesting that activation of PI3K/Akt or Erk1/2 signaling is sufficient to restore inferior ossification in Nf2 mutant osteoblasts.

Nf2 deficiency in postnatal suture stem cells retarded bone regeneration of cranial defects

In order to further investigate the function of Nf2 in mesenchymal stem cell in repairing the cranial bone defects, we generated the cranial bone defect models to drill an injury area about 2 mm² in the frontal and parietal bone^11,68 using inducible Prx1-CreER⁴⁰, which has been generally used to label suture stem cells. Prx1⁺ suture stem cells reside particularly in the sutures and are accountable for osteogenic potential and bone regeneration^4,40,41. After the injection of tamoxifen in adult mice (2 m) to induce knockout of Nf2 in suture stem cell for five days, the cranial defects on frontal and parietal bones were generated, different stages post cranial bone defects (D0, D7, D14, D21, n = 3) were collected to visualize the healing efficacy of the damaged bones in Nf2 mutants using micro-CT imaging (Fig. 8a, b). At post-injury D7, Nf2 mutants exhibited 84.6% and 88.36% nonhealing region in frontal and parietal bones compared to the controls (frontal bone p < 0.001; parietal bone p = 0.0153). At post-injury D14, Nf2 mutants displayed 67.2% and 70.5% nonhealing region in frontal and parietal bones, and Nf2 controls exhibited 18.2% and 40.1% of nonhealing region in frontal and parietal bones (frontal bone p < 0.001; parietal p < 0.001). At post-injury D21, Nf2 controls presented almost complete restoration of cranial bones defects, Nf2 mutants displayed 17.1% and 29.5% of nonhealing area in frontal and parietal bones (frontal bone p < 0.001; parietal bone p < 0.001) (Fig. 8c), suggesting that Nf2 deletion in suture stem cell in vivo retarded bone regeneration of cranial defects.

**Fig. 8: Disruption of *Nf2* in suture stem cell impaired the regeneration of cranial bone defects.**

To trace the contribution of migrated cells at injury sites, R26-tdTomato was introduced to label the migrated suture stem cells/progeny. We found that Nf2 mutants exhibited remarkably reduced migrated MSC/progeny in frontal and parietal bones compared to the control (frontal bone p < 0.001; parietal bone p < 0.001, Fig. 8d, e, n = 3) at post-injury D10, suggesting that the contribution of migrated MSC and its progeny to injury site was considerably defective in Nf2 mutants. EdU⁺/R26-tdTomato⁺ cells were found distributed extensively at frontal injury sites, and still more cells could be observed at parietal injury sites (Fig. 8d, arrow), suggesting that superb capability of these EdU⁺/R26-tdTomato⁺ cells to actively proliferate and contribute to the repair and growth of the new bone. On the contrary, less EdU⁺/R26-tdTomato⁺ cells were found in Nf2 mutant frontal and parietal injury sites, indicating an inferior osteogenic potential in Nf2 mutant injury sites. At post-injury D7 in adult mice (2 m, n = 3) using skull whole-mount immunofluorescent imaging, we found that R26-tdTomato⁺ suture stem cells/progeny were projecting in abundance towards (Fig. 8f, arrow) and at injury site in control mice (Fig. 8f, dotted line). In Nf2 mutants, R26-tdTomato⁺ suture stem cells/progeny were projecting less towards (Fig. 8f, arrow) and at the injury sites (Fig. 8f, dotted line) compared to the controls, suggesting that Nf2 deficiency in Prx1⁺ suture stem cells caused delayed cell migration of suture stem cells/progeny towards bone injury site (Fig. 8f, arrow). Collectively, Nf2-FAK interaction is of great importance in regulating the migration of suture stem cells/progeny and their osteogenic capacity to regenerate new bone tissues and restore the injury of cranial bones (Fig. 8g).

Discussion

In this study, we disclosed an unrecognized role of Nf2-FAK signaling axis in skeletal mesenchymal stem cells that critically required for normalizing cranial osteoblast lineage development, cranial bones development, and bone regeneration of cranial defects. The correlations between Nf2 expression and the sensitivity of FAK inhibitors have been established in preclinical models⁶⁹ and clinical courses⁷⁰ in tumors. Nf2 negative tumors were susceptible to FAK inhibitors^69,71. While in serous ovarian tumor, reduced Nf2 expression occasionally did not cause the sensitivity to the inhibition of FAK action⁷². The reciprocal role between Nf2 and FAK has been implied in several tumor cells^71,73,74. However, in some Nf2 mutant tumors, the integrins and Ras were activated, and then provoked autophosphorylation of FAK⁷⁵. Nf2 was found directing the phosphorylation of Src and its downstream FAK⁷⁶, suggesting that the interactive role between Nf2 and FAK was varied, directly or indirectly, in tumor cells. FAK deletion in early osteoprogenitor generated low bone mass and declined number of osteoblasts¹⁶, flawed collagen synthesis¹⁸, and attenuated Runx2 expression⁷⁷. However, it remains unknown on the upstream factors governing the activity of FAK in osteoblasts. We found that Nf2 mutants exhibited significantly decreased phospho-397FAK. Nf2 overexpression can sufficiently rescue diminished phospho-397FAK and Runx2 in Nf2 mutant osteoblasts, what is more, Nf2 physically interacts with FAK in osteoblasts, suggesting that Nf2/merlin represents an upstream governor in modulating FAK activity in osteoblasts.

FAK and its mediated downstream signaling comprised PI3K/Akt^21,22, mTOR¹⁶ and MAPK pathways²⁰. Erk1/2⁵⁸, p38 MAPK⁸ and JNK pathways⁵⁹ are prominent MAPK signaling centers for osteoblast differentiation. We manifested that Nf2-FAK interaction preferentially mediated cellular Erk1/2 and Akt1/2 signaling pathways instead of JNK and p38 MAPK signaling pathways. However, in tumor cells, Nf2 deficiency led to intense expression of ErbB2, and thus caused increasing activities of Erk1/2 and Akt/S6 in human schwannoma⁷⁸, or gave rise to the trigger of Ras-Raf-MEK-ERK1/2 signaling in tumor cells⁷⁵. Hence, the reciprocal mechanism and the downstream signaling of FAK in tumor cells is clearly different from our discoveries that FAK mediated Erk1/2 and Akt pathway in osteoblasts. Moreover, PI3K is a heterodimer comprised of a p85 regulatory subunit and a p110 catalytic subunit⁶⁰, and engaged in osteoblasts differentiation^61,62. In fibroblasts, FAK was found to serve as upstream of PI3K/Akt⁵⁷ and binds to PI3K-p85 regulatory subunit⁶³. However, in human cancer cell lines, FAK was found negatively governed by p110⁷⁹, indicating that the action between PI3K and FAK varied in response to the types of the cells. In our study, we revealed that FAK interacts with the catalytic subunit p110-PI3K instead of p85 regulatory subunit, and Nf2 mutant osteoblasts attenuate the association between FAK and subunit p110/PI3K, implying that Nf2-FAK positively regulates PI3K action in osteoblasts via the interaction with p110 catalytic subunit, uncovering a new scaffolding role for Nf2-FAK as upstream factor in controlling PI3K/Akt and Erk1/2 action in osteoblasts.

During the repair of cranial defects, suture stem cells will be activated, migrated, and differentiated to develop new bone and restore the injury. Modulations of BMP, FGF, Wnt, TGF signaling pathways in suture stem cells were competent in modifying osteogenic potential and the capability of cranial regeneration^4,9,10,11. Gli1⁺^80,81, Axin2⁺^82,83, and Prx1⁺⁴⁰ were used to label suture stem cells. Prx1⁺ suture stem cells were found resident in cranial sutures⁴⁴ and postnatal periosteum⁴⁰. Prompt migration of suture stem cells towards the injury site was essential for the repair of cranial defects. FAK was involved in gathering at the budding FAs earlier than Paxillin at cell front²⁷. Variation of Paxillin-FAK connection resulted in mitigated cell adhesion⁸⁴. Recent evidence also showed that Nf2 functions as a component of focal adhesions in tumor cells^85,86. We found that disturbed Nf2-FAK activity in MSC contributed to malformed focal adhesion and notably delayed MSC migration. In vivo, R26-tdTomato⁺ suture stem cells/progeny were projecting much less towards and at the injury sites in Nf2 mutant mice compared to the controls, implying delayed cell migration towards bone injury site upon Nf2 deficiency in Prx1⁺ suture stem cells.

FAK was also closely associated with bone formation¹⁹, and acute injuries in adult skeleton can initiate FAK signaling¹⁹, while FAK deficiency gave rise to abnormal bone matrix and delayed bone regeneration¹⁹. Likewise, we found that Nf2 mutant osteoblasts exhibited diminished activities of OPN and ColI in bone matrix deposition, and impaired FAK in Nf2 mutant mice markedly retarded bone regeneration of cranial defects. Comparably, FAK was shown critical in trafficking MSCs⁸⁷, deciding cell shape, cell motility and the physiological role of osteoblast in response to the extracellular stimuli²⁶, implying that Nf2-FAK in suture stem cells could produce considerable influences similarly on MSCs homing, motility, differentiation and physiological role of osteoblasts reacting to cranial injury. In sutures, diverse populations of suture stem cells were reported to be accountable for the cranial regeneration^11,88,89, and our finding provides a potential direction targeting classic signaling to modulate cell migration and differentiation for a better cranial bone regeneration.

In conclusion, our studies manifest that Nf2 plays a pivotal role in normalizing osteogenic lineage development, cranial bone formation and regeneration through the modulation of FAK activity, especially pronounced in the physical Nf2-FAK interaction and its mediated cellular Erk1/2 and p110-PI3K/Akt signaling being an indispensable scaffold in the skeletal system.

Methods

Generation of Nf2 cKO mice

The use of animals was approved by the Institutional Animal Care and Use Committee at the Zhejiang SCI-TECH University (ethical approval number #201911001). The study was performed in compliance with the guidelines for the care and use of animals in research. All mice were on a pure C57BL/6 background and housed at 20 °C-22 °C on a 12 hr light/dark cycle with 45-55% humidity in the institutional animal facility of the Hangzhou Normal University and Zhejiang SCI-TECH University.

The Nf2 floxed allele (f/f) was provided by Inserm⁴². Prx1-Cre was kindly provided by Prof. Bo Zhou (Shanghai Institute of Biochemistry and Cell Biology). Inducible Prx1-Cre^ER was purchased by Jacksonlab (Stock #007914). ROSA-R26^tdTomato mouse (R26^tomato) was kindly provided by Prof. Yanmei Tao (Hangzhou Normal University) to genetically label derived cells under Prx1-Cre^ER transgenic mouse line. Nf2 ^f/f mice and mice with tissue-specific promoter-driven Cre (Prx1-Cre or Prx1-Cre^ER) were crossed to generate heterozygous mice, which were intercrossed to obtain homozygous cKO (conditional knockout) mice (Prx1-Cre/Nf2^f/f/R26^tomato or Prx1-Cre/Nf2-cKO). Cre-negative littermates served as controls. Prx1-Cre/Nf2^f/+/R26^tomato was used as a negative control to label migrated suture stem cell in cranial injury model.

Tail or toe tissue of each animal was collected for genotyping. The following primers were used for genotyping⁹⁰: 5’-CTTCCCAGACAAGCAGGGTTC-3’ and 5’-GAA GGC AGC TTC CTT AAG TC-3’ for Nf2 ^f/f (~442 bp) and WT ( ~ 305 bp) fragments; 5’-GAA GGC AGC TTC CTT AAG TC-3’ and 5’-CTC TAT TTG AGT GCG TGC CATG-3’ for the deleted allele (338 bp) driven by Prx1-Cre and Prx1-Cre^ER. 5′-TCC AAT TTA CTG ACC GTA CACC-3′ and 5’-CGT TTT CTT TTC GGA TCC-3’ for the Cre gene product (~372 bp). R26^tomato mutant was determined using R26Tomato-M3: 5’-GGC ATT AAA GCA GCG TAT CC-3’; R26Tomato-M5: 5’-CTG TTC CTG TAC GGC ATG-3’, and a fragment of ~196 bp can be visualized. Primers R26Tomato-WT5: 5’-AAG GGA GCT GCA GTG GAG TA-3’ and R26Tomato-WT3: 5’-CCG AAA ATC TGT GGG AAG TC-3’ were used to determine phenotype of wildtype at a fragment of ~297 bp.

Inducible knockout of Nf2 and cranial bone defects model

To induce the knockout of Nf2 in suture mesenchymal stem cell, Nf2^ff/Prx1-Cre^ER or Prx1-Cre/Nf2^f/f/R26^tomato animals were treated with tamoxifen in sterile oil for 5 days (40 mg/kg, intraperitoneally), and the control were Nf2^ff or Prx1-Cre/Nf2^f/+/R26^tomato animals (2 m). For the cranial bone defects, it is performed based on publication^9,10,11. Briefly, after mice anesthesia, disinfection was made at the surgical site of the mice, an incision was made laterally to the sagittal midline to expose the frontal and parietal bones. The pericranium was removed using a sterile cotton swab. Using diamond coated trephine bits and saline irrigation, a 2-mm² defect was created carefully (so as not to damage the underlying dura) in the right frontal bone and left parietal bones of the recipient mice with no damage to the underlying dura mater. Post-surgery care was made to animals to lower the pain and disinfection. EdU was injected intraperitoneally at a single dose of 100 mg/kg to each animal 2 hr before the tissues collection on day 10 and day 21.

Skeletal staining

Alizarin Red-Alcian Blue staining was used to determine the skeletal structure in embryos. Skeletons in gestation at different developmental stages were double stained for cartilage and bone using Alcian Blue and Alizarin Red solution All carcasses were skinned and fixed in 95% ethanol for 24 h. Samples were then placed in 95% ethanol-Alcian Blue (#A5268, Sigma) and Alizarin red solution (#A5533, Sigma) (24, 48 h respectively) for cartilage and bone staining, followed by a 95% ethanol wash (8 h), and maceration in 1% KOH overnight at 4 °C. Samples were cleared in 20%, 50%, 80% glycerol in 1% KOH for 12 h for each step. Samples were stored in 100% glycerol for subsequent imaging.

μ-CT analysis

Micro-computed tomography (μ-CT) was performed using Bruker Skyscan1272. For newborns, bones were scanned at 30 keV, 150 μA, and 5 μm. For adult mice, bones were scanned at 70 keV, 140 μA, and 12 μm. The scan and analysis of the trabecular bones were performed from the growth plate and consisted of 150 slices. The scan and analysis of the cortical bone were performed at the midshaft of the bone and consisted of 100 slices (12 mm per slice). Analysis was performed using the Bruker CTAn Micro-CT Software. Three-dimensional images were reconstructed using Amira-Avizo Software (Thermofisher).

Histological tissue preparation

All female and male mice were used for tissue collection. Histology and tissue preparation were performed based on our previous publications^90,91. Briefly, pregnant mice and adult mice (2 m) were euthanized with CO₂ followed by cervical dislocation. Embryos (E12.5–E18.5) were dissected from the uterus under a microscope. E0.5 was designated as noon of the day on which the dam was positive for the vaginal plug. Murine femurs and tibiae were harvested, skinned, and eviscerated before fixing in 4% paraformaldehyde (PFA) in 1x PBS overnight. For paraffin sections, specimen was twice dehydrated in 50% and then 70% ethanol solution respectively and embedded in paraffin. Sections were cut at a thickness of 6 μm and mounted on Superfrost Plus slides (Fisher). For frozen sections, PFA-fixed tissues were cryoprotected in 30% sucrose in 0.1 M PBS at 4 °C for at least 24 hr, embedded in O.C.T. compound (Tissue Tek), and rapidly frozen for cryostat sectioning at 10 μm using Leica microtome (Leica LM 3095), and then mounted on Superfrost Plus slides (Fisher).

H&E staining

Cranial bone paraffin sections were stained with Hematoxylin buffer (BBI E607317) and Eosin buffer (BBI E607312) according to protocol, rehydrated and stained the slides in hematoxylin. Rinsed using acid alcohol (1% HCl in 50% EtOH) and counterstained with Eosin. Slides dehydrated in 100% EtOH and Xylene. Safranin O staining was performed by staining in Weigert’s iron hematoxylin solution for 3 minutes, followed by Fast Green (0.001%) and Safranin O (0.1%) for 5 minutes each.

von Kossa staining

To examine mineralization, sections from E15.5 embryos were incubated in 1% silver nitrate for 30 min while under sun light. Unreacted silver was washed with 5% sodium thiosulfate and distilled water for 5 min each. The slides then incubated in 1% Alcian Blue solution for 30 min, followed counterstained with nuclear fast red solution. Sections were dehydrated and coversliped using permanent mounting medium. For cell staining and sections by von Kossa staining, firstly to wash the cells with Ca/Mg-free PBS and then fixed the slides in 4% formalin/PBS. Added silver nitrate solution and incubated the cells sequestered from any UV exposure. The unincorporated silver nitrate was removed via 5% sodium thiosulfate.

Immunostaining

For immunohistochemistry staining based on our publication^92,93, the paraffin sections were used for the experiments, sections were treated with 6% H₂O₂ in 100% methanol at room temperature for 5 h to block endogenous peroxidase activity followed by rehydration through a descending methanol series (50, 25, and 0% in 0.1 M PBS) at room temperature for 30 min each. Antigen retrieval was performed by heating the tissues at 92–95 °C for 5 min in Universal Antigen Retrieval Agent (CTS015; R&D Systems, Minneapolis, MN). PBST (0.1 M PBS and 0.1% Triton X-100) was used to block nonspecific staining. Sections were then incubated with primary antibody in 10% normal donkey serum (D9663; Sigma Aldrich, St Louis, MO) in PBST at 4 °C overnight (Supplemental Table 1). After rinses with PBS for three time, sections were incubated at 4 °C for 24 h with biotin-conjugated secondary antibody (BA-5000; Vector Laboratories, Burlingame, CA) in 1% NDS in PBST, and then incubated at room temperature for 30 min with peroxidase-conjugated streptavidin in blocking solution (PK6200; Vector Laboratories, Burlingame, CA). After three rinses in PBS, sections were preincubated in nickel-intensified DAB solution (SK4100; Vector Laboratories, Burlingame, CA) without H₂O₂ at room temperature for 30 min followed by incubation with DAB solution containing 0.0003% H₂O₂. The reaction was stopped by PBS rinses twice and photographed in 0.1 M PBS. Counterstaining by hematoxylin was dependent on sections.

For immunofluorescence (IF) staining, frozen sections were air-dried at room temperature for 1 h and rehydrated in 0.1 M PBS. Blocking of non-specific staining was carried out by incubation with 10% normal donkey serum in 0.1 M PBS containing 0.3% Triton ×-100 (×100, Sigma Aldrich, St. Louis, MO) at room temperature for 30 min. Then, the sections were incubated with primary antibodies (Supplemental Table 1) in the carrier solution (1% normal donkey serum, 0.3% Triton ×-100 in 0.1 M PBS) at 4 °C for overnight. Sections without a primary antibody treatment were used as negative control. Following three rinses in 0.1 M PBS, sections were incubated with Alexa Fluor® 488- or 647-conjugated secondary antibody (1:500, Jackson immune research, West Grove, PA) in carrier solution at room temperature for 1 h. Following rinses with 0.1 M PBS, sections were counterstained with DAPI (200 ng/ml in PBS, D1306; Life Technologies, Carlsbad, CA) at room temperature for 10 min. After thorough rinsing in 0.1 M PBS, sections were air-dried and cover slipped with Prolong®Diamond antifade mounting medium (P36970; Fisher Scientific, Waltham, MA). The sections were photographed using a laser scanning confocal microscope (Zeiss LSM 710, Zhejiang SCI-TECH University).

EdU analysis

To collect tissues for EdU immunoreactions, EdU (B5002, Beyotime, Shanghai, China) was dissolved in Dulbecco’s phosphate-buffered saline (DPBS, Cytiva, Marlborough, MA) at 10 mg/ml and injected intraperitoneally at a single dose of 100 mg/kg 2 h before embryo collection. EdU staining was performed based on the BeyoClick™ EdU-594 (#C0078S, Beyotime, Shanghai, China), and EdU positive osteoprogenitor cells cells were calculated from three different samples using Imaging J.

Apoptosis assay

The apoptosis was determined using terminal deoxynucleotidyl trans-ferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). The frozen sections were fixed and permeabilized, followed by TUNEL labeling using a One Step TUNEL Apoptosis Assay Kit (#C1089, Beyotime, China) based on the protocol. The TUNEL positive cells were imaging using confocal microscope and counted calculated from three different samples using Imaging J.

Cell culture and differentiation assay

Primary calvarial osteoblast culture, embryonic mesenchymal stem cells (MSCs) culture, and osteogenic induction were performed as described^49,94. Freshly sorted MSCs were seeded in 10 cm dish at a density of 500 cells/cm² in aMEM medium supplemented with 20% FBS and 100 IU/ml Penicillin/Streptomycin (GIBCO, Invitrogen, Carlsbad, CA, United States) based on publication^49,50,95, and the MSCs were then used for scratch assay to test the cell migration. For osteoblast culture, calvarial bones were isolated from newborn mice or embryonic stage of E18.5 from Prx1-Cre/Nf2-cKO mutants and Cre⁻/Nf2^f/f littermates using a dissecting microscope^96,97. After the removal of the periosteum and dura mater from the skull, the bones were then minced and digested using 0.2% dispase II and 0.1% collagenase in serum-free medium at 37 degrees. Digestion was carried out three times, and the first two digestions for 30 min were discarded, the last digestion for 90 min was pooled, centrifuged, and resuspended in aMEM supplemented with 10% FBS and 100 IU/ml Penicillin/Streptomycin (GIBCO, Invitrogen, Carlsbad, CA, United States) to reach cell confluence.

For the osteogenic differentiation assay, the confluence osteoblasts were digested using 0.25% trypin/EDTA (Thermofisher, 25200056) and the cells were seeded at in six-well plates at 2 × 10⁵ per well. Subsequently, cells were induced using osteogenic differentiation medium and BGJb medium (12591, Gibco) supplemented with 10% (vol/vol) FBS, 50 μg/mL ascorbic acid (A4544; Sigma-Aldrich), and 5 mM glycerolphosphate (G9891; Sigma-Aldrich) and 100 IU/ml Penicillin/Streptomycin (GIBCO, Thermo, United States). Osteoblast were collected at different differentiation stage (d0, d7, d14). Osteoblastogenesis was analyzed by ALP staining at d7 according to manufacturer’s manual (A2356; Sigma-Aldrich). Osteoblast mineralization was examined by Von Kossa staining at D14. RNA and proteins isolated for the analysis of osteogenic markers. During the osteogenic differentiation, activators such as Adhesamine, dumbbell-shaped molecule, activates MAPK/FAK pathway⁶⁵. SC79 (cat # HY-18749, MCE), a unique specific AKT activator to activate AKT in the cytosol⁶⁶. Pamoic acid disodium, which (cat # HY-W010907, MCE) induces ERK1/2 activation⁶⁷ were used to treat osteoblasts cells to evaluate the activities of osteoblast differentiation.

Cell adhesion, detach and migration assay

The cells were pretreated with precoated with fibronectin (10 µg/ml) plates. For adhesion examination, primary osteoblasts were trypsinized and diluted to a concentration of 1 × 10⁵ cells/ml with the same medium. Then 100 µl of cells was added to each well of a 24 well plate, and the cells were incubated for different time points at 0 min, 15 min and 30 min. After discarding of the floating cells, the attached cells were fixed with 4% paraformaldehyde and stained with methylene blue; and the number of attached cells was examined by microscopy. For the detach assay, osteoblasts were grown to 90% confluency on culture plates prior to trypsinization for 2 min at 37 °C. The cells were then washed, and the remaining adherent cells were incubated for 1 h at 37 °C and the cells were counted. Two experiments were used for the migration assay, MSCs isolated from control and Nf2-cKO mice were used for migration assay. What is more, Lenti-shNf2 and Lenti-shctrl viruses were transfected into MSC cells using the Lipo8000 transfection reagent (Beyotime, C0533) following manufacturer’s instructions to reduce the effect of cell proliferation. After 24 h, scratches were made in each well using tips to create the space for cell migration, and the FBS-free aMEM medium was used for the sequent calculation of the migrated distances of the cells at different time points 0 h, 3 h, 6 h, 9 h, 12 h, 24 h. Each time points were biologically repeated at least 3 times. The migrated distances of cells were examined by microscope.

Live cell imaging

Live cell imaging was performed as described⁹⁸. Nf2 ff and Nf2-cKO primary osteoblasts were were transiently transfected with mouse paxillin-GFP and were plated on fibronectin (10 μg/mL)-coated glass-bottom cell culture dishes before the use of a time-lapse fluorescence microscope (Olympus). Images were taken at 2.5-minute intervals and were processed for estimation of various parameters. Live cell imaging was performed in live-cell imaging buffer (Thermofisher) containing ProLong Live antifade Reagent (Thermofisher, P36974) using FV3000 Confocal laser scanning microscope (Olympus).

NanoBiT protein:protein interaction assay

NanoBiT assay was performed using Nano-Glo® Live Cell Assay System following manufacturers’ instructions (N2015, Promega). Briefly, mouse Nf2 and FAK cDNA sequences were cloned into pBiT1.1-C[TK/LgBiT], pBiT2.1-C[TK/SmBiT], pBiT1.1-N[TK/LgBiT] and pBiT2.1-N[TK/SmBiT] vectors by homologous recombination using the Seamless Cloning kit (Beyotime, D7010). For experimental group, 3T3 cells plated on black-wall 96-well plates were transfected with paired plasmids (pBiT-Nf2/LgBiT+pBiT-FAK/SmBit, pBiT-Nf2/SmBiT+pBiT-FAK/LgBit). For the control group, 3T3 cells on the same plate were transfected with pBiT-Nf2/LgBit or pBiT-FAK/LgBit with HaloTag-SmBiT® (negative control plasmid). Luminescence signal was detected 48 h post-transfection immediately after adding 25 μl Nano-Glo® Live-cell Reagent (N1661, Promega) using Varioskan Flash Full Wavelength Scanning Multifunction Reader (Thermofisher) at ambient temperature and 1000 ms intervals. Primers sequences are presented in Supplemental Table 2.

Lentivirus package and cell lines

Nf2 shRNA was purchased from Santa Cruz Biotechnology (sc-36053-SH, Santa Cruz, China), Nf2 cDNA was constructed by inserting the cDNA sequence into the pLentiPGK-puro vector using Gateway Cloning (Invitrogen). For lentivirus packaging, the 2^nd generation packaging plasmids (psPAX2 and pMD2.G) and lentivirus vector were co-transfected into 293 T cells using the Lipo8000 transfection reagent (Beyotime, C0533) following manufacturer’s instructions as described⁹³, and the virus was harvested 48 and 72 h post-transfection. Viral titers were determined in 293 T cells, and lentivirus infection was performed by incubating primary cells with 5 infection units (IU) per cell (about 1 × 10⁵ IU per well in a 24 well plate) in the presence of 10 μg/ml polybrene overnight. For the stable shNf2 MC3T3-E1 (3T3) cell line screening (#1101MOU-PUMC000012, MC3T3-E1, NICR, China), puromysin (4 µg/ml, Byotime, China) was used to screen the stable transfect cells, which further maintained in puromysin (2 µg/ml, Byotime, China) in aMEM media supplemented with 10% FAS and 1% gentamysin.

Western blot assay

Proteins from cultured osteoblast at different differentiated stages from Prx1-Cre/Nf2^cKO mutants and Cre⁻/Nf2^f/f littermate controls were extracted using radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 150 mmol/L NaCl, 50 mmol/L Tris-HCI, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, pH 7.4). The concentration of extracted proteins was determined using Pierce™ BCA protein assay kit (#23225, Thermo Fisher Scientific) and Synergy™ 4 microplate reader (#7161000, BioTek Instruments, Winooski, VT). An amount of total protein was loaded into each lane. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve the protein bands and transferred to the nitrocellulose membrane. Non-specific binding was blocked using blocking buffer containing 3% bovine serum albumin (A-420–100, Gold Biotechnology, St Louis, MO) in Tris-buffered saline and Tween-20 buffer (TBST) buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h. The membranes were incubated with primary antibodies (Supplemental Table 1) in blocking buffer at 4 °C for 24 h. Following three rinses in TBST (15 min/each), membranes were incubated with conjugated secondary antibodies (#111-035-144, Jackson ImmunoResearch, West Grove, PA) in blocking buffer at room temperature for 2 h. ChemiDocMP Imaging System (Bio-Rad ChemiDoc, Hercules, CA) was used to image the Western blot bands as described⁹³.

co-immunoprecipitation (co-IP) assay

For co-IP, cell lysates were prepared from cells cultured on one 10 cm dish in 500 μl co-IP buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% Glycerol, pH7.4) supplemented with proteinase and phosphatase inhibitor cocktail (P1050, Beyotime, China). Cell lysates were incubated with co-IP antibody or normal IgG with rotation overnight at 4 °C. Then, Dynabeads (10001D, Thermo) were incubated for 30 min and then wash using DynaMag-2 magnet (12321D, Thermo) and eluted in SDS elution buffer (50 mM Tris, 2% SDS, 10% glycerol) at 60 °C for 10 min. The supernatant containing co-IP products was supplemented with 100 mM DTT and 0.1% Bromophenol Blue, denatured at 95 °C for 10 min, and submitted for western blot analysis.

GTPase assay

RhoA activities were evaluated using kit from Cytoskeleton (BK030, Cytoskeleton, Inc.). Briefly, the Nf2 mutant and control MSC were pretreated with fibronectin coating (10 µg/ml), when the cells reach to about 30% confluence, the cells were lysate collected, and rhotekin-RBD beads were added to lysate to pull down the RhoA based on the instruction, and Western Blotting were performed to visualize the activity of RhoA in Nf2 mutant MSC.

Quantitative real-time reverse transcription (qRT)-PCR

Total RNA was extracted from cultured cells with TRIzol reagent (15596018, Life Technologies). Mouse cDNA was reversed-transcribed from 0.5 g total RNA with Hifair® III 1^st Strand cDNA Synthesis SuperMix for qPCR (#11141ES10, YEASNE, China). The qRT-PCR was performed using one step RT-PCR System with Hieff® qPCR SYBR Green Master Mix (#11202ES03, YEASNE, China) on the ABI7500 platform. Expression levels of each gene were given relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Primers sequences are presented in Supplemental Table 3.

Statistical analysis

All data are expressed as mean ± standard deviation (SD) with at least three biological replicates. The differences between two groups were examined by two-tailed Student’s unpaired t-test. One-way analysis of variance (ANOVA) was used to evaluate a statistical difference across multiple groups. A P-value less than 0.05 (P < 0.05) is taken as statistical significance. Prism version 9.5.1 software was used to perform statistical analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The authors confirm that all relevant data are available in the paper and/or its Supplementary Information files. Source data are provided within this paper. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants by Zhejiang Provincial Natural Science Foundation of China (LZ23H140001 to GQ CHEN), funds from National Natural Science Foundation of China (81400489, to GQ CHEN, 81900806 to CHZHOU, 32070814 to MR WU), Zhejiang Qianjiang Talent Program (21040040-E, to GQ CHEN), a startup grant from Zhejiang Sci-Tech University (18042290-Y, to GQ CHEN), the Fundamental Research Funds of Zhejiang Sci-Tech University (2021Q031), Jiaxing Science Technology Foundation (2023AY11045 & 2020AY10001, to XG ZHANG). We thank Professor Zefu Lu (Chinese Academy of Agricultural Sciences) and Ms. Rexhina Vlashi for their critical review and discussions with the manuscript. We apologize to many researchers whose work could not be cited due to space limitations.

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These authors contributed equally: Junguang Liao, Yuping Huang.

Authors and Affiliations

Department of Biopharmaceutics, Zhejiang Provincial Engineering Research Center of New Technologies and Applications for Targeted Therapy of Major Diseases, College of Life Science and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
Junguang Liao, Yuping Huang, Fuju Sun, Chenggong Zheng, Yifeng Yao & Guiqian Chen
Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, China
Cui Zhang
Department of Orthopedics, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
Chenhe Zhou
Department of Orthopedics, Jiaxing Key Laboratory for Minimally Invasive Surgery in Orthopaedics & Skeletal Regenerative Medicine, Zhejiang Rongjun Hospital, Jiaxing, 314001, China
Xingen Zhang
Department of Cell and Developmental Biology, College of Life Sciences, Zhejiang University, Hangzhou, China
Mengrui Wu

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Junguang Liao
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Yuping Huang
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Fuju Sun
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Chenggong Zheng
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Yifeng Yao
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Cui Zhang
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Chenhe Zhou
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Xingen Zhang
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Mengrui Wu
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Guiqian Chen
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Contributions

Conceptualization: G.C., M.W., X.Z. Methodology: J.L., Y.H., F.S., C.Z., Y.Y., X.Z., C.Z. Investigation: J.L., Y.H., F.S., C.Z., Y.Y., X.Z., C.Z. Visualization: J.L., Y.H., F.S., C.Z., X.Z., C.Z., M.Wu., G.C. Writing—original draft: J.L., G.C. Writing—review & editing: G.C., M.Wu., J.L., Y.H. Supervision: G.C., M.Wu., X.Z. All the authors reviewed and approved the paper.

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Correspondence to Xingen Zhang, Mengrui Wu or Guiqian Chen.

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Liao, J., Huang, Y., Sun, F. et al. Nf2-FAK signaling axis is critical for cranial bone ossification and regeneration. Nat Commun 16, 2478 (2025). https://doi.org/10.1038/s41467-025-57808-4

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Received: 19 April 2023
Accepted: 05 March 2025
Published: 12 March 2025
DOI: https://doi.org/10.1038/s41467-025-57808-4