Hepatocyte growth factor signaling regulates tooth germ development, inducing proliferation of mesenchymal cells via fission of mitochondria

Hepatocyte growth factor signaling regulates tooth germ development, inducing proliferation of mesenchymal cells via fission of mitochondria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hepatocyte growth factor signaling regulates tooth germ development, inducing proliferation of mesenchymal cells via fission of mitochondria Prodhan MD Rubayet Alam, Nobuo Takeshita, Wei Jiang, Koji Miyazono, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7493229/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tooth morphogenesis is regulated by epithelial–mesenchymal interactions and cellular metabolism through intricate signaling pathways. Here, we identify a novel mechanism by which hepatocyte growth factor (HGF) signaling promotes mesenchymal cell proliferation during tooth germ development via the regulation of mitochondrial dynamics. Immunohistochemistry revealed spatiotemporal expression of c-Met, the HGF receptor, in both the epithelial and mesenchymal compartments of developing mouse molars. Organ culture showed recombinant HGF increased tooth germ size and mesenchymal proliferation, whereas the c-Met inhibitor PHA-665752 suppressed these effects. Mitochondrial staining and Western blotting demonstrated that HGF stimulated mitochondrial fission through increased phosphorylation of Drp1, whereas PHA-665752 or Mdivi-1, a Drp1 inhibitor, blocked this response and attenuated proliferation. The inhibition of mitochondrial fission also reduced HGF-induced tooth germ growth ex vivo. HGF activated MAPK pathways, particularly the ERK, p38, and JNK pathways, with JNK inhibition (SP600125) most effectively suppressing Drp1 phosphorylation, defining the HGF-JNK-Drp1 axis as central to this process. Epithelial proliferation was largely unaffected, underscoring a mesenchyme-specific mechanism. These findings reveal a previously unrecognized role of mitochondrial fission as a downstream effector of HGF signaling and highlight mitochondrial dynamics as a potential therapeutic target in developmental anomalies of the dentition. Biological sciences/Cell biology Health sciences/Diseases Odontogenic mesenchymal cells mitochondrial fission tooth development hepatocyte growth factors Drp-1 Mdivi-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Epithelial‒mesenchymal interaction (EMI) is essential for the early development of various organs composed of the epithelium and mesenchyme, including teeth, hair, lungs and kidneys[ 1 ]. Teeth contribute not only to mastication but also to pronunciation/speech and aesthetics, and these essential roles must be properly maintained with normal occlusion and appropriate tooth morphology. Multiple signaling pathways, including the hepatocyte growth factor (HGF) [ 2 , 3 ], wingless (Wnt) [ 4 ], bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and epidermal growth factor (EGF) pathways, are known regulators of EMI [ 5 ] and contribute to organogenesis in the abovementioned organs. These growth factors and cytokines are known to regulate the proliferation of epithelial and mesenchymal cells during tooth development through EMI. Among them, HGF is a ligand of c-Met, a transmembrane receptor containing a tyrosine kinase domain that plays a significant role in organogenesis[ 2 ]. During tooth development, both HGF and c-Met are expressed in epithelial and mesenchymal tissues, suggesting their potential role in regulating tooth morphogenesis [ 6 ]. While this finding implies that HGF signaling regulates the proliferation of precursor cells and their differentiation into specific dental cells, the precise role of HGF signaling in the proliferation of mesenchymal cells within tooth germs during tooth development remains largely unknown. Mitochondria are central regulators of cellular homeostasis, linking energy metabolism with developmental and differentiation programs by their dynamic fission and fusion, acting as key determinants of cell fate[ 7 ]. Fission is largely mediated by Drp1 which supports cell cycle progression and progenitor proliferation[ 8 , 9 , 10 ], while fusion sustains mitochondrial integrity during differentiation and tissue morphogenesis[ 11 , 12 ]. Inhibition of Drp1 in developing teeth accelerates odontoblast differentiation and dentinogenesis [ 13 ], highlighting that excessive fission limits maturation, whereas regulated remodeling is required for morphogenesis. These findings support the concept that mitochondrial dynamics is crucial for development and balancing mesenchymal proliferation. Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that link extracellular cues to intracellular responses, regulating proliferation, differentiation, and survival[ 14 ]. In craniofacial and tooth development, ERK signaling is essential for epithelial proliferation and enamel knot activity, serving as a central regulator of cusp morphogenesis[ 15 ]. p38 MAPK contributes to odontoblast differentiation and dentin matrix secretion through BMP-2–driven transcriptional programs[ 16 ]. JNK, initially characterized in apoptosis and stress responses[ 17 ], has since been linked to mitochondrial remodeling and bioenergetic control, processes relevant to proliferative expansion during organogenesis. Since HGF-c-Met activates multiple MAPK branches[ 18 ], dissecting their contribution to mitochondrial remodeling provides a mechanistic basis for understanding how growth factor signaling drives mesenchymal proliferation during tooth germ development. The purpose of the present study was to examine the function of HGF signaling in the proliferation of mesenchymal cells during tooth germ development and to clarify its underlying mechanism, with a focus on mitochondrial fission and fusion. We first analyzed the expression of c-Met during tooth germ development in embryonic mice. Next, we administered HGF signaling inhibitors in an organ culture of mouse tooth germs to analyze the roles of HGF signaling during tooth germ development. The results indicated that the HGF signaling inhibitor suppressed the growth of tooth germs and the proliferation of mesenchymal cells in tooth germs. We examined whether mitochondrial dynamics are involved in the HGF signaling-modulating proliferation of tooth germ mesenchymal cells. Furthermore, we explored the downstream activation of MAPK cascades to determine their regulatory contribution to HGF-induced mitochondrial remodeling and mesenchymal cell proliferation during odontogenesis. In conclusion, our data revealed that HGF signaling induces the proliferation of mesenchymal cells via the regulation of mitochondrial dynamics during tooth germ development. Results Development of tooth germs expressing c-Met It is known that mouse mandibular molar tooth germs at embryonic day (ED) 13.5 are in the bud stage [ 19 ]. At this stage, strong fluorescent signals of c-Met were detected in the epithelium; however, the signals were faintly detected in the mesenchyme (Fig. 1 a − c). At ED 14.5, the tooth germs are in the cap stage [ 19 ]. The fluorescent signals of c-Met were still detected in the epithelium and the signals in the mesenchyme are more clearly observed than at ED 13.5. At ED 16.5, the tooth germs are in the bell stage [ 19 ]. The signals of c-Met were observed in the enamel organ and the dental papilla (Fig. 1 g − i).At ED 18.5, the development of tooth germs further progressed, with a dramatic increase in size [ 19 ]. The fluorescent signals of c-Met at this stage were still detected in the enamel organ and dental papilla (Fig. 1 j − o). The negative controls for c-Met staining produced no signals (Fig. 1 p − r). Recombinant murine HGF increased while PHA-665752 decreased the size of tooth germs in organ culture To analyze the function of HGF signaling during tooth germ development, we first isolated mouse mandibular molar tooth germs at the bud stage (ED 14.5) and cultured them ex vivo to administer recombinant HGF. The sizes of the tooth germs appeared to be same in size between the groups on day 0 (Fig. 2 a) In both groups, tooth germs showed cusp development and an increase in the size of tooth germs on day 6 (Fig. 2 a). The size of the tooth germs in the HGF group was greater than that in the control group (Fig. 2 a). Quantitative measurements revealed significant increases in height and width in the HGF group compared with those in the control group (Fig. 2 b). To further validate the functional involvement of HGF signaling, tooth germs were cultured with PHA-665752, a selective inhibitor of c-Met/HGF signaling. The size of the tooth germs in the PHA-665752 group was smaller than that in the control group (Fig. 2 d). Quantitative analysis revealed significant decreases in height and width in the PHA-665752 group compared with those in the control group (Fig. 2 e, f). These findings demonstrate that the activation of HGF signaling promotes, whereas its inhibition suppresses, the growth of developing tooth germs. Recombinant murine HGF was induced, while PHA-665752 inhibited the proliferation of mesenchymal cells during tooth germ development The proliferation of cells is closely associated with organ growth during embryogenesis [ 20 ]. We therefore hypothesized that HGF signaling induced the growth of tooth germs by increasing cellular proliferation. To evaluate this hypothesis, BrdU incorporation was assessed in organ-cultured tooth germs treated with recombinant HGF and PHA-665752. HGF did not affect the BrdU-stained area in the epithelium but significantly increased the stained area in the mesenchyme (Fig. 3 a − c). PHA-665752 also did not affect the proliferation of epithelial cells but significantly reduced the proliferation of mesenchymal cells (Fig. 3 d − f). Furthermore, to examine the effect of HGF signaling on the proliferation of dental epithelial and mesenchymal cells, the number of cells was quantified after treatment with recombinant HGF and PHA-665752. SF2 cells are known as precursor ameloblasts [ 20 ]. HGF and PHA-665752 did not significantly affect the number of SF2 cells (Fig. 3 g, h). Mouse dental papilla (mDP) cells are derived from mouse dental papilla and are widely used as a model for odontoblast precursor cells [ 21 ]. Compared with control cells, HGF- and PHA-665752-treated mDP cells presented significantly greater and lower cell numbers, respectively (Fig. 3 i, j). These results indicated that HGF signaling promoted the proliferation of mesenchymal cells during tooth germ development. Recombinant murine HGF and PHA-665752 induced fission and fusion, respectively, of mitochondria in mDP cells. To assess the role of HGF signaling in mitochondrial dynamics in mDP cells, we performed mitochondrial staining and evaluated the mitochondrial area relative to the total cell area. In addition, we analyzed the phosphorylation status of dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fission. mDP cells stained with MitoTracker revealed the morphology of the mitochondrial network under control and HGF-treated conditions (Fig. 4 a). Quantification of the ratio of the area of stained mitochondria to the total cell area revealed a significant decrease in the mitochondrial area upon HGF treatment (Fig. 4 b), suggesting enhanced mitochondrial propagation. Treatment with the HGF signaling inhibitor PHA-665752 led to a more fragmented mitochondrial morphology, as observed via MitoTracker staining (Fig. 4 c). Quantification revealed a significant increase in the mitochondrial area relative to the total cell area in the PHA-treated group compared with the control group (Fig. 4 d). Western blot analysis of p-Drp1 (phospho-Drp1) levels in control and HGF-treated mDP cells revealed that HGF significantly increased Drp1 phosphorylation (Fig. 4 e). Densitometric analysis normalized to total Drp1 confirmed a greater p-Drp1/Drp1 ratio in the HGF-treated group (Fig. 4 f), indicating HGF-induced activation of mitochondrial fission signaling. In contrast, cells treated with PHA-665752 displayed reduced phosphorylation of Drp1 (Fig. 4 g). Compared with that in the control group, quantitative analysis of the p-Drp1/Drp1 ratio revealed a significant decrease in Drp1 activation (Fig. 4 h), suggesting that inhibition of HGF signaling suppresses mitochondrial fission. Mdivi-1 suppressed HGF-induced mitochondrial dynamics, cell proliferation and tooth morphogenesis. The involvement of mitochondrial fission in HGF-induced responses in dental mesenchymal cells and developing tooth germs was assessed by treating mDP cells with Mdivi-1, a selective inhibitor of Drp1-mediated mitochondrial fission, in the presence or absence of HGF. mDP cells demonstrated that HGF treatment markedly increased the area of mitochondrial staining, which is indicative of mitochondrial expansion or biogenesis (Fig. 5 a). Compared with that in the control group, the ratio of the mitochondrial-stained area to the total cell area in the HGF group was significantly greater, whereas cotreatment with Mdivi-1 attenuated this increase, suggesting that HGF-induced mitochondrial expansion requires active mitochondrial fission (Fig. 5 b). Western blot analysis revealed that HGF increased the level of phosphorylated Drp1, whereas total Drp1 levels remained unchanged (Fig. 5 c). Densitometric quantification confirmed a significant increase in the p-Drp1/Drp1 ratio in the HGF group, which was suppressed by cotreatment with Mdivi-1 (Fig. 5 d). These results indicate that HGF induced mitochondrial fission through the phosphorylation of Drp1 and that this process is inhibited by Mdivi-1. Furthermore, we examined whether the inhibition of mitochondrial fission affects the morphogenesis of developing tooth germs. Mandibular molar tooth germs isolated at E14.5 were cultured ex vivo in the presence or absence of HGF and Mdivi-1. Compared with those in the control group, the HGF increased size of the tooth germ whereas Mdivi-1 inhibited the effect of HGF (Fig. 5 e). Morphometric analysis revealed that both the width and height of the tooth germs were significantly increased by HGF, and these effects were suppressed by Mdivi-1 cotreatment (Fig. 5 f,g). mDP cell proliferation was also assessed by the incorporation of Mdivi-1 and HGF. The changes were significant between the HGF group and the Mdivi-1-treated group (Fig. 5 h). Mesenchymal cell proliferation within the tooth germs was evaluated via BrdU incorporation during tooth germ development. The number of BrdU-positive mesenchymal cells per tooth germ was significantly greater in the HGF-treated group than in the control group (Fig. 5 i,j). This increase was markedly reduced by Mdivi-1, suggesting that mitochondrial fission is required for HGF-induced proliferation of dental mesenchymal cells. On the other hand, the number of BrdU-stained epithelial cells was almost the same in all the groups (Fig. 5 k). HGF induces Drp1 phosphorylation through MAPK signaling. We next investigated the upstream signaling pathways mediating this HGF–Drp1 axis focusing MAP kinase signaling pathway. HGF stimulation significantly increased phosphorylation of ERK1/2, p38, and JNK in mDP cells, with differences in the magnitude of response between pathways. ERK phosphorylation showed a modest but significant increase (Fig. 6 a, b), p38 activation was moderate (Fig. 6 c, d), and JNK phosphorylation was the most robust (Fig. 6 e, f). Inhibition of c-Met signaling with PHA-665752 revealed pathway selectivity, as ERK and p38 phosphorylation were only partially reduced (Fig. 6 g–j), whereas JNK activation was strongly suppressed (Fig. 6 k, l). These data indicate that all three MAPKs are activated downstream of HGF, with JNK representing the most c-Met–dependent branch. To determine the contribution of these pathways to Drp1 activation, the effect of pathway-specific inhibitors on HGF-induced Drp1 phosphorylation was examined. ERK inhibition with U0126 significantly reduced HGF-induced Drp1 phosphorylation (Fig. 6 m, n). Inhibition of p38 with SB203580 also decreased Drp1 phosphorylation (Fig. 6 o, p), though the reduction was less pronounced. In contrast, JNK inhibition with SP600125 almost completely abolished HGF-induced Drp1 phosphorylation (Fig. 6 q, r). Discussion In our study, we revealed an important mechanism through which HGF signaling regulates tooth germ development by promoting mesenchymal cell proliferation via mitochondrial fission. By linking an extracellular growth factor pathway to intracellular organelle remodeling, our work provides new insight into the molecular and cellular processes underlying odontogenesis. Our immunofluorescence analysis demonstrated that c-Met is expressed throughout the sequential phases of tooth development with spatial and temporal changes in localization. During the bud (ED 13.5) and cap (ED 14.5) stages, c-Met expression was mainly localized in the epithelium, while weak mesenchymal signals at ED13.5 became more pronounced by ED 14.5. This expression pattern suggests that HGF/c-Met signaling contributes to early epithelial morphogenesis and progressively intensifies in the mesenchyme, supporting the epithelial–mesenchymal interactions that drive bud elongation and cap formation. By the bell stage (ED16.5), c-Met expression became more broadly distributed, with detectable signals in both the enamel organ and the dental papilla. This shift indicates that mesenchymal tissues progressively acquire responsiveness to HGF signaling, consistent with the increased proliferation and differentiation requirements of the papilla as the tooth germ matures. At ED18.5, c-Met signals remained in the dental papilla, in line with the substantial increase in tooth germ size and cellular complexity. The persistence of c-Met expression into late developmental stages suggests that HGF signaling is not restricted to early morphogenesis but continues to regulate proliferation and tissue remodeling during bell stage differentiation. Earlier studies, however, were restricted to single developmental windows or focused primarily on either epithelial or mesenchymal compartments, leaving the sequential progression of c-Met expression during odontogenesis incompletely understood [ 22 , 23 , 24 ]. Moreover, previous work has suggested a role for HGF in dental morphogenesis, but most studies were limited to correlative expression analyses or cell culture systems that c-Met is expressed in enamel epithelium and HGF in dental mesenchyme at the cap stage, implying a paracrine role in epithelial-mesenchymal interactions[ 23 ]. Other showed c-Met immunolocalization in human fetal tooth germs, with strong epithelial staining and weaker mesenchymal signals[ 24 ]. Our findings provide a stage resolved analysis of c-Met expression in mouse molars, showing its transition from epithelial restriction to combined epithelial and mesenchymal distribution. This mapping confirms that HGF/c-Met signaling persists across critical stages of tooth development. Exogenous HGF stimulation significantly promoted the growth of molar tooth germs, while pharmacological inhibition of c-Met with PHA-665752 suppressed their expansion. Importantly, tooth germs in all groups were comparable in size at day 0 confirming that the observed differences arose during the culture period. By day 6, HGF-treated tooth germs exhibited greater dimensions, whereas PHA-665752 treatment significantly reduced both height and width. These results suggests that HGF-c-Met signaling directly contributes to tooth germ growth during the bud-to-cap transition. Previous studies identified the presence of HGF/c-Met components[ 23 , 24 ], they did not provide direct functional evidence of how manipulating the pathway alters tooth germ growth. Our findings extend this knowledge by providing functional validation in an ex vivo organ culture model, demonstrating that activation of HGF signaling accelerates, while inhibition retards, the growth of developing tooth germs. A similar HGF/c-Met-dependent epithelial-mesenchymal mechanism operates in other branching organs, including the lung, kidney, mammary gland, and salivary gland, where exogenous HGF promotes, and HGF inhibition suppresses, branching morphogenesis in organ culture. In these systems, genetic and biochemical evidence consistently positions c-Met on epithelial cells receiving mesenchymal HGF signals, highlighting a paracrine mode of action [ 25 , 26 , 27 , 28 , 29 ]. BrdU incorporation assays revealed that HGF treatment significantly increased the proliferation of mesenchymal cells within the tooth germ, whereas PHA-665752 treatment markedly suppressed this process. These findings collectively highlight that HGF signaling enhances mesenchymal cell proliferation in an autocrine manner that contributes to the morphological expansion of the developing tooth germ. Mitochondrial fission and fusion are highly regulated processes that collectively regulate mitochondrial morphology and function, profoundly impacting cell proliferation by influencing cellular bioenergetics and cell cycle progression [ 30 , 31 , 32 ]. In our study, MitoTracker staining and Western blot analysis revealed that HGF treatment in mDP cells increased mitochondrial area and elevated phosphorylation of Drp1, a key mediator of fission, indicating enhanced mitochondrial fragmentation and activation of proliferative bioenergetic states. Conversely, inhibition of HGF signaling with PHA-665752 decreased Drp1 phosphorylation and reduced mitochondrial staining, consistent with the induction of fusion. Notably, previous studies in dental systems have emphasized the opposite trend: Drp1 inhibition was shown to enhance odontoblast differentiation and accelerate dentinogenesis in tooth germ organ cultures and dental papilla cells[ 33 ]; in dental pulp stem cells exposed to inflammatory stress, excessive fission promoted senescence, whereas fusion preserved proliferative and regenerative potential[ 34 ]; and during root morphogenesis, mitochondrial fusion mediated by OPTN/NRF2 signaling supported odontoblast maturation [ 35 ]. Together, these findings suggest that fusion supports differentiation and long-term survival, while our present data identify a distinct requirement for fission during the proliferative expansion of the dental mesenchyme in early odontogenesis. To further identify the functional importance of mitochondrial fission in HGF-mediated mesenchymal proliferation and tooth germ morphogenesis, we utilized Mdivi-1, a selective inhibitor of Drp1. Treatment with Mdivi-1 suppressed HGF-induced mitochondrial fission in mDP cells, as evidenced by a significant reduction in the area of mitochondrial staining and decreased phosphorylation of Drp1. These findings indicate that mitochondrial fission is not merely a consequence of proliferation but also a necessary step for HGF-driven bioenergetic activation in mesenchymal cells. In agreement with previous reports where Drp1-mediated mitochondrial fission was shown to be essential for ERK-dependent cell cycle progression and proliferation [ 36 ], our results demonstrate that the inhibition of mitochondrial fission is related to HGF-induced cell proliferation during tooth development. In ex vivo organ culture, Mdivi-1 treatment suppressed the HGF-induced increase in tooth germ size, particularly width, and significantly reduced the number of proliferating cells within the mesenchymal compartment, as shown by BrdU staining. These results support the hypothesis that mitochondrial fission is required for mesenchymal proliferation and proper tooth morphogenesis. This observation aligns with earlier findings in which mitochondrial fragmentation promoted the Warburg effect and supported rapid cell division in other proliferative systems [ 37 ]. Notably, underscoring the mesenchyme-specific response to HGF signaling and mitochondrial remodeling, which is consistent with our earlier data using mDP and SF2 cell lines. In contrast, multiple mesenchymal systems report different outcomes of Drp1 inhibition: osteogenic and chondrogenic lineages favor differentiation when fission is restrained [ 38 , 39 ], vascular smooth muscle and pulmonary adventitial fibroblasts reduce pathological proliferation with Mdivi-1 [ 40 ], and hepatic stellate cells show reduced activation in fibrogenic paradigms [ 41 , 42 ]. This finding establishes a mechanistic link between growth factor-mediated signaling and organelle remodeling, which is essential for developmental morphogenesis. To elucidate the downstream mechanisms by which HGF regulates mitochondrial dynamics and mesenchymal proliferation, we examined MAPK signaling in HGF-treated mDP cells. Western blot analysis revealed that HGF stimulation enhanced phosphorylation of ERK1/2, p38, and JNK, confirming broad MAPK pathway activation. This observation aligns with prior work showing that growth factors, including HGF, can activate multiple MAPK branches to regulate proliferation and morphogenesis in developing tissues [ 43 , 44 ]. HGF stimulation activated all three MAPKs in mDP cells, with ERK showing a modest increase, p38 a moderate response, and JNK the most robust activation. Inhibition of c-Met signaling revealed partial dependency for ERK and p38, but strong dependency for JNK, identifying JNK as the most c-Met–responsive MAPK branch. Using pathway-specific inhibitors further demonstrated distinct contributions of these kinases to Drp1 regulation. ERK inhibition significantly reduced HGF-induced Drp1 phosphorylation, p38 inhibition also decreased phosphorylation though to a lesser extent, while JNK inhibition almost completely abolished it. These findings establish JNK as the principal mediator linking HGF–c-Met signaling to Drp1-dependent mitochondrial fission, with ERK and p38 acting as additional contributors. This result is consistent with previous reports in hepatic and cardiac systems showing that JNK-dependent phosphorylation of Drp1 is critical for mitochondrial fragmentation and stress adaptation [ 45 , 46 ]. Our data extend this paradigm to tooth development, providing evidence that extracellular HGF signals regulate mitochondrial remodeling through a JNK–Drp1 axis. Importantly, while ERK has been reported as a key regulator of epithelial proliferation and enamel knot activity during cusp patterning [ 15 ], our findings suggest that ERK also contributes to mesenchymal mitochondrial regulation, highlighting a broader role for this pathway beyond classical epithelial morphogenesis. Likewise, p38 MAPK, previously implicated in stress and differentiation responses [ 47 ], appears to act in a supportive capacity in HGF-mediated mitochondrial remodeling. By integrating these observations, our study identifies for the first time an HGF–JNK–Drp1 signaling axis as a regulator of mitochondrial fission in dental mesenchyme. This represents a novel mechanism by which growth factor signaling can coordinate epithelial–mesenchymal interactions with mitochondrial dynamics, thereby advancing our molecular understanding of odontogenesis. Future work using conditional knockout models for JNK and Drp1, along with single-cell and spatial approaches, will be necessary to confirm the in vivo relevance of this pathway and to determine how mitochondrial remodeling transitions from proliferative to differentiative stages during tooth morphogenesis. Methods Ethics statement ICR mice were purchased from Japan SLC, inc (Shizuoka, Japan). All animal experiments were approved by the ethics committee of the Kyushu University Animal Experiment Center (protocol numbers: A21-397-1 and A23-110-0). All methods were carried out in accordance with relevant guidelines and regulations, and the study was performed in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Organ culture of tooth germs Organ culture of tooth germs was performed following the procedure outlined in our previous report [ 27 ]. Molar tooth germs were dissected from the mandibles of ED 14.5 ICR mice. Tooth germs dissected from 21 embryos were divided randomly into 2 groups: the control, HGF-treated, and PHA-665752 (Sigma-Aldrich, USA) -treated groups. A tooth germ was injected into a 35 µL collagen gel drop of Cellmatrix type I-A, incubated for 10 min at 37°C, and transferred to a cell culture insert (BD Biosciences, San Jose, CA, USA) placed in a 24-well cell culture plate. Tooth germs were cultured with 250 µL/well Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, and 100 µg/mL ascorbic acid at 37°C in a humidified atmosphere of 5% CO 2 . The medium was changed every other day. To stimulate HGF (PeproTech, USA), 0.5 ng/ml HGF and to inhibit HGF signaling, 2 µM PHA-665752 (Sigma‒Aldrich, USA) was added to the collagen gel and the medium in randomly selected tooth germs. In the control group, HGF and PHA-665752 were replaced with bovine serum albumin (BSA; Sigma‒Aldrich, USA) in 0.1% FBS and dimethyl sulfoxide (DMSO; Sigma‒Aldrich, USA), respectively. Measurement of the size of tooth germs Tooth germs were imaged on day 6 of organ culture via a KEYENCE BZ-X810 fluorescence microscope (KEYENCE Corporation, Osaka, Japan). Images were captured at 40X magnification using standardized exposure settings across all samples to ensure comparability. Images were analyzed via BZ-X analyzer software (ver. BX-X800. 1.1.2.4), and the image contrast and brightness were uniformly adjusted without altering the signal intensity. Tissue processing and sectioning The tooth germs were cultured for 6 days. The head tissues, which included mandibular molar tooth germs, were dissected from ED 13.5, 14.5, 16.5, and 18.5 ICR mice. The samples were fixed with 40 mg/mL paraformaldehyde(PFA). The tooth germs were demineralized for 24 h at 4°C using 45 mg/mL ethylenediaminetetraacetic acid. The tooth germs and head tissues were embedded in paraffin, and the tissue sections were cut into 6 µm wide pieces. Bromodeoxyuridine (BrdU) assay The BrdU assay was performed via the BrdU In Situ Detection Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol. The cultured tooth germs were incubated for 4 h with medium supplemented with BrdU labeling reagent (1:100). The tissue sections of the tooth germs were treated with hydrogen peroxide for 10 min, followed by BD Retrievagen A at 89°C for 10 min. The tissue sections were reacted with anti-BrdU antibody for 1 h at room temperature, streptavidin-HRP for 30 min at room temperature, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) for signal detection. The images obtained from the BrdU assay were analyzed via ImageJ software (NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/ ); thresholds of 0 and 200 were used to quantify the BrdU-positive area. We calculated the percentages of the inner enamel epithelium and dental follicle area that were BrdU positive. Immunohistochemistry Deparaffinized sections were immersed in 0.01 M citrate solution, microwaved for 2 min, and then incubated at room temperature for 30 min. The sections were treated with 10% donkey serum (Sigma‒Aldrich) in PBS at room temperature for 1 h and incubated with rabbit anti-c-Met (1:250; Abcam, Cambridge, England) antibody at 4°C overnight. For the negative controls, the primary antibody was omitted. After washing with PBS, the sections were incubated with donkey anti-rabbit IgG Alexa Fluor 568 (1:500; Invitrogen, Carlsbad, CA, USA) at RT for 1 h. 4’,6-Diamidino-2-phenylindole (DAPI; SeraCare, Milford, MA, USA) was used for nucleus detection. The fluorescent signals were visualized via a confocal laser scanning microscope system (C2si; Nikon, Tokyo, Japan). Cell culture mDP cells and SF2 ameloblast-like cells were cultured at 37°C in a humidified incubator with 5% CO₂ in DMEM/F-12 supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were passaged via 0.05% trypsin-EDTA when 70–80% confluence was reached. The cells were serum-starved in DMEM/F-12 containing 0.1% FBS for 4 hours. The cells were subsequently treated with 0.5 ng/mL HGF to activate HGF signaling. To inhibit the HGF/c-Met pathway, cells were treated with 2 µM PHA-665752, a selective c-Met tyrosine kinase inhibitor, according to the manufacturer's protocol. In the control group, HGF was replaced with BSA in 0.1% FBS, and PHA-665752 was replaced with an equivalent volume of DMSO. Mitochondrial staining To evaluate mitochondrial dynamics in mDP cells, MitoBright LT Red (Dojindo Laboratories, Japan), a live-cell fluorescent dye that selectively stains active mitochondria, was used. Prior to cell seeding, 35-mm glass-bottom dishes (Matsunami, Japan) were coated with fibronectin (10 µg/mL in PBS) and incubated for 1 hour at room temperature to enhance cell attachment. The dishes were then rinsed with sterile PBS. mDP cells were seeded at a density of 2.1 × 10⁴ cells/mL (2 ml of cell suspension/dish) and cultured overnight at 37°C in a humidified incubator with 5% CO₂. The following day, the cells were starved for 4 hours in DMEM/F-12 medium containing 0.1% FBS, followed by treatment with 0.5 ng/mL HGF (PeproTech, USA) or PHA-665752; 2 µM/ml (Sigma‒Aldrich, USA) was used for no starved cells. The control group received bovine serum albumin (BSA; Sigma‒Aldrich, USA ) in 0.1% FBS and DMSO/H₂O (1:1, v/v) in place of HGF and PHA-665752, respectively. On Day 2, the cells were washed with HBSS and incubated with MitoBright LT Red working solution, which was prepared by diluting 2 µL of 0.1 mmol/L stock solution into 2 mL of fresh culture medium with a final concentration of 0.1 µmol/L. The cells were incubated for 20 minutes at 37°C and then fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After fixation, the cells were washed twice with HBSS and mounted with Fluoromount-G (Thermo Fisher Scientific, USA). A 20–25 µL drop of mounting medium was applied to the glass-bottom area, which was covered with a round coverslip and allowed to set. Fluorescence images were acquired via the confocal laser scanning microscope system. The signals were quantified via ImageJ software. Western blot Western blotting was performed following the procedure outlined in our previous report [ 25 ]. mDP cells were seeded at a density of 29.5 × 10⁵ cells/100 mm dish and cultured for 2 days. Whole-cell lysates were prepared via CelLytic™ M lysis reagent (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, USA), phosphatase inhibitor cocktail 2 (Sigma-Aldrich, USA), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, USA) at a 1:100 dilution. The protein concentration was measured via the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA), and equal amounts of protein (10 µg for total proteins or 30 µg for phosphoproteins) were denatured in 2-mercaptoethanol–containing SDS sample buffer (Fujifilm), boiled at 95°C for 5 min, and subjected to SDS‒PAGE via 4–15% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad). Proteins were transferred to PVDF membranes via the Trans-Blot® Turbo™ system (Bio-Rad). The membranes were blocked with Block Ace (DS Pharma Biomedica, Japan) containing 0.02% sodium azide for 1.5 h at room temperature and incubated overnight at 4°C with the following primary antibodies diluted in fresh Block Ace: rabbit anti-phospho-ERK1/2 (1:1000), rabbit anti-phospho-p38 MAPK (1:500), rabbit anti-phospho-JNK (1:1000), rabbit anti-Drp1 (1:1000), and mouse anti-GAPDH (1:2000) (all from Cell Signaling Technology). The next day, the membranes were washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse; Jackson ImmunoResearch, USA) diluted 1:10,000 in 5% skim milk for 1 h at room temperature. Bands were visualized via EzWestLumiOne chemiluminescence substrate (ATTO, Japan), and images were acquired via the Amersham™ ImageQuant™ 800 imaging system (Cytiva, Tokyo, Japan). Densitometric analysis of band intensities was performed via ImageJ software (NIH, USA). 8. Statistical analysis BellCurve for Excel (version 3.21; Social Survey Research Information, Tokyo, Japan) and GraphPad Prism (version 10.0; GraphPad Software, San Diego, CA, USA) were used for statistical analysis. All the data are presented as the means ± standard deviations (SDs). The data were statistically analyzed by Student’s t test to compare differences between two groups. For more than two groups, we used analysis of variance (ANOVA) followed by the Tukey‒Kramer test. P values less than 0.05 were considered statistically significant. Declarations Acknowledgments We would like to thank Keigo Yoshizaki and Yao Fu (Orthodontics and Dentofacial Orthopedics, Faculty of Dental Science, Kyushu University) for their technical support. Declaration of conflicts of interest All the authors state that they have no conflicts of interest. Funding This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [21K10156 to NT and 23H03116 to IT]. Author contributions Prodhan MD Rubayet Alam contributed to the data acquisition and interpretation; performed the statistical analyses; drafted the manuscript; Nobuo Takeshita contributed to the conception, design, data acquisition, and interpretation; performed the statistical analyses; and drafted and finished the manuscript; Wei Jiang contributed to the data acquisition; Koji Miyazono contributed to the data acquisition; Satoshi Fukumoto provided mDP and SF2 cells and gave valuable advice on this study; Ichiro Takahashi contributed valuable advice on this study and interpretation; and drafted and finished the manuscript. All the authors gave their final approval and agreed to be accountable for all aspects of the work. 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1","display":"","copyAsset":false,"role":"figure","size":1541105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeveloping tooth germs expressed c-Met. \u003c/strong\u003eMouse mandibular molar tooth germs at ED 13.5 (a−c), 14.5 (d−f), 16.5 (g−i), and 18.5 (j−l) were analyzed via c-Met (a, d, g, j) (red), DAPI labeling (b, e, h, k) (blue), and merged (c, f, i, l). DAPI (blue) is a nucleus leveling probe. m, n and o are magnified images of the rectangular areas in j, k and l, respectively. Negative controls for immunohistochemistry are shown in p, q, and r. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/18c19e14d3aff174d768601d.png"},{"id":94369669,"identity":"acda65d3-5ded-4bd1-80fc-fb6f04bd5c64","added_by":"auto","created_at":"2025-10-27 13:18:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":768680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecombinant murine HGF increased while PHA-665752 decreased the size of tooth germs in organ culture. \u003c/strong\u003eHGF and its inhibitor PHA-665752 were added to the organ culture of molar tooth germs isolated from ED 14.5 mouse mandibles. (a) Phase contrast images of tooth germs at days 0 and 6 of organ culture in the control and HGF-treated groups. (b) Phase contrast images of tooth germs at days 0 and 6 of organ culture in the control and PHA-665752-treated groups. Scale bar, 200 μm. The width (b, e) and height (c, f) of tooth germs on day 6 were measured in the control, HGF-treated, and PHA-665752-treated groups (n=11−13), respectively. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/8e54dceef2a30b19774af71d.png"},{"id":94489079,"identity":"5e23caac-6538-449b-b92b-aca30cd9b9c0","added_by":"auto","created_at":"2025-10-27 17:02:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1145715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecombinant murine HGF was induced, while PHA-665752 inhibited the proliferation of mesenchymal cells during tooth germ development. \u003c/strong\u003e(a, d) BrdU assay images of mandibular molar tooth germs on day 6 of organ culture in the control, HGF-treated (a) and PHA-665752-treated (d) groups. Scale bar, 200 μm. (b, c) Quantitative data of the BrdU assay of the epithelial stained area (b) and mesenchymal stained area (c). **p \u0026lt; 0.01, n=5. (e, f) Quantitative data of the BrdU assay of the epithelial stained area (e) and mesenchymal stained area (f). **p \u0026lt; 0.01, n=5. (g, h, i, j) SF2 and mDP cells were cultured for 1 day and then starved and cultured with HGF (g, i) and without starvation cultured with PHA-665752 (h, j) for another 1 day. The cells were cultured independently three times. *p \u0026lt; 0.05, n=3.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/65f96710c6f4c43002924900.png"},{"id":94369677,"identity":"60dc28f1-7e09-4af6-9896-a7b3862cb63a","added_by":"auto","created_at":"2025-10-27 13:18:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":593923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGF and PHA-665752 induce fission and fusion, respectively, of mitochondria in mDP cells. \u003c/strong\u003e(a, b) MitoTracker LT Red was used to stain mitochondria in control and HGF-treated mDP cells (a), and quantification of the mitochondrial-stained area relative to total cells was performed (b). *p \u0026lt; 0.05, n=20. (c, d) MitoTracker LT Red-stained mitochondria in control and PHA-665752-treated mDP cells (c). Quantification analysis of the mitochondrial stained area relative to total cells was performed (d), \u003cstrong\u003e**\u003c/strong\u003ep \u0026lt; 0.01, n=20. (e, f) Western blot analysis of phosphorylated Drp1 (p-Drp1) and total Drp1 levels in control and HGF-treated cells (e), and quantification of the intense signal was performed (f). \u003cstrong\u003e*\u003c/strong\u003ep \u0026lt; 0.05, n=3. (g, h) Western blot analysis (g) and quantification (h) of p-Drp1 and total Drp1 levels in control and PHA-treated cells. n=3.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/13efb8173f296661a749a242.png"},{"id":94369809,"identity":"df803656-085c-4000-afc7-9665c84697a2","added_by":"auto","created_at":"2025-10-27 13:19:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1275537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMdivi-1 suppressed mitochondrial dynamics, cell proliferation, and tooth morphogenesis\u003c/strong\u003e. (a, b) MitoTracker LT Red-stained mitochondria of mDP cells treated with HGF and Mdivi (a), and quantification of the mitochondrial stained area relative to that of total cells was performed (b). ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, n=20. (c, d) Western blot analysis of phosphorylated Drp1 (p-Drp1) and total Drp1 expression after HGF and/or Mdivi-1 treatment; GAPDH served as the loading control (c). Densitometric quantification of the p-Drp1/Drp1 ratio via Western blot analysis (d), n = 3. (e,f,g) Phase contrast images of tooth germs at days 0 and 6 of organ culture in the control and HGF- and Mdivi-1-treated groups (e), width (f) and height (g). Scale bar, 200 μm. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, n = 6. mDP cells were cultured with HGF and Mdivi-1. **p \u0026lt; 0.01(h), n=3. Images of the BrdU assay of the mandibular molar tooth germs on day 6 of organ culture in HGF- and Mdivi-1-treated cultures (i). Scale bar, 200 μm. (j, k) Quantitative data from the BrdU assay of the mesenchymal-stained area (j) and epithelial-stained area (k). *p \u0026lt; 0.05, (n=6).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/d7c19a54610d092ca01444d9.png"},{"id":94369595,"identity":"66aefabc-82af-44d9-9740-d96a8a071f98","added_by":"auto","created_at":"2025-10-27 13:18:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":964565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGF induces Drp1 phosphorylation through MAPK signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a–f) Western blot analysis and quantification of phosphorylated and total MAPKs in mDP cells treated with HGF. (a, b) ERK1/2; (c, d) p38; (e, f) JNK. GAPDH was used as a loading control. The quantitative data are presented as the ratio of phosphorylated protein levels to total protein levels. *p \u0026lt; 0.05, ***p \u0026lt; 0.001, (n = 5). (g–l) Effects of c-Met inhibition by PHA-665752 on ERK (g, h), p38 (i, j), and JNK (k, l). The quantitative data are presented as the ratio of phosphorylated protein levels to total protein levels. *p \u0026lt; 0.05, ***p \u0026lt; 0.001, (n = 5). (m–r) Western blot analysis of phosphorylated Drp1 (p-Drp1) and total Drp1 after HGF treatment in the presence or absence of specific MAPK inhibitors: U0126 (ERK inhibitor; m, n), SB203580 (p38 inhibitor; o, p), and SP600125 (JNK inhibitor; q, r). GAPDH was used as the loading control. The quantification of thep-Drp1/Drp1 ratio is shown for each condition. Statistical analysis revealed that the number of mDp cells treated with SB203580 (p38 inhibitor; o, p) was significantly different between the HGF-treated group and the p38 inhibitor-treated group, and the number of SP600125 (JNK inhibitor; q, r) was significantly different between the control, HGF-treated and the JNK inhibitor-treated HGF groups. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001 (n = 5).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/32f4f1939c07ed8bc8c54684.png"},{"id":99796812,"identity":"15570cb9-14b5-4303-a40c-e972fd406773","added_by":"auto","created_at":"2026-01-08 13:43:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7429794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7493229/v1/e9fbcb22-a77b-49b9-ba31-b08d48b24740.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hepatocyte growth factor signaling regulates tooth germ development, inducing proliferation of mesenchymal cells via fission of mitochondria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpithelial‒mesenchymal interaction (EMI) is essential for the early development of various organs composed of the epithelium and mesenchyme, including teeth, hair, lungs and kidneys[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Teeth contribute not only to mastication but also to pronunciation/speech and aesthetics, and these essential roles must be properly maintained with normal occlusion and appropriate tooth morphology. Multiple signaling pathways, including the hepatocyte growth factor (HGF) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], wingless (Wnt) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and epidermal growth factor (EGF) pathways, are known regulators of EMI [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and contribute to organogenesis in the abovementioned organs. These growth factors and cytokines are known to regulate the proliferation of epithelial and mesenchymal cells during tooth development through EMI. Among them, HGF is a ligand of c-Met, a transmembrane receptor containing a tyrosine kinase domain that plays a significant role in organogenesis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During tooth development, both HGF and c-Met are expressed in epithelial and mesenchymal tissues, suggesting their potential role in regulating tooth morphogenesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While this finding implies that HGF signaling regulates the proliferation of precursor cells and their differentiation into specific dental cells, the precise role of HGF signaling in the proliferation of mesenchymal cells within tooth germs during tooth development remains largely unknown.\u003c/p\u003e\u003cp\u003eMitochondria are central regulators of cellular homeostasis, linking energy metabolism with developmental and differentiation programs by their dynamic fission and fusion, acting as key determinants of cell fate[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Fission is largely mediated by Drp1 which supports cell cycle progression and progenitor proliferation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while fusion sustains mitochondrial integrity during differentiation and tissue morphogenesis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Inhibition of Drp1 in developing teeth accelerates odontoblast differentiation and dentinogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], highlighting that excessive fission limits maturation, whereas regulated remodeling is required for morphogenesis. These findings support the concept that mitochondrial dynamics is crucial for development and balancing mesenchymal proliferation.\u003c/p\u003e\u003cp\u003eMitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that link extracellular cues to intracellular responses, regulating proliferation, differentiation, and survival[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In craniofacial and tooth development, ERK signaling is essential for epithelial proliferation and enamel knot activity, serving as a central regulator of cusp morphogenesis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. p38 MAPK contributes to odontoblast differentiation and dentin matrix secretion through BMP-2\u0026ndash;driven transcriptional programs[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. JNK, initially characterized in apoptosis and stress responses[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], has since been linked to mitochondrial remodeling and bioenergetic control, processes relevant to proliferative expansion during organogenesis. Since HGF-c-Met activates multiple MAPK branches[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], dissecting their contribution to mitochondrial remodeling provides a mechanistic basis for understanding how growth factor signaling drives mesenchymal proliferation during tooth germ development.\u003c/p\u003e\u003cp\u003eThe purpose of the present study was to examine the function of HGF signaling in the proliferation of mesenchymal cells during tooth germ development and to clarify its underlying mechanism, with a focus on mitochondrial fission and fusion. We first analyzed the expression of c-Met during tooth germ development in embryonic mice. Next, we administered HGF signaling inhibitors in an organ culture of mouse tooth germs to analyze the roles of HGF signaling during tooth germ development. The results indicated that the HGF signaling inhibitor suppressed the growth of tooth germs and the proliferation of mesenchymal cells in tooth germs. We examined whether mitochondrial dynamics are involved in the HGF signaling-modulating proliferation of tooth germ mesenchymal cells. Furthermore, we explored the downstream activation of MAPK cascades to determine their regulatory contribution to HGF-induced mitochondrial remodeling and mesenchymal cell proliferation during odontogenesis. In conclusion, our data revealed that HGF signaling induces the proliferation of mesenchymal cells via the regulation of mitochondrial dynamics during tooth germ development.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDevelopment of tooth germs expressing c-Met\u003c/h2\u003e\u003cp\u003eIt is known that mouse mandibular molar tooth germs at embryonic day (ED) 13.5 are in the bud stage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. At this stage, strong fluorescent signals of c-Met were detected in the epithelium; however, the signals were faintly detected in the mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c). At ED 14.5, the tooth germs are in the cap stage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The fluorescent signals of c-Met were still detected in the epithelium and the signals in the mesenchyme are more clearly observed than at ED 13.5. At ED 16.5, the tooth germs are in the bell stage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The signals of c-Met were observed in the enamel organ and the dental papilla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u0026thinsp;\u0026minus;\u0026thinsp;i).At ED 18.5, the development of tooth germs further progressed, with a dramatic increase in size [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The fluorescent signals of c-Met at this stage were still detected in the enamel organ and dental papilla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej\u0026thinsp;\u0026minus;\u0026thinsp;o). The negative controls for c-Met staining produced no signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ep\u0026thinsp;\u0026minus;\u0026thinsp;r).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRecombinant murine HGF increased while PHA-665752 decreased the size of tooth germs in organ culture\u003c/h3\u003e\n\u003cp\u003eTo analyze the function of HGF signaling during tooth germ development, we first isolated mouse mandibular molar tooth germs at the bud stage (ED 14.5) and cultured them \u003cem\u003eex vivo\u003c/em\u003e to administer recombinant HGF. The sizes of the tooth germs appeared to be same in size between the groups on day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) In both groups, tooth germs showed cusp development and an increase in the size of tooth germs on day 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The size of the tooth germs in the HGF group was greater than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Quantitative measurements revealed significant increases in height and width in the HGF group compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). To further validate the functional involvement of HGF signaling, tooth germs were cultured with PHA-665752, a selective inhibitor of c-Met/HGF signaling. The size of the tooth germs in the PHA-665752 group was smaller than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Quantitative analysis revealed significant decreases in height and width in the PHA-665752 group compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). These findings demonstrate that the activation of HGF signaling promotes, whereas its inhibition suppresses, the growth of developing tooth germs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecombinant murine HGF was induced, while PHA-665752 inhibited the proliferation of mesenchymal cells during tooth germ development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe proliferation of cells is closely associated with organ growth during embryogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We therefore hypothesized that HGF signaling induced the growth of tooth germs by increasing cellular proliferation. To evaluate this hypothesis, BrdU incorporation was assessed in organ-cultured tooth germs treated with recombinant HGF and PHA-665752. HGF did not affect the BrdU-stained area in the epithelium but significantly increased the stained area in the mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026thinsp;\u0026minus;\u0026thinsp;c). PHA-665752 also did not affect the proliferation of epithelial cells but significantly reduced the proliferation of mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026thinsp;\u0026minus;\u0026thinsp;f). Furthermore, to examine the effect of HGF signaling on the proliferation of dental epithelial and mesenchymal cells, the number of cells was quantified after treatment with recombinant HGF and PHA-665752. SF2 cells are known as precursor ameloblasts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. HGF and PHA-665752 did not significantly affect the number of SF2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h). Mouse dental papilla (mDP) cells are derived from mouse dental papilla and are widely used as a model for odontoblast precursor cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Compared with control cells, HGF- and PHA-665752-treated mDP cells presented significantly greater and lower cell numbers, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, j). These results indicated that HGF signaling promoted the proliferation of mesenchymal cells during tooth germ development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecombinant murine HGF and PHA-665752 induced fission and fusion, respectively, of mitochondria in mDP cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the role of HGF signaling in mitochondrial dynamics in mDP cells, we performed mitochondrial staining and evaluated the mitochondrial area relative to the total cell area. In addition, we analyzed the phosphorylation status of dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fission. mDP cells stained with MitoTracker revealed the morphology of the mitochondrial network under control and HGF-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Quantification of the ratio of the area of stained mitochondria to the total cell area revealed a significant decrease in the mitochondrial area upon HGF treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), suggesting enhanced mitochondrial propagation. Treatment with the HGF signaling inhibitor PHA-665752 led to a more fragmented mitochondrial morphology, as observed via MitoTracker staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Quantification revealed a significant increase in the mitochondrial area relative to the total cell area in the PHA-treated group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Western blot analysis of p-Drp1 (phospho-Drp1) levels in control and HGF-treated mDP cells revealed that HGF significantly increased Drp1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Densitometric analysis normalized to total Drp1 confirmed a greater p-Drp1/Drp1 ratio in the HGF-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), indicating HGF-induced activation of mitochondrial fission signaling. In contrast, cells treated with PHA-665752 displayed reduced phosphorylation of Drp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Compared with that in the control group, quantitative analysis of the p-Drp1/Drp1 ratio revealed a significant decrease in Drp1 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), suggesting that inhibition of HGF signaling suppresses mitochondrial fission.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMdivi-1 suppressed HGF-induced mitochondrial dynamics, cell proliferation and tooth morphogenesis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe involvement of mitochondrial fission in HGF-induced responses in dental mesenchymal cells and developing tooth germs was assessed by treating mDP cells with Mdivi-1, a selective inhibitor of Drp1-mediated mitochondrial fission, in the presence or absence of HGF. mDP cells demonstrated that HGF treatment markedly increased the area of mitochondrial staining, which is indicative of mitochondrial expansion or biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Compared with that in the control group, the ratio of the mitochondrial-stained area to the total cell area in the HGF group was significantly greater, whereas cotreatment with Mdivi-1 attenuated this increase, suggesting that HGF-induced mitochondrial expansion requires active mitochondrial fission (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Western blot analysis revealed that HGF increased the level of phosphorylated Drp1, whereas total Drp1 levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Densitometric quantification confirmed a significant increase in the p-Drp1/Drp1 ratio in the HGF group, which was suppressed by cotreatment with Mdivi-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These results indicate that HGF induced mitochondrial fission through the phosphorylation of Drp1 and that this process is inhibited by Mdivi-1. Furthermore, we examined whether the inhibition of mitochondrial fission affects the morphogenesis of developing tooth germs. Mandibular molar tooth germs isolated at E14.5 were cultured ex vivo in the presence or absence of HGF and Mdivi-1. Compared with those in the control group, the HGF increased size of the tooth germ whereas Mdivi-1 inhibited the effect of HGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Morphometric analysis revealed that both the width and height of the tooth germs were significantly increased by HGF, and these effects were suppressed by Mdivi-1 cotreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,g). mDP cell proliferation was also assessed by the incorporation of Mdivi-1 and HGF. The changes were significant between the HGF group and the Mdivi-1-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Mesenchymal cell proliferation within the tooth germs was evaluated via BrdU incorporation during tooth germ development. The number of BrdU-positive mesenchymal cells per tooth germ was significantly greater in the HGF-treated group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei,j). This increase was markedly reduced by Mdivi-1, suggesting that mitochondrial fission is required for HGF-induced proliferation of dental mesenchymal cells. On the other hand, the number of BrdU-stained epithelial cells was almost the same in all the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHGF induces Drp1 phosphorylation through MAPK signaling.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated the upstream signaling pathways mediating this HGF\u0026ndash;Drp1 axis focusing MAP kinase signaling pathway. HGF stimulation significantly increased phosphorylation of ERK1/2, p38, and JNK in mDP cells, with differences in the magnitude of response between pathways. ERK phosphorylation showed a modest but significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b), p38 activation was moderate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d), and JNK phosphorylation was the most robust (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). Inhibition of c-Met signaling with PHA-665752 revealed pathway selectivity, as ERK and p38 phosphorylation were only partially reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg\u0026ndash;j), whereas JNK activation was strongly suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek, l). These data indicate that all three MAPKs are activated downstream of HGF, with JNK representing the most c-Met\u0026ndash;dependent branch.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the contribution of these pathways to Drp1 activation, the effect of pathway-specific inhibitors on HGF-induced Drp1 phosphorylation was examined. ERK inhibition with U0126 significantly reduced HGF-induced Drp1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003em, n). Inhibition of p38 with SB203580 also decreased Drp1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eo, p), though the reduction was less pronounced. In contrast, JNK inhibition with SP600125 almost completely abolished HGF-induced Drp1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eq, r).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we revealed an important mechanism through which HGF signaling regulates tooth germ development by promoting mesenchymal cell proliferation via mitochondrial fission. By linking an extracellular growth factor pathway to intracellular organelle remodeling, our work provides new insight into the molecular and cellular processes underlying odontogenesis.\u003c/p\u003e\u003cp\u003eOur immunofluorescence analysis demonstrated that c-Met is expressed throughout the sequential phases of tooth development with spatial and temporal changes in localization. During the bud (ED 13.5) and cap (ED 14.5) stages, c-Met expression was mainly localized in the epithelium, while weak mesenchymal signals at ED13.5 became more pronounced by ED 14.5. This expression pattern suggests that HGF/c-Met signaling contributes to early epithelial morphogenesis and progressively intensifies in the mesenchyme, supporting the epithelial\u0026ndash;mesenchymal interactions that drive bud elongation and cap formation. By the bell stage (ED16.5), c-Met expression became more broadly distributed, with detectable signals in both the enamel organ and the dental papilla. This shift indicates that mesenchymal tissues progressively acquire responsiveness to HGF signaling, consistent with the increased proliferation and differentiation requirements of the papilla as the tooth germ matures. At ED18.5, c-Met signals remained in the dental papilla, in line with the substantial increase in tooth germ size and cellular complexity. The persistence of c-Met expression into late developmental stages suggests that HGF signaling is not restricted to early morphogenesis but continues to regulate proliferation and tissue remodeling during bell stage differentiation. Earlier studies, however, were restricted to single developmental windows or focused primarily on either epithelial or mesenchymal compartments, leaving the sequential progression of c-Met expression during odontogenesis incompletely understood [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, previous work has suggested a role for HGF in dental morphogenesis, but most studies were limited to correlative expression analyses or cell culture systems that c-Met is expressed in enamel epithelium and HGF in dental mesenchyme at the cap stage, implying a paracrine role in epithelial-mesenchymal interactions[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Other showed c-Met immunolocalization in human fetal tooth germs, with strong epithelial staining and weaker mesenchymal signals[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our findings provide a stage resolved analysis of c-Met expression in mouse molars, showing its transition from epithelial restriction to combined epithelial and mesenchymal distribution. This mapping confirms that HGF/c-Met signaling persists across critical stages of tooth development.\u003c/p\u003e\u003cp\u003eExogenous HGF stimulation significantly promoted the growth of molar tooth germs, while pharmacological inhibition of c-Met with PHA-665752 suppressed their expansion. Importantly, tooth germs in all groups were comparable in size at day 0 confirming that the observed differences arose during the culture period. By day 6, HGF-treated tooth germs exhibited greater dimensions, whereas PHA-665752 treatment significantly reduced both height and width. These results suggests that HGF-c-Met signaling directly contributes to tooth germ growth during the bud-to-cap transition. Previous studies identified the presence of HGF/c-Met components[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], they did not provide direct functional evidence of how manipulating the pathway alters tooth germ growth. Our findings extend this knowledge by providing functional validation in an ex vivo organ culture model, demonstrating that activation of HGF signaling accelerates, while inhibition retards, the growth of developing tooth germs. A similar HGF/c-Met-dependent epithelial-mesenchymal mechanism operates in other branching organs, including the lung, kidney, mammary gland, and salivary gland, where exogenous HGF promotes, and HGF inhibition suppresses, branching morphogenesis in organ culture. In these systems, genetic and biochemical evidence consistently positions c-Met on epithelial cells receiving mesenchymal HGF signals, highlighting a paracrine mode of action [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. BrdU incorporation assays revealed that HGF treatment significantly increased the proliferation of mesenchymal cells within the tooth germ, whereas PHA-665752 treatment markedly suppressed this process. These findings collectively highlight that HGF signaling enhances mesenchymal cell proliferation in an autocrine manner that contributes to the morphological expansion of the developing tooth germ.\u003c/p\u003e\u003cp\u003eMitochondrial fission and fusion are highly regulated processes that collectively regulate mitochondrial morphology and function, profoundly impacting cell proliferation by influencing cellular bioenergetics and cell cycle progression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, MitoTracker staining and Western blot analysis revealed that HGF treatment in mDP cells increased mitochondrial area and elevated phosphorylation of Drp1, a key mediator of fission, indicating enhanced mitochondrial fragmentation and activation of proliferative bioenergetic states. Conversely, inhibition of HGF signaling with PHA-665752 decreased Drp1 phosphorylation and reduced mitochondrial staining, consistent with the induction of fusion. Notably, previous studies in dental systems have emphasized the opposite trend: Drp1 inhibition was shown to enhance odontoblast differentiation and accelerate dentinogenesis in tooth germ organ cultures and dental papilla cells[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; in dental pulp stem cells exposed to inflammatory stress, excessive fission promoted senescence, whereas fusion preserved proliferative and regenerative potential[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]; and during root morphogenesis, mitochondrial fusion mediated by OPTN/NRF2 signaling supported odontoblast maturation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Together, these findings suggest that fusion supports differentiation and long-term survival, while our present data identify a distinct requirement for fission during the proliferative expansion of the dental mesenchyme in early odontogenesis.\u003c/p\u003e\u003cp\u003eTo further identify the functional importance of mitochondrial fission in HGF-mediated mesenchymal proliferation and tooth germ morphogenesis, we utilized Mdivi-1, a selective inhibitor of Drp1. Treatment with Mdivi-1 suppressed HGF-induced mitochondrial fission in mDP cells, as evidenced by a significant reduction in the area of mitochondrial staining and decreased phosphorylation of Drp1. These findings indicate that mitochondrial fission is not merely a consequence of proliferation but also a necessary step for HGF-driven bioenergetic activation in mesenchymal cells. In agreement with previous reports where Drp1-mediated mitochondrial fission was shown to be essential for ERK-dependent cell cycle progression and proliferation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], our results demonstrate that the inhibition of mitochondrial fission is related to HGF-induced cell proliferation during tooth development. In ex vivo organ culture, Mdivi-1 treatment suppressed the HGF-induced increase in tooth germ size, particularly width, and significantly reduced the number of proliferating cells within the mesenchymal compartment, as shown by BrdU staining. These results support the hypothesis that mitochondrial fission is required for mesenchymal proliferation and proper tooth morphogenesis. This observation aligns with earlier findings in which mitochondrial fragmentation promoted the Warburg effect and supported rapid cell division in other proliferative systems [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, underscoring the mesenchyme-specific response to HGF signaling and mitochondrial remodeling, which is consistent with our earlier data using mDP and SF2 cell lines. In contrast, multiple mesenchymal systems report different outcomes of Drp1 inhibition: osteogenic and chondrogenic lineages favor differentiation when fission is restrained [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], vascular smooth muscle and pulmonary adventitial fibroblasts reduce pathological proliferation with Mdivi-1 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and hepatic stellate cells show reduced activation in fibrogenic paradigms [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This finding establishes a mechanistic link between growth factor-mediated signaling and organelle remodeling, which is essential for developmental morphogenesis.\u003c/p\u003e\u003cp\u003eTo elucidate the downstream mechanisms by which HGF regulates mitochondrial dynamics and mesenchymal proliferation, we examined MAPK signaling in HGF-treated mDP cells. Western blot analysis revealed that HGF stimulation enhanced phosphorylation of ERK1/2, p38, and JNK, confirming broad MAPK pathway activation. This observation aligns with prior work showing that growth factors, including HGF, can activate multiple MAPK branches to regulate proliferation and morphogenesis in developing tissues [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. HGF stimulation activated all three MAPKs in mDP cells, with ERK showing a modest increase, p38 a moderate response, and JNK the most robust activation. Inhibition of c-Met signaling revealed partial dependency for ERK and p38, but strong dependency for JNK, identifying JNK as the most c-Met\u0026ndash;responsive MAPK branch. Using pathway-specific inhibitors further demonstrated distinct contributions of these kinases to Drp1 regulation. ERK inhibition significantly reduced HGF-induced Drp1 phosphorylation, p38 inhibition also decreased phosphorylation though to a lesser extent, while JNK inhibition almost completely abolished it. These findings establish JNK as the principal mediator linking HGF\u0026ndash;c-Met signaling to Drp1-dependent mitochondrial fission, with ERK and p38 acting as additional contributors.\u003c/p\u003e\u003cp\u003eThis result is consistent with previous reports in hepatic and cardiac systems showing that JNK-dependent phosphorylation of Drp1 is critical for mitochondrial fragmentation and stress adaptation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our data extend this paradigm to tooth development, providing evidence that extracellular HGF signals regulate mitochondrial remodeling through a JNK\u0026ndash;Drp1 axis. Importantly, while ERK has been reported as a key regulator of epithelial proliferation and enamel knot activity during cusp patterning [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], our findings suggest that ERK also contributes to mesenchymal mitochondrial regulation, highlighting a broader role for this pathway beyond classical epithelial morphogenesis. Likewise, p38 MAPK, previously implicated in stress and differentiation responses [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], appears to act in a supportive capacity in HGF-mediated mitochondrial remodeling.\u003c/p\u003e\u003cp\u003eBy integrating these observations, our study identifies for the first time an HGF\u0026ndash;JNK\u0026ndash;Drp1 signaling axis as a regulator of mitochondrial fission in dental mesenchyme. This represents a novel mechanism by which growth factor signaling can coordinate epithelial\u0026ndash;mesenchymal interactions with mitochondrial dynamics, thereby advancing our molecular understanding of odontogenesis. Future work using conditional knockout models for JNK and Drp1, along with single-cell and spatial approaches, will be necessary to confirm the in vivo relevance of this pathway and to determine how mitochondrial remodeling transitions from proliferative to differentiative stages during tooth morphogenesis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003eICR mice were purchased from Japan SLC, inc (Shizuoka, Japan). All animal experiments were approved by the ethics committee of the Kyushu University Animal Experiment Center (protocol numbers: A21-397-1 and A23-110-0). All methods were carried out in accordance with relevant guidelines and regulations, and the study was performed in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eOrgan culture of tooth germs\u003c/h2\u003e\u003cp\u003eOrgan culture of tooth germs was performed following the procedure outlined in our previous report [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Molar tooth germs were dissected from the mandibles of ED 14.5 ICR mice. Tooth germs dissected from 21 embryos were divided randomly into 2 groups: the control, HGF-treated, and PHA-665752 (Sigma-Aldrich, USA) -treated groups. A tooth germ was injected into a 35 \u0026micro;L collagen gel drop of Cellmatrix type I-A, incubated for 10 min at 37\u0026deg;C, and transferred to a cell culture insert (BD Biosciences, San Jose, CA, USA) placed in a 24-well cell culture plate. Tooth germs were cultured with 250 \u0026micro;L/well Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 \u0026micro;g/mL streptomycin, 2 mM L-glutamine, and 100 \u0026micro;g/mL ascorbic acid at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was changed every other day. To stimulate HGF (PeproTech, USA), 0.5 ng/ml HGF and to inhibit HGF signaling, 2 \u0026micro;M PHA-665752 (Sigma‒Aldrich, USA) was added to the collagen gel and the medium in randomly selected tooth germs. In the control group, HGF and PHA-665752 were replaced with bovine serum albumin (BSA; Sigma‒Aldrich, USA) in 0.1% FBS and dimethyl sulfoxide (DMSO; Sigma‒Aldrich, USA), respectively.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMeasurement of the size of tooth germs\u003c/h3\u003e\n\u003cp\u003eTooth germs were imaged on day 6 of organ culture via a KEYENCE BZ-X810 fluorescence microscope (KEYENCE Corporation, Osaka, Japan). Images were captured at 40X magnification using standardized exposure settings across all samples to ensure comparability. Images were analyzed via BZ-X analyzer software (ver. BX-X800. 1.1.2.4), and the image contrast and brightness were uniformly adjusted without altering the signal intensity.\u003c/p\u003e\n\u003ch3\u003eTissue processing and sectioning\u003c/h3\u003e\n\u003cp\u003eThe tooth germs were cultured for 6 days. The head tissues, which included mandibular molar tooth germs, were dissected from ED 13.5, 14.5, 16.5, and 18.5 ICR mice. The samples were fixed with 40 mg/mL paraformaldehyde(PFA). The tooth germs were demineralized for 24 h at 4\u0026deg;C using 45 mg/mL ethylenediaminetetraacetic acid. The tooth germs and head tissues were embedded in paraffin, and the tissue sections were cut into 6 \u0026micro;m wide pieces.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBromodeoxyuridine (BrdU) assay\u003c/h2\u003e\u003cp\u003eThe BrdU assay was performed via the BrdU In Situ Detection Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. The cultured tooth germs were incubated for 4 h with medium supplemented with BrdU labeling reagent (1:100). The tissue sections of the tooth germs were treated with hydrogen peroxide for 10 min, followed by BD Retrievagen A at 89\u0026deg;C for 10 min. The tissue sections were reacted with anti-BrdU antibody for 1 h at room temperature, streptavidin-HRP for 30 min at room temperature, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) for signal detection. The images obtained from the BrdU assay were analyzed via ImageJ software (NIH, Bethesda, MD, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); thresholds of 0 and 200 were used to quantify the BrdU-positive area. We calculated the percentages of the inner enamel epithelium and dental follicle area that were BrdU positive.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eDeparaffinized sections were immersed in 0.01 M citrate solution, microwaved for 2 min, and then incubated at room temperature for 30 min. The sections were treated with 10% donkey serum (Sigma‒Aldrich) in PBS at room temperature for 1 h and incubated with rabbit anti-c-Met (1:250; Abcam, Cambridge, England) antibody at 4\u0026deg;C overnight. For the negative controls, the primary antibody was omitted. After washing with PBS, the sections were incubated with donkey anti-rabbit IgG Alexa Fluor 568 (1:500; Invitrogen, Carlsbad, CA, USA) at RT for 1 h. 4\u0026rsquo;,6-Diamidino-2-phenylindole (DAPI; SeraCare, Milford, MA, USA) was used for nucleus detection. The fluorescent signals were visualized via a confocal laser scanning microscope system (C2si; Nikon, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003emDP cells and SF2 ameloblast-like cells were cultured at 37\u0026deg;C in a humidified incubator with 5% CO₂ in DMEM/F-12 supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. The cells were passaged via 0.05% trypsin-EDTA when 70\u0026ndash;80% confluence was reached. The cells were serum-starved in DMEM/F-12 containing 0.1% FBS for 4 hours. The cells were subsequently treated with 0.5 ng/mL HGF to activate HGF signaling. To inhibit the HGF/c-Met pathway, cells were treated with 2 \u0026micro;M PHA-665752, a selective c-Met tyrosine kinase inhibitor, according to the manufacturer's protocol. In the control group, HGF was replaced with BSA in 0.1% FBS, and PHA-665752 was replaced with an equivalent volume of DMSO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial staining\u003c/h2\u003e\u003cp\u003eTo evaluate mitochondrial dynamics in mDP cells, MitoBright LT Red (Dojindo Laboratories, Japan), a live-cell fluorescent dye that selectively stains active mitochondria, was used. Prior to cell seeding, 35-mm glass-bottom dishes (Matsunami, Japan) were coated with fibronectin (10 \u0026micro;g/mL in PBS) and incubated for 1 hour at room temperature to enhance cell attachment. The dishes were then rinsed with sterile PBS. mDP cells were seeded at a density of 2.1 \u0026times; 10⁴ cells/mL (2 ml of cell suspension/dish) and cultured overnight at 37\u0026deg;C in a humidified incubator with 5% CO₂. The following day, the cells were starved for 4 hours in DMEM/F-12 medium containing 0.1% FBS, followed by treatment with 0.5 ng/mL HGF (PeproTech, USA) or PHA-665752; 2 \u0026micro;M/ml (Sigma‒Aldrich, USA) was used for no starved cells. The control group received bovine serum albumin (BSA; Sigma‒Aldrich, USA\u003cb\u003e)\u003c/b\u003e in 0.1% FBS and DMSO/H₂O (1:1, v/v) in place of HGF and PHA-665752, respectively. On Day 2, the cells were washed with HBSS and incubated with MitoBright LT Red working solution, which was prepared by diluting 2 \u0026micro;L of 0.1 mmol/L stock solution into 2 mL of fresh culture medium with a final concentration of 0.1 \u0026micro;mol/L. The cells were incubated for 20 minutes at 37\u0026deg;C and then fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After fixation, the cells were washed twice with HBSS and mounted with Fluoromount-G (Thermo Fisher Scientific, USA). A 20\u0026ndash;25 \u0026micro;L drop of mounting medium was applied to the glass-bottom area, which was covered with a round coverslip and allowed to set. Fluorescence images were acquired via the confocal laser scanning microscope system. The signals were quantified via ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eWestern blotting was performed following the procedure outlined in our previous report [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. mDP cells were seeded at a density of 29.5 \u0026times; 10⁵ cells/100 mm dish and cultured for 2 days. Whole-cell lysates were prepared via CelLytic\u0026trade; M lysis reagent (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, USA), phosphatase inhibitor cocktail 2 (Sigma-Aldrich, USA), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, USA) at a 1:100 dilution. The protein concentration was measured via the Pierce\u0026trade; BCA Protein Assay Kit (Thermo Scientific, USA), and equal amounts of protein (10 \u0026micro;g for total proteins or 30 \u0026micro;g for phosphoproteins) were denatured in 2-mercaptoethanol\u0026ndash;containing SDS sample buffer (Fujifilm), boiled at 95\u0026deg;C for 5 min, and subjected to SDS‒PAGE via 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Gels (Bio-Rad). Proteins were transferred to PVDF membranes via the Trans-Blot\u0026reg; Turbo\u0026trade; system (Bio-Rad). The membranes were blocked with Block Ace (DS Pharma Biomedica, Japan) containing 0.02% sodium azide for 1.5 h at room temperature and incubated overnight at 4\u0026deg;C with the following primary antibodies diluted in fresh Block Ace: rabbit anti-phospho-ERK1/2 (1:1000), rabbit anti-phospho-p38 MAPK (1:500), rabbit anti-phospho-JNK (1:1000), rabbit anti-Drp1 (1:1000), and mouse anti-GAPDH (1:2000) (all from Cell Signaling Technology). The next day, the membranes were washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse; Jackson ImmunoResearch, USA) diluted 1:10,000 in 5% skim milk for 1 h at room temperature. Bands were visualized via EzWestLumiOne chemiluminescence substrate (ATTO, Japan), and images were acquired via the Amersham\u0026trade; ImageQuant\u0026trade; 800 imaging system (Cytiva, Tokyo, Japan). Densitometric analysis of band intensities was performed via ImageJ software (NIH, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003e8. Statistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBellCurve for Excel (version 3.21; Social Survey Research Information, Tokyo, Japan) and GraphPad Prism (version 10.0; GraphPad Software, San Diego, CA, USA) were used for statistical analysis. All the data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs). The data were statistically analyzed by Student\u0026rsquo;s t test to compare differences between two groups. For more than two groups, we used analysis of variance (ANOVA) followed by the Tukey‒Kramer test. P values less than 0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank\u0026nbsp;Keigo Yoshizaki and Yao Fu (Orthodontics and Dentofacial Orthopedics, Faculty of Dental Science, Kyushu University)\u0026nbsp;for their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors state that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [21K10156 to NT and 23H03116 to IT].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProdhan MD Rubayet Alam contributed to the data acquisition and interpretation; performed the statistical analyses; drafted the manuscript; Nobuo Takeshita contributed to the conception, design, data acquisition, and interpretation; performed the statistical analyses; and drafted and finished the manuscript; Wei Jiang contributed to the data acquisition;\u0026nbsp;Koji Miyazono\u0026nbsp;contributed to the data acquisition; Satoshi Fukumoto provided mDP and SF2 cells and gave valuable advice on this study; Ichiro Takahashi contributed valuable advice on this study and interpretation; and drafted and finished the manuscript. All the authors gave their final approval and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePuthiyaveetil JS, Kota K, Chakkarayan R, Chakkarayan J, Thodiyil AK. Epithelial\u0026ndash;mesenchymal interactions in tooth development and the significant role of growth factors and genes with emphasis on mesenchyme: a review. J Clin Diagn Res. 2016;10(9):ZE05\u0026ndash;ZE09.\u003c/li\u003e\n\u003cli\u003eSchmitt R, Fausser J.L, Lesot H, Ruch J.V. Effects of hepatocyte growth factor antisense oligodeoxynucleotides or met D/D genotype on mouse molar crown morphogenesis. \u003cem\u003eInt. J. Dev. 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PMID: 30080297.\u003c/li\u003e\n\u003cli\u003eHuang CY, Lai CH, Kuo CH, Chiang SF, Pai PY, Lin JY, Chang CF, Viswanadha VP, Kuo WW, Huang CY. Inhibition of ERK-Drp1 signaling and mitochondria fragmentation alleviates IGF-IIR-induced mitochondria dysfunction during heart failure. J Mol Cell Cardiol. 2018 Sep;122:58-68. doi: 10.1016/j.yjmcc.2018.08.006. Epub 2018 Aug 9. PMID: 30098987.\u003c/li\u003e\n\u003cli\u003eChang CR, Blackstone C. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1. \u003cem\u003eScience\u003c/em\u003e. 2010;330(6005):1530\u0026ndash;1533.\u003c/li\u003e\n\u003cli\u003eHan XJ, Lu YF, Li SA, Kaitsuka T, Sato Y, Tomizawa K, Nairn AC, Takei K, Matsui H, Matsushita M. CaM kinase I\u0026alpha;-induced phosphorylation of Drp1 regulates mitochondrial morphology. J Cell Biol. 2008;182(3):573-85. doi:10.1083/jcb.200802164. \u003c/li\u003e\n\u003cli\u003eZhuang S, Schnellmann RG. A death-promoting role for extracellular signal-regulated kinase. \u003cem\u003eJ Biol Chem\u003c/em\u003e. 2000;275(7):4375\u0026ndash;4382.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Odontogenic mesenchymal cells, mitochondrial fission, tooth development, hepatocyte growth factors, Drp-1, Mdivi-1","lastPublishedDoi":"10.21203/rs.3.rs-7493229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7493229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTooth morphogenesis is regulated by epithelial\u0026ndash;mesenchymal interactions and cellular metabolism through intricate signaling pathways. Here, we identify a novel mechanism by which hepatocyte growth factor (HGF) signaling promotes mesenchymal cell proliferation during tooth germ development via the regulation of mitochondrial dynamics. Immunohistochemistry revealed spatiotemporal expression of c-Met, the HGF receptor, in both the epithelial and mesenchymal compartments of developing mouse molars. Organ culture showed recombinant HGF increased tooth germ size and mesenchymal proliferation, whereas the c-Met inhibitor PHA-665752 suppressed these effects. Mitochondrial staining and Western blotting demonstrated that HGF stimulated mitochondrial fission through increased phosphorylation of Drp1, whereas PHA-665752 or Mdivi-1, a Drp1 inhibitor, blocked this response and attenuated proliferation. The inhibition of mitochondrial fission also reduced HGF-induced tooth germ growth ex vivo. HGF activated MAPK pathways, particularly the ERK, p38, and JNK pathways, with JNK inhibition (SP600125) most effectively suppressing Drp1 phosphorylation, defining the HGF-JNK-Drp1 axis as central to this process. Epithelial proliferation was largely unaffected, underscoring a mesenchyme-specific mechanism. These findings reveal a previously unrecognized role of mitochondrial fission as a downstream effector of HGF signaling and highlight mitochondrial dynamics as a potential therapeutic target in developmental anomalies of the dentition.\u003c/p\u003e","manuscriptTitle":"Hepatocyte growth factor signaling regulates tooth germ development, inducing proliferation of mesenchymal cells via fission of mitochondria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 23:30:32","doi":"10.21203/rs.3.rs-7493229/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12bf2655-e156-4c29-8f5e-0b6198fcc5b2","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56592061,"name":"Biological sciences/Cell biology"},{"id":56592062,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-01-07T11:40:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-24 23:30:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7493229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7493229","identity":"rs-7493229","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}