Abstract
Background
Craniofacial malformations, caused by dysregulated neural crest cell (NCC) differentiation, affect approximately one-third of newborns worldwide. Although TGFB3 mutations were recently associated with human coronoid process hypertrophy (CPH) and other craniofacial disorders, the mechanisms by which TGF-β3 regulates NCC fate determination through cell-cell communication remains unknown.
Methods
A zebrafish model was used to investigate the impact of tgfb3 on craniofacial cartilage and bone development. Additionally, Tgfb3 was knocked down in neural crest stem cells (NCSCs) in vitro to observe cellular changes and effects on chondrogenic and osteogenic differentiation.
Results
Knockdown of tgfb3 in zebrafish resulted in impaired cartilage development and bone formation, which was associated with the TGF-β signaling pathway. A reduction in the expression of markers for neural crest cell formation, migration, and differentiation was observed. Although Tgfb3 knockdown did not affect the proliferation capacity of NCSCs, it led to increased apoptosis, reduced migration, and decreased chondrogenic and osteogenic differentiation. The expression of osteogenesis-related proteins and TGF-β/Smad pathway-related proteins was also reduced in NCSCs to varying degrees.
Conclusion
Silencing the Tgfb3 gene in zebrafish led to significant impairment in craniofacial cartilage and bone development, clearly highlighting the critical role of TGF-β3 in regulating NCSC fate. These findings underscore the importance of TGF-β3 in maintaining NCSC migration and differentiation.
Key words: TGF-β signaling, Neural crest cell, Craniofacial development, Zebrafish, Osteochondrogenic differentiation
Introduction
Craniofacial developmental anomalies affect approximately one-third of newborns worldwide and stem primarily from dysregulated neural crest cell (NCC) differentiation and migration.1 Among the signaling pathways that govern NCC behavior, the transforming growth factor-β (TGF-β) family – particularly the TGFB3 isoform – plays a crucial role in chondrogenesis and osteogenesis.2 Although TGFB3’s involvement in palatogenesis through medial edge epithelium (MEE) degeneration is well established,3 its specific regulatory mechanisms in craniofacial skeletal morphogenesis remain poorly understood. Our previous clinical study identified compound heterozygous TGFB3 mutations (p.I111T and p.P291L) in a family with autosomal recessive mandibular coronoid process hyperplasia (CPH), marking the first genetic association between TGFB3 and human craniofacial dysmorphogenesis.4 However, the molecular pathways by which TGFB3 regulates NCC-driven osteochondral differentiation remain unknown. Zebrafish, with their well-conserved craniofacial developmental mechanisms,2,5 provide an ideal model to investigate this gap. Previous studies suggest that TGFB3 dysregulation impairs cranial NCC (CNCC) formation and migration,6 yet the downstream effects on Smad-dependent signaling and NCC differentiation trajectories require further elucidation. In this study, we integrate tgfb3-knockdown zebrafish models with in vitro NCSC assays to delineate TGFB3's role in craniofacial development. Our findings provide mechanistic insights that may translate into therapeutic advancements for congenital craniofacial disorders.
Materials and methods
Morpholino injection
The wild-type TU zebrafish strain was obtained from the China Zebrafish Resource Center. Three morpholino (MO) oligonucleotides – Ctrl MO (5′-CCTCTTACCTCAGTTACAATTTAT-3′), e1i1-MO (5′-CATCATCCCTAAGGGAAACTTACTG-3′), and i1e2-MO (5′-TCATCTGCAAGGTCAGAGGTCAGCG-3′) – were purchased from Gene Tools. The Ctrl MO dose was 8 ng, while the tgfb3-targeting DMOs (e1i1-MO + i1e2-MO) were injected at 4 ng each (total 8 ng).
Western blot
Total protein lysates were mixed with loading buffer, denatured (100 °C, 5 minutes), separated by SDS-PAGE (200 V, 1 hour), and transferred to PVDF membranes. After blocking and washing, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 unless noted) against: TGFB3, ALP, Runx2, OSX (Abcam); Smad2, pSmad2, Smad4 (CST); Col1 (Affinity); GAPDH (1:5000; Proteintech); and β-actin (1:5000; Origene). Goat anti-rabbit IgG (1:5000, Proteintech) served as the secondary antibody (1 hour, RT). Protein bands were visualized via ECL and quantified using ImageJ.
Quantitative PCR (qPCR)
Total RNA was reverse-transcribed into cDNA, and qPCR was performed (AG instrument, China) under the following conditions: 95 °C (2 minutes), 40 cycles of 95 °C (15 s), 60 °C (30 s), and 72 °C (30 s), followed by 72 °C (5 minutes). mRNA expression was normalized to Gapdh (primers from Shanghai Bioengineering Co., Ltd.), and other primer sequences are provided in Table S1.
Alcian blue (AB) staining in zebrafish
After fixation in 4% paraformaldehyde (PFA) at 4 °C overnight, 5-day-post-fertilization (dpf) zebrafish larvae were stained in 0.2% Alcian blue (RT, 17 hours), bleached in 6% H₂O₂/1% KOH (1 hour), and cleared through graded glycerol/KOH solutions (25% → 50%). Images were captured in glycerol.
Alizarin red (AR) staining in zebrafish
Following fixation in 4% PFA (4 °C, overnight), 5-dpf zebrafish larvae were processed using the Zebrafish Bone Staining Kit (Murui Biotech, China). After washing, bleaching, and staining for 1.5 hours, specimens were preserved in storage solution and photographed.
In situ hybridization (ISH) in zebrafish
Tissue samples were digested with proteinase K, pre-hybridized, and hybridized overnight with digoxigenin-labeled oligonucleotide probes. Post-hybridization, washes were performed with SSC buffer, followed by blocking and incubation with anti-digoxigenin-AP antibody (Sigma-Aldrich, America). Staining was developed using BM Purple AP Substrate (Roche, Switzerland), and specimens were stored in glycerol prior to imaging. Probe sequences are listed in Table S2.
Zebrafish RNA-Seq analysis
Total RNA was extracted from 48 hpf zebrafish embryo heads and sequenced on the Illumina NovaSeq 6000 platform. Differential expression analysis was performed using DESeq2, followed by Gene Ontology (GO) enrichment analyses via ClusterProfiler.
Primary NCSC culture and Tgfb3 knockdown
Neural tubes from E9.5 mouse embryos were digested in 0.15% Dispase for 5 minutes and plated in Matrigel-coated 24-well plates. Growth medium (DMEM/F12 containing 0.5% N2, 1% B27, 100 ng/mL EGF, and 100 ng/mL bFGF) was replaced at 12 hours, and neural tubes were removed at 48 hours. NCSCs(1 × 10⁶ cells/mL) were electroporated (Celetrix LE+/EX+) with 2 μM siRNA targeting Tgfb3.
Cell proliferation assay
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CCK-8 Assay:Transfected NCSCs (5 × 10³ cells/well) were seeded in a 96-well plate and cultured for 1 to 4 days. Subsequently, 10 μL of CCK-8 solution was added to each well, followed by a 2-hour incubation at 37°C. Absorbance was measured at 450 nm (OD450) to assess cell viability.
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EdU Assay:Transfected NCSCs cultured in a 24-well plate for 48 hours were pulsed with 2 × EdU for 2 hours. After fixation with 4% paraformaldehyde (15 minutes) and permeabilization, cells were labeled via Click reaction (30 minutes). Nuclei were counterstained with Hoechst 33342 (10 minutes) prior to imaging for proliferation analysis.
Apoptosis analysis by flow cytometry
Following 48 hours of transfection, NCSCs (5 × 10⁵ cells/mL) were stained with Annexin V-FITC and propidium iodide (PI, KGI, China) for 10 minutes in the dark. Apoptotic rates were quantified using flow cytometry.
Scratch assay for migration
NCSCs cultured in 6-well plates were scratched with a 200 μL pipette tip to create a uniform wound. After washing with PBS, images were captured at 0, 12, and 24 hours. The migration rate (%) was calculated as: Migration rate (%) = ([0 hour scratch area – 24 hours scratch area] / 0 hour scratch area) × 100.
Chondrogenic and osteogenic differentiation
Chondrogenesis: NCSCs were cultured in chondrogenic induction medium (OriCell) for 21 days, then fixed and stained with Alcian blue to evaluate glycosaminoglycan deposition.Osteogenesis:For osteogenic differentiation, cells were induced for 7 days (alkaline phosphatase [ALP] staining) or 21 days (Alizarin red staining) in osteogenic medium (α-MEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate).
Statistical analysis
Data are presented as mean ± SD and analyzed using SPSS 20.0. Student’s t-test or 1-way ANOVA was used for comparisons. A P-value <.05 was considered statistically significant (ns = non-significant, P > .05).
Results
tgfb3 knockdown inhibits chondrogenesis and osteogenesis in zebrafish
To investigate the biological function of tgfb3 in craniofacial development, we constructed a tgfb3 knockdown zebrafish model using a double morpholino oligonucleotide approach. Quantitative polymerase chain reaction (qPCR) and Western blot analyses confirmed that the expression levels of tgfb3 mRNA (Figure 1A) and protein (Figure 1B) in the embryos at 48 hpf were significantly reduced compared to the control group (P < .01), indicating successful model construction. In vivo morphological observations revealed that tgfb3 knockdown led to significant developmental abnormalities in 76.67% of the embryos, primarily characterized by a reduction in tail fin size, axial curvature, and notable decreases in head and eye size, with an enlarged heart (Figure 1C).
Fig. 1.
Effects of tgfb3 knockdown on zebrafish craniofacial cartilage and bone formation. A, qPCR validation of tgfb3 mRNA reduction. B, Western blot confirming Tgfb3 knockdown efficiency. C, Morphological and statistical analysis of zebrafish after Tgfb3 knockdown (5 × magnification). D–E. Impaired cartilage formation assessed by Alcian blue staining and in situ hybridization. F-G, Disrupted bone formation visualized via Alizarin red staining and in situ hybridization (P < .05, *P < .01).
Alcian blue staining of 5 days post-fertilization (dpf) zebrafish compared to the control group showed more severe cartilage malformations in the tgfb3 DMOs group. These malformations included a shortened Meckel's cartilage, mandible and hyoid arch. We could not observe the branchial arches (Figure 1D), indicating a significant reduction in cartilage formation area (P < .05). Further investigation using in situ hybridization for col2a1a revealed a decrease in the expression levels of cartilage differentiation markers in 72 hpf embryos (P < .01) (Figure 1E), confirming that tgfb3 deficiency significantly inhibits chondrocyte differentiation.
Regarding osteogenesis, Alizarin red staining indicated insufficient ossification in the cranial bones (including the presphenoid), the anterior notochord, the operculum, and the branchial arches in the experimental group at 5 dpf (Figure 1F). Additionally, the area of ossification in the tgfb3 DMOs group was significantly reduced (P < .01). In situ hybridization for the osteogenic differentiation marker osx in 4 dpf zebrafish demonstrated a reduction in osx expression following tgfb3 knockdown compared to the control group (Figure 1G). These results align with the findings from Alizarin red staining, indicating that tgfb3 deficiency also significantly inhibits osteoblast differentiation.
These phenotypes are highly reminiscent of craniofacial malformations associated with human TGFB3 mutations, suggesting the effectiveness of the zebrafish model.
Tgfb3 knockdown impacts gene expression in zebrafish embryos
To investigate the effects of tgfb3 knockdown on gene expression, we performed RNA sequencing (RNA-seq) on head tissues of zebrafish embryos at 48 hours post-fertilization (hpf). The analysis revealed a total of 6470 differentially expressed genes (DEGs) in the tgfb3 DMOs group compared to the control group, with 3036 genes downregulated and 3434 upregulated (Figure 2A). GO enrichment analysis indicated that these differential genes were significantly enriched in pathways related to NCC migration, development, and differentiation (Figure 2B). Heatmap analysis further demonstrated that several key NCC marker genes, including hoxb3a, hoxd3a, gbx2, dlx6a, gsc, b3gat1, id2a, tfap2a, tfap2b, and otx2b, exhibited significant downregulation in the DMOs group (Figure 2C).
Fig. 2.
Effects of Tgfb3 Knockdown on Neural Crest Cells in Zebrafish. A, Volcano plot of DEGs between the Control Morpholino (CMO) and the DMO)groups. Red dots represent upregulated genes, while blue dots indicate downregulated genes. B, GO enrichment analysis of the differentially expressed genes, with red boxes highlighting enriched entries in pathways related to neural crest cells. C, Heatmap showing expression levels of neural crest cell-related genes (hoxb3a, hoxd3a, gbx2, dlx6a, gsc, b3gat1a, id2a, tfap2a, tfap2b, and otx2b) in both groups. D, qPCR results for neural crest cell-related genes in embryos at different developmental stages. E. In situ hybridization analysis showing changes in the expression of neural crest markers (foxd3 and sox9a) and the pharyngeal mesoderm marker (gsc) (magnified 5 times) (P < .05, *P < .01, **P < .001).
To validate the RNA-seq results, we conducted quantitative polymerase chain reaction qPCR analyses on zebrafish head tissues at 3 time points: 12, 24, and 48 hpf. The results showed that the expression of the early NCC formation marker gene foxd3 was significantly reduced at 12 hpf. At 24 hpf, the expression of the NCC migration-related gene sox9a also noticeably decreased (P < .05). At 48 hpf, multiple NCC differentiation-related genes (hoxb3a, hoxd3a, gbx2, dlx6a, gsc, b3gat1a, id2a, tfap2a, tfap2b, and otx2b) were significantly downregulated, which was highly consistent with the RNA-seq data (Figure 2D).
Further examination of NCC dynamic changes was performed using in situ hybridization experiments. The results showed a marked reduction in the expression area of the NCC formation marker gene foxd3 in the tgfb3 DMOs group at 12 hpf (Figure 2E), indicating that tgfb3 knockdown affects early NCC formation. At 24 hpf, the expression area of the NCC migration marker gene sox9a decreased, although the signal was not completely lost (Figure 2E). This suggests that tgfb3 knockdown partially obstructed the migration of cranial neural crest cells. At 48 hpf, the expression area of the pharyngeal mesoderm marker gene gsc also diminished (Figure 2E), indicating that tgfb3 knockdown interfered with the homing and differentiation processes of pharyngeal mesodermal cells.
In summary, tgfb3 knockdown not only affects the early formation of neural crest cells but also significantly inhibits their migration and differentiation capabilities, which may be one of the key mechanisms leading to craniofacial developmental defects.
TGFB3 knockdown impairs the function of neural crest stem cells in mice
To investigate the role of TGFB3 in NCSCs, we first isolated NCSCs from embryonic day 9.5 (E9.5) ICR mouse embryos. After dissection, the embryos displayed typical morphology (Figure 3A, upper left), followed by isolation of the neural tube tissue (Figure 3A, upper middle). After 24 hours of in vitro culture, numerous spindle-shaped NCSCs migrated from the neural tube and adhered to the culture plate (Figure 3A, upper right). After removing the neural tube tissue 48 hours later, purified NCSCs were obtained (Figure 3A, lower left). Continuous subculturing resulted in NCSCs of passage 1 (P1) (Figure 3A, lower middle) and passage 3 (P3) (Figure 3A, lower right) maintaining stable morphological characteristics.
Fig. 3.
Effects of Tgfb3 Knockdown on NCSC Proliferation, Apoptosis, and Migration. A, Observation of neural crest stem cell culture in vitro. B, Immunofluorescence identification of neural crest stem cells. C, Expression of NCSC-specific genes (P75, Pax3, Snail, Hnk1) in passages P0 to P5. D, mRNA expression levels of Tgfb3 in NCSCs following knockdown. E, Protein expression levels of Tgfb3 in NCSCs following knockdown. F, Proliferation of NCSCs assessed by the CCK-8 assay. G, EdU incorporation assay after 48 hours to detect NCSC proliferation. H, Apoptosis levels of NCSCs detected by flow cytometry. I, Scratch assay evaluating NCSC migration at 0, 12, and 24 hours (magnified 10 times) (P < .05, *P < .01, **P < .001, ns P > .05).
To confirm the stem cell properties of NCSCs, we performed immunofluorescence detection of neural crest markers HNK1 and neural stem cell marker PAX6. The results showed that the isolated cells were positive for both HNK1 and PAX6 (HNK1⁺/PAX6⁺) (Figure 3B), confirming their typical NCSC phenotype. Additionally, qPCR analysis demonstrated that NCSCs from passages P0 to P5 consistently expressed NCSC-specific genes (P75, Hnk1, Pax3, Snail), with the most stable gene expression observed in passages P3 to P5 (Figure 3C). Therefore, we selected P3 to P5 NCSCs for subsequent experiments.
Subsequently, we knocked down Tgfb3 in NCSCs. The results from qPCR (Figure 3D) and Western blot (Figure 3E) showed that siRNA-1077 effectively silenced the expression of Tgfb3 in NCSCs and was used in subsequent experiments. CCK-8 assays indicated that there was no significant difference in the proliferation ability of siTGFB3 group cells compared to control group NCSCs (Figure 3F) (P > .05). The results from the EdU incorporation assay after 48 hours showed a slight decrease in proliferation in the siTGFB3 group compared to the control group, but this difference was not statistically significant (Figure 3G) (P > .05). However, the apoptosis results after 48 hours indicated a significantly increased apoptosis rate in the siTGFB3 group compared to the control group (P < .01) (Figure 3H). Meanwhile, the wound healing assay showed a decreased migratory capability of siTGFB3 group cells compared to the control group (P < .05) (Figure 3I). While TGFB3 knockdown did not significantly affect the proliferation of NCSCs, it notably promoted cell apoptosis and inhibited migratory ability, suggesting that TGFB3 plays a critical role in maintaining the survival and migration of NCSCs.
Tgfb3 knockdown inhibits chondrogenic and osteogenic differentiation of NCSCs
After 21 days of chondrogenic induction culture of NCSCs, we assessed cartilage matrix formation using Alcian blue staining. The results showed a significant reduction in cartilage matrix formation in the siTGFB3 group compared to the control group (P < .05) (Figure 4A), indicating that TGFB3 knockdown markedly impaired the chondrogenic differentiation capacity of NCSCs.
Fig. 4.
Tgfb3 knockdown impairs chondrogenic and osteogenic differentiation of NCSCs in vitro. A, Alcian blue staining after 21-day chondrogenic induction. B-C, Osteogenic differentiation markers (ALP: day 7; Alizarin red: day 21). D, Western blot analysis of osteogenic markers (P < .05).
ALP staining revealed (Figure 4B) that after 14 days of induction, alkaline phosphatase (ALP) activity in the siTGFB3 group was significantly reduced (P < .05). Alizarin red staining demonstrated (Figure 4C) that after 21 days of induction, the amount of calcium nodule formation in the siTGFB3 group was significantly decreased (by 63.5% ± 7.3%, P < .001).
Western blot analysis indicated that the expression levels of osteogenic-related proteins, including ALP, Col1, Runx2, and OSX, were significantly downregulated in the siTGFB3 group (Figure 4D) (P < .05). Additionally, the express level of the key signaling molecule Smad2 and Smad4 were also markedly decreased (Figure 4D) (P < .05).
Discussion
This study systematically clarifies the core mechanisms by which TGFB3 regulates craniofacial development through an integration of clinical genetics and molecular embryology methods. As a key regulatory factor, TGFB3 coordinates craniofacial morphogenesis through an interaction network: first, by maintaining the survival and migration of NCCs, it ensures the directed movement of these pluripotent embryonic cells to form the craniofacial primordia; second, by precisely regulating chondrogenic and osteogenic differentiation, it guides the fate determination of precursor cells, thereby progressively advancing skeletal morphogenesis.
This regulatory logic, which consists of NCC homeostasis maintenance and differentiation within the chondrogenic and osteogenic lineages, not only reveals the molecular mechanisms underlying TGFB3′s action but also elucidates the pathological basis by which mutations can lead to craniofacial malformations such as cleft palate and hypospadias. Consequently, this study provides a novel theoretical framework for targeted therapies related to these congenital malformations.
TGFB3 regulates osteochondral differentiation of neural crest stem cells
TGFB3 can modulate the chondrogenic and osteogenic differentiation of NCSCs through the TGF-β/Smad4-Sox9a signaling pathway.2 The TGFB signaling pathway is well-known for its key role in bone development and tissue homeostasis7 Upon binding to cell membrane receptors, TGFB – particularly the TGFB3 subtype – activates the canonical Smad-dependent pathway by binding to cell membrane receptors, inducing Smad2/3 phosphorylation. Phosphorylated Smad2/3 then translocates to the nucleus to regulate downstream gene expression, including the formation of the pSmad2/Smad5 complex, which drives Runx2 upregulation and promotes osteoblast differentiation.8, 9, 10 Beyond bone formation, this signaling axis also plays a crucial role in neural crest cell development and neural system function.2,11 Our experimental findings demonstrate that TGFB3 knockdown in zebrafish leads to a 76% decrease in cartilage area (P < .05) and a 68% reduction in bone mineralization (P < .01) – phenotypes mirroring human craniofacial deformities.12,13 RNA sequencing further confirmed significant alterations in TGF-β/Smad pathway-related gene expression (P < .001), accompanied by reduced expression of genes essential for NCSC migration. While TGFB3 knockdown did not affect NCSC proliferation, it significantly increased apoptosis, impaired cell migration, and severely compromised chondrogenic and osteogenic differentiation potential.14,15 Mechanistically, TGFB3 depletion resulted in the downregulation of osteogenic markers (ALP, Col1, Runx2, OSX) and key TGF-β/Smad pathway components (Smad2, pSmad2, and Smad4), indicating that TGFB3 primarily regulates NCSC differentiation through Smad-dependent signaling. The diminished pSmad2 and Smad4 levels likely disrupt Runx2/OSX activity, thereby suppressing osteogenesis,16 while impaired Sox9 activation via Smad signaling contributes to defective chondrogenesis.17 Additionally, TGFB3 knockdown-associated apoptosis and migration defects suggest that TGFB3 sustains NCSC survival and motility through Smad-mediated mechanisms. Collectively, these findings underscore the central role of the TGFB3-Smad axis in regulating NCSC pluripotency and offer promising targets for NCSC-based regenerative therapies in bone and cartilage disorders.18, 19, 20
The context-dependent role of TGF-β3 in craniofacial development
Our results demonstrate that TGFB3 knockdown significantly impairs craniofacial cartilage formation in zebrafish, as indicated by reduced cartilage area and diminished expression of the differentiation marker Col2a1a. In addition to the general impairment of craniofacial cartilage, our analysis revealed that TGF-β3 knockdown specifically disrupts the development of Meckel’s cartilage, leading to a pronounced reduction in its size. This phenotype aligns with the observations of Reeck et al,21 who reported that Col11a1a deficiency similarly perturbs Meckel’s cartilage morphogenesis, likely due to altered extracellular matrix (ECM) composition and compromised biomechanical properties. Notably, our data show that TGF-β3 knockdown reduces Col2a1a expression, suggesting that TGF-β3-mediated regulation of ECM remodeling – particularly collagen organization – is a key mechanism underlying cranial cartilage development and integrity.
Additionally, this genetic perturbation hinders NCC migration and increases apoptosis. These observations align with certain published findings but also highlight notable discrepancies. On one hand, prior studies support a chondrogenic role for TGF-β3, showing that it promotes cartilage formation via SMAD2/3 pathway activation in synergy with BMP signaling.14,22 For instance, combining TGF-β3 with BMP-2 in scaffold materials enhances cartilage repair.23,24 On the other hand, some reports indicate that TGF-β3 suppresses osteoclast differentiation by modulating autophagy,25 suggesting its role varies across cell types and contexts. Interestingly, while our study found that TGF-β3 depletion impedes NCC migration, other research suggests that the broader TGF-β signaling pathway exerts dual effects – protective in uterine inflammation but detrimental in cranial neural crest development.26 Further complicating its role, TGF-β3 appears to exhibit stage-specific functions, promoting migration at early stages while inhibiting proliferation later.27,28 These inconsistencies may stem from species-specific sensitivities, as zebrafish likely exhibit heightened responsiveness to TGF-β3 regulation compared to mammals, where TGF-β3 activity is more microenvironment-dependent (eg, influenced by growth factors).29,30 Additionally, disparities between in vitro and in vivo models – such as differing TGF-β3 concentration effects in 3D cultures – could contribute to variable outcomes.31 Collectively, these findings underscore the context-dependent nature of TGF-β3 in craniofacial morphogenesis.
Clinical translation potential
This study holds significant clinical translational value, particularly in supporting the inclusion of TGFB3 in craniofacial deformity gene screening panels based on evidence from animal models and human familial data. Therapeutically, small-molecule activators of the TGF-β/Smad pathway (eg, SRI-011381) show promise in ameliorating osteochondral developmental disorders linked to TGFB3 deficiency, though further validation in conditional knockout models remains necessary.
Limitations and future directions
While this study provides valuable insights, several limitations should be acknowledged. First, the zebrafish model system cannot fully replicate the genetic and phenotypic heterogeneity associated with human TGFB3 mutations. Second, the absence of tissue-specific knockout models restricts our ability to precisely examine developmental mechanisms in distinct craniofacial regions. To bridge these gaps, future investigations should prioritize 2 key strategies: (1) generating conditional knockout mouse models to better mimic human disease phenotypes, and (2) employing single-cell sequencing to elucidate spatiotemporal variations in TGFB3-mediated regulation. Implementing these approaches will refine our understanding of craniofacial development mechanisms and enhance the translational potential of this research.
Conclusion
This study confirms that Tgfb3 regulates the migration and survival of cranial neural crest cells, while also influencing osteogenic and chondrogenic differentiation. These findings provide new insights into the pathogenesis of craniofacial developmental disorders. Future research should aim to optimize experimental models and explore non-canonical pathways to advance clinical applications in this field.
Author contributions
Hongrong Zhang, Shiying Shen, and Yemei Qian designed the study, performed experiments, and wrote the original manuscript. Liqin Zhang, Jiantong Pu and Xue Zhou assisted with methodology and data collection. Na Yang conducted and analyzed zebrafish experiments. Weihong Wang supervised the project, revised the manuscript, and coordinated publication. Hongrong Zhang, Shiying Shen, and Yemei Qian contributed equally. All authors participated in data interpretation, critically reviewed the manuscript, approved the final version, and consented to data publication.
Conflict of interest
None disclosed.
Funding
This study was financially supported by the National Natural Science Foundation of China (No. 82160321), Yunnan Province Xing Dian Talent (No. XDYC-MY-2022-0052) and Kunming Medical University First-Class Discipline Team Construction Project (No. 2024XKTDPY09).
Footnotes
Supplementary material associated with this article can be found in the online version at doi:10.1016/j.identj.2025.103874.
Contributor Information
Na Yang, Email: yangna@mail.ynu.edu.cn.
Weihong Wang, Email: wangweihong@kmmu.edu.cn.
Appendix. Supplementary materials
Data availability
The data in the figures are available in the published article and its online supplemental material. Further information and requests for resources and reagents are available from corresponding authors upon request.
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Data Availability Statement
The data in the figures are available in the published article and its online supplemental material. Further information and requests for resources and reagents are available from corresponding authors upon request.