A novel perspective of calvarial development: the cranial morphogenesis and differentiation regulated by dura mater.

There are lasting concerns on calvarial development because cranium not only accommodates the growing brain, but also safeguards it from exogenous strikes. In the past decades, most studies attributed the dynamic expansion and remodeling of cranium to the proliferation of osteoprecursors in cranial primordium, and the proliferation of osteoprogenitors at the osteogenic front of cranial suture mesenchyme. Further investigations identified series genes expressed in suture mesenchymal cells as the markers of the progenitors, precursors and postnatal stem cells in cranium. However, similar to many other organs, it is suggested that the reciprocal interactions among different tissues also play essential roles in calvarial development. Actually, there are increasing evidence indicating that dura mater (DM) is indispensable for the calvarial morphogenesis and osteogenesis by secreting multiple growth factors, cytokines and extracellular matrix (ECM). Thus, in this review, we first briefly introduce the development of cranium, suture and DM, and then, comprehensively summarize the latest studies exploring the involvement of ECM in DM and cranium development. Eventually, we discussed the reciprocal interactions between calvarium and DM in calvarial development. Actually, our review provides a novel perspective for cranium development by integrating previous classical researches with a spotlight on the mutual interplay between the developing DM and cranium.

Aplasia Cutis Congenita Pathomechanisms Reveal Key Regulators of Skin and Skin Appendage Morphogenesis.

Aplasia cutis congenita (ACC) manifests at birth as a defect of the scalp skin. New findings answer 2 longstanding questions: why ACC forms and why it affects mainly the midline scalp skin. Dominant-negative mutations in the genes KCTD1 or KCTD15 cause ACC owing to loss of function of KCTD1/KCTD15 complexes in cranial neural crest cells (NCCs), which normally form midline cranial suture mesenchymal cells that express keratinocyte growth factors. Loss of KCTD1/KCTD15 function in NCCs impairs the formation of normal midline cranial sutures and, consequently, the overlying skin, resulting in ACC. Moreover, KCTD1/KCTD15 complexes in keratinocytes regulate skin appendage morphogenesis.

Overexpression of Fgf18 in cranial neural crest cells recapitulates Pierre Robin sequence in mice.

The pivotal role of FGF18 in the regulation of craniofacial and skeletal development has been well established. Previous studies have demonstrated that mice with deficiency in Fgf18 exhibit severe craniofacial dysplasia. Recent clinical reports have revealed that the duplication of chromosome 5q32-35.3, which encompasses the Fgf18 gene, can lead to cranial bone dysplasia and congenital craniosynostosis, implicating the consequence of possible overdosed FGF18 signaling. This study aimed to test the effects of augmented FGF18 signaling by specifically overexpressing the Fgf18 gene in cranial neural crest cells using the Wnt1-Cre;pMes-Fgf18 mouse model. The results showed that overexpression of Fgf18 leads to craniofacial abnormalities in mice similar to the Pierre Robin sequence in humans, including abnormal tongue morphology, micrognathia, and cleft palate. Further examination revealed that elevated levels of Fgf18 activated the Akt and Erk signaling pathways, leading to an increase in the proliferation level of tongue tendon cells and alterations in the contraction pattern of the genioglossus muscle. Additionally, we observed that excessive FGF18 signaling contributed to the reduction in the length of Meckel's cartilage and disrupted the development of condylar cartilage, ultimately resulting in mandibular defects. These anomalies involve changes in several downstream signals, including Runx2, p21, Akt, Erk, p38, Wnt, and Ihh. This study highlights the crucial role of maintaining the balance of endogenous FGF18 signaling for proper craniofacial development and offers insights into potential formation mechanisms of the Pierre Robin sequence.

Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

amer1 Regulates Zebrafish Craniofacial Development by Interacting with the Wnt/ß-Catenin Pathway.

Microtia-atresia is a rare type of congenital craniofacial malformation causing severe damage to the appearance and hearing ability of affected individuals. The genetic factors associated with microtia-atresia have not yet been determined. The AMER1 gene has been identified as potentially pathogenic for microtia-atresia in two twin families. An amer1 mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. The phenotype and the development process of cranial neural crest cells of the knockdown zebrafish were examined. Components of the Wnt/ß-catenin pathway were examined by qPCR, Western blotting, and immunofluorescence assay. IWR-1-endo, a reversible inhibitor of the Wnt/ß-catenin pathway, was applied to rescue the abnormal phenotype. The present study showed that the development of mandibular cartilage in zebrafish was severely compromised by amer1 knockdown using CRISPR/Cas9. Specifically, amer1 knockdown was found to affect the proliferation and apoptosis of cranial neural crest cells, as well as their differentiation to chondrocytes. Mechanistically, amer1 exerted an antagonistic effect on the Wnt/ß-catenin pathway. The application of IWR-1-endo could partially rescue the abnormal phenotype. We demonstrated that amer1 was essential for the craniofacial development of zebrafish by interacting with the Wnt/ß-catenin pathway. These findings provide important insight into the role of amer1 in zebrafish mandibular development and the pathology of microtia-atresia caused by AMER1 gene mutations in humans.

Identification of an adverse outcome pathway (AOP) for chemical-induced craniofacial anomalies using the transgenic zebrafish model.

Craniofacial anomalies are one of the most frequent birth defects worldwide and are often caused by genetic and environmental factors such as pharmaceuticals and chemical agents. Although identifying adverse outcome pathways (AOPs) is a central issue for evaluating the teratogenicity, the AOP causing craniofacial anomalies has not been identified. Recently, zebrafish has gained interest as an emerging model for predicting teratogenicity because of high throughput, cost-effectiveness and availability of various tools for examining teratogenic mechanisms. Here, we established zebrafish sox10-EGFP reporter lines to visualize cranial neural crest cells (CNCCs) and have identified the AOPs for craniofacial anomalies. When we exposed the transgenic embryos to teratogens that were reported to cause craniofacial anomalies in mammals, CNCC migration and subsequent morphogenesis of the first pharyngeal arch were impaired at 24 hours post-fertilization. We also found that cell proliferation and apoptosis of the migratory CNCCs were disturbed, which would be key events of the AOP. From these results, we propose that our sox10-EGFP reporter lines serve as a valuable model for detecting craniofacial skeletal abnormalities, from early to late developmental stages. Given that the developmental process of CNCCs around this stage is highly conserved between zebrafish and mammals, our findings can be extrapolated to mammalian craniofacial development and thus help in predicting craniofacial anomalies in human.

vwa1 Knockout in Zebrafish Causes Abnormal Craniofacial Chondrogenesis by Regulating FGF Pathway.

Hemifacial microsomia (HFM), a rare disorder of first- and second-pharyngeal arch development, has been linked to a point mutation in VWA1 (von Willebrand factor A domain containing 1), encoding the protein WARP in a five-generation pedigree. However, how the VWA1 mutation relates to the pathogenesis of HFM is largely unknown. Here, we sought to elucidate the effects of the VWA1 mutation at the molecular level by generating a vwa1-knockout zebrafish line using CRISPR/Cas9. Mutants and crispants showed cartilage dysmorphologies, including hypoplastic Meckel's cartilage and palatoquadrate cartilage, malformed ceratohyal with widened angle, and deformed or absent ceratobranchial cartilages. Chondrocytes exhibited a smaller size and aspect ratio and were aligned irregularly. In situ hybridization and RT-qPCR showed a decrease in barx1 and col2a1a expression, indicating abnormal cranial neural crest cell (CNCC) condensation and differentiation. CNCC proliferation and survival were also impaired in the mutants. Expression of FGF pathway components, including fgf8a, fgfr1, fgfr2, fgfr3, fgfr4, and runx2a, was decreased, implying a role for VWA1 in regulating FGF signaling. Our results demonstrate that VWA1 is essential for zebrafish chondrogenesis through effects on condensation, differentiation, proliferation, and apoptosis of CNCCs, and likely impacts chondrogenesis through regulation of the FGF pathway.

Ectomesenchymal Six1 controls mandibular skeleton formation.

Craniofacial development requires intricate cooperation between multiple transcription factors and signaling pathways. Six1 is a critical transcription factor regulating craniofacial development. However, the exact function of Six1 during craniofacial development remains elusive. In this study, we investigated the role of Six1 in mandible development using a Six1 knockout mouse model (Six1 -/- ) and a cranial neural crest-specific, Six1 conditional knockout mouse model (Six1 f/f ; Wnt1-Cre). The Six1 -/- mice exhibited multiple craniofacial deformities, including severe microsomia, high-arched palate, and uvula deformity. Notably, the Six1 f/f ; Wnt1-Cre mice recapitulate the microsomia phenotype of Six1 -/- mice, thus demonstrating that the expression of Six1 in ectomesenchyme is critical for mandible development. We further showed that the knockout of Six1 led to abnormal expression of osteogenic genes within the mandible. Moreover, the knockdown of Six1 in C3H10 T1/2 cells reduced their osteogenic capacity in vitro. Using RNA-seq, we showed that both the loss of Six1 in the E18.5 mandible and Six1 knockdown in C3H10 T1/2 led to the dysregulation of genes involved in embryonic skeletal development. In particular, we showed that Six1 binds to the promoter of Bmp4, Fat4, Fgf18, and Fgfr2, and promotes their transcription. Collectively, our results suggest that Six1 plays a critical role in regulating mandibular skeleton formation during mouse embryogenesis.

The etiology, clinical features, and treatment options of hemifacial microsomia.

The second most frequent craniomaxillofacial congenital deformity is hemifacial microsomia (HFM). Patients often accompany short mandible, ear dysplasia, facial nerve, and soft tissue dysplasia. The etiology of HFM is not fully understood. To organize the possible up-to-date information on the etiology, craniofacial phenotypes, and therapeutic alternatives in order to fully comprehend the HFM. Reviewing the potential causes, exploring the clinical features of HFM and summarizing the available treatment options. Vascular malformation, Meckel's cartilage abnormalities, and cranial neural crest cells (CNCCs) abnormalities are three potential etiology hypotheses. The commonly used clinical classification for HFM is OMENS, OMENS-plus, and SAT. Other craniofacial anomalies, like dental defects, and zygomatic deformities, are still not precisely documented in the classification. Patients with moderate phenotypes may not need any treatment from infancy through adulthood. However, patients with severe HFM require to undergo multiple surgeries to address facial asymmetries, such as mandibular distraction osteogenesis (MDO), autologous costochondral rib graft (CCG), orthodontic and orthognathic treatment, and facial soft tissue reconstruction. It is anticipated that etiology research will examine the pathogenic mechanism of HFM. A precise treatment for HFM may be possible with thoroughly documented phenotypes and a pathogenic diagnosis.

Mechanical dissection and culture of mouse cranial neural crest cells.

Owing to the contribution of cranial neural crest cells (CNCCs) to the majority of craniofacial structures, they have been studied extensively for the pathogenesis of craniofacial diseases. To investigate and summarize how to isolate and culture the CNCCs from wild-type mice, a literature search was performed in online databases (PubMed and Web of Science) using optimized keywords "mouse," "cranial neural crest cell" and "culture." The literature was checked by two investigators according to the screening and exclusion criteria. Initially, 197 studies were retrieved from PubMed and 169 from Web of Science, and after excluding replicate studies, 293 articles were considered. Finally, 17 studies met all the criteria and were included in this review. The results showed that obtaining purified stem cells and balancing the need to promote cell growth and prevent unwanted early cell differentiation were the two key points in the isolation and culture of CNCCs. However, no standard criteria are available for answering these questions. Thus, it is important to emphasize the necessity for standardization of CNCC isolation, culture, and identification in research on craniofacial diseases.

Targeting fibroblast growth factor receptors causes severe craniofacial malformations in zebrafish larvae.

Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2, as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish (Danio rerio) tempted us to investigate effects of the mutated fgfr2 sa10729 allele in zebrafish on cartilage and bone formation. Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf. Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a, fgfr1b, fgfr4, sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2. Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations.

A Neural Crest-specific Overexpression Mouse Model Reveals the Transcriptional Regulatory Effects of Dlx2 During Maxillary Process Development.

Craniofacial morphogenesis is a complex process that requires precise regulation of cell proliferation, migration, and differentiation. Perturbations of this process cause a series of craniofacial deformities. Dlx2 is a critical transcription factor that regulates the development of the first branchial arch. However, the transcriptional regulatory functions of Dlx2 during craniofacial development have been poorly understood due to the lack of animal models in which the Dlx2 level can be precisely modulated. In this study, we constructed a Rosa26 site-directed Dlx2 gene knock-in mouse model Rosa26 CAG-LSL-Dlx2-3xFlag for conditionally overexpressing Dlx2. By breeding with wnt1 cre mice, we obtained wnt1 cre ; Rosa26 Dlx2/- mice, in which Dlx2 is overexpressed in neural crest lineage at approximately three times the endogenous level. The wnt1 cre ; Rosa26 Dlx2/- mice exhibited consistent phenotypes that include cleft palate across generations and individual animals. Using this model, we demonstrated that Dlx2 caused cleft palate by affecting maxillary growth and uplift in the early-stage development of maxillary prominences. By performing bulk RNA-sequencing, we demonstrated that Dlx2 overexpression induced significant changes in many genes associated with critical developmental pathways. In summary, our novel mouse model provides a reliable and consistent system for investigating Dlx2 functions during development and for elucidating the gene regulatory networks underlying craniofacial development.

Different Ectopic Hoxa2 Expression Levels in Mouse Cranial Neural Crest Cells Result in Distinct Craniofacial Anomalies and Homeotic Phenotypes.

Providing appropriate positional identity and patterning information to distinct rostrocaudal subpopulations of cranial neural crest cells (CNCCs) is central to vertebrate craniofacial morphogenesis. Hox genes are not expressed in frontonasal and first pharyngeal arch (PA1) CNCCs, whereas a single Hox gene, Hoxa2, is necessary to provide patterning information to second pharyngeal arch (PA2) CNCCs. In frog, chick and mouse embryos, ectopic expression of Hoxa2 in Hox-negative CNCCs induced hypoplastic phenotypes of CNCC derivatives of variable severity, associated or not with homeotic transformation of a subset of PA1 structures into a PA2-like identity. Whether these different morphological outcomes are directly related to distinct Hoxa2 overexpression levels is unknown. To address this issue, we selectively induced Hoxa2 overexpression in mouse CNCCs, using a panel of mouse lines expressing different Hoxa2 ectopic expression levels, including a newly generated Hoxa2 knocked-in mouse line. While ectopic Hoxa2 expression at only 60% of its physiological levels was sufficient for pinna duplication, ectopic Hoxa2 expression at 100% of its normal level was required for complete homeotic repatterning of a subset of PA1 skeletal elements into a duplicated set of PA2-like elements. On the other hand, ectopic Hoxa2 overexpression at non-physiological levels (200% of normal levels) led to an almost complete loss of craniofacial skeletal structures. Moreover, ectopic Hoxa5 overexpression in CNCCs, while also resulting in severe craniofacial defects, did not induce homeotic changes of PA1-derived CNCCs, indicating Hoxa2 specificity in repatterning a subset of Hox-negative CNCCs. These results reconcile some discrepancies in previously published experiments and indicate that distinct subpopulations of CNCCs are differentially sensitive to ectopic levels of Hox expression.

Disruption of Trip11 in cranial neural crest cells is associated with increased ER and Golgi stress contributing to skull defects in mice.

BACKGROUND: Absence of Golgi microtubule-associated protein 210 (GMAP210), encoded by the TRIP11 gene, results in achondrogenesis. Although TRIP11 is thought to be specifically required for chondrogenesis, human fetuses with the mutation of TRIP11 also display bony skull defects where chondrocytes are usually not present. This raises an important question of how TRIP11 functions in bony skull development. RESULTS: We disrupted Trip11 in neural crest-derived cell populations, which are critical for developing skull in mice. In Trip11 mutant skulls, expression levels of ER stress markers were increased compared to controls. Morphological analysis of electron microscopy data revealed swollen ER in Trip11 mutant skulls. Unexpectedly, we also found that Golgi stress increased in Trip11 mutant skulls, suggesting that both ER and Golgi stress-induced cell death may lead to osteopenia-like phenotypes in Trip11 mutant skulls. These data suggest that Trip11 plays pivotal roles in the regulation of ER and Golgi stress, which are critical for osteogenic cell survival. CONCLUSION: We have recently reported that the molecular complex of ciliary protein and GMAP210 is required for collagen trafficking. In this paper, we further characterized the important role of Trip11 being possibly involved in the regulation of ER and Golgi stress during skull development.

Progress on the regulation of neural crest and the genetics in craniofacial development.

The craniofacial features endow vertebrates with unparalleled evolutionary advantages. The craniofacial is composed of bone, cartilage, nerves, and connective tissues mainly developed from cranial neural crest cells (cNCCs). These tissues form complex organs which enable vertebrates to have powerful neural and sensory systems. NCCs are groups of migratory and pluripotent cells that are specific to vertebrates. The specification, premigration and migration, proliferation, and fate determination of the NCCs are precisely and sequentially controlled by gene regulatory networks, to ensure the ordered and accurate development of the craniofacial region. The craniofacial region represents a combined set of highly heritable phenotypes, which could be illustrated by the inherited facial features between relatives but perceptible differences among non-relatives. Such phenomena are termed heredity and variation, which are in accordance with the precision and plasticity of cNCCs gene regulatory network, respectively. Evidence has shown that genetic variations within the regulatory network alter the proliferation and differentiation of NCCs within a tolerable range, while deleterious mutations will lead to craniofacial malformations. In this review, we first summarize the development procedure of NCCs and their gene regulatory networks and then provide an overview on the genetic basis of the facial morphology and malformations. This review will benefit the understanding of craniofacial development and the prevention of craniofacial diseases.

Research progress on the regulation of craniofacial development and malformation by fibroblast growth factor 8 / 口腔疾病防治

@#Fibroblast growth factor 8 (FGF8) is a kind of secretory polypeptide that has crucial roles in the development of various tissues and organs. Current studies have found that FGF8 can regulate the differentiation of cranial neural crest cells by activating the mitogen-activated protein kinase (MAPK) signaling pathway and affect the establishment of mandibular arch polarity and the development of craniofacial symmetry by regulating the expression of target genes. Cleft lip with or without cleft palate, ciliopathies, macrostomia and agnathia are four developmental malformations involving the craniofacial region that seriously affect the quality of life of patients. The abnormal FGF8 signal caused by gene mutation, abnormal protein conformation or expression is closely related to the occurrence of craniofacial malformations, but the molecular mechanism and signaling pathway underlying these malformations have not been fully elucidated. Craniofacial development is a complex process mediated by a variety of signaling molecules. In the future, the role of various signaling molecules in craniofacial development and malformations need to be explored to provide a new perspective and vision for the prevention and treatment of these craniofacial malformations.

A case of lipoma, calcification, and brain malformations in the midline of the skull.

Few cases of pericallosal lipoma with several other lesions, including specific forms of calcification and brain malformations, have been reported. We present the case of an asymptomatic 83-year-old man with a pericallosallipoma with peculiar symmetrical morphology in the midline of the skull. We posit that the lesions began forming in the very early embryonic period and were closely associated with the cranial neural crest cells. We report the neuroradiological findings of this characteristic lesion and discuss several literature reviews on the process of its formation.

The molecular complex of ciliary and golgin protein is crucial for skull development.

Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.

Augmented BMP4 signal impairs tongue myogenesis.

Tongue muscles are derived from mesodermal cells, while signals driven by cranial neural crest cells (CNCCs) regulate tongue myogenesis via tissue-tissue interaction. Based on such mechanisms of interaction, congenital tongue defects occur in CNC-related syndromes in humans. This study utilized a pathologic model for the syndrome of congenital bony syngnathia, Wnt1-Cre;pMes-Bmp4 mouse line, to explore impacts of enhanced CNCCs-originated BMP4 signal on tongue myogenesis via tissue-tissue interaction. Our results revealed that microglossia, a clinical phenotype of congenital bony syngnathia in humans exhibited in Wnt1-Cre;pMes-Bmp4 mice due to impaired myogenesis. The augmented BMP4 signal affected the distal distribution, proliferation, and differentiation of myogenic cells as well as tendon patterning, resulting in disarrangement and atrophy of tongue muscles and the loss of the anterior digastric muscle. This study demonstrated how a CNCCs-originated ligand impaired tongue myogenesis via a non-autonomous way, which provided potential formation mechanisms for understanding tongue abnormalities in CNC-related syndromes.

Inhibition of Cyclooxygenase-2 Alters Craniofacial Patterning during Early Embryonic Development of Chick.

A recent study from our lab revealed that the inhibition of cyclooxygenase-2 (COX-2) exclusively reduces the level of PGE2 (Prostaglandin E2) among prostanoids and hampers the normal development of several structures, strikingly the cranial vault, in chick embryos. In order to unearth the mechanism behind the deviant development of cranial features, the expression pattern of various factors that are known to influence cranial neural crest cell (CNCC) migration was checked in chick embryos after inhibiting COX-2 activity using etoricoxib. The compromised level of cell adhesion molecules and their upstream regulators, namely CDH1 (E-cadherin), CDH2 (N-cadherin), MSX1 (Msh homeobox 1), and TGF-ß (Transforming growth factor beta), observed in the etoricoxib-treated embryos indicate that COX-2, through its downstream effector PGE2, regulates the expression of these factors perhaps to aid the migration of CNCCs. The histological features and levels of FoxD3 (Forkhead box D3), as well as PCNA (Proliferating cell nuclear antigen), further consolidate the role of COX-2 in the migration and survival of CNCCs in developing embryos. The results of the current study indicate that COX-2 plays a pivotal role in orchestrating craniofacial structures perhaps by modulating CNCC proliferation and migration during the embryonic development of chicks.