Treacher Collins Syndrome (TCS) is an autosomal dominant disorder. Features of TCS include microtia with conductive hearing loss, slanting palpebral fissures with possible coloboma of the lateral part of the lower eyelids, midface hypoplasia, micrognathia, as well as sporadically cleft palate and choanal atresia or stenosis. TCS is caused by pathogenic variants in the TCOF1, POLR1D, POLR1C, and POLR1B genes, with mutations in TCOF1 accounting for more than 90% of cases (72, 73). The TCOF1 gene codes for the nucleolar phosphoprotein Treacle (known as xtreacle in Xenopus), which is essential for ribosomal DNA transcription. Mutations in TCOF1 lead to impaired rRNA synthesis, nucleolar stress, and apoptosis of neural crest cells (74, 75). In Xenopus, knockdown of xtreacle results in rDNA transcriptional defects consistent with the ribosome biogenesis abnormalities observed in human TCS patients. The resulting reduction in ribosome production mimics the nucleolar stress caused by TCOF1 haploinsufficiency in TCS (76).

Auriculocondylar syndrome (ARCND) can be inherited in either an autosomal dominant or autosomal recessive manner. Features of ARCND include congenital ear clefts, mandibular condyle hypoplasia, temporomandibular joint abnormalities, micrognathia, small mouth, round facial appearance, and prominent cheeks. ARCND is caused by pathogenic variants in GNAI3, PLCB4, or EDN1 (77–79). Xenopus models have demonstrated that both wild-type and ACS-mutant forms of gαi3 can result in embryonic developmental defects. The mutant gαi3, which is unable to bind GTP, interferes with downstream signaling and disrupts neural crest differentiation by antagonizing Gαq activity. These findings highlight a conserved cross-species mechanism underlying ACS pathogenesis (80).

Floating-Harbor Syndrome (FHS) is an autosomal dominant disorder. Features of FHS include short stature, facial dysmorphism, delayed bone mineralization, speech impairment, and intellectual disability. FHS is caused by heterozygous truncating variants in the SRCAP gene (81, 82). A Xenopus model of FHS was generated using morpholino oligonucleotides targeting srcap to mimic human C-terminal truncating mutations, inducing typical craniofacial malformations. SRCAP mutations impaired CNCCs migration and downregulated key genes, such as twist1 and sox9, suggesting neural crest dysfunction underlies the phenotype. The defects were rescued by wild-type SRCAP but not by the FHS-associated mutant. Mechanistically, SRCAP mutations disrupted nuclear localization, H2A.Z.2 deposition, and AT-rich enhancer activity, highlighting the role of H2A.Z.2 in neural crest development (8).

CHARGE syndrome is an autosomal dominant disorder that presents with a broad range of congenital malformations affecting multiple organ systems. Common clinical features include external ear malformations, cranial nerve dysfunction (often with facial palsy), semicircular canal dysplasia or aplasia, choanal atresia, and craniofacial abnormalities such as cleft lip and/or palate. In addition, anosmia, genital hypoplasia, congenital heart defects, and tracheoesophageal anomalies are frequently observed. The syndrome is caused by heterozygous loss-of-function mutations in the CHD7 gene (83). Patients often exhibit severe feeding difficulties, motor developmental delays, intellectual disabilities, and growth retardation. The majority of CHARGE syndrome cases result from haploinsufficiency of CHD7, a chromatin remodeling protein (84). In Xenopus embryos, knockdown of chd7 leads to impaired expression of NCCs effector genes (e.g., twist1, sox9), disruption of its interaction with the PBAF complex, and aberrant pharyngeal arch development, recapitulating phenotypes such as choanal atresia. These effects are associated with dysregulation of guidance molecules such as Sema3a (85, 86).

Axenfeld-Rieger Syndrome (ARS) is an autosomal dominant disorder. Features of ARS include anterior segment dysgenesis, glaucoma, dental anomalies, craniofacial dysmorphism, growth retardation, cardiovascular defects, redundant periumbilical skin, and pituitary defects leading to secondary endocrine disorders. ARS can be caused by pathogenic variants in the PITX2, FOXC1, and FOXO1A genes (87, 88). Studies using Xenopus models have demonstrated that foxc1 expression is regulated by VegT via the Nodal signaling pathway. Loss of foxc1 results in downregulation of adhesion molecules such as E-cadherin and the Ephrin/Eph system, causing mesodermal cell dissociation, axial shortening, and neural tube malformations. Injection of mutant FOXC1 mRNA can partially rescue these phenotypes (89).

Mowat-Wilson Syndrome (MWS) is an autosomal dominant disorder that presents with neural crest defects and distinctive craniofacial features, accompanied by a broad spectrum of multisystem abnormalities. Common clinical features include mild to severe intellectual disability, epilepsy, and congenital Hirschsprung disease. In addition, congenital malformations such as genital anomalies, congenital heart defects, corpus callosum agenesis, and ocular abnormalities are frequently observed. The syndrome results from heterozygous deletions or truncating mutations of the ZFHX1B gene (90). Patients typically exhibit a characteristic facial phenotype accompanied by a spectrum of multisystem abnormalities including mild to severe intellectual disability (ID), epilepsy, congenital Hirschsprung disease (HSCR), and frequently associated congenital malformations such as genital anomalies (most commonly hypospadias), congenital heart defects, corpus callosum agenesis, and ocular abnormalities (91). Knockdown of mi-2β in Xenopus reduces the expression of neural marker genes induced by zfhx1b and attenuates repression of BMP signaling pathway genes. Mutant ZFHX1B fails to effectively suppress BMP signaling, indicating that defective recruitment of the NuRD complex is a key pathogenic mechanism underlying craniofacial developmental defects (92).

Cardiofaciocutaneous syndrome (CFC) is an autosomal dominant disorder that presents with multisystem congenital anomalies and moderate to severe intellectual disability. Common clinical features include postnatal growth retardation with relative macrocephaly, characteristic craniofacial features such as prominent forehead, bitemporal narrowing, absent eyebrows, downslanting palpebral fissures with epicanthal folds, depressed nasal bridge, bulbous nasal tip, and distal limb anomalies. In addition, skin abnormalities such as dry, hyperkeratotic, scaly skin, sparse curly hair, and capillary malformations are frequently observed, along with congenital heart defects, most commonly pulmonary valve stenosis and hypertrophic cardiomyopathy. The syndrome is the result of heterozygous pathogenic variants in BRAF, MAP2K1/MEK1, MAP2K2/MEK2, KRAS, or YWHAZ (93). In Xenopus models, injection of the S230W variant of YWHAZ mRNA induces severe craniofacial malformations. Co-expression with Craf enhances GFP-Erk2 phosphorylation and rescues mesodermal defects, implicating sustained activation of the RAF–ERK signaling pathway in the pathogenesis (94).

DDX3 syndrome is an X-linked dominant disorder that presents with neurodevelopmental delay, and intellectual disability, accompanied by a spectrum of multisystem abnormalities. Common clinical features include motor and language delay, autism spectrum disorder, epilepsy, and congenital brain and cardiac malformations (95), craniofacial anomalies are observed in the majority of affected individuals (96). The syndrome is the result of pathogenic variants in the DDX3 gene. In Xenopus embryos, ddx3 is specifically expressed at the neural plate border, pharyngeal arches (regions of neural crest cell migration), and head tissues. Knockdown of ddx3 results in craniofacial cartilage hypoplasia, such as reduced size of the ceratohyal cartilage, along with downregulation of neural crest cell markers, indicating impaired neural crest induction (97).

Fetal Alcohol Spectrum Disorders (FASD) affect 1%−5% of newborns in high-risk populations, characterized by smooth philtrum, thin vermilion border, and micrognathia. These craniofacial dysmorphisms correlate with neurocognitive deficits and require lifelong supportive care. Prenatal alcohol exposure is a critical factor causing FASD, which are characterized primarily by neurodevelopmental abnormalities and may be accompanied by craniofacial malformations, congenital organ defects, and growth retardation among other multisystem impairments (98). Exposure of Xenopus embryos to ethanol disrupts the expression of genes associated with later-stage NCCs migration. Supplementation with 5-methyltetrahydrofolate can partially rescue the observed phenotypes (99).

Kabuki Syndrome (KS) is a rare neuro-developmental disorder caused by variants in genes of histone modification, including KMT2D and KDM6A (100) it is considered a Mendelian disorder of epigenetic regulation, affecting multiple organ systems including the nervous, sensory, immune, cardiac, renal, and skeletal systems, significantly impacting patients' quality of life (101). Knockdown of the kmt2d gene in Xenopus models recapitulates phenotypes of mandibular, hyoid, and pharyngeal arch cartilage hypoplasia. The underlying mechanism involves defects in CNCCs formation and migration, evidenced by downregulation of marker genes such as twist, abnormal cell dispersal capacity, and decreased levels of histone modifications H3K4me1 and H3K27ac. Further studies revealed that kmt2d regulates CNCCs migration by modulating the secreted factor Sema3F, and overexpression of Sema3F can partially rescue the migration defects (102).

Wolf-Hirschhorn syndrome (WHS) is araredisorderwithan estimated prevalence being around 1 in 50,000 births. The syndrome is caused by the deletion of a critical region (Wolf-Hirschhorn Syndrome Critical region—WHSCR) on chromosome 4p16.3. WHS is clinically characterized by pre-and postnatal growth restriction, hypotonia, intellectual disability, craniofacial dysmorphismand congenital fusion anomalies (103, 104), which are associated with aberrant development of CNCCs. Modeling this in Xenopus revealed that whsc1/tacc3 knockdown disrupts neural crest migration, directly linking genetic loss to craniofacial pathogenesis (105).

Musculocontractural Ehlers–Danlos syndrome (MC-EDS) is an autosomal recessive disease characterized primarily by increased connective tissue fragility, craniofacial structural abnormalities, congenital contractures, and impaired growth and development (106). It is caused by mutations in DSE or CHST14, which result in defects in craniofacial cartilage development. In Xenopus models, dse knockdown results in the downregulation of key EMT regulators in neural crest cells, including snail2 and twist1, and impairs cell migration on fibronectin substrates, providing a cellular dynamic basis for the craniofacial anomalies associated with the disease (107).

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder typically characterized by branchial cleft anomalies, ear developmental defects, and renal malformations. Additional clinical features may include abnormalities of the face, maxilla, ureters, and bladder, lacrimal system dysfunction, otitis media, and shoulder anomalies (108). About 40% of patients with BOS carry aberrations of EYA1 gene which is the most important cause of BOS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements (109). Knockdown of the eya1 gene in Xenopus leads to defects in otic vesicle development (inner ear structural abnormalities) and hypoplasia of branchial arch cartilages, such as Meckel's cartilage, thereby recapitulating the craniofacial skeletal malformations observed in patients. Mechanistically, eya1 deficiency downregulates neural crest cell marker genes, including sox10 and snail2, suggesting that eya1 regulates craniofacial development through modulation of neural crest cell migration and differentiation. Furthermore, wild-type human EYA1 mRNA can partially rescue the phenotype (110, 111).

Campomelic Dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9 (112). It is caused by SOX9 haploinsufficiency, manifesting as mandibular hypoplasia and craniosynostosis. Knockdown of sox9 in Xenopus suppresses the expression of neural crest effector genes such as snail2 and foxd3, resulting in complete loss of pharyngeal arch cartilages, including Meckel's cartilage, and malformation of the otic capsule, thereby recapitulating sensorineural hearing loss phenotypes (35).

Oral-facial-digital (OFD) represent a heterogeneous group of rare developmental disorders affecting the mouth, the face and the digits. Additional signs may involve brain, kidneys and other organs thus better defining the different clinical subtypes. With the exception of OFD types I and VIII, which are X-linked, the majority of OFDS is transmitted as an autosomal recessive syndrome. A number of genes have already found to be mutated in OFDS and most of the encoded proteins are predicted or proven to be involved in primary cilia/basal body function (113). Injection of morpholino oligonucleotides targeting ints13 into Xenopus embryos to knock down ints13 expression results in developmental defects including microcephaly, shortened body axis, and tail curvature. Additionally, differentiation of multiciliated cells (MCCs) is impaired, accompanied by reduced cilia number and functional abnormalities, thereby recapitulating craniofacial hypoplasia phenotypes (114)

Hamamy syndrome is a rare genetic disorder characterized by intellectual disability, sensorineural hearing loss, congenital cardiac anomalies with intraventricular conduction delay, hypopigmented microcytic anemia, and skeletal abnormalities of the long bones with recurrent fractures. Craniofacial features include severe hypertelorism and craniofacial skeletal dysmorphisms (115). Homozygous mutations in IRX5 are associated with this syndrome. Knockdown of irx5 in Xenopus disrupts the expression of the key chemokine cxcl12 involved in cranial neural crest cell migration, impeding pharyngeal arch mesenchymal condensation. Moreover, knockdown of cxcl12 partially rescues the phenotype, suggesting that IRX5 regulates neural crest migration through negative modulation of chemotactic signaling (1).

Nager syndrome is a rare human developmental disorder characterized by craniofacial defects including the downward slanting of the palpebral fissures, cleft palate, limb deformities, mandibular hypoplasia, hypoplasia or absence of thumbs, microretrognathia, and ankylosis of the temporomandibular joint. There is evidence of autosomal dominant and autosomal recessive inheritance for Nager syndrome, suggesting genetic heterogeneity. The majority of the described causes of Nager syndrome include pathogenic variants in the SF3B4 gene, which encodes a component of the spliceosome; therefore, the syndrome belongs to the spliceosomopathy group of diseases (116). Knockdown of sf3b4 in Xenopus results in downregulation of neural crest marker genes (sox10, twist1) and induces ectodermal apoptosis, suggesting that aberrant RNA splicing leads to craniofacial defects by impairing the survival of neural crest progenitor cells (117).

Craniosynostosis is one of the most common congenital cranial malformations affecting approximately 6/10,000 live births (118), with increasing incidence trends reported over the recent decades (119). The condition, which is characterized by premature fusion of one or more cranial sutures, is classified according to the type (e.g., sagittal, coronal, lambdoid) and/or number (single/multiple) of affected sutures. Involvement of the coronal suture is relatively rare and accounts for only 11%−13% of all cases of single-suture craniosynostosis. Injection of mRNA encoding human ZIC1 mutations (such as p. Ser388^* and p. Glu402) into Xenopus embryos enhances the expression of the downstream target gene engrailed-2 (en-2), leading to aberrant neural crest signaling. Truncating variants, such as p. Ala437, retain the zinc finger domain and aberrantly activate En-2, thereby recapitulating craniosynostosis-like phenotypes in the model organism (120).

Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by distinctive physical features, developmental delay, cognitive impairment, and a typical behavioral phenotype. SMS is caused by interstitial 17p11.2 deletions (90%), encompassing multiple genes and including the retinoic acid-induced 1 gene (RAI1), or by pathogenic variants in RAI1 itself (10%). RAI1 is a dosage-sensitive gene expressed in many tissues and acting as transcriptional regulator. The majority of individuals exhibit a mild-to-moderate range of intellectual disability (121). Knockdown of ra11 has been shown to reduce the expression of the neural crest cell marker tfap2a, leading to decreased craniofacial cartilage formation and increased apoptosis in the forebrain (122).

The Xenopus model has been employed to faithfully recapitulate a wide spectrum of craniofacial disease phenotypes—including cartilage hypoplasia, craniosynostosis, and hypertelorism—thus enabling precise in vivo and in vitro functional analyses such as gene knockdown/overexpression, phenotype rescue assays, pathway interrogation, and cellular behavior studies. These investigations have collectively revealed a shared pathological basis underlying craniofacial malformations: disrupted developmental programs of CNCCs, and the central roles of key signaling pathways including FGF, Wnt, BMP/TGFβ, RA, and Shh. Furthermore, critical cellular processes such as EMT, directed migration, differentiation, and apoptosis are shown to be perturbed. The Xenopus model therefore provides robust in vivo evidence and a unique experimental platform for elucidating disease mechanisms, identifying pathogenic genes, and developing therapeutic strategies.