Screening of genes with similar trajectories between mice and zebrafish during craniofacial development. (A,B) Violin plots of scaled log2 normalized gene expression of Sox9 orthologs in (A) mice and (B) zebrafish. (C) Criteria for identifying highly correlated orthologs with Sox9 and sox9b. (D) Enrichment analysis results of the gene ontology terms, including “skeletal” for the 86 mouse orthologs, including Sox9.

Comparing the expression pattern of the selected genes during craniofacial morphogenesis in mouse and zebrafish embryos. (A–D) Schematic illustrations of the craniofacial anatomy of the maxillary (A) and mandibular (B) regions of mice at E12.5 and zebrafish at 48 hours postfertilization (hpf) (C,D). (E–X) The expression pattern of Sox9 (sox9b), Zfhx4 (zfhx4), Zfhx3 (zfhx3b), Cjun (cjun), and Six1 (six1b) were examined using whole‐mount in situ hybridization. The samples are shown at the top, and the genes are shown on the left. (E′–X′) Schematic summary of the expression pattern of the selected genes. Black arrows, black arrowheads, and white arrowheads indicate Meckel's cartilages, ethmoid plate, and ceratohyal, respectively. Scale bars: 500 μm in V; 200 μm in X.

Protein localization in the developing head of mice and zebrafish. (A–D) Immunohistochemistry of selected transcription factors involved in chondrogenesis and osteogenesis using frontal sections of E12.5 mice head (A–C) and ventral view of 72 hours postfertilization (hpf) zebrafish larva (D). Samples are shown at the top, and proteins are shown on the left. White arrows indicate the protein detected in the nasal septum (A) and palate (B,C). (D) Immunofluorescence staining with anti‐Zfhx4 antibody in zebrafish embryos at 72 hpf. The developing ethmoid plate (zebrafish palate, dashed line) and Meckel's cartilage (mandibular, blacked) were stained with the antibody. Zfhx4 was also detected in the eye, ceratobranchial, ceratohyal, and hyosymplectic. (E,F) Similar expression domains of Cjun in both species were revealed using immunohistochemistry in the frontal section of E12.5 mice head, (E) cjun mRNA in the lateral view of E12.5 mice head, (E′) and the lateral view of 72 hpf zebrafish (F). Arrowheads indicate the expression surrounding the ocular region. E, eye; ep, ethmoid plate; MAN, mandibular; MAX, maxillary; m, Meckel's cartilage; NS, nasal septum; PS, palatal shelf; T: tongue. Scale bars: 500 μm in (E); 100 μm in (D,F).

Morpholino (MO)‐based zfhx4 function loss led to craniofacial malformation in zebrafish. (A) Pre‐mRNA of zfhx4 and splicing MO design. Splicing MO (E2I2) was designed to bind to the exon 2 and intron 2 boundary. Presumptive zfhx4 mRNA inhibited by the MO (E2I2) showed splice site skipping between exon 2 and intron 2. (B) Splice‐blocking efficiency confirmation of the MO (E2I2) using reverse transcription‐polymerase chain reaction (RT‐PCR). Total RNA was isolated from the control and zfhx4 morphants at 1 day postfertilization. Forward and reverse primers were designed to target exons 2 and 3, respectively (A). PCR product length of the control was 285 bp, and the length of the splice‐inhibited products was 1305 bp. A single band was detected at the 285 bp length in the control. An unspliced product containing intron 2 was detected at the 1.3 kbp length in the zfhx4 morphants. This band was observed in the same position as the genomic PCR product. (C,D) Typical zfhx4 morphants exhibited moderate and severe craniofacial anomalies. (D). Frequency of the craniofacial anomalies in the zfhx4 morphants. (Control: N = 46, MO [E2I2] of 2 ng/embryo: N = 62, MO [E2I2] of 4 ng/embryo: N = 28). Phenotypic severity was observed with MO (E2I2) in a dose‐dependent manner, corresponding to the RT‐PCR result in Figure 4B. (E,F′) Three‐dimensional reconstructed maxillary (green) and palatine bones (red) in E15.5 control and Zfhx4 null embryos. Smaller maxillary and deformed palatine bones were observed in Zfhx4 null mice compared with their littermate controls. (G–H″) zfhx4 morphants displayed cleft palate and micrognathia (G,H). Effective functional zfhx4 inhibition was confirmed by decreased Zfhx4 expression via immunofluorescence staining (G–H″, Control: N = 46, MO [E2I2]: N = 78). ATG MO of zfhx4 showed craniofacial defects similar to those of E212 MO (I–L). (M) Overall morphology of zfhx4 morphant at 80 hpf. (Control: N = 45, ATG MO: N = 30 in I–J″). (Control: N = 55, ATG MO: N = 38 in K–M). ep, ethmoid plate; m, Meckel's cartilage. Scale bars: 100 μm.

Lineage tracing of cranial neural crest cells (CNCCs) in sox10:Dendra2. (A,B) For time‐lapse imaging of the prospective frontonasal and anterior maxillary prominence, photoconversion of the prospective frontonasal prominence (A) and prospective anterior maxillary prominence (B) was performed in sox10:Dendra2 transgenic zebrafish at 15 ss. As photoconvertible fluorescent protein Dendra2 is photoactivated by UV light (405 nm) from green to red, specific Dendra2‐expressing cells were labeled irreversibly and traced. The time‐lapse imaging was performed by 30 ss (Movies [Link], [Link], [Link]). (C–C″) To investigate the cell lineage of the prospective frontonasal prominence after 30 ss, photoconversion of the frontonasal prominence at 24 hpf (30 ss) were performed (C). The lineage was subsequently examined at 48 (C′) and 72 hpf (C″). The frontonasal prominence was labeled at 24 hpf (C) and differentiated into the medial part of the ethmoid plate at 48 hpf (C′) and 72 hpf (C″). (D–D″) To investigate the cell lineage of the prospective anterior maxillary prominence after 30 ss, photoconversion of the anterior maxillary prominence at 24 hpf (30 ss) were performed (D). The lineage was subsequently examined at 48 hpf (D′) and 72 hpf (D″). The anterior maxillary prominence was labeled at 24 hpf (D) and differentiated into a lateral part of the ethmoid plate (trabeculae) at 48 hpf (D′) and 72 hpf (D″). e, eye; ch, ceratohyal; ep, ethmoid plate; gc, gill cartilage; m, Meckel's cartilage; PA1, first pharyngeal arch. Scale bars: 100 μm.

zfhx4 morphants resulted in defective cranial neural crest cell (CNCC) development and pharyngeal arch formation. (A–D″) Immunofluorescence images of the control and zfhx4 MO (E2I2)‐injected sox10:EGFP. Samples were stained with anti‐GFP antibody (NCCs), anti‐zfhx4 antibody, and 4′,6‐diamidino‐2‐phenylindole (DAPI, nucleus) at the 10‐somite stage (ss) and 24 hpf. (A–A″) zhfx4 protein demonstrated nuclear localization in the NCCs. Insets show a magnified view of the first pharyngeal arch (PA1, white dotted lines). Yellow arrowhead shows the co‐localization of the CNCCs with the zfhx4 protein. (B–B″) zfhx4 morphants showed decreased zfhx4 expression in the CNCCs. (C,D) zfhx4 morphants showed a decreased number of NCCs, leading to PA1 morphological defects. White dotted lines represent PA1 at 24 hpf. (C′–D″) Magnified view of PA1 in (C,D). (Control: N = 72, MO [E2I2]: N = 89). (E–F′) CNCC migration defect in zfhx4 morphants. zfhx4 MO was injected into sox10:EGFP embryos. Migrating CNCCs were visualized using sox10:EGFP at the 10 ss. The yellow arrowhead represents the position of the anteriormost midbrain and hindbrain, which are the starting sites for migrating CNCCs. The white arrowhead indicates the front edge of the migrating CNCCs. (Control: N = 55, MO [E2I2]: N = 38). e, eye; PA1, first pharyngeal arch. Scale bars: 100 μm in (C, D, E–F'); 50 μm in (A–B'', C'–D'').