Identification of CTDSP2 and its homologous gene ctdsp2 in zebrafish: (A) Schematic representation of CTDSP2 sequence variants in families with hemifacial microsomia. The variant c.C332A:p.T111N was identified in probands with sporadic hemifacial microsomia (black), but it was absent in their unaffected parents (blank). Protein structure predictions suggest significant changes from the wildtype (bottom-left image, left: wildtype CTDSP2; right: mutant type), and pathway analysis indicates an interaction between CTDSP2 and the TGF-β pathway. (B) BLAST analysis reveals sequence homology and conserved domain characteristics of zebrafish ctdsp2, which is homologous to human CTDSP2. (C) The expression profile of zebrafish ctdsp2, as determined by the Spatial Transcript Omics DataBase, shows dynamic expression patterns during embryonic development.

Temporal and spatial expression patterns of ctdsp2 in developing zebrafish embryos: (A−D) ctdsp2 is broadly expressed throughout the entire embryo from the 1-cell stage to 10 h post-fertilization (hpf). (E,F) By 1 day post-fertilization (dpf), ctdsp2 expression is notably elevated in the head, eyes, and otic vesicle (inset). (G–L) From 2 dpf, ctdsp2 expression becomes concentrated in the mandibular region.

Knockout of ctdsp2 and associated phenotypic changes: (A) Schematic diagram illustrating the four ctdsp2 gRNA targets in the CRISPR/Cas9 knockdown system. (B–E) Representative efficiencies of the four gRNA targets in ctdsp2 knockout. (F–I) Bright-field images of wildtype and ctdsp2-knockout zebrafish at 4 dpf (F,G) and 5 dpf (H,I), demonstrating mandibular deformity in ctdsp2-knockout embryos.

(A–C) Phenotypes, (D–F) Sanger sequencing chromatograms, and (G–I) genotypes of F2 embryos. The F2 generation displayed two distinct phenotypic categories and three different genotypes: embryos without deformities had either wildtype or heterozygous ((−3+1) bp indels) genotypes, while those with deformities were exclusively homozygous for the (−3+1) bp indels. The red box denotes the sequence of gRNA (exon 1) (GGT TGT CTT CGA AAA CAG GT AGG), and the blue box indicates the 3 bp deletion sequence (GGT) and the 1 bp insert (A).

Phenotypic comparison between ctdsp2^−/− mutants and wildtype zebrafish embryos from 1 to 5 days post-fertilization (dpf): (A–D) No significant phenotypic differences were observed between the two groups at 1 dpf and 2 dpf. (E–J) ctdsp2^−/− embryos abnormally lacked visible signs of mandible development and exhibited cranial developmental abnormalities and microphthalmia compared to wildtypes from 3 dpf. The differences persisted until 5 dpf (I,J), with all ctdsp2^−/− embryos dying of swallowing difficulties and cardiac edema.

Staining results of pharyngeal arch cartilages using Alcian blue in various groups: (A,E) Wildtype embryos, (B,F) ctdsp2^−/− embryos, (C,G) ctdsp2^−/− embryos injected with ctdsp2 mRNA, and (D,H) tp53-knockout ctdsp2^−/− embryos. Abbreviations: m, Meckel’s cartilage; pq, palatoquadrate cartilage; ch, ceratohyal cartilage; cb, ceratobranchial cartilage; hs, hyosymplectic cartilage; e, ethmoid plate cartilage.

Comparative analysis of chondrocyte morphology in (A,E,I) control siblings, (B,F,J) ctdsp2^−/− mutants, (C,G,K) ctdsp2^−/− embryos injected with ctdsp2 mRNA, and (D,H,L) tp53-knockout ctdsp2^−/− embryos. The craniofacial cartilage in control siblings demonstrates a consistent, elongated, and slender chondrocyte structure, forming a “stack of pennies” organization. In ctdsp2^−/− embryos, chondrocytes within the cartilage are noticeably smaller, and the overall cartilage structures are markedly deformed. Injection of ctdsp2 mRNA or knockout of tp53 in mutants partially rescued chondrocyte morphology.

Analysis of somatic cell apoptosis in (A,E,I,M) control siblings, (B,F,J,N) ctdsp2^−/− mutants, (C,G,K,O) ctdsp2^−/− embryos injected with ctdsp2 mRNA, and (D,H,L,P) tp53-knockout ctdsp2^−/− embryos at 24 h post-fertilization (hpf). TUNEL staining signals, indicative of apoptotic cells, were observed in the head, pharyngeal arch, and trunk in ctdsp2^−/− mutants at 24 hpf. The signals were reduced after ctdsp2 mRNA injection. Control siblings and ctdsp2^−/− embryos with tp53 knockout showed no apparent apoptotic signals. The dotted boxes highlight areas of TUNEL staining, emphasizing specific areas where apoptosis was detected.

Immunofluorescence results depicting NCC proliferation through antiphosphohistone H3 (PH3) staining in (A,E,I,M) control siblings, (B,F,J,N) ctdsp2^−/− mutants, (C,G,K,O) ctdsp2^−/− embryos injected with ctdsp2 mRNA, and (D,H,L,P) tp53-knockout ctdsp2^−/− embryos at 48 h post-fertilization (hpf). The merged images highlight the anti-PH3 signals in the NCCs located in the pharyngeal arch region, marked by a dotted box. (Q,R) Number of proliferating NCCs among each group. Note: Immunofluorescence experiments were performed in triplicate, statistical analyses were carried out using 3 randomly picked embryos in each group, and statistical significance was assessed by unpaired t-tests, with p < 0.05 considered statistically significant; **** p < 0.0001.

The impact of ctdsp2 knockout on pharyngeal pouches, neural crest cells (NCCs), and pharyngeal cartilage development in zebrafish embryos: (A–D) ISH results with crestin and foxd3 probes, markers for NCCs, at 24 h post-fertilization (hpf) and 28 hpf. The staining patterns show no noticeable differences between mutant embryos and their control siblings. (E,F) Fluorescence imaging of sox10-labeled NCCs in the pharyngeal arch region. Green fluorescence signals indicating NCCs are similar in both ctdsp2^−/− embryos and siblings. (G,H) Expression of dlx2a at 30 hpf appears similar in both ctdsp2^−/− and control embryos. (I–N) Expression of tbx1, fgf3, and nkx2.3 at 48 hpf shows no significant differences in the segmentation and number of pharyngeal pouches between ctdsp2^−/− embryos and siblings. (O,P) ISH with the barx1 probe at 48 hpf. No significant variation was observed between mutant and control embryos. (Q–X) ISH with sox9a and col2a1a probes at 72 hpf; sox9a expression is notably reduced in mutants, and the expression of col2a1a, a marker for cartilage, is absent in the hypopharyngeal arches of the mutant embryos compared to controls.

qPCR analysis of gene expression in ctdsp2^−/− mutant and control sibling zebrafish embryos. Note: The comparison was conducted using an unpaired t-test, and a p-value less than 0.05 was considered a significant difference; * p < 0.05, ** p < 0.01.