Identification of AMER1 and its homologous gene in zebrafish. (A) Schematic diagram of AMER1 sequences in families of patients with dichotomous monozygotic twins with microtia. The AMER1 variant (c.61C>T: p.R21C) was detected in two patients with sporadic microtia (shaded), as well as in their normal twins and parents (blank). The predicted protein structure showed dramatic changes from wildtype, and pathway prediction showed a relationship between AMER1 and the Wnt pathway. (B) BLAST analysis of sequences homologous to human AMER1 in zebrafish. (C) Expression profile of zebrafish amer1, as determined using the Spatial Transcript Omics DataBase.

Expression of amer1 in zebrafish. (A) RT-PCR analysis, showing that amer1 was constitutively expressed in zebrafish embryos, beginning at the 1-cell stage to 7 dpf. (B–K) In situ hybridization of amer1, showing that (F) amer1 was expressed in otic vesicles (white circle) at 18 hpf; and that (H–K) concentrated expression of amer1 in the mandible region (white circle) was observed beginning at 2 dpf.

Knockdown of amer1 and phenotypic changes. (A) Schematic diagram showing the four amer1 gRNA targets of the CRISPR/Cas9 knockdown system. (B–E) PCR analysis, showing representative amer1 knockdown efficiencies of the four gRNA targets. (F-M) Brightfield images of embryos of (F–I) wildtype and (J–M) amer1 knockdown zebrafish at (F,J) 2, (G,K) 3, (H,L) 4, and (I,M) 6 dpf, showing malformed mandibles in the amer1 knockdown embryos.

Phenotypic changes induced by amer1 knockdown, as shown by Alcian and WGA staining. (A–D) Alcian staining of embryos of (A,C) wildtype and (B,D) amer1 knockdown embryos at 4 dpf, showing malformation of Meckel’s and palatoquadrate cartilages in the amer1 knockdown embryos. (E–P) Alcian staining of tissue sections of embryos of (E–J) wildtype and (K–P) amer1 knockdown zebrafish at 4 dpf, showing that Meckel’s cartilage was severely deformed in amer1 knockdown embryos (red rectangles in (M–O)). (Q,R) WGA staining of wildtype embryos, showing regularly distributed, well-shaped chondrocytes, with Meckel’s cartilage and ceratohyal cartilage normally not observed in the same plane. (S–U) WGA staining of amer1 knockdown embryos, showing Meckel’s cartilage (red arrowheads) and palatoquadrate and ceratohyal cartilage (long red arrows) in the same plane. All cartilage was deformed and the arrangement of chondrocytes was disordered.

Genotypes and phenotypes of F2 zebrafish. (A) Sequencing results and brightfield images of F2 zebrafishes at 3 and 4 dpf with five different genotypes/treatments: wildtype, amer1^+/−, amer1^−/−, and amer1^−/− + upf3a MO, wildtype + upf3a MO. amer1^−/− F2 embryos treated with upf3a MO showed malformation of the mandible. Red suqares indicate mutated sites. NA: not applicable. (B) Fluorescence staining showing that chondrocytes were regularly distributed in wildtype and amer1^−/− F2 embryos, but were disarranged in amer1^−/− F2 embryos treated with upf3a MO.

Proliferation and apoptosis of CNCCs in zebrafish embryos. (A) Proliferation of CNCCs, as shown by PHH3 staining, in wildtype and F0 amer1 knockdown embryos. (B) Quantitation of CNCC proliferation in wildtype and amer1 knockdown embryos (n = 3 each). (C) Apoptosis of CNCCs, as shown by TUNEL staining. (D) Quantitation of CNCC apoptosis in wildtype and amer1 knockdown embryos (n = 3 each). ** p < 0.01; *** p < 0.001.

Expression of CNCC related markers in zebrafish embryos. In situ hybridization, showing the expression of (A) crestin and (B) foxd3 at 12 hpf, (C) dlx2a at 30 hpf, and (D) barx1 at 48 hpf in wildtype and F0 amer1 knockdown embryos. (A–C) The levels of expression of crestin, foxd3, and dlx2a showed no apparent differences in wildtype and amer1 knockdown embryos. (D) The expression pattern of barx1 was smaller in amer1 knockdown than in wildtype embryos. (E,F) The expression of sox9a at 72 hpf was increased in amer1 knockdown than in wildtype embryos (for quantitative analysis, n = 3 each). (G,H) The expression of col2a1a at 72 hpf was decreased in amer1 knockdown than in wildtype embryos (for quantitative analysis, n = 3 each). * p < 0.05; ** p < 0.01; *** p < 0.001.

Expression and localization of components of the wnt pathway in zebrafish embryos. (A,B) Western blotting and quantitation showing that β-catenin levels were higher in amer1 knockdown than in wildtype embryos at 4 dpf. (C) qPCR showing that the levels of expression of lef1, jun, and fosl1a were significantly higher in amer1 knockdown embryos. (D–G) Validation of the expression of (D) lef1 and (F) jun by in situ hybridization and their quantitation (E,G) (for quantitative analysis, n = 3 each). (H) Immunofluorescence staining of the ceratohyal cartilage using WGA, β-catenin, and DAPI in wildtype and amer1 knockdown embryo sections at 4 dpf. (I) Manual counting of co-localization of β-catenin with cell nuclei (n = 3 each). * p < 0.05; ** p < 0.01; *** p < 0.001.

Phenotypic rescue of F0 amer1 knockdown embryos using IWR-1-endo. (A) Schematic diagram of the rescue and observation process. Different numbers indicate different treatment. (B) Proportions of malformed wildtype, amer1 knockdown and IWR-treated amer1 knockdown embryos, showing that the proportion of malformed IWR-treated amer1 knockdown was significantly lower than that of amer1 knockdown embryos. (C) Representative brightfield images of wildtype, amer1 knockdown embryos, and IWR-treated amer1 knockdown embryos. (D) Representative Alcian staining images of wildtype (first row, left), IWR-treated wildtype (first row, middle), amer1 knockdown (first row, right), and IWR-treated amer1 knockdown embryos showing normal development of pharyngeal arch cartilage (second row, left), partial loss (second row, middle), and complete loss (second row, right). ** p < 0.01; *** p < 0.001.

Effects of IWR-1-endo on components of the Wnt pathway in F0 amer1 knockdown embryos. (A,B) Western blotting and quantitation showing that β-catenin expression was lower in IWR-treated amer1 knockdown embryos than in amer1 knockdown embryos at 4 dpf. (C) qPCR showing that the levels of expression of lef1, jun, and fosl1a were lower in IWR-treated than in untreated amer1 knockdown embryos. (D) Co-localization of β-catenin with the cell nucleus in the ceratohyal cartilage was reduced in amer1 knockdown embryos treated with IWR. (E) Manual counting of co-localization of β-catenin with cell nuclei in embryos with different treatments (n = 3 each). * p < 0.05; ** p < 0.01; *** p < 0.001.