Tcof1 knockdown in zebrafish recapitulates TCS features. (a) RT-qPCR of rRNA transcripts (by ETS and ITS quantification) in samples from control (STD) and TRA-MO-injected 256 cell stage and 24 hpf embryos. Bars represent the mean of relative abundance and S.E.M., n=3, *P<0.05, **P<0.01 (t-test). AU: arbitrary units. (b and c) Ventral views of a typical control (b; STD-MO-injected) and a representative TCS-like (c; TRA-MO-injected) 4 dpf larvae stained with Alcian Blue. Meckel area (MA), area of the inner triangle defined by the Meckel cartilage, PQ length: length of palatoquadrate+hyosymplectic cartilages, ceratohyal length (CL): length of ceratohyal cartilage, CD: distance between ceratohyal cartilages joint and lateral fins, cranial distance (Cr): distance between the most anterior Meckel and lateral fins

Craniofacial phenotypes generated by tcof1 knockdown were partially rescued by Tp53 depletion. (a) Western blotting showing the Tp53 abundance in control (STD), TRA and TRA+^ATGTp53 24 hpf embryos. Molecular weight markers are shown at the right. Tubulin was analyzed as a loading control. The graph shows the densitometric quantification of western blotting band (normalization of Tp53 signal with respect to tubulin). Bars represent mean of relative abundance and S.E.M., n=3, *P<0.05 (t-test) AU: arbitrary units. (b) Expression analysis of Tp53 targets: ccng1 (encoding cyclin G1), pmaip1 (encoding Noxa) and tp53inp1 (encoding tumor protein p53-inducible nuclear protein 1) in control (STD), TRA and TRA+ATGTp53 24 hpf embryos. Bars represent mean of relative abundance and S.E.M., n=3, *P<0.05, (ANOVA test). (c) Acridine Orange staining in vivo performed in 24 hpf embryos. Representative photos of embryos displaying normal (+), intermediate (++) or extensive (+++) cell death. The bar graph shows the distribution of each phenotype among the groups (STD, TRA and TRA+^ATGTp53 embryos). ey=eye, n=50 embryos in each of three independent experiments. ***P<0.001 versus STD; ^###P<0.001 versus TRA; NS=non-significant P>0.05 (chi-square test). (d–h) Quantification of craniofacial parameters (as defined in Figure 1) measured in STD, TRA-MO TRA+^ATGTp53 larvae (4 dpf), stained with Alcian Blue. (d) Meckel Area, (e) PQ, (f) Ceratohyal length, (g) CD, (h) Cr. Bars represent means in AU and S.E.M. n=47 for STD, n=79 for TRA, n=30 for TRA+^ATGTp53. *P<0.05, **P<0.01, ***P<0.001 versus STD; ^#P<0.05, ^##P<0,01 ^###P<0.001 versus TRA; NS=non-significant P>0.05 (t-test)

TCS-like phenotype is ameliorated in cnbp-overexpressing transgenic zebrafish. (a) Relative abundance of cnbp mRNA determined by RT-qPCR using total RNA from 24 hpf zebrafish control (STD) and TRA-embryos. Bars represent mean of relative abundances and S.E.M., n=3 AU: arbitrary units. (b) Western blotting analysing cnbp expression in 24 hpf embryos microinjected with TRA-MO. Controls were injected with STD-MO. Actin detection was used as a loading control. (c) Densitometric analysis of Cnbp normalized to Actin. The bars are means±S.E.M. of three experiments. n=5, **P<0.01 (t-test) (d) Fluorescence images of wild-type (WT) and transgenic zebrafish Tg(XIa.Eefiai:cnbpa-EGFP) lines (L-Cnbp and H-Cnbp). (e) Levels of Cnbp-eGFP detected by western blotting in 24 hpf transgenic zebrafish embryos by using anti-GFP antibodies. (*) Non-specific bands. Actin was assessed as a loading control. (f–j) Bar charts representing the quantification of craniofacial parameters measured in controls (STD), wild-type tcof1-knockdown larvae (TRA) and transgenic tcof1-knockdown larvae overexpressing low (TRA+L-Cnbp) or high (TRA+H-Cnbp) levels of Cnbp. (f) Meckel Area, (g) PQ length, (h) Ceratohyal length; (i) CD, (j) Cr. In TRA+L-Cnbp larvae, only the CD was restored (P<0.05 versus TRA and not different from STD), the Meckel area was indistinguishable from TRA and the rest of parameters showed in-between statistics (not displayed). Bars represent means in AU and S.E.M. n=47 for STD, n=79 for TRA, n=15 for TRA+L-Cnbp, n=36 for TRA+H-Cnbp. ***P<0.001 versus STD, ^###P<0.001 versus TRA NS=non-significant P>0.05 (t-test)