Photoreceptor degeneration and defective protein transport in tulp1-dKO zebrafish.A Sections were stained with rod-specific marker Rhodopsin, green cone-specific marker Opn1mw1, red cone-specific marker Opn1lw1, blue cone-specific marker Opn1sw2, and UV cone-specific marker Opn1sw1 at 3 dpf, 5 dpf, 20 dpf, and 4 mpf. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 15 µm. B TEM of the photoreceptors of wt and tulp1-dKO zebrafish at 5 dpf. The higher-magnification images within the rectangles (in a and b) are shown on the right (a’ and b’). Scale bar: 2 µm. C Relative mRNA expression of gnat1 and gnat2 in wt and tulp1-dKO zebrafish at 4 dpf. Mean ± SD (n = 3). ****P < 0.0001. D Gnat1 and Gnat2 were detected by western blot.

Overall phenotypes of chd7 mutants. (A) Expression of chd7 in chd7 sr5 and chd7 sr6 larvae at 4 dpf. The fold change of chd7 expression compared to WT was shown. (B) Percentage of the surviving offspring from chd7 sr5 incross (n > 40 for each time point). (C) Bright field images of chd7 sr5 and MZchd7 sr5 mutants at 7 dpf. MZchd7 sr5 fish have a smaller swim bladder (yellow arrowhead), pericardial edema (red asterisk), and small eyes (blue arrowhead). (D) Quantification of heart edema. Nearly 40% of MZchd7 sr5 fish have heart edema; however, chd7 sr5 fish do not show heart edema phenotypes. WT: n = 879, chd7 sr5 : n = 747, MZchd7 sr5 : n = 751. (E) Quantification of the small eye phenotype. The longest diameter of the dissected eye was measured. The eye size of the MZchd7 sr5 mutants is significantly reduced (n = 22 for each group). Statistical analyses were performed using the ordinary one-way ANOVA test for (A,E) and chi-square test for (B,D). ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

(A) Transmission electron micrographs showing posterior lateral line nerve (pLLN) axons myelinated by Schwann cells (SCs) at 7 days post fertilization (dpf). Myelin is shaded in blue, and myelinated axons are shaded in orange. (B) Immunofluorescence (IF) showing NaV channels (cyan) along mbpa:tagrfp-caax-positive pLLNs (orange) at 7 dpf. Diffuse NaV channel staining along unmyelinated nerves was not quantified. White dashed lines outline the pLLN. White dashed boxes correspond with the insets on the right. (C) IF showing NF186 clusters (cyan) along mbpa:tagrfp-caax-positive pLLNs (orange) at 7 dpf. White dashed lines outline the pLLN, and the white dashed boxes correspond to the insets on the right. Representative images in (B) and (C) depict somites 11–13 (~320 µm). (D) Average number of myelin wrappings per pLLN axon plotted relative to the average area of axon cross-section at 7 dpf. Data were collected from three sections per fish separated by 100 µm. Significance was determined by comparing the average number of myelin wraps divided by the average axon area for each fish with Student’s t-test using GraphPad Prism (average number of myelin wraps per fish mean ± SEM: WT: 3.95 ± 0.19, cd59uva48: 3.27 ± 0.20; average axon area per fish mean ± SEM: WT: 0.51 ± 0.03, cd59uva48: 0.52 ± 0.03; average number of myelin wraps/average axon area per fish mean ± SEM: WT: 0.13 ± 0.01, cd59uva48: 0.16 ± 0.002; p=0.0568; dot = 1 fish). Data quantified in (D) were determined from electron micrographs in (A). (E) Scatter plot of the number of NaV channel clusters along mbpa:tagrfp-positive pLLN nerves at 7 dpf (mean ± SEM: WT: 17.3 ± 0.7, cd59uva48: 9.9 ± 0.9; p<0.0001; dot = 1 fish). (F) Scatter plot of the number of NF186 clusters along mbpa:tagrfp-positive pLLN nerves at 7 dpf (mean ± SEM: WT: 24.0 ± 0.7, cd59uva48: 18.2 ± 1.3; p=0.0011; dot = 1 fish). Data were collected from somites 3–13 (~320 µm) and normalized to units per 100 µm for (E) and (F). Images shown in (A) were acquired with transmission electron microscopy. Images shown in (B) and (C) were acquired with confocal imaging. Each dataset was compared with Student’s t-test using GraphPad Prism. Scale bars: (A) 1 µm; (B, C) 25 µm.

Nucleolin regulates Fgf8a expression. (A) Immunostaining of 36 hpf ncl+/+ and ncl−/− embryos with an Fgf8a-specific antibody reveals reduced expression of Fgf8a in ncl−/− embryos (n=15). (B) qPCR using craniofacial tissues from ncl+/+ and ncl−/− embryos at 18, 24, 30 and 36 hpf indicates that ncl and fgf8a expression gradually reduce over time (n=10 per replicate). (C) RNA immunoprecipitation followed by qPCR indicates higher binding of fgf8a mRNA to Nucleolin compared with the IgG control. actb was used as a negative control (n=100 per replicate per condition). The y-axis indicates fold change of RNA pulldown compared with its absolute expression in the embryos. (D-F) Skeletal preparations of 5 dpf ncl+/+ and ncl−/− larvae as controls (D) for 0.25 µg/µl (E) and 1 µg/µl (F) FGF8 exogenous treatment. Exogenous FGF8 rescued the cranioskeletal phenotype of ncl−/− larvae (n=45). Black arrows indicate the improvement of the basihyal phenotype in FGF8-treated larvae. All experiments were performed three times. Scale bars: 100 µm (A); 70 µm (D-F). Data are represented as mean±s.d. ns, not significant; *P<0.05 (two-tailed, paired Student's t-test).