Figure 3.

Figure 3.

Cone outer segment generation is reduced in tmem216 knockout fish. Cryosections of whole zebrafish heads were stained with cone OS markers GNAT2 (green) and 1D4 (red) at 3-, 7- and 14-dpf. All sections were counterstained with DAPI to visualize nuclei. Knockout panels shown were from the tmem216^snyR8Δ60 line. Similar results were observed in the tmem216^snyΔ175 line. (A–F) GNAT2 immunostaining of wild-type retina at 3-, 7-, and 14-dpf, respectively. (G–L) GNAT2 staining of tmem216^snyR8Δ60 homozygous retina at 3-, 7-, and 14-dpf, respectively. (M–R) 1D4 immunostaining of wild-type retina at 3-, 7-, and 14-dpf, respectively. (S–X) 1D4 labeling of tmem216^snyR8Δ60 homozygous retina at 3-, 7-, and 14-dpf, respectively. (Ya, Yc, Ye) Quantification of GNAT2 fluorescence intensity between wildtype and knockout retinas at 3-, 7-, and 14-dpf, respectively. (Yb, Yd, Yf) Quantification of 1D4 fluorescence intensity between wildtype and tmem216^snyR8Δ60 homozygous retina at 3-, 7- and 14-dpf, respectively. Note that GNAT2 and 1D4 immunoreactivity was significantly reduced at 3-, 7- and 14-dpf (n = 3, Student's t-test). (Z) Western blotting using anti-GNAT2 was performed on whole head lysates collected from three 7-dpf wildtype and tmem216^snyR8Δ60 homozygous zebrafish larvae. The intensity ratio of GNAT2/-β-actin between wildtype and knockout was reduced from 0.88 to 0.27. Scale bar in X: 55 µm for A, G, M, and S; 110 µm for C, I, E, K, O, U, Q, and W; 6 µm for B, H, D, J, F, L, N, T, P, V, R, and X. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.