(A–C) Lateral view; rnf152 was abundantly expressed in the zebrafish embryos at early stages. (D) Image from the animal pole of shield stage demonstrated thin germ ring with embryonic shield (arrowhead). (E–G) Lateral view of the embryos at tail bud, 14 hpf and 18 hpf. (H) Dorsal view of the embryo at 18 hpf found rnf152 transcripts in the eyes, CNS and spinal cord. (I&J) Lateral and (K) anterior view of the embryos at 24 hpf showed rnf152 transcripts were present in the eyes and CNS and some parts of posterior region. (L&M) Lateral view, (N) dorsal view of the embryos at 48 hpf demonstrated that rnf152 transcripts were in the eyes, midbrain and hindbrain boundary (MHB), and rhombomeres. All embryos were collected synchronously from WT zebrafish for WISH analysis at the corresponding stages. Abbreviations: de- Diencephalon, ES- Embryonic shield, Fb-Forebrain, hb-hind brain, PSM- presomitic mesoderm, ret-eye, SC- spinal cord (n=3). Scale bars A-N: 50 μm.

(A) WT zebrafish embryo, (B) vehicle control injected with the same volume of phenol red dye in distilled water as the volume of the mRNAs injected, and (C) rnf152 mRNA injected embryo. Microinjection of rnf152 mRNA (200 pg) into embryos at 1- or 2 cell stages for overexpression of rnf152. All images are in lateral view (n = 3). (D) WT, (E) 5′ mismatch MO, and (F) rnf152 MO that 5 ng of rnf152 morpholino was injected into embryos at 1-cell stage for knockdown of rnf152. All images (D–F) are in lateral view of embryos at 24 hpf. (G–J) Graphs showing diameter of the eyes and width of the neural tubes. (G) Diameter of the eyes, (H) width of the neural tube of WT, vehicle control, and rnf152 overexpressed embryos. (I) Diameter of the eyes, (J) width of the neural tube of WT, 5′ mismatch control, and rnf152 MO. Red arrowheads indicate the rhombomeres (r1–7) while dotted red lines indicate the width of the neural tube. Area of the eyes was marked with red dotted lines. The graphs were obtained using ImageJ, error bars depicting standard errors. Unpaired t-test was executed to obtained p-values, which is p<0.0001 (n=3). Abbreviations: fb- forebrain, hb- hindbrain, mb- midbrain, NT- neural tube, ret- eye, SC- spinal cord, and TB- tail bud. Scale bars A–F: 50 μm.

Two color WISH analysis with neuroD- and pax6–specific probes detected changes in their transcripts in the retinal layers of embryos at 27 hpf. (A, D) WT, (B, E) 5′ mismatch MO control, and (C, F) rnf152 MO. neuroD transcripts (dark blue) were abundant in the INL and ONL of eyes, telencephalon, dorsal diencephalon, octaval/statoacustic ganglia and posterior lateral line ganglia of WT as well as the 5′ mismatch control, but remarkably reduced in rnf152 MO. Levels of pax6 transcripts (tomato color) in the telencephalon, dorsal diencephalon, hindbrain, anterior spinal cord, and eyes of the WT were similar those of rnf152 MO at 27 hpf. Red arrowheads indicates the areas of significant differences in the level of neuroD transcripts between WT and rnf152 MO. Abbreviations: ad/av/f- Anterodorsal/anteroventral lateral line/facial placodes/ganglia, dd- Dorsal diencephalon, GCL- Ganglion cell layer, INL- Inner nuclear layer, o- Octaval/statoacustic ganglia. ONL- outer nuclear layer, p- Posterior lateral line ganglia, RPE- retinal pigment epithelium, T- Telencephalon (n = 3). Scale bars (A–F): 50 μm.

WISH analysis of rnf152 MO using deltaD as a probe. (A, D) WT embryos, (B, E) embryos injected with 5′ mismatch as control, and (C, F) rnf152 MO. deltaD transcripts were abundant in the ventral midbrain and rhombomeres of the brain but low in the eyes of WT (A, D) and 5′ mismatch (B, D). However, level of deltaD transcripts was significantly reduced in the respected tissues (C, F). Red arrowheads indicate the eye, midbrain, and hindbrain (n = 3). Scale bars (A–F): 50 μm.

WISH analysis of rnf152 MO using notch1a and notch3 as probes. (A, D) notch1a transcripts in the eye, forebrain, midbrain, and hindbrain of WT embryos at 24 hpf. (B, E) Embryos injected with 5′ mismatch at 24 hpf showed similar patterns to those of WT embryos. (C, F) rnf152 MO shows remarkable reduction of notch1a transcripts in the eyes, forebrain, midbrain, and hindbrain at 24 hpf. Eyes, mid-hindbrain were shown in red arrowheads. Transcripts of notch3 in (I, L) rnf152 MO, (G, J) WT, and (H, K) 5′ mismatch control at 24 hpf. notch3 transcripts were present in the olfactory bulbs, midbrain, and hindbrain in WT and 5′ mismatch control whereas they were significantly diminished in the corresponding areas of rnf152 MO. Red arrowheads indicate eyes, mid-hindbrain tissues (n = 3). Scale bars (A–L): 50 μm.

(A–F) Dorsal view of embryos at 29 hpf. WISH analysis with her4 detected the transcripts in the eyes, forebrain, mid-hindbrain, and spinal cord of the WT (A) and 5′ mismatch control (B) while they disappeared in the eye and rhombomeres of rnf152 MO (C). Red arrowheads indicate her4 transcripts in the eyes and anterior rhombomeres (n = 3). WISH analysis with ascl1a found that ascl1a transcripts in the eyes, forebrain and mid-hindbrain of the two control groups but were significantly reduced in those of rnf152 MO. Red arrowheads indicate the eyes and rhombomeres (n = 3). Abbreviations: fb- forebrain, mb- midbrain, hb- hindbrain, ret- retina. Scale bars (A–F): 50 μm.

Level of deltaC transcripts is elevated in rnf152 morphants.

rnf152 MO were analyzed with WISH using deltaC as a probe. (A&D) WT embryos, (B&E) embryos injected with 5' mismatch as control, and (C&F) embryos injected with rnf152 MO. deltaC transcripts were abundant in the ONL and INL of the eyes, but low in the forebrain, midbrain, and hindbrain of WT (A&D) and 5' mismatch (B&D). However Level of deltaC transcripts was significatnly elevated in the ONL, INL, and GCL of the eye (C&F). Red arrows indicate the layers of eyes where level of deltaC transcripts were elevated at 24 hpf (=3). Scale bars A-F: 50 μm.

Transcripts of notch1b is not effected by rnf152 knock-down.

WISH analysis with notch1b transcripts in rnf152 MO. (A&D) WT embryos, (B&E) Embryos injected with 5' mismatch MO control, and (C&F) Embryos injected with rnf152 MO. Level of notch1b transcripts was similar among the three groups. dotted red circles indicate eyes. Red arrowheads indicate eyes, midbrain and hindbrain at 24 hpf (n=3). Scale bars A-F: 50 μm

WISH analysis of WT and rnf152 MO with rx2- specific probe identified alteriations in the optic vesicles at 12,15, and 18 hpf.

(A-C) 12 hpf, (D-F) 15 hpf, and (G-I) 18 hpf. (A,D,&G) WT embryos, (B,E,&H) Embryos injected with 5' mismatch control morpholino, and (C,F,&I) Embryos injected with rnf152 morpholino. Level of rx2 transcripts did not show significant differences in the optic vesicles among WT, 5' mismatch control, and rnf152 MO at 12, 15, and 18 hpf. Red arrowheads indicate optic vesicle of the embryo (n=3). Scale Bars A-I: 50 μm.