Retinoic acid regulates krt18a.1, but not krt8, expression in the eye during early embryogenesis. (A?F) Treatment of Casper zebrafish embryos at 24 and 27 hpf with DEAB and RA, respectively, followed by wholemount colorimetric in situ analysis shows decreased krt18a.1, but not krt8, expression in the developing eye in response to pharmacological insult. (G) Quantification of these effects, as measured in the anterior chamber (orange dashed box) and hyaloid (yellow dashed box) regions of the eyes of 48-hpf zebrafish, further shows that compared with the DMSO vehicle control, treatment with either DEAB or ATRA significantly decreased krt18a.1 expression, while that of krt8 was not affected. *, p-value < 0.05.

Fertility and embryonic development of the established paqr5b?/? line. (A) DNA sequence and predicted protein structure of an established paqr5b gene-edited strain. (Uppest panel) DNA sequences around the target site (in red) for CRISPR/Cas9 digestion in wild-type (paqr5b+/+) and paqr5b?/? mutants are indicated. A 13-nucleotide deletion was induced in the target site in the selected mutant. (Middle panel) The predicted protein sequences of the paqr5b+/+ and paqr5b?/? mutants are indicated. It was expected that a peptide of 74 amino acids in length with an N-terminal 18 amino acid sequence produced in the paqr5b?/? mutant was the same as that produced in wild-type Paqr5b zebrafish. Thus, only an N-terminal short fragment without a transmembrane region is present in the Paqr5b protein produced in the paqr5b?/? mutant. (Lowest panel) Diagrams of the predicted protein structures of the paqr5b+/+ and paqr5b?/? mutant strains are shown. The predicted transmembrane regions and the altered amino acid regions in the paqr5b?/? mutant are indicated by black boxes and diagonal boxes, respectively. (B) The relative expression levels of paqr5b in the olfactory rosette (OR) of the wild-type and the paqr5b mutants were compared. mRNA abundance was measured in triplicate for each sample from 10 fish, and all data from three preparations were normalized by the number of elongation factor 1? (EF1?) transcripts in each sample. The data indicated the expression level of the wild type set as 100. (C) Fecundity of paqr5b+/+ and paqr5b?/? zebrafish. The total number of eggs crossing each line was counted (n = 4, ** P ? 0.001). (D) Representative photograph of developing embryos with different sizes of chorions. The scale bar is 1 mm. Comparison of choroidal diameter between paqr5b+/+ and paqr5b?/? embryos (n = 4, **** P ? 0.00001). (E) Representative photograph of embryos at 5 dpf. Typical head abnormalities and spine curvatures in the paqr5b?/? embryos are indicated by arrows. The scale bars in the upper panels are 1 mm. The scale bars in the lower panels are 500 ?m. (F) Comparison of the percentage of abnormal embryos between the paqr5b+/+ and paqr5b?/? embryos (n = 4, **** P ? 0.00001). (G) Comparison of brain transverse sections of paqr5b+/+ and paqr5b?/? embryos. The ventricle of the embryo brain is indicated by arrows. The scale bars are 20 ?m.

sacs-/- mutant retinas showed Müller glia activation and inflammation (A and C) Eye confocal images of control and sacs-/- larvae obtained after immunostaining with antibody 4C4, which is specific for zebrafish microglia. (B and D) cell digital image. After random selection of cells from the eye picture (A and C), the noise was removed by filtering the overall background to get a shape extraction. Next, the image was changed to grayscale, and then transformed into a binary image. The binary image was edited to clear the background and to join all branches (B′ and D′). (E) Quantification of the roundness index revealed activated microglia in sacs-/- larvae which appeared ameboid in shape. *p < 0.05 was calculated by two-tailed Mann-Whitney U Test (N = 3 for each group). (F) The up-regulation of GFAP in sacs-/- retinas as detected by immunostaining. (G) Relative mRNA expression of inflammatory factors evaluated in sacs-/- larvae at 120 hpf. qRT-PCR analysis revealed an increase in the levels of inflammatory genes, normalized to β-actin. Three independent RNA samples from sacs-/- mutant larvae at 120 hpf and from controls. *p < 0.05, calculated by two-tailed Student's t-test. Error bars indicate mean ± s.e.m.

Increasingly more specific expression pattern of Gv suggests manifold roles during ontogenesis. Expression of Gv was examined by whole mount in situ hybridization in six developmental stages, 6, 12, 24, and 48 hpf (embryos), and 3 and 5 dpf (larvae). Organ abbreviations: bst, brain stem; c, cerebellum; ch, ceratohyal; e, eye; hg, hatching gland; ES, embryonic shield; hs, hyosymplectic; k, Kupffer’s vesicle; m, mandibular (Meckel’s + palatoquadrate); mb, midbrain; p.a., pharyngeal arches; sb, swim bladder; tb, tail bud. Symbols and line styles: white dotted line, hatching gland; white stippled/dotted line, hypoblast; red stippled line, notochord (axial mesoderm at 12 hpf); red asterisk, midbrain/hindbrain boundary; red arrow head, inner ear; green line, midbrain; green arrow head, gut; cyan stippled line, first and second pharyngeal arches (mandibular: Meckel’s cartilage and palatoquadrate and hyoid: ceratohyal and hyosymplectic); blue line, pharyngeal arches; gray dotted line, epiblast; gray asterisk, proximal convoluted tubule (PCT); black stippled line, pronephros (lateral plate mesoderm at 12 hpf); black line, yolk extension; black arrow, pectoral fin bud; black ‘v’, somites. For better visibility some lines are contrast adapted. a) 6 hpf; b), d), e) 12 hpf; d) dorsal view; e) dorsolateral view; c), f), g) 24 hpf; c) lateral view; f) dorsal view; g) dorsolateral view; h), j), k), l) 48 hpf; h) lateral view; j) dorsal view; k) ventral view; l) ventrolateral view; i) 72 hpf, lateral view; m) 120 hpf, lateral view