Expression pattern of aldh7a1 in zebrafish.

Whole-mount in situ hybridization of aldh7a1 at (A) 24 hpf and (B) 48 hpf. L, lens; OF, optic fissure; PF, pectoral fin. Scale bar: 65 μm in A; 60 μm in B.

Aldh7a1 is important for optic fissure closure.

(A–D) Injection of Aldh7a1 morpholino results in failure of optic fissure to close at 28 hpf and sustained at 5 dpf. (A) Eye of control morpholino (MO) injected embryo at 28 hpf, (B) Eye of aldh7a1 morphant at 28 hpf; (C) Ventral view of eye in control MO embryo at 5 dpf, (D) Ventral view of eye in embryos injected with 7.5 ng Aldh7a1 MO at 5 dpf, black bars indicate edges of optic fissures; (E) Bar graph demonstrate distribution of eye phenotypes 0, I, II, and III at 28 hpf following 7.5 ng Aldh7a1 MO injection followed by partial rescue of phenotype upon co-injection of two doses of aldh7a1 mRNA. All control MO injected embryos displayed “0” phenotype. (F) zn-5 staining of control MO injected embryos at 48 hpf compared to (G) aldh7a1 morphant which displays optic nerve hypoplasia, arrow-heads indicate optic nerve. Scale bar: 65 μm in A,B; 125 μm in C,D; 75 μm in F,G.

Aldh7a1 morpholino knockdown embryos show defects in pectoral fin and cartilage development.

(A–B2) Fin phenotypes long (l), medium (m), and short (s) classified by length at 5 dpf, marked by black asterisks. All control MO embryos displayed “Long” fins (A) and Aldh7a1MO injected embryos develop medium (B, 6%) or short (B2, 10%) fin. (C–D) Ventral view of Alcian blue staining of jaw- cartilages in control MO (C) and aldh7a1morphant (D) embryos. Scale bar: 350 μm in A–B2 125 μm in C,D.

Expression pattern of genetic eye development markers in control and aldh7a1 morphant embryos.

(A) Expression of nlz1 in optic fissure is down-regulated in (B) aldh7a1 morphant fish. vax2 and pax2.1 do not seem to show significant change in expression between control MO (C,E) and nlz1 morphant (D, F) fish. (G) Co-injection of nlz1 mRNA resulted in partial rescue of aldh7a1 MO phenotype, examined at 28 hpf, while co-injection of vax2 mRNA showed no change. Scale bar: 65 μm.

Aldh7a1 is required for retinal cell proliferation.

(A–B) Dividing cells in developing eye were labeled with phosophohistone-3 antibody (H3P) in (A) Control MO and (B) alh7a1morphant embryos at 28 hpf. (A) and (B) are projection images of z-stacks through the depth of the eye. (C) Average number of dividing cells per eye quantified for control MO (n = 6), Nlz1 MO (n = 7), nlz1 mRNA (n = 6) rescued Statistical significance indicated above columns *P<0.05, **P<0.01, ***P<0.0001. Scale bar: 65 μm.

aldh7a1 expression.

Whole-mount in situ hybridization of shows expression of aldh7a1 at (A) 9hpf, (B) 18hpf,(C) 36hpf, and (D) 72hpf.e,eye; t, tectum; ce,cerebellum; ov, otic vesicle; fb, fin bud; pf, pectoral fin. Arrow head indicates pharyngeal arches.

Grades of severity for eye development.

Classification of phenotype severity following morpholino injections. Four grades of coloboma (0, least severe; I, II,and III, most severe) classified by distance between edges of optic fissure at ~28 hpf, marked by white asterisks. Scale bar: 65 μm

aldh7a1 loss-of-function phenotype.

(A,D) Aldh7a1MO1 injected embryos developed bent tail compare to control MO injected embryos. (A-F) Lateral view of zebrafish eye at 50 hpf. Co-injection of Aldh7a1MO1 and Aldh7a1MO2 developed coloboma(I n=36/41,J n=25/31), compared to(G,H) control MO injected embryos.(C-F) Sagittal section of eye. Asterisks indicate edges of optic fissure, arrow indicates ventral defect in the eye at 4d.

Coloboma in aldh7a1morphant fish is not due to apoptosis.

Active Caspase3 staining of control (C,E) and aldh7a1 morphants embryos (D,F). White lines in C, D demarcates the eye.