Fig. 4

The wave of retinal neurogenesis is affected in smu mutant and Gli-MO-injected embryos. (A-B′) Plastic sectioning of wild-type (A) and smu^b577–/– (B,B′) retinas at 48 hpf. In smu^b577–/– retinas, lamination defects vary. No lamination is observed in the most severe phenotype (B) and a nearly normal lamination occurs in the mildest phenotype (B′). (C-D′) ath5 expression in wild-type (C) and smu^b577–/– (D,D′) retinas at 33 hpf. ath5 expression is initiated at the ventro-nasal retina (D, arrowhead) but does not spread to the entire eyes in smu^b577–/– embryos. This phenotype varies from severe (D) to mild (D′, asterisk), and the defect in ath5 expression is proportional to that of a cyclopic phenotype. (E-F′) Labelling of 48-hpf wild-type (E,E′) and smu^b577–/–(F,F′) retinas with the anti-BrdU antibody. (E,F) Central retinas of wild-type (E) and smu^b577–/– embryos (F). These sections correspond to no.7 (wild-type) and no.2 (smu^–/–) shown in G, respectively. (E′,F′) Peripheral retinas of wild-type (E′) and smu^b577–/– (F′) embryos. These sections correspond to no.13 (wild-type) and no.10 (smu^–/–) shown in G, respectively. (G) Spatial profile of the ratio of BrdU-positive area to total area in wild-type and smu^b577–/– retinas. Solid and dotted lines indicate smu^–/– and wild-type sibling retinas, respectively. Numbers represent anterior-posterior locations of cryosections, which are shown in the left schematic drawing. (H) Western blot analysis of forskolin-treated and smu^b577–/– heads using antibodies against Ser133-phosphorylated CREB (upper) and CREB (lower). The phosphorylation of CREB is more than 70 times higher in forskolin-treated heads than in control DMSO-treated heads. However, in smu^b577–/– heads, CREB phosphorylation is decreased to one-third of the normal level. (I,J) Plastic sectioning of wild-type (I) and Gli-MO-injected (J) retinas at 3 dpf. In the severe case of Gli-MO-injected embryos, retinal lamination is severely delayed. (K-L′′) In situ hybridisation of 33-hpf wild-type (K) and Gli-MO-injected (L-L′′) embryos with an ath5 RNA probe. The progression of ath5 expression is perturbed to different degrees in Gli-MO-injected retinas, from no initiation (L), to severe (L′) or mild (L′′) inhibition. In the severe case, ath5 expression is only observed in the ventronasal retina (L′, arrowhead). (M) Quantitative assessment of ath5 expression in wild-type (orange), smu mutant (light blue), Gli-MO-injected (blue) and forskolin-treated (dark blue) embryos. Embryos were classified into four groups according to the severity of the defect in ath5 expression, as shown in (L-L′′) and counted in number. Numbers of examined embryos were n=11 for wild type, n=21 for the smu mutant, n=63 for Gli-MO-injected embryos and n>100 for forskolin-treated embryos. Almost all of the forskolin-treated embryos show the `severe′ phenotype. FK, forskolin.