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Figure 1. Sprouty2 is required for eye development through regulating the population of retina. (a) Whole-mount in situ hybridization reveals that sprouty2 is expressed in the optic vesicle at late neurula stage. Black arrowhead indicates optic vesicle on stage 18 embryo. The eye field was outlined with white dotted line. (b) Embryos were injected with wild type Sprouty2 mRNA (WT) or morpholino-resistant (MOR) form of Sprouty2 alone, or plus Sprouty2 morpholino at one cell stage, and analyzed by Western blot at late gastrula stage. Sprouty2 MO blocks the expression of Sprouty2 WT but not the Sprouty2 MOR. (c) Embryos were injected with Sprouty2 WT RNA or sprouty2 MO or a combination of Sprouty2 MO and Sprouty2 MOR RNA at one cell stage. Phosphorylation of ERK was analyzed by Western blot using embryo lysate from late gastrula stage. (d) D1.1.1 blastomere was injected with Sprouty2 MO with or without Sprouty2 MOR RNA, and the eye size was analyzed at stage 36. Dorsal view of the eyes in Sprouty2 morphant shows a significant reduction in diameter which could be well rescued by Sprouty2 MOR RNA co-injection. Quantification of eye diameter with one-way ANOVA (Dunnett’s multiple comparison), ****P < 0.0001, ***P < 0.001, Error bars indicate ± SD. (e) The D1.1.1 blastomere was injected with GFP mRNA plus Sprouty2 WT or Sprouty2Y55F RNA alone or Sprouty2 MO with or without Sprouty2 MOR RNA. Embryos were sectioned and immunostained with GFP antibody (Green) on the eye region at stage 33. The retina was outlined with an oval dotted line. (f) The Histograms indicate the percentage of embryos with D1.1.1 progeny in the retina from three biological repeats. Quantification with one-way ANOVA (Sidak's multiple comparison), ****P < 0.0001, Error bars indicate ± SD. |
