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Fig. 7
Inhibition of apoptosis with Bcl‐xL RNA rescues cell death within the MHB and POM and partially rescues coloboma in pax2a and pax2a; pax2b mutants. (A‐D) Whole‐mount immunofluorescence for cell death (green; activated Caspase‐3) and nuclei (magenta; TO‐PRO‐3) at 24 hpf for (A) wild‐type, (B) pax2a tu29a , (C) pax2b sa10953 , (D) and pax2a tu29a ; pax2b sa10953 mutant embryos injected with 100 pg Bcl‐xL RNA. Images are lateral views of three‐dimensional renderings. (E) Quantification of total number of activated Caspase‐3‐positive cells throughout the depth of the optic fissure within the optic cup in Bcl‐xL‐injected embryos, 24 hpf. n (embryos) shown at base of graphs. Schematic (E) shows the region of the optic fissure through the optic cup in which activated Caspase‐3‐positive cells were quantified. (F) Table summarizing number of Bcl‐xL‐injected embryos at 24 hpf containing five or more activated Caspase‐3‐positive cells located in the forebrain, MHB, and POM for each genotype. (G‐J) Phenotype of (G) wild‐type, (H) pax2a tu29a , (I) pax2b sa10953 , (J) and pax2a tu29a ; pax2b sa10953 mutant embryos injected with Bcl‐xL RNA at 55 hpf. In each example, the eye is evenly pigmented and does not exhibit coloboma. Zoomed insets show the optic nerve head and medial optic fissure where closure has occurred in each genotype. The fraction in the bottom right corner represents the number of embryos without coloboma over the total number of embryos for each genotype. (K) Penetrance of coloboma phenotype in uninjected embryos (light gray bars) compared to embryos injected with 100 pg Bcl‐xL RNA (dark gray bars), 55 hpf. n (embryos) shown at base of graphs. P‐values were calculated using an unpaired Student's t‐test