Fig 2: JNK3 is expressed in regressing hyaloid vessels and mediates EphrinB2-dependent endothelial cell death. (a) STAT1 silencing augments cell viability in HUVEC expressing eB2-5Y. Results reflect the means±SD from 3 experiments; error bars: ±SD. (b,c) eB2-shRNA augments JNK3 mRNA (b; means±SD from 3 experiments; error bars: ±SD) and protein (c) expression in HUVEC. (d) Transduction of eB2-5Y but not eB2-WT augments JNK3 expression in HUVEC; means±SD from 3 experiments; error bars: ±SD (e) Time-dependent JNK3 and cJUN activation in serum-starved HUVEC. Cell lysates from serum-starved HUVEC were tested at the indicated time-points for kinase activity and content of p-JNK1, p-JNK2 and p-JNK3 after immunoprecipitation with anti-p-JNK antibody. (f) JNK3 transduction reduces HUVEC viability; means±SD from 3 experiments; error bars: ±SD (g) JNK3 silencing improves HUVEC viability compromised by eB2-shRNA and B2-5Y transduction; means±SD from 3-5 experiments; error bars: ±SD (h) JNK3 is detected in p5 and p7 WT hyaloid vessels. Degenerating hyaloid vessels are marked by anti-JNK3 antibody staining (red); p-EphrinB immunostaining (green) is absent from JNK3+ hyaloid vessels; DAPI (blue). Scale bars: 200µm top panels, 50µm bottom panels. (i–k) Immunostaining reveals an inverse relationship between JNK3 and p-EphrinB fluorescence intensity in individual IB4+ cells within p5 and p7 WT hyaloid vessels. Cell distribution based on JNK3 and p-EphrinB2 mean fluorescence intensity; each dot represents 1 cell (i); the bar graphs show (j) the means (n=100–300/group) and range (±SD error bars) of p-EphrinB fluorescence intensity in JNK3+ cells and (k) the geographical distribution within hyaloid vessels of JNK3+ (mean fluorescence intensity =3.5)/p-EphrinB+ (mean fluorescence intensity =30) cells (means±SD; n=4 areas/group); fluorescence intensity values in each region are normalized by DAPI. P values from two-tailed Student t-test: N.S. not significant, *P<0.05, **P< 0.01, ***P<0.001.

Fig 3: Phenotypes of JNK3-/- eyes. (a) JNK3-/- mice display microphthalmia and abnormal retinal folding. Front and rear view of p5 eyes from JNK3+/+ and JNK3-/- mice. Arrows: abnormal retinal folds and pockets. Scale bars: 1mm (b) Measurements of eye and lens size in eye sections of JNK3+/+ and JNK3-/- p5 mice. The results reflect the means ±SD; n=3 mice/group; P values from two-tailed Student t-test: **P<0.01 and *** P<0.001. (c) Presence of an abnormal retrolental mass in the vitreous region of JNK3-/- mice. IB4 staining of eye sections in representative p5 JNK3+/+ and JNK3-/- pups; scale bars: 500µm. IB4: red; DAPI: blue. Insets are enlarged in panel d. (d) The retrolental mass in JNK3-/- mice is stained with IB4 (red), p-EphrinB (green) and DAPI (blue). Arrowheads: p-EphrinB+/IB4+ cells; arrows: p-EphrinB-/IB4+ cells; scale bars: 50µm. (e) Retrolental mass from a JNK3-/- mouse at p5 contains NG2+ pericytes (cyan) and F4/80+ macrophages (green) within an IB4+ vascular-type structure (red). A littermate JNK+/+ mouse displays no retrolental mass; hyaloid vessels are located on the lens. Images are from sections of the entire eyeball. Scale bars: 50µm. (f) The retrolental mass of JNK3-/- mice is Cleaved caspase3-negative. Cleaved Caspase3: green; IB4: red; DAPI: blue. Arrowheads: Cleaved Caspase3+/IB4+ cells; arrows: Cleaved caspase3-/IB4+ retrolental mass. Scale bars: 50µm. (g) A pathological retrolental mass is detected in the eye of a 16 week-old JNK3-/- mouse but not in a JNK3+/+ littermate (scale bars: 1mm); inset is magnified on the right (scale bar: 500µm); tissues were stained with colloidal Comassie G-250. (h) Phase contrast imaging of the unstained or DAPI-stained (blue) retrolental mass shown in g; scale bar: 100µm.