JAGGED1-NOTCH regulates RPE proliferation and specification.

<p>(<b>A–F</b>) Optic cups of the indicated genotypes were harvested at the indicated times and stained for JAGGED1. Note that in wild type and all indicated mutants, JAGGED1 is expressed in the dorsal distal retina and in (<b>F</b>) also in the E12.0 ventral distal retina and RPE (arrow), in addition to its prominent expression in the lens vesicle. Also note that a dorsal RPE subdomain in <i>Mitf</i> single or <i>Vax1/2/Mitf</i> triple mutants expresses JAGGED1 at both E10.5 and E12.0. (<b>G</b>) Western blots for the indicated proteins from wild-type OV cultures eight hours after exposure to human FGF2 and/or MEK1/2 inhibitor (MEKi). Note decrease in the level of JAGGED1 in the presence of MEKi. (<b>H–J</b>) JAGGED1 expression in sections of E10.5 <i>Mitf ^−/−</i> optic cups 36 hours after exposure to control beads (<b>H</b>, marked by o), MEKi bead (<b>I</b>, marked by +) or after co-implantation of an FGF2 bead (<b>J</b>, marked by *) and a bead coated with both FGF2 and MEKi (<b>J</b>, marked by +). Note that these are horizontal sections and that FGF2 and FGF2+MEKi have differential effects within the same OV culture. (<b>K–N</b>) Effects of antibody neutralization of JAGGED1 on VSX2 expression in <i>Vax2^−/−</i>;<i>Mitf ^−/−</i> cultures. Compared to control antibodies (<b>K</b>), anti-JAGGED1 antibodies modestly increased VSX2 expression in the RPE (<b>L</b>) and further increased it when the beads were double-coated with FGF2 (<b>N</b>). Note that the anti-JAGGED1 effect in (<b>L</b>) is seen only in a subdomain of the RPE, consistent with the fact that JAGGED1 expression was confined to the thickening portion of the <i>Mitf</i> single and <i>Vax/Mitf</i> compound mutant RPE. (<b>O</b>) Quantitation of VSX2-positive cells in optic cup cultures. Box plots show minimal, 25^th percentile, median, 75^th percentile and maximal percentages of VSX2-positive cells in the RPE per section (one section per embryo, and 8–9 embryos for each treatment). Significance determined by Student’s <i>t-</i>test: p<0.01 for control versus FGF2, and FGF2 versus FGF2+anti-JAGGED1; p<0.001 for control versus anti-JAGGED1, anti-JAGGED1 versus FGF2+anti-JAGGED1. (<b>P–Y</b>) Wild-type whole embryo cultures after 36-hour exposure to DMSO or NOTCH antagonist γ-secretase inhibitor 1. Note that under control conditions, RPE markers MITF (<b>P</b>) and TYROSINASE (TYR)(<b>Q</b>) were normally expressed, the numbers of phosphorylated histone H3 (p-H3) positive (<b>R</b>) and Ki67 positive (<b>S</b>) cells in the RPE were low, and JAGGED1 expression (<b>T</b>) was normal in the distal future retina. After γ-secretase 1 incubation, however, MITF and TYR expression was greatly reduced, the number of proliferative cells was elevated, and the expression territory of JAGGED1 expanded into the distal RPE (<b>U–Y</b>). Scale bar: 80 µm (<b>A–F</b>), 60 µm (<b>H–N</b>), 25 µm (<b>P–Y</b>).