Figure 7.

Six3 is a direct activator of Pax6 and most likely of Sox2 lens lineage expression. (A) Bacterially expressed Six3 protein (full-length) and [³²P]-labeled Pax6 EE, Pax6 SIMO, Sox2 N3, and Sox2 N4 enhancer fragments were subjected to EMSA. Six3-bound DNA fragments are indicated by solid arrowheads and super-shifted bands (following addition of anti-Six3 antibody), by open arrowheads. The binding specificity was determined by the ability of the complex to be competed by 400 × excess of nonradioactive probes. Position of free probe is indicated by the solid arrows. (B) Six3 protein was enriched in selected regions of the Pax6 EE, Pax6 SIMO, and Sox2 N4 enhancers in vivo. Chromatin prepared from 11- to 17-somite stage heads and trunks was used for the ChIP assays and real-time PCR analysis. For Pax6 EE, Pax6 SIMO, and Sox2 N4 enhancers, the calculated ratio when using the heads as a DNA source was significantly higher (>5) than the ratio obtained when using the trunks (1.2–2.97), a result indicating that Six3 protein was bound to those regions. For the Sox2 N3 enhancer, the ratio for the head (1.52) was relatively similar to that for the trunk (1.2–2.97), suggesting that Six3 protein was not bound to that region, at least at that particular stage. (C) Luciferase transcriptional reporter assays showed that Six3 activates Pax6 lens lineage expression in the context of the Pax6 EE. The pCAB-Six3 (blue bars) plasmid activated the Pax6 lens lineage reporter plasmid (Pax6-EE-luc); pCAB-Six3VP16 (green bars) enhanced this activation, and pCAB-Six3EnR (red bars) attenuated it. Each bar represents six replicates (±s.d.). (D) Luciferase reporter assay also showed that Pax6 EE regulates Pax6 lens lineage expression by Six3. Pax6-ΔEE-luc (red bars) attenuated the activation of luciferase activity (blue bars). Each bar represents six replicates (±s.d.). (E) Six3 might also regulate Sox2 lens lineage expression. The pCAB-Six3 slightly increased RL-TK-N4 reporter activity (green bars), and slightly affect that of RL-TK-N3 (red bars), as compared to the activity of the control RL-TK reporter (blue bars). Each bar represents four replicates (±s.d.). (F) Electroporation of pCAB-Six3-IRES-GFP into the SE of chick embryos at HH8 stage (4- to 5-somite stage) specifically expanded the Pax6 expression domain only on the electroporated side (arrow). (G) Proposed gene-regulation network for lens cell fate specification. Solid arrows indicate direct regulation, and dashed arrows indicate potential direct regulation that has not yet been proven.