Figure 5.

The LMO4-Peak is activated by the Isl1-Lhx3 complex. A, HxRE-Long and HxRE-Short sequences identified by ChIP-Seq de novo motif analysis, and the HxRE-Long and HxRE-Short sequences in the LMO4-Peak. B, LMO4-Peak HxRE-Long mutant used for GFP-reporter experiments. C, GFP-reporter experiments for LMO4-Peak variants. Embryos were electroporated with LMO4-Peak-GFP reporter constructs plus ubiquitously expressing LacZ to mark electroporated cells. Sections were immunostained for GFP and Mnr2 to mark MNs. Images are representative of electroporations from multiple embryos. LMO4-Peak-wt: n = 13, LMO4-Peak-ΔHx-L: n = 12. D, Embryos electroporated with LMO4-Peak-GFP reporter construct plus Isl1-Lhx3 fusion protein construct. Sections were immunostained for GFP and Mnr2. Images are representative of electroporations from multiple embryos. LMO4-Peak-wt: n = 4, LMO4-Peak-ΔHx-L: n = 5. E, Luciferase assays testing LMO4-Peak-wt and mutants. Luciferase assays performed in cultured P19 cells. Results show the luciferase activation fold upon the addition of Isl1 plus Lhx3, compared to empty vector; n = 5 independent experiments. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis, comparing each reporter construct to control reporter (no enhancer). **p < 0.01. Error bars represent the SEM.