Genetic requirement for Prep1 in lens induction. (A) Whole-mount control or Prep1^i/− mutants at E10.5, E12.5, and E14.5. The inset shows high magnification of eye region (boxed). Arrows show the absence of eyes. (B) Sections through E12.5 control or Prep1^i/− mutant eyes stained with hematoxylin and eosin to show histology, or stained with antibodies to Foxe3, γ-crystallin, or Pax6 as indicated. (C) Sections through E9.5 or E10.5 control or Prep1^i/− mutant embryos stained with antibodies for Pax6 or Foxe3. Arrows indicate presumptive lens ectoderm, and arrowheads indicate nonlens head ectoderm. Bars: B,C, 100 μm. (l) Lens; (r) retina.

Prep1 is required for Pax6 EE activation and binds multiple highly conserved sites upstream of Pax6. (A) Whole-mount GFP visualization of E10 control or Prep1^i/− mutant eyes expressing the P0-3.9-GFPCre transgene, or section through the pancreas of the same embryo stained with antibodies for Pdx1 and GFP. Dotted line marks the optic vesicle. (B) Logo of the Prep1 DNA-binding site motif (Berger et al. 2008), with a box showing the CTGTCA core sequence. (C) Schematic of the Pax6 P0-3.9 genomic region used in the transgenic mouse line. P0 box represents the location of the Pax6 P0 promoter. Shown in the University of California at Santa Cruz Genome Browser view are PBM scores (enrichment score, ≥0.37) from a sliding 8-bp window. Hits in the minimal essential region of the EE (cyan) or Pancreas enhancer (magenta) are labeled L1, L2, and P1; sequences are shown boxed below. Red nucleotides indicate divergence from the boxed CTGTCA core sequence.

L1 and L2 are essential lower-affinity Prep1-binding sites. (A) SPR dose response curves for GST-Prep1 on L1, L2, or P1. (B) Kd values of Prep1 for L1-, L2-, and P1-binding sites and fold change in affinity relative to the P1 site (numbers >1 indicate decreased affinity) as determined by SPR and PBM data. (C) Whole-mount view of β-galactosidase-stained E10.5 embryos expressing wild-type P0-3.9-L1L2 or P0-3.9-L1ΔL2 transgenes. The arrow points to the lens, while the arrowhead points to the pancreas. A summary is shown for Pax6 reporters lacking either L1 or L2 sites (21) or genetically deficient for Prep1 as assayed at E10.5. Only nucleotides that diverge from the wild-type EE sequence are shown. Lightly shaded L1 or L2 boxes indicate lower-affinity sites; white boxes indicate ablated binding sites. Binding site ablations for L1 and L2 are genetically indicated with Δ for deletion, although they are specific point mutations that have been tested by PBM and biochemical analyses to have no detectable Prep1 binding. Activity indicates relative β-galactosidase (name in blue) or GFP (name in green) reporter activity in the lens.

L1 and L2 interact synergistically to activate the EE. (A) Summary and examples of Pax6 reporter constructs with mutations in L1 or L2 sites that create high-affinity binding sites (asterisks and black boxes). The ratio indicates the number of transgenic embryos with lens β-galactosidase staining as a fraction of all transgenic embryos. Activity indicates relative β-galactosidase reporter activity in the lens. (B) Modeling of EE activation versus Prep1 concentration (log₂ scale) for L1L2 modeled with synergy (black solid line) or without synergy (black dashed line), single-site mutations (ΔL1L2, L1ΔL2, gray dashed line), or high-affinity site mutations (ΔL1L2*, L1*ΔL2, gray solid line; L1*L2*, blue solid line). (C) Ratio of activation levels for reporter constructs modeled in B to facilitate the comparison of model predictions and the ratio of relative reporter levels. Single high-affinity site mutations (L1*ΔL2) versus wild-type (L1L2) reporter modeled with synergy (black line) or single-site ablation (L1ΔL2, gray line); double high-affinity site mutation (L1*L2*) versus wild-type (L1L2) reporter modeled with synergy (red line). We conservatively estimate the ratio differences between L1*ΔL2 and L1ΔL2 to be at least twofold, between L1L2 and L1*ΔL2 to be at least fourfold, and between L1*L2* and L1L2 to be at most 1.3-fold. These estimates are based on β-galactosidase staining intensities and histochemical development times. The shaded area defines the predicted range of physiological concentration for Prep1. (D) β-Galactosidase staining of an eye from an E9.5 L1*L2* transgenic embryo was more intense than from wild-type (P0-3.9-L1L2) embryos of increasingly older ages up to E10.25.