Sox2 and Pou2f1 interact to control lens and olfactory placode development

Abstract

Sox2, which encodes an SRY-like HMG box transcription factor, is critical for vertebrate development. Sox2 mediates its transcriptional effects through the formation of complexes with specific co-factors, many of which are unknown. In this report, we identify Oct-1, encoded by the Pou2f1 gene, as a co-factor for Sox2 in the context of mouse lens and nasal placode induction. Oct-1, Sox2, and Pax6 are co-expressed during lens and nasal placode induction and during subsequent developmental stages. Genetic combination of Sox2 and Pou2f1 mutant alleles results in impaired induction of the lens placode, an ocular phenotype that includes anophthalmia, and a complete failure of nasal placode induction. These ocular and nasal phenotypes closely resemble those observed in Pax6 null embryos. Moreover, we identify DNA-binding sites that support the cooperative formation of a complex between Sox2 and Oct-1 and mediate Sox2/Oct-1-dependent transactivation of the Pax6 lens ectoderm enhancer in vitro. We demonstrate that the same Sox- and Octamer-binding sites are essential for Pax6 enhancer activity in the lens placode and its derivatives in transgenic mouse embryos. Collectively, these results indicate that Pou2f1, Sox2 and Pax6 are interdependent components of a molecular pathway utilized in both lens and nasal placode induction.

FIG. 1

Pax6, Sox2, and Pou2f1 are broadly co-expressed in anterior pre-placode stage E8.5 embryos. Whole mount in situ analysis of Pax6 (A–B), Sox2 (C–D) and Pou2f1 (E–F) in E8.5 embryos displayed in a frontal view (A, C, E), lateral view (B, D, F), or in cross section (E′). The white line in E indicates plane of section for E′. Abb: nf neural folds; ov optic vesicle; se surface ectoderm. Scale bars: 100 microns in all figures.

FIG. 2

Pax6 precedes Sox2 and Oct-1 in the PLE. Immunofluorescence for Pax6 (red, A–C), Sox2 (green, D–F), and Oct-1 (red, G–I) are shown for embryos at the 12-somite (s) (A, D, G), 15s (B, E, H), or 17s stages (C, F, I). Arrows highlight nuclei positive for Sox2 or Oct-1 expression in the PLE. Abb: OV optic vesicle; PLE presumptive lens epithelium.

FIG. 3

Sox2, Pax6 and Oct-1 are co-expressed during lens placode induction and early eye morphogenesis. Immunofluorescence and nuclear co-localization (DAPI-blue) of Pax6 (red, A, C, D, F), Sox2 (green, B–C, E–F), and Oct-1 (red, G–I) at E9.5 (A–C, G), E11.5 (D–F, H) and E13.5 (I) in the ocular region. (For Oct-1 antibody controls, see Fig. S3). Abb: ael anterior epithelial layer; fc fiber cell compartment; lp lens placode; lv lens vesicle; nr neural retina; ov optic vesicle.

FIG. 4

Sox2, Pax6 and Oct-1 are co-expressed in the nasal placode, nasal epithelium and vomero nasal organ (VNO). Immunofluorescence and nuclear co-localization (DAPI, blue, D–F) of Pax6 (red, A, D) (green G, J), Sox2 (green, B, E, H, K), and Oct-1 (red, C, F, I, L) at E9.0 (AC), E10.5 (D–F) and E12.5 (G–L). Abb: ne nasal epithelium; np nasal placode; npt nasal pit; p-lp pre-lens placode; vno vomero nasal organ.

FIG. 5

Compound Oct1^−/−, Sox2^+/− mutant embryos display severe ocular defects. Histological analyses of E10.5 wild type (A), Oct1^−/− (B), Oct1^−/−, Sox2^+/− (C, F), Pax6^Sey/+ (D) and Pax6^Sey/Sey (E) embryos. Histological analyses of E12.5 Oct^+/−, Sox2^+/− (G) and Oct1^−/−, Sox2^+/− (H–I) embryos. * in panel C indicates lens primordium (see Fig. 6) and * in panel I indicates a lack of lens tissue (see Fig. S4 for marker analyses). Abb: ael anterior epithelial layer; fc fiber cell compartment; pt lens pit; nr neural retina; ov optic vesicle; se surface ectoderm.

FIG. 6

Lens placode induction fails in severely affected Oct1^−/−, Sox2^+/− embryos. Immunofluorescence of E10.5 Oct1^−/− (A–C), Pax6 ^Sey/+(D–F), Oct1^−/−, Sox2^+/− (G–L), and Pax6 ^Sey/Sey (M–O) embryos for Pax6 (green, A, D, G, J, M), E-cadherin (red, B, E, H, K, N), and Sox2 (green, C, F, I, L, O). DAPI nuclear stain is blue. Circles indicate small lenses. * in panels J and M indicate non-specific Pax6 antibody staining in embryos that have lost Pax6 expression. Arrowheads in panels L and O indicate the domain devoid of Sox2 expression in the distal OV, while the arrows in panels J–L indicate the surface ectoderm. Abb: pt lens pit; nr neural retina; ov optic vesicle; se surface ectoderm.

FIG. 7

Nasal placode induction fails in Oct1^−/−, Sox2^+/− mutant embryos. (A–D) Photographs of the heads of E10.5 wild type (A), or Oct1^−/−, Sox2^+/− mutant embryos (B–D). (E) Diagram illustrating inappropriate location of a small lens placode in an Oct1^−/−, Sox2^+/− embryo between the normal sites of lens and nasal placode formation. Immunofluorescence for Pax6 (red, F) and Sox2 (green, G) overlaid with DAPI (blue, F–G) in Oct1^−/− E10.5 nasal pits. Immuno-fluorescence for E10.5 Pax6 (red, H–I), Sox2 (green, J–K), and E-cadherin (red, J–K) in Pax6^Sey/Sey (H, J) and Oct1^−/−, Sox2^+/− (I, K) embryos. DAPI nuclear stain is blue (H–J). (L–O) Epithelial derivatives of the nasal placode are absent at E12.5. E-cadherin (red, L–O) immunofluorescence is shown with DAPI nuclear stain for Oct1^+/−, Sox2^+/− (L), Pax6^Sey/+ (M), Oct1^−/−, Sox2^+/− (N), and Pax6^Sey/Sey (O) embryos. Abb: lp lens placode; ne nasal epithelium; npt nasal pit; tg tongue; vno vomero nasal organ.

FIG 8

Sox2 and Oct-1 proteins interact cooperatively with the Pax6 EE. (A) Schematic representation of Pax6 showing the two major promoters (P₀, P₁), the 5′ ectoderm enhancer (EE) and the 3′ SIMO lens enhancer. (B) Purified Sox2-HMG protein forms monomeric and dimeric protein-DNA complexes when titrated onto radiolabeled EE-3 (for sequence, see Table 1B) in EMSA (lanes 1–16). Purified Oct1-GST forms a protein-DNA complex with EE-3 (Oct1). This complex is diminished and super-shifted (SS) by an Oct-1 specific antibody (lanes 18–19) but unaffected by a Brn3a antibody (lane 20). (C) Titration of Oct1-GST onto EE-3 in the absence (lanes 1–8) or presence of constant amounts of Sox2-HMG protein (lanes 9–15). Shifts due to Oct1-GST binding (Oct1), Sox2-HMG (Sox2) and the co-complex (Oct1/Sox2) are indicated.

FIG. 9

Oct-1 and Sox2 synergistically transactivate the Pax6 EE in epithelial cells. Fold induction of luciferase activity in the presence of increasing concentrations of plasmids encoding Sox2 (a 100ng, b 200ng, c 400 ng) and Oct-1 (a 200 ng, b 400 ng, c 800 ng), in isolation or together on EE109, SOXX, OCTX, IR, and DR luciferase reporter constructs in 293t epithelial cells.

FIG. 10

Sox and Octamer DNA binding elements are required for the in vivo transcriptional activity of the Pax6 EE. (A) Schematic representation of transgenic constructs utilized. The Pax6 EE directs expression of LacZ through the TATA element of the human β-globin (βg) gene. (B) Whole mount photo of EE526 transgenic embryo at E10.0 and representative sections of E12.5 embryos transgenic for EE526, SOXAM4, SOXBM4, OCTPM, and OCTHM. The arrows indicate the lens at E10.0, the non-lens epithelium of EE526, SOXAM4 AND SOXBM4 sections and the AEL of OCTPM and OCTHM images.