DEV041202 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Distribution of pHH3-positive cells. (A-F) pHH3 is green and DAPI is blue. (A,B) The morphology of the OVs and distribution of pHH3 are similar in control (A) and Lhx2^−/− (B) embryos at E8.75. (C,D) At E9.0, the control optic neuroepithelium exhibits pHH3 throughout the optic neuroepithelium and adjacent dorsal diencephalon (C). In the mutant, pHH3-positive cells are fewer in the optic neuroepithelium (D). (E,F) The control optic neuroepithelium has a well-formed OC and pHH3-positive cells distributed along its entire extent at E10.25 (E). In the mutant, which fails to form OCs, pHH3-positive cells are few in number and clustered at the distal tip (F). (G) Quantification of the proportion of pHH3-positive cells out of the total number of cells (DAPI positive). Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Lens and OC formation are normal in Tg(Le-cre); Lhx2^f/− mice. (A) X-Gal staining is not detectable in E9.25 control OC, which contains an allele of the lacZ cDNA in the Rosa26 locus, but lacks Cre expression. (B) X-Gal staining is predominantly confined to the lens placode region of the E9.25 surface ectoderm when Cre is expressed from the Le enhancer of Pax6. (C) LHX2 expression in the E10.5 control OC. (D) LHX2 expression is unaffected by Cre deletion of the Lhx2 gene in the lens ectoderm. (E,F) DAPI staining in control (E) and lens ectoderm-specific Lhx2 inactivation (F) OCs reveal no effect on OC development. Scale bars: 50 µm in A for A,B; 100 µm in C for C-F.

• Supplemental Figure S3 -

  Fig. S3. Expression of dorsal diencephalon markers is unchanged in Lhx2^−/− embryos. (A-D) Wnt3a mRNA expression patterns in E9.5 control (A) and Lhx2^−/− (B) embryos. Lines in A and B indicate the location and planes of section shown in C and D. (E,F) Axin2 mRNA expression patterns in E9.5 control (E) and Lhx2^−/− (F) embryos. (G-L) Dbx1 mRNA expression patterns in E9.5 control (G) and Lhx2^−/− (H) embryos. Lines in G and H indicate locations and planes of section for I,K and J,L, respectively. Scale bars: 500 µm in A for A,B,G,H; 100 µm in C for C-F,I-L.

• Supplemental Figure S4 -

  Fig. S4. Fgf15 is not detected in the Lhx2^−/− optic neuroepithelium by whole-mount in situ hybridization. (A-H) Fgf15 mRNA is detected in the control OV and OC at E9.0 (A,C) and E10.0 (E,G), but is not detected in Lhx2^−/− optic neuroepithelium at either stage (B,D,F,H). Arrows point to optic tissues. (A,B,E,F) Lateral views; (C,D,G,H) frontal views. Scale bar: 500 µm.

• Supplemental Figure S5 -

  Fig. S5. Mosaic reporter activity in E10.5 optic neuroepithelium of Hes1^creERT2/+ mice. (A-D′) β-gal immunoreactivity on sections obtained from embryos exposed to TM at E8.5. (A) Control embryo (wild-type at Hes1 locus). Positive immunoreactivity outside of optic neuroepithelium is due to cross reactivity with secondary antibody (data not shown). (B) Examples of β-gal-positive cells (arrowheads) in neural retina. (B′) Magnified view of positive cells in B. (C) Examples of β-gal-positive cells (arrowheads) in retinal pigment epithelium (within dashed lines). (C′) Magnified view of positive cells in C. (D) Examples of β-gal-positive cells (arrowheads) in optic stalk. (D′) Magnified view of positive cells in D. Scale bars: 100 µm in A for A-D and in B′ for B′-D′.

• Supplemental Table S1 -