DEV02764 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Autoregulation of Pax6 demonstrated with the YAC reporter. (A-F) All embryos carry the YAC reporter and express GFP. (A,B) Comparison between E11.5 Pax6^+/+ and Pax6^−/− embryos: absence of functional Pax6 protein causes upregulation of expression from the reporter transgene. (C,D) Comparison between E10.5 embryos with and without the PAX77 transgene: overexpression of PAX6 causes a strong reduction in GFP expression from the reporter transgene, in particular in the telencephalon. (E,F) Comparison between E17.5 brains dissected from embryos with and without the PAX77 transgene: both dorsal (E) and ventral (F) views show a dramatic reduction of GFP expression in the cortex and olfactory bulbs.

• Supplemental Figure 2 -

  Fig. S2. Pax6 overexpression does not affect early neurogenesis. (A) Coronal sections through the cortex of wild-type and PAX77^+/+ E12.5 embryos stained for the expression of β-tubulin III (green) and counterstained with TOPRO3 (blue). Scale bar: 50 μm. (B) Percentages of cells expressing β-tubulin III in the rostral (RCx), middle (MidCx) and caudal (CCx) cortex of E12.5 wild-type and PAX77⁺ embryos, as determined by flow cytometry. No significant difference was found between PAX77 and wild-type embryos.

• Supplemental Figure 3 -

  Fig. S3. Pax6 overexpression does not alter the topography of thalamocortical axonal connections. (A) DiI (red) and DiA (green) fluorescent carbocyanine dyes were injected, respectively, in the somatosensory and visual cortex of wild-type and PAX77 E18.5 embryos, and were left to diffuse for several weeks. (B,C) The panels show confocal images of the thalamic nuclei backlabeled by DiI and DiA on coronal sections through the brain. Normal topography is maintained in the mutants with no obvious differences between (B) the wild-type and (C) the PAX77 somatosensory and visual thalamocortical connections. dLG, dorsal lateral geniculate; VP, ventro-posterior geniculate. Scale bar: 220 μm.

• Supplemental Figure 4 -

  Fig. S4. Pax6^−/− embryos lack the olfactory placode signalling centre. Whole-mount in situ hybridization for Fgf8 on wild-type, Pax6^−/− and PAX77^+/+ embryos at E10.5. Although Pax6^−/− embryos retain the anterior telencephalic source of Fgf8 (black arrow), they lack the olfactory placodes (white arrow), which are a site of mesenchymal/epithelial induction, mediated by retinoic acid, Shh, Fgf8 and the BMPs. Scale bar: 1 mm.