DEV01524 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Dorso-ventral patterning in the telencephalon of mice harboring mutations in the distinct DNA-binding domains of Pax6. Micrographs of coronal vibratome sections of the lateral telencephalon stained for Gsh2 in WT (A,C) and the PD mutant Pax6^Aey18–/– (B) or HD mutant Pax6^4Neu–/– (D). Note that Gsh2-immunoreactivity is restricted to the ganglionic eminence (GE) of the ventral telencephalon, in WT (A,C), while it expands ectopically into the cortex (CTX) in the PD mutant Pax6^Aey18–/– (B), but not in the HD mutant Pax6^4Neu–/– (D). CTX, cortex; GE, ganglionic eminence. Scale bars: 100 mm.

• Supplemental Figure 2 -

  Fig. S2. Expression of the Pax6 downstream target gene Ngn2 in the caudo-medial cortex of mice harboring mutations in the distinct DNA-binding domains of Pax6. Micrographs of coronal sections of the caudo-medial cortex from embryonic day (E) 14 mice immunostained for Ngn2 (red) and Pax6 (green). Note that Ngn2-immunoreactive cells are a subset of Pax6-positive cells that are severely reduced, but not completely absent in the medial cortex of the PD mutant Pax6^Aey18–/– (B) and in the functional null allele Pax6^Sey–/– (E) compared to WT (A). Normal numbers of Ngn2-positive cells persist in the cortex of Pax6(5a)–/– (C) and the HD mutant Pax6^4Neu–/– (D). The dashed white line (A,B,C,E) indicates the ventricular surface. CTX, cortex; HC, hippocampus. Scale bar:100 mm.

• Supplemental Figure 3 -

  Fig. S3. Expression levels of Pax6 isoforms in the cortex of WT and Pax6(5a)–/– embryos. (A) A schematic drawing of the Pax6 and Pax6(5a) (yellow insert into paired domain, PD) mRNA with the position of the primers used for the detection of all Pax6 isoforms containing a PD (red) and the Pax6(5a) isoform (orange). (B) The expression levels obtained by real-time RT-PCR analysis of Pax6 and Pax6(5a) mRNA relative to GAPDH in tissue isolated from cortex (ctx) of WT or Pax6(5a)–/– mice at embryonic day 10, 11, and 12. RNA was isolated with the quiagen RNeasy Kit. cDNA synthesis was done with the invitrogen cDNA Synthesis Kit. Real time RT-PCR was performed as previously described by (Hack et al., 2004). Note that the expression of the canonical form of Pax6 (=all isoforms of Pax6 (red) minus Pax6(5a) isoform) is increased in the Pax6(5a)–/– cortex compared to WT levels 1,8 fold at E10 and 1,4 fold at E11 and 12. This increase may  therefore be sufficient to compensate the loss of Pax6(5a) mRNA that comprises only 15-25% of the total Pax6 containing a PD mRNA in WT cortex. (The following primers were used: Paired forward, 5¢-CAGCTTGGTGGTGTCTTTGT; Paired reverse, 5¢-GCAGAATTCGGGAAATGTCG; Splice forward, 5¢-GCAGATGCAAAAGTCCAGGT; Splice reverse, 5¢-CTCGTAATACCTGCCCAGAA; GAPDH sense, 5¢-ATTCAACGGCACAGTCAAGG; and GAPDH antisense, 5¢-TGGATGCAGGGATGATGTTC).