DEV031872 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. MAP2K protein sequence comparison. (A) Alignment of the murine MAP2K1 and MAP2K2 protein sequences. The boxed sequences are divergent between MAP2K1 and MAP2K2. Green and red bold residues can be phosphorylated and their phosphorylation influences MAP2K activity positively or negatively, respectively. Both proteins contain the two serine residues in the activation loop at positions 218 and 222 (green), and the serine residue at position 212 (red) that regulates negatively the activity of the MAP2Ks (Gopalbhai et al., 2003). Both threonine residues at positions 286 and 292 (red), which are involved in negative regulation and are phosphorylated by CDK5 and ERK, respectively, are missing in Map2k2 (Sharma et al., 2002). The threonine 386 (red), phosphorylated by ERK and also involved in negative feedback, is present in Map2k1 and Map2k2 proteins (Brunet et al., 1994). Finally, serine 298 (green), phosphorylated by PAK1, is present in both Map2ks, even though only Map2k1 is a PAK1 substrate (Frost et al., 1997; Park et al., 2007). (B) Alignment of MAP2K1 protein sequences from mouse, human and frog. The boxes indicate the sequence divergence between MAP2K1 and MAP2K2. The '*', ':' and '.' symbols under the sequence indicate identical residues, or conserved and semi-conserved substitution, respectively. Protein sequence alignment was performed with CLUSTAL W (1.83) multiple sequence alignment software.

• Supplemental Figure S2 -

  Fig. S2. Normal development of the labyrinth region in Map2k1^+/− placentas. Hematoxylin and Eosin staining of E12.5 wild-type and Map2k1^+/− placenta sections (n=7). The development of the Map2k1^+/− placenta is undistinguishable from that of wild-type specimens. Scale bars: in A, 100 µm for A,C; in B, 50 µm for B,D.

• Supplemental Figure S3 -

  Fig. S3. Efficient deletion of the Map2k1^flox allele in embryonic tissues by the Sox2Cre transgene. Map2k1^flox/floxMap2k2^−/−×Tg^Sox2Cre intercrosses were used to generate the E12.5 Map2k1^+/floxMap2k2^+/−Tg^Sox2Cre specimens analyzed for Fig. 7G. To genotype the specimens by Southern (A) or PCR (B) analyses, DNA was isolated from yolk sac (A) and embryo (B). A representative litter is shown. (A) Yolk sac DNA was digested with StuI, BamHI or EcoRV, and analyzed by Southern blot with probes specific for Map2k1, Map2k2 or SoxCre, respectively. The position of the expected fragments is indicated on the right side of each panel. The yolk sac DNA sample for specimen #4 was lost during the purification. (B) Embryonic DNA was also used for embryo genotyping by PCR. DNAs from Map2k1^+/flox and Map2k1^Δ/Δ mice were used as controls. The position of the respective alleles is indicated on the right side. This breeding only produced double heterozygotes (Map2k1^+/floxMap2k1^+/−). Those carrying the Sox2Cre transgene had embryonic tissues that were Map2k1^+/ΔMap2k2^+/−, whereas the yolk sac, composed of embryonic and extra-embryonic tissues, was Map2k1^+/flox/Δ. This confirms that, as expected, Sox2Cre deleted completely the Map2k1^flox allele in the embryo, but not in the extra-embryonic structures, which have a dual origin.