DEV032714 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Figure S1 -

  Fig. S1. ES-cell targeting of MFCS4. (A) Homologous recombination was used to replace a 999 bp fragment of mouse MFCS4 with a neomycin (Neo) cassette. (B) The p1 and p2 PCR primers were used for screening recombined ES cell clones and genotyping germline chimeric mice. Two ES cell lines were transmitted to germ lines. (C) Genotyping of progeny from the germline chimeric mice was carried out using a mixture of three primers: p2, p3 and p4.

• Supplemental Figure S2 -

  Fig. S2. Reporter expression in MFCS3 transgenic embryos. (A) X-gal staining of a MFC3 transgenic embryos at E11.5. Signal is detected in the brain and motor neurons (mn). (B) Cross-section of A at the level of ventrolateral cord (vc) and spinal roots (sr).

• Supplemental Figure S3 -

  Fig. S3. Sequence analysis by evolutionary rigidity assay (ERA). ERA was performed focusing on a 217 bp (nucleotide 571-787) sequence in MFCS4, which is conserved from mammals to teleost fish medaka (Fig. 1B). Evolutionary conserved sequences named NPMMs (nearly perfect match motifs) were extracted by previously reported method considering phylogenetic relationship (Sumiyama et al., 2001). Here, NPMM is defined as a region covered by consecutive 6 bp windows with one or no nucleotide substitution along total phylogenetic tree branches of all sequences compared. Vertebrate species (human, rhesus macaque, marmoset, mouse, rat, guinea pig, horse, cow, dog, opossum, platypus, chicken, lizard, frog, medaka, fugu, tetraodon, stickleback and zebrafish) were used to extract four NPMMs (MFCS4-MF-A, B, C, D).

  Reference

  Sumiyama, K., Kim, C. B. and Ruddle, F. H. (2001). An efficient cis-element discovery method using multiple sequence comparisons based on evolutionary relationships. Genomics 71, 260-262.

• Supplemental Figure S4 -

  Fig. S4. Activation of Meis/Pbx, Sox and Wnt and Notch signaling pathways in the epiglottis and arytenoid swelling. Upper left insets are images of the lacZ reporter signals in the epiglottis and arytenoid swelling at E13.5 (left) and the dissected tissue of the epiglottis and arytenoid swelling, which was used for the RT-PCR (right). Results of RT-PCR of the Meis/Pbx, Sox, Wnt and Hes family genes showed that the many genes are expressed in the epiglottis and arytenoid swelling as well as the tongue and lung.