DEV003855 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Posterior pharyngeal phenotype of Sox2^EGFP/LP compound mutants. In the tongue at P2 (A), Sox2EGFP is expressed in the circumvallate papilla (cv) and lingual gland ducts (arrowheads) of the intact organ (anterior to the bottom) and in a cryosection through the lingual glands (lg) (B). (C) Histology of wild-type P15 posterior pharynx to indicate the location of palatine and lingual glands, soft palate and epiglottis. SMG, submucosal glands of the upper trachea. In the hypomorphic mutants note the absence of lingual glands (D,E), palatine glands (F,G) and the presence of circumvallate papilla but without branched Ebner’s glands (H,I). Note also the abnormal position of the mutant epiglottis relative to the soft palate in I. Scale bar: 50 μm.

• Supplemental Figure S2 -

  Fig. S2. Phenotype of Sox2 hypomorphic mutant esophagus. (A) Fluorescence microscopy of foregut of Sox2^EGFP/COND mutant without TEF. This shows a narrowing of the esophagus, a condition known as isolated esophageal atresia (Brunner and van Bokhoven, 2005). (B,C) Ectopic expression of mucin-producing and ciliated cells in the distal esophagus (fistula) of Sox2^EGFP/COND mutant with TEF. Note ciliated cells marked by beta-tubulin staining and Alcian blue-positive cells in the monolayer epithelium (here, black). The section also shows a region in which the epithelium is multilayered (asterisk). Nuclei counterstained with DAPI. Scale bar: 50 μm. fi, fistula. (D) Western blot analysis of Sox2 protein levels in E15.5 esophagus of mutants of different genotypes. Control sample is from limb bud mesenchyme that does not express Sox2.

• Supplemental Figure S3 -

  Fig. S3. Epithelial proliferation in conditional mutants. E18.5 embryos from a cross between Sox2^EGFP/+ and Sox2^COND/COND mice were labeled in utero with BrdU for 1 hour. Sections were stained with antibody to BrdU and the number of positive nuclei/total nuclei in the epithelium counted from a total of 22-40 sections from three independent embryos of each class. Note that in Sox2^COND/+ esophagus most of the labeled nuclei are in the basal layer of the stratified epithelium, whereas in the Sox2^EGFP/COND esophagus of embryos without TEF there are apparently more positive nuclei in the suprabasal layers. Proliferation in the Sox2^EGFP/+ trachea was determined to compare with proliferation in mutant fistula. Scale bar: 50 μm.

• Supplemental Figure S4 -

  Fig. S4. Abnormal morphology of the forestomach of Sox2^EGFP/LP compound mutants. (A,B) Fluorescence microscopy of intact stomach from wild type and compound mutant at E18.5. (C,F) Wild-type forestomach from two different embryos/pups showing stratified squamous keratinized epithelium. (D,E,G,H) Sections through the forestomach of two different compound mutants. (E) An enlargement of the region boxed in D. Arrowheads indicate regions where the epithelium is columnar and has formed shallow pits within the mesenchyme. (I,J) Posterior stomach from wild type and mutant showing no significant change in the latter. Scale bar: 50 μm.