DEV030544 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Sox2 mRNA expression in wild-type and Fst^−/− tongues. Analysis of Sox2 expression by ISH in wild-type and Fst^−/− mice at e18.5. In the tongue of Fst^−/− mice, invaginated patches of Sox2-expressing epithelial cells can be seen in both the anterior (Ant) and posterior (IE) regions. These patterns of Sox2 expression are similar to the patterns of X-Gal staining observed in the tongues of Sox2-βgeo and Fst^−/−; Sox2-βgeo mice (see Fig. 2, main text). We made this comparison because this Sox2 reporter allele (Avilion et al., 2003) was generated by gene targeting, and minor phenotypes have been observed in some, but not all, tissues that express only one allele of Sox2 (Bakrania et al., 2007; Kelberman et al., 2008; Que et al., 2007; Taranova et al., 2006). Since the phenotypes we observe in Fst^−/− tongue are the same whether or not they are on a Sox2-βgeo background, we conclude that Sox2 heterozygosity has no obvious effect on taste papilla/bud development, and have used the Sox2-βgeo allele as a Sox2 reporter throughout these studies. Asterisks denote artifacts from the ISH procedure. Scale bar: 20 µm.

  References

  Bakrania, P., Robinson, D. O., Bunyan, D. J., Salt, A., Martin, A., Crolla, J. A., Wyatt, A., Fielder, A., Ainsworth, J., Moore, A. et al. (2007). SOX2 anophthalmia syndrome: 12 new cases demonstrating broader phenotype and high frequency of large gene deletions. Br. J. Ophthalmol. 91, 1471-1476.

  Kelberman, D., de Castro, S. C., Huang, S., Crolla, J. A., Palmer, R., Gregory, J. W., Taylor, D., Cavallo, L., Faienza, M. F., Fischetto, R. et al. (2008). SOX2 plays a critical role in the pituitary, forebrain, and eye during human embryonic development. J. Clin. Endocrinol. Metab. 93, 1865-1873.

  Que, J., Okubo, T., Goldenring, J. R., Nam, K. T., Kurotani, R., Morrisey, E. E., Taranova, O., Pevny, L. H. and Hogan, B. L. (2007). Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development 134, 2521-2531.

  Taranova, O. V., Magness, S. T., Fagan, B. M., Wu, Y., Surzenko, N., Hutton, S. R. and Pevny, L. H. (2006). SOX2 is a dose-dependent regulator of retinal neural progenitor competence. Genes Dev. 20, 1187-1202.

• Supplemental Figure S2 -

  Fig. S2. Circumvallate and foliate papillae and lingual glands appear normal in Fst^−/− tongue. (A) Coronal sections through the posterior tongue of control and Fst^−/−; Sox-2βgeo tongues stained with X-Gal reveal no obvious changes in the circumvallate papilla (CV) or in the developing taste buds along the lateral edges of the CV (insets, Example 1). Scale bars: 20 µm, low magnification; 10 µm, insets. (B) Foliate papillae appear normal in Fst^−/−; Sox2-βgeo tongues. Scale bar: 20 µm. (C) Lingual serous glands are present in the posterior tongue, near the CV papilla, of Fst^−/− tongue and appear normal as compared with wild-type controls. Glandular epithelium is detected by ISH to Bmp7. Scale bar: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Keratin expression in wild-type and Fst^−/− tongues. Sagittal sections through wild-type and Fst^−/− tongues (e18.5) were stained with a wide-spectrum antibody to keratins (see Materials and methods); this antibody recognizes all keratins described as being expressed in tongue epithelium and taste papillae/buds e.g. keratins 5, 8, 19 (Luo et al., 2008; Mbiene and Roberts, 2003; Wong et al., 1994). Invaginated regions of lingual surface tissue, as well as ectopic 'taste papillae', immunostain with anti-keratins in anterior and posterior tongue, revealing the epithelial character of these cells. Scale bars: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Increased density of fungiform papillae on the anterior surface of Fst^−/− tongue. Tongues from e14.5 BAT-Gal and Fst^−/−; BAT-Gal mice, or e17.5 Sox2-βgeo and Sox2-βgeo; Fst^−/− mice, were stained with X-Gal and photographed. Images of X-Gal-stained tongues were converted to black and white, background noise was subtracted, and measurements were made in Adobe Photoshop 10.0. The number of fungiform papillae (defined as structures protruding from the surface of the epithelium that are >20 µm in diameter and stained with X-Gal) in the distal third of the tongue was counted for each tongue, and this number normalized to the surface area circumscribed by the counted papillae on the tongue dorsum (diagrams, lower panels, A and B). (A) There is a significant increase in the number of papillae/mm² along the tongue dorsum in Fst mutants: Control (BAT-Gal), 28.4±1.8 (s.e.m.) papillae/mm²; Fst^−/−; BAT-Gal, 47.7±3.8 (s.e.m.) papillae/mm²; P<0.024, Student’s t-test (Glantz, 2005). There is a higher concentration of papillae towards the medial sulcus. A total of three tongues of each genotype were analyzed. (B) Tongues from e17.5 Control (Sox2βgeo) and Fst^−/−; Sox2βgeo mice were analyzed as described above. In Fst mutant tongues there is a strong trend towards an increased number of papillae/mm², although this difference is not statistically significant: Control (Sox2βgeo), 37.3±1.3 (s.e.m.) papillae/mm²; Fst^−/−; Sox2βgeo, 45.3±4.9 (s.e.m.) papillae/mm². A total of three tongues of each genotype were analyzed.

  Reference

  Glantz, S. A. (2005). Primer of Biostatistics. New York: McGraw-Hill.

• Supplemental Figure S5 -

  Fig. S5. Bmp7 expression during tongue development. Sagittal sections through wild-type tongue showing expression of Bmp7 at different developmental ages. (A) At e11.5, Bmp7 is expressed in the epithelium of the developing tongue (asterisk, inset) where the first branchial arches fuse forming what will become the anterior two-thirds of the tongue. We also observed Bmp7 expression in the epithelium of what will become the pharyngeal tongue (arrow), consistent with previous reports of Bmp7 expression patterns (Krimm, 2007). (B) At e12.5, Bmp7 is expressed at low levels throughout the lingual epithelium and at higher levels in regions of developing taste placodes (asterisk; inset shows higher magnification). (C,D) At later stages, Bmp7 expression is highest in cells of the developing taste buds, with some expression in lingual mesenchyme. Scale bars: 100 µm.

• Supplemental Figure S6 -

  Fig. S6. Effect of added BMP7 on Wnt−β-catenin activity and SHH expression. (A) Tongues (e12.0) were cultured for 2 days in control medium or medium with recombinant BMP7 added, as described in Materials and methods. Changes in papilla development were assessed using either X-Gal staining (for tongues cultured from BAT-Gal reporter mice) or SHH immunohistochemistry (for tongues cultured from CD1 mice). Tongues cultured in control medium show a typical array of fungiform papillae along the anterior two-thirds of the tongue. Treatment with BMP7 results in a reduction in both X-Gal staining (Wnt−β-catenin activity) and SHH immunoreactivity. (B) Quantification of results shown in A. Black bars represent data from control tongues and white bars are data from tongues cultured in the presence of BMP7. (Left) Quantification of pixel intensities of BAT-Gal-positive clusters was performed using ImageJ (http://rsbweb.nih.gov/ij/) (Grider et al., 2006). Images of cultured tongues were converted to 16-bit images, inverted, and background noise subtracted. Papillae were evaluated in a 36×36 ellipse, and the pixel intensity was calculated for each. Shown graphed are the percentages of taste papillae, of a given pixel intensity, for each condition (n=80 for control, n=67 for BMP7 addition). For statistical analysis, the mean intensity (±s.d.) was calculated for all papillae counted in each condition. X-Gal staining intensity was significantly reduced in the papillae of tongues cultured in the presence of BMP7 (P<0.001, Student’s t-test). (Right) The total number of SHH-immunopositive taste buds per tongue was counted from images of tongue explants cultured with (n=5) or without (n=3) BMP7. Mean±s.e.m. is graphed for each condition. Tongues grown in BMP7 had significantly fewer SHH-immunopositive taste buds on the tongue dorsum (P=0.00022, ANOVA).

  Reference

  Grider, M. H., Chen, Q. and Shine, H. D. (2006). Semi-automated quantification of axonal densities in labeled CNS tissue. J. Neurosci. Methods 155, 172-179.

• Supplemental Figure S7 -

  Fig. S7. Expression of Shh and Sox2 in Bmp7^−/− tongue. Tongues from wild-type and Bmp7^−/− littermates were sectioned and ISH performed for Shh and Sox2 as described in Materials and methods. (A) Shh expression is localized within taste buds in both wild-type and Bmp7^−/− tongues. No expression is seen in the intermolar eminence (IE) region in tongues from mice of either genotype. Furthermore, filiform papillae develop normally in the IE of both wild-type and Bmp7^−/− tongues. (B) Expression of Sox2 at e17.5 in Bmp7^−/− animals is localized to the taste bud, and is not expanded into the intermolar eminence like in the Fst^−/− tongue. Scale bars: 25 µm.

• Supplemental Figure S8 -

  Fig. S8. Reaction-diffusion simulation of FST and BMP7. The reaction-diffusion lab package by Selwyn Hollis was run in Mathematica 5 to model the functions of FST and BMP7 in pattering the developing tongue, as described in Materials and methods.

• Supplemental Table S1 - Adobe PDF