Nature Reference# 2005-03-03640B

Nature reference# 2005-03-03640B

Titles of SI files:

Corbit SuppFig1

Corbit SuppFig2

Corbit SuppFig3

Corbit SuppFig4

SI summaries:

Supplementary Figure 1

-Smo antibodies are specific. a, Western blots of 10g of E9.5 embryo lysates separated by 8% SDS-PAGE with -Smo A2666 (1:500) and -actin (1:1000, Sigma) antibodies. The -Smo antibody recognizes a band of the predicted size in the lysate from the wild-type embryo that is absent in the lysate from the Smo mutant embryo. b, Ventral view of the node of Smo mutant late head fold stage embryo stained for acetylated Tubulin (green) and Smo (red). Nuclei are visualized with DAPI staining (blue). No residual Smo staining is observed in cilia, as demonstrated with comparison to Fig.1a. c, High magnification view of Smo mutant nodal primary cilia (Photoshop; 133 KB).

Supplementary Figure 2

Ciliary localization of Smo occurs in Shh-expressing nodal cells. A ventral view of the node stained for Shh (-Shh 5E1, 1:20, green) and Smo (red). Nuclei are visualized with DAPI staining (blue). Two cilia are indicated with arrowheads. The immunohistochemical protocol followed only detects Shh in producing cells, and not secreted Shh1The-Shh antibody was obtained from the Developmental Studies Hybridoma Bank under contract NO1-HD-7-3263 from the NICHD (Photoshop; 167 KB).

Supplementary Figure 3

Smo localization to cilia in populations of MDCK cells. a-d, MDCK expression of acetylated Tubulin (green) and the Myc tag of wild-type Smo or CLDSmo (red). a, Smo-expressing MDCK cells cultured in the presence of cyclopamine. b, Smo-expressing MDCK cells cultured in the presence of Shh-conditioned medium. Cilia are denoted with arrowheads. c, CLDSmo-expressing MDCK cells cultured in the presence of cyclopamine. d, CLDSmo-expressing MDCK cells cultured in the presence of Shh-conditioned medium (Photoshop; 123 KB).

Supplementary Figure 4

The presence of cilia correlates with Hedgehog responsiveness, and Shh induces the movement of Smo to the primary cilia of different cell types. a, MEFs were plated at different densities in a 24-well plate. MEFs plated at high density reached confluency and almost always possess primary cilia. Sub-confluent MEFs possess cilia at lower frequencies. Error bars, 1 s.d. b, MEFs were transfected with wild-type Smo-myc, Renilla luciferase, and Gli-luc plasmids, then stimulated with Shh-conditioned media as described above. The amount of normalized Gli-luciferase induction correlates with the presence of primary cilia. c, Transfected confluent MEFs stained for acetylated Tubulin (green) and Smo-myc (red). Arrows indicate cilia. Nuclei are visualized with DAPI staining (blue). Smo-myc moves to MEF cilia in response to Shh. d, Endogenous Smo visualized in IMCD-3 cells (ATCC) grown on transwell plates similarly to MDCK cells. Shh stimulation increases the localization of endogenous Smo on IMCD-3 primary cilia (arrow). e, Apical-basal optical sections of confluent MEFs stained for endogenous Smo. MEFs also show a relocalization of endogenous Smo to primary cilia (arrows) upon Shh stimulation (Photoshop; 98 KB).

Supplementary Table 1

Summary of zebrafish injection results

Totals are from four independent experiments. *Overexpression phenotype.

Supplementary Reference

1.Gritli-Linde, A., Lewis, P., McMahon, A. P. & Linde, A. The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides. Dev Biol 236, 364-86. (2001).