Files in this Data Supplement:
Fig. S1. Sequence similarity among Dachsous proteins. Solid lines show radial depiction of relative distance among Drosophila Ds (NP_523446.2), mouse Dchs1 (NP_001156415.1) and mouse Dchs2 (XP_143371.6), generated using NCBI multiple sequence alignment and tree view. Numbers within rectangles indicate percent sequence identity in pairwise alignments. The similarity is higher in the extracellular domains, which consist mostly of cadherin domains, than in the intracellular domains.
Fig. S2. Molecular and cellular analysis of kidneys. Examination of signaling pathway components in embryonic kidneys by western blot or confocal microscopy. No significant differences were detected for any of the proteins we examined. (A-C) Western blots on E12.5 kidneys dissected from Fat4 mutants and wild-type littermates. Kidneys were frozen in liquid nitrogen, and later lysed in RIPA supplemented with proteinase inhibitor cocktail (Roche) and phosphatase inhibitor cocktail Set II (Calbiochem). Antibodies used were mouse anti-β-catenin (BD Biosciences, 1:2,000), mouse anti-MYPT (BD Biosciences, 1:1,000), rabbit anti-phospho-MYPT (Cell Signaling, 1:500) and rabbit anti-Lats1 (Bethyl lab, 1:1000). α-Tubulin and GAPDH are used as loading controls. (A) Lats1, a component of the Hippo pathway, examined because Drosophila fat regulates levels of the Lats homologue Warts (Cho et al., 2006). (B) β-Catenin, a component of canonical Wnt signaling pathways, was examined because of evidence that Wnt pathways influence kidney development (Majumdar et al., 2003) and that Hippo pathway components can regulate Wnt signaling (Varelas et al., 2010). (C) Phospho-Mypt, a Rho-kinase substrate, was examined because it was reported to be affected in a Fz-PCP pathway mouse mutant (Suriben et al., 2009). (D-I) Panels show higher magnifiations of E11.5 kidneys in Fat4 mutants (E,G,I) or wild-type littermates (D,F,H) stained for E-cad (green) and Pax2 (blue) with (D,E) β-catenin, (F,G) Yap or (H,I) Kit (red). An area of more intense staining (asterisk) is a consequence of focal pane and curvature of the epithelium. (F,G) Yap (red), a transcription factor of the Hippo pathway (E11.5), is detected at high levels in the nucleus of epithelial cells at this stage. (H,I) Kit (red), a receptor implicated in kidney stromal/interstitial cell function (Schmidt-Ott et al., 2006), is expressed in these cells. The lack of staining in the center (arrow), reflects the incomplete migration of these cells at this stage (E12.0) in mutants (e.g. see Foxd1 stain in Fig 4I,J), consistent with the general delay in early kidney development.
References
Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and Irvine, K. D. (2006). Delineation of a Fat tumor suppressor pathway. Nat. Genet. 38, 1142-1150.
Majumdar, A., Vainio, S., Kispert, A., McMahon, J. and McMahon, A.P. (2003). Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development 130, 3175-3185.
Schmidt-Ott, K. M., Chen, X., Paragas, N., Levinson, R. S., Mendelsohn, C. L. and Barasch, J. (2006). c-kit delineates a distinct domain of progenitors in the developing kidney. Dev. Biol. 299, 238-249.
Suriben, R., Kivimäe, S., Fisher, D. A. C., Moon, R. T., Cheyette, B. N. R. (2009). Posterior malformations in Dact1 mutant mice arise through misregulated Vangl2 at the primitive streak. Nat. Genet. 41, 977-985.
Varelas, X., Miller, B. W., Sopko, R., Song, S., Gregorieff, A., Fellouse, F. A., Sakuma, R., Pawson, T., Hunziker, W., McNeill, H., Wrana, J. L. and Attisano, L. (2010). The Hippo pathway regulates Wnt/beta-catenin signaling. Dev. Cell 18, 579-591.
Supplemental Figure 3
Fig. S3. Cell death, and Dchs1 and Fat4 protein expression, in lungs. All panels show lungs (E11.5 for A,B; E12.5 for C-L) stained for E-cad (green) and either Cas3, Dchs1 or Fat4 (red), as indicated. Panels marked by prime symbols show the Cas3, Dchs1 or Fat4 channel only of the stain on the left. Scale bars are in the top right-hand corner. (A) Wild-type litter mates from Fat4 mutant stock, stained for Cas3. Some apoptosis is detected, especially within the epithelium. (B) Fat4 mutant stained for Cas3. Some apoptosis is detected, especially within the epithelium. (C) Wild-type littermates from Dchs1 mutant stock, stained for Dchs1. (D) Wild-type littermates from Fat4 mutant stock stained for Fat4. (E) Dchs1 mutant stained for Dchs1. (F) Fat4 mutant, stained for Fat4. Some faint staining is detected, which we infer represents non-specific background from the antisera. C,E and D,F are matched pairs, dissected from the same litters, and fixed, stained and imaged in parallel using identical conditions. (G) Higher magnification of Dchs1 staining. (H) Higher magnification of Fat4 staining. (I) Wild-type littermates from Fat4 mutant stock stained for Dchs1. (J) Wild-type littermates from Dchs1 mutant stock stained for Fat4. (K) Fat4 mutant stained for Dchs1; note increased expression relative to I. (L) Dchs1 mutant, stained for Fat4; note increased expression relative to J. I,K and J,L are matched pairs, dissected from the same litters, and fixed, stained and imaged in parallel using identical conditions.
Fig. S4. Comparative analysis of Dchs2 expression. (A-C) Transverse sections through the body of E9.5 embryos, showing expression of (A) Fat4, (B), Dchs1 and (C) Dchs2 by in situ hybridization. Both Fat4 and Dchs1 are expressed in the sclerotome (arrowed in A) and neural tube. The arrow in B indicates the expression of Dchs1 in the neural tube, which is low compared with expression in the sclerotome. Dchs2 expression is confined to the ventral neural tube. (D-H) Vibratome slices through (D) the brain and cochlea (arrowed), (E) kidney, (F) lungs and (G) gastrointestinal tract of an E12.5 embryo, showing expression of Dchs2. Dchs2 transcripts were only detectable in the brain. nt, neural tube.
Fig. S5. Dchs1 and Fat4 protein are not preferentially localized to cilia. Panels show higher magnification of E12.5 kidneys, stained for acetylated α-tubulin (green) to mark cilia, and either Dchs1 or Fat4 (red). Panels marked by prime symbols show the Dchs1 or Fat4 channel only; panels marked by double prime symbols show the α-tubulin only.