DEV02861 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. The BMP antagonist noggin induces ectopic epithelial buds, outgrowth and branching in cultured Grem1-deficient kidney primordia. (A) Culture of a Grem1-deficient metanephric primordia in control medium for 60 hours. (B) Culture of a Grem1-deficient (G1^Δ/Δ) metanephric primordia in noggin-supplemented medium. After 48 hours, several ectopic epithelial buds have formed (red asterisks). The ureteric epithelium (white asterisk) has undergone its first branching by 48 hours and the second branching is initiated after about 60 hours (n=6).

• Supplemental Figure S2 -

  Fig. S2. Expression of Wnt11 in the ureteric bud epithelium depends on gremlin 1. (A-D) The expression of Wnt11 is lost from the ureteric bud epithelium of Grem1-deficient metanephric kidneys. Detection of Wnt11 transcripts at E11.0 (A,B) and E11.5 (C,D) in wild-type (A,C) and Grem1-deficient (B,D) kidney primordia. The approximate position of the mutant ureteric bud is indicated by a broken line in D. (E) Genetic reduction of Bmp4 activity in the context of a Grem1 deficiency (Grem1^Δ/Δ; Bmp4^Δ/+) restores Wnt11 expression. Arrowheads point to the ureteric epithelial tips. ub, ureteric bud; ue, ureteric epithelial tip region expressing Wnt11 after first branching.

• Supplemental Figure S3 -

  Fig. S3. Semiquantitative RT-PCR analysis Gdnf and Grem1 expression. (A) Determination of the relative Gdnf expression levels in wild-type (Wt) and Grem1-deficient (G1^Δ/Δ) kidney primordia isolated at E10.75-11.0 (38-40 somites). RNA was isolated either at the time of dissection (Non-cultured) or after 48 hours in culture (Cultured) in medium alone or supplemented with recombinant gremlin 1 (5 μg/ml; lane G1^Δ/Δ +GREM1). Relative Gdnf transcript levels (upper row) after normalization for β-Actin mRNA content (lower row). As expected, the relative Gdnf levels and kidney primordia sizes are similar when wild-type (Wt) and Grem1 (G1^Δ/Δ)-deficient kidney primordia are isolated at E10.75. After 48 hours in culture, one would expect lower levels of Gdnf transcripts in mutant kidneys (see Fig. 2B). However, due to the developmental arrest, the overall size of the mutant kidney primordia is significantly smaller than those of wild-type controls and mutants treated with gremlin 1 (G1^Δ/Δ +GREM1; see Fig. 2B and compare with Fig. 2A,C; and data not shown). The normalization for β-Actin transcript content will also adjust for the smaller overall size; therefore, the relative Gdnf transcript levels of untreated mutants appear similar to wild-type controls after 48 hours in culture. Most importantly, treatment of Grem1 mutants with recombinant gremlin 1 protein (G1^Δ/Δ +GREM1) increases Gdnf expression levels about twofold (1.9±0.4, n=3) in comparison with untreated mutants (G1^Δ/Δ) and wild-type controls. (B) Grem1 expression in wild-type and Gdnf-deficient kidney primordia isolated at E11.0 (40-44 somites). Note that Grem1 remains expressed at similar levels in Gdnf mutant (Gdnf^Δ/Δ) kidney primordia as in wild-type controls (Wt).

• Supplemental Figure S4 -

  Fig. S4. The metanephric mesenchyme of Grem1-deficient embryos remains competent to condense and initiate nephrogenesis. (A-C) Distribution of the PAX2 protein in kidney primordia cultured for 72 hours. (A) PAX2 is present in both the epithelium and condensing mesenchyme of wild-type metanephric kidney primordia. (B) PAX2 is only retained in the epithelium of Grem1-deficient metanephric kidney primordia (arrowhead points to the tip of ureteric bud). (C) Culturing Grem1-deficient metanephric kidney primordia in the presence of 15 μM LiCl (+LiCl) induces mesenchymal condensation (Davies et al., 1995; Oxburgh and Robertson, 2002) as revealed by PAX2. LiCl induces nephrogenesis probably by activating canonical Wnt signal transduction (as it acts at the level of GSK3β) (Klein and Melton, 1996). Note that the ureteric epithelium has neither grown nor branched. Arrowheads (B,C) point to the distal tip of the ureteric bud. (D-F) 15 μM LiCl induces the isolated metanephric mesenchyme to condense as revealed by activation of the Wilm’s tumor-1 (WT1) protein. (D) Isolated wild-type and Grem1-deficient (data not shown) mesenchyme cultured without LiCl is eliminated by apoptosis. (E,F) LiCl treatment of both wild-type (E) and Grem1-deficient (F) mesenchyme induces the WT1 protein. (G-I) Wnt4 expression is activated in isolated metanephric mesenchyme cultured with 15 μM LiCl for 72 hours. (G) Wnt4 is not detected in mutant mesenchyme cultured without LiCl. (H,I) LiCl treatment induces Wnt4 expression in to a similar extent in wild-type (H) and Grem1-deficient (I) mesenchyme, which indicates that nephrogenesis has been initiated. For immunodetection of PAX2 and WT1, kidney primordia were incubated overnight in a methanol:DMSO solution (4:1, 4°C) and incubated again overnight with either PAX2 (Covance, 1/500) or WT1 (Santa Cruz, 1/50) primary antibodies diluted in FBS:DMSO (4:1, 4°C). After washing in PBT/FBS/DMSO, samples were incubated overnight with secondary antibodies coupled to specific fluorochromes (Cy3 for WT1 and Cy2 for PAX2; Amersham) and washed extensively the following day.