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Supplementary Material

DEV051938 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Figure S1 -

    Fig. S1. Conditional Nog and Grem1 alleles and summary of compound mutant classes. (A) Wild-type mouse fetus at E16.5. (B) E16.5 Grem1fx/fx;ACTB-Cre Tg fetus displaying characteristic deformed limbs. (C) E16.5 Nogfx/fx;ACTB-Cre Tg fetus displaying characteristic paddle-shaped autopods and cranial malformation. (D) A description of the compound mutant breeding scheme and summary of embryo genotypes obtained from E8.5 and E9.5 dissections.

  • Supplemental Figure S2 -

    Fig. S2. Additional markers indicating sclerotome agenesis in Nog;Grem1 double-mutant embryos. (A-P) Whole-mount E9.5 in situ hybridizations for sclerotome markers Pax9 (A-D), Nkx3.2 (E-H), Uncx4.1 (I-L) and Tbx18 (M-P); lateral views, embryos facing left. Nogfx/+;Grem1fx/+ (A,E,I,M) and Nogfx/+;Grem1fx/fx (B,F,J,N) embryos display normal Pax9 (A,B), Nkx3.2 (E,F), Uncx4.1 (I,J) and Tbx18 (M,N) expression. (C) Pax9 expression is faint but detectable in Nogfx/fx;Grem1fx/+ embryos (arrowheads). (D) A Nogfx/fx;Grem1fx/fx embryo. No somitic Pax9 expression was detected. (G) Nkx3.2 was expressed in the anterior somites of Nogfx/fx;Grem1fx/+ embryos. (H) No somitic Nkx3.2 expression appeared in Nogfx/fx;Grem1fx/fx embryos. Nkx3.2 transcript was detected in the gut of all classes (E-H, arrows). (K) Uncx4.1 expression is reduced posteriorly in Nogfx/fx;Grem1fx/+ embryos. (L) Faint, discontinuous Uncx4.1 expression could be detected in this Nogfx/fx;Grem1fx/fx embryo (arrowheads). (O) Tbx18 expression becomes disorganized posteriorly in Nogfx/fx;Grem1fx/+ embryos. (P) Tbx18 expression was detected in this Nogfx/fx;Grem1fx/fx embryo.

  • Supplemental Figure S3 -

    Fig. S3. Dermomyotomal derivatives are induced in Nog;Grem1 embryos. (A-H) Whole-mount E9.5 in situ hybridizations for the myoblast progenitor marker Myf5 (A-D) and the presumptive limb musculature marker Lbx1 (E-H); lateral views, embryos facing left. (A,E) Nogfx/+;Grem1fx/+ controls. (B,F) Nogfx/+;Grem1fx/fx embryos. (C,G) Nogfx/fx;Grem1fx/+ embryos. (D,H) Nogfx/fx;Grem1fx/fx embryos. Myf5 and Lbx1 are expressed in all genetic classes.

  • Supplemental Figure S4 -

    Fig S4. Nog;Grem1 somite tissue fails to form cartilage in vitro. Representative micromass preparations of E10.5 somite tissue stained with Alcian Blue to detect cartilage following 6 days of culture. (A,C) Chondrogenic nodules form in both high-density (A) and low-density (C) cultures derived from somites. Somite tissue from three Nogfx/+;Grem1fx/+ embryos was separately dissociated and plated. (B,D) No Alcian Blue aggregates formed in cultures from Nogfx/fx;Grem1fx/fx somites at either high (B) or low (D) density. Somite tissue from three Nogfx/+;Grem1fx/+ and two Nogfx/fx;Grem1fx/fx embryos was separately dissociated and plated in at least four 20 µl drops at the indicated cell densities. Scale bars: 1 mm.

  • Supplemental Figure S5 -

    Fig. S5. Conditional ablation of Bmpr1a does not alter Pax1 expression levels. Quantitative PCR analysis of Bmpr1a, Pax1 and Lbx1 in Bmpr1afx/− embryos either with (white) or without (black) the Tg(Cre/Esr1) allele. Tamoxifen was administered at E7.5 and animals were analyzed at E9.5, resulting in a significant reduction in Bmpr1a transcript levels. Whereas this results in a strong reduction in Lbx1, Pax1 expression is not significantly affected. A minimum of three embryos was used for each condition. Error bars indicate 2× s.e. *, P<0.05 between the relative expression recorded for embryos with or without Cre.