DEV02210 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression and processing of Gdf3 protein in cell culture. (A) Expression of Gdf3 (lanes 1-5) and bf-Gdf3 (lanes 6-9) proteins in conditioned media (CM; lanes 1-3, 6, 7) and cell lysates (lysate; lanes 4, 5, 8, 9) of transfected 293T cells, detected by western blotting using an antibody that recognizes the mature Gdf3 ligand. Owing to inefficient processing of the native Gdf3 proprotein, the mature Gdf3 ligand is detected only in conditioned media (asterisk) after long film exposure (lane 1, overexposure of lane 3). The multiple Gdf3 isoforms detected are likely to be due to differences in N-linked glycosylation. Positions of molecular standards are indicated. (B) Gdf3 activity in an assay for Nodal signaling. 293T cells were transfected with expression constructs for Nodal or Gdf3, Cripto, the Foxh1 transcription factor and the Nodal/activin-responsive A3-luc reporter; luciferase activities are normalized to those of controls. Control samples correspond to cells transfected with Foxh1 without Nodal or Gdf3. The assay was performed in triplicate; error bars represent one s.d. Levels of Gdf3 and bf-Gdf3 protein are as shown in A.

• Supplemental Figure 2 -

  Fig. S2. Generation of Gdf3 mutant mice. (A) Replacement of the wild-type (Gdf3) allele with the Gdf3tm1Zuk (null) allele in embryonic stem cells. Gdf3 has 2 exons. Exon 2 (E2) encodes 162 amino acids of the propeptide and all of the mature Gdf3 peptide (114 amino acids). Homologous recombination between the targeting construct and the Gdf3 wild-type allele replaces exon 2 with a Pgk1-HPRT expression cassette, resulting in functional inactivation of the gene. BamHI (B); SalI (S); HindIII (H); HpaI (Hp); XbaI (X). (B) Southern blot analysis of DNA from Gdf3 mutant mice. Genomic DNA was digested with BamHI and SalI. The presence of 10.8 kb and 3.1 kb fragments are diagnostic for the wild-type and null alleles, respectively. The same Southern blot was stripped and hybridized with an exon 2-specific probe. The diagnostic 10.8 kb fragment is absent in Gdf3^–/– mice.

• Supplemental Figure 3 -

  Fig. S3. Expression levels of Nodal and Gdf1 in Gdf3 mutants. (A) Variable levels of Nodal expression in Gdf3 homozygotes at 6.0 dpc. Real-time PCR analysis was carried out on first-strand cDNA prepared from individual embryos from wild-type or Gdf3 homozygous embryos in the C57BL/6 strain background. Results for Nodal expression were normalized against Gapdh expression as an internal standard, and the average relative ratio for seven wild-type embryos was set to 1; relative values within a twofold range are shaded. Gdf3^–/– embryos 3, 11 and 24 show more than twofold higher levels of normalized Nodal expression, and are considered to show Nodal upregulation; Gdf3^–/– embryos 4, 16, 19, 20 and 25 show Nodal downregulation. Each value corresponds to a triplicate assay of independent first-strand cDNA preparations from a single embryo; error bars correspond to one s.d. (B) Levels of Gdf1 expression in Gdf3 homozygotes at 6.0 dpc. Real-time PCR analysis was carried out on individual embryos as in A.