DEV02519 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. Expression of an inactive, truncated Gata4 protein by a conditional Gata4 allele after Cre recombination. (A) As a result of the use of a cryptic splice site, the Gata4^Δex2 allele continues to express a modified Gata4 transcript. The transcript contains an in frame start codon just upstream of the start of exon 3. The truncated transcript lacks the N-terminal 46% of the coding region, including both transcriptional activation domains. (B) Western blotting of E10.5 whole heart lysates. Embryo genotypes were Gata4^flox/flox with the indicated Cre allele. A truncated protein is present at reduced levels compared with wild type. Expression of this protein is stage specific, as it is present in embryonic but not adult Gata^wt/Δex2 hearts (data not shown). (C) The truncated protein failed to activate the ANF promoter (Lee et al., 1998) and a Bmp-responsive chick Nkx2-5 enhancer (Lee et al., 2004), which were both responsive to wild-type Gata4. Results are from triplicate samples and are representative of at least three independent experiments.

• Supplemental Figure 2 -

  Fig. S2. Contribution of neural crest to the outflow tract cushion. The bulk of the outflow tract cushion (red arrows) is derived from neural crest, as indicated by fate mapping of cells that express the Wnt1-Cre transgene. Neural crest does not contribute significantly to the AV cushions (black arrows). Recombined R26RstoplacZ cells were visualized in an E11.5 embryo by whole-mount X-gal staining, followed by paraffin sectioning. The boxed region is shown at a higher magnification in the lower panel. OT, outflow tract; A, atrium; V, ventricle. Scale bar: 100 μm.

• Supplemental Figure 3 -

  Fig. S3. Liver hypoplasia in Gata4^T2del embryos. (A,B) E12.5 embryos from Fig. 2B were saggitally sectioned and stained with Hematoxylin and Eosin. Sections with the maximal amount of liver (L) are shown. Inset shows a higher magnification of the boxed region. Scale bar: 1 mm. (C,D) In situ hybridization for the hepatocyte marker Alb1 (red pseudocolor) on transversely sectioned E10.5 embryos. Blue, DAPI counterstain. Scale bar: 200 μm. Normal expression of Alb1 in mutant embryos suggests that hepatocytes are specified normally.

• Supplemental Figure 4 -

  Fig. S4. Myocardial hypoplasia in a subset of Gata4^T2del mutant embryos. (A-D) Transverse sections of E11.5 control and Gata4^T2del littermates. Boxed areas in A and C are shown at a higher magnification in B and D. Six out of 17 systematically examined Gata4T2del hearts displayed significant myocardial thinning in addition to hypocellularity of the AV cushions (arrow).

• Supplemental Figure 5 -

  Fig. S5. Conserved GATA sites within Erbb3 regulatory elements are not required for transcriptional activation by Gata4 in vitro. Purple boxes indicated conserved non-coding sequences, and red lines indicated evolutionarily conserved GATA binding sites. The yellow box is the SV40 minimal promoter. Coordinates are relative to the 5′ end of the 5′ most expressed sequence tag (blue hatch). We mutated the conserved GATA binding sites (red ‘X’) and measured the degree to which the co-transfected Gata4 expression plasmid stimulated reporter activity, compared with a co-transfected empty vector. Mutation of the conserved GATA binding sites did not significantly alter the degree of activation when compared with the wild-type promoter or enhancer reporter constructs. NS, not statistically significant by t-test. n=3, promoter; n=5, enhancer.