Zebrafish Fog proteins bind and repress GATA4. Zebrafish Fog proteins can physically interact with GATA4. In panel A, in vitro translated, ³⁵S-labeled Fog1, Fog2a, or Fog2b proteins (column 1) were incubated with bacterially produced GST (column 2) or GST-GATA4 (column 3) fusion proteins and purified using glutathione sepharose beads. The resultant complexes were resolved by 7% SDS-PAGE and detected by autoradiography. A physical interaction of GATA4 and Fog proteins is indicated by the presence of ³⁵S-labeled protein in column 3. Zebrafish Fog proteins can repress GATA4 mediated transcriptional activation. In panel B, NIH 3T3 fibroblasts were transfected with a reporter plasmid containing 638 bp of the ANF promoter driving expression of human growth hormone (hGH). In addition, expression plasmids for murine GATA4 (columns 2–5) and zebrafish fog1 (column 3), fog2a (column 4), or fog2b (column 5) were included. Forty-eight hours post-transfection, cells were harvested and assayed for hGH. Results are reported as the mean ± SEM (n = 12).

Expression of Fog genes during zebrafish development. Whole-mount in situ hybridization using digoxigenin-labeled antisense cRNA fog1, fog2a, and fog2b probes on zebrafish embryos from 19 hpf to 72 hpf. An asterisk in the top left panel indicates intermediate cell mass expression of fog1. In all panels, arrows indicate heart staining; arrowheads indicate brain staining.

Fog1 is required for cardiac looping. Anti-fog morpholinos inhibit translation or splicing of fog mRNA. In panel A, in vitro transcription and translation of Fog1 (top panel), Fog2a (middle panel), or Fog2b (bottom panel) in the absence (column 1) or presence of gene-specific morpholino (columns 2–4) or mismatch control (column 5). In panel B, RT-PCR of RNA from 48 hpf zebrafish embryos uninjected (-) or injected (+) with morpholinos directed against the exon 4 splice donor site of fog1 (left panel), fog2a (middle panel), or the exon 5 splice donor site of fog2b (right panel) using primers from flanking exons. Arrowheads indicate the correctly spliced product; asterisks indicate aberrantly spliced products. Developmental defects in morpholino injected embryos. In panel C, morpholinos directed against fog1 (a and b), fog2a (c and d), fog2b (e and f) or a mismatch control (g and h) were injected into single-cell zebrafish embryos and embryos photographed 48 h post-fertilization (a, c, e, g). Arrows indicate a pericardial effusion and blood pooling in the atrium. Panels on the right (b, d, f, h) are in situ hybridizations using the cmlc2 probe on 48 hpf morphants.

Fog1 morphants have a hypoplastic ventricular wall. Gross morphology of a 4.5 days post-fertilization (a and d) or 7 days post-fertilization (b and e) embryo uninjected (a, b) or injected with an anti-fog1 morpholino at the single-cell stage (d, e). Note the extensive pericardial effusion in the Fog1 morphant. Below, longitudinal sections through the heart of an uninjected embryo (c) or a fog1 morphant (f) at 7 days post-fertilization. The ventricle is indicated by “v”, the atrium by “a”. Scale bar is 50 μm.

Gene expression in fog1 morphants. Whole-mount in situ hybridizations were performed using probes specific for tbx5 (left column) or tbx20 (right column) on 48 hpf wild-type embryos (rows 1 and 3) or fog1 morphants (rows 2 and 4). Arrows indicate heart expression of each of these genes.

Expression of murine FOG-1 rescues the fog1 morphant phenotype. Shown above are 48hpf zebrafish embryos uninjected (a), injected with an anti-fog1 morpholino (b), injected with an anti-fog1 morpholino and mRNA encoding murine FOG-1 (c), or injected with an anti-fog1 morpholino and mRNA encoding an N-terminal truncation of murine FOG-1 (ΔFOG-1) (d). Atrial and yolk sac blood pooling is indicated by the arrows.