Transcriptional and Translational Control of Zebrafish Mesodermal Development

Establishment of the mesodermal germ layer is a process dependent on the integration of multiple transcriptional and signaling inputs. Here I investigate the role of the transcription factor FoxD3 in zebrafish mesodermal development. FoxD3 gain-of-function results in dorsal mesoderm expansion and body axis dorsalization. FoxD3 knockdown results in axial defects similar to Nodal loss-of-function, and was rescued by Nodal pathway activation. In Nodal mutants, FoxD3 did not rescue mesodermal or axial defects. Therefore, FoxD3 functions through the Nodal pathway and is essential for dorsal mesoderm formation. The FoxD3 mutant, sym1, previously described as a null mutation with neural crest defects, was reported to have no mesodermal or axial phenotypes. We find that Sym1 protein retains activity and induces mesodermal expansion and axial dorsalization. A subset of sym1 homozygotes display axial and mesodermal defects, and penetrance of these phenotypes is enhanced by FoxD3 knockdown in mutant embryos. Therefore, sym1 is a hypomorphic allele, and reduced FoxD3 function results in reduced cyclops expression and subsequent mesodermal and axial defects. The sym1 molecular lesion, a point deletion leading to a seven codon frameshift and premature termination, is predicted to be a truncated protein lacking part of the DNA-binding domain and an essential Groucho corepressor interaction domain (GEH). Strongly predicted to be functionally inactive, the hypomorphic character of sym1 suggested that a -1 translational frameshift may correct the reading frame and produce functional protein. Consistent with this hypothesis, mutation of the distal GEH ablated sym1 cDNA activity. Within the frameshift region are three rarely used codons predicted to cause ribosomal pausing and promote translational frameshifting. Conversion of these codons to highly used codons encoding the same residues rendered the sym1 cDNA inactive. Biochemical analyses confirm that a full length FoxD3 protein is produced from sym1 cDNA, but not when the rare codons are replaced. These results indicate that the null character of the sym1 mutation is suppressed by a novel translational frameshifting mechanism, and support the conclusion that FoxD3 is a Nodal-dependent regulator of zebrafish mesodermal development. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Daniel S. Kessler