Function and regulation of the Strongylocentrotus purpuratus gatae gene

The Strongylocentrotus purpuratus gatae is orthologous to vertebrate gata4/5/6 genes. gatae is expressed throughout embryogenesis, beginning in the 15 h blastula in presumptive mesoderm cells, and at mesenchyme blastula, in endoderm and mesoderm cells of the veg2 lineage. During gastrulation, gatae is expressed in the midgut, hindgut and mesoderm, while in the pluteus expression it is limited to the midgut and coelomic pouches. Perturbation of gatae expression resulted in the lowered RNA levels for many endomesoderm transcription factors, including foxA, brachyury, and [beta]1/2-otx, highlighting Gatae’s role as a regulator of transcription factors. gatae occupies an important node in the endomesoderm gene regulatory network, using its cross-regulatory interactions with otx to stabilize the endomesoderm gene expression program. Cis-regulatory analysis of gatae identified two modules responsible for its embryonic expression. Module 10 drives endomesoderm expression in the blastula, while module 24 activates gut expression in the gastrula and pluteus. Deletion of module 10 from a gatae GFP BAC resulted in a complete loss of blastula stage expression, demonstrating its necessity and sufficiency for early activity. Global cis-regulatory analysis of the gatae locus suggests that module usage is exclusionary; only one module can associate with the basal transcriptional apparatus and affect gene transcription at any given time. The endomesoderm gene regulatory network predicts that gatae is downstream of Otx and Notch signaling. Analysis of the sequence of module 10 identified Otx and Suppressor-of-Hairless (Su(H)) binding sites. Injection of Otx-engrailed RNA repressed the expression of module 10:GFP reporter; the effect is abolished when Otx binding sites were mutated. Gel shifts demonstrated that the Otx protein binds to module 10. Module 10 expression was reduced under perturbation of Notch signaling. Mutations of either Otx or Su(H) binding sites resulted in lowered GFP RNA levels with no effect on spatial expression. Mutations of both Otx and Su(H) binding sites led to a further reduction but not elimination of reporter expression, suggesting that another input is involved. This unknown input was determined to be also downstream of Notch signaling and that gatae regulation functions via OR logic.