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Morphogen gradients play fundamental roles in patterning and cell specification during development by eliciting differential transcriptional responses in target cells. In Drosophila, Decapentaplegic (Dpp), the BMP2/4 homolog, downregulates transcription of the nuclear repressor brinker (brk) in a concentration-dependent manner to generate an inverse graded distribution. Both Dpp and Brk are crucial for directing Dpp target gene expression in defined domains and the consequent execution of distinct developmental programs. Thus, determining the mechanism by which the brk promoter interprets the Dpp activity gradient is essential for understanding both Dpp-dependent patterning and how graded signaling activity can generate different responses through transcriptional repression. We have uncovered key features of the brk promoter that suggest it uses a complex enhancer logic not represented in current models. First, we find that the regulatory region contains multiple compact modules that can independently drive brk-like expression patterns. Second, each module contains binding sites for the Schnurri/Mad/Medea (SMM) complex, which mediates Dpp-dependent repression, linked to regions that direct activation. Third, the SMM repression complex acts through a distance-dependent mechanism that probably uses the canonical co-repressor C-terminal Binding Protein (CtBP). Finally, our data suggest that inputs from multiple regulatory modules are integrated to generate the final pattern. This unusual promoter organization may be necessary for brk to respond to the Dpp gradient in a precise and robust fashion.

Bone Morphogenetic Proteins (Bmps) are secreted growth factors that play crucial roles in animal development across the phylogenetic spectrum. Bmp signaling results in the phosphorylation and nuclear translocation of Smads, downstream signal transducers that bind DNA. In Drosophila, the zinc finger protein Schnurri (Shn) plays a key role in signaling by the Bmp2/Bmp4 homolog Decapentaplegic (Dpp), by forming a Shn/Smad repression complex on defined promoter elements in the brinker (brk) gene. Brk is a transcriptional repressor that downregulates Dpp target genes. Thus, brk inhibition by Shn results in the upregulation of Dpp-responsive genes. We present evidence that vertebrate Shn homologs can also mediate Bmp responsiveness through a mechanism similar to Drosophila Shn. We find that a Bmp response element (BRE) from the Xenopus Vent2 promoter drives Dpp-dependent expression in Drosophila. However, in sharp contrast to its activating role in vertebrates, the frog BRE mediates repression in Drosophila. Remarkably, despite these opposite transcriptional polarities, sequence changes that abolish cis-element activity in Drosophila also affect BRE function in Xenopus. These similar cis requirements reflect conservation of trans-acting factors, as human Shn1 (hShn1; HIVEP1) can interact with Smad1/Smad4 and assemble an hShn1/Smad complex on the BRE. Furthermore, both Shn and hShn1 activate the BRE in Xenopus embryos, and both repress brk and rescue embryonic patterning defects in shn mutants. Our results suggest that vertebrate Shn proteins function in Bmp signal transduction, and that Shn proteins recruit coactivators and co-repressors in a context-dependent manner, rather than acting as dedicated activators or repressors.

We report the identification of a Drosophila Pax gene, eye gone (eyg), which is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression, (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg, (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. Our results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription.