JNK Signaling Antagonism: The role of Raw during Drosophila dorsal closure. Molly C. Jud, Melissa Ratcliffe, Anthea Letsou. Human Genetics, University of Utah, Salt Lake City, UT.

   A surprisingly small number of conserved signaling pathways are used in development, and their tight regulation is necessary for embryogenesis to occur normally. We study two sequentially acting signaling pathways necessary for dorsal closure in the fruit fly Drosophila melanogaster; the Jun-N-terminal kinase/AP-1 pathway (JNK/AP-1; MAPK family member) is specifically activated in leading edge (LE) epidermal cells and transcriptionally activates the Decapentaplegic pathway (Dpp; TGF- family member). Of particular interest to our lab are antagonists of the JNK and Dpp signaling pathways, including the novel gene, raw, an antagonist of the JNK signaling pathway. raw is a member of a dorsal-open subgroup, known as the raw group; this group includes three other genes (puckered, ribbon, and mummy) and is characterized by three shared, loss-of-function phenotypes: (1) a dorsal closure defect observed as a dorsal hole or pucker, (2) hypotrophy of ventral denticle belts, and (3) ectopic dpp expression in the lateral epidermis beyond the LE. We have previously shown that raw is expressed broadly throughout embryogenesis and is required to suppress zygotic Basket (JNK)-independent Jun activity in embryos undergoing dorsal closure. We hypothesize that Raw functions to silence basal levels of epidermal JNK signaling and is therefore a master regulator in the complex circuitry of the developing Drosophila embryo. Here, we show biochemical evidence that activated phospho-Jun accumulates in raw and raw bsk mutant embryos relative to wild types. As Jun is active in raw embryos, even without zygotic basket, another kinase must be responsible for Jun activation in raw mutant embryos. Here we show: (1) raw is necessary to define a LE, (2) a maternal basket-encoded JNK likely activates Jun in the epidermis of raw mutants, and (3) multiple levels of JNK/Dpp antagonism are necessary to restrict dpp to LE cells.