The family of TGF-beta signalling molecules play inductive roles in various developmental contexts. One member of this family, Drosophila Decapentaplegic (Dpp) serves as a morphogen that patterns both the embryo and adult. We have now isolated a gene, Daughters against dpp (Dad), whose transcription is induced by Dpp. Dad shares weak homology with Drosophila Mad (Mothers against dpp), a protein required for transduction of Dpp signals. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signalling pathway, overexpression of Dad blocks Dpp activity. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by each protein alone. Dad can also antagonize the activity of a vertebrate homologue of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. We conclude that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development.

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

Developmental patterning relies on morphogen concentration gradients, which generally provide invariable positional information despite genetic fluctuations. Theoretical studies have predicted robust patterning; however, little experimental evidence exists to support this idea. In this report, we examine the robustness of the Decapentaplegic (Dpp) (a Drosophila homologue of bone morphogenetic protein [BMP]) activity gradient in the presence of fluctuations in Dpp receptor levels. Dpp activity can be measured by the degree of phosphorylation of Mothers against dpp (Mad), a major signal transducer. We determined that phosphorylated Mad (pMad) levels remain constant when an extra copy of thickveins (tkv), which encodes the receptor, is introduced into the wild-type background. Higher Tkv levels, expressed under the control of an artificial promoter, result in constant pMad levels. This prompted us to study the mechanisms that underlie pMad level maintenance even when Tkv levels are increased. We focused on the inhibitory Smad, daughters against dpp (dad), which is induced by Dpp signaling and negatively regulates Dpp activity. In the absence of dad, pMad levels significantly increase when Tkv levels increase. These results suggest that Dpp activity gradient robustness when Tkv levels increase depends, at least in part, on negative feedback regulation by dad.

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-medi- Mating in moths is limited to a specific phase of the photoperiod and developmental stage. Accordingly, the biochemical processes that comprise sex pheromone biosynthesis in female moths must be precisely regulated. In most moth species these processes are regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), 4 a 33-amino acid peptide that originates in the subesophageal ganglion and that is characterized by a core C-terminal FSPRLamide sequence (1, 2). After adult emergence, PBAN is released into the hemolymph during a species-specific period and acts on the pheromone gland (PG) to trigger the production and release of species-specific sex pheromones (3, 4).PG is a functionally differentiated organ in close proximity to the terminal abdominal tip that originates in the intersegmental membrane between the 8th and 9th abdominal segments (5-7). In the silkmoth, Bombyx mori, the sex pheromone, E,Z-10,12-hexadecadien-1-ol, commonly known as bombykol, is synthesized de novo within PG cells from acetyl-CoA via the conventional long chain fatty acid biosynthetic pathway (8, 9). The straight chain fatty acyl intermediate, palmitate, is converted stepwise to bombykol by the actions of a bifunctional Z11-10/12 fatty acyl desaturase, Bmpgdesat1, and a PG-specific fatty acyl reductase, pgFAR (10 -12). On the day before adult emergence, B. mori PG cells rapidly accumulate numerous lipid droplets (LDs) within the cytoplasm (13). These LDs play an essential role in bombykol biosynthesis by acting as a reservoir for the de novo synthesized bombykol precursor, ⌬10,12-hexadecadienoate, which is deposited in the LDs in the form of triacylglycerols (TAGs) with the precursor predominantly sequestered at the sn-1 and sn-3 positions of the glycerides (14). After adult emergence, the stored fatty acid is cleaved and converted to bombykol in response to PBAN (7,15).The pheromonotropic effects of PBAN are dependent on extracellular Ca 2ϩ (3,4) and are mediated by the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family (16 -18