David Stein

slalom encodes an adenosine 3′‐phosphate 5′‐phosphosulfate transporter essential for development in Drosophila

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high‐energy sulfate donor adenosine 3′‐phosphate 5′‐phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3′‐phosphate 5′‐phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycos aminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal–ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal–ventral determinant pipe.

Drosophila Pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3′-phosphoadenosine 5′-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.

Dorsoventral Patterning: A Direct Route from Ovary to Embryo

Recent data indicate that Gurken-mediated activation of the EGF receptor in the somatic follicle cells of the Drosophila ovary - required for dorsoventral patterning of the fly embryo - leads to cell-autonomous repression of pipe expression, suggesting that the EGF receptor signaling pathway acts directly to control pipe transcription.

Localization and Activation of the Drosophila Protease Easter Require the ER-Resident Saposin-like Protein Seele

Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity.

Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torso-like in receptor activation

Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase Torso. Both Torso and Torsos presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torso-like, which is expressed by follicle cells adjacent to the ends of the developing oocyte. We find that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torso-like or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torso-like were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torso-like were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torso-like alone were ineffective but acted synergistically to stimulate Torso signaling. Our results suggest that Torso interacts with both Trunk and Torso-like, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo. Summary: Evenly distributed Trunk protein and terminally localized Torso-like combine to promote spatially restricted dimerization and activation of the receptor tyrosine kinase Torso.

The Torso Ligand, Unmasked?

When a transmembrane receptor protein tyrosine kinase (RTK) is expressed throughout the plasma membrane, yet only a specific handful of them must be activated, whats a ligand to do? During the development of the anterior and posterior termini of the Drosophila embryo, uniformly secreted ligand precursors are activated by proteolysis near the location of the receptors that must be activated. Stein and Stevens discuss the recent publication by Casali and Casanova that describes the mechanism of activation of the Drosophila RTK called Torso. In addition, Casali and Casanova may have identified a physiologically relevant ligand for Torso called Trunk. Proteolytic cleavage of the Trunk precursor can activate Torso-dependent signaling, but the existence of cleaved Trunk has not yet been demonstrated in vivo for Drosophila. Stein and Stevens discuss the ramifications of such a highly regulated process of ligand activation, and also proffer alternative scenarios for Torso activation.

Developmental biology. Post-expressionist flies.

The signalling pathways underlying the development of multicellular organisms have come under a lot of scrutiny. For a long while, the focus was on the regulation of genes involved in these pathways. But a recent meeting saw a shift towards looking at how proteins are regulated and localized.

The 30th anniversary Crete fly meeting: from individual molecules to 12 genomes, and beyond.

The 16th biennial meeting of the EMBO-sponsored International Workshop on the Molecular and Developmental Biology of Drosophila was held in Kolymbari, Crete from June 23 to June 28, 2008. As in previous years, the weather was hot, the Mediterranean Sea was clear and blue and the presentations were exciting and illuminating.