Investigations of the role of the Pipe sulfotransferase in the establishment of Drosophila embryonic dorsal-ventral polarity

textThe Drosophila dorsal group gene pipe provides the crucial link that transmits dorsal-ventral (DV) polarity information from the ovary to the embryo. Females homozygous for mutations in pipe produce dorsalized embryos. pipe encodes ten protein isoforms with amino acid sequence similarity to vertebrate glycosaminoglycan 2-O-sulfotransferases, suggesting that Pipe functions by modifying a carbohydrate-bearing molecule that controls embryonic DV patterning. Two major components of my project have been to examine the functional specificities of different Pipe isoforms and to identify Pipe's enzymatic substrate and learn how it participates in DV pattern formation. I have used two approaches to investigate whether the various Pipe isoforms share the same functional specificities. In one approach, I expressed each isoform in the follicle cells and found that the expression of only one of them was able to rescue the pipe mutant phenotype or ventralize progeny embryos. In a second set of transgenic studies, three of the other isoforms were individually shown to restore the production of a pipe-dependent sulfated epitope when expressed in the salivary glands of otherwise pipe null mutant embryos. These data indicate that distinct functional specificities are associated with the various Pipe protein isoforms. In addition, these studies allowed me to determine that embryos from females lacking endogenous pipe expression nevertheless retain polarity along their dorsal-ventral axis, suggesting the existence of a second polarizing signal in addition to the ventral transcription of pipe. To identify Pipe’s substrate, I developed a technique for metabolic labeling which enabled me to identify a molecule exhibiting Pipe-dependent sulfation. This molecule was identified as the protein Vitelline Membrane-Like (VML), a putative component of the vitelline membrane layer of the eggshell. The involvement of VML in dorsalventral patterning was demonstrated on the basis of the enhancing effects of a vml mutation on the severity of dorsalization of embryos from females of a sensitized genetic background. Thus, VML represents a bona fide substrate of Pipe that participates in the establishment of dorsal-ventral polarity. In these studies I was also able to show Pipedependent sulfation of other vitelline membrane components which may also influence embryonic dorsal-ventral patterning.Cellular and Molecular Biolog