Molecular Genetic Techniques for the Proteoglycan Functions in Drosophila.

Several classes of heparan sulfate proteoglycan (HSPG) core proteins and all HS biosynthetic/modifying enzymes are evolutionarily conserved from human to Drosophila melanogaster. This genetically tractable model offers highly sophisticated techniques to manipulate gene function in a spatially and temporally controlled manner. Thus, Drosophila genetics has been a powerful system to explore functions of HSPGs in vivo. In this chapter, we will introduce three genetic techniques available in Drosophila: TARGET (temporal and regional gene expression targeting), MARCM (mosaic analysis with a repressible cell marker), and FLP-Out.

Generation of Drosophila Heparan Sulfate Mutant Cell Lines from Existing Fly Strains.

Genetic studies using a model organism, Drosophila melanogaster, have been contributing to elucidating the in vivo functions of heparan sulfate proteoglycans (HSPGs). On the other hand, biochemical analysis of Drosophila glycosaminoglycans (GAGs) has been limited, mainly due to the insufficient amount of the material obtained from the animal. Recently, a novel in vitro system has been developed by establishing mutant cell lines for heparan sulfate (HS)-modifying enzyme genes. Metabolic radiolabeling of GAGs allows us to assess uncharacterized features of Drosophila GAGs and the effects of the mutations on HS structures and function. The novel in vitro system will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

Drosophila MOV10 regulates the termination of midgut regeneration.

The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. The Drosophila midgut intestinal stem cell (ISC) offers an excellent model system to study the molecular basis for stem cell inactivation. Here, we show that a novel gene, CG6967 or dMOV10, is induced at the termination stage of midgut regeneration, and shows an inhibitory effect on ISC proliferation. dMOV10 encodes a putative component of the microRNA (miRNA) gene silencing complex (miRISC). Our data, along with previous studies on the mammalian MOV10, suggest that dMOV10 is not a core member of miRISC, but modulates miRISC activity as an additional component. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including Daughter against Dpp (Dad), a known inhibitor of BMP/TGF-ß signaling. We show that RNAi knockdown of Dad significantly impaired ISC division during regeneration. We also identified six miRNAs that are induced at the termination stage and their potential target transcripts. One of these miRNAs, mir-1, is required for proper termination of ISC division at the end of regeneration. We propose that miRNA-mediated gene regulation contributes to the precise control of Drosophila midgut regeneration.

Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila.

Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. In this study, using a recently developed glycoproteomic method, we found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Here we show that Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. The wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNA interference knockdown and mutant clone analyses showed that loss of wdp leads to the up-regulation of Hh signaling. Altogether, our study demonstrates a novel role of CSPGs in regulating Hh signaling.