Leslie M. Stevens

Spatially Restricted Expression of pipe in the Drosophila Egg Chamber Defines Embryonic Dorsal–Ventral Polarity

Expression of pipe in the somatic tissue of the Drosophila ovary is required for the formation of embryonic dorsal-ventral polarity. pipe, which encodes an enzyme similar to the glycosaminoglycan-modifying enzyme heparan sulfate 2-O-sulfotransferase, is expressed in a spatially restricted domain of follicle cells on the ventral side of the egg chamber. Mutations that affect follicular polarity correspondingly alter the spatial pattern of pipe expression. Directed expression of pipe in otherwise pipe mutant females restores embryonic lateral and ventral pattern elements and can orient the dorsal-ventral axis of the embryo. Thus, the localized expression of pipe and the spatially restricted modification of carbohydrate chains play pivotal roles in the mechanisms that establish embryonic pattern and integrate follicular and embryonic polarity.

Drosophila dMyc is required for ovary cell growth and endoreplication.

Although the Myc oncogene has long been known to play a role in many human cancers, the mechanisms that mediate its effects in both normal cells and cancer cells are not fully understood. We have initiated a genetic analysis of the Drosophila homolog of the Myc oncoprotein (dMyc), which is encoded by the dm locus. We carried out mosaic analysis to elucidate the functions of dMyc in the germline and somatic cells of the ovary during oogenesis, a process that involves cell proliferation, differentiation and growth. Germline and somatic follicle cells mutant for dm exhibit a profound decrease in their ability to grow and to carry out endoreplication, a modified cell cycle in which DNA replication occurs in the absence of cell division. In contrast to its dramatic effects on growth and endoreplication, dMyc is dispensable for the mitotic division cycles of both germline and somatic components of the ovary. Surprisingly, despite their impaired ability to endoreplicate, dm mutant follicle cells appeared to carry out chorion gene amplification normally. Furthermore, in germline cysts in which the dm mutant cells comprised only a subset of the 16-cell cluster, we observed strictly cell-autonomous growth defects. However, in cases in which the entire germline cyst or the whole follicular epithelium was mutant for dm, the growth of the entire follicle, including the wild-type cells, was delayed. This observation indicates the existence of a signaling mechanism that acts to coordinate the growth rates of the germline and somatic components of the follicle. In summary, dMyc plays an essential role in promoting the rapid growth that must occur in both the germline and the surrounding follicle cells for oogenesis to proceed.

Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster.

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins (FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)] are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates. Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLODm). FLODm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLODm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLODm message in D. melanogaster embryos reveals that expression of FLODm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLODm gene is localized to chromosomal region 52 B1-B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.

The Drosophila Embryonic Patterning Determinant Torsolike Is a Component of the Eggshell

The development of the head and tail regions of the Drosophila embryo is dependent upon the localized polar activation of Torso (Tor), a receptor tyrosine kinase that is uniformly distributed in the membrane of the developing embryo. Trunk (Trk), the proposed ligand for Tor, is secreted as an inactive precursor into the perivitelline fluid that lies between the embryonic membrane and the vitelline membrane (VM), the inner layer of the eggshell. The spatial regulation of Trk processing is thought to be mediated by the secreted product of the torsolike (tsl) gene, which is expressed during oogenesis by a specialized population of follicle cells present at the two ends of the oocyte. We show here that Tsl protein is specifically localized to the polar regions of the VM in laid eggs. We further demonstrate that although Tsl can associate with nonpolar regions of the VM, the activity of polar-localized Tsl is enhanced, suggesting the existence of another spatially restricted factor acting in this pathway. The incorporation of Tsl into the VM provides a mechanism for the transfer of spatial information from the follicle cells to the developing embryo. To our knowledge, Tsl represents the first example of an embryonic patterning determinant that is a component of the eggshell.

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.

Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation.

The establishment of Drosophila embryonic dorsal-ventral (DV) polarity relies on serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective cleaves and activates Snake, which processes and activates Easter, which cleaves Spätzle to form the activating ligand for the Toll receptor. Ventral restriction of ligand formation depends on the Pipe sulfotransferase, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. Pipe modifies components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane. This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space. By examining the processing of transgenic, tagged versions of the perivitelline proteins during DV patterning, we find that the proteolysis of Easter by Snake is the first Pipe-dependent step and therefore the key ventrally restricted event in the protease cascade. We also find that Snake and Easter associate together in a complex in both wild-type and pipe mutant-derived embryos. This observation suggests a mechanism in which the sulfated target of Pipe promotes a productive interaction between Snake and Easter, perhaps by facilitating conformational changes in a complex containing the two proteins.

Sulfation of Eggshell Components by Pipe Defines Dorsal-Ventral Polarity in the Drosophila Embryo

Drosophila embryonic dorsal-ventral (DV) polarity is controlled by a group of sequentially acting serine proteases located in the fluid-filled perivitelline space between the embryonic membrane and the eggshell, which generate the ligand for the Toll receptor on the ventral side of the embryo. Spatial control of the protease cascade relies on the Pipe sulfotransferase, a fly homolog of vertebrate glycosaminoglycan-modifying enzymes, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. Here we show that the vitelline membrane-like (VML) protein undergoes Pipe-dependent sulfation and, consistent with a role in conveying positional information from the egg chamber to the embryo, becomes incorporated into the eggshell at a position corresponding to the location of the follicle cells from which it was secreted. Although VML influences embryonic DV pattern in a sensitized genetic background, VML is not essential for DV axis formation, suggesting that there is redundancy in the composition of the Pipe enzymatic target. Correspondingly, we find that additional structural components of the vitelline membrane undergo Pipe-dependent sulfation. In identifying the elusive targets of Pipe, this work points to the vitelline membrane as the source of signals that generate the Drosophila DV axis.

Twin Peaks: Spitz and Argos Star in Patterning of the Drosophila Egg

EGFR signaling is Argos, a secreted protein that contains an EGF repeat but which acts as an inhibitor of the EGFR (Schweitzer et al., 1995a). Interestingly, argos expression is induced in response to EGFR activation, thereby initiating a negative feedback cycle to dampen Leslie Stevens Department of Developmental and Molecular Biology Albert Einstein College of Medicine 1300 Morris Park Avenue signaling (Golembo et al., 1996b). Bronx, New York 10461 EGFR Signaling during Oogenesis The maturation of the Drosophila oocyte requires extensive collaboration between the germline cells, which Recent reviews have highlighted the many roles played consist of the oocyte and the nurse cells, and the soduring development by the Drosophila epidermal growth matic follicle cells, which form an epithelium around the factor receptor (EGFR) (Perrimon and Perkins, 1997; cluster of germline cells (Figure 1, left). The follicle cells Schweitzer and Shilo, 1997). Now the list must be exprovide yolk to the oocyte, secrete the eggshell, and tended to include a novel patterning activity in the ovartransmit patterning information required for the developian follicular epithelium that transforms a single domain ment of the future embryo. Dorsal–ventral patterning of of EGFR signaling into twin peaks of activation that are the follicle initiates during stage 8 of oogenesis, after required for morphogenesis of the Drosophila egg shell the oocyte nucleus comes to lie at an anterior corner (Wasserman and Freeman, 1998 [this issue of Cell ]). of the oocyte (reviewed in Ray and Schüpbach, 1996). Our understanding of the regulation of EGFR activamRNA encoding Gurken, another TGFa homolog, is tion has been greatly facilitated by studies of the patassociated with the oocyte nucleus and the protein terning of the ventral ectoderm in the embryo and the is secreted at the oocyte membrane overlying the nudifferentiation of photoreceptor cell clusters in the eye. cleus (Figure 1, left; Neuman-Silberberg and SchüpIn these tissues, the major activating ligand for the EGFR bach, 1996). It is not known whether Gurken is cleaved, is Spitz, a homolog of the vertebrate EGFR ligand transas proposed for Spitz, or whether it acts as a membraneforming growth factor a (TGFa). Although Spitz is probound ligand. The EGFR is expressed uniformly in the duced as a transmembrane protein, it has been shown follicle cell layer (Sapir et al., 1998), but it is specifically to function when expressed as a secreted protein, sugactivated by Gurken in the follicle cells opposed to the gesting that it may be cleaved and act as a diffusible oocyte nucleus, resulting in the establishment of dorsal ligand (Schweitzer et al., 1995b; Golembo et al., 1996a). fate in these cells. Females carrying strong mutant alBased on the results of genetic epistasis experiments, it leles of gurken and EGFR produce eggs in which both has been proposed that the processing of Spitz requires the embryonic pattern and the eggshell are ventralized Rhomboid, a novel protein with multiple transmembrane (Schüpbach, 1987). The eggshell is characterized in the domains, which has also been implicated in EGFR sigdorsal-anterior region by the operculum, a specialized naling. However, the mechanism of Rhomboid action region that permits the larva to exit from the egg, and the dorsal appendages, two filamentous structures on has not yet been defined. Acting to shape the profile of

Maternal control of the Drosophila dorsal–ventral body axis

The pathway that generates the dorsal–ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)‐α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral‐to‐dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity.

Synthesis of the sulfate donor PAPS in either the Drosophila germline or somatic follicle cells can support embryonic dorsal-ventral axis formation

The establishment of dorsal-ventral (DV) polarity in the Drosophila embryo depends upon a localized signal that is generated in the perivitelline space of the egg through the action of a serine proteolytic cascade. Spatial regulation of this pathway is determined by the expression of the pipe gene in a subpopulation of ventral follicle cells in the developing egg chamber. The Pipe protein exhibits homology to vertebrate glycosaminoglycan sulfotransferases. In a previous study, we demonstrated that embryonic DV polarity depends upon the sulfotransferase activity of Pipe. Surprisingly, however, our results also indicated that formation of the embryonic DV axis does not require the synthesis of the high-energy sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) in the follicle cells in which Pipe is presumed to function. Here, we resolve this apparent paradox by demonstrating that dorsalized embryos are only produced by egg chambers in which both germline and follicle cells lack PAPS synthetase activity. Thus, PAPS produced either in the germline or in the follicular epithelium can support the requirement for Pipe sulfotransferase activity in embryonic DV patterning. This finding indicates the existence of a conduit for the movement of PAPS between the germline and the follicle cells, which highlights a previously unappreciated mechanism of soma/germline cooperation affecting pattern formation.