The EMBO Journal Vol.17 No.23 pp.6769–6771, 1998

EMBO WORKSHOP REPORT

Flies over Crete: Drosophila Molecular BiologyKolymbari, Crete, July 12–18, 1998

Ben-Zion Shilo

Department of Molecular Genetics, Weizmann Institute of Science,Rehovot 76100, Israel

e-mail: lvshilo@weizmann.weizmann.ac.il

Introduction

For the past 20 years, leading scientists studying a broadrange of aspects ofDrosophilagenetics and developmentmeet every two years at the remote and magical spot ofKolymbari, off the north-west coast of Crete. Thesemeetings have always been ‘trend setters’ in the field dueto the intense discussions and open exchange of ideas,prompted by the sheer beauty and isolation of the placeand the informal atmosphere it fosters. The workshop isunique amongDrosophila meetings in that it covers abroad range of topics and yet maintains a small forum. Ithas been interesting to follow the change in emphasis inthese meetings over the years, from discussions on heat-shock response to gene cloning and expression, and finallytowards a more comprehensive biological analysis ofgenes and pathways determining embryonic and post-embryonic development. A clear trend can be observed,from more descriptive molecular studies towards thebiological understanding of the context in which individualgenes and entire pathways function. It is therefore alwaysexciting to identify new themes that come up in thesemeetings and are likely to shape future work in the field.

It is impossible to provide a comprehensive review of ameeting which included 124 talks and covers an extremelywide range of topics in developmental and cell biology,neurogenesis, organogenesis, gene expression and chromo-some structure. This review will therefore highlight onlysome of the exciting themes of the meeting; those per-taining to signaling, transcription and technicalinnovations.

Broad inhibition of signaling

A central motif emerging from the recent meeting wasthe description of a plethora of mechanisms which inhibitmorphogenetic signaling pathways. Past efforts were con-cerned with the capacity of diffusible morphogenes toreach and activate their targets many cell diameters fromthe source. It now seems that a variety of mechanisms areinvolved in executing the opposite task, namely acting torestrict and reduce the overall level of signaling, makingit harder for patterning molecules to induce their respectivetargets and thus restricting the expansion of morphogengradients. This mechanism can be regarded as a constitu-tive one, attenuating signaling uniformly, and may becompared to the elevation of the water level around anisland that will determine the extent of its exposed

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pinnacles (i.e. the domains which respond to peaks ofsignaling), an appropriate comparison for a meeting heldin Crete.

Steve Cohen (EMBL, Heidelberg, Germany) describedthe role of Nubbin, a POU-domain protein expressed inthe wing pouch that restricts Notch activity and allows itto induce Wingless (Wg) expression only in the wingmargin, where Notch signaling is maximal and can over-come the repression by Nubbin. Nubbin-binding sites onNotch-target genes have been identified, so Nubbin maybe repressing the activity of Su(H) triggered by Notch.

Another broad repressive activity operating at the levelof transcription was described for theDrosophila TCF/Pangolin transcription factor, representing the nucleartarget for activation by the Wg pathway. Amy Bejsovec(Northwestern University, Evanston, IL), in collaborationwith Mark Peifer (University of North Carolina, NC),presented genetic and molecular evidence that TCF isassociated with Groucho (a universal transcriptionalrepressor, see below). As a rule, TCF is associated withGroucho in an inhibitory complex, and only in responseto high levels of Wg signaling can the activator Armadilloreplace or overcome the Groucho effect, and TCF caninduce its target genes. Another component involved inTCF repression was described by Mariann Bienz (MRC,Cambridge, UK). TheDrosophila CBP/p300 protein(which was previously identified as an activator of tran-scription with histone acetylase activity) associates withTCF and functions, in this context, as a repressor. Inmutants for the locus (nejire), expression of theUbxmidgut enhancer expands on both sides of the Wg source.Acetylation of TCF by CBP at the N-terminal domain(which is responsible for Armadillo binding) was demon-strated, suggesting that CBP activity reduces Armadillo-binding to TCF. CBP may be involved in transcriptionalrepression in other contexts, as Hiroshi Akimaru (TsukubaLife Science Center, Japan) described a requirement forthe protein to assist Dorsal in repressing genes likezen,in the ventral and lateral parts of the embryo. Anotherpossible chromatin associated protein is Eyelid (JessicaTreisman, NYU, NY), which appears to antagonize Wing-less signaling.

The champion general repressor of transcription isclearly Groucho, originally identified as a member of theNotch pathway and previously described to be involvedin segmentation, sex determination, neurogenesis andterminal patterning. Additional facets of Groucho activitywere presented. Al Courey (UCLA, Los Angeles, CA)described a requirement for Groucho in converting theDorsal activator into a repressor of target genes. As Dorsalis a transcriptional activator of several promoters, simplebinding of Groucho to Dorsal can not sufficiently explainrepression specificity. It is probable that in promotersundergoing repression by Dorsal, additional DNA-binding

B.-Z.Shilo

factors which are also in contact with Groucho providethe right context for negating gene expression. At thetermini of the early embryo, Groucho functions at severalsuccessive steps. First, it uniformly represses expressionof genes liketailless and huckebein, and this repressionis relieved only at the termini where high levels of Torsosignaling take place. Subsequently, Groucho associateswith Huckebein, to repress the transcription ofsnail at thetermini (Ze’ev Paroush, Hebrew University, Jerusalem).Additional putative transcriptional repressors weredescribed by Susan Parkhurst, Hutchison Cancer Center,Seattle, WA). Using the Hairy protein, which played amajor role in the original identification of Groucho’srepressive properties, to identify associated proteins, aDrosophila homolog of a vertebrate tumor suppressorgene that interacts with Adenovirus E1a protein (CtBP)and a RING finger protein with similarity to BRCA1 wereidentified.

Restricted inhibition

The above mechanisms represent ubiquitous repressivesystems. Another class of inhibitors involves elementsthat carry spatial or temporal information, and thus shapemorphogen gradients in an instructive manner. Brinkerrepresents a novel protein which has antagonistic inter-actions with the Dpp pathway (Siegfried Roth, Max PlanckInstitute, Tubingen, Germany, and Chris Rushlow, NYU,NY). Brinker is expressed in a complementary pattern toDpp at several stages, including the early embryo, wingdisc and ovary. This pattern is maintained by mutualantagonism in the expression of the two genes, thussharpening the borders of Dpp activity. The questions ofwhere and how Brinker intersects with the Dpp pathwayremain open.

Activation of the EGF receptor pathway in neighboringcells is used to recruit these cells to follow the samedevelopmental pathway as the founder cells. Two pat-terning inhibitors associated with this pathway weredescribed. In the R8 eye photoreceptor cell and theembryonic chordotonal organs, cells expressing Atonalprovide a source for activation of the EGF receptorpathway in neighboring cells, culminating in the inductionof the ETS domain transcription factor Pointed P2. It isnecessary to block the activity of Pointed P2 in thesesource cells, and this is achieved by expression of aprotein termed Edl in the same cells (Yasushi Hiromi,National Institute of Genetics, Mishima, Japan). Edl actsas an antagonist of Pointed P2; it contains a ‘Pointeddomain’ found in Pointed P2, but does not includethe ETS DNA-binding domain. Edl binds Pointed P2,preventing its activity as a transcriptional activator. Itseems that Pointed P2 carries out two opposing activities,i.e. repressing the expression ofrhomboid (which isessential for triggering the EGF receptor pathway) andinducing response genes. By inhibiting Pointed P2, Edlthus increasesrhomboid expression and represses theinduction of Pointed target genes in the Atonal-expressing cells.

Another inhibitor of the same pathway is Argos, asecreted protein with an EGF domain, acting in manytissues. In the ovary, the EGF receptor, expressed in thefollicle cells, is activated in a broad region in the dorsal

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anterior corner of the egg chamber lying above the oocytenucleus. In contrast,argosis induced by the pathway onlyin a narrow stripe immediately above the nucleus, wheremaximal levels of receptor activation take place. As aresult of inducing the inhibitor Argos, signaling is reducedin this central region. This splits the single peak of EGFRsignaling into two peaks, which specify the position of thedorsal appendages (Matthew Freeman, MRC, Cambridge,UK). In this case Argos inhibits signaling close to itssource; in other instances, such as the eye or embryonicectoderm, where Argos and an activating ligand of thepathway (Spitz) emanate from the same focus, Argosfunctions to reduce overall signaling, thus affecting primar-ily cells that are distant from the signaling source.

Novel signaling components

While no novel signaling pathways were described inthe meeting, several new components and regulatoryinteractions in the known pathways were reported. GerryRubin (University of California at Berkeley, Berkeley,CA) presented two new components in receptor tyrosinekinase signaling, identified by suppressor screens in theeye. PTP-ER is a protein tyrosine phosphatase that dephos-phorylates MAP kinase, and appears to be negativelyregulated by MAP kinase phosphorylation. Another com-ponent is Cnk, an adaptor protein that associates with Raf,and may target it to cell–cell contact regions.

No Drosophila meeting is complete without detaileddiscussion of the Notch signaling pathway. Kuzbanian(Kuz), a metalloprotease, was previously shown to be anessential component in Notch signaling. It now appearsthat a cardinal molecule modified by Kuz is Delta (Dl), aNotch ligand (Spyros Artavanis-Tsakonas, Yale University,CT), in collaboration with Tian Xu (Yale University, CT).Genetic interactions point to a more direct interactionbetween Kuz and Dl rather than between Kuz and Notch.The appearance of a cleaved extracellular Dl fragment inS2 cells was noted, and shown to be absent followinginduction of a dominant-negative Kuz or addition ofmetalloprotease inhibitors. This fragment, which can bindNotch-expressing cells, is also missing inkuz mutantembryos. These observations may change our view of theNotch pathway, in the sense that diffusible Dl signalscould be capable of affecting not only the cells immediatelyadjacent to the Dl-expressing cells.

Notch not only directs individual cell fates, but also incollaboration with other genes can influence implementa-tion of entire developmental programs leading to theformation of appendages (Walter Gehring, Biozentrum,Basel, Switzerland). Normally, activation of Notch in theeye imaginal disc is responsible for induction of the geneseyelessand twin of eyeless, which are at the top of thehierarchy of eye development, as demonstrated by theinduction of ectopic eyes following their misexpression.Combinations of activated Notch with other proteins caninduce different appendages. For example, in associationwith Antennapedia, ectopic wings and legs are inducedon the head.

It was assumed that the major molecules transducingthe signals from activated receptor tyrosine kinases hadbeen identified, until the work of Markus Affolter (Biozen-trum, Basel, Switzerland, in collaboration with Maria

Drosophila molecular biology

Leptin, Koln University, Germany) presented a new sur-prise. Thedof gene (downstream of FGF) is essential forsignaling by bothDrosophila FGF receptors, Breathlessand Heartless. It encodes a novel adaptor protein that maybe phosphorylated by the receptor and serve as a dockingsite for Grb2 and other proteins. Dof is expressed only inthe tissues where activity of FGF receptors is required,i.e. trachea and mesoderm. The fact that Dof is absolutelynecessary and specific for FGF receptor signaling mayprovide another tier of regulation for restricting or localiz-ing the activities of different receptor tyrosine kinases thatare activated in overlapping cell populations.

While all receptor tyrosine kinases studied so far inDrosophila were dedicated to morphogenesis or celldivision, Ernst Hafen (Zurich University, Switzerland)described a new function for theDrosophila insulinreceptor cascade. Mutations in the insulin receptor or inthe downstream component IRS1, result in smaller flieshaving smaller cells which are normally patterned (hencethe IRS1 locus is termedchico). This pathway may thusadjust cell size after completion of the morphogeneticprocesses, according to the level and availability of meta-bolic sources, which could induce different levels of thestill unknown activating ligand(s).

Morphogenetic cell movements

New approaches to study how activation of signalingpathways is translated to morphogenetic cell movementshave been described. In the cluster of 16 germ cells, thefuture oocyte is always located at the posterior pole ofthe egg chamber, raising the issue of the mechanismresponsible for this localization, which in turn will allowthe oocyte to be in contact with the posterior follicle cellsand trigger activation of the EGF receptor. It turns outthat the follicle cells on the posterior side of the germlinecyst transiently upregulate the expression of the adhesionmolecule E-Cadherin. In parallel and independently, thecell which subsequently will become the oocyte alsoincreases its Cadherin-dependent adhesiveness. Homo-philic interactions between the two cell layers, thus posi-tion the future oocyte at the posterior end of the cyst(Daniel St Johnston, Wellcome/CRC, Cambridge, UK).

The role of Hedgehog signaling in controlling differen-tial cell affinities was presented by Peter Lawrence (MRC,Cambridge, UK) and Seth Blair (University of Wisconsin,Madison, WI). Marked clones of wild-type cells in seg-ments or adult appendages are ‘happy’, as reflected bytheir capacity to mix with neighboring cells and formwiggly borders. Mutant clones that do not resemble theirneighbors in terms of reception of the Hedgehog signal(for example smoothened-mutant cells just anterior tothe A/P compartment boundary, which lack Hedgehogreception in a region of high Hedgehog signaling) are‘unhappy’, forming a tight cluster with their kin, andgenerate straight and minimal borders with their neighbors.Such cells react by moving away from the A/P boundaryregion, either anteriorly, where there is less receptionof Hedgehog signal (abdomen), or into the posteriorcompartment, where Hedgehog reception is absent (wing).This illustrates the potential of a morphogen to changecell affinities and subsequent patterning.

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Innovative techniques

Many of the quantum jumps in theDrosophila field arebased on innovative techniques. It is impossible to imaginewhat the field would look like today in the absence of Pelements or the Gal4/UAS inductive system. Pernille Rorth(EMBL, Heidelberg, Germany) presented a modificationof the UAS vectors utilizing the promoter of the Ptransposase, which now allows them to be induced in thegermline, thus opening the door to misexpression in thistissue which was previously refractive to it. In manyinstances it is desirable to activate Gal4 and its targetgenes only in a restricted population of cells, in order tocarry out lineage tracing, induce cell death or otherprocesses. Jon Minden (Carnegie Mellon University, Pitts-burgh, PA) described the injection of caged Gal4-VP16protein into early embryos. Local UV activation of theprotein can induce targeted gene expression in a smallpopulation of cells, for example the dorsal head cellsgiving rise to the optic lobe. Norbert Perrimon (Harvard,Boston, MA) presented the development of a temperature-sensitive version of Intein, a protein catalyzing its ownprotein splicing. By inserting this version of the Inteinwithin the coding region of transgenes, it may be possibleto inactivate or activate Intein splicing by temperatureshifts, and hence the activity of these proteins. This maycreate a temperature-sensitive allele for any given gene.Finally, Andrea Brand (Wellcome/CRC, Cambridge, UK)and Carl Thummel (University of Utah, UT) presentedthe use of GFP to follow planes of cell division in theembryo or metamorphosis in living pupae, respectively.

The progress in theDrosophila genome sequencingproject and in the annotation of accumulating data is likelyto change many of our experimental approaches, byallowing rapid cloning of genes or identification ofmutations, and more global examination of gene families.In two years, at the next Crete meeting, the genomesequencing should be nearing completion (Gerry Rubin,University of California at Berkeley, Berkeley, CA), geneknockouts for a majority of genes will have been generated(Alan Spradling, Carnegie Institute, Baltimore, MD) andcDNA probe arrays to monitor differential gene expression(David Hogness, Stanford, CA) will be widely utilized.With more powerful tools to address old problems suchas cell polarity, cell migration and establishment of celland tissue identity, we should be able not only to accumu-late much more data, but also to obtain a deeper under-standing of these central and still enigmatic processes.