The NOTCH receptor and its ligands

In 1970, T. Wright wrote the first review o n NOTCH arid started thus: 'If one was asked to choose the single, m o s t important genetic variation.., during embryogenesis in Drosophila melanogaster, the answer would have to be the Notch locus '1. Today, this statement m i g h t be regarded as a hyperbole, but it illustrates the degree to which Notch (N) has intrigued developmental biologists and geneticists over the years. Studies of this gene were confined to arduously complicated and esoteric genetics until Notch was s h o w n to encode a surface receptor with an extracellular d o m a i n carrying epidermal growth factor (EGF)-like repeats z. NOTCH-like molecules have n o w been identified from Caenorhabditis elegans to humans, and they play imporl~ant, and apparently conserved, functional roles in development. Several reviews on the subject have appeared in the past few years3,4; therefore, our intention here is to discuss the NOTCH signalling p a t h w a y briefly and instead emphasize recent developments on the biochemistry of the receptor as well as Ehe action of the ligands. The developmental role of Notch Notch was classified originally as a neurogenic gene since m u t a n t embryos displayed abnormalities of neural versus epidermal cell lineages s,6. However, even the first characterization of tile m u t a n t phenotypes made it clear that Notch affected a vast spectrum of tissues, n o t just neurogenesis 7. In fact, hardly any tissue in Drosophila is unaffected by Notch mutations. Wright, in his review, was puzzled by the fact that the gene manifests itself by so m a n y distinct m u t a n t phenotypes, posing a challenge to identify a unitary function of NOTCH. Accumulated molecular and genetic studies of the past decade from C. elegans to humans have documented the very broad role NOTCH signalling plays in development..Activation of the NOTCH pathway is utilized throughout development to modulate the ability of non-terminally differentiated cells to acquire or maintain a particular cell fate. Cell-interaction events resulting in the segregation of developmentally equivalent precursors into distir:ct cell types are often formally distinguished as either lateral inhibition/specification or inductive signalling. NOTCH has been s h o w n to affect b o t h these types of transition. Activation of the receptor has been described in developmental terms to define a 'permissive' m e c h a n i s m and, in certain cases, an 'instructive' role. BLOTCH also affects differentiation events and can profoundly affect proliferation. The developmental consequences of NOTCH signalling in a distinct cell population depend on the developmental history of those cells. Since the phenotypes of NOTCH malfunction are context dependent, it is difficult to describe the developmental action of NOTCH in an all-encompassing manner, even t h o u g h the underlying biochemical mechanism m a y be the same. A general characterization of the developmental role of NOTCH emerged from studies with truncated, constitutively actiwtted forms of the receptor in several species 8-~1. Activation of NOTCH in non-terminally differentiated cells renders t h e m recalcitrant to differentiation cues. Once NOTCH signalling activity ha:; subsided, however, the cells can differentiate or trends in CELL BIOLOGY (Vol. 7) November 1997

The NOTCH receptor and its ligands

An intricate interplay of signalling pathways dictates the acquisition of specific cell fates during development. The NOTCH receptor is the central element in a cell-interaction mechanism that controls the fate of a very broad spectrum of precursor cells. Conservation across species implies that signalling through this receptor is a tool frequently used by metazoans to modulate the fate of precursor cells. This article describes recent advances in the genetic and molecular dissection of this developmentally fundamental pathway that have provided new insights into the mechanism by which extracellular signals act through the NOTCH receptor to determine or alter cellular fate.

respond to later developmental signals. This has led to the n o t i o n that NOTCH controls the ability of precursor cells to progress to the next differentiated state. Such a role could explain m a n y of the k n o w n m u t a n t phenotypes and offers a plausible explanation of w h y Notch is pleiotropic: activation of the NOTCH receptor could arrest cells at their current state of differentiation. The elements of the NOTCH pathway Our current understanding of NOTCH signalling has come primarily from genetic analyses. Two ligands, DELTA (DL) and SERRATE (SER), bind to a subset of the 36 EGF-like repeats present in the extracellular domain of NOTCH. lntracellularly, NOTCH contains six t a n d e m ankyrin-like repeats, which bind to DELTEX (DX), a positive regulator of NOTCH signalling (Fig. 1). In addition to its ability to bind to NOTCH, DX is also capable of h o m o t y p i c interactions lz. This cytoplasmic protein contains a functional Src-homology 3 (SH3)-binding domain, and mutations in dx display a multitude of genetic interactions with both gain-offunction and loss-of-function mutations in Notch 13. A second downstream effector of NOTCH signalling is SUPPRESSOR OF HAIRLESS [SU(H)]. This protein is a transcription factor that can also bind to the intracellular d o m a i n of the NOTCH receptor. Experiments involving cultured cells have shown that SU(H) is sequestered in the cytoplasm w h e n coexpressed with NOTCH and is translocated to the nucleus when NOTCH binds to its ligand DL. Moreover, in the same

Copyright © 1997 ElsevierScienceLtd. All rights reserved.0962-8924/97/$17.00 PIl: s0962-8924(97)01161-6

Robert Fleming is in the Dept of Biology, The University of Rochester, Hutchinson HallRiver Campus, Rochester, NY 14627, USA; and Karen Purcell and Spyros Artavanis-Tsakonas are at the Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, Dept of Cell Biology, Yale University, New Haven, CT 06536-0812, USA. 437

cell-culture system, the binding of NOTCH to DL resuits in the recruitment of DX to the NOTCH-DL complexes and in the nuclear translocation of SU(H), indicating a potential interplay between NOTCH, DX and SU(H) 1:'~.These in vitro studies obviously suggest that the ligand-dependent translocation of SU(H) is part of NOTCH signalling, but in vivo studies have not yet confirmed such a model, and immunocytochemical analysis of NOTCH and SU(H) during bristle differentiation, a NOTCH-dependent event, did not show colocalization of the two proteins 14. The final pathway component, the Enhancer of split [E(spl)] gene complex, encodes several basic helixloop-helix (bHLH) proteins. In vivo and in vitro studies in Drosophila and mammals have identified the promoter of this complex as a site of SU(H) binding that is requisite for NOTCH signalling ~s,16. In general, bHLH proteins are capable of forming heterodimers, each of which has distinct specificities ~7. A system that controls the activity of a variety of bHLH genes would have a pleiotropic action and could explain how NOTCH signalling results in the control of downstream targets that influence such a diverse set of undifferentiated cells. It is not known whether direct NOTCH targets other than the bHLH genes exist, but several new lines of evidence indicate the presence of such Su(H)-independent NOTCH signalling events ~8,19.

Proteolytic cleavage of NOTCH Until recently, models for signal transduction via the NOTCH pathway have depicted the receptor as a full-length molecule on the cell surface. However, biochemical analyses have now shown that NOTCH is synthesized in the endoplasmic reticulum and is then cleaved in the trans Golgi network (TGN) 2°. This is an evolutionarily conserved step that takes place in Drosophila, human and rat tissues. Proteolysis results i n a C-terminal transmembrane fragment and an N-terminal fragment containing most of the extracellular domain. These fragments are tethered together on the plasma membrane, forming a heterodimeric receptor (Fig. 1). As only heterodimeric NOTCH is present at the plasma membrane, this is the probably the form to which the ligands bind. Supporting this notion, inhibition of cleavage by null mutations or a d o m i n a n t negative form of the ADAM meta110protease, KUZBANIAN,alters NOTCH signallingZk Significantly, kuzbanian loss-of-function mutations resemble Notch loss-of-function phenotypes 21,22. Further experiments are needed to determine whether other proteases are involved in the cleavage of NOTCH. Demonstrating that this cleavage site is functionally unique will be important, as smaller proteolytic products exist across species. A series of observations over the past decade has raised the provocative hypothesis that NOTCH signalling may depend on cleavage of the NOTCH intracellular domain 8,~°,23-zs. Truncated NOTCH proteins without transrnembrane and extracelhflar domains are nuclear and constitutively activate the pathway, as measured by upregulation of downstream genes ls,26. At present, there are no data showing that wild-type Drosophila NOTCH is ever found in the nucleus 27,2s. Vertebrate NOTCH I 438

immunoreactivity has been detected in the nuclei of h u m a n cervical tissues and rat retina z9,3°, but t h e biochemical nature of these nuclear antigens and their functional significance, if any, are also unclear. Therefore the intriguing issue of nuclear NOTCH remains open for further analysis.

NOTCH ligands Two NOTCH ligands, DL and SER, have been identified, which are at least partial functional homologues. Homozygous loss-of-function mutations i n Dl show neuronal hypertrophy (neurogenic phenotype) similar to Notch null mutations and dosagesensitive interactions with Notch 31-33.Loss-of-function mutations in Ser do not have a neurogenic phenotype, but dominant mutations mimic the haploinsufficient N/+ phenotype characterized by loss of adult wing-margin tissue. SER and DL proteins have a relatively short intracellular sequence and a single membrane-spanning domain. Their extracellular sequences contain EGF-like repeats and N-terminal regions with significant homology to each other, termed DSL domains 34 (Fig. 1). In conjunction with other N-terminal sequences, the DSL domain forms the extracellular binding domain (EBD) of DL and SER3s. This domain appears to be both necessary and sufficient to bind to a comm o n region of the extracellular domain of NOTCH. Two distinct ligand types have also been identified in C. elegans and in vertebrates. One of the h u m a n SERRATE-like genes, ]AGGED-1 (Ref. 36), recently has b e e n linked to an autosomal dominant pleiotropic disorder known as Alagille syndrome 37,38. All known NOTCH ligands contain a DSL domain, but the number of EGF repeats varies widely from o n e ligand type to another. The repeat number does not appear crucial for receptor activation. For example, Drosophila DL and SER have 9 and 14 repeats, respectively, whereas the vertebrate DL homologues contain only eight EGF-like repeats and the SER homologues each have 16 EGF-like repeats. The intracellular domains (ICs) of SER and DL have no apparent homology with one another, and the Drosophila ligands do not share significant IC homology with their vertebrate counterparts. Experiments in which the C-terminal portions of Drosophila or vertebrate ligands were deleted showed that membrane-bound and secreted forms of the ligands can antagonize NOTCH signalling 26,39-41. All of these d o m i n a n t negative mutants retained the EBD region. While the mutations giving rise to Alagille syndrome have not been shown to reflect dominant-negative forms of JAGGED-I, their sequences predict truncated secreted forms of the h u m a n ligand that contain the EBD region. The phenotypes of all these mutations could reflect simple unproductive binding of the mutant form to NOTCH or perhaps homotypic interactions with wild-type ligand forming inactive complexes. Interestingly, the ligand IC domain does not appear to be necessary for signalling in C. elegans. The functionally interchangeable apx-1 and lag-2 ligands lacking the transmembrane and IC domains are able to substitute for endogenous lag-2 activity4z. Why the C. elegans ligands behave differently compared with trends in CELL BIOLOGY (Vol. 7) November 1997

NOTCH

other NOTCH family ligands remains unclear. Given the primary sequence differences between the C. elegans ligands and those of other species, the observed difference might simply be clue to such deletions changing the stmctn2re and function of the EBD region in Drosophila and vertebrates, but not in C. elegans.

trer~ds in CELL BIOLOGY (Vol. 7) November 1997

•

Ligand-binding EGF repeats

•

Ankyrin-likerepeatsinteract with DX and Su(H)

Liganc DELTJ SERF

Ligands: DELTA and SERRATE

The action of DELTA and SERRATE

Ill the past few years, important :insights into the mechanism of action of the two ligands have been obtained by studying wing blade development in Drosophila. Even though DL and SER can function equivalently during Drosophila neurogenesis, they have distinct roles during wing formation. The wing originates as an epithelium known as an imaginal dis,: (Fig. 2). The dorsal (D) and ventral (V) compartments of this tissue are divided by the presumptive wing margin, which acts as an organizing centre to control cell proliferation and patterning 43~s. Activation of NOTCH along this D-V boundary triggers expression of wingless (wg) and vestigial (vx). WG, synergistically with NOTCH actiwltion, then controis the expression of the genes of the achaete-scute complex and might act to reinforce expression of cut a wing-margin-specific gene. F.oth DL and SER are required for proper wing development, but they show compartmentally restricted abilities to activate NOTCH 44-47. SER is required only along the dorsal side of the presumptive wing margin and acts as a dorsal-to-ventral signal through NOTCH. By contrast, DL activity is required in the ventral marginal zone and acts as the ventral-to-dorsal signal. Ectopic DL expression causes cell proliferation in 30th the dorsal and ventral wing compartments but: induces significant levels of margin-specific gene expression only in the dorsal side 48,49. Similar experiments with ectopic SER expression reveal an even more restricted response; SER does not activate NOTCH in the dorsal wing but causes cell proliferation and margin gene expression in ventral regions. Since ligand-independent activated forms of NOTCH are sufficient to control both cell proliferation and margin-gene induction on both sides of the D-V boundary, the compartmentally restricted actions of SER and DL suggest: that they have access to NOTCH oniy in one compartment. [low the two ligands acquire differential access to NOTCH can be explained by a third player, FRINGE (FNG) s°. Two recent studies demonstrate that FNG has a bifunctional role in the regulation of NOTCHligand interactions ~8,49. One role is to inhibit SERNOTCH interactions. FNG is normally found throughout the dorsal compartment where ectopic SER expression has little detectable effect. However, by reducing endogenous FNG levels, cell proliferation can be induced by SER expression in the dorsal compartment. Likewise, SEE activity can be blocked by ectopic FNG expression in the ventral wing compartment or when SER is expressed ectopically during neurogenesis. This inhibitory natTure of FNG on SER-NOTCH interactions is specific, as the second role of FNG appears to facilitate signalling by DL. Ectopic DL expression on]y induces margin-specific gene expression in the presence of FNG (Ref. 48; R. Fleming, unpublished).

Receptor: NOTCH g EGF repeats in receptor

•

EGF-repeats in ligand

I

NOTCH-binding domain of ligand

Positive regulators of pathway:

Su(H): bindsto the intracellular region of NOTCH DX:

interacts homotypically and with the intracellular region of NOTCH

FIGURE 1 The NOTCH signalling pathway. The NOTCH receptor has two ligands, DELTA and SERRATE, which contain epidermal growth factor (EGF)-Iike repeats and homologous N-terminal domains (DSLs) known to bind to the same region of the receptor. The NOTCH protein is a heterodimer, consisting of an extracellular fragment with 36 EGF-like repeats and an intracellular fragment containing six ankyrin-like repeats that are involved in DELTEX (DX) and SUPPRESSOROF HAIRLESS[SU(H)] interactions. DX and SU(H) are two positive regulators of NOTCH signalling, with SU(H) activating transcription of the basic helix-loop-helix ENHANCER OF SPLIT [E(SPL)] proteins.

FIGURE 2 Interplay between NOTCH and WINGLESS signalling: a secreted, dominant-negative form of SERRATE,which is expressed in a patched pattern (blue), downregulates the expression of wing-margin genes, including wingless (red), in a third instar Drosophila wing imaginal disc and interferes with morphogenesis, creating an ectopic constriction along the anterior-posterior boundary of the disc41. 439

FNG is a secreted protein with homology to Lexl biosynthetic enzymes, suggesting it may function as a galactosyltransferase to modify carbohydrate moieties sl. However, since no biochemical function has as yet been demonstrated for FNG, its mode of action remains a matter of conjecture. We do know, however, that the effects of FNG on NOTCH-ligand interactions occur at the protein level, affecting the receiving (NOTCH-expressing) rather than the sending (ligand-expressing) celP 9,48,49. Replacement of the SER EBD with the corresponding DL EBD generates a chimeric molecule that is capable of activating NOTCH in the presence of FNG (i.e. becomes like DL). Since the EBD region of these ligands can bind to the NOTCH receptor, it appears likely that FNG alters the interaction properties of NOTCH with SER and DL. These interaction mechanisms appear to have been conserved in vertebrate development. Recent findings show that the vertebrate homologues of Notch, Serrate and fringe are expressed in similar positions along the D-V boundary during formation of the chick limb bud sz,s3. Future questions

Many questions remain regarding the action of NOTCH ligands in development. The difference in the molecular action of the ligands in embryonic versus postembryonic development is of particular interest. For instance, DL appears to require FNG to activate NOTCH in the wing, yet FNG is not expressed during embryonic neurogenesis, when DL is known to activate NOTCH (K. D. Irvine, pers. commun.). It is also unclear whether the differential action of the two ligands in proliferation versus wing-margin-specific gene induction discussed earlier reflects qualitative or quantitative differences involving additional partners on the surface or distinct downstream effectors. There is much we still do not understand regarding the interaction between NOTCH and its ligands, especially how they influence the expression and therefore the activity of NOTCH pathway elements. There are several lines of evidence that suggest the existence of feedback mechanisms affecting NOTCH signalling between NOTCH- and ligand-expressing cells, although little is understood about the nature of these mechanisms. Reciprocal expression changes in receptor- and ligand-expressing cells have been documented in both Drosophila and C. elegans 4s,s4,ss. Expression of specific intracellular NOTCH fragments in a particular cell has been reported to result in nonautonomous downregulation of NOTCH signalling in a neighbour 19. In addition, recent studies suggest that NOTCH signalling can be autonomously downregulated in cells expressing high levels of SER and/ or DLs6,sT. Finally, genetic studies involving suppressors and enhancers of phenotypes associated with activated forms of NOTCH have also indirectly implied the existence of feedback mechanisms sT. There is little doubt that the NOTCH pathway components identified so far do not tell the entire story. Further studies will determine whether additional ligands exist and how many more nuclear or cytoplasmic effectors respond to extracellular signals transm i r e d through NOTCH. Perhaps the most important 440

issue, however, is to gain a better insight into the biochemistry of NOTCH signalling. The molecular nature of this mechanism, which seems to be capable of controlling the ability of precursor cells from C. elegans to humans to progress from one developmental state to the next, is still largely obscure. References 1 WRIGHT,T. R. F. (1970) Adv. Genet. 15, 261-359 2 WHARTON, K. A., JOHANSEN, K. M., XU, T. and ARTAVANIS-TSAKONAS,S. (1985) Ce1143, 567-581 3 ARTAVANIS-TSAKONAS,S., MATSUNO, K. and FORTINI,M. E. (1995) Science268, 225-232 4 BLAUMUELLER,C. M. and ARTAVANIS-TSAKONAS,S. (1997) Perspec Dev. Neurobiol. 4, 325-343 5 POULSON,D. F. (1968) Proc. 12th Int. Congr.Genet. Tokyo 1,143 6 LEHMANN, R., DIETRICH,U., JIMENEZ,F. and CAMPOS-ORTEGA,J. A. (1981 ) Roux's Arch. Develop. Biol. 190, 226-229 7 WELSHONS,W. J. (1965) Science 150, 1122-1129 B REBAY,I., FEHON, R. and ARTAVANIS-TSAKONAS,S. (1993) Cell 74, 318-329 9 STRUHL,G., FITZGERALD,K. and GREENWALD, I. (1993) Cell 74, 331-345 10 LIEBER,T., KIDD, S., ALCAMO, E., CORBIN, V. and YOUNG, M. W. (1993) Genes Dev. 7, 1949-1965 11 COFFMAN,C. R., SKOGLUND, P., HARRIS,W. A. and KINTNER, C. R. (1993) Cell 73, 659-671 12 MATSUNO, K., DIEDERICH,R. J., GO, M. J., BLAUMUELLER,C. M. and ARTAVANIS-TSAKONAS,S. (1995) Development 121, 2633-2644 13 XU, T. and ARTAVANIS-TSAKONAS,S. (1990) Genetics 126, 665-677 14 GHO, M., LECOURTOIS,M., GERAUD,G., POSAKONY,J. and SCHWEISGUTH, F. (1996) Development 122, 1673-1682 15 JENNINGS,B., PREISS,A., DELIDAKIS,C. and BRAY,S. (1994) Development 120, 3537-3548 16 JARRIAULT,S., BROU,C., LOGEAT, F., SCHROETER,E. H., KOPAN, R. and ISRAEL,A. (1995) Nature 377, 355-358 17 JAN,Y. N. and JAN, L. Y. (1993) Ce1175,827-830 18 SHAWBER,C. J., BOULTER,J. and WEINMASTER,G. (1996) Dev. Biol. 180, 370-376 19 MATSUNO, K., GO, M. J., SUN, X., EASTMAN,D. S. and ARTAVANIS-TSAKONAS,S. Development (in press) 20 BLAUMUELLER,C. M., QI, H., ZAGOURAS,P. and ARTAVANIS-TSAKONAS,S. (1997) Cell 90, 281-291 21 PAN, D. and RUBIN,G. M. (1997) Cell90, 271-280 22 ROOKE,J., PAN, D., XU, T. and RUBIN,G. M. (1996) Science 273, 1227-1231 23 XU, T. (1990) PhD Thesis, Yale 24 FORTINI,M. E., REBAY,I., CARON, L. A. and ARTAVANIS-TSAKONAS,S. (1993) Nature 365, 555-557 25 KOPAN, R., NYE,J. S. and WEINTRAUB, H. (1994) Development 120, 2385-2396 26 SUN, X. and ARTAVANIS-TSAKONAS,S. (1996) Development 122, 2465-2474 27 KIDD, S., BAYLIES,M. K., GASIC,G. P. and YOUNG, M. W. (1989) Genes Dev. 3, 1113-1129 28 FEHON,R. G., JOHANSEN, K., REBAY,I. and ARTAVANIS-TSAKONAS,S. (1991 ) J. Cell Biol. 113, 657-669 29 ZAGOURAS,P., STIFANI, S., BLAUMUELLER,C. M., CARCANGIU, M. L. and ARTAVANIS-TSAKONAS,S. (1995) Proc. NatL Acad. Sci. U. S. A. 92, 6414-6418 30 AHMAD, I., ZAGOURAS,P. and ARTAVANIS-TSAKONAS,S. trends in CELL BIOLOGY (Vol. 7) November 1997

(1995) Mech. Dev. 53, 73-85 31 V~,SSIN,H., VIELME]-I-ER,J. and CAMPOS-ORTEGA,J. A. (1985) J. Neurogenet.2, 291-308 32 de la CONCHA, A., DIETRICH,U., WEIGEL, D. and CAMPOS-ORTEGA,J. A. (1988) Genetics 118, 499-508 33 XU, T., REBAY,I., FLEMING, R. ]., SCOlqGALE,T. N. and ARTAVANIS-TSAKONAS,S. (1990) GenesDev. 4, 464-475 34 TAX, F. E., YEARGERS,J. J. and THOMAS, J. H. (1994) Nature 368, 150-154 3.~ MUSKAVITCH,M. A. T. (1994) Dev. BioL 166, 415-430 36 SHAWBER,C. etal (1996) Development122, 3765-3773 32 LI, L. etaL (1997)Nat. Genet. 16, 243-251 38. OAD, R. et al. (1997) Nat. Genet. 16, 235-242 39 HUKRIEDE,N. A. and FLEMING, R. J. (1997) Genetics145, 359-374 4C CHITNIS,A., HENRIQUE,D., LEWIS,J., ISH-HOROWlCZ, D. and KINTNER, C. (1995) Nature 375, 761-766 41 SUN,X. and ARTAVANIS-TSAKONAS,S. Development (in press) 42 FITZGERALD,K. an~dGREENWALD, I. (1995) Development121, 4275-4282 43 DOHERTY,D., FEGER,G., YOUNGER-SHEPHERD,S., JAN, L. Y. and JAN, Y. N. (19'96) GenesDev. 10, 421-434 44 DIAZ-BENJUMEA,F. and COHEN, S. (1995) Development121, 4215-4225

45 RULIFSON,E. I. and BLAIR,S. S. (1995) Development121, 2813-2824 46 de CELLS,J. F., de CELLS,J., LIGOXYGAKIS,P., PREISS,A., DELIDAKIS,C. and BRAY,S. (1996) Development122, 2719-2728 47 COUSO,J. P., KNUST, E. and MARTINEZ-ARIAS,A. (1995) Curr. Biol. 5, 1437-1448 48 PANIN, V. M., PAPAYANNOPOULOS,V., WILSON, R. and IRVINE,K. D. (1997) Nature 387, 908-912 49 FLEMING,R. J., GU, Y. and HUKRIEDE,N. A. (1997) Development 124, 2973-2981 50 IRVINE,K. and WEISCHAUS, E. (1994) Cell 79, 595-606 51 YUAN, L., BARRIOCANAL,J. G., BONIFACINO, I. S. and SANDOVAL, I. V. (1987) J. CellBioL 105, 215-227 52 RODRIGUEZ-ESTEBAN,C., SCHWABE,J. W. R., de la PENA,J., FOYS, B., ESHELMAN,B. and IZPISUABELMONTE,J. C. (1997) Nature 386, 360-366 53 LAUFER,E. et aL (1997) Nature 386, 366-373 54 WILKINSON, H. A., FITZGERALD,K. and GREENWALD, I. (1994) Cell 79, 1187-1198 55 HUPPERT,S. S., JACOBSEN,T. L. and MUSKAVITCH,M. A. T. Development(in press) 56 MICCHELLI,C. A., RULIFSON,E. J. and BLAIR,S. S. (1997) Development 124, 1485-1495 57 VERHEYEN,E., PURCELL,K., FORTINI,M. and ARTAVANIS-TSAKONAS,S. (1996) Genetics 144, 1127-1141

Pictures in cell biology Rho-family

proteins control polarized cytoskeietal rearran~ents d u r i n g w i n g h a i r f o r m a t i o n in Drosophilo

Drosophilawing epithelial cells display polarity both along their apical-basal axis and along a second axis in the plane of the epithelium. Planarpolarization is reflected in the uniform distal orientation of wing hairs, one of which isformed by each epithelial cell. Dudng wing hair formation, actin and microtubuleslocated in the apical region of the cellsundergo polarized rearrangements. Initially, the actin is distributed uniformly around the apical junctions. Later, it begins to polymedze at the distal vertex of each cell and then assemblesinto filaments that fill the growing wing hair1(Fig. la; actin is red). Micro. tubules are organized initially in a planar web at the level of apical junctions [(b), green; red shows the junctional marker CORACLE]. At about the time actin begins to polymerize distally, microtubules begin to accumulate between the centre of the cell and its distal vertex (c). They subsequently elongate into the growing hair (d). Two Rho-family G-1Pasescontrol different aspects of these cytoskeletal rearrangements. CDC42 moves from a uniform junctional distribution to the distal side of the cell where it is required for polymerization of actin in hairs [(a), CDC42 is green; actin is red]. Cells expressing dominant-negative CDC42 either fail to make hairs altogether or make deformed hairs like that shown in the scanning electron micrograph (e) - compare with the wild-type hairs (0. Racl is needed to limit hair formation to a single /al Ib/ ~cl td~ site. Expressing a dominantnegative Racl protein causes gaps in apical junctional actin, disorganization of the planar microtubule web and formation of multiple wing hairs by one cell (g), suggesting that localization of the machinery for hair fgl synthesis may depend on the (e) f~ organization of one or both of these cytoskeletal elements2. References

FIGURE 1

trends in CELL BIOLOGY (Vol. 7) November 1997

1 WONG, L. and ADLER, P. (1993) J. Cell Biol. 123, 209-221 2 EATON, S., WEPF, R. and $1MONS, K. (1996) J. Cell Biol. 135,1227-1289

Copyright © 1997 S. Eaton. Publishedby ElsevierScienceLtd. 0962-8924/97 PIl: s0962-8924(97)01157-4

Contributed by Suzanne Eaton, EMBL, Heidelberg, Germany. 441