Cell Differentiation and Development, 32 (1990) 391-400 391 © 1990 Elsevier Scientific Publishers Ireland, Ltd. 0922-3371/90/$03.50

CELDIF 99923

Genetic analysis of the Drosophila PS integrins

Michael Wilcox

M.R.C Laboratory of Molecular Biology, Cambridge, U.K.

Integrin; Drosophila integrin mutant; Wing morphogenesis

Introduction

Integrins function at the center of a complex multicomponent system. They act as mechanical connectors linking extracellular molecules to the cytoskeleton and probably also as signal trans- ducers, mediating the transmission of signals from the extracellular matrix to the interior of the cell (for review see Hynes, 1987; Ruoslahti and Pierschbacher, 1987). There is evidence that pro- tein tyrosine and serine/threonine kinases can each modulate integrin function (Hirst et al., 1986; Dustin and Springer, 1990) and it seems likely that other controlling factors will be found. Genetic analysis of such a complex system is likely to prove extremely valuable. I will describe here the early findings of a genetic analysis of the Drosophila PS integrin family. The study has dem- onstrated important integrin functions during fly development (which I will review briefly) and is beginning to reveal interacting genes which may encode other components of the integrin system or possible constituents of related morphogenetic processes.

There are two members of the PS integrin family (Brower et al., 1984) which, like vertebrate in- tegrins, are aft dimers (Wilcox et al., 1984; Leptin et al., 1987). They share a common fl subunit (PSfl) but have different a subunits (PSla and

Correspondence address: M. Wilcox, M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.

PS2a). Alternative splicing may add further com- plexity to the family (Brown et al., 1989). The proteins each show a high level of sequence and structural homology to vertebrate subunits (Bo- gaert et al., 1987; MacKrell et al., 1988; M. Wehrti et al., unpublished data).

PS integrins: distribution and function during em- bryogenesis

The integrins show varied and dynamic distri- butions during development (Wilcox et al., 1981; Brower et al., 1985 and Figs. 1 and 2). Although the subunits are transcribed early in embryogene- sis (the PSfl subunit is translated initially from maternal transcripts), the complexes do not ap- pear on the surface until after the onset of gastru- lation, which begins with the rapid invagination of the presumptive mesoderm along the ventral side of the embryo. This event appears to be equivalent to the 'bottle cell' invagination which initiates gastrulation in amphibians (Leptin and Grune- wald, 1990). Fly gastrulation continues with the extension of the two- (and later, three-) layered germ band from the ventral round to the dorsal side, leading to the invagination of the gut rudi- ments. At the same time, mesodermal cells migrate laterally over the underside of the ectoderm. (The equivalent step in vertebrates, the migration of the mesoderm in and around the interior of the em- bryo, appears to require integrin function (see e.g. Darrib~re et al., 1990). During this stage, the PS1

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complex (expressed in embryonic ectoderm and endoderm) is first seen on the cell surface, on the basal side of the ectodermal sheet, adjacent to the underlying mesoderm (Fig. 1). Loss of zygotic integrin function (null mutations in the PSfl gene, lethal (1) myospheroid or mys; Table I) does not affect this process.

However, embryos in which the maternal PSfl transcripts are also absent, often fail in the subse- quent retraction of the germ band (Wieschaus and Noell, 1986; Leptin et al., 1989). The PS2 integrin complex appears somewhat later, initially on myoblasts, and eventually becomes confined to the basal surface of visceral and pharyngeal muscles and to the ends of body wall muscles where they attach to the ectoderm (Bogaert et al.,

Fig. 1. The earliest expression of a PS integrin on the cell surface. A section of the middle region of an extending germ band embryo stained with an anti-PSfl antibody (in this print from a colour transparency, antibody staining shows as white). At this time, the antibody identifies the PS1 complex on the basal surface of the ectoderm (e) adjacent to underlying mesoderm (m), and on cells of the presumptive hind-gut at the point where the gut is invaginating (arrowheads). Anterior is to

the left, dorsal to the top.

1987). Within the ectoderm, the PS1 complex be- comes progressively restricted to the tendon cells, to which the body wall muscles attach, so that both here and in the gut and pharynx, the two integrins become directly opposed on either side of the muscle attachments (Leptin et al., 1989). rays null mutants show dramatic failures in these attachments (Leptin et al., 1989).

Genetic analysis of wing morphogenesis

The two integrins are expressed on a variety of tissues throughout the larval and pupal stages of development, including the imaginal discs which eventually give rise to much of the adult fly. They no longer respect their initial germ layer restric- tions. On the wing disc, they are strongly ex- pressed, again in a complementary way, on sep- arate epithelial regions which eventually become apposed to create the adult wing (Brower et al., 1984; Fig. 2b-d).

Flies homozygous for the viable PSfl mutation, rays n j42 or non- jumper , s h o w defects in the jump muscle (Costello and Thomas, 1981), the largest muscle in the adult fly. They also show, at low frequency, a phenotype in which the wings are held out slightly away from the body, suggesting that the mutation may also have some effect on the indirect flight muscles. Both the penetrance (the percentage of wings showing the defect) and expressivity (the extent of the defect; in this case, the angle at which the wings are held out) of this are greatly increased in flies heterozygous for rays n j42 and a deficiency for the rays gene, D f(1) sn c~28 (Table I; Wilcox et al., 1989). The wings of these flies also often show blistering or venation defects, indicative of problems with wing morpho- genesis. A viable mutation in the PS2a ( in f la ted or i f ) gene shows a similar disruption of wing mor- phogenesis (Wilcox et al., 1989; Fig. 3), while viable somatic clones homozygous for a null m y s allele disrupt both eye and wing development (Brower and Jaffe, 1989; Zusman et al., 1990). We find also that different combinations of the vari- ous m y s and i f alleles (Table I) lead to lethality during both larval and pupal stages, confirming a

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Fig. 2. Localized integrin expression in larval and pupal tissues. (a) In a section of a larval brain, PS2a mRNA is expressed (black) in the optic lobe of the brain (br) and in a repeated (segmental?) pattern in the ventral ganglion (vg). The transcript, and its product, are expressed on the majority of cells in the larval differentiating eye, but not on the axons going from the eye to the optic lobe. We do not know whether the expression seen here is on the termini of those axons or on other specialized nerve cells. (b) Bright field and (c) dark field images showing PS2a mRNA localization in a longitudinal section of a wing imaginal disc from a 3rd instar larva; mRNA is seen as white grains in (c). The disc is a continuous sac with a columnar epithelium (ep) on one side, a thinner squamous epithelium (the peripodial membrane; pm) on the other. The message is abundant in the cells of the presumptive lower wing surface (above the arrowheads indicating the region which will differentiate the wing margin). Cells immediately below the arrowheads will make the upper wing surface. These cells express the PS1 integrin. Outside these areas, the expression of each integrin is complex; discrete patches of PS2a transcription can be seen elsewhere in this section (arrows). (d) A cross-section of a developing adult wing from a pupa, stained with anti-PSfl antibody. The disc has evaginated and folded around the presumptive wing margin (large arrowheads; see previous pictures), bringing the upper (right-hand) and lower (left-hand) wing blade epithelia together. The integrins are most strongly expressed on the basal surfaces of the epithelia, especially where the two are tightly apposed (small arrowheads). The surfaces are held together by adherens junction-like structures (Mogenson and Tucker, 1988) which may be where the integrins are

localized. At various points the two epithelia are not joined; these mark the positions of the differentiating veins.

role for the integrins throughout development (author, unpubl i shed data).

We know noth ing as yet of the other compo- nents of the PS integrin system. Both the predomi- nan t ly basal localization of the complexes in epi- thelia (Brower et al., 1984; Lept in et al., 1989) and

certain features of the PS2a sequence suggest that the integrins are probably matrix receptors of the /31 type. However, no l igands have been identified. Al though m a n y contracti le and cytoskeletal genes have been identif ied in Drosophila (Fyrberg, 1989), genes encoding tal in and vinculin, two proteins

involved in integr in-cytoskeleton l inking in verte- brates, have not yet been found. [A nul l lethal m u t a n t in a gene encoding ot-actinin, a third potent ia l a t t achment protein, shows defects in muscle sarcomeric organiza t ion (Fyrberg et al.,

1990)]. We are using genetic analysis to look for these and other componen t s of the fly integrin system, and, at the same time, for putat ive genes encoding other modu la t ing factors. Our screen is based on the idea that genes coding for proteins that are par t of a m u l t i c o m p o n e n t complex should show genetic in teract ions; specifically, we are

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TABLE I

The PS integrin genes and mutants

Mutant Type of mutation Effect on PS References components

PSfl = 1(1) myospheroid IX chromosome 7D1-5]; maternal component

D r ( l ) 8n C128 embryonic lethal (null) rays xG43 (muscle, sheet migration defects)

rays xR°4 embryonic lethal

mys n j4: viable (muscle, wing defects)

PS2a = inflated [X chromosome 15A4-11]; zygotic expression only?

i f k27e embryonic lethal (null?) (muscle, gut defects)

i f 3 viable (wing defects)

no protein 1, 2, 3

protein present 2, 4

altered protein 4, 5, 6

no protein detected 7, 4

low level of protein 4, 8 in imaginal discs

References: 1, Digan et al. 1986; 2, Wieschaus et al. 1984; 3, Leptin et al. 1989; 4, Wilcox et al. 1989; 5, Costello and Thomas, 1981; 6, De la Pompa et al. 1989; 7, Falk et al. 1984; 8, Brower and Jaffe, 1989.

looking for genes whose products are involved, together with the integrins, in wing morphogene- sis.

The wing is an ideal system for genetic analy- sis: it is big so that mutations affecting its devel- opment can be identified readily and, like the eye, it is completely expendable (in the laboratory any- way). Further, viable mutants exist, at many dif- ferent chromosomal locations which perturb the development either of the wing or of the adult thoracic muscles, (many of which also derive from precursor cells in the wing imaginal disc) (Linds- ley and Grell, 1968). Some alleles at these loci also perturb processes other than wing morphogenesis. The many wing mutants we are analysing can be broadly classified into those which cause either excessive or insufficient venation, those which cause blistering of the wing surface, those which lead to bigger or smaller wings, those which affect only thoracic muscles, and so on (see Fig. 3 for examples). In a recent paper, Diaz-Benjumea and Garcia-Bellido (1990) have categorised these phe- notypes in a more precise way. As pointed out by these authors, many of the mutant phenotypes are regionalized, confined to fairly restricted regions of the wing (e.g. see Fig. 3b and i). Nevertheless, and this is not surprising in view of the complex

integrated series of events required to produce the wing (Waddington, 1940), some mutations cause pleiotropic effects: b a l l o o n , for example, produces extra venation, frequent wing blistering and an extreme wings-held-out phenotype (Fig. 3h). The strategy we have adopted involves looking for synergistic interactions in double mutant combi- nations of the viable integrin mutants i f 3 a n d

rays n j42 with other of the wing mutant alleles. The weak character of the two wing integrin mutations - remember, m y s n j42 flies show a wing phenotype only when the number of copies of the mutant allele is halved, while the i f 3 phenotype is weak [only some 20% of wings show blisters, which are usually small] (Wilcox et al., 1989, Fig. 3b) - makes the detection of synergism relatively straightforward. As a test for the double mutant approach, we first combined the two viable in- tegrin mutations, that is, created a homozygous m y s ~j42 i f 3 fly (Wilcox et al., 1989). The two mutants interact to give a phenotype which is much enhanced in terms of both penetrance and expressivity; the flies show, at high frequency (> 90%), very severely defective wings which fail either to unfold properly or to expand after the hatching of the fly from the pupal case.

We chose to analyse first those mutants which

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Fig. 3. Representative wings from wild-type and a variety of mutant fries. (a) Wild-type. The shape and venation pattern (veins are numbered I-V; crossveins indicated by arrowheads) are almost invariant. (b and c) iff; arrows indicate a typical small centrally-located blister in (b) and a large blister (found only infrequently) in (c). (d) ueinlet; The distal tip of vein III and most of veins IV and V are missing. (e) plexus j and (f) net, showing typical extra venation (arrowheads) and abnormal vein positioning. Note that the defects affect predominantly the distal part of the wings. (g) blistered 2, showing vein abnormalities (arrowheads). A small proportion of such wings also have small blisters, usually in the region of veins IV and V. (h) balloon; distorted and extra venation (arrowheads) together with a blister (arrow). Fries of this genotype also show a wings-held-out phenotype. (i) oesiculated 1, showing a typical small distal blister (arrow). Blisters form frequently also between veins IV and V. (j) uesiculated ~ if 3. The wing is badly distorted and largely unexpanded: some 90% of wings have this appearance. Wings from rays "j~2 i f f (Wilcox et al., 1989) and if3; blistered 2 flies have a

similar appearance.

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TABLE II

Double mutant interactions between i f (PS2a) and rays n j42 (PSfl) alleles and other wing defect mutan ts

With i f ~ With rays nj42

Degree of Degree of Degree of Degree of wing held-out wing held-out defect defect

rnys n j42 + + + severe weak

vs i + + + severe none net + + + severe none

bs 2 + + + severe none bs 2//+ + + severe none

p x ~ weak none p x J / + weak none p x ~ / bs 2 + moderate none p x pB + + moderate none

+ + moderate mod.-sev. none

+ + mod.-sev, severe + *

?

No obvious interaction was detected between i f ~ and dx, shf2; ho, tkv, ex, el, P f d / + ; ri, eg, D I / + , nor between rnys "j4e and ewg vtw; sr. The level of synergism (second and fifth columns) was calculated in terms of wing blistering (for i f 3 double mutants) and both blistering and the degree of wings-held-out (for rays "j42 double mutants) , assessing both penetrance (the proportion of wings showing the phenotype) and expressivity (the severity of the phenotype): + + + indicates a very strong enhancement of the integrin mutan t phenotype, + + a weaker enhancement, - - - a strong suppression of phenotype, and so on. The degree of wing bhstering and of the held-out phenotype (between 0 o and 90 o from the body) was assessed as none, weak, moderate (mod.) or severe (sev.). * This was determined by demonstrat ing an enhancement of the mysnj42/l(1)mysxG43 phenotype by one copy of bs 2. There is necessarily a certain level of subjective assessment; like i f ~, a number of other mutants , vs ~ and bs 2 for example, show blistering, usually weak and regionalized (see Fig. 3), while rays "J42 flies display a weak held-out phenotype at low penetrance. No at tempt has been made here to assess the effect of the interaction on the other mutan t phenotype; antagonistic or mutually suppressive interactions between certain mutat ions can occur (Diaz-Benjumea and Garcia-BeUido (1990). Since mutan t stocks often carry modifiers which reduce the phenotype, most were outcrossed before use. Each double mutan t combinat ion was made at least twice: similar results were obtained in each case. All crosses were carried out at 25 o. Mutan t abbreviations not explained in the text can be found in Lindsley and Grell (1968).

are either weakly expressed or weakly penetrant or which show quite different phenotypes to those displayed by the PS integrin mutants. The results are summarised in Table II. Many of the combina- tions we have tested show only additive pheno- types (see bottom of Table II). However, a num- ber of wing mutants do interact synergistically with i f 3. Four of these are listed in Table II. Three, vesiculated (vs), net and blistered (bs), in- teract with if 3 to cause, at high frequency, very severely affected wings similar to those seen in the double r a y s n j42 if 3 mutant (Fig. 3). Further analy- sis has shown that blistered 2, but not net or oesiculated ~, acts as a dominant enhancer; that is, if3; blistered2~ + flies also show severe wing phe- notypes, although at a lower frequency than in doubly homozygous i f ; blistered 2 flies.

Two of the three mutations which interact with i f 3 to cause severe defects in the wing, interact also with mys "J42, but here the major effect is seen in a separate and quite different structure, the thoracic musculature. The rays "j42 mutation pro- duces an altered PSfl protein (Wilcox et al., 1989) and so might cause defects at any point in devel- opment. However, the failure in the jump muscle is all that is seen normally, except for the low frequency weak wings-held-out phenotype. This latter phenotype is strongly enhanced in flies dou- bly homozygous for mys n j42 and vesiculated I or blistered 2 (Table II). We are presently analysing mutant thoraces to confirm that the held-out phe- notypes do reflect deterioration of the indirect flight muscles relative to those of the single muta- tions.

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It is interesting, but not unexpected, that little evidence of a wings-held-out phenotype is seen in if 3 double mutant combinations. In if 3 homo- zygotes, PS2a protein levels are much reduced in the imaginal discs but appear essentially normal at other places and times in development, suggesting that the mutation probably affects the regulation of PS2a gene expression in the discs (Brower and Jaffe, 1989; Wilcox et al., 1989).

One allele at another locus, plexus (px), shows a quite different interaction with if 3. The wings of homozygous if3; plexus I flies retain the extra venation typical of a plexus phenotype (Fig. 3f) but are blistered only rarely (0-5%). Other studies show that plexus ! acts as a dominant suppressor of the if 3 phenotype: p lexus1 /+ strongly sup- presses the frequency and severity of blistering not only in if 3 flies, but also in i f 3 / i f k27e and if3; blistered2/+ combinations, each of which nor- mally leads to a much higher frequency of blister- ing than is shown by i f 3 flies. The suppressive effect of plexus on if 3 is allele specific: a second allele, plexus ps, enhances the frequency of if 3 blistering (Table II).

Discussion

The double mutant analysis has yielded a num- ber of genes which might encode other compo- nents of the integrin system. The best candidates are blistered and vesiculated, mutants in each of which interact with the integrin mutants to cause defects in two separate locations, the wing and the adult muscles, which seem to be 'weak spots' in the integrin system. Further, blistered 2 is a domi- nant enhancer of phenotype, while vesiculated ~ and blistered 2 also interact strongly with each other (author, unpublished data). It is perfectly plausible, however, that mutations in molecules that are not part of the integrin system might also cause such enhancement. The spatial organization of the wing disc and its subsequent morphogenesis and differentiation require the coordinated func- tioning of a large variety of genes. It would not be surprising, then, if mutations in genes for parallel adhesive or recognition systems, for example, in

combination with the integrin mutations led to apparently synergistic effects. The cloning of the interacting genes we have already identified should give clues about their functions which may help to resolve these possibilities.

It is unlikely that plexus is exclusively a com- ponent of the integrin system. One px allele acts as a dominant suppressor, another as an enhancer of the if phenotype. Plexus has been shown to act also in a mutually suppressive system with veinlet (Fig. 3d), and to exhibit, among other interactions, both positive and negative synergisms with differ- ent alleles of the neurogenic genes Notch and Delta, which function in the developing wing (Diaz-Benjumea and Garcia-Bellido, 1990). veinlet appears to be an allele of rhomboid, an essential embryonic gene which encodes a transmembrane protein with no clear homology to known proteins (Bier et al., 1990), while the neurogenic genes have no obvious involvement with the integrin system (for review, see Simpson, 1990). Thus, plexus in- teracts with a number of apparently quite separate morphogenetic systems, and it seems likely that it may function in a more general kind of mecha- nism (see, for example, a discussion of 'informa- tional suppressors', which appear to affect processes other than translation, in the nematode, Caenorhabditis (Hodgkin et al., 1989).

The analysis is still at an early stage. We are beginning alternative mutagenesis screens in the expectation that we can find different classes of interaction. For example, a screen in the presence of a weak integrin mutant background, homo- zygous mys n j42 say, should lead to the detection of 'dominant enhancer' mutations, one mutant copy of which (like blistered 2) is sufficient to cause an enhanced wing defect phenotype. Alter- natively, one might screen for mutations in new genes which fail to complement stronger integrin mutant alleles (a technique used successfully by Fuller et al. (1989) in a search for genes encoding proteins which interact with a fly tubulin). Among the existing integrin lethal alleles, rays xR°4 or if k27e may prove useful in such a screen, since they fail to complement one another: the doubly hetero- zygous mys xR°4 + / + if k27e combination causes pupal lethality.

398

Acknowledgements

I am grateful to Richard Smith for his excellent assistance, to many colleagues, especially Maria Leptin and Marcel Wehrli, for help and advice and to Luis Garcia-Alonso (Berkeley), Kathy Mat- thews (Indiana University Stock Collection), Edwin Stephenson (Rochester) and the Bowling Green Stock Center for stocks.

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