boundaries in the drosophila wing imaginal disc organize vein … · 4246 membrane protein (bier et...

4245Development 125, 4245-4257 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV8536

Boundaries in the Drosophila wing imaginal disc organize vein-specific

genetic programs

Brian Biehs 1, Mark A. Sturtevant 1,2 and Ethan Bier 1,*1Department of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA92093-0349, USA2Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA*Author for correspondence (e-mail: [email protected])

Accepted 18 August; published on WWW 30 September 1998

Previous studies have suggested that vein primordia inDrosophila form at boundaries along the A/P axis betweendiscrete sectors of the larval wing imaginal disc. Genesinvolved in initiating vein development during the thirdlarval instar are expressed either in narrow stripescorresponding to vein primordia or in broader ‘provein’domains consisting of cells competent to become veins. Inaddition, genes specifying the alternative intervein cell fateare expressed in complementary intervein regions. Theregulatory relationships between genes expressed innarrow vein primordia, in broad provein stripes and ininterveins remains unknown, however. In this manuscript,we provide additional evidence for veins forming in narrowstripes at borders of A/P sectors. These experimentsfurther suggest that narrow vein primordia producesecondary short-range signal(s), which activate expressionof provein genes in a broad pattern in neighboring cells. We

also show that crossregulatory interactions among genesexpressed in veins, proveins and interveins contribute toestablishing the vein-versus-intervein pattern, and thatcontrol of gene expression in vein and intervein regionsmust be considered on a stripe-by-stripe basis. Finally, wepresent evidence for a second set of vein-inducingboundaries lying between veins, which we refer to asparavein boundaries. We propose that veins develop at bothvein and paravein boundaries in more ‘primitive’ insects,which have up to twice the number of veins present inDrosophila. We present a model in which different A/Pboundaries organize vein-specific genetic programs togovern the development of individual veins.

Key words: Drosophila, Wing vein, Paravein, Boundaries, Lateralinhibition, rhomboid, hedgehog

SUMMARY

f angthe

ntgce

7;/Pent

des,’ein

ral

INTRODUCTION

Wing vein development in Drosophila can be separated intotwo temporally distinct periods: an initiation phase, whictakes place during the third larval instar when the winprimordium consists of a monolayer of cells, and maintenance/refinement phase, which occupies prepupal early pupal development when the wing is a bilayer of ce(Waddington, 1940; García-Bellido and de Celis, 199Sturtevant and Bier, 1995). In third larval instar wing disclongitudinal vein primordia arise as a series of parallel narrstripes of cells (Sturtevant et al., 1993). Several vein primorlie at known boundaries between discrete sectors along anterior-posterior (A/P) axis of the wing primordium(Sturtevant and Bier, 1995; Sturtevant et al., 1997; Lunde et1998). For example, the L2 vein primordium runs along tanterior border of a broad domain of spalt-m(salm)-expressingcells (Sturtevant et al., 1997). In the case of L2, there is strevidence that salm-expressing cells induce their neighborsalmnon-expressing neighbors to become the L2 primordiu(Sturtevant et al., 1997). Also, the L3 and L4 primordia for

hgaandlls2;s,owdiathe

al.,he

ongingm

m

respectively along the anterior and posterior boundaries ocentral stripe of cells that are engaged in Hh signali(Sturtevant et al., 1997). These Hh-signaling cells express patched(ptc) and decapentaplegic(dpp) genes. The posteriorborder of ptc expression corresponds to the A/P compartmeboundary, which is the primary source of A/P patternininformation in all imaginal discs. There is suggestive evidenlinking the anterior border of ptc expression to formation ofthe L3 primordium (Phillips et al., 1990; Sturtevant et al., 199Strigini and Cohen, 1997), and signaling across the Aboundary to formation of the L4 vein at the border of thposterior compartment (García-Bellido et al., 1994; Sturtevaet al., 1997).

Genes involved in initiating wing vein development in thirlarval instar wing discs are expressed either in narrow stripcorresponding to vein primordia, or in broader ‘proveinstripes, consisting of cells that are competent to become vcells. For example, rhomboid (rho) and argos (aos) areexpressed in narrow vein stripes, while Dl, achaete(ac), scute(sc), caupolican (caup) and araucan (ara) are expressed inbroader provein domains. rho, which encodes an integ

4246

set,at

re

d ink

)l

s

int)r

,seant

ndys,

ion ar,re

lalin

toryion

e

al

g

B. Biehs, M. A. Sturtevant and E. Bier

membrane protein (Bier et al., 1990; Sturtevant et al., 199is expressed in all vein primordia and promotes vein formatthroughout wing development by locally activating the EGR-signaling pathway (Sturtevant et al., 1993; Noll et al., 199aos encodes an EGF-R antagonist (Freeman et al., 19Schweitzer et al., 1995), which feeds back negatively to inhEGF-R activity (Golembo et al., 1996a). caup and theneighboring gene ara encode related homeobox genes thpromote expression of vein genes such as rhoand proneuralgenes such as acand sc(Gomez-Skarmata et al., 1996). Delta(Dl) encodes a ligand (Dl) for the Notch (N) receptor (reviewin Muskavitch, 1994; Campos-Ortega, 1995), which medialateral inhibitory interactions among cells in vein-competedomains during pupal development (Shellenbarger and Moh1978; Parody and Muskavitch, 1993; Kooh et al., 1993).

The vein pattern is also reflected by the complementintervein expression patterns of blistered(bs), which encodesthe Drosophila homologue of the Serum Response Fact(DSRF) (Montagne et al., 1996) and vein (vn), which encodesa putative EGF-R ligand of the neuregulin/heregulin cla(Schnepp et al., 1996). bs provides an essential generafunction for intervein development (Fristrom et al., 199Montagne et al., 1996) and is strongly downregulated in vein primordia relative to intervein regions (Montagne et a1996). vn promotes vein development and is expressed insingle strong intervein stripe running between the L3 and primordia in third instar larval discs (Simcox et al., 1996).

Initiation of vein development during the third larval instais followed by a period of vein maintenance and refinemeduring prepupal and pupal stages. At least three different tyof cell-cell communication contribute to the refinement proce(García-Bellido, 1977; Díaz-Benjumea and García-Bellid1990; García-Bellido and de Celis, 1992; Sturtevant and B1995): (1) lateral inhibitory signal(s) elaborated bpresumptive vein cells restrict vein formation to the centerbroad vein-competent domains, (2) dorsal-to-ventral signamaintain vein fates in cells on the ventral surface of the wand (3) vein continuity signal(s) promote vein formation straight lines along the vein axis. These various signcollaborate to insure that the dorsal and ventral componentnarrow veins are strictly aligned and uninterrupted.

In this manuscript, we address three major issues. First,provide new evidence in favor of the ‘veins forming at A/boundaries’ hypothesis. We show that expansion acontraction of the Hh-signaling domain coordinately shifts tpositions of L3 vein, provein and intervein markers relativeL4, which remains tightly associated with the A/P boundaSecond, we examine the unknown regulatory relationsbetween genes expressed in sharp vein stripes and gexpressed in broader provein domains during the early peof vein initiation. We show that gene expression in broprovein stripes is centered over narrower stripes of geexpression in vein primordia and depends on the functionrho in narrow vein stripes. We also show that different Aboundaries organize distinct patterns of gene expressionparticular veins and that crossregulatory interactions amovein, provein and intervein genes play a significant role establishing the vein pattern. We propose a model in whboundaries between A/P sectors induce the expressionputative ‘vein-organizing’ genes in narrow vein stripes, whithen orchestrate vein development in and around veins. Fin

6),ionF-4).92;ibit

at

edtesntler,

ary

or

ssl4;alll., aL4

rnt

pessso,ier,y ofl(s)inginalss of

wePnd

he tory.hipenesriodadne of/P innginich of

chally,

we address the evolutionary origin of the Drosophila veinpattern. We provide evidence for the existence of a second of vein-inducing boundaries running between vein primordiawhich we refer to as paravein boundaries. We suggest thveins form at both vein and paravein boundaries in moprimitive insects than Drosophila.

MATERIALS AND METHODS

Fly stocksAll genetic markers and chromosome balancers used are describeLindsley and Grell (1968) and Lindsley and Zimm (1992). We thanDr Tom Kornberg (UCSF, San Francisco) for the GAL4-enstock andthe en11 allele, Dr Matthew Scott (Stanford) for the UAS-ptc lines, DrWalter Gehring (Biozentrum, University of Basel, Basel, Switzerlandfor the A405.1M2 salm-lacZ enhancer trap stock, and Dr IssabeGuerrero for the UAS-hhstocks. Other stocks were obtained from theBloomington, Indiana and Bowling Green, Ohio Drosophila StockCenters. All crosses of GAL4 driver lines with UAS responding line(Brand and Perrimon, 1993) were performed at 25°C.

Mounting fly wingsWings from adult flies were dissected in isopropanol and mountedCanadian Balsam mounting medium (Gary’s magic mountanfollowing the protocol of Lawrence and others (in Roberts, 1986) oin 50% glycerol.

In situ hybridization to whole-mount embryos or discsIn situ hybridization to whole-mount wing discs and pupal wingscarried out alone or in combination with antibody labeling, waperformed with digoxigenin-labeled RNA probes (visualized as a blualkaline phosphatase precipitate) as previously described (Sturtevet al., 1993; O’Neill and Bier, 1994). Wings were mounted inPermount or 50% glycerol and photographed under a compoumicroscope using Nomarski optics. The anti-Dl antibody was kindlprovided by Marc Muskavitch (Indiana University) and the anti-Bantibody was kindly provided by Marcus Affolter (Biocenter, BaselSwitzerland).

RESULTS

Vein primordia are centered within broader vein-competent domainsTo determine the precise relationships between the expresspatterns of vein, provein and intervein genes, we performedseries of double-label experiments. Our primary vein markerho, is expressed in five sharp stripes 1-2 cells wide, which alikely to correspond to the primordia for the L1-L5 longitudinawing veins (Fig. 1A). We have previously shown that neuronprecursor cells for sensory organs located along the L3 vealign with the L3 stripe of rho expression in third instar wingdiscs (Sturtevant et al., 1993). To generalize this finding other veins, which normally are not decorated with sensoorgans, we determined the relationship between the expresspatterns of rhoand the A101 neuronal precursor cell marker inwing primordia of Hw49c mutants, which have ectopic sensillarunning along each longitudinal vein. Consistent with thpremise that each stripe of rhoexpression in third instar wingdiscs corresponds to a vein primordium, ectopic neurprecursors in Hw49c mutants coincide with rho-expressing cellsin third instar wing discs (data not shown) and in early evertinprepupal wings (Fig. 1B).

4247Boundaries in the Drosophila wing imaginal disc

inor).ge

ind

erince

heeor

iornth

ls

of4

positions of vein and intervein gene expression. (A) rho expression innstar disc. In this, and subsequent panels, vein primordia L1-L5 are the margin is indicated by M. (B) Double label for rhoRNA expressionprotein (brown) in a Hw49c; A101 (neuralized-lacZ) prepupal disc.ed-lacZ (neu-lacZ)-expressing neural precursors form along each rho- primordium. The ectopic neu-lacZ-expressing cells along the L2ust out of focus in this image. (C) Double label for rhoRNA (blue) andbrown) expression in a wild-type third instar disc. Insets in C-F:f the dorsal component of the L3 primordium. As double-labeleal that Dl RNA and Dl protein are expressed in coincident patterns), we conclude that the sharp stripes of rho expression run up the centerstripes. Double-label experiments with antisense rho and Dl

eled RNA probes confirm this conclusion (data not shown). (D) DoubleA (blue) and anti-Bs (= DSRF) protein (brown) expression in a wild- disc. (E) Double label for anti-Dl protein (brown) and caupRNA (blue) wild-type third instar disc. (F) Double label for anti-Dl protein (brown)e) expression in a wild-type third instar disc.

Having confirmed that each stripe of rho expressioncorresponds to a longitudinal vein primordium, we determinthe relative expression patterns of various genes expressenarrow vein stripes or broader provein stripes by double-laexperiments. We first compared the expression patterns ofrhoand Dl. In mid-to-late third instar larvae, Dl is expressed inseries of four stripes 4-6 cells wide. Double-label experimereveal that the broader stripes of Dl protein expression centered over the narrower L1, L3, L4 and L5 rho stripes (Fig.1C). Additionally, double-label experiments with the anti-Dantibody and antisense RNA probes for aos, caup and acrevealthat the three stripe of aosexpression coincide with the L3, L4and L5 Dl stripes (data not shown), that the three broad caupstripes straddle the narrower L1, L3 and L5 Dl stripes (F1E), and that the single dorsally restricted stripe of acexpression is coincident with the dorsal component of the Dl stripe (Fig. 1F).

We also determined the relationship between the expressof rho and intervein markers. bs RNA and Bs protein areexpressed ubiquitously in the wing pouch, but are strondownregulated in a pattern of four stripes (Montagne et 1996). The L2-L5 rho stripes are centered within the troughof Bs downregulation (Fig. 1D), which tendto be one or two cells wider than the rhostripes (e.g. there are single rows of cellsflanking rho-expressing cells not expressingeither rho or high levels of Bs). These data areconsistent with the previous observation thatL3 sensory organ precursor cells lie within theL3 trough of Bs downregulation (Montagne etal., 1996). Finally, in accordance withpreviously reported double-label experiments(Simcox et al., 1996), we observed that strongexpression of vnis confined to the regionbetween the L3 and L4 stripes of Dlexpression (data not shown).

An important feature of these variousdouble-labeling experiments is that thecenters of all vein and provein stripescoincide. For example, the narrow stripes ofrho expression run up the middle of thebroader Dl stripes, and the yet broaderdomains of caupexpression (7-8 cells wide)symmetrically straddle the odd-numbered Dlstripes. Also, as mentioned above, the stripesof Bs downregulation are centered over thenarrower stripes of rho expression in veins.The nearly perfect nested registration ofseveral vein, provein and intervein markers inthird instar wing discs suggests that commonpositional cues coordinate expression of thesegenes in and around each vein primordium.

Altering the level of Hh signalingshifts the position of the L3primordiumHh is produced in the posterior compartmentand diffuses a short distance into the anteriorcompartment where it activates target genessuch as ptc and dppin stripes running up thecenter of the wing disc (reviewed in Lawrence

Fig. 1. Relative wild-type third ilabeled 1-5 and(blue) and β-gal Ectopic neuralizexpressing veinprimordium lie janti-Dl protein (magnification oexperiments rev(data not shownof the broader Dldigoxigenin-lablabel for rhoRNtype third instarexpression in aand acRNA (blu

edd inbel antsare

l

ig.

L3

ion

glyal.,s

and Struhl, 1996). As mentioned previously, the L3 and L4 veprimordia form respectively along the anterior and posteriborders of the Hh-signaling domain (Sturtevant et al., 1997In addition, a variety of evidence suggests that the anterior edof the Hh-signaling domain defines the position of the L3 ve(Phillips et al., 1990; Sturtevant et al., 1997; Strigini anCohen, 1997).

To investigate the relationship between the anterior bordof the Hh-signaling domain and the expression of vein, proveand intervein genes, we manipulated the level of Hh and henthe width of the Hh-signaling domain. To this end, we used tGAL4/UAS system (Brand and Perrimon, 1993) to drivexpression of Ptc (a Hh antagonist) or Hh in the postericompartment. The GAL4-endriver line activates expression ofUAS-transgenes in a large domain comprising the postercompartment and in a narrow strip of anterior compartmecells (data not shown). Thus, to reduce the amount of Hliberated from the posterior compartment, we used GAL4-ento misexpress a UAS-ptctransgene and to increase the leveof Hh, we overexpressed a UAS-hhtransgene with the sameGAL4 driver. Consistent with the proposition that the edges the Hh-signaling domain define the positions of the L3 and L

4248

-


position of the L3 primordium by altering the levels of Hh signaling.g. Veins L1-L6 are labeled 1-6. (B) A GAL4-en; UAS-ptcwing. Note veins are closer to each other than in wild-type discs. The proximal sectionften missing in these wings (asterisk). (C) A GAL4-en; UAS-hhwing. Note veins are spread apart relative to wild-type discs. (D) dppRNA expression imaginal disc. Inset shows part of a wild-type wing disc double labeledrown) and hhRNA (blue) in a dpp-lacZ wing disc. (E) dppRNA

AL4-en; UAS-ptcwing imaginal disc. Inset shows part of a GAL4-en/dpp- disc double labeled for β-gal protein (brown) and hhRNA (blue). Notes narrower than in wild-type discs, but that it abuts the posterioroes the wild-type dppstripe (compare with inset in panel D). An alternativeion for the posterior shift in the L3 primordium in GAL4-en; UAS-ptcfliesc expression in cells lying just anterior to the A/P boundary could preventells from responding to Hh (Chen and Struhl, 1996). If this were the case, gap between the A/P boundary and the domain of Hh signaling. Since theis expressed in a narrower than wild-type stripe (Fig. 2E, compare withne-the-less abuts the posterior compartment as normal in GAL4-en; UAS-et in Fig. 2E, compare with inset Fig. 2D), we do not favor this latterwe believe that sequestration of Hh in the posterior compartment is thehe posterior displacement of the L3 primordium in GAL4-en; UAS-ptcexpression in a GAL4-en; UAS-hhwing imaginal disc. Note that thean that observed in wild-type discs. (G) Double label for anti-Dl (brown)

expression in a wild-type third instar wing disc. Consistent with the Dl expression being centered over the narrower stripes of rho expression,tends 1-2 cells into the anterior compartment (inset) in contrast to the rhostrictly confined to the posterior compartment (Sturtevant et al., 1997).or anti-Dl (brown) and hh RNA (blue) expression in a GAL4-en; UAS-ptcisc. (I) Double label for β-gal protein (brown) and rho RNA (blue)-lacZ; GAL4-en; UAS-hh third instar wing disc. Inset: magnification of thet of the L4 primordium.

vein primordia, it has been observed previously that GAL4-en;UAS-ptc flies have a diminished distance between L3 and veins (Johnson et al., 1995; Fig. 2B) relative to wild-type (F2A), while GAL4-en; UAS-hh flies haveincreased distance between these veins(Mullor et al., 1997; Fig. 2C). As expectedfrom these final wing phenotypes, the widthof dpp stripe is decreased in GAL4-en;UAS-ptc wing discs (Fig. 2E) and isexpanded in GAL4-en; UAS-hh discs (Fig.2F) relative to wild-type (Fig. 2D).

In the above experiments, it was not clearwhether the L3 or L4 vein was beingdisplaced relative to the A/P compartmentboundary. To address this question, weperformed a series of double-labelexperiments from which we conclude thatthe position of the L3 primordium shiftswhen the levels of Hh signaling aremanipulated, but that the L4 primordiumremains tightly associated with the A/Pboundary. For example, the L4 primordiumforms directly along the posterior edge ofthe A/P compartment boundary in GAL4-en; UAS-ptc wing discs (Fig. 2H) andGAL4-en; UAS-hh wing discs (Fig. 2I) asit does in wild-type discs (Fig. 2G; see alsoFig. 2C,E in Sturtevant et al., 1997). Inthese experiments, the A/P compartmentboundary is marked by either the anteriorborder of the hh expression domain (Fig.2G,H) or the posterior border of the dppexpression domain (Fig. 2I). These resultssupport the view that the anterior border ofthe Hh-signaling domain induces formationof the L3 vein in neighboring cells.

Manipulation of Hh levels leads tothe coordinate displacement of geneexpression in L3To determine whether vein and interveinmarkers respond in concert to alterations inthe level of Hh, we performed a series ofdouble-label experiments. In GAL4-en;UAS-ptcwing discs, which have a reduceddistance between the L3 and L4 veinprimordia, the L3 expression patterns of Dland rho (Fig. 3A), Dl and ac (Fig. 3B), andDl and caup (data not shown) shiftcoordinately in a posterior direction. Therelative positions of vein and interveinmarkers also are preserved in these discs asrevealed by the concerted shift in theexpression of rhoand Bs (data not shown).Reciprocally, in GAL4-en; UAS-hhwingdiscs, which have an increased distancebetween the L3 and L4 vein primordia,there is a coordinate anterior displacementof the vein markers Dl and rho (Fig. 3C), Dland ac (Fig. 3D), and Dl and caup(Fig. 3E).Downregulated Bs expression in L3 also

Fig. 2. Shifting the(A) A wild-type winthat the L3 and L4of the L4 vein is othat the L3 and L4in a wild-type wingfor β-gal protein (bexpression in a GlacZ; UAS-ptcwingthat the dppstripe icompartment as dpotential explanatis that elevated Ptthis thin band of cthere should be aHh target gene dppFig. 2D), which noptcwing discs (insalternative. Thus, primary cause of tflies. (F) dppRNA staining is wider thand hh RNA (blue)broader stripes ofthe Dl L4 stripe exL4 stripe which is (H) Double label fthird instar wing dexpression in a dppventral componen

L4ig.

shifts anteriorly in register with rho in GAL4-en; UAS-hhwingdiscs (Fig. 3F). As in the case of wild-type discs, the midpoints of vein and provein stripes are coincident in GAL4-en;


gl

lly

te

e

)e

ith

3. Effects of shifting the anterior border of the Hh-signaling domain onession of vein and intervein genes in the L3 primordium. (A) Dln) and rho (blue) expression in a GAL4-en; UAS-ptc third instar wing

Note that the L3 and L4 vein primordia are closer together than intype discs and that Dl and rho expression in L3 shift in register. (B) Dln) and ac (blue) expression in a GAL4-en; UAS-ptcthird instar wing

also shift together posteriorly. (C) Dl (brown) and rho (blue) expressionGAL4-en; UAS-hhthird instar wing disc. Note that the L3 and L4 veinordia are spread apart and that Dl and rho expression in L3 shift inter. (D) Dl (brown) and ac (blue) expression in a GAL4-en; UAS-hh instar wing disc also shift anteriorly together. (E) Dl (brown) and caup) expression in a GAL4-en; UAS-hh third instar wing disc are

dinately displaced. (F) Bs (brown) downregulation and rho (blue)ession in the L3 primordium of a GAL4-en; UAS-hhthird instar wingalso shift anteriorly in register.

UAS-ptc and GAL4-en; UAS-hhwing discs. Cumulatively,these data suggest that gene expression in and around thprimordium is organized by a single positional cue specifiby the anterior limit of the Hh-signaling domain.

In addition to the coordinate displacement of Dl and rhoexpression in GAL4-en; UAS-ptc wing discs (Fig. 3A), weobserved that the levels of Dl and rho expression in L4 aresignificantly reduced relative to wild type (Figs 2H, 3A) anthat adult wings generated from these discs often have gapthe L4 vein (Fig. 2B, asterisk). This reduction in Dl and rhoexpression in L4 suggests that the Hh-signaling domain in anterior compartment normally sends a signal to adjacposterior compartment cells to initiate L4 formation. Sincfewer cells would express this putative L4-inducing signal GAL4-en; UAS-ptcwing discs than in wild-type discs, thelevel of signal might fall below that required to reliably inducL4 formation. In accord with this possibility, loss of vnfunction in the anterior compartment non-autonomously leato loss of the L4 vein (García-Bellido et al., 1994). This noautonomous effect on L4 formation and the observation tthe L4 primordium invariantly abuts the A/P boundarreinforces the view that signaling across the A/P boundinduces the formation of L4.

Crossregulation between vein and interveingenes consolidates vein boundariesSince the expression of vein, provein and interveingenes is initiated almost simultaneously in third larvalinstar wing discs, it is possible that crossregulatoryinteractions among these early acting genes, as well ascontinued signaling from boundaries, are important forestablishing the vein pattern. To address this question,we examined the expression of vein, provein andintervein genes in early acting vein mutants (Fig. 4),which have been previously shown to disrupt initiation,rather than maintenance, of vein development(Sturtevant et al., 1995; Gomez-Skarmata et al., 1996).Early acting loss-of-vein mutants include the recessivemutants rhove, a cis-regulatory allele of rho that lacksdetectable rho expression in vein primordia (Fig. 4A),vn1 (Fig. 4B), rhove vn1 double mutants (Fig. 4C),iroDFM2 (which behaves as an L3-specific loss-of-function allele of the irolocus and does not survive toadulthood), radius incompletus(ri), which is a likelyregulatory allele of the knirps/knirps-related locus(Lunde et al., 1998) (Fig. 4D) and abrupt(ab) (Fig. 4E).We also examined expression of markers in early actingextravein mutants such as the recessive net mutant (Fig.4F) and the dominant rhoSld enhancer piracy line (Nollet al., 1994; Fig. 4H).

The results of analyzing the initial expression patternsof vein and intervein genes in early vein mutants aretabulated in Table 1 and summarized schematically inFig. 9. Examples of these crossregulatory interactionsare presented in Fig. 5. Two major conclusions can bedrawn from these results. First, crossregulatoryinteractions do play a significant role in establishing theinitial sharp vein-versus-intervein pattern. For example,in vn1 wing discs, which lack detectable expression ofthe EGF-R ligand vn (Simcox et al., 1996), expressionof Dl (Fig. 5A) and rho (Sturtevant and Bier, 1995) is

Fig. expr(browdisc.wild-(browdisc in a primregisthird(bluecoorexprdisc

e L3ed

ds in

theentein

e

dsn-hatyary

virtually eliminated in the L4 primordium. rho-mediatedactivation of EGF-R signaling also contributes to establishinthe vein pattern since both the L3 and L4 stripes of Dexpression are severely compromised in rhove vn1 doublemutants (Fig. 5B). rhoand vnalso collaborate to activate acand sc expression in the L3 primordium as expression of acand sc in broad L3 stripes is lost in rhove vn1 double mutantsdiscs (Fig. 5C,D). The presence of isolated sc-expressing cellsin rhove vn1 discs (Fig. 5D, arrows), likely to be L3 sensoryorgan precursors, may explain why L3 sensilla are usuapresent in rhove vn1 wings (Díaz-Benjumea and García-Bellido, 1990). Finally, rho function is necessary and sufficienfor initiating argosexpression throughout the wing disc (Tabl1).

The iro locus is known to play a central role in establishing thvein pattern in odd-numbered veins. For example, the iroDFM2

mutation causes severe reduction in rho (Gomez-Skarmata et al.,1996), ac (Gomez-Skarmata et al., 1996) and Dl (Fig. 5Eexpression in the L3 primordium. Interestingly, however, thpattern of Bs downregulation in L3 is normal in iroDFM2 mutantdiscs (Fig. 5F). This wild-type expression of Bs contrasts wthe weakened pattern of Bs downregulation in rhove vn1 double

4250

o

rof

othe


Table 1. Expression of vein and intervein genes in early acting vein mutantsMutant

Gene ri1 iroDFM2 ab1 rhove vn1 rhove vn1 net rhoSld

rho −L2 −L3 −L5 −L1-L5 ↓L2,L4 −L1-L5 ect. L2:L3 ect. L2:L3ect. L4:L5 ect. L4:L5

argos NA −L3 −L5 −L1, L3-L5 ↓L4 −L1, L3-L5 ect. L2:L3 ect. L2:L3ect. L4:L5 ect. L4:L5

Delta NA −L3 −L5 wt −L4 −L3, L4 ect. L2:L3 ±ect. L4:L5ect. L4:L5

caup NA −L3 −L5 wt wt wt wt wt

ac NA −L3 NA wt wt −L3 wt wt

sc NA −L3 NA wt wt ↓ L3 wt wt

Bs filled L2 wt filled L5 wt wt filled L2 ↓L2:L3 wt±L3, L4 ↓L4:L5

vn wt wt wt wt −I.V.3 −I.V.3 wt wt

The expression of genes expressed in vein and intervein patterns (left-most vertical column) was examined in various mutant genetic backgrounds (tophorizontal row). Entries in the table indicate whether the expression pattern of a given gene is wild-type (wt) or abnormal in a particular mutant. Examples ofsymbols indicating abnormal gene expression are as follows: −L3, L3 expression is lost; −I.V.3, expression of vn in the intervein region between L3 and L4 inthird instar wing discs is lost; ↓L2, L2 expression is reduced; ect.L2,L3, ectopic veins form between L2 and L3; filled L2, downregulated expression of Bs in L2is not observed (i.e. vein expression = intervein expression); ±L3, L4 = downregulated expression of Bs in L3 and L4 is less pronounced than in wild-type discs;NA = not applicable (e.g. the gene is normally not expressed in a vein affected by the particular mutant in question). Bold entries are shown as data in figures.

mutant wing discs (Fig. 5G). Bs expression in intervein regiois partially dependent on net function since the area of Bsdownregulation in net mutant wing discs is enlarged (Fig. 5Hbrackets) in regions corresponding to those ectopicaexpressing rho (compare with Fig. 6A).

The second major point regarding crossregulatointeractions among vein, provein and intervein genes is tindividual stripes of gene expression may represeindependent units of regulation. This point is mostobvious for the ri and abmutants in which expressionof all relevant vein, provein and intervein markers (e.g.downregulated Bs expression) is strictly dependent onri function in L2 and on ab function in L5. The distinctbehaviors of the L3, L4 and L5 Dl stripes in vn1 versusrhove vn1 mutants described above is another exampleof stripe-dependent regulation of gene expression. Thedifferential requirement for EGF-R signaling to activateexpression of genes in particular veins presumablyreflects differing threshold requirements for EGF-Rsignaling.

Analysis of plexate versus solid ectopic veinphenotypesThe mutant analysis discussed above raises an apparent

Fig. 4. Adult wings of early acting wing vein mutants. (A) Arhove/rhove adult wing. (B) A vn1/vn1 adult wing. (C) A rhove

vn1/rhove vn1 adult wing. (D) An ri1/ri1 adult wing. (E) Anab1/ab1 adult wing. (F) A net/netadult wing. Solid arrowsindicate the P1 and P6 paraveins and the open arrow points tothe P2 paravein. In other netwings, veins can be found in allparavein positions (i.e. P1-P6). (G) A N/+; net/netadult wing.Brackets indicate domains of solid ectopic veins between L2and L3 and between L4 and L5. Note the absence of ectopicveins between L3 and L4. H) A rhoSld/+ adult wing. Note thatectopic veins (brackets) form in the same position as in N/+;net/netwings (G).

ns

,lly

ryhatnt

paradox. The rhoSld mutant has intervein sectors converted intsolid veins (Fig. 4H), while net mutants have a reticulum ofectopic veins of normal thickness running through similaregions of the wing (Fig. 4F). The pattern and duration ectopic rho expression in net(Fig. 6A) and rhoSld (Fig. 6C)third larval instar wing discs and prepupal wings (data nshown) are very similar, however. A clue to the basis for tdifferent adult vein phenotypes of rhoSld versus netflies is


dL4or.us

5

he

irdet

;ar

ed

ns,

interactions among early acting vein and intervein genes. (A) Dlarval instar disc. The intensity of the L4 stripe is reduced (arrow) to expression in other veins in the same disc. Similar results wereprobe indicating that the loss of expression occurs at the level ofression in a rhove vn1 double mutant third larval instar disc. Note thatd L4 stripes is greatly reduced (arrows). Similar results were obtained

) ac in expression in arhove vn1 third larval instar disc is eliminated in(D) sc in expression in arhove vn1 third larval instar disc is lost in theetained in isolated cells likely to be sensory organ precursor cells (theell in the focal plane of the image and the dotted arrow indicates twol plane). (E) Dl expression in a iroDFM2/iroDFM2 third larval instar disc.

the L3 primordium is severely reduced (arrow). iroDFM2/iroDFM2

re identified from a iroDFM2/TM6,βTb,Hu balanced stock based on theer. (F) Bs expression in a iroDFM2/iroDFM2 third larval instar disc isild-type. (G) Bs expression in a rhove vn1 third larval instar disc. Notein L2 is undetectable (arrowhead) and that downregulation in L3 and L4ounced than in wild-type (arrows; compare with Fig. 5F). Bs expression3 and L4 also may be elevated relative to wild-type. H) Bs expression in ar disc. The area of Bs downregulation, particularly anterior to L3 ands), is expanded relative to wild-type (compare with Fig. 5F).

provided by N/+; net double mutants (Díaz-Benjumea et al1990; Sturtevant et al., 1995; Fig. 4G) in which laterinhibition also is compromised. N/+; net flies and rhoSld flieshave solid ectopic veins forming throughout the same intervsectors (Fig. 4G, brackets – compare with Fig. 4H), suggesthat the netsingle mutant phenotype results from the ectopactivation of two competing genetic pathways: a vepromoting pathway and a lateral inhibitory pathway whicrestricts vein formation.

Because Dl encodes a ligand for N and is expressed earlyvein competent domains, we examined the pattern of Dlexpression in net mutant discs. Consistent with Dl playing arole in limiting vein formation in net mutants, Dl RNA (datanot shown) and Dl protein (Fig. 6B) are mis-regulated mulike rho in net mutant wing discs (compare brackets in Fig6A,B). In contrast, Dl RNA (data not shown) and Dl protein(Fig. 6D) are expressed relatively normally in rhoSld wing discs(compare with Fig. 6B). Thus, if we assume that ectopexpression of rho in the absence of effective lateral inhibition(e.g. as in rhoSld flies or N/+; netdouble mutants) generates solidectopic vein phenotypes, thecoordinate mis-expression of rho andDl in net discs and the solitary mis-expression of rho in rhoSld discs couldaccount for the different resulting veinphenotypes of net versus rhoSld

mutants. The observation that genessuch as caup and ac are expressednormally in net mutant discsreinforces the view that the netphenotype results from the selectivecoordinate mis-expression of rho andDl.

Cryptic ‘paraveins’ run betweenvein boundariesA variety of evidence indicates thatbiologically meaningful boundariesalso run between and parallel tolongitudinal vein primordia. We referto these cryptic borders as paraveinboundaries since ectopic veins(paraveins) have a strong tendency toform in these positions in a variety ofextravein mutants (Fig. 4F, arrows;Thompson, 1974). Four likelyparavein boundaries (P2, P4, P5 andP6; see Fig. 7J) can be observed inthird instar wing discs. The position ofthe putative P4 paravein between theprimordia for L3 and L4 can berevealed by a stripe of rho mis-expression in fused mutant wing discs(Fig. 7A). P4 also is marked by a shortectopic vein between L3 and L4 invarious extravein mutants (Sturtevantet al., 1993; Sturtevant and Bier, 1995;see below) and a true vein, which isfound in this position in primitiveinsects, forms along the anterior

Fig. 5. Crossregulatory expression in a vn1 third lrelative to wild-type andobtained using a Dl RNA transcription. (B) Dl expexpression in the L3 anusing a DlRNA probe. (Cthe L3 stripe (bracket). broad L3 stripe, but is rsolid arrow indicates a ccells in an adjacent focaNote that expression inhomozygous larvae weabsence of the Tb markindistinguishable from wthat Bs downregulation is significantly less pronin the sector between Lnet/net third larval instaposterior to L4 (bracket

.,al

eintingicinh

in

ch.

ic

boundary of en expression (see below; Fig. 7H). The proposeP5 paravein boundary runs between the primordia for the and L5 veins in the approximate location of the posteriborder of the spaltexpression domain in third instar discs (Fig7B; Sturtevant et al., 1997). In pupal wings, it is unambiguothat P5 borders the posterior edge of salmexpression. Thus, ashort ectopic section of vein (P5) running between L4 and Lin net/+ adult wings (Fig. 7C) can be visualized in net/+ pupalwings as an ectopic segment of Dl expression abutting tposterior edge of the spaltdomain (Fig. 7D). The positions ofthe P2 and P6 paraveins also are likely to be defined in thinstar discs as revealed by ectopic expression of rho in nmutants. In mid-third instar wing discs, ectopic rho expressionis bounded by L2 anteriorly and by L5 posteriorly (Fig. 6ASturtevant and Bier, 1995). Shortly thereafter in late third instdiscs, however, rhoexpression expands anteriorly beyond L2and posteriorly behind L5 (data not shown). These enlargborders of rho mis-expression in netdiscs are likely tocorrespond to the positions of the P2 and P6 paravei

4252

ryeraid

ertod

nginghe

tis

tsain toPl.,he

ly

l.,sinhets

inntiortofd

owrstsandcouldedrn.mnthis

gte;

in-


Fig. 6.Dl and rhoare mis-expressed in netmutant discs. (A) rhoexpression in a netthird larval instar disc. rhois mis-expressed insolid sectors (brackets) between L2 and L3 and between L4 and but is excluded from the intervein region between L3 and L4. (B) expression in a netthird larval instar disc. Dl is mis-expressed insolid sectors (brackets) similar to rho (see A). Dl RNA expressionsimilarly mis-regulated in netmutant discs, indicating that ectopicexpression occurs at the level of transcription. (C) rhotransgeneexpression in arhoSld third larval instar disc visualized with a 3′untranslated cDNA probe that hybridizes only to the transgenetranscript. Endogenous rho expression is wild type. The transgene iexpressed in solid sectors (brackets) between L2 and L3 and betL4 and L5, but is excluded from the intervein region between L3 aL4, similar to the pattern of endogenous rho mis-expression observedin netmutant discs (A). (D) Dl expression in arhoSld third larvalinstar disc is essentially wild type, although there may be a smalldegree of elevated Dl expression over background between L4 aL5. Dl RNA expression also is largely normal in rhoSld discs.

respectively, since there are ectopic veins that run betweenmargin and L2 (i.e. P2) and between L5 and L6 (i.e. P6) in netadult wings (see solid arrows in Fig. 4F).

The P4 and P5 paraveins also can be marked by rowsectopic bristles in wings of AS-CHw49c or h1 mutants. Thesearrays of ectopic bristles often are associated with fragmeof ectopic vein and line up with particular regularity along thP4 and P5 paraveins in h1 rhove double mutants (Fig. 7E). TheP4 row of ectopic bristles forms at the anterior edge of theenexpression domain in h1 rhove pupal wings (Fig. 7F), whichexpands beyond the A/P lineage compartment boundary the anterior compartment beginning in the late third larvinstar (Blair, 1992). Similarly, an ectopic vein segment presein flies carrying a gain-of-function rho allele (rhoHS-Mod) formsalong the en boundary (data not shown). Consistent with enboundaries having vein-inducing potential, en− clonesgenerated in the posterior compartment of the wing aencircled by ectopic veins that run along the inside border

the

of

ntse

intoalnt

re of

the en− clones (data not shown). The P4 paravein boundaappears to have been conserved during the evolution of Diptsince a bonafide vein, which forms in this location in syrphflies (Fig. 7G: large arrow), abuts the enexpression domainduring pupal stages (Fig. 7H: large arrow). Interestingly, othmorphological features of the syrphid wing also correspond sharp en boundaries in the pupa (Fig. 7G,H: small arrow anarrowhead) suggesting that late enboundaries organize variouslinear features of the adult wing.

DISCUSSION

Boundaries along the A/P axis initiate vein formationThe data presented in this study reinforce the view that wiveins form at boundaries between discrete sectors in the w(Sturtevant and Bier, 1995; Sturtevant et al., 1997). When tlocation of a vein-inducing boundary is alteredexperimentally, the positions of vein markers shifcorrespondingly in register (Sturtevant et al., 1997; thstudy). For example, when the anterior edge of the salmexpression domain is altered, the position of the L2 shifcorrespondingly (Sturtevant et al., 1997). Because the domof salmexpression is determined by a threshold responseDpp emanating from a stripe of cells running along the A/compartment boundary (Nellen et al., 1996; Lecuit et a1996; Lawrence and Struhl, 1996; Singer et al., 1997), tposition of L2 depends most directly on Dpp activity. Incontrast to L2, the positions of the L3 and L4 veins are liketo be determined primarily by Hh signaling along the A/Pcompartment boundary rather than by Dpp (Sturtevant et a1997; Strigini and Cohen, 1997). Consistent with thihypothesis, the positions of several L3 vein and provemarkers shift concertedly in response to displacement of tanterior edge of the Hh-signaling domain. These experimenalso lend further support to the proposal that the L4 veforms along the anterior border of the posterior compartmein response to a signal such as Vn produced in the antercompartment. An important invariant in wild-type and mutandiscs is that narrow vein stripes run through the centers broader domains of provein gene expression andownregulated expression of intervein genes.

A hierarchical model for vein formation atboundariesFour classes of models for activating gene expression in narrversus broad stripes can be entertained (Fig. 8). In the fimodel (Fig. 8A), crude A/P patterning information activategene expression in large A/P sectors such as the anterior posterior compartments. In analogy to embryonisegmentation, genes expressed in these large A/P sectors ccollaborate to activate gene expression in more restrictprovein stripes, which then refine to the final sharp vein patteDuring later pupal stages, veins are indeed selected frobroader vein-competent domains by such a refinememechanism. However, for several reasons, we believe that ttype of model is unlikely to be applicable to initiation of veindevelopment during larval stages. Firstly, there is stronevidence that veins form along borders between discresubdivisions of the A/P axis (Sturtevant and Bier, 1995Sturtevant et al., 1997; this study). Secondly, the broad ve

L5,Dl

is

sweennd

nd


l.,

ersers as inder

Fig. 7.Paravein boundaries run between veinprimordia. (A) rho expression in a fu1 mutantwing disc. fu1 adult wings have L3 and L4shifted closer together. The arrow indicates thelocation of a stripe of rho expression runningbetween L3 and L4. Similar adult and earlypupal phenotypes are observed inknotmutants, which often have a vein segmentrunning between L3 and L4 (data not shown).(B) The pattern of lacZexpression driven froma sal-lacZ enhancer trap insertion (brown)relative to rho expression in veins (blue). Theanterior boundary of this staining territoryabuts L2 and the posterior edge falls half waybetween the primordia of L4 and L5. Thislatter boundary is in the approximate positionexpected for paravein P5. (C) A net/+ adultwing. The arrow indicates an ectopic P5‘paravein’ which frequently forms in thislocation. (D) sal-lacZ (brown) and Dl (blue)expression in developing veins of a net/+heterozygous pupal wing. Dl expression in theP5 paravein (arrow) forms at the posterioredge of the spaltdomain. (E) A h1 rhove/h1

rhove adult wing. Arrows indicate rows ofectopic bristles running between longitudinalveins along the putative P4 and P5 boundaries.(F) A pupal h1 rhove/h1 rhove wing doublestained for En (mAb4D9) and mAb22C10(mAb22C10 labels differentiating neurons).Neurons form at the anterior edge of the Endomain (arrows). En expression is alsodownregulated in posterior compartment veins(marked 4 and 5). (G) An adult syrphid flywing has a vein in the P4 position (largearrow). Other morphological features of thewing include a double vein corresponding toP6 (small arrow) and a wedge shaped fold inthe wing (arrowhead). (H) A pupal syrphidwing stained with Mab4D9 (Mab4D9 cross-reacts with En proteins in various insectspecies). The anterior boundary of Enexpression borders the P4 vein (large arrow).En also is sharply downregulated in posteriorveins and sharp En boundaries in the pupalwing mark other morphological features of theadult wing form (small arrow and arrowhead – compare with G). (I) An adult cranefly wing has veins in locations corresponding toDrosophila paraveins as well as veins.(J) Diagram of an archetypal wing vein patternhypothesized to be ancestral to diverse moderninsects (redrawn from Colless and McAlpinein The Insects of Australia). We propose acorrespondence between our nomenclature foralternating veins and paraveins (L0-L6 and P0-P7) and the standard nomenclature for theprimitive vein pattern (in parentheses).

competent patterns of Dl, ac and sc expression and Bsdownregulation depend on rho, which is expressed in narrowstripes along well-defined A/P boundaries. Finally, expressof Dl, ac, sc and rho is initiated contemporaneously and noaccording to a sequence of broad to narrow stripes. We canhowever, rule out the possibility that a progressive refinemmechanism leads to the pattern of caupand araexpression in

iontnot,ent

the odd-numbered vein primordia (Gomez-Skarmata et a1996; Gomez-Skarmata and Modolell, 1996).

In the second model, A/P patterning generates sharp bordbetween discrete sectors of the wing disc. These sharp bordserve as sources for locally acting signals that function indose-dependent fashion to activate expression of vein genenarrow vein stripes and expression of provein genes in broa

4254

/Prpte

atent

gtep-al).

hegenein isinhe

es.

erl.,

ineinry

ofle,

edofy ete

t

ticve innt

red

al,

nin-ofn-r

nes

e

in


Fig. 8.Models for initiating wing vein development. (A) Progressivrefinement model. Genes expressed in broad domains interact toregulate gene expression in narrower vein-competent domains, wthen refine the vein pattern to sharp lines. (B) A boundary betweeadjacent A/P sectors produces a short-range signal, which activaexpression of genes in narrow vein (black) versus broad provein(blue) stripes based on different threshold responses to the signa(C) A boundary between adjacent A/P sectors induces expressioan intermediate ‘vein-organizing’ gene in a narrow stripe (black).The vein-organizing gene activates expression vein genes, supprexpression of intervein genes and leads to the production of asecondary signal (signal 2), which activates expression of genes broad provein domains (blue) centered over the vein-organizer.(D) Crossregulatory interactions among vein, provein and intervegenes are required to establish the primary vein pattern.

vein-competent domains (Fig. 8B). Activation of Hhresponsive genes in the anterior compartment in broad str(e.g. dpp) versus narrow stripes (e.g. ptc) along the A/Pcompartment border is an example of this kind of mechani(Strigini and Cohen, 1997). According to this model, genexpressed in broad vein-competent domains are activatedlow to high levels of this signal, while genes expressed narrow vein primordia are activated only by high levels signal. One prediction of this model is that broad and narrstripes of gene expression should all be in register along sharp border. As we observe that vein-competent domainscentered precisely over narrower stripes of rhoexpression invein primordia in wild-type and mutant discs, we do not favthis model either.

ipes

smes byin

ofowone are

or

In the third model, signals passing between adjacent Asectors activate expression of ‘vein-organizing genes’ in shavein stripes (Fig. 8C). These vein-organizing genes activaexpression of secondary short-range signals that activexpression of provein genes in broader vein competedomains. Diffusion of Dpp from a narrow stripe of cells alonthe A/P compartment boundary of the wing disc to activasalm expression in a broad central sector centered over dpexpressing cells is an example of this type of symmetricinductive mechanism (Nellen et al., 1996, Lecuit et al., 1996According to this model, sharp vein stripes should run up tmiddle of broader provein domains rather than along one edof them. Another consequence of the third model is that geexpression in broad provein domains would be disrupted mutants failing to produce the secondary signal. This modelconsistent with double-label experiments showing that provedomains are centered over narrow vein primordia and with tobservation that expression of provein genes such as Dl, ac andscare dependent on rho, which is expressed in narrow stripThe non-autonomous action of rho to influence fates ofneighboring cells has also been observed in a variety of othdevelopmental contexts (García-Bellido, 1977; Golembo et a1996b; Bier, 1998; Guichard et al., unpublished data).

Finally, a fourth model is that genes that are initiallyexpressed imprecisely in vein primordia, in proveins and intervein domains crossregulate to establish the sharp vpattern. In this study, we show that several crossregulatointeractions are indeed important in establishing the patternvein-versus-intervein expression of other genes. For examprho and vn, which promote EGF-R activity, are required for theproper expression of Dl, ac, sc and bs(a complete summaryof the observed crossregulatory interactions is presentschematically in Fig. 9). Furthermore, strong expression dominant negative and activated forms of EGF-R pathwacomponents alters gene expression in vein primordia (Rochal., 1998). Another example of coordinated regulation is thsuppression of rho and Dl expression by netin alternatingintervein sectors of the wing. One implication of nefunctioning as a negative regulator of both rho and Dl is thatthese two opposing genes may form a meaningful genesubmodule during vein development. Similar mechanisms habeen shown to underlie patterning of the nervous systemwhich proneural genes both promote neural developmewithin broad competent domains and activate genes requifor lateral inhibition, which restrict the number of cellsassuming neuronal fates (Hinz et al., 1994; Signson et 1994).

In our favored model (Figs 8C,D, 9), boundaries betweediscrete sectors along the A/P axis initiate expression of veorganizing genes, which in turn orchestrate expression various genes in and around veins. A mutant in a veiorganizing gene should lack any hint of vein, provein ointervein features in the position of that vein primordium ithird instar larvae. Candidate mutants in vein-organizing genare ri for the L2 primordium and ab for the L5 primordium.Additional evidence that ri functions as a vein-organizing geneis that this mutation is likely to be a regulatory allele of thknirp/knirps-related locus, which specifically eliminatesexpression of theknirp and knirps-relatedtranscription factorsin a narrow stripe of cells corresponding to the L2 primordium(Lunde et al., 1998). The coordinate regulation of vein, prove

e

hichntes

l. n of

esses

in

in


to therly

insition,nts

etoinve

inghip

isinhebyberint,

Fig. 9.Model for initiating and maintaining vein development on avein-by-vein basis. We propose that vein formation is initiated atboundaries between discrete A/P sectors of the wing disc (indicatedin blue type). The vein-inducing boundary for the L2 primordium islikely to be the border between salm-expressing and salm non-expressing (or weakly expressing) cells (Sturtevant et al., 1997). TheL2 primordium forms within the salmnon-expressing domain ofcells. The vein-inducing boundary for the L3 primordium may be theborder between Hh responding cells expressing high/moderate levelsof ptcand cells expressing very low levels of ptc. The L3 primordiumforms within the domain of very low ptc expression (Phillips et al.,1990; Sturtevant et al., 1997). With respect to the L4 primordium, thevein-inducing boundary is likely to be the A/P compartmentboundary itself. Although the L4 vein is displaced posteriorly by afew cell diameters from the A/P compartment boundary in adult flies,the L4 primordium initially abuts the A/P boundary in third instarwing discs (Sturtevant et al., 1997; this study). Currently, there is nota good candidate border known in the position of the L5 primordium.Vein-inducing boundaries might act directly to regulate geneexpression in and around vein primordia, or might act throughintermediate vein-organizing genes (indicated in red type) toorchestrate gene expression. Mutants lacking the function of a vein-organizing gene should lack expression of all vein markers andshould not downregulate expression of intervein markers in that vein.Based on this criterion, candidate vein-organizing genes are ri for theL2 vein and ab for the L5 vein. For further evidence that rifunctionsas a vein-organizing gene see Lunde et al. (1998). Whether there aresimilar genes acting to organize gene expression in L3 and L4remains to be determined. As depicted in Fig. 8C, we propose thatvein-inducing boundaries and/or vein-organizing genes activateexpression of vein genes (e.g. rho) in narrow stripes, initiate theproduction of locally acting signals that activate gene expression inbroader vein-competent regions centered over veins (e.g. Dlandac/sc) and suppress expression of intervein genes (e.g. bs). These genes then engage in various vein-specific crossregulatory interactions(indicated in black type). Symbol key:Arrows in the diagram, activating interactions; barred lines, suppressive interactions; I.V.1-I.V.5,intervein regions; L1-L5, longitudinal vein primordia; the thick red line labeled A/P, the anterior-posterior compartment boundary.

and intervein genes in L3 suggests that there also may bvein-organizing gene for L3. This stripe-by-stripe form of geregulation in veins is reminiscent of the independent regulatof primary pair-rule genes by distinct stripe-specific enhancelements. The restricted pattern of rho mis-expression in netmutant discs suggests that intervein sectors also may repreautonomous domains of gene expression. Identification aanalysis of cis-regulatory sequences driving gene expressiomultiple veins will be required to determine whetheindependent enhancer subelements control gene expressioindividual veins.

According to our preferred model, boundaries in third instwing discs induce gene expression in narrow vein primordwhich produce short-range signal(s) to activate expressiongenes in broader provein domains. Subsequently, veins selected by lateral inhibition from the broad vein-competedomains during pupal development. It may seecounterintuitive to generate broad provein stripes from narrstripes and then refine them to narrow stripes again. This isthe case, however, since the period of vein initiation and vrefinement represent two very different developmencontexts. During vein initiation, the developmental task is define the locations of vein primordia and to center these vecrudely within broader vein competent domains and regionsintervein gene downregulation. In contrast, during pupdevelopment, dorsal→ventral inductive signals and latera

e aneioner

sentnd

n inrn in

aria, ofarentmow noteintaltoins ofall

inhibition within broad vein-competent domains collaborate assure that the dorsal and ventral components of veins ontwo surfaces of the wing become precisely aligned. Thus, eapatterning events provide an approximate location for veand subsequent refinement processes, such as lateral inhibadjust the fine positions of the dorsal and ventral componeof veins with respect to one another.

Paravein boundaries in Drosophila are likely sites ofvein formation in primitive insectsBoundaries such as the posterior salm border run betweenexisting vein primordia in third instar larvae and early pupa.In various wing vein mutants, ectopic veins, which we refer as paraveins, preferentially form at these mid-intervelocations (Thompson, 1974). In primitive insects, which haup to twice the number of veins as Drosophila, it is likely thatvein development is initiated along boundaries correspondto paraveins as well as veins (Fig. 7I). A possible relationsbetween veins and paraveins in Drosophila and the vein patternin primitive insects is presented in Fig. 7J. According to thmodel, veins form at all vein and paravein positions primitive insects. Vein patterns in insects with fewer than tfull complement of veins generally have been interpreted assuming that ancestral veins are fused into a reduced numof veins (e.g. R2 and R3 being fused to generate L2 Drosophila) (e.g. see Colless and McAlpine, 1970; Nijhou

4256

F

l

ry

nd

s

y

o

c

cy.


1991; García-Bellido and de Celis, 1992). We propose, instethat vein formation is initiated at a subset of vein and paravboundaries present in all insects. According to this view, tpattern of veins generated in a given insect species dependwhich of these boundaries initiates vein formation. Aattractive aspect of this hypothesis is that it provides a reaexplanation for the curious evolutionary fact that, in nearly major orders of insects, there are examples of species that the primitive archetypal vein pattern illustrated in Fig. 7J. Suapparently ‘primitive’ species exist in many orders where itclear that the founding member of that group must have hfewer veins (i.e. because the great majority of species witthat order share a particular subpattern of veins in additionother more advanced characteristics). According to tvein/paravein boundary model, the archetypal vein pattecould re-emerge from an insect lineage having a simplified vpattern by virtue of an atavistic mutation that relievesuppression of vein formation at certain paravein boundarFuture experiments in other insect species will be requiredaddress the origins of different venation patterns.

We thank Raffi Aroian, Dan Lindsley, Karen Lunde, MargareRoark and the helpful reviewers for comments on the manuscript. Twork was supported by NIH Grant No. RO1-NS29870-01, NSF #IB9318242 and NSF #IBN-9604048.

REFERENCES

Biehs, B., François, V. and Bier, E. (1996). The Drosophila short gastrulatiogene prevents Dpp signaling from autoactivating and suppressneurogenesis in the neuroectoderm. Genes Dev. 10, 2922-2934.

Bier, E., Jan, L. Y. and Jan, Y. N. (1990). rhomboid, a gene required fordorsoventral axis establishment and peripheral nervous system developin Drosophila melanogaster. Genes Dev. 4, 190-203.

Bier, E. (1998). Localized activation of RTK/MAPK pathways duringdevelopment. (1998). BioEssays20, 189-194.

Blair, S. S. (1992). Engrailed expression in the anterior lineage compartmof the developing wing blade of Drosophila. Development115, 21-34.

Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a meaof altering cell fates and generating dominant phenotypes. Development118,401-415.

Campos-Ortega, J. A. (1995). Genetic Mechanisms of Early Neurogenesis Drosophila melanogaster. Mol. Neurobiol. 10, 75-89.

Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering antransducing Hedgehog. Cell 87, 553-563.

Colless, D. H. and McAlpine, D. K. (1970). Chapter 34 – Diptera. In TheInsects of Australia, Carlton, Victoria, Melbourne University Press.

Díaz-Benjumea, F. J. and García-Bellido, A. (1990). Genetic analysis of thewing vein pattern of Drosophila. Wilhelm Roux’s Arch. Dev. Biol. 198, 336-354.

Freeman, M., Klambt, C., Goodman, C. S. and Rubin, G. M. (1992). Theargosgene encodes a diffusible factor that regulates cell fate decisionthe Drosophilaeye. Cell 69, 963-975.

Fristrom, D., Gotwals, P., Eaton, S., Kornberg, T. B., Sturtevant, M. A.,Bier, E. and Fristrom, J. W. (1994). Blistered; a gene required forvein/intervein formation in wings of Drosophila. Development120, 2661-2686.

García-Bellido, A. (1977). Inductive mechanisms in the process of wing veformation in Drosophila. Wilhelm Roux’s Arch. Dev. Biol. 182, 93-106.

García-Bellido, A., Cortes, F. and Milan, M. (1994). Cell interactions in thecontrol of size in Drosophilawings. Proc. Natl. Acad. Sci. USA91, 10222-10226.

García-Bellido, A. and de Celis, J. F. (1992). Developmental genetics of thevenation pattern of Drosophila. Annu. Rev. Genet. 26, 277-304.

Golembo, M., Schweitzer, R., Freeman, M. and Shilo, B. Z. (1996a). argostranscription is induced by the DrosophilaEGF-receptor pathway to forman inhibitory feedback loop. Development 122, 223-230.

ad,einhes onndy

allhavech isad

hin tohern

eind

ies. to

this

N-

ning

ment

ent

ns

in

d

s in

in

Golembo, M., E. Raz and Shilo, B. Z. (1996b). The Drosophilaembryonicmidline is the site of Spitz processing and induces activation of the EGreceptor in the ventral ectoderm. Development122, 3363-3370.

Gomez-Skarmata, J. L., del Corral, R. D., de la Calle-Mustienes, E., Ferre-Marco, D. and Modolell, J. (1996). araucanand caupolican, two membersof the novel iroquoiscomplex, encode homeoproteins that control proneuraand vein-forming genes. Cell85, 95-105.

Gomez-Skarmata, J. L. and Modolell, J. (1996). araucanand caupolicanprovide a link between compartment subdivisions and patterning of sensoorgans and veins in the Drosophila wing. Genes Dev. 10, 2935-2945.

Hinz, U., Giebel, B., Campos-Ortega, J. A. (1994) The basic-helix-loop-helix domain of Drosophilalethal of scute protein is sufficient for proneuralfunction and activates neurogenic genes. Cell 76, 77-87.

Johnson, R. L., Grenier, J. K. and Scott, M. P. (1995). patchedoverexpression alters wing disc size and pattern: transcriptional apost-translational; effects on hedgehogtargets. Development121, 4161-4170.

Kooh, P. J., Fehon, R. G., Muskavitch, M. A. (1993). Implications ofdynamic patterns of Delta and Notch expression for cellular interactionduring Drosophiladevelopment. Development117, 493-507.

Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments, andpattern: lessons from Drosophila? Cell85, 951-961.

Lecuit, T., Brook, W. J., Ng., M., Calleja, M., sun, H. and Cohen, S. M.(1996). Two distinct mechanisms for long range patterning bDecapentaplegic in the Drosophilawing. Nature381, 387-393.

Lindsley, D. L. and Grell, E. H. (1968). Genetic variations in Drosophilamelanogaster. Carnegie Institute of Washington, Washington, D. C.

Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophilamelanogaster. San Diego, CA: Academic Press Inc.

Lunde, K., Biehs, B. and Bier, E. (1998). The knirpsand knirps-relatedgenesorganize development of the second wing vein in Drosophila. Development(In press).

Montagne, J., Groppe, J., Guillemin, K., Krasnow, M. A., Gehring, W. J.and Affolter, M. (1996). The DrosophilaSerum Response Factor gene isrequired for the formation of intervein tissue of the wing and is allelic tblistered. Development122, 2589-2597.

Mullor, J. L., Calleja, M., Capdevila, J. and Guerrero, I. (1997). Hedgehogactivity, independent of decapentaplegic, participates in wing dispatterning. Development124, 1227-1237.

Muskavitch, M. A. (1994). Delta-Notch signaling and Drosophilacell fatechoice.

Dev. Biol. 166, 415-30. Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long range

action of a Dpp morphogen gradient. Cell 85, 357-368. Nijhout, H. F. (1991). Development and Evolution of Butterfly Wing Patterns,

Smithsonian Institution Press. Noll, R., Sturtevant, M. A., Gollapudi, R. R. and E. Bier. (1994). New

functions of the Drosophila rhomboidgene during embryonic and adultdevelopment are revealed by a novel genetic method, enhancer piraDevelopment120, 2329-2338.

O’Neill, J. W. and Bier, E. (1994). Double label in situ hybridization usingbiotin and digoxigenin tagged RNA probes. BioTechniques17, 870-875.

Parody, T. R. and Muskavitch, M. A. (1993). The pleiotropic function ofDelta during postembryonic development of Drosophila melanogaster.Genetics135, 527-539.

Phillips, R. G., Roberts, I. A. H., Ingham, P. W. and Whittle, J. R. S. (1990).The Drosophila segment polarity genepatchedis involved in a position-signalling mechanism in imaginal discs. Development110, 105-114.

Roberts, D. B. (1986). Basic Drosophilacare and techniques. In Drosophila:A Practical Approach(ed. D. B. Roberts), pp. 1-38. Washington, D. C., IRLPress.

Roch, F., Baonza, A., Martin-Blanco, E. and Garcia-Bellido A. (1998).Genetic interactions and cell behaviour in blistered mutants duringproliferation and differentiation of the Drosophilawing. Development125,1823-1832.

Schnepp, B. G., Grumbling, Donaldson, T. and Simcox, A. (1996). veinisa novel component in the Drosophila Epidermal Growth Factor Receptorpathway with similarity to the neuregulins. Genes Dev. 10, 2302-2313.

Schweitzer, R., Howes, R., Smith, R., Shilo, B. Z. and Freeman, M. (1995).Inhibition of Drosophila EGF receptor activation by the secreted proteinArgos. Nature376, 699-702.

Shellenbarger, D. L. and Mohler, J. D. (1978). Temperature-sensitive periodsand autonomy of pleiotropic effects of l(1)Nts1, a conditional Notch lethalin Drosophila. Dev. Biol. 62, 423-446.


ts

t

e

ic

Simcox, A., Grumbling, G., Schnepp, B., Bennington-Mathias, C.,Hersperger, E. and Shearn, A. (1996). Molecular, phenotypic, andexpression analysis of vein, a gene required for growth of the Drosophilawing disc. Dev. Biol. 177, 475-489.

Singer, M. A., Penton, A., Twombly, V., Hoffmann, F. M. and Gelbart, W.M. (1997). Signaling through both type I Dpp receptors is required anterior-posterior patterning of the entire Drosophila wing. Development124, 79-89.

Singson, A., Leviten, M. W., Bang, A. G., Hua, X. H. and Posakony, J. W.(1994). Direct downstream targets of proneural activators in the imagdisc include genes involved in lateral inhibitory signaling. Genes Dev.2058-2071.

Strigini, M. and Cohen, S. M. (1997). A Hedgehog activity gradientcontributes to AP axial patterning of the Drosophilawing. Development124, 4697-4705.

Sturtevant, M. A., Roark, M. and Bier, E. (1993). The Drosophila rhomboid

for

inal 8,

gene mediates the localized formation of wing veins and interacgenetically with components of the EGF-R signaling pathway. Genes Dev.7, 961-973.

Sturtevant, M. A. and Bier, E. (1995). Analysis of the genetic hierarchyguiding wing vein formation in Drosophila. Development 121, 785-801.

Sturtevant, M. A., Roark, M., O’Neill, J. W., Biehs, B., Colley, N. and Bier,E. (1996). The DrosophilaRhomboid protein is concentrated in patches athe apical cell surface. Dev. Biol. 174, 298-309.

Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E. (1997). The spaltgenelinks the A/P compartment boundary to a linear adult structure in thDrosophilawing. Development124, 21-32.

Thompson, J. N. Jr. (1974). Studies on the nature and function of polygenloci in Drosophila. II. The subthreshold wing vein pattern revealed inselection experiments. Heredity 33, 389-401.

Waddington, C. H. (1940). The genetic control of wing development inDrosophila. J. Genet. 41, 75-139.

boundaries in the drosophila wing imaginal disc organize vein … · 4246 membrane protein (bier et...

Documents