Hedgehog-Dependent Patterning in theDrosophilaEye Can Occur in the Absence of Dpp Signaling

DEVELOPMENTAL BIOLOGY 179, 360–368 (1996)ARTICLE NO. 0267

Hedgehog-Dependent Patterning in the DrosophilaEye Can Occur in the Absence of Dpp Signaling

Richard Burke and Konrad BaslerZoologisches Institut, Universitat Zurich, Winterthurerstrasse 190,CH-8057 Zurich, Switzerland

The progression of retinal morphogenesis in the Drosophila eye is controlled to a large extent by Hedgehog (Hh), a signalingprotein emanating from differentiating photoreceptor cells. Adjacent, more anterior cells in the morphogenetic furrowrespond to Hh by expressing decapentaplegic (dpp), suggesting that the relationship between Hh and Dpp might be similarto that in the limb imaginal discs where Dpp mediates the organizing activity of Hh. In this study we show that this isnot the case. Analysis of somatic clones of cells lacking the Dpp receptors Punt or Tkv reveals that Dpp plays only a minorrole in furrow progression and no critical role in subsequent ommatidial development. In contrast, Hh-independent dppexpression around the posterior and lateral margins of the first and second instar eye discs is important for the growth ofthe eye disc and for initiation of the morphogenetic furrow at these margins. q 1996 Academic Press, Inc.

INTRODUCTION ectopic dpp expression produces similar effects to ectopichh expression in the anterior compartment (Basler andStruhl, 1994; Zecca et al., 1995; Ingham and Fietz, 1995).A common feature in the development of multicellular

In the eye primordium there is no division of the disc intoorganisms is the presence of subsets of cells that have thecompartments by cell lineage restriction. Rather, pattern isability to organize the patterning and polarity of sur-controlled by a mobile organizer region, the morphogeneticrounding tissue, possibly by acting as sources of secretedfurrow. Visible as a narrow dorsal-to-ventral indentationsignaling molecules. Classic examples of such organizingdue to apical constriction of the cells constituting the fur-centers include the Spemann organizer in the Xenopus em-row, it is initiated at the posterior margin of the eye discbryo and the ‘‘zone of polarizing activity’’ of the developingat the beginning of the third instar larval stage and sweepsvertebrate limb bud. In the Drosophila larva, two examplesacross the disc in an anterior direction. Within and posterior

of such organizing regions are the compartment boundariesto the furrow, a process of neuronal differentiation is set in

of the wing and leg imaginal discs and the morphogenetic motion, which eventually leads to the development andfurrow of the eye imaginal disc. assembly of the array of identical ommatidial units which

In the wing imaginal discs, the TGFb-like ligand Deca- make up the adult eye (Wolff and Ready, 1991; reviewed inpentaplegic (Dpp; Padgett et al., 1987) acts as the key or- Wolff and Ready, 1993).ganizing molecule controlling patterning along the antero- In analogy to the limb imaginal discs, cells posterior toposterior axis (Gelbart, 1989; Posakony et al., 1991; Basler the morphogenetic furrow in the eye disc express Hh, andand Struhl, 1994; Capdevila and Guerrero, 1994; Zecca et this Hh expression is vital for the forward progression ofal., 1995). The expression of dpp in a row of anterior cells the furrow (Heberlein et al., 1993; Ma et al., 1993). An eye-along the anteroposterior (A/P) compartment boundary (Ma- specific hh allele causes the premature halting of the furrowsucci et al., 1990; Blackman et al., 1991), vital for proper (Heberlein et al., 1993), and raising larvae mutant for a tem-wing development, is maintained by the juxtaposition of perature-sensitive hh allele to the nonpermissive tempera-Hedgehog (Hh)-responsive anterior cells and Hh-producing ture also stops the furrow (Ma et al., 1993). Furthermore,posterior cells (Ingham and Fietz, 1995; Capdevila and Guer- ectopic activation of the Hh pathway anterior to the furrow,rero, 1994; Zecca et al., 1995). The continuous separation either by ectopic hh expression (Heberlein et al., 1995) orof these two cell populations allows Hh secreted from poste- by removal of patched (ptc) or Protein kinase A (Pka) activ-rior cells to induce dpp expression in the adjacent Hh-re- ity, is sufficient to induce ectopic furrows and subsequentsponsive cells of the anterior compartment. Indeed this ap- neuronal differentiation (Ma and Moses, 1995; Pan and Ru-

bin, 1995; Strutt et al., 1995).pears to be Hh’s primary role in wing disc patterning, as

360

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AID DB 8339 / 6x13$$$101 10-20-96 19:29:34 dba AP: Dev Bio

361Role of Dpp in Drosophila Eye Development

The similarity to the wing disc is strengthened by the MATERIALS AND METHODSobservation that Hh induces dpp expression within the fur-row, just anterior to the Hh-expressing cells (Heberlein et Induction of Somatic Clonesal., 1993; Ma et al., 1993). This late expression of dpp is

Adult eyes. Marked clones of cells homozygous mutant for tkv,genetically separable from the dpp expression in the poste-punt, and dpp were generated in a Minute background by Flp-medi-

rior and lateral margins of the eye disc which is present ated recombination (Xu and Rubin, 1993). To induce such clones,throughout early larval development (Blackman et al., 1991; first and second instar larvae of the genotypes w1118 hsp70-flp,Treisman and Rubin, 1995). dpp expression in the furrow tkvstrII FRT40/M(2)25A P[w/] FRT40, w1118 hsp70-flp, dppH61

is lost when the furrow stops (Heberlein et al., 1993; Ma et FRT40 p20 (dpp minigene)/M(2)25A P[w/] FRT40, w1118 hsp70-al., 1993) and is always present in ectopic furrows induced flp, FRT82 punt135, and puntP/FRT 82 M(3)67C P[w/] were subjected

to heat shock (30 min at 357C) to trigger Flp-mediated mitoticanterior to the endogenous furrow (Heberlein et al., 1995;recombination.Ma and Moses, 1995; Pan and Rubin, 1995; Strutt et al.,

Eye imaginal discs. To generate punt, tkv, and dpp homozy-1995). Thus, it appears possible that the relationship be-gous mutant clones marked in the imaginal discs by the loss of thetween Hh and Dpp function in the eye disc is similar to thatPMyc epitope, either in a wild-type or a Minute background, first

in the limb discs, with Hh exerting its role at the organizing and second instar larvae of the genotypes w1118 hsp70-flp, tkva12

center by inducing dpp expression in neighboring cells (He- FRT40/2PMyc FRT40, w1118 hsp70-flp, tkva12 FRT40/M(2)25Aberlein and Moses, 1995). However, a direct functional role PMyc FRT40, w1118 hsp70-flp, dppH61 FRT40 P20/M(2)25A PMycfor Dpp in the furrow is yet to be demonstrated. dpp mutant FRT40, w1118 hsp70-flp, FRT82 puntP/FRT 82 2PMyc, and w1118

hsp70-flp, FRT82 puntP/FRT 82 M(3)67C PMyc were subjected toclones cause a reduction in the size of the adult eyes (He-heat shock (30 min at 34 –357C). Resulting third instar larvae wereberlein et al., 1993), and an eye-specific dppblink allele causessubjected to a second severe heat shock (60 min at 377C) to inducedramatically reduced eyes (Masucci et al., 1990). WhetherPMyc expression. Imaginal discs were fixed after a 1-hr recoverysuch phenotypes are caused by disruption of Dpp activityperiod and stained for PMyc and Elav expression.

at the disc margins or in the furrow remains unclear. The To generate clones expressing the activated tkv constructgoal of this study is to clarify the role of Dpp in the develop- tkvQ253D and marked in the imaginal discs by the loss of the CD2ment of the eye. epitope, first instar larvae of the genotype yw118 hsp70-flp,

TGFb-like ligands are known to mediate their signaling UASúCD2, y/útkvQ253D/C765 were subjected to heat shock (30min at 357C). Third instar larvae were fixed and stained for CD2activities via transmembrane serine–threonine kinase re-and Elav expression.ceptors, of which two forms, termed type I and type II,

Discs were stained for PMyc or CD2 o/n at 47C, then for Elavrespectively, are required to act in concert (Massague et al.,the following day for 2 hr at room temperature.1994). In Drosophila, two such receptors, encoded by the

genes thickveins (tkv) and punt, are both essential for allaspects of Dpp signaling so far examined in the embryo Immunohistochemistry(Nellen et al., 1994; Penton et al., 1994; Brummel et al.,

Staining for PMyc, CD2, and Elav expression was performed1994; Ruberte et al., 1995; Letsou et al., 1995) and in theusing the mouse monoclonal antibodies 9E10 (Evan et al., 1985)wing imaginal disc (Nellen et al., 1996; Lecuit et al., 1996;and OX34 (Serotec), and the rat monoclonal anti-Elav antibody

Burke and Basler, 1996). Thus, we would expect any role of (Robinow and White, 1991), respectively. Donkey anti-mouse FITCDpp in eye development to also be mediated by these two and anti-rat Texas Red conjugated antibodies were used. Expressionreceptors. of the dpp lacZ BS3.0 construct was visualized with a rabbit poly-

Here we have analyzed clones of cells unable to produce clonal anti-bGal antibody (Cappel). Immunofluorescent signalswere analyzed on a confocal laser scanning microscope (MolecularDpp, or lacking tkv or punt activity, and have demonstratedDynamics).that dpp expression in the posterior and lateral margin re-

gions of the eye discs is vital for proper eye development.This expression is needed during the first larval instar for Adult Eye Sectionsthe growth of the disc. Cells lacking the Dpp receptor Tkv

Adult eyes were fixed, embedded, and sectioned as described byshow a strong proliferation disadvantage during this stage,Tomlinson and Ready (1987). Photographs of whole adult headsand eye discs with reduced Dpp activity along the marginswere taken by video microscopy under a normal light microscopeare reduced in size. In addition, initiation of photoreceptorilluminated by an external light source.differentiation and morphogenetic furrow movement at the

posterior margin is blocked by punt and tkv mutant clones,indicating that Dpp signaling is required for these processes. RESULTSIn contrast, we found that the expression of dpp withinthe furrow does not serve an essential role. Neither furrow

1. The Dpp Receptors Tkv and Punt Are Notprogression nor neuronal differentiation is blocked if DppRequired for Photoreceptor Cell Differentiationsignaling is prevented in the furrow. Thus, surprisingly, un-

like in the limb imaginal discs, Dpp does not appear to The provocative expression of the signaling molecule Dppin the morphogenetic furrow has lead to speculation as tomediate the role of Hh signaling in eye development.

Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.


362 Burke and Basler

FIG. 1. Photoreceptor differentiation occurs normally in the absence of Punt or Tkv activity. (A) Section of an adult eye containing alarge punt mutant clone. The clone is marked by the absence of the pigment normally surrounding the ommatidia. Photoreceptordifferentiation occurs normally within the clone. Vacuoles are occasionally observed between mutant ommatidia. In all adult eyes andimaginal discs shown, anterior is to the left. (B) High-magnification view of a tkv null mutant clone. Photoreceptor differentiation occursrelatively normally within such clones.

FIG. 2. Morphogenetic furrow progression is retarded but not halted within tkv and punt mutant clones. All imaginal discs shown areeye imaginal discs from third instar larvae. Left panels show clones visualized by the loss of the anti-PMyc staining (in green). Rightpanels show the same discs stained with anti-Elav (in red). Middle panels show the two stainings superimposed. (A) High-magnificationview of a tkv mutant clone predominantly posterior to the furrow in the disc. Neuronal differentiation (red anti-Elav staining) occursnormally within the clone. The morphogenetic furrow (arrow) has already passed through the main body of the clone. (B) Eye disccontaining two large tkv mutant clones generated in a Minute background. The position of the morphogenetic furrow is again indicatedwith arrows. There is a retardation of neuronal differentiation within these clones. B is shown at half the magnification of A.


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FIG. 3. Dpp signaling is required for growth of the first instar eye imaginal disc. (A) Eye disc containing a tkv mutant clone (arrow, lackof green anti-PMyc staining) and several wild-type twinspot clones (brighter green staining) induced in first instar larvae. (B) Eye disccontaining several punt mutant clones and their twinspots induced at a similar time point to the clones in A. (C) Eye disc containing alarge lateral dpp null clone causing a reduction in the size of the disc in the side containing the clone.

what role Dpp might play in the development of the omma- critical requirement for reception of the Dpp signal in thedifferentiation of cells into neuronal photoreceptor cells.tidial clusters present in the adult Drosophila eye. Dpp may

be required to initiate the neuronal differentiation into pho-toreceptor cells that takes place posterior to the furrow. Ithas previously been shown that photoreceptor differentia-

2. Tkv and Punt Play a Nonessential Role intion can take place in clones of cells homozygous for dppMorphogenetic Furrow Progressionhypomorphic mutations (Heberlein et al., 1993). However,

Dpp is a secreted signaling molecule, and as such, dpp mu-Although ommatidial development takes place normallytant cells could conceivably be rescued by Dpp secreted

within tkv and punt mutant clones, the Dpp receptors mayfrom surrounding cells. To test the cell autonomous re-be required transiently within the furrow as it progressesquirement for Dpp input, we generated clones of cells ho-across the eye disc. Mild defects in this role may not bemozygous for loss-of-function alleles of the genes encodingdetected in adult mutant clones so we generated mutantthe Dpp receptors Tkv and Punt.clones that could be observed in the imaginal disc, theClones of cells homozygous for a hypomorphic punt al-clones being marked by the absence of the PMyc marker.lele, or a null tkv allele, in both cases marked in the adultWe employed a neuronal antibody against the Elav proteineye by the absence of a white transgene, were generated in(Robinow and White, 1991), which stains the differentiatinga Minute background, giving the mutant clones a growthphotoreceptor cells behind the furrow, as an indirect markeradvantage. Very large punt mutant clones were obtained,for furrow progression.sometimes covering almost the entire adult eye. A posterior

Mutant clones anterior to the furrow show no detectablerequirement for punt was observed which will be discussedphenotype since Elav is not expressed in these cells nor-later, but these eyes appeared generally wild-type. Whenmally. Clones posterior to the furrow show normal Elavsectioned, these clones displayed perfectly normal omma-staining (Fig. 2A), consistent with the adult clones whichtidia (Fig. 1A). The only defects observed were occasionalshowed that photoreceptor differentiation occurs normallyvacuoles between the ommatidia, not seen in neighboringin mutant cells and demonstrating that the furrow can pro-wild-type tissue. Only small tkv mutant clones were ob-gress normally through and beyond a mutant clone. How-tained, but again photoreceptor differentiation occurs rela-ever, within clones traversing the furrow at the time oftively normally within these clones (Fig. 1B). Approxi-dissection, neuronal differentiation, as shown by Elav stain-mately 25% of tkv mutant ommatidia are missing one oring, is somewhat retarded, especially in the middle of largemore photoreceptor cells, usually outer photoreceptor cells,

and the normally precise hexagonal array of the ommatidia clones (Fig. 2B). This effect is visible within both punt andis disrupted, both within the tkv mutant clones and in sur- tkv mutant clones, suggesting that the Dpp receptors, androunding wild-type tissue. We attribute the differences in therefore Dpp itself, are required in some aspect of furrowthe effects caused by tkv and punt mutant clones described progression. However, this function must be nonessentialhere and further on to the fact that the punt alleles used or redundant as the furrow is only slightly slowed, but notare not defined molecular nulls, and thus some residual stopped. Interestingly, the slowing effect is more markedpunt activity may be present. in the middle of clones than at the edges, suggesting partial

rescue by surrounding wild-type tissue.From these results it can be concluded that there is no




3. Tkv and Dpp Mutant Clones Reveal a Role for containing the clone (Fig. 3C). The large size of dpp mutantclones described here and later stands in contrast to theDpp in the Early Growth of the Eye Imaginal Discsmall tkv mutant clones and could be due to nonautono-

Dpp is thought to play a role not only in the patterning mous rescue of dpp mutant cells by surrounding wild-typeof the imaginal disc-derived structures, but also in the cells in the small first instar discs where we propose thatgrowth of the imaginal discs, and we have shown previously dpp plays a proliferation role.that Tkv and Punt are absolutely required for cell prolifera- We conclude that early expression of dpp around the lat-tion in the early developing wing pouch of the wing imagi- eral and posterior margins of the eye disc is necessary ini-nal disc (Burke and Basler, 1996). We now present several tially for the normal growth of the disc, supporting ournew observations indicating that Dpp is also necessary for previous proposal that Dpp input is necessary for cell prolif-eye imaginal disc growth. eration in the early developmental stages of some imaginal

As mentioned above, only small tkv mutant clones are tissues (Burke and Basler, 1996).observed in the adult eye, even when induced in a Minutebackground. In fact, visible white clones are only ever seenalong the anterior half of the equator of the eye. In the rest 4. Tkv and Punt Are Required for Initiation of theof the eye black spots and lines, which are actually small Morphogenetic Furrow at the Posterior Marginwhite clones, are observed (not shown). These results indi-cate that tkv mutant clones are severely restricted in their So far we have demonstrated that neither Tkv nor Puntability to grow, implying a strong requirement for the Dpp is critically required within the eye disc for progression ofsignal for cell proliferation in the early eye disc, although the morphogenetic furrow or for the differentiation eventswe cannot rule out the possibility of a second ligand that that occur afterwards. However, as mentioned above, therealso acts through Tkv and is necessary for proliferation. is a posterior requirement for punt function in eye develop-

Growth of punt and tkv clones was compared to that ment, which suggests a role for Dpp signaling in the initia-of their wild-type twinspots in eye imaginal discs. When tion of the furrow at the posterior margin.induced in first instar larvae, punt mutant clones were ob- Adult eyes containing predominantly punt mutant tissueserved throughout the discs in roughly equivalent number are regularly observed, but such eyes always have someand size to their twinspots (Fig. 3B). tkv mutant clones, on wild-type tissue at the posterior margin (Fig. 4A). Eyes com-the other hand, were observed predominantly in the center pletely lacking posterior wild-type tissue are never found,of the disc (Fig. 3A), consistent with such clones in the but flies lacking entire eyes are found (Fig. 4B). This suggestsadult eye. This differential growth was not observed when that reception of the Dpp signal is required at the posteriortkv clones were induced later in second instar larvae. The margin for eye development to occur. No tkv mutant clonesdifferential growth is not due to differences in proliferation are ever visible at the posterior margin of the adult eye, butbehavior in different regions of the disc, as wild-type twin- flies containing tkv mutant clones sometimes have eyesspot clones are able to grow normally and to similar size reduced in size (Fig. 4C) or are missing eyes completely. Inthroughout the disc. Interestingly, the region in which tkv these cases head cuticle is also lost, something not causedclones can grow corresponds to the area furthest away from by punt mutant clones.the source of Dpp in the early discs, at the posterior and Unlike the internal tkv mutant clones described above,lateral margins. The different behavior of tkv and punt mu- clones lacking tkv at the posterior or lateral margins of thetant clones could reflect the unequal strengths of the two eye discs act as absolute barriers to neuronal differentiationalleles. A similar disparity was previously observed in the (Figs. 4D and 4F), indicating that reception of the Dpp signaldeveloping wing pouch where punt clones survive much is necessary for initiation of this process at the posteriormore readily than tkv mutant clones (Burke and Basler, margins. Tkv mutant clones on the posterior margin can1996). Similarly, weaker tkv alleles do not show the prolifer- also cause autonomous overproliferation (Fig. 4F). The tis-ation disadvantage that null tkv alleles display in the wing. sue in these margin clones must be lost as loss of headHowever, we cannot rule out the possibility that a second cuticle and eye structures is observed in adult eyes con-type II receptor is also active and that punt function is par- taining tkv clones.tially redundant. Small punt mutant clones restricted to the posterior mar-

Reduction in eye size correlated with posterior and lateral gin can also cause local overproliferation and block neu-dpp mutant clones has previously been reported (Heberlein ronal differentiation (Fig. 4G). However, in contrast to tkvet al., 1993). We extended this work by generating dpp null clones, neuronal differentiation does occur in large lateralclones in a Minute background in the eye imaginal disc, punt mutant clones (Figs. 4E and 4H). It appears that puntthe clones again marked by loss of the PMyc marker. Discs function is necessary only for the first initiation of the fur-with only a small amount of wild-type tissue at the poste- row at the central stalk region—neuronal differentiation isrior margin (the remainder of the disc being dpp mutant) always centered on wild-type tissue at the stalk (Fig. 4H).were considerably smaller than wild-type discs (not shown). One case was observed where there was no wild-type tissueEye discs were also observed with lateral dpp mutant clones in the stalk region, only at the two lateral margins, and in

this case two areas of neuronal differentiation were ob-that caused a reduction in size only of the half of the disc




served centered on the wild-type tissue at the margins, but dpp expression in the furrow is not required for normal eyedevelopment.not in the central stalk region (not shown).

Again, there is a difference between the effects of puntand tkv mutant clones. It could be that different levels of

6. Ectopic Activation of the Dpp Pathway DoesDpp signal are required at different positions along the mar-Not Lead to Ectopic Neuronal Differentiationgin. Central posterior margin cells must require high levels

of Dpp signaling as furrow initiation in the stalk region is So far we have presented strong evidence that Dpp playsnever observed in the absence of wild-type tissue. At the no critical role in furrow movement or in subsequent differ-more lateral posterior margins, however, the requirement entiation events. One possibility is that Hh mediates itsfor Dpp signal might be lower and could possibly be sup- role in the furrow by inducing two redundant responses,plied by punt mutant cells which still have low remaining one of which is dpp expression. If this were the case, thenpunt activity. activation of the Dpp pathway alone may be sufficient to

Our results indicate that Punt and Tkv activity is required induce responses equivalent to those induced by activationalong the posterior margin, most likely for the initiation of of the Hh pathway, bypassing Hh signaling altogether.the morphogenetic furrow. It has been observed that in spe- Expression of Hh just anterior to the endogenous morpho-cial situations the morphogenetic furrow can move without genetic furrow is able to induce ectopic neuronal differentia-subsequent neuronal differentiation (Jarman et al., 1995). tion by stimulating an additional furrow (Heberlein et al.,However, when eye discs containing punt mutant clones 1995). To test whether ectopic activation of the Dpp path-are double stained for Elav expression and expression of way has the same effect, we generated clones expressing anthe dpp lacZ reporter BS3.0 which marks the progressing activated form of the tkv gene, tkvQ253D, under the controlfurrow, BS3.0 expression is always tightly associated with of the Gal4 driver line C765 which is expressed throughoutneuronal differentiation (Fig. 4I). This indicates that in the the third instar eye imaginal disc (Nellen et al., 1996). Thiscases above, not only neuronal differentiation but also fur- tkvQ253D construct has previously been shown to be capablerow initiation at the posterior margin are being inhibited of eliciting Dpp responses in the embryo and in the wingby lack of punt or tkv. imaginal disc (Nellen et al., 1996). Clones were visualized

by the absence of the CD2 marker, and ectopic neuronaldifferentiation was assayed by monitoring Elav expression.5. Normal Ommatidial Development Occurs in the

While clones expressing hh under the control of C765Complete Absence of Dppwere associated with ectopic Elav expression (not shown),

It has previously been reported that ommatidia form nor- tkvQ253D-expressing clones in analogous positions did notmally within anterior clones homozygous for dpp hypomor- induce any ectopic neuronal differentiation (Fig. 6). Thisphic alleles (Heberlein et al., 1993). However, low re- result indicates that ectopic Dpp signaling cannot bypassmaining levels of dpp expression in cells of such clones the requirement for the Hh signal. Hh appears to act incannot be ruled out, as only hypomorphic alleles were used, the furrow via a mechanism other than induction of dppand potential rescue of mutant cells by Dpp secreted from expression.surrounding tissue must also be considered. In addition,although our results with punt and tkv mutant clonesstrongly indicate that the Dpp signal is not essential in the

DISCUSSIONfurrow, a further possibility is that Dpp utilizes other ser-ine–threonine kinase receptors to mediate its role in thefurrow. In Drosophila wing development, the organizing activity

of the secreted signaling molecule Hh is mediated throughTo address the ambiguities described above, we generatedclones of cells homozygous for dpp null alleles, again in a its ability to induce dpp expression in the Hh-responsive

cells of the anterior compartment. Although the eye imagi-Minute background in order to obtain large clones. Theproblem of haplo-insufficiency of dpp null alleles was cir- nal disc is not divided into compartments, Hh activity is

also essential for the maintenance and forward movementcumvented by using an FRT chromosome containing on theleft arm the dpp null allele H61. On the right arm is a dpp of the mobile organizing center of eye development, the

morphogenetic furrow. Again, Hh induces dpp expressionminigene which rescues the embryonic lethality of the dppallele, but lacks the disc regulatory regions. Using this sys- in adjacent, more anterior cells, leading to the idea that Dpp

may mediate the effects of Hh signaling also in the eyetem we were able to generate large clones in the anteriorof the eye, completely devoid of Dpp production (Fig. 5A). imaginal disc (Heberlein and Moses, 1995). However, our

results provide compelling evidence that this Hh-inducedThe size of the clones effectively rules out the possibilityof rescue by surrounding wild-type tissue as cells in the dpp expression is not essential for the propagation of the

furrow or for subsequent differentiation events. Only a mi-middle of such clones are further away from any wild-typetissue than the most ambitious estimates of Dpp’s range of nor requirement for Dpp is indicated by the slowing of the

furrow in large Dpp receptor mutant clones. This impliesaction. These clones display normal ommatidia throughoutthe entire clone (Fig. 5B), providing further evidence that that in the eye disc, Hh acts mainly via a mechanism other




FIG. 4. Reception of the Dpp signal at the posterior margin is required for eye development. (A) Adult eye containing predominantlypunt mutant tissue (white), with a small amount of wild-type tissue (red) at the posterior border. The eye appears phenotypicallynormal. (B) Head of an adult fly which contains punt mutant clones. No eye structures are seen where the eye normally forms, onlyhead cuticle. Eye loss is presumably caused by a punt mutant clone. (C) Eye of an adult fly which contains tkv mutant clones. Nomutant tissue is observed in the retina but the eye is significantly reduced in size, and surrounding head cuticle is missing. (D, E)Eye discs containing lateral tkv and punt mutant clones, respectively. Clones are marked by the absence of green anti-PMyc staining.Photoreceptor differentiation (anti-Elav staining in red) can extend into punt mutant tissue (E, arrow) but not into tkv mutant tissue(D, arrow). (F, G) Eye discs containing small posterior tkv and punt mutant clones, respectively (arrows). In both cases the mutantclones cause local overproliferation, and neuronal differentiation does not occur within the mutant tissue. (H) High-magnificationview of the posterior-most stalk region of a disc containing predominantly punt mutant tissue. Neuronal differentiation is centeredon the wild-type tissue (green anti PMyc staining) at the stalk but can extend well into mutant tissue, similar to the adult eye shownin A. (I) Double staining of dpp (BS3.0 lacZ reporter line, green staining against bGal) and Elav (red) in a disc containing punt mutantclones. BS3.0 staining in these discs is always just anterior to the differentiating photoreceptors, as in the wild-type situation,indicating that no separation of furrow movement and photoreceptor differentiation is occurring in these discs.

than induction of dpp expression, in contrast to its mode static compartment boundaries of the limb imaginal discs,the organizing center of the eye disc is mobile and in factof action in the limb imaginal discs.

dpp expression in the furrow may be of no great functional ‘‘contacts’’ all areas of the disc at one stage or another,removing the necessity for a mechanism to convey posi-significance, merely reflecting the regulation of dpp tran-

scription by Hh signaling which is so essential in the limb tional information to distant cells.Our results do, however, demonstrate a strong require-discs. As long as dpp expression in the furrow is not deleteri-

ous to eye development, it might have been maintained or ment for the Hh-independent expression of dpp around theposterior and lateral margins of the first and second instareven coopted to play the minor role revealed by our results.

Patterning along the anteroposterior axis of the eye disc eye discs. This early dpp expression is important in thegrowth of the entire young eye disc, implying that Dpp mustmay not even require long-range signals such as the Dpp

signal in the wing disc (Nellen et al., 1996). Unlike the be secreted from its source. This requirement for Dpp is




FIG. 5. Ommatidia development occurs normally in the complete absence of dpp expression. (A) Adult eye section containing a largedpp null clone. (B) High-magnification view of a dpp null clone similar to that shown in A. Photoreceptor differentiation and ommatidialassembly are perfectly normal, even in the center of such large clones.

similar to that in the growth of the wing primordium, which formed in these regions at the expense of head cuticle. Possi-bly punt and tkv mutant clones in the posterior marginas we have previously proposed may reflect a role for Dpp

in the promotion of cell proliferation (Burke and Basler, regions have the same effect on eye development as Wgexpression in the lateral margin, i.e., prevention of furrow1996).

Dpp signaling at the posterior margin also appears to be initiation and formation of head cuticle instead. Wg andDpp may mutually regulate each other’s expression nega-necessary for initiation of neuronal differentiation and the

endogenous morphogenetic furrow. It has previously been tively to inhibit, or promote, respectively, furrow initiationat different positions along the margin.proposed that wg expression at the lateral eye disc margins

serves to inhibit Dpp action and thus furrow initiation in To conclude, we propose that the more relevant dpp ex-pression during eye development appears to be that alongthese regions. When Wg activity is temporarily removed

from these regions using a temperature-sensitive allele, ec- the lateral and posterior margins of the disc rather than thedomain of dpp expression induced by Hh in the morphogen-topic furrows appear at both lateral margins (Ma and Moses,

1995; Treisman and Rubin, 1995), and extra ommatidia are etic furrow.

FIG. 6. Ectopic activation of the Dpp signaling cascade is insufficient to induce ectopic neuronal differentiation. Eye imaginal disccontaining two adjacent clones both expressing an activated form of the tkv gene, tkvQ253D, under the control of the Gal4 driver C765(Nellen et al., 1996). The clones which are marked by the absence of CD2 staining (in green) are positioned on and anterior to the furrow.No ectopic neuronal differentiation (red anti-Elav staining) is observed in such clones.




Ma, C., Zhou, Y., Beachy, P. A., and Moses, K. (1993). The segmentACKNOWLEDGMENTSpolarity gene hedgehog is required for progression of the morpho-genetic furrow in the developing Drosophila eye. Cell 75, 927–We thank Maria Dominguez and Ernst Hafen for comments on938.the manuscript. This work was supported by a grant from the Swiss

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Accepted July 26, 1996121, 2279–2289.



Hedgehog-Dependent Patterning in theDrosophilaEye Can Occur in the Absence of Dpp Signaling

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Transcript of Hedgehog-Dependent Patterning in theDrosophilaEye Can Occur in the Absence of Dpp Signaling