Role of knot (kn) in Wing Patterning in Drosophila

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Copright Q 1997 by the Genetics Society of America



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Wing Patterning



Katerina Nestoras, Helena Lee




Department of Biological Sciences, Barnard College, New York, New York 10027 Manuscript received January 17, 1997

Accepted for publication August 8, 1997


We have undertaken a genetic analysis of new strong alleles of knot ( k n ) . The original kn' mutation causes an alteration of wing patterning similar to that associated with mutations of fused


an apparent fusion of veins 3 and 4 in the wing. However, unlike fu, strong kn mutations do not affect embryonic segmentation and indicate that kn is not a component of a general Hh (Hedgeh0g)signaling pathway. Instead we find that kn has a specific role in those cells of the wing imaginal disc that are subject to ptc- mediated Hhsignaling. Our results suggest a model for patterning the medial portion of the Drosophila wing, whereby the separation of veins 3 and 4 is maintained by kn activation in the intervening region in response to Hhsignaling across the adjacent anterior-posterior compartment boundary.


ATTERN formation along the anterior-posterior (A- P) axis of the Drosophila wing provides an excel- lent opportunity to study mechanisms of Hedgehog- mediated control of developmental patterning. The patterning along the A-P axis of the wing is especially amenable to analysis, because of the array of visible distinct markers on the wing surface. These include five distinct wing veins (numbered 1-5 in anterior to posterior order) and a number of distinguishable bris- tle-types along the wing margin. A large number of mutations affecting wing venation and other aspects of wing morphology have been isolated over the years


Secretion of the Hh protein plays a major role in establishing the organization of the wing imaginal disc (BASLER and STRUHL 1994), which differentiates to form the dorsal mesothorax including the wing. Hedge- hog protein is synthesized and secreted from the cells of the posterior compartment of the wing, under the control of the engruiled selector gene (LEE et al. 1992;

TABATA et al. 1992; TABATA and KORNBERG 1994). Secre- tion of the Hedgehog protein across the anterior-poste- nor compartment boundary results in the apparent in- activation of the Patched protein in the immediately anterior cells (INGHAM et al. 1991; STONE et al. 1996).

Inactivation of the Patched protein leads to the tran- scription of the dpp gene (TABATA and KORNBERG 1994;

ZECCA et al. 1995), which encodes a long-range morpho- gen, Dpp. Diffusion of the Dpp morphogen away from the center of the wing imaginal disc organizes the rest of the wing, notably by activating at different thresholds concentrations of genes encoding the transcription fac- tors, Omb and Sal (DE CELIS et al. 1996; GRIMM and PF'LUGFELDER 1996; LECUIT et al. 1996; NELLEN et al. 1996).

Corresponding author: Jym Mohler, Barnard College, 3009 Broadway, New York, NY 10027. E-mail:

Genetics 147: 1203-1212 (November, 1997)

Activation of transcription of specific target genes by the Hedgehog signal involves a signal transduction cascade including at least two protein kinases [protein kinase A (PKA)


JIANG and STRUHL 1995; LEPAGE et al.

1995; LI et al. 1995; and the product of the fu gene (INGHAM 1993; THEROND et al. 1993)l and the transcrip tion factor, Ci (SLUSARSKI et al. 1995). All of these pro- teins have been implicated in Hedgehog signaling in the embryo (where instead wgis the key activated target gene) as well as the wing (where dpp is the key target gene), although whether they are all required for activa- tion of every target gene is not clear. Although the pathway for activation of dpp transcription is incom- pletely elucidated, one possible hypothesis (STONE et al.

1996) is that the binding of Hedgehog signal to the Patched surface protein, mediated through activation of the membrane protein Smoothened (ALCEDO et al. 1996; VAN DEN HEUVEL and INGHAM 1996), results in the inactivation of PKA and the activation of the Fu

kinase (THEROND et al. 1996). These alterations in ki- nase activities in turn activate the Ci transcription factor and allow for transcription of the target genes, such as dpp. Viable mutations of fu result in a specific wing phenotype: the reduction of the area between veins 3

and 4 and the partial fusion of those two veins ( BUSSON


1204 K. Nestoras, H. Lee and J. Mohler


Strains used for clonal analysis

Strains used for analysis of mutant clones in adult cuticle

"" P l y + = hsl?LP}l; P l y + = neoFRT}42D knKNC#)/P{y+= neoFRT}42D Ply+ J (for knm', knm3, knm4)

w1118 Ply'= hsFLP}l; P{T+ = neaFRT}42D pt? knm(#)/P{y+= neoFRTJ42D Ply+} (for knmz, knm3, knm4)

w'118 Ply' = hsFLP} 1; P l y + = neoFRT}42D ptds2/P{y' = neoFRT}42D Ply+ J

W"'R Ply' = hsFLP} 122; Ply' = neoFRTJ42D knm(" sha/P{ry' = neoFRTJ42D (for knmz)

w1118 P/y+=hsl?LP}122; P{y'=neoFRTJ420 pt2' knm(#) sha/P{yf=neoFRTJ42D (for knm', knm4)



P{y'=hsl?LP}122; P{y'=neoFRTJ42Dpt~?~ shu/P{y'=neoFRT}4W

Strains used of analysis of ptc mutant clones in imaginal discs

P{y'=hsFLp1122; P{y'=neoFRT}42D pt1?/P{z+=neoFRT}42D Ply+ J; P{w", dpp:lacZ}/+

w1118 P{y'=hsFLP}122; P{y'=neoFRT}42Dpt~' kn (#'/P{y+=neoFRT}420 Ply'}; P{w', dpp:lacZ}/+ (for knm3, knm4) w 1 1 1 8 P{y'=hSFLpl122; P{y+=neoFRT}4W pt1?/P$+=neoFRT}42D Ply'}; Plw', ptc:lacZ}/+

:'"8 P l y + = hsFLp/122; Ply'= neoFRT}42D pt6S2 kn (#)/P{y'= neoFRTl42D Ply'}; P{w', ptc:lucZ}/


(for knm3, knm4)

The classical mutation, knoe (kn', as described by BRIDGES a n d BREHME 1944), has a similar wing pheno- type to fu. In this study, we have isolated additional, stronger alleles of kn to determine whether kn, like fu, is also generally required for Hedgehog signal transduc- tion. Instead, we find that kn has a specific role in Hedgehog-mediated patterning of the wing with no a p preciable effect on general Dppmediated wing organi- zation. We find that kn acts in the Hh-responsive cells at the posterior edge of the anterior compartment of the wing, primarily by preventing vein formation by these cells, thus forming a distinct intervein region be- tween veins 3 a n d 4.


Generation of new knot alleles: y w; Dp(1;2)TE90 adult males were fed 12.5 mM EMS in 1% sucrose for 24 hr. The mutagenized males were mated to w; L' kn' females and the F, progeny were screened for wing vein abnormalities. The non-Lobe second chromosome was recovered from mutant

F1 flies.

Mapping of lux kn was mapped by recombination to <0.1 cM to the right of L. kn complemented all deficiencies tested in polytene region 51 that includes L, indicating that it is located in 51BGC3 between the right breakpoint of Df(2R)trix and the left breakpoint of Df(2R)Jpl. This is con- sistent with the knot locus being associated with the left breakpoint of the inversion (51C1-6, 53A) present in an X-

ray-induced mutation, kn" (S. AGARWAL, personal communi- cation).

Generation of germ-he knot mosaics: Germ-line clones were generated by the technique of CHOU and PERRIMON

(1996). Adult females (w"" P { y + = hsFLPJ1; PIw" = wFRT}G13 knm(#)/P{w'c = wFRT}Gl3 P{w" = ovoDl/2Rl P{w+' = ovoDl}ZR2 for knm', knmz and knm4), heterozygous for each of the strong kn alleles and copies of the o v 8 muta- tion transposed to ZR, were collected as adults following a 1 hr 37" heat pulse 5 days post egglay and allowed to lay eggs (presumably homozygous for the strong kn allele). Sibling females that received no heat pulse failed to lay eggs.

Generation of knot clones in adult cuticle and imaginal

discs: The linked FRT-FLP system of XU and RUBIN (1993) was used to generate mosaic clones. Larvae heterozy ous for a strong kn allele either with or without a linked

P t F

allele, homozygous for a FRT element at the base of 2 R , were heats- hocked at 37" for 1 hr between 24 and 36 hr post-egglay. Homozygous clones were identified either by a y- character

caused by the loss of a y+-transposon on the reciprocal chro- mosome, or by a shu- character due to the homozygosity of a linked shu' allele. In general, precise clonal boundaries for both sides of the wing could only be reliably ascertained in s h d clones (which were done for only some alleles), however generally consistent observations were obtained with the yel- low-marked clones in all other alleles. Strains used are shown in Table 1.

To examine the ability of ptc kn mutant clones to activate downstream gene expression, similar heterozygous larvae were generated that also contained a EucZreporter gene under the control of either dpp or ptc. These larvae were heat- shocked to induce clones identically to generation of adult clone and the wing imaginal discs were recovered from ma- ture third-instar larvae and stained for &galactosidase activity. Strains examined for 0-galactosidase expression in wing disc clones are indicated in Table 1.


Isolation and phenotypic characterization of new knot

alleles: The principal effect of the kn' mutation is the narrowing of the intervein space between veins 3 and 4 of the wing a n d the fusions of these two veins in the position of the anterior cross-vein (Figure 1B). Four new EMS-induced alleles of kn were isolated by failure to complement the original kn' allele (named knm',

knmz, knm3 and knm4). Unlike the original kn' allele, each of these four alleles is embryonic lethal when ho- mozygous. The similarity of the phenotype of kn' a n d known fused vu) mutations suggested that kn, like fu, might play a role in Hh-signal transduction. Because the best characterized function of hh in Drosophila de- velopment is in embryonic segmentation (MOHLER


Wing Patterning by knot 1205

FI(;c:RE: 1.-Effect of kn mutations o n vein patterning in the Drosophila wing. Numbers near the wing margin identify wing veins 2-5. (A) Wild type. (B) kn' homozygote. (C) knK.y4/ kn transheterozygote.

to fully viable offspring when outcrossed to kn+ sperm and did not alter the apparent zygotic lethal-kn pheno- type (dead, morphologically normal larvae) when fertil- ized with knK'""' sperm. Because the combination of germ-line and zygotic loss of kn in these three mutants does not disrupt embryonic segmentation, we conclude that kn is not an element in a developmentally con- served Hh-signaling pathway, but instead must play a specific role in medial wing patterning.

In heterozygotes of all four new alleles with kn', the kn phenotype is enhanced such that the intervein space between veins 3 and 4 is highly reduced with extensive fusion of veins 3 and 4, especially in the region normally containing the anterior cross-vein and in the distal re- gions of the wing (Figure 1C). The enhancement of the kn' phenotype when heterozygous with knl, and their homozygous lethality, indicates that these four al- leles represent stronger alleles at the kn locus. The wings from these transheterozygous flies are 80-90% of wild-type size in both length and width; this reduction in wing size appears to reflect the simple loss of the intervein region between veins 3 and 4, since other

regions are approximately the same size. In normal wings, all marginal bristles posterior to vein 3 are un- socketed, while further anterior are interspersed sock- eted, chemosensory bristles. This lack of socketed bris- tles posterior to vein 3 appears to be due to the activity of en, which is expressed in the 3-4 intervein region during late imaginal disc development (BLAIR 1992; HI- DALGO 1995). In these kn transheterozygous wings, both the position of the third vein and the transition point between socketed and unsocketed bristles are shifted coordinately to the posterior. The strict correlation with the transition of socketed to unsocketed bristles with vein 3, found also associated with ectopically induced third veins in ptc or pka mutants (JIANG and STRUHL 1995; TABATA et al. 1995), suggests that this represents a distinct biological domain with a unique character and is not simply a region encompassed by specific wing veins. A principal effect of kn mutants is therefore to reduce the size of this 3-4 intervein domain.

Adult cuticular clones of kn mutants suggest kn is specifically required just anterior to the anterior-poste- nor compartment boundary of the wing: Mosaic clones of the four lethal kn alleles were generated in adult cuticle to determine which imaginal tissues required kn

function for normal patterning. kn mutant clones are normal in all regions of the adult cuticle, unless in- duced in the anterior compartment of the wing in the intervein region between veins 3 and 4. Clones gener- ated just anterior to the A-P boundary are associated either with ectopic veins, characteristic of vein 3 (dorsal veins bearing campaniform sensilla), running within the clone or with the shifting of vein 3 posteriorly to run through the clone (Figure 2). In contrast, clones generated anterior to vein 3 or in the posterior com- partment never gave rise to ectopic veins, even in the case of large posterior compartment clones encom- passing all of vein 4 and running the length of anterior- posterior compartment boundary. The most extreme effect was found associated with extensive kn clones at the posterior edge of the anterior compartment, which resulted in complete loss of the intervein region be- tween veins 3 and 4 (Figure 2A). In general, ectopic veins were induced in the region regardless of whether the clone was located on the dorsal or ventral surface, although dorsal clones generally produced morpholog- ically more robust veins. Veins formed within these clones were often either broader than normal or formed a network or plexus within the clones (Figure 2, B and D). kn clones obey the A-P compartment boundary; however, when the kn clone is induced along the A-P boundary, resulting in an ectopic or shifted vein, there was an associated loss of vein 4 in the non- mutant posterior compartment (Figure 2, B-D). The phenotypes associated with these clones suggest that kn

functions just anterior to the A-P compartment bound- ary of the wing to prevent ectopic vein production.


1206 K. Nestoras, H. Lee and J. Mohler

FIGURE 2.-Effect of strong kn clones on patterning near the A-P compartment boundary. (A and B) Large clones near the A-P compartment boundary. (Clones marked with y-, dashed lines indicate approximate clone boundary.) (A) An extensive knm3 clone near the A-P boundary. (B) A knmf4 clone, the vein formed in the proximal portion of the clone is broader than normal, whereas the distal end of the clone contains two normal-width veins running near either edge of the clone. (C) Close- up of a dorsal knK."2 clone generated near the A-P boundary. (Clone marked with sha, dashed line indicates clone boundary.) The proximal portion of the clone is offset from the A-P boundary and vein 3 is slightly shifted to run along the anterior margin of the clone. The distal portion of the clone runs along the A-P boundary (the marginal bristles in the clone are anterior in character), which contains an ectopic vein along its posterior margin supplanting a normal vein 4 in the adjacent posterior compartment. (D) Close up of a plexus of veins formed in a knK"Z clone, marked with sha. The clone covers all of the ventral anterior compartment shown in the picture and the areas depleted of hairs in the dorsal anterior compartment (---). Note that vein 4 disa ears where the ectopic plexus widens to approach the A-P compartment boundary. (E) Marginal bristles associated with a knK"clone marked with y- (---). Virtually the enure clone, except on its edges, has developed into a broad weak vein, observed as thickened cuticle and an high density of wing hairs. Only a single socketed bristle is present at the anterior edge of the clone (arrow). (F) Socketed bristle associated with the terminus of an ectopic vein induced in a knK." clone (marked with sha-, ---).

sional socketed bristles are found in kn clones in the

3-4 intervein region, most frequently one such bristle at the anterior margin of the clone, but their frequency is far lower than normally observed anterior to vein 3

(Figure 2E). Because socketed bristles are also fre- quently observed on the terminus of ectopic veins that do not reach the margin (Figure 2F), we suspect that the few unsocketed bristles we observe in kn clones may represent "terminal vein 3 bristles" rather than true marginal bristles. However, in these wings, socketed

bristles are always found on the anterior side of vein 3,

despite the fact that the kn clone has invariably reduced the distance between vein 3 and the A-P compartment boundary.

kn mutants do not effect dpp or


activation in ptc


Wing Patterning by k ~ 7 d 1207



reporter gene expression (P-galactosidase) in ptc mutant clones. (A) P-galactosidase expression in dpP:LucZ imaginal discs lacking a ptc- clone. P-gal expression is seen only along the A-P boundary. (E5 and C) &gal expression in the ptc- clone induced in the anterior compartment matches the level of expression along the A-P boundary regardless of whether the clone is also mutant for /in. (E) pic" clone. (C) ptc" kn""\'' clone.

in the center of the wing. Because Dpp is required both for growth of the wing imaginal disc and for patterning along the A-P axis, insufficient activation of Dpp would be expected to result in both a smaller wing and loss

of pattern elements associated with the highest Dpp levels. Such pattern elements would be those normally just anterior to the A-P compartment boundary, which are essentially those affected in kn mutants. To test whether kn mutations disrupt Hh signal transduction in the responsive cells of the wing that might result in lower Dpp activation, we examined P-galactosidase in a reporter construct under dpp or Ptc control in kn'/ lethal-kn transheterozygous flies. For each of our four new lethal alleles, no differences from wild type were detected in either the level of P-galactosidase induction or the pattern of cells expressing the P-galactosidase reporter for either the d f ~ / ~ h c % o r ptc:lncZreporter lines

(data not shown).

Because the pattern defects associated with the trans- heterozygous kn mutations are rather minor, they might be caused by rather modest alterations in Dpp levels that could be difficult to detect. To determine whether the homozygous lethal-kn mutations might affect Hh signal transduction, we compared the ability of p t c and of ptckn double mutant clones to activate Hh-responsive target genes. ptc mutant cells in the anterior compart- ment mimic Hh-responsive cells along the A-P bound- ary, where the p t c receptor is inactivated by Hh and the same array of target genes is activated. P-galactosidase reporter genes either under d/$ control (Figure 3) or ptc control (not shown) were activated to the same de- gree in ptc mutant clones as the endogenous activation along the A-P boundary, regardless of whether the clone was simultaneously mutant for a kn-lethal muta- tion. These data suggest that kn does not cause the loss

of medial wing pattern elements by the reduction of dpp transcription in the medial portion of the wing.

kn mutants affect local, but not global, alterations in

wing patterning associated with ptc inactivation: Be- cause kn. might function to control some posttranscrip tion event in Dpp activation (such as translation, pro- cessing or secretion), we examined cuticular structures associated with doubly mutant p t c kn clones to more critically ascertain the effect of kn mutations on the ability of Dpp induced by p t c mutant clones to reorga- nize the adult wing. Clones doubly mutant for pt? (a strong ptc allele) and lethal kn alleles ( knKAv2, knK,V3 and

knK."4) are fully capable of reorganizing the wing when induced in the anterior compartment. Based on com- parison of clones of similar size and position, the extent of wing expansion and reorganization associated with the ptr kn double mutant clones was similar to clones mutant for p t ~ " alone (compare for example, Figure 4,

A with B and Figure 4, D with E). Furthermore, the double mutant ptc kn clones induced near the anterior margin of the wing are fully capable of inducing a com- plete duplication of the anterior wing blade similar to that of p l c mutant clones (Figure 4C). These results substantiated our conclusion that these strong kn muta- tions do not significantly affect the induction of the Dpp morphogen associated with ptc inactivation.

While the potential for p t c mutant clones to affect global reorganization was unchanged by kn mutations, these mutations radically altered the local patterning around ptc mutant clones. As has been noted previously


1208 K. Nestoras, H. Lee andJ. Mohler



FIGURE 4.-Clonal analysis of strong kn alleles on wing vein patterning. (A-C) Effect of strong kn alleles on reorganization of the wing by ptc clones. (Clones marked with 31.) (A) A small

p d 2

clone between veins 2 and 3 shows a slight expansion of the surrounding tissue (compare with Figure 1A) and an ectopic vein 3 surrounding the clone. (B) A ptcY2 kn".v4 clone, similar in size and position to the plcs2 clone in A, shows a similar expansion of surrounding tissue, but has no surrounding vein, instead a large patch of vein material is in the center of the clone. ( C ) A plcs2 kn""anterior to vein 1 generates a mirror-image "winglet," centered upon a wide vein 3. (D and E) Effect of strong kn alleles on reorganization of the wing by ptc clones. (Clones marked with sha, clone on dorsal surface marked with dashed lines.) (D) A large


clone generated between veins 2 and 3. An ectopic vein 3 forms on either side of the clone. (E) A large ptcs2 kn"." clone, similar in size and position to the ptc clone in D. The center of the clone develops as a wide, thick vein 5 and no ectopic veins are induced outside of the clone. (F and G ) Effect of kn on margin bristles in plr" clones generated in the region between veins 2 and 3. (Clones are marked with y-, approximate boundaries of the clones are indicated by dashed lines.) (F) ptr'? clone, arrows indicate socketed bristles in region flanking the clone but are absent within the clone itself. (G) pie" knK,'" clone. Arrows indicate socketed bristles within the clone.

and possess a few socketed margin bristles (Figure 4G),

characteristic of the margin of the wing at the terminus of vein 3 or slightly anterior. Thus, it appears that the function of kn is to define the 3-4 intervein space, in major part by blocking vein production in Hh-respon- sive cells, as mimicked here by Ptc inactivation.

kn mutants suppress the vein-loss phenotype associ- ated with the weak


allele: While most p t c mutants are embryonic lethal, one weak viable allele of ptc, ptc""',

is characterized by a distortion of the anterior wing and the loss of vein 2 (PHILLIPS et al. 1990). Assuming the role of kn is to block vein formation in ptc-inactivated wing cells, the loss of vein 2 in this weak, loss-of-function


Wing Patterning by knot 1209

FIGURE 5."Suppression of loss-of-vein phenotype of tccZo.

(A) Homo~~ous~/c':';'"wing. (B) Homozygous kn' ptc"'"!ving. ( C ) kn' ptk"/kn'~


ptcf:';z'l wing.

a function of the strength of the kn mutations, in which homozygous kn' partially and heterozygous kn'/knKN2

completely suppress the vein loss caused by ptcG2*. This suggests that the vein loss phenotype of ptccZo is depen- dent on kn activity.


This analysis of strong kn mutations suggests that the

knot gene plays a specific role in Hedgehog-mediated patterning to specify the intervein region between veins

3 and 4. We find that knot plays this role primarily by blocking wing vein formation by the Hedgehog-respon- sive cells near the


boundary in the middle of the wing and secondarily by promoting 3-4 intervein spe- cific differentiation of structures such as the edge mar- gin bristles. The new, strong kn alleles we have gener- ated have no effect on segmentation in the embryo, either when they are lost zygotically or maternally, indi- cating that it is unlikely that knot has a role in Hedge- hog-mediated patterning processes during early em- bryogenesis (although knot has some other role in em-

bryogenesis judging by the embryonic lethality of the four strong alleles). Furthermore, clonal analysis of ho- mozygous clones for these strong kn alleles failed to find any requirement for knot in any adult cuticular structure other than the wing. This suggests that knot

is not required for patterning imaginal discs in general and instead has a specific role in patterning the wing imaginal disc.

The function of knot appears to be primarily to s u p

press vein formation in the 3-4 intervein region and secondarily to allow the elaboration of a developmen- tally distinct 3-4 intervein region. These functions of

knot appear to be dependent on Hedgehog-mediated signaling. knot normally prevents vein production in Hh-responsive cells just anterior to the


compart- ment boundary and in ptc mutant cells elsewhere in the anterior compartment, which mimic normal Hh- responsive cells in the wing. In addition, the vein-sup- pression phenotype of a weak loss-of-function ptc allele,

ptc';20, is dependent on knot function. This dependence upon Hh-signaling for the vein suppression function of

knot suggests that it may be activated as a direct response to Hedgehog-mediated signaling in the wing. Such an activation of knot might work at any level of control, either by direct transcriptional induction, by transla- tional or post-translational control, or by indirect activa- tion such as the induction of necessary cofactors. As a result of its vein-inhibiting potential, knot acts in the Hh-responsive cells to restrict them to an intervein fate. In a normal wing, the Hh-responsive cells along the posterior edge of the anterior compartment therefore form intervein and veins 3 and 4 are induced on either side of this strip of cells.

The sole effect of kn mutant clones on the develop- ment of the adult wing is the specific loss of the in- tervein region between veins 3 and 4, completely if the clone encompasses all of this region as in Figure


This 3-4 intervein region has a distinct developmental character from the other intervein regions in the ante- rior compartment, as defined by the nature of the mar- ginal bristles. Regardless of the position of vein 3 rela- tive to the


compartment boundary, anterior to vein

3 the marginal bristles in intervein regions are primarily socketed, whereas bristles in the 3-4 intervein region,

as in the posterior compartment, are unsocketed. While

knot is necessary to establish this distinct 3-4 intervein domain, it does not by itself define the nature of this region. In kn mutant clones generated posterior to vein

3, except for sporadic bristles that may represent "ter- minal vein 3 bristles," the marginal bristles retain their unsocketed, intervein 3-4 character.

Medial wing patterning mediated by Hh-signaling and its associated knot activation does not appear to depend on modulation of dpp expression. Extreme pat- tern alterations in the medial wing caused by kn mutants do not appear to affect levels of dpp expression, either when assayed by reporter gene expression under dpP


1210 K Nestoras, H. Lee and J. Mohler

of the wing. This suggests that the medial portion of the wing may be patterned essentially independently of Dpp signaling, which acts morphogenically to establish the global pattern of the wing, and implicates a more direct role for Hh signaling in medial wing patterning. The independent role of hh in medial wing patterning is also supported by the observation that local ectopic expression of hh in an anterior clone results in the ectopic production of all anterior compartment struc- tures, whereas ectopic expression of d@ results in pro- duction of only those structures normally anterior to vein 3 (BASLER and STRUHL 1994; ZECCA et aZ. 1995;

MULLOR et aZ. 1997). A similar conclusion has been advanced by MULLOR and colleagues (1997), who have found that uniformly high d@ expression eliminates most pattern of the Drosophila wing with the exception of veins 3 and 4, which are positioned properly with respect to each other. Significantly, modulation of Hh activities in these fixed Dpplevel wing discs results in duplication and altered positioning of medial wing ele- ments. Our results confirm that, while dpp may act to globally pattern the Drosophila wing, hh acts as local organizer of medial wing patterning in which knot plays an important function.

Establishment of the venation pattern in the wing has been proposed to occur in three discrete phases

(STURTEVANT and BIER 1995): (I) global wing pat- terning during early wing disc development, (2) estab- lishment of competent “provein” regions during the third larval instar and (3) restriction of the provein region into discrete vein precursors by lateral inhibition across the width of the provein and vein extension along the long axis of the provein. The establishment of the provein can be detected by the localized expres- sion of rhomboid (rho), a membrane effector of EGF signaling (BIER et al. 1990; STURTEVANT et al. 1993).

Establishment of the proveins requires the “balance” of two classes of genes (STURTEVANT and BIER 1995),

vein promotion genes (including rho itself and compo- nents of the EGF-signaling cascade) and vein suppres- sion genes (which prevent ectopic vein formation in intervein space). Lateral inhibition to restrict the vein precursors within this provein competency region re- quires the function of the Notch/DeZtu class of neur- genic genes; mutations in these genes are associated with thickened veins (Dm-BENJUMEX and GARCU-BE LLIDO 1990). Significantly, mutants of the vein suppres- sion class (such as net or px) form ectopic veins through- out the wing blade except in the intervein region be- tween veins 3 and 4 where we propose knot acts to block vein production.

The phenotype of the knot clones induced in the wing is consistent with the hypothesis that knot inhibits provein activation in the posterior portion of the ante- rior compartment of the wing. knot clones in this region are consistently associated with veins (either ectopic or shifted vein 3), suggestive of provein activation within the knot clone. The veins induced in kn clones are often

thicker than normal veins and in some instances a net- work of veins may run randomly through the clone (as seen in Figure 3D). But the veins induced in kn clones take up only a portion of the clone, consistent with a subsequent lateral inhibition event within the ectopic provein. Furthermore, clones generated near the nor- mal location of veins 3 or


result in suppression of the normal vein and the formation of a vein on the edge of the clone, a result that is consistent with a single vein formed following lateral inhibition within an enlarged, fused provein region (as in Figure 3C).

Loss of Knot function also causes vein formation in

ptc mutant clones in the anterior wing, which mimic Hh-responsive cells. ptc mutant clones are normally as-

sociated with an ectopic vein skirting the margin of the clone (PHILLIPS et aZ. 1990), which indicates that cells of a ptc mutant clone secrete a vein-promoting factor that causes vein formation in the adjacent tissue. Clones mutant for PKA, which also cause ectopic activation of the Hh-signaling cascade, are similarly skirted by ec- topic veins


and STRUHL 1995; LI et al, 1995), suggesting that the secretion of this vein-promoting fac- tor is a consequence of Hh-signaling in the wing. When the ptc mutant clones are simultaneously mutant for

knot, an ectopic vein is still induced, but the ectopic vein covers a wide region in the center of the clone flanked within the clone by intervein material. This re- sult is consistent with the hypothesis that, in the absence of knot, an exceedingly large provein is induced that includes both the entire ptc mutant clone and the flank- ing region around the clone that would normally be induced when wild type for kn. Because lateral inhibi- tion generally acts in clusters


10 cells wide ( SIMPSON

1990), the approximate width of a normal provein (STURTEVANT et aZ. 1993), this exceedingly broad provein would be expected to exceed the normal range of lateral inhibition. A broad vein, or a network of veins, centered within the ptc kn clone would then develop from almost all of the induced provein, except at its peripheral margins as defined by the extent of lateral inhibition. We propose that the knot gene blocks vein production in Ptc-inactivated cells (either mutationally or as a result of Hh-signaling) , making these cells refrac- tory to their own vein-promoting signal. In the normal wing patterning, the Hh-responsive cells just anterior to the A-P compartment boundary would secrete the proposed vein-promoting factor and thereby induce vein 3 anterior to this region of Hh-responsive cells

(where knot is not activated) and vein 4 to the posterior (just inside the posterior compartment).


Wing Patterning by knot 1211

Vein 3



vein2 ----#"

FIGURE 6.-Model for the role of knot in patterning the 3-

4 intervein region. Hh inactivation of the Ptc receptor in posterior-most anterior compartment cells of the wing leads

to the activation of the Ci transcription factor. Activated Ci leads to the transcription/translation or indirect activation of three proteins necessary for vein patterning: Dpp, X, Kn. Dpp is a long-range morphogen that patterns the rest of the wing, specifying the positions of distant veins (veins 1, 2 and 5).

Factor X is a local vein inducer that activates initial step of vein development (provein activation). Kn acts to block the activity of factor X in the cells from which it was secreted (the Hh-responsive cells) and prevent vein development by those cells. As a result, veins 3 and 4 flank the Hh-responsive cells at the posterior margin of the anterior compartment.

for dpp in promotion of vein differentiation within the activated provein region during pupal wing develop ment. However, clones mutant for PKA continue to possess a skirting vein even if also mutant for dpp

(though not the global repatterning associated with


clones), suggesting that secretion of Dpp is not necessary to induce the flanking vein around such clones (JIANG and STRUHL 1995). An alternative candi- date is vein ( v n ) , which encodes an EGF-like ligand that is synthesized at the midline of the dorsal imaginal discs and that is required for the proper formation of vein

4 (SCHNEPP et al. 1996; SIMCOX et al. 1996). Because vein is thought to be a direct ligand for the EGF-recep tor, whose activity is required for provein specification,

vein is likely to have a function in provein formation. However, because vein expression overlaps the poste- rior compartment (and the primordia of vein 4) where

ptc is not normally inactivated, the expression pattern of vein suggests that it might itself be a candidate for a target gene for the proposed vein-promoting factor rather than the factor itself. Ultimately, we know of no clear candidate for the proposed vein-promoting factor. In Figure 6 we propose our model for the role of Hh-mediated signaling in patterning the venation of the medial wing, focusing on the role of kn in that

process. Secretion of Hh across the A-P compartment boundary inactivates the Ptc-receptor in the adjacent anterior compartment cell, leading to the activation of the Ci transcription factor. Activated Ci causes the in- duction or activation of three proteins important for vein patterning: Dpp, which is responsible for global patterning of the wing and activation of distant vein primordia; X, our proposed vein-promoting factor that activates provein development in the flanking cells; and Kn, which blocks induction of vein development by fac- tor X in the cells in which Kn is active (and X is ex- pressed).

It has been proposed that veins are induced in the Drosophila wing just outside of domains defined by the expression of particular selector genes (STURTEVANT et al. 1997). The best characterized example involves the induction of veins 2 and 5 adjacent to the domain de- fined by the expression of the spalt selector genes (salm

and salr), which are activated by moderate to high levels of Dpp (DE CELLS et al. 1996). These spult expressing cells have been hypothesized to secrete a vein-promot- ing factor, to which the spalt expressing cells are refrac- tory and that induces veins in adjacent, non-spaltex- pressing cells (STURTEVANT et al. 1997). In some ways, we hypothesize that knot functions in the medial wing analogously to spalt to induce veins 3 and 4. knot is required for the establishment of a region in the medial wing with distinct developmental identity, as defined by the marginal bristles. knot causes the cells to be refrac- tory to a vein-promoting signal those cells themselves secrete, such that veins 3 and 4 form adjacent to the region of knot activity. However, unlike the case with

spalt, knot is not required for the synthesis of the vein- promoting activity factor itself, which appears to be in- dependently activated in parallel with knot by Hh-signal- ing. Furthermore, while knot is required to establish the 3-4 intervein domain, unlike spalt it does not appear to define the identity of the marginal bristles character- istic for that domain. While knot does not appear to be a selector gene for this domain, nevertheless, like the case for veins 2 and 5 formed adjacent to the spalt

domain, veins 3 and 4 appeared to be positioned in the wing adjacent to a domain of unique developmental character in the wing defined and established, at least in part, through knot function.

The authors thank DAN KALDERON for shemarked chromosomes of our kn mutants, for confirming our observations, for his continuing discussions of medial wing patterning and for his comments on vari-

ous versions of the manuscript. We also thank SHRADHA AGARWAL for the use of her results. This study was fttnded by a grant from the National Institutes of Health (R01HD-22751).


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FIGURE 2.-Effect  of A-P compartment boundary. (Clones marked with vein with a knK"clone with The proximal portion character), which contains an ectopic vein along its posterior margin supplanting  a normal vein of the clone (arrow)

FIGURE 2.-Effect

of A-P compartment boundary. (Clones marked with vein with a knK"clone with The proximal portion character), which contains an ectopic vein along its posterior margin supplanting a normal vein of the clone (arrow) p.4
FIGURE 4.-Clonal  analysis vein surrounding tissue (compare with Figure 1A) and  an ectopic vein of the wing by size and position to the with center of the  clone develops centered  upon a large patch boundaries of the clones are indicated by dashed lines.

FIGURE 4.-Clonal

analysis vein surrounding tissue (compare with Figure 1A) and an ectopic vein of the wing by size and position to the with center of the clone develops centered upon a large patch boundaries of the clones are indicated by dashed lines. p.6
FIGURE 5."Suppression (A) (C) of loss-of-vein phenotype of tccZo. Homo~~ous~/c':';'"wing

FIGURE 5."Suppression

(A) (C) of loss-of-vein phenotype of tccZo. Homo~~ous~/c':';'"wing p.7
FIGURE 6.-Model for the role of vein development (provein activation). Factor cells. activity of factor is a long-range morphogen that  patterns the rest of the wing, specifying the positions of distant veins (veins Hh-responsive cells)  and prevent vein d

FIGURE 6.-Model

for the role of vein development (provein activation). Factor cells. activity of factor is a long-range morphogen that patterns the rest of the wing, specifying the positions of distant veins (veins Hh-responsive cells) and prevent vein d p.9