Induction of a mirror-image duplication of anterior wing structures by localized hedgehog expression in the anterior compartment of Drosophila melanogaster wing imaginal discs

Gene, 148(1994)211-217 0 1994 Elsevier Science B.V. All rights reserved.

0378-l

211

119/94/$07.00

GENE 08145

Induction of a mirror-image duplication of anterior wing structures by localized hedgehog expression in the anterior compartment of Drosophila melanogaster wing imaginal discs (Patched; decapentaplegic;

appendage formation; proximal/distal axis; posterior compartment; positional information)

Tetsuya Kojima, Tatsuo Michiue, Minako Orihara and Kaoru Saigo Department of Biophysics and Biochemistry, School ofScience, Received by Y. Sakaki: 30 March

1994; Revised/Accepted:

Universityof Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan

14 April/l5

April 1994; Received at publishers:

2 June 1994

SUMMARY

The segment polarity gene hedgehog (hh) encodes a secretory protein involved in cell-cell communication in Drosophila melanogaster. The hh gene is expressed in the posterior compartment and is essential for the establishment and maintenance of the anterior/posterior-compartment boundary of each embryonic parasegment [Ingham, P.W., Nature 366 (1993) 560-5621. To clarify possible hh functions in adult appendage formation, we isolated a fly line ( h9D) associated with a wing malformation from among fly lines with an hh transgene whose expression is under the control of trapped enhancers. In h9D flies, the ectopic expression of hh occurred in the anterior edge of wing pouch in the wing disc. This abnormal hh expression resulted in not only a mirror-image duplication and ectopic outgrowth in the anterior wing compartment, but also the ectopic expression of patched and decapentaplegic, strongly suggesting that the hh product serves as a morphogen or an inducer essential for wing development, including the proximal/distal axis formation.

INTRODUCTION

Adult appendages of Drosophila develop from imaginal discs, derivatives of the embryonic epithelium. During development, most discs are subdivided into compartments. In particular, the border between the anterior and posterior compartments is established at an early embryonic stage (for a review, see Jim et al., 1993). As with patterning in embryos, segmentation genes encoding sigCorrespondence Biochemistry,

to: Dr. School

K. Saigo,

of Science,

Department

University

of Biophysics

of Tokyo,

Bunkyo-ku, Tokyo 113, Japan. Tel. (81-3) 3812-2111, (8 1-3) 5684-2394; e-mail: [email protected] Abbreviations:

A/P, anterior/posterior;

bp, base pair(s);

7-3-l

and

Hongo,

ext. 4406; Fax

dpp, decapen-

taplegic gene encoding Dpp; DR, double row; D/V, dorsal/ventral; hh, hedgehog gene encoding Hh; h9D, fly line with the hh minigene; kb, kilobase or 1000 bp; NBNH, non-anterior bristle and non-posterior hair; nt, nucleotide(s); P-D, proximal-distal; PR, posterior row; ptc, patched gene encoding Ptc; TR, triple row; wt, wild type. SSDI 0378-1119(94)00356-W

naling molecules play essential roles in the formation of spatial patterns in imaginal discs. D/V polarity in wing and leg discs is established by the ventral restriction of wg expression (Struhl and Basler, 1993; Williams et al., 1993). Concerted actions of dpp (Spencer et al., 1982; Padgett et al., 1987) and wg with the homeobox gene aristaless at the point where their domains of expression are juxtaposed may be required to specify a distal focus for leg outgrowth (Campbell et al., 1993). Genetic analysis showed another segment polarity gene, hh,to be involved in both embryonic development and adult appendage formation (Mohler, 1988). In a current model concerning the establishment and maintenance of the A/P borders in embryos, Hh is presumed to serve as an extrinsic signal antagonizing the repressive activity of the ptc gene protein on its own and wg transcription (Ingham et al., 1991; Ingham, 1993). In eye discs, developing retinal cells were suggested to drive the progression of morphogenesis utilizing Dpp and Hh

Fig. I. Dosage effects of the cctopic /I/I and endo~enou\ indicates the regions found in hY” mutants. ha\ing in h9” mutants. Anterior

/JIM genes on duplication

up. The wt wing (a) contains five longitudinal

TR (inset 2) and DR (inset 3) are specific to the anterior

male fly hemizygous for hY” (hY”; Y ). L2’ indicates :I duplicate the absence of TR,DR shows campaniform-like show trichomc obtained

( h9U ‘+I.

of the anterior-most

structures found in a broad vein. L3’. (f, g) Enlargements Note

200 pm for (f).

that cxtcnsive change in polarity

Arrowheads

The 23kb

phsphh4. to the blunt-ended

fragment

containing

Srrll site of pCarX723.

scnsilla

show the regions lacking 111TR. (cl A wing of :I

A wing

region of \ein 2. (d) for ptc

an

of ;I female fy hctcl-o/ygou& for both

enlargement

( h9D ‘Y:

of Ihe NBNH

pt~““‘~ ‘+I.

region. Note

Arron head5 in the lnvcl

of the anterior wing margIn of a 11) hemizygous fat- h9”. Arrowheads

of bristles occurs along the eclopic vein L2’. A acalc bar shown in (c) t-cprchenlh

100 pm for (9) and the inset in (d). and 50 pm for insets in (a) and the inset in (e). Methods:

by screening flies with the hsp-/I/I mini-gene constructed fragment).

for hY”

NBNH

protrusion

along with three type\ of bristle ro\vs sllualcd along its ma~-gln.

L2’ and L3’ show duplicates of veins 2 and 3. respectively. The inset i<

Tashiro et al.. 1993) was inserted into the filled-in BMIHI X/WI-XMII

veins. LI-L5.

and PR. (e) A wing of a malt fly hemizygous for hY” and hetero7ygous

directions.

400 mm for (a-e).

+).

wing struc1ure\ in Ries having ~hc h9” mutation.

margin, while PR (inset 4). the posterior margin. Only vein 3 contains campaniform

(see inset I and arrow heads). (b) A wing of :I female Ry heterozygous h9” and ptc (h9” !X: ptc?“”

of anterior

neither sensory brittles nor posterior hairs. Thick arro\4s show the location of ahnormal

as follows. After end-filling.

site of pBlue-hsp-Sm. the /l/r cDNA

a derivative

the 1.9.kb EuJRI

insert of chhl2

of Bluscript SK contaimng

associated with the llsp70 promoter.

a modified Carnegie 20 construct (Rubin and Spradling.

The h9” fy lint wa\ (an 1111cDNA

an hsp70 promoter

clone:

(the 0.4-kb

was removed from the resultant plasmid. 19X3: our unpublished data) after end-filling.

213 (Heberlein action

et al., 1993; Ma et al., 1993). Genetic

between

hh and ptc was also suggested

volved in eye development Recently,

inter-

to be in-

(Ma et al., 1993).

hh was cloned

by four groups

(Mohler

and

Vani, 1992; Lee et al., 1992; Tabata

et al., 1992; Tashiro

et al., 1993). Molecular

analyses

to encode

a secretory

to generate

and cellular protein,

two secretory

possibility

cleavable plural

revealed

suggesting

signalling

and Kornberg,

hh

near the center

polypeptides,

that hh generates

(Lee et al., 1992; Tabata

I

the

molecules

1994; our unpub-

lished data). The aim of the present of ectopic

hh expression

study was to analyze in the anterior

the wing disc, and to determine the formation

of a mirror-image

that of the normal

RESULTS

a possible

the effect

compartment

of

mechanism

for

duplication

along with

P/D axis.

AND DISCUSSION

(a) Isolation of a fly line with the /z/i transgene expressed in the anterior compartment of wing primordia The expression of hh is normally restricted to the posterior compartment of embryonic parasegments and many imaginal discs (Lee et al., 1992; Tabata et al., 1992; Tashiro et al., 1993). Since Hh is presumed to be secretory (Lee et al., 1992; Tabata et al., 1992; Tashiro et al., 1993) and its presumed receptor, Ptc (Hooper and Scott, 1989; Nakano et al., 1989), is distributed throughout the anterior compartment (Phillips et al., 1993; Capdevila et al., 1994), one interesting approach to examine roles of hh in the development of adult appendages may be to ectopitally express hh in a limited number of cells in the anterior compartment within a desired imaginal disc and to learn what morphological changes and/or changes in gene expression are induced. During the course of generating transgenic flies with an hsp-hh minigene, in which an hh cDNA is connected to the hsp promoter, a fly line (h9D) having a pair of wings abnormal in their anterior structures was found (Fig. lb and c). The h9D insertion, whose effect is semi-dominant, occurred at 15A on the X chromosome. During development, no appreciable morphological defects were detected in tissues other than the wings. No appreciable reduction in viability and fertility was detected. Genetic analysis suggested the h9D wing phenotype and ry + eye color ( h9D insertion) to be tightly linked to each other. As described below, the ectopic expression of hh occurred in primordia for the anterior proximal region (the region exhibiting morphological defects) of the h9D wing blade (see Fig. 3b and c). No ectopic expression of the reporter 1acZ gene of Q50, an enhancer trap line of hh, was observed in h9D wing discs (data not shown). This may suggest that the chromosomal inter-

d e

-

_

A

NBNHl

I

P

Fig. 2. Ectopic expression of hh, dpp and gtc in the wing pouch of the h9D/Y wing disc (I) and duplication of anterior structures along the wing margin

in mutant

wing blades

(II) are schematically

shown.

Ptc protein is presumed to be an hh receptor and to function negative regulator for its own transcription and dpp transcription.

(I)

as a Cells

expressing ptc, dpp and hh or other unknown gene(s) in the anterior terminus may form the NBNH region (cross-hatched). The Hh protein secreted from the NBNH

region is presumed

to generate

an A/P-border-

like zone just outside of the NBNH region (left stripe hatched by thin lines), consisting of cells expressing dpp and ptc but not hh. As with the intersection between the A/P-border and the D/V-border, the intersection A/P-border-like

(right stripe hatched by thin lines) between the D/V-border and the

zone may serve as a center for organization

of spatial

pattern along the second P/D axis (see two thick arrows). (II) Distribution of putative positional values along the wing margin in wing discs expressing positional position

value

along

hh ectopically.

The ordinate

the wing margin,

from the proximal

shows

a putative

while the abscissa,

end. Correspondence

between

relative positional

values and adult structures along the anterior wing (or D/V) margin is shown in the right margin. In the lower margin, duplication patterns along

the anterior

(a) to (d) correspond

D/V margin of the discs ectopically expressing hh. to structures shown in Fig. lb to d. Presumed

duplications are indicated by thin arrows. Change in positional value in a-d and that of the wt disc are shown in the graph. TR, triple row. DR, double row. PR, posterior row. L2, vein 2. L3, vein 3. L2’, a duplicate of vein 2. L3’, a duplicate of vein 3. NBNH, the region having neither anterior sensory bristles nor posterior hairs.

ruption itself, generated by the h9D insertion, is unrelated to the ectopic hh expression, and hence, wing defects. The h9D phenotype required no heat treatment. Taken to-

214

215 gether, these results indicate that the h9D phenotype is due to the expression of the hh transgene, which had entrapped an enhancer necessary for the expression in primordia of the wing anterior compartment. (b) A mirror image duplication in the anterior compartment

of the h9D wing blade

In the wt wing, the anterior margin is covered by two types of sensory bristle rows (Fig. la); DR (inset 3), specific to the anterior distal region, and TR (inset 2), characterizing the remaining. The posterior margin of the wt wing is associated with a row of non-sensory hairs (PR; inset 4). In flies heterozygous for the h9D insertion, apparent outgrowth occurs at a point labeled by the arrow in Fig. lb. No appreciable change in morphology was detected in the anterior distal and posterior regions. TR in the proximal anterior margin of the wing blade is often interrupted by regions sparse in sensory bristles and lacking in vein 1 (see arrowheads in Fig. lb). In h9D homozygotes (or hemizygotes), the anterior proximal protrusion was more extensive and vein 2 is bifurcated (Fig. lc). Furthermore, internal hairs (trichomes) in the anterior proximal half, which normally turn toward the distal-most margin, turn toward anterior-most and/or anterior proximal margins (Fig. lg). Trichomes in the anterior distal half are turned toward the distal-most margin as in the case of wt. In an appreciable fraction of homozygotes and hemizygotes, TR or interrupted TR in the anterior proximal margin is replaced by the region having neither sensory bristles nor PR (Fig. Id, inset; hereafter, we will call this region the NBNH region). In the region immediately adjacent to the NBNH region, TR is frequently replaced by a continuous stretch of DR, which was followed by a normal TR stretch in the medial region and the distal DR stretch (Fig. If). Costa1 structures (labeled c in Fig. la) also often disappeared. No apparent change in morphology was observed in the pos-

terior compartment in all homozygotes and hemizygotes for h9D insertion so far examined. Taken together, these results indicate that h9D insertion simultaneously results in abnormal outgrowth in the anterior compartment, a partial mirror-image duplication of the anterior structures, and transformation of the anterior proximal region to one having neither sensory bristles nor PR. (c) Enhancement of the h9D phenotype by dosage reduction of the endogenous ptc gene

The h9D phenotype is enhanced by reducing the copy number of the endogenous ptc gene (Fig. Id and e), whose gene product is presumed to be a negative regulator of its own transcription and to serve as the receptor for Hh in embryos (Ingham et al., 1991; Ingham, 1993). In wings of flies heterozygous for both h9D insertion and ptc’N’08 or ptc7M59 (presumptive functional null alleles of ptc; Hooper and Scott, 1989) the area surrounded by bifurcated vein 2 reduced in size and a new vein-like structure (L3’) appears in the anterior proximal region (Fig. Id). In ptc/+ flies homozygous or hemizygous for h9D, TR expression is restricted only to a small area on the margin, where the plexated vein 2 terminates (Fig. le). The veinlike structure in the proximal anterior region is broadened and is associated with campaniform sensilla (Fig. le, inset), characteristic of the wt vein 3 (Fig. la, inset l), suggesting it to be a broadened duplicate of vein 3. The morphological changes found in the hh and ptc mutants are schematically summarized in the lower margin of Fig. 211. It is apparent that the h9D phenotype becomes progressively severe in the following order: +/+
Fig. 3. Expression patterns of hh, ptc and dpp in wing discs of h9D/Y flies. Compartment structures and location of the wing pouch are illustrated in (c). All discs are oriented anterior to the left. In the wt (a), hh expression occurs only in the posterior compartment. In a h9”/Y wing disc (b), ectopic expression

of hh can be seen in the anterior

discs. (d), second

instar;

tip of the wing pouch (see an arrowhead),

(e), early third instar; (f), middle

which appears

protruding.

third instar; (g), late third instar; (h), a pupal

(d-b) dpp expression

stage. Note that (i) ectopic

in h9D/Y wing dpp expression

becomes evident at an early third instar (e), (ii) cells not expressing dpp emerge in the medial region of the anterior instar and (iii) ectopic outgrowth (see an open thick arrow in h) occurs in the region adjacent to the ectopic-expression

compartment at middle third center of dpp (the arrowhead

in II). The second-instar

hh expression

wing disc of h9D exhibits

no appreciable

hh expression

(i). The first apparent

signal for ectopic

can be detected

at an early third instar (j). The arrowhead in (j) shows a cell-cluster expressing hh in the anterior compartment. (k, I) The wt control of dpp expression. (k), early third instar; (I), late third instar. (m-q) ptc expression in wt and mutant wing discs. (m) ptc expression in h9D/Y at a pupal stage. The open thick arrow shows the location of the ectopic outgrowth, whereas the arrowhead indicates the ectopic-expression center of dpp. (n) Wt ptc expression at an early third instar. (0) ptc expression in the h9D mutant at an early third instar. The arrowhead indicates ptc to be ectopically expressed almost evenly in the anterior compartment, except for the strong expression along the A/P border. (p) ptc expression of the wild type at late third instar. (q) ptc expression in the h9D mutant at late third instar. The arrowhead indicates the location of the region ectopically expressing ptc. Methods: The hh expression was detected by in situ hybridization using in vitro transcription products of the 1.9-kb EcoRI fragment of chhl2 (Tashiro et al., 1993) as a probe (Shishido et al., 1993). The expression of dpp and ptc, respectively, were examined using a fly line carrying lacZ under the control of dpp disc enhancer (Blackman et al., 1991) and r48, an enhancer trap line of ptc (Tashiro et al., 1993). A scale bar in (a) represents 65 mm for (a-c), 25 mm for (d) and (i), 50 mm for (e), (j-k) and (n-o), and 100 mm for (f-h), (l-m), (p) and (q).

216 that a concerted nous and ectopic positional

action

of signals

generated

h/z determines

by endoge-

the final distribution

of

values along the A/P axis in the h9” wing disc

as detailed

in section

Anterior

cells not expressing

in number

with disc development.

that Hh and/or exposure

(d) Induction of the expression ofptc and dpp by hh

the

compartment.

The expression from late third hybridization.

pattern instar

b). As shown

was examined

of hh occurs

(Fig. 3c), corresponding

by in situ

end of the wing pouch of h9D

projecting

by the arrowhead

topic expression end

distribution

(compare

Fig. 3a and

in Fig. 3b, apparent at the protruding to the anterior

ec-

pouchproximal

In h9D wings, region

the second

immediately

NBNH strongest

region

adjacent

values

in

determine

in the

anterior

experiments

in anterior

compartment

and, hence adjacent

cells situated to posterior

cells expressing hh (Fig. 31 and p). Thus, examination of expression patterns of ptc and dpp was made in h9D wing discs using an enhancer trap line of ptc and a line with a 1acZ gene driven by the dpp disc enhancer (Fig. 3g and q). Both ptc and dpp were found to be ectopically expressed in or near the region expressing /z/r ectopically, suggesting that Hh is capable of inducing expression of ptc and dpp in wing discs. In contrast to the expression of ptc and dpp, no appreciable ectopic expression was detected in the case of wg, which expressed along the D/V border in the wt wing primordia at third instar (data not shown). Ectopic expression of dpp, ptc and hh genes became discernible at early third instar, prior to fold formation (Fig. 3e, j and 0). No appreciable ectopic expression of these genes could be detected in second instar discs (Fig. 3d and i). At early third instar, the ectopic hh expression is restricted only to a few small patches within the anterior compartment (see the arrowhead in Fig. 3j), whereas ectopic expression of ptc and dpp occurs in the entire anterior compartment (see arrowheads in Fig. 3e and o), suggesting that Hh is capable of diffusing at least over short distances (several-cell wide) on the surface of the early third-instar wing disc. Note that, at this stage, the dpp stripe in the wt disc (Fig. 3k) is about 55%cells wide and no morphological defect is evident in h9D discs (Fig. 3e). As partly shown in Fig. 3e-g, the area expressing neither dpp, ptc nor hh emerges and progressively expands in the middle of the anterior compartment as the third-instar discs grow older. By the end of third instar, the expression patterns of hh, ptc and dpp in or near the region ectopically expressing hh become similar to those along the A/P border. In the case of the wt disc, about one-third of the cells for the future anterior wing blade expressed dpp at the second instar (see Fig. 3d).

be required fragments

outgrowth

occurs

to the primordia

of dpp/ptc/hh

transplantation

to be

zone by hh in the for the

(Fig. lc). Since Fig. 3h and m show the

misexpression

1975). In the wt wing disc, ptc and dpp are known expressed

could

misexpression

to the NBNH

the A/P border

gradient

and difference

signal

(e) Formation of an A/P-horder-like

corresponding

along

distances

of positional

wing blade and costal regions on the wt fate map (Bryant, strongly

These results suggest

of hh in h9D wing discs dissected larvae

The anterior

wing discs is abnormally

over short

time to the HhiDpp

ectopically expressed in the h9” wing disc

increase

Dpp can form a concentration

by the diffusion

e (see Fig. 211).

r//‘~ progressively

for regeneration (Bryant,

to occur in the area

region showed

in pupal

discs and

the A/P border

and distal outgrowth

1975; Karlsson,

to

of disc

1988) it is reasonable

to presume that an A/P-border-like zone is formed near the second protrusion center of the h9D wing disc, as illustrated in Fig. 21. In normal wing development, the A/P border may be established by the Hh secreted from the posterior compartment, antagonizing the Pet activity to induce dpp and probably some other genes, and to determine the distribution of positional values in the anterior compartment. A mirror-image duplication in h9D wings is likely to be brought about by combining two types of positional information. one from the authentic A/P border and the other due to the A/P-border-like zone generated by the ectopic Hh protein (Fig. 211). We presume that the intersection between the A/P-borderlike zone and the D/V-border, as well as that between the authentic A/P-border and the D/V-border serves as a center for organization of the P/D axis. (f ) Conclusions The hh misexpression in the anterior wing compartment resulted in not only a mirror-image duplication and ectopic outgrowth, but also the ectopic expression of ptc and dpp. This may indicate that Hh serves as a morphogen or an inducer essential for wing development including the P/D axis formation. During the course of preparation of this manuscript, a paper was published by Basler and Struhl (1994). in which the effect of ectopic expression of hh on the formation of wings and legs in Drosophila. Their results are consistent with ours.

ACKNOWLEDGEMENTS

We thank J. Kassis and R. Blackman for Drosophila strains 17en40 and H l-l, respectively. This research was

217 funded in part by grants from the Ministry of Education, Science and Culture of Japan to K.S.

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