Mechanisms of Development, 38 (1992) 143-156 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/92/$05.00

143

MOD 00104

Hairless, a Drosophila gene involved in neural development, encodes a novel, serine rich protein Dieter Maier a, Gabriele Stumm b, Karin Kuhn

a

and Anette Preiss

a

" Biozentrum, Department of Cellbiology, Universityof Basel, Basel, Switzerlandand b Institute of Neuropathology, University of Heidelberg, HeMelberg, F.R.G. (Received 5 May 1992; accepted 12 May 1992)

Hairless is a dominant loss of function mutation in Drosophila affecting the formation of adult sensory organs. In the mutants, neuronal precursor cells do not differentiate, suggesting that Hairless might be involved in specifying or realizing neuronal fate in the fly, similar to the 'pro- neural' genes of the achaete.scute complex. As highlighted by the manifold phenotypic interactions of Hairless with most of the neurogenic loci, the gene might play an important role in nervous system development. Therefore, we initiated a molecular analysis of the Hairless locus in order to elucidate the function of its gene product and gain insight into the biochemical nature of the observed genetic interactions in which it participates. Here, we report the molecular cloning of the Hairless locus, confirmed by breakpoint and transformation analysis. Unexpectedly, Hairless activity peaks during embryogenesis, where transcripts accumulate primarily in endo- and mesodermal cell layers, and is lowest during larval stages, the lethal phase of Hairless mutants. The putative Hairless protein deduced from DNA sequencing is extremely basic and highly enriched in serine residues. Hairless appears to encode a novel protein without compelling homology to other known proteins which function in specifying peripheral nervous system development in Drosophila. Drosophila; Hairless; Peripheral nervous system (PNS); Development; Serine rich protein

Introduction The Hairless gene was first described in 1923 by Bridges and Morgan as a haplo-insufficient mutation in Drosophila. In heterozygous flies, a large number of bristles on the head and thorax are lost, and the wing veins, mostly the fourth and fifth longitudinals, are shortened (Lindsley and Grell, 1968; see also Fig. 7). Recently, it was shown that Hairless is required for the differentiation of the precursor cells from which adult sensory organs arise (Bang et al., 1991). Therefore, Hairless function might be analogous to the 'pro-neural' genes of the achaete-scute complex which control sensory organ formation by specifying neuronal fate (Garcia-Bellido and Santamaria, 1978; Dambly-Chaudi~re and Ghysen, 1987; Jan and Jan, 1990). In accordance, a small percentage of Hairless mutant embryos display deletions in the central nervous system (De la

Correspondence to: D. Maier, Biozentrum, Department of Cell Biology, Universityof Basel, CH 4056 Basel, Switzerland.

Concha et al., 1988; Preiss et al., in preparation), reminiscent of the 'anti-neurogenic' phenotype observed in lethal of scute mutant embryos (Jim6nez and Campos-Ortega, 1987). Thus, mutations in Hairless appear to have the opposite effect of mutations in several loci refered to as the neurogenic genes. Mutations in any of these loci, Notch (N), Delta (DI), mastermind (mam), neuralized (neu), big brain (bib) and Enhancer of split (E(spl)) cause a misrouting of presumptive epidermoblasts into the neural pathway resulting in an embryonic lethal hypertrophy of the nervous system (Lehmann et al., 1983). The gene products derived from some of the neurogenic loci, operationally classified as the 'Notch group' of genes (Artavanis-Tsakonas and Simpson, 1991), appear to be involved in a cell communication mechanism which underlies neuronal cell fate choice in the central and peripheral nervous system (reviewed in CamposOrtega, 1988; Artavanis-Tsakonas et al., 1991). While the participation of Hairless in the cell communication mechanism remains to be elucidated, it is clear that Hairless displays dramatic genetic interac-

144 tions with m e m b e r s of the Notch g r o u p of n e u r o g e n i c loci. P h e n o t y p i c i n t e r a c t i o n s a r e o b s e r v e d , for e x a m p l e , in d o u b l e m u t a n t s of Hairless a n d Notch, w h e r e the d o m i n a n t Notch wing p h e n o t y p e is c o m p l e t e l y supp r e s s e d . S t r o n g e n h a n c e m e n t effects are seen b e t w e e n Hairless a n d c e r t a i n Notch alleles, for e x a m p l e split (spl) a n d Abruptex (Ax). M o r e strikingly, c o m b i n a t i o n s of Hairless with c e r t a i n A x alleles a r e even semi-lethal. In fact, t h e r e is a r e m a r k a b l e similarity b e t w e e n A x a n d Hairless m u t a n t s c o n c e r n i n g m a n y p h e n o t y p i c traits ( L i n d s l e y a n d Grell, 1968; L i n d s l e y a n d Z i m m , 1985). A n o t h e r m e m b e r o f the Notch g r o u p of genes, Delta i n t e r a c t s with Hairless in a similar m a n n e r as Notch, s t r e n g t h e n i n g the n o t i o n t h a t the Notch a n d Delta g e n e p r o d u c t s act in a c o m m o n g e n e t i c circuitry ( r e v i e w e d in A r t a v a n i s - T s a k o n a s et al., 1991): Hairless m u t a t i o n s r e p r e s s the d o m i n a n t Delta wing p h e n o t y p e a n d vice v e r s a ( L i n d s l e y a n d G r e l l , 1968). M o r e d r a matically, h o m o z y g o u s Delta cell clones a r e lethal in the a d u l t wild type cuticle, b u t can survive w h e n also m u t a n t for Hairless ( D i e t r i c h a n d C a m p o s - O r t e g a , 1984). Briefly, t h e c o n s e q u e n c e s o f m u t a t i o n s in e i t h e r Notch or Delta a r e relieved by a r e d u c t i o n o f Hairless g e n e activity, suggesting t h a t Hairless c o u n t e r a c t s t h e s e two n e u r o g e n i c genes. In a similar way, m u t a t i o n s in Hairless might cause a r e g r e s s i o n of the n e u r a l h y p e r p l a s i a in n e u r o g e n i c

embryos. This r e m a r k a b l e effect o f Hairless has b c c n r e p o r t e d to occur on the n e u r o g e n i c loci Notch, Delta, mam a n d neuralized with the e x c e p t i o n of Enhancer c~[ split. T h e s e results led to the hypothesis that E(spl) is the m a j o r t a r g e t of Hairless which, t h e r e f o r e , s e e m s to act as n e g a t i v e r e g u l a t o r in the p r o c e s s of n e u r o g e n e s i s only by d e r e g u l a t i n g Enhancer of split (V~issin et al., 1985; D e la C o n c h a et al., 1988). In fact, the gene p r o d u c t s of b o t h loci a p p e a r to function in a c o m m o n d e v e l o p m e n t a l pathway, r e f l e c t e d by a synergistic enh a n c e m e n t of certain p h e n o t y p e s c a u s e d by the d o m i n a n t gain of function allele E(spl) D (Knust et al., 1987). B o t h m u t a t i o n s have, in a d d i t i o n , a very similar e n h a n c e m e n t effect on the Notch allele split (see above; L i n d s l e y a n d Grell, 1968). A l t o g e t h e r , t h e s e o b s e r v a t i o n s suggest a close functional r e l a t i o n s h i p b e t w e e n the g e n e p r o d u c t s of Hairless a n d the Notch g r o u p of the n e u r o g e n i c loci. In o r d e r to e l u c i d a t e the function o f the Hairless g e n e p r o d u c t s a n d gain insight into the m o l e c u l a r basis u n d e r l y i n g t h e d e s c r i b e d g e n e t i c i n t e r a c t i o n s we have i n i t i a t e d a g e n e t i c a n d m o l e c u l a r c h a r a c t e r i z a t i o n of the Hairless locus. In this p a p e r we d e s c r i b e t h e p h e notypic, c y t o g e n e t i c a n d m o l e c u l a r analysis o f a large set of Hairless m u t a n t s . T h e Hairless gene has b e e n cloned, s e q u e n c e d a n d its t r a n s c r i p t i o n a l activity a n a lyzed t h r o u g h o u t d e v e l o p m e n t with an e m p h a s i s on e m b r y o g e n e s i s . S e q u e n c e analysis p r e d i c t s a novel, ex-

TABLE 1

Hairless alleles Allele

Origin

Cytology

Phenotype

Source

Hi H2 H3

spont, spont,

H 57c

X-ray X-ray X-ray EMS ENU spont. X-ray X-ray X-ray X-ray X-ray X-ray

normal normal normal Tp(3R) (not correlated with Hairless) normal Df (3R) 91F5/9-92EI2/15 plus Tr (3; Y) Inv (3R) 92E12/15-93A1/2 normal Df (3R) 92B/C-93A1/2 Df (3R) 92E9-E 15 normal normal normal normal Tr (2/3) 90 F 4/11-56F/57A (Tr not correlated with Hairless) normal (no P-element in Hairless) normal (no P, but hobo in Hairless) normal (no P-element in Hairless) Df (3R) 92B2/3-92F13/93A1

strong weak/int. weak/int. nd nd amorph intermediate strong amorph/sterile * amorph/sterile strong nd nd/(sterile) nd nd/(sterile)

1 1 1 1 2 3 4 5 6 7 7 7 7 7 7

weak (weak) intermediate amorph

8 8 8 8

H wl1 H Kx2 H 8sd H b,,h

H 89c27 H sl H99 H 107 H t33 H t3~ H 16~ H A120

H B8 H 94a

H B79

dysgenic dysgenic dysgenic dysgenic

Phenotype: refers to pupal survival rate (see Table 2) and dominant female sterility observed in most (or only certain) genetic backgrounds. Strong, behaves like amorphs; intermediate, larval lethal up to 40% heterozygous pupae in trans over weak alleles; weak/int., larval lethal, more than 40% trans-heterozygous pupae observed; weak, homozygous pupal lethal (not fully penetrant); nd, not determined, * lost. Source: (1) Lindsley and Zimm, (1985). (2) Jimenez, F. and Campos-Ortega, J.A., unpublished. (3) Schronz, H. and Campos-Ortega, J.A., unpublished. (4) Welshons, W., unpublished. (5) Fleming, R.J. and Artavanis-Tsakonas, S., unpublished. (6) Lewis, P. and Kankel, D., unpublished. (7) Preiss, A. and Artavanis-Tsakonas, S., unpublished. (8) Tepass, U. and Campos-Ortega, J.A., unpublished.

145 tremely basic gene product of about 110 kDa which is highly enriched in serine residues. Transformation with wild type genomic D N A rescues the Hairless mutation and indicates that overexpression causes an anti-Hairless phenotype.

Results

Genetic analysis of Hairless The Hairless (H) gene (3-69.5) has been placed cytogenetically on the right arm of the third chromosome between 92 D and 93 B (V~issin et al., 1985). In order to define the locus more precisely, additional Hairless alleles were isolated from an X-ray mutagenesis screen and analyzed for cytological rearrangements as were all other Hairless alleles available to us. Only four out of 19 alleles are deficient for the Hairless region (Table 1) and three of these deficiencies involve a pronounced dominant female sterility in most genetic backgrounds (see also Materials and Methods). The fourth deficiency, H T M could not be tested since it includes a translocation to the Y-chromosome and, therefore, only males are mutant (Table 1). Sterile females show no overt defects in the ovaries, however, lay only few, apparently unfertilized eggs. Dominant female sterility was also observed in other, cytologically and molecularly normal alleles (e.g. HI33, H 169, Table 1; see below) suggesting that this phenotype is closely linked to Hairless. The deficiencies H T M and H sl are lethal in trans, allowing the gene to be m a p p e d to the interval of 92 E5-14, which also contains the proximal inversion breakpoint of H ssd at 92 E12/14. Three cytologically normal Hairless alleles which were derived from a hybrid-dysgenic screen (gift from U. Tepass and J.A. Campos-Ortega) were analyzed by in situ hybridization

to polytene chromosomes for insertions of either P- or hobo-elements as the latter can also be mobilized in P-M dysgenic conditions (Blackman et al., 1987). No P-elements were detected in the Hairless region. However, among the approximately 70 hobo elements found in H B8 one is located in the Hairless gene, as confirmed by the molecular analysis (see below). The cytological position of this hobo-insertion coincides with the H 88d inversion breakpoint, mapping Hairless into (3R) 92 E12/14. All Hairless alleles listed in Table 1 are recessive lethals. The lethal phase of H 2 was determined to lie at the transition from first to second instar larval stage with a slight delay in larval development. Some of the mutant H 2 animals developed to third instar larvae. H A I 2 ° homozygotes even developed to bald pharate adults, which did not emerge from the pupal case. These could be also observed for H ~s in rather low numbers (Table 2). In order to rule out that lethality is only a consequence of second site mutations a complementation analysis was performed. For these experiments, Hairless alleles were balanced over TM6B to be able to distinguish unambiguously the homozygous Hairless larvae and pupae from their siblings by the dominant Tubby marker (Craymer, 1984). In fact, most combinations allowed pupal development of trans-heterozygotes at a low frequency. However, only crosses with H BS, H2 and H3, respectively, resulted in high numbers of pupae and also of pharate adults, indicating that these three alleles are rather weak (Table 2). These observations are in contrast to the report of Bang et al. (1991) who considered the H 2 and H 3 alleles as amorphic mutations. Besides the complete female sterility of the two deficiencies HB79 and H s~ when balanced over TM6B no marked difference in the direction of the crosses was observed. The complementation analysis, based on the development of Hairless pupae and pharate adults allowed

TABLE 2 Hairless c o m p l e m e n t a t i o n H 1

H 2

H 3

H Be

H 88d

H 99

H 94a

H b°h

H a79

HSl

_ ++ + + + + + +

+ -++ + + + + + + + +

++ ++ + + + + + +

+ ++ ++ + + + + + +

+

+ + ++ +

+

+ +

+

+

+ +

+ + + +

+ (_ )

++ + + + + + +

H B79

S

S

S

S

S

S

S

S

S

S

H sl

S

S

S

S

S

S

S

S

H 1 H2 H 3 H Bs

H H H H

ssd 99 94a bob

+ + + + + +

+ + +

- , 0 - 3 % larval ( p u p a l ) lethal; + , 3 - 4 0 % Hairless p u p a e ; + + , > 4 0 % Hairless p u p a e ; (bold), 1 0 % - 2 2 % Hairless p h a r a t e adults; S, sterile (no viable larvae). This t a b l e gives the p e r c e n t a g e o f the e x p e c t e d n u m b e r of p u p a e w h e n c o m p a r e d to t h e n u m b e r o f p u p a l cases f r o m the T M 6 B b a l a n c e d siblings. P u p a e : includes p r e p u p a e a n d p u p a e . P h a r a t e adults: r e f e r s to a n i m a l s w h e r e eyes a r e d i s c e r n i b l e ( f r o m stage P 8 / 9 on; A s h b u r n e r , 1989).

146 us to establish the following phenotypic series: H AI2°

For example, H l has on average less bristles at most sites than H 2 (Ashburner, 1982), but the variations are minor. W e a k alleles like H AI2° overlap with wild type as they miss at most 3 m a c r o c h a e t a e (on average 1.5).

> H -~ > H 2 > H B8 > H 88d > H 94a > H bob > H 9') = H > > H s~ = H B79. Phenotypic differences that fit the above series can also be found in heterozygous adults.

a proximal

Phage clones

distal

30

20

I

I

10 I

0

physical map cDNA clones

-20

I

-30

I

HB-I HA 2

-40

i

-50

I

-60

I

K/Oli - 1

-70

I

-80

I

K)

HBR-1

Cosmid clones

-10

I

i

K/Lu IA

Gap - 1

K 3/6

K/Lu 6-1

cos Lu 10

cos Hair cos May 81

cosMo3-5

t ........ l ...........

-_.2LLJ ...... I

t i

.............!-' ~,! L'.......... r:~..... t

I

[]

/

adm

h

rea

\

/

\

/

b

\

\

/

\ \ R

/_//94a

H

X

\

/

~ B8 / PC R

x

x

I

"H 99

~

I

\ N

Sc

Sc

III

\

/s'ue/mic

./

I I

I I

a~

I N I

I

H

/

~ H

-* . . . . . . . . . . . . . . . . . . . . . . | . . . . . . . .

HR

BNN '1

l

I

H

X

X

J,~-...., .,~ -....,. ~ ....,

R

,88d

\

R -,. i b,..._ \ r 1 H 1 kb

P

Fig. 1. Cloning of the Hairless region and breakpoint mapping of Hairless alleles. (a) About 110 kb of genomic D N A of the Hairless region were cloned in a chromosomal walk. A physical map with EcoRl restriction sites is shown (dashes; starred sites are polymorphic). The entry point to walk (point 0), the adm124BlO locus, is indicated. Genomic D N A clones were isolated from different phage libraries and a cosmid library as detailed in Materials and Methods; only representative clones are depicted. In the region from + 30 to - 5 0 two repetitive areas were detected (hatched bars underneath the map). Transcriptional activity during embryonic and larval stages in the same region, as reflected by a 'reverse Northern' experiment, is indicated above the map (shaded boxes). Restriction fragments hybridizing to isolated cDNAs (adm, rea, h, rnic, sue) a r e depicted. The orientation of the walk in relation to the chromosome was determined by in situ hybridization of cosmid clones to polytene salivary gland chromosomes. (b) Hairless alleles as listed in Table 1 were scanned for genomic alterations. Only seven alleles revealed a n a b e r r a n t genotype and all breakpoints were mapped within the about 6 kb h transcription unit indicated by the black arrow (open boxes: introns; arrow indicates direction of transcription). Both dysgenic Hairless alleles H 94a and H n8 contain insertions, as do the spontaneous alleles H I and H 2. In H ns an incomplete type 102 hobo element is inserted in the 5' untranslated leader opposite to the direction of Hairless transcription. Very close by is the insertion of an undescribed element of approximately 6.9 kb in H 2. Also H 94a bears a novel element of about 4 kb. H I appears to carry a complete copia element (Mount and Rubin, 1985) in the second intron of the Hairless transcription unit. In the X-ray induced allele H 99 a deletion of about 1.5 kb might affect the very 3' portion of the putative Hairless protein. The EMS induced allele H ssd has a small, cytologically visible distal inversion and seems to carry, in addition, a proximal deletion of undefined length. The origin of the alleles is listed in Table 1. Restriction sites are: B, BarnHI; H, Hindlll; N, Notl; R, EcoRI; Sc, SacI; X, Xhol. NotI and Sacl sites are only shown in the genomic wild type DNA; PstI sites are presented only in the copia element. The adjacent mic transcription unit is indicated by the black bar.

147 On the other hand, H A12° flies show a distinct Hairless wing venation phenotype. Although this wing phenotype is very much background dependent (in preparation) it is an excellent marker for weak Hairless alleles (see also Fig. 7).

Molecular cloning and transcriptional analysis of the Hairless region The Hairless region was cloned by classical chromosomal walking using the adm124BlO cDNA clone as an entry point (generously provided by M. Wolfner and previously mapped into the cytological interval of 92 E) (Wolfner, 1980). The cloned region comprises about 110 kb in overlapping clones isolated from three different lambda phage libraries and a cosmid library to cover the various gaps and repetitive stretches (Fig. la; libraries are detailed in Materials and Methods). Cosmid clones were used along the way for in situ hybridization on polytene chromosomes to monitor the advancement and direction of the walk. This allowed us at the same time to determine the orientation of our physical map with respect to the chromosome (Fig. la). Mutations in Hairless cause larval lethality, indicating that the gene is active during larval life and presumably also during (late) embryogenesis. In the hope of detecting the Hairless transcription unit by these criteria within the cloned region a 'reverse Northern' with embryonic and larval cDNA was performed. However, no significant difference was observed in the activity of the transcribed regions during these stages (Fig. la).

Breakpoint mapping of Hairless mutants All Hairless mutants as listed in Table 1 were assayed for molecular alterations when compared to their parental and various wild type strains in genomic Southerns. At least four different restriction enzymes were used to distinguish mutations from restriction site polymorphisms which, however, occurred so rarely that also alleles of undefined genetic origin could be inspected (Fig. la; compare with Preiss et al., 1988). We found that all four cytologically visible deficiencies completely remove the entire cloned region and verified this by in situ hybridization to polytene chromosomes (not shown). As shown in Fig. lb, seven of the remaining 15 alleles revealed molecular rearrangements which could be mapped into a single transcription unit, represented by the h-cDNA clones. Most useful for this analysis were spontaneous and dysgenic alleles which all appear to carry insertions of transposable elements in the gene. H Bs, which was known from the in situ analysis to be loaded with hobo elements, carries an incomplete hobo element from the type 102 (Streck et al., 1986) in reverse orientation close to the

Hairless transcription start (see below). In H 2 an insertion of about 6.9 kb which does not match other known transposable elements (Ashburner, 1989) was mapped at a very similar position. The foreign DNA inserted in the second intron of the H j mutant Hairless gene matches a copia transposable element (Mount and Rubin, 1985). An insertion approximately 4 kb in length, which shows no similarity to either P- or hoboelements was detected in the dysgenic line H 94a. The allele H AI20 displays very complex polymorphisms affecting fragments of the Hairless gene and the downstream region (between - 4 0 and -50). The EMS induced H 88~ inversion might carry an additional proximal deletion of undefined length starting in the Hairless transcription unit. Surprisingly, from the nine X-ray induced Hairless alleles, only H 81 and H 99 involve deletions, the latter of only about 1.5 kb, while all the others appear normal in our analysis (compare with Table 1). This finding could be explained by the elimination of large Hairless deletions as they tend to confer dominant female sterility. To summarize, genomic alterations detected in the various Hairless mutants were found to map within a single transcription unit which, as we show below, corresponds to the Hairless gene. Transcriptional activity of the Hairless gene As indicated already by the 'reverse Northern' analysis (Fig. la) the Hairless gene is expressed during embryonic and larval development. In order to study Hairless activity more carefully, a Northern blot containing all stages of Drosophila development was probed with different Hairless genomic and cDNA probes, respectively. A group of at least three mRNAs in the size range of 4-6 kb was detected (see Fig. 2a-c) represented by the different cDNAs we isolated (compare with Fig. 3). Expression of these transcripts is highest during early embryogenesis (0-2 h and 3-11 h). It drops during mid/late embryogenesis and is lowest in the second larval instar. Transcriptional activity resumes in third instar larvae to reach a second peak in adult flies. Male and female flies show little differences with the exception of the ~ 5.5 kb transcripts less expressed in males (Fig. 2a, middle arrowhead). These transcripts are the only ones expressed throughout development (Fig. 2b). The shortest, about 4 kb messages are mainly expressed in the adult flies and in the young embryos. They are most prominent in the 0-2 h embryos, which at this time have not yet resumed zygotic transcription suggesting that these are maternal transcripts (lowest arrowhead in Fig. 2a). These short transcripts appear to be represented by the cDNA clones NB1 and NB3 since probes downstream of their 3' end no longer light up the 4 kb band (compare Fig. 2a with 2b and 2c, respectively). The longest transcripts

148 of ~ 6 kb,~though expressed in females, seem not to be maternally deposited since they are not present in 0-2 h young embryos (compare lanes 1 and 9 in Fig. 2c). The ~ 6 kb band (top arrowhead in Fig. 2a) is the only signal detected with the most 3' located probe which only contains trailer sequences (Fig. 2c, compare with Figs. 3 and 4). Apparently, size differences of Hairless transcripts arise from the usage of different polyadenylation signals and not from differential splicing. As a result, transcripts vary in the length of their untranslated trailer. Accordingly, only 5' located probes

tx\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\N\\\\\\\\~\\\\\\\\\\\\\\\\\\\~ Sc

R 1~

N

R

C

i

"I(

I

_L

I

I H

It

I H

NJa

h4

V

R

Nla X Na ~,~aX

IH

v

h7 119

B N N Na

I rl I,iI................... r. . . . ! : '

v-'x,/

~

h i _.~,,.,,,,,.,-~

1 kb

h2

D.

h5 ~ V

h3__

V

b

h l 2 ~ hll

V NB3

1

2

3

4

5 6 7 8

NBI

9

NBI3

a

V NBI5

e M N 17

i~ m,

V eMN 20

D8 - - V D 14

b

C

1

2

3

4 5 6

7

8

9

d

Fig. 2. Developmental Northern. About 5 p.g of poly(A) + R N A isolated from 0 - 2 h (1); 3-11 h (2) and 12-24 h (3) embryos; first (4), second (5) and third instar larvae (6); pupae (7); adult males (8) and females (9) were loaded in each lane. Equity was controlled with a ras probe (Mozer et al., 1985) as shown in (d) which detects a strong signal of 1.6 kb length and two weak bands (2.1 kb; 2.6 kb). This revealed a slight underrepresentation of embryonic R N A (lanes 1 and 2) and some overloading of first instar larval R N A (lane 4). (a) The same blot was rehybridized with various Hairless genomic and c D N A probes, respectively (see also Figs. 1 and 3). c D N A h2 recognizes at least three longer transcripts in the size of approximately 6 kb, 5.5 kb and 4 kb, respectively (open triangles). The 4 kb transcripts are the predominant messages in very young embryos ( 0 - 2 h) and also present in females, indicating maternal contribution. (b) c D N A NB15 detects only the two longer transcripts. This c D N A starts downstream of the second poly-adenylation site where c D N A NB1 and NB 3 end in the untranslated trailer (compare with Figs. 3 and 4). (c) Only the longest transcripts can be detected with a genomic 0.7 kb HindIII/EcoRI fragment located at the very 3' end of the transcription unit indicating differential poly-adenylation of the different sized transcripts. This longest message is not found in the young embryos, suggesting its absence from maternal RNA.

V

Fig. 3. Hairless c D N A clones and transformation. A set of c D N A clones was isolated from four different c D N A libraries, one prepared of imaginal discs R N A [D-clones] and three of embryonic R N A [eMN, NB- and h-clones]. The latter clones were prepared by random priming (for details see Materials and Methods). The clones h2, h7, h9, NB1 and NB13 were sequenced completely from either strand, all other c D N A clones were mapped and sequenced in part, in order to confirm their exact locations. Genomic D N A was sequenced to determine the positions of introns 1-3, the 3' end of the transcription unit as well as in vivo usage of putative poly-adenylation signals. The fourth intron was mapped with various restriction digests. Three classes of c D N A s were isolated that end at different poly-adenylation sites (compare with Fig. 4). Direction of transcription is indicated by arrows. The resultant transcription unit is shown above the cDNAs. Black boxes, translated regions; open boxes, introns; shaded boxes, untranslated leader and trailer, respectively. Restriction sites are: B, BamHI; C, ClaI; H, HindIIl; N, Notl; Na, NarI; R, EcoRI (polymorphic site is outlined); Sc, SacI; V, EcoRV; X, XhoI. The hatched bar above the map represents an 8.1 kb genomic HindllI/SacI fragment covering the entire Hairless transcription unit which was used for the transformation experiments.

detect all three sizes, and probes from the 3' end only the longer transcripts (Fig. 2b,c). We have examples for the three major transcripts in our collection of cDNAs (see below and Fig. 3). In some hybridizations we could also detect rather rare transcripts of about 1 kb expressed mainly during larval stages. No corresponding cDNA could be recovered from various different cDNA libraries, suggesting that the signals resulted from non-specific cross-hybridization. Hairless cDNA and sequence analysis Hairless cDNAs were recovered from various embryonic and larval cDNA libraries (Fig. 3; for details see Materials and Methods). Hairless cDNA clones shown in Fig. 3 were mapped with restriction enzymes and

149 sequenced from either end to unambiguously align them with the genomic map and the established Hairless sequence. They seem identical in their intron/exon structure indicating the absence of differential splicing. Apparently, all the cDNAs represent in part the large Hairless transcripts as shown in Figs. 2, 3 and 4. None of the isolated cDNA clones was full length, and most were rather short, maybe as a consequence of the unusually high GC content in the center of the transcription unit (highlighted by the accumulation of Not I and NarI sites in Fig. 3). Only the cDNAs prepared by random priming (Hovemann et al., 1991) allowed us to cover the 5' region of the gene. Clone h9 extends into the 5' untranslated leader and ends presumably quite close to the transcription start since the composite sequence (see below) matches very well the mRNA sizes observed in the Northern analysis. The two cDNA clones NB1 and NB3 seem to represent the shortest ~ 4 kb transcripts. These cDNAs end with about 110 bases unusually far away from the preceding polyadenylation signals (Birnstiel et al., 1985). The other cDNAs appear to correspond to the longer transcripts and have a conventional 3' end (see Figs. 3 and 4). The Hairless sequence in Fig. 4 was composed of the complete sequences of cDNA clones h9, h7, h2, NB1, NB13 and the genomic 0.7 kb EcoRI/HindlII fragment ( - 4 2 . 9 / - 43.6; Fig. la) located at the very 3' end of the transcription unit. Only four nucleotide polymorphisms were identified, all occuring in the untranslated trailer (indicated in Fig. 4). The position of the introns, determined by restriction mapping and sequencing of genomic DNA, respectively, is indicated in Figs. 3 and 4.

The putative Hairless protein is highly enriched in serine residues The Hairless sequence has a single large open reading frame encoding 1076 amino acids. In Fig. 4 the AUG at bp 264 is indicated as the putative translation start. However, the sequence preceding the second AUG at bp 318 more closely conforms to the Drosophila translation start consensus (Cavener, 1987), If the second AUG would be used, the expected protein would be composed of 1058 amino acids. The predicted amino acid sequence does not impart much information concerning the function of the Hairless gene product as it shares no compelling homologies to any proteins currently published in the SwissProt and EMBL databanks. Furthermore, the deduced polypeptide of about 110 kDa displays no obvious structure, although it has a rather unusual amino acid composition. About 17% of the amino acids in this product consist of serine, and 25% are composed of serine plus threonine. This is more than twice the normal amount in a common Drosophila protein (Smoller et al., 1990). Further, the

content of proline residues is higher than average. These amino acids are scattered more or less randomly across the sequence (Fig. 5). A conspicuous cluster of asparagines is located at the N-terminus of the peptide, and the center is highly acidic due to a culmination of glutamate residues (Figs. 4 and 5). The unusual amino acid composition renders the putative Hairless polypeptide extremely basic with a conceptual pI of 10.4. However, the two rather acidic regions, one of about 70 amino acids in the very N-terminal region and one of about 150 amino acids in the center of the protein, render the N-terminal half of the protein neutral (Fig. 5). Overall, the protein appears rather hydrophilic.

Temporal and spatial activity of Hairless during embryogenesis The spatial and temporal distribution of Hairless transcripts was determined during embryogenesis, since this is the stage of highest Hairless gene activity (Figs. 2 and 6). At first, Hairless transcripts are detected in an uniform distribution in embryos until blastoderm stage (Fig. 6a) most likely corresponding to maternal RNA deposited in the egg. With the onset of gastrulation zygotic expression appears in the entire germ band where RNA clearly accumulates basally in the cells (Fig. 6b,c). During germ band extension Hairless transcripts rapidly fade from the ectodermal cell layer and become enriched in the mesodermal sheath (Fig. 6c,d,h) and in the invaginating anterior and posterior midgut (Figs. 6d-g). With the flattening of the mesoderm, this staining becomes less evident, but can be still detected. Midgut staining is most prominent particularly in older embryos (Fig. 6g) where expression also in the central nervous system and body wall is discernible. Other mountants such as glycerol permit a more superficial view of the embryo and ectodermal expression can be followed throughout development. In embryos cleared in methyl salicylate this weak expression is obscured by stronger underlying staining. With the resolution of the whole mount in situ hybridization technique we cannot tell whether the late body wall staining is of ectodermal or mesodermal origin. We are currently addressing this question by analyzing tissue sections.

Transformation An independent proof was sought for the successful cloning of the Hairless gene. Therefore, we performed a stable germ line transformation with Hairless wild type DNA and tested the transformants for their ability to rescue Hairless mutant phenotypes. An 8.1 kb genomic HindlII/SacI DNA fragment, overlapping the h-transcription unit (indicated in Fig. 3) was used for the transformation. Two independent transformant

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151 lines, [HBS ry+]7 and [HBS ry÷]10, were established and insertion sites checked via in situ hybridization (both inserted on 3L, in 79 A and 75 B, respectively). Both transformant lines were tested for their ability to rescue the dominant phenotype of different Hairless alleles. All mutants listed in Table 2 were crossed with heterozygous [HBS ry+]10 balanced over TM3 ry RK Sb. Trans-heterozygous Hairless/[HBS ry+] l° offspring which could be distinguished unambiguously from their siblings by the markers on the balancer chromosomes, were completely normal with respect to the Hairless dominant bristle and wing phenotypes. This was also found for the Hairless allele H r'x2, a deficiency removing both Hairless and the neurogenic gene Delta (Table 1; V~issin and Campos-Ortega, 1987). Mutations in Delta cause a dominant vein thickening and formation of deltas at the margin of the adult wings, a phenotype that can be suppressed by simultaneous mutations in Hairless and vice versa (Lindsley and Grell, 1968). In accordance, H r,x2 adults show a suppressed Delta and no Hairless wing venation phenotype (Fig. 7d, compare with 7b,c). However, in trans over [HBS ry÷] 1° the Hairless mutation is rescued and, therefore, the suppression on Delta lifted resulting in a fully developed Delta wing phenotype (Fig. 7e). The same effect is found with [HBS ry÷]7. As both transformant lines carry the transgene on the third chromosome which is also the location of Hairless they could not be used directly to rescue the recessive lethality of the Hairless mutation. Therefore, the [HBS ry+]t0 transgene was recombined onto the H 99 chromosome. About 10% of the offspring were homozygotes that could be kept as a stock. This strain was subsequently crossed to several other Hairless alleles (1, 2, 88d, B8, 94a) to assay for the viability of the hetero-allelic combinations which was normal in all cases. It was rather unexpected to us to find any viable [HBS ry+]t0 H99 homozygotes, since the [HBS rye-]10 line itself is lethal. The lethality might be a result of the four Hairless doses which are reduced to normal in the recombinants. However, it is also conceivable that a lethal mutation arose on the third chromosome during transformation and was lost in the course of recom-

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bination with the H 99 chromosome. The second line, [HBS ry+] 7 is viable with four Hairless copies. Such flies express a hairy phenotype, as they produce multiple bristles on the head and thorax. In addition, extra wing vein material can be observed, predominantly in the marginal cell (Fig. 7f). A similar phenotype can be found with low expressivity in heterozygous [HBS ry+]~0 transformants suggesting that it is a result of an overexpression of the Hairless gene.

Fig. 4. Hairless sequence. The sequence is a composite derived from the different c D N A clones and genomic D N A as indicated in the text and in Fig. 3. The c D N A s cover about 6000 bases with an open reading frame of 1076 amino acids. The second start codon at 318 (encircled) conforms better with the Drosophila consensus sequence of translation initiation (Cavener, 1987) yielding a polypeptide of 1056 amino acids. The deduced Hairless amino acid sequence is depicted u n d e r n e a t h the D N A sequence. O p e n triangles indicate e x o n / i n t r o n junctions. Hairless m R N A s of various lengths appear to differ only in their 3' end by making use of different poly-adenylation signals (underlined). In the case of c D N A s NB1 and NB3 the poly(A) tail is unusually far downstream of the poly-adenylation signal, also from a degenerate one (dotted underlined, positions 3865-3870). These two c D N A s NB1 + 3 appear to correspond to the transcripts of about 4 kb length, c D N A s NB13 + 15 to the ~ 5.5 kb, and c D N A s e M N 1 + 13 and D 8 + 14 to the ~ 6 kb RNAs, respectively (compare with Figs. 2 and 3). The 3' ends of different c D N A clones are indicated. Four polymorphic bases were identified in the 3' untranslated trailer in a different wild type strain (Oregon R) and in two c D N A clones when compared with the Canton S wild type D N A as indicated above the sequence. Underlined are three homopolymeric alanine stretches. Two short P R D boxes (Frigerio et al., 1986) in the three prime end, as well as a novel KX repeat which follows the glutamate rich central region and a short, perfect N G D L repeat are boxed.

152 Taking all results together, we conclude that the HBS construct is capable to partially rescue the recessive lethality of Hairless mutations and to completely

rescue the dominant effects on adult sensory organ development. Furthermore, we have indications that Hairless overexpression results in an anti-Hairless phe-

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Fig. 6. Hairless expression during embryogenesis. Distribution of Hairless" 'transcripts during Drosophila embryogenesis was detected by whole mount in situ hybridization (Tautz and Pfeifle, 1989). Embryos (a-g) are oriented anterior left and dorsal up and shown in the same magnification (scale bar in (a): 50 ~zm). Stages are as described in Campos-Ortega and Hartenstein (1985). (a) In early embryos until stage 4 (syncytial blastoderm) Hairless m R N A is uniformly distributed and seems to be maternally derived. (b) A little later (stage 6), the overall expression fades and presumably zygotic transcripts become discernible mainly in the invaginating mesoderm, and basally in the remaining blastoderm cells. (c) During germ band extension (stage 7) Hairless message is present in the entire germ band. The basal accumulation in the ectodermal cells is prominent, mesodermal cells appear to stain overall. Conspicuous staining is observed in the cephalic furrow. (d) In stage 8, when the germ band is still extending, Hairless message is strongest in the mesodermal cell layer. Expression also appears in the posterior midgut anlage. (e) In the extended germ band (stage 9/10) neuroblasts appear to stain weakly. Much stronger is the expression in the invaginating anterior and posterior midgut and the mesodermal cell layer. The ectodermal cell layer appears void of Hairless transcripts, however, faint expression can be seen in a tangential focal plane (not shown). (f) The general distribution of Hairless message remains stable until germ band retraction (stage 12). Highest accumulation is found in the midgut. The flattened mesoderm still expresses Hairless, but the signals are difficult to spot. The weak ectodermal staining often gives the embryos a striped or patchy appearance (not shown). (g) At the time of dorsal closure in late embryogenesis (stage 13), Hairless expression remains in the anterior and posterior midgut during its fusion, as well as thereafter. During this stage Hairless expression in the dorsally growing ectoderm can be seen best as faint staining overlying the fusing midgut (half open arrowhead). Hairless is also weakly active in the central nervous system (ventral chord and brain, arrowhead). Weak expression is apparent in all tissues at any stage of development, especially after prolonged staining. (h) Close up of the ventral germ band of a stage 10/11 embryo. The two germ layers, ectoderm (most ventral) and mesoderm (cell row indicated by the arrow) are separated by delaminating neuroblasts (large, single cells). There is a clear gradient in Hairless transcript accumulation: the ectoderm is nearly void of transcripts (open arrowhead), neuroblasts stain faintly (arrowheads) and the mesodermal layer strongest (arrow). Scale bar: 200 ~zm.

153

il

U

U

|

Fig. 7. Transformation experiments. Wingsof Hairless flies in trans over wild type and transformants are shown in comparison to wild type. Scale bar in (a): 250 p.m. (a) Wing of a wild type fly. Note that wing veins never show gaps and always reach the wing margin. (b) Wing of HSl/+with the typical shortened fifth longitudinal vein, L5. (c) Wing of the weak allele H88d/TM6B with extreme venation defects (L2 and posterior crossvein disrupted, L5 incomplete). (d) Hr'x2/+, deficient for Delta as well as Hairless: the Hairless wing phenotype is completelysuppressed by the Delta and the Delta wing phenotype almost fully by the Hairless mutation. Typically, only a small delta is left at the posterior crossvein. (e) Hr'xz/[HBS ry+ ]l°ry; the same mutation as above combined with the extra Hairless copy of the transposant [HBS ry + ix0 line: the Hairless dominant phenotype is completely rescued by the transgene, and, therefore, the suppression of the Delta penotype is lifted. As a consequence, these flies develop a strong Delta wing phenotype recognizedby vein thickening in L2, large deltas at the posterior crossvein and a small one in L5 at the wing margin. (f) Wing of a homozygous[HBS ry+ ]7ry/[HBS ry÷ ]7ry transformant fly with a total of four Hairless wild type copies. Note the additional wingvein material in the marginal cell caused by Hairless overexpression. notype suggesting that normal fly development quantitatively depends on Hairless and, therefore, responds highly sensitively to Hairless dosage.

Discussion

Hairless is required for the formation of adult sensory organs, reflected by the loss of:bristles in Hairless mutants. This dominant phenotype is remarkably uniform in the large set of Hairless alleles we analyzed, indicating that t h e y all affect the same developmental process. However, we also discovered a dominant female sterility which, to our current knowledge, is a feature of all Hairless deficiencies. It is, therefore, not surprising that only so few rearrangements in the Hairless region were collected. The Hairless female sterility is highly background dependent and most pronounced in combinations with third chromosome balancers. It can be rescued by the H R I chromosome which bears a recessive lethal in close proximity to Hairless, maybe corresponding to a small duplication or a hypermorphic mutation of the gene itself (A. Preiss, unpublished observations). Similar to many Minute mutations, which also tend to be of low fertility (Lindsley and Grell, 1968), Hairless might affect more g e n e r a l aspects of development, enhanced by the sublethal defects of balancer chromosomes. However, reduced fertility of apparent point mutations implies that the sterility resides within the Hairless locus. Unable to separate the dominant female sterility from Hairless, we speculate

that it reveals an aspect of Hairless gene function. An involvement of Hairless in oogenesis is consistent with the strong maternal expression of the gene. Only three unambiguous Hairless null alleles, the deficiencies exist. The sterility of the deficiencies prohibited to test them in the same thorough scheme as the other alleles, particularly concerning the exact lethal phase. A hypomorphic allele, H 2 causes death predominantly at the transition of 1st to 2 no larval instar. However, some of the mutant animals develop even further, maybe reflecting the activity of residual maternal transcripts. Persisting activity of maternal messages is not unusual in Drosophila, as illustrated, for example by fused and dishevelled, late larval lethals in the presence, and embryonic lethals in the absence of maternal product (Perrimon and Mahowald, 1987). The molecular analysis of the Hairless locus indicated that all the other Hairless alleles might still produce some gene products. They would, therefore, classify as hypomorphic mutations, consistent with the fact that most of these alleles behaved rather weakly in the complementation analysis. As it turned out, the best marker for weak Hairless alleles are the wing venation defects. In contrast, Hairless deficiencies overlap with wild type (compare Fig. 7b and c). These observations suggest that the Hairless wing phenotype is an antimorphic feature, supported by the notion that Suppressor o f Hairless (Su(H)) alleles, which repress the Hairless loss of bristle phenotype, tend to enhance the Hairless wing vein phenotype (Ashburner, 1982). Therefore, this wing phenotype seems a rather inap-

154 propriate criterion for judging the severity of Hairless mutations (compare with Bang et al., 1991). The requirement of Hairless for the formation of the adult peripheral nervous system would predict high Hairless activity during larval and pupal life. However, Hairless activity is lowest in second instar larvae, although most of H 2 mutant animals die at this stage. This puzzling observation could be explained by maternally derived Hairless transcripts that would allow the mutants to develop beyond embryonic stages, supported by the strong maternal contribution of Hairless transcripts. Despite the high activity of Hairless during embryogenesis, in situ analysis of the transcript distribution was rather uninformative regarding Hairless' function. This might be due to the fact that our probes recognized all three different Hairless transcripts collectively. On the other hand, the activity pattern does not necessarily disclose the genes function. The best example is the Notch locus, with which Hairless strongly interacts: it is also rather generally expressed in the embryos and by far not restricted to the neuroblasts which require Notch gene products for cell fate choice (Hartley et al., 1987). Neither homology searches nor scanning of the Hairless open reading frame has revealed any apparent motifs from which Hairless functions could be inferred. The most conspicuous observation is the fact that the putative Hairless gene product is highly basic. This, in conjunction with the high proline content, is characteristic for a DNA binding protein, although no motifs typical for nucleotide binding or nuclear localization are present in the deduced Hairless gene product. As Hairless appears to specify neuronal fate (Bang et al., 1991) it might be required to repress neurogenic genes in neuronal precursor cells. There, the major target could be E(spl) which, based on genetic arguments, is regulated negatively by Hairless (V~issin et al., 1985). On the other hand, Hairless is the only known gene which phenotypically interacts with both classes of neurogenic genes (Lindsley and Grell, 1968), with the membrane bound gene products of Notch and Delta, and the nuclear products of E(spl). Hairless could transmit developmental signals from the membrane into the nucleus and execute them by acting on E(spl). However, with the little information we have to date any notions regarding Hairless function must remain purely speculative. Hopefully, details on Hairless subcellular localization and its biochemical partners will answer some of these questions. Materials and Methods

Drosophila strains Descriptions of genetic markers, balancer chromosomes and fly strains are found in Lindsley and Greil

(1968) and Lindsley and Zimm (1985, 1990), respectively. The Hairless mutant alleles w l l , KX2, A120, B8, B79, and 94a were obtained from U. Tepass and D. Godt in J.A. Campos-Ortega's laboratory; H b°b w a s received from R. Fleming, H 88d from W. Welshons and H 89c'27 from P. Lewis. All other mutant alleles described in this work were from the S. ArtavanisTsakonas stock collection or selected during an X-ray mutagenesis performed in his laboratory.

X-ray mutagenesis In an F1 screen for E(spl) alleles (Preiss et al., 1988), more than 80,000 offspring of irradiated rucuca males ( ~ 4000 R) and gro females were examined for the dominant Hairless phenotype. Two mutant females and 7 males were collected. Three of these Hairless alleles were female sterile and were lost, and many of the other alleles stabilized only over time and by using different balancer chromosomes (see also Table 1). Df (3R) H sl could be only kept stable and fertile over HRI, which is derived originally from an E(spl) al parental chromosome (Lehmann et al., 1983) that lost the deletion/inversion associated with the E(spl) mutation (Preiss et al., 1988). Female sterility was also observed in alleles obtained from different laboratories: Df (3R) H B79 w a s found to be only fertile over TM2 and not over TM3 Sb or TM6B balancers; H 89c'27 was only fertile over wild type chromosomes and was eventually lost.

Lethal phase and complementation analysis For the determination of the lethal phase H e / T M 6 B flies were allowed to lay eggs on apple juice plates (Wieschaus and NiJsslein-Volhard, 1986). Eggs were counted, and hatched larvae observed until their death. H e homozygous larvae could be distinguished from their siblings being wild type for the dominant Tubby marker (Craymer, 1984). For the complementation analysis, Hairless stocks balanced over TM6B were crossed in trans and pupal cases from all the offspring counted. The frequency of normal looking Hairless pupae was determined in relation to the Tubby pupae from the balanced siblings, and is given as percentage of the expected number.

Genomic library screening and breakpoint mapping Genomic libraries were screened as outlined in Sambrook et al. (1989). Libraries from four different sources were used; three lambda libraries: Oregon R [H clones] (Preiss et al., 1985); KrSm°/SM1 [K clones] (Baumgartner et al., 1987); and E(spl) D, partially EcoRI digested [Gap clones] (A. Preiss, unpublished) and a cosmid library: Canton S in Cos 828 (Haenlin et

155 al., 1985). For the 'reverse' Southern and Northern experiments, cloned D N A fragments representing the walk between + 30 and - 50 were hybridized with total genomic and cDNA (embryonic 0-18 h; 5 - 6 day second instar larvae (gift of S. Baumgartner)), respectively, which was radio-labelled by random priming (Feinberg and Vogelstein, 1983). Signal intensity reflects the degree of repetition and transcriptional activity of a given fragment, since it is linear to the proportions of the corresponding sequences in the probe. Breakpoints of Hairless mutant alleles were mapped as described in Preiss et al. (1988). In situ hybridizations to polytene chromosomes were performed as described by Ashburner (1989).

BamHI/SacI

Transcriptional analysis

We acknowledge R. Fleming, P. Lewis, W. Welshons, and U. Tepass and D. Godt from J. Campos-Ortega's laboratory for providing us with Hairless alleles. We thank S. Artavanis-Tsakonas for the opportunity to perform the X-ray mutagenesis screen in his laboratory and his support and interest throughout this work. We are very grateful to M. Wolfner for the adm124BlO c D N A clone, and J. Carlson for his help to spot it. Genomic and cDNA libraries, respectively, were obtained from S. Baumgartner, B. Hovemann, M. Noll and C. Wilson, which we greatly acknowledge. We thank R. Blackman for a hobo probe and P. Cron for its in situ hybridization. We are indebted to S. Baumgartner for his invaluable help and U. Walldorf for the Carnegie 20.1 transformation vector. Many thanks go to R. Ramos for sharing the Cosmid library and the NB-cDNA library with us. We acknowledge I. Dawson, K. Cadigan and S. Artavanis-Tsakonas for helpful discussions and critical comments on the manuscript. We thank R. Doelz for his help in the computer analysis and L. Miiller for the excellent photographs. D.M. and G.S. received a post-doctoral fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by the Louis-Jeantet Foundation and by a grant from the Swiss National Science Foundation (3126257.89) to A.P.

Total R N A was isolated from frozen animals of the different stages according to Chirgwin et al. (1979). Selection of poly (A) + RNA, the separation in denaturing formaldehyde gels and further processing was performed as explained in Sambrook et al. (1989). Whole mount in situ hybridization in embryos was done according to the protocol of Tautz and Pfeifle (1989) with modifications taken from Lehner and O'Farell (1990).

Sequencing of genomic and cDNA clones Four different cDNA libraries were screened using genomic DNA fragments or inserts of earlier isolated cDNA clones: 0-16 h embryonic, random primed [h clones] (Hovemann et al., 1991); 8-12 h embryonic, poly(A) primed [NB clones] (Brown and Kafatos, 1988); 8-12 h embryonic, poly(A) primed [eMN clones] (Dambly-Chaudi~re et al., 1992); imaginal discs, poly(A) primed [D clones] (C. Wilson, unpublished). cDNAs and genomic fragments, cloned into Bluescript vectors (Stratagene) were further subcloned and nested deletions created using exonuclease III (Henikoff, 1984). Standard dideoxy sequencing of double and single stranded DNA, respectively, was performed using the Sequenase Version 2.0 Kit (United States Biochemical). Sequence was compiled and analyzed using the GCG-package programs (University of Wisconsin; Devereux et al., 1984). Fig. 5 was assembled using the W I N D O W and S T A T P L O T programs (Devereux et al., 1984; Smith et al., 1983) and database searches were performed using the FastA and TFastA programs (Pearson and Lipman, 1988).

Transformation The HBS construct was derived from a ligation and subsequent subcloning of two genomic D N A fragments, a 3.2 kb HindlII/BamHI and a 4.8 kb

fragment into a Bluescript vector (Stratagene). After excision, the whole was inserted into the Carnegie 20.1 transformation vector (Walldorf, unpublished; Rubin and Spradling, 1983). For injections (Santamaria, 1986) cn, ?.y506 embryos were used to later facilitate the detection of ry ÷ transformants in the cn mutant background and to help follow the transgene during chromosomal segregation. Wings of adult flies were mounted in Hoyer's medium (Van der Meer, 1977) incubated over night at 60°C and pictures taken in bright field in a standard Zeiss microscope.

Acknowledgements

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