A simple and efficient method to identify replacements of

P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in

the bithorax complex of Drosophila

Luis de Navas, David Foronda, Magali Suzanne* and Ernesto Sánchez-

Herrero#

Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.) Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain Telephone number:34-914978497 Fax number: 34-914974799 e-mail: esherrero@cbm.uam.es *Present address: Institute of Signalling, Developmental Biology and Cancer UMR6543-CNRS University of Nice Sophia-Antipolis Parc Valrose 06108 Nice cedex 2 France

#Corresponding author

Keywords: Drosophila, P-element, bithorax complex, Gal4 lines, Ubx, Abd-B

2

Abstract

The functional replacement of one gene product by another one is a powerful method

to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has

been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that

inactivate a gene and reproduce its expression pattern are required, and they can be

frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We

have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4

lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and

Abdominal-B Hox genes. We have used these lines to study the functional replacement of a

Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace

Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the

formation of the genitalia.

3

Introduction

The Gal4/UAS system of Drosophila is a versatile method which can be used for the

study of different biological problems (Brand and Perrimon, 1993). One of its applications is

the possibility of finding out if a certain gene product can substitute for another one during

development. Perfect replacement requires the introduction of a gene in the same position as

the gene to be replaced and the in vivo analysis of this substitution. In Drosophila this is

difficult to achieve, but the Gal4/UAS system has been used to allow functional replacement

studies without actually replacing a gene at its chromosomal location. For this analysis it is

necessary to isolate Gal4 lines inserted in the gene of interest that reproduce exactly (or very

closely) its spatial and temporal expression. If the insertion is also mutant for the gene, by

introducing in a mutant fly a cDNA linked to UAS sequences, one can study the

consequences of replacing the protein encoded by this gene by another one, by a modified

version of the protein or by a protein encoded by an homologous gene from another species

(see, for example, Pfeiffer et al., 2000).

Such approach has been successfully used, for instance, in the study of the apterous

(ap) gene, which specifies the dorsal compartment of the wing disc (Díaz-Benjumea and

Cohen, 1993). A Gal4 insertion in the ap gene is mutant for ap and reproduces faithfully ap

expression in the wing disc (Calleja et al., 1996). With such line it has been possible to

dissect the function of the Ap protein domains (O'Keefe et al., 1998; Rincón-Limas et al.,

2000), to demonstrate that the homologous ap gene of human can substitute for the

Drosophila one (Rincón-Limas et al., 1999) and to show that the fringe and Serrate genes

can accomplish some of the ap functions (Milán and Cohen, 1999).

4

We wanted to use similar experiments in the study of the functional specificity of

homeotic (Hox) genes. These genes determine different segments or body regions in

Drosophila and other species by controlling the expression of particular downstream genes

(Graba et al. 1997). In some cases, one homeotic protein can substitute, to a great extent, for

another one (Greig and Akam, 1995; Casares et al., 1996; Hirth et al., 2001; Greer et al.,

2000; Zhao and Potter, 2001; 2002; Wellik and Capecchi, 2003; Sprecher et al., 2004). In

Drosophila, however, most of these experiments have been confined to the embryo, and our

interest is to extend this analysis to the adult. Previous results carried out at this stage have

shown, for instance, that the Ultrabithorax (Ubx) and abdominal-A (abd-A) Hox genes are

equally able to develop halteres (Casares et al., 1996), the dorsal metathoracic appendages

whose normal development requires Ubx (Lewis, 1963). These experiments, however, were

done with Gal4 lines that do not reproduce the pattern of expression of Ubx. We reasoned

that if the expression of a certain Hox gene is directed by its own regulatory sequences, the

functional replacement studies would approach the spatial and temporal situation of the

wild-type gene and would lead to better founded conclusions.

To fulfil these requirements, P-GAL4 lines inserted in homeotic genes are required,

and for our studies we would like to use Gal4 lines located in the Ubx and Abdominal-B

(Abd-B) Hox genes of the bithorax complex (BX-C). Many P-lacZ lines inserted in these

genes have been isolated (Galloni et al., 1993; McCall et al., 1994; Casares et al., 1997a;

Zhou and Levine, 1999; Barges et al., 2000; Bender and Hudson, 2000; Fitzgerald and

Bender, 2001; Estrada et al., 2002), but only two BX-C Gal-4 lines, both in the Ubx gene,

have been reported (Herranz and Morata, 2001; Pallavi and Shashidara, 2003). These lines,

however, are not suitable for functional replacements in adults: they drive strong expression

in the whole parasegment (PS) 5, whereas Ubx only shows weak expression in some cells of

PS5 in wildtype embryos (Akam and Martínez-Arias, 1985); thus, attempts to use the Ubx-

5

Gal4782 line (Herranz and Morata, 2001) to functionally replace the endogenous Ubx gene

led to embryonic lethality (E. S-H., unpublished results).

A method to substitute P-Gal4 for P-lacZ lines has been described (Sepp and Auld,

1999) and this could be applied to the BX-C P-lacZ. In this method, the Gal4 lines are

identified by the different eye color markers (rosy and white) included in the two types of

transposons; it requires the P-lacZ insertions not to carry the white transformation marker

and also the genetic characterization, among all the presumptive Gal4 line isolated, of the

true substitution events (Sepp and Auld, 1999).

We have designed a simple method to identify replacement of P-lacZ by P-Gal4

lines, independently of the marker inserted in either P-element, and applied it successfully to

obtain Ubx-Gal4 and Abd-B-Gal4 lines that are also mutants for Ubx or Abd-B. These lines

have allowed testing some cases of functional replacement of one homeotic gene product by

another one in adult structures. The method could be useful to replace any lacZ line that is

expressed in any tissue or stage of development.

Results and Discussion

The genetic scheme used for replacing P-lacZ by P-Gal4 lines is described in Fig. 1A

for an Ubx-Gal4 line. It is based in the identification, in a yellow (y) mutant background, of

the adult pattern of expression driven by the Gal4 line as a patch of y+ cuticle (Calleja et al.,

1996). We used a Gal4 line as "donor" P-element (4.7-Gal4 line) that jumps effectively but

does not give any particular expression pattern in the adult (Calleja et al., 1996). y mutant

6

males carrying this line, the lacZ line and the ∆2-3 transposase source are crossed to UAS-

y+ flies (also mutant for y). The line of interest is identified by the dark y+ patch of cuticle

in a particular region. In our experiments, the lines were selected because of the dark color

in the halteres (Ubx-Gal4 lines) or in the female genitalia and posterior abdomen (Abd-B-

Gal4 ones; Figs 2A, B and 3A, B). A summary of the P-Gal4 lines obtained by this method

is shown in Fig. 1B and Table 1. The Gal4 insertions replaced P-lacZ lines that were

selected because they reproduce totally or in part the pattern of expression of the Ubx or

Abd-B genes and are also mutants for either of these genes (McCall et al., 1994; Bender and

Hudson, 2000; Estrada et al., 2002).

Three Ubx-Gal4 lines were obtained (see Table 1). The Ubx-Gal4LDN insertion

substitutes for the UbxPlac(-31) transposon (McCall et al., 1994), is homozygous lethal and is

located at the same position as the P-lacZ line (Table 1). However, contrary to UbxPlac(-31),

the Ubx-Gal4LDN line does not drive expression in the embryo; it directs expression in the

haltere pouch (Fig. 2C) and larval central nervous system (not shown) and in patches of

cells in the third leg disc (Fig. 2D). Thus, it reproduces only partially the Ubx expression

pattern (Akam and Martínez-Arias, 1985; Beachy et al., 1985; White and Wilcox, 1985; Fig.

2K-M). A possible explanation for the discrepancy in the embryonic expression between the

two insertions is that the UbxPlac(-31) line includes 17 kb of DNA from the bithorax region of

Ubx, which are probably responsible for the embryonic lacZ pattern (McCall et al., 1994).

The Ubx-Gal4M1 and Ubx-Gal4M3 insertions were obtained by replacing the HC174B P-lacZ

line (Bender and Hudson, 2000). They are inserted almost at the same position (4 bp apart),

although with opposite orientation (Fig. 1B and Table 1). The pattern they drive reproduces

the Ubx wildtype pattern in the embryonic epidermis and, with some differences, in the

haltere and third leg discs (White and Wilcox, 1984; 1985; Beachy et al., 1985; Fig. 2E-G

7

and H-J, compare with the Ubx wildtype expression in Fig. 2K-M). Ubx-Gal4M1 and Ubx-

Gal4M3 are also mutant for Ubx: Ubx-Gal4M3 is a strong bithoraxoid (bxd) mutation whereas

Ubx-Gal4M1 hemizygotes also shows a bxd phenotype and, in addition, are weakly mutant

for the bithorax function. The two different phenotypes may be related to the opposite

orientation of the insertions, but may also be attributed to a small DNA sequence

modification found 422bp upstream of the Ubx-Gal4M1 insertion point: 41bp are deleted and

10bp from a different region of the genome are inserted, so that a 31 bp deletion remains.

Two Abd-B-Gal4 lines were obtained by replacing previously characterized Abd-

B-lacZ lines (Bender and Hudson, 2000; Estrada et al., 2002; Fig. 1B and Table 1). They

were recognized by the y+ pattern in the female genitalia and posterior abdominal segments

(Fig. 3A, B). The pattern driven by the Abd-B-Gal4LDN insertion resembles the pattern of

Abd-B m (or A transcript) in driving strong expression in parasegment (PS) 13; however, the

signal in PS10-12 is absent or much weaker than that of Abd-B m (Sánchez-Herrero and

Crosby, 1988; Kuziora and McGinnis, 1988; Boulet et al., 1991; Fig. 3C, compare with the

Abd-B m expression in Fig. 3E). The expression of Abd-B-Gal4LDN also differs from the

lacZ line it replaces, and this is also probably due to Abd-Blac1 including a genomic fragment

of Abd-B regulatory regions. When this fragment is removed, as in certain Abd-Blac1

derivatives, the lacZ expression pattern resembles the Abd-B-Gal4LDN expression pattern

(Estrada et al., 2002 and this report). In the genital disc, and similarly to the Abd-B m

transcription (Casares et al., 1997b; Foronda et al., 2006), Abd-B-Gal4LDN is expressed in

A8 but not in A9 of female or male discs (Fig. 3F, G). The Abd-B-Gal4199 insertion also

drives expression in PS13 in the embryo (Fig. 3D) and in the A8 in the female and male

genital discs, although we also observe a weak signal in the A9 of these discs (Fig. 3H, I;

8

compare with the wildtype Abd-B pattern in 3J, K). Both Abd-B insertions are mutant for

Abd-B.

The Ubx-Gal4 and Abd-B-Gal4 insertions carry the white (w) gene as a

transformation marker (Brand and Perrimon, 1993). It was predicted that w transcription

would be repressed when inserted in the BX-C (McCall et al., 1994): the anterior limit of

expression of the BX-C genes is the posterior compartment of the second thoracic segment

and, therefore, any gene expressed more anteriorly in the wildtype, and acting cell

autonomously like w, should be repressed when located in this complex. However, our Ubx-

Gal4 and Abd-B-Gal4 lines (except Abd-B-Gal4199) have pigmented eyes, ranging from pale

yellow to light orange (see also Hittinger et al., 2005). Two lines of evidence suggest the

color is not due to a second transposon inserted on the third chromosome during the P-

element mobilization: first, only one band was obtained by inverse PCR with all the Gal4

insertions; second, we could not separate by recombination the eye color and the mutant

phenotype in two lines tested, Ubx-Gal4LDN and Ubx-Gal4M1. Ubx and Abd-B expressions

are controlled throughout development by the Polycomb-group and trithorax-group genes

(reviewed in Ringrose and Paro, 2004). Therefore, we have tested the variations in eye color

of the Abd-B-Gal4LDN

insertion in flies heterozygous for a mutation in either the Polycomb

(Pc) or the trithorax (trx) genes. In Pc3/Abd-B-Gal4LDN flies, the eye color is darker (Fig.

4A), and in trxE2/Abd-B-Gal4LDN flies slightly lighter (Fig. 4B), than in Abd-B-

Gal4LDN/TM6B flies, suggesting that the w gene is partially repressed by Pc and activated

by trx in these insertions. Thus, it is possible that not all the regions in the BX-C are equally

repressed by the Pc system, and that P-Gal4 insertions far from the Polycomb response

elements show only partial repression in the w gene. Curiously, the colour of the eyes

9

occasionally varies in some outcrosses of the Abd-B-Gal4LDN or Ubx-Gal4LDN lines. This

suggests they may be some variation in the repression by Pc-group genes.

The Ubx-Gal4 and Abd-B-Gal4 lines are mutant for Ubx and Abd-B, respectively.

Although none of the mutations is amorph, they produce clear transformations or abnormal

development in the adult in hemizygous or trans-heterozygous condition and, therefore, can

be used for functional gene replacement studies. To explore this possibility, we analyzed if

we could restore a wildtype pattern in the adult by expressing Ubx or Abd-B proteins in an

Ubx- or Abd-B- mutant background, respectively. Ubx-Gal4LDN/abx bx3 pbx flies have a

mutant phenotype in the haltere (Fig. 5B; compare with the wildtype in 5A), and by

introducing a UAS-Ubx cDNA in this mutant background we recovered the haltere wildtype

pattern (Fig. 5C). Some rescue of the mutant phenotype is also observed when we carry out

similar experiments in an Abd-B mutant background: Abd-B-Gal4LDN/Abd-B- females have

no genitalia (Fig. 5F; compare with the wildtype in Fig. 5E), but UAS-Abd-B m/+; Abd-B-

Gal4LDN/Abd-B- females bear genitalia with some vaginal teeth (Fig. 5G). There is also

rescue of the mutant phenotype in the dorsal abdomen: in the wildtype, males have no

seventh tergite (T7) and females have a reduced T7 (Fig. 5I); by contrast, Abd-B-

Gal4LDN/Abd-B- females or males have a big T7 (Fig. 5J). In UAS-Abd-B m/+; Abd-B-

Gal4LDN/Abd-B- adults (Fig. 5K), the T7 is smaller than in Abd-B-Gal4LDN/Abd-B- animals,

thus showing the rescue of the mutant phenotype by the Abd-B protein.

It has been reported (Casares et al., 1996) that abd-A can fulfil the role of Ubx in the

development of the haltere. We have tested this in flies of the UAS-abdA/+; Ubx-

Gal4LDN/abx bx3 pbx genotype. As shown in Fig. 5D, there is an almost complete rescue of

10

the haltere morphology in these flies, similar to that obtained by the Ubx product (compare

Fig. 5D with 5C). By contrast, abd-A cannot substitute for Abd-B in any aspect of genitalia

or abdominal development: UAS-abd-A/+; Abd-B-Gal4LDN/Abd-B- flies do not present

genitalia (Fig. 5H) and develop dorsally a big A7, similar to that of Abd-B-Gal4LDN/Abd-B-

flies, and even a small A8 (Fig. 5L; compare with Fig. 5J).

The method described to identify substitutions of a Gal4 line for a lacZ insertion has

some advantages over a previous technique (Sepp and Auld, 1999): first, fewer generations

are required; second, it allows the lacZ line to carry the white marker; in fact, any pair of P-

elements may be used, independently of the dominant markers they carry; finally, the

replacement can be recognized directly, and the experiment can be done en masse. It does

not require the gene to be replaced to be expressed in the adult cuticle since the UAS-GFP

line can be used instead of the UAS-y+ and the expression scored in internal tissues (D. F.

and E. S., unpublished).

The efficiency in obtaining the Ubx-Gal4 and Abd-B-Gal4 lines varied significantly:

we obtained Abd-B-Gal4LDN after scoring only about 80 flies but had to score more than

1,500 to isolate Abd-B-Gal4199. The jumping efficiency of P-elements depends, among other

variables, on the size of the transposons: large elements do not mobilize so efficiently as

small ones (Robertson et al., 1988). However, most of the Ubx-lacZ and Abd-B-lacZ

transposons that we have replaced by GAL4 insertions are very large, ranging from about

19kb (Abd-Blac1, HCJ199 and HC174B) to nearly 30kb (UbxPlac (-31). This suggests that the

large size of an insert is not an impediment for obtaining Gal4 lines in a relatively short

time.

11

Material and Methods

Genetics. The mutants used and the UAS-lacZ and UAS-GFP lines are described in

Flybase (Drysdale and Crosby, 2005). The UAS-abdA stock has been generously provided

by A. Michelson (Michelson, 1994), and the UAS-Abd-B (m ) and UAS-Ubx lines are gifts

of M. Akam (Castelli-Gair et al., 1994).

In situ hybridization. It was done as described in Cubas et al., 1991. The probe for

the Abd-B m (alpha) transcript is a BamHI genomic probe from 50,702 to 48,864,

approximately, in the bithorax complex DNA map (Martin et al., 1995).

Inmunohistochemistry. It was done as previously described (Sánchez-Herrero,

1991; Estrada and Sánchez-Herrero, 2001) with slight modifications. The antibodies used

are mouse and rabbit anti-β-galactosidase (Cappel), mouse anti-Ubx (White and Wilcox,

1984) and rabbit anti-Abd-B (DeLorenzi and Bienz, 1990). Secondary antibodies are

coupled to Red-X, FITC and Cy5 fluorochromes (Jackson Immunoresearch).

Adult cuticle analysis: Flies were kept in a mixture of ethanol: glycerol (3:1)

macerated in 10% KOH-at 60ºC for ten minutes, dissected, washed with water, dehydrated

with ethanol and mounted in Euparal for inspection under a compound microscope.

PCR analysis: The P-Gal4 lines were localized by inverse PCR

(http://www.fruitfly.org/about/methods/inverse.pcr.html).

12

Acknowledgements

We thank G. Morata for support and comments on the manuscript, B. Estrada for

help in the initial location of Abd-B-Gal4LDN, W. Bender for providing P-lacZ lines, M.

Akam, A. Busturia, A. Michelson and G. Morata for stocks, M Bienz and R. White for

providing antibodies, A. Busturia and C.T. Hittinger for discussions and R. González for

technical assistance. This work was supported by grants from the Ministerio de Educación y

Ciencia (BMC2002-00300), the Comunidad Autónoma de Madrid (nº 08.1/0031/2001.1 and

GR/SAL/0147/2004.) and an Institutional Grant from the Fundación Ramón Areces.

13

References

Akam, M. E. and Martínez-Arias, A. 1985. The distribution of Ultrabithorax

transcripts in Drosophila embryos. EMBO J. 4, 1689-1700.

Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Müller, M., Shanower, G.,

Schedl, P., Gyurkovics, H. and Karch, F. 2000. The Fab-8 boundary defines the distal limit

of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a

PRE in the adjacent iab-8 domain. Development 127, 779-790.

Beachy, P. A., Helfand, S. L. and Hogness, D. S. (1985). Segmental distribution

of bithorax complex proteins during Drosophila development. Nature 313, 545-551.

Bender, W. and Hudson, A. 2000. P element homing to the Drosophila bithorax

complex. Development 127, 3981-3992.

Boulet, A.M., Lloyd, A. and Sakonju, S. 1991. Molecular definition of the

morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila

Abd-B homeotic gene. Development 111, 393-405.

Brand, A. and Perrimon, N. 1993. Targeted gene expression as a means of

altering cell fates and generating dominant phenotypes. Development 118, 401-415.

Calleja, M., Moreno, E., Pelaz, S. and Morata, G. 1996. Visualization of gene

expression in living adult Drosophila. Science 274, 252-255.

14

Casares, F., Calleja, M. and Sánchez-Herrero, E. 1996. Functional similarity in

appendage specification by the Ultrabithorax and abdominal-A Drosophila Hox genes.

EMBO J. 15, 3934-3942.

Casares, F., Bender, W., Merriam, J. and Sánchez-Herrero, E. 1997a. Interactions

of Drosophila Ultrabithorax regulatory regions with native and foreign promoters. Genetics

145, 123-137.

Casares, F., Sánchez, L., Guerrero, I. and Sánchez-Herrero, E. 1997b. The genital

disc of Drosophila melanogaster. I. Segmental and compartimental organization. Dev.

Genes Evol. 207, 216-228.

Castelli-Gair, J., Greig, S., Micklem, G. and Akam, M. (1994). Dissecting the

temporal requirements for homeotic gene function. Development 120, 1983-1995.

Cubas, P., de Celis, J. F., Campuzano, S. and Modolell, J. (1991). Proneural

clusters of achaete-scute expression and the generation of sensory organs in the Drosophila

imaginal wing disc. Genes Dev. 5, 996-1008.

DeLorenzi, M. and Bienz, M. (1990). Expression of Abdominal-B homeoproteins

in Drosophila embryos. Development 108, 323-329.

Díaz-Benjumea, F. and Cohen, S. M. 1993. Interaction between dorsal and

ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75, 741-752.

15

Drysdale, R. A., Crosby, M. A. and the FlyBase Consortium (2005). FlyBase:

genes and gene models. Nucl. Acid. Res. 33, D390-D395.

Estrada, B. and Sánchez-Herrero, E. (2001). The Hox gene Abdominal-B

antagonizes appendage development in the genital disc of Drosophila. Development 128,

331-339.

Estrada, B., Casares, F., Busturia, A. and Sánchez-Herrero, E. (2002). Genetic

and molecular characterization of a novel iab-8 regulatory domain in the Abdominal-B gene

of Drosophila melanogaster. Development 129, 5915-5204.

Fitzgerald, D. P. and Bender, W. 2001. Polycomb group repression reduces DNA

accessibility. Mol. Cell Biol. 21, 6585-6597.

Foronda, D., Estrada, B., de Navas, L. F. and Sánchez-Herrero, E. 2006.

Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila

breaks the posterior down-regulation rule. Development 133, 117-127.

Galloni, M., Gyurkovics, H. Shedl, P. and Karch, F. 1993. The bluetail

transposon: evidence for independent cis-regulatory domains and domain boundaries in the

bithorax complex. EMBO J. 12, 1087-1097.

Graba, Y., Aragnol, D. and Pradel, J. 1997. Drosophila Hox complex

downstream targets and the function of homeotic genes. Bioessays 19, 379-388.

16

Greer, J. M., Puetz, J., Thomas, K. R. and Capecchi, M. (2000). Maintenance of

functional equivalence during paralogous Hox gene evolution. Nature 403, 661-665.

Greig, S. and Akam, M. 1995. The role of homeotic genes in the specification of

the Drosophila gonad. Curr. Biol. 5, 1057-1062.

Herranz, H. and Morata, G. 2001. The functions of pannier during Drosophila

embryogenesis. Development 128, 4873-4846.

Hirth, F., Loop, T., Egger, B., Miller, D. F. B., Kaufman, T. C. and Reichert, H.

2001. Functional equivalence of Hox gene products in the specification of the tritocerebrum

during embryonic brain development of Drosophila. Development 128, 4781-4788.

Hittinger, C. T., Stern, D. L. and Carroll, S. B, (2005). Pleiotropic functions of a

conserved insect-specific Hox peptide motif.

Kuziora, M.A. and McGinnis, W. 1988 . Different transcripts of the Drosophila

Abd-B gene correlate with distinct genetic sub-functions. EMBO J. 7, 3233-3244.

Lewis, E. B. (1963). Genes and developmental pathways. Amer. Zool. 3, 33-56.

Martin, C. H., Mayeda, C. A., Davis, C. A., Ericsson, C. L., Knafels, J. D.,

Mathog, D. R., Celniker, S., Lewis, E. B. and Palazzolo, M. J. (1995). Complete sequence of

the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. U.S.A. 92, 8398-8402.

17

McCall, K., O'Connor, M.B. and Bender, W. 1994. Enhancer traps in the

Drosophila bithorax complex mark parasegmental domains. Genetics 138, 389-399.

Michelson, A. M. 1994. Muscle pattern diversification in Drosophila is

determined by the autonomous function of homeotic genes in the embryonic mesoderm.

Development 120, 755-768.

Milán, M. and Cohen, S. M. 1999. Notch signalling is not sufficient to define the

affinity boundary between dorsal and ventral compartments. Mol. Cell 4, 1073-1078.

O'Keefe, D. D., Thor, S. and Thomas, J. B. 1998. Function and specificity of LIM

domains in Drosophila nervous system and wing development. Development 125, 3915-

3923.

Pallavi, S. K. and Shashidhara, L. S. 2003. Egfr/Ras pathway mediates

interactions between peripodial and disc proper cells in Drosophila wing discs.

Development 130, 4931-41.

Pfeiffer, S., Alexandre, C., Calleja, M. and Vincent, J.-P. 2000. The progeny of

wingless-expressing cells deliver the signal at a distance in Drosophila embryos. Curr. Biol.

10, 321-324.

Rincón-Limas, D. E., Lu, C. H., Canal, I., Calleja, M., Rodriguez-Esteban, C.,

Izpisúa-Belmonte, J. C. and Botas, J. 1999. Conservation of the expression and function of

apterous orthologs in Drosophila and mammals. Proc. Natl. Acad. Sci. USA 96, 2165-2170.

18

Rincón-Limas, D. E., Lu, C. H., Canal, I. and Botas, J. 2000. The level of

DLDB/CHIP controls the activity of the LIM homeodomain protein Apterous: evidence for

a functional tetramer complex in vivo. EMBO J. 19, 2602-2614.

Ringrose, L. and Paro, R. (2004). Epigenetic regulation of cellular memory by the

Polycomb and Trithorax group proteins. Ann. Rev. Genet. 38, 413-443.

Robertson, H. M., Preston, C. R., Phillis, R. W., Johnson-Schlitz, D. M., Benz,

W. K. and Engels, W. R. (1988). A stable source of P element transposase in Drosophila

melanogaster. Genetics 118, 461-470.

Sánchez-Herrero, E. (1991). Control of the expression of the bithorax complex

genes abdominal-A and Abdominal-B by cis-regulatory regions in Drosophila embryos.

Development 111, 437-449.

Sánchez-Herrero, E. and Crosby, M. A. 1988. The Abdominal-B gene of

Drosophila melanogaster: overlapping transcripts exhibit two different spatial distributions.

EMBO J. 7, 2163-2173.

Sepp, K. J. and Auld, V. J. 1999. Conversion of lacZ enhancer trap lines to GAL4

lines using targeted transposition in Drosophila melanogaster. Genetics 151, 1093-1101.

Sprecher, S. G., Muller, M., Kammermeier, L., Miller, D. F., Kaufman, T. C.,

Reichert, H. and Hirth, F. (2004). Hox gene cross-regulatory interactions in the embryonic

brain of Drosophila. Mech. Dev. 121, 527-536.

19

Wellik, D. M. and Capecchi, M. R. (2003). Hox10 and Hox11 genes are required

to globally pattern the mammalian skeleton. Science 301, 363-367.

White, R. A. H. and Wilcox, M. (1984). Protein products of the bithorax complex

in Drosophila. Cell 39, 163-171.

White, R. A. H. and Wilcox, M. (1985). Distribution of Ultrabithorax proteins in

Drosophila. The EMBO J. 4, 2035-2043.

Zhao, Y. and Potter, S. S. (2001). Functional specificity of the Hoxa13

homoebox. Development 128, 3197-3207.

Zhao, Y. and Potter, S. S. (2002). Functional comparison of the Hoxa4, Hoxa10

and Hoxa11 homoeoboxes. Dev. Biol. 244, 21-36.

Zhou, J. and Levine, M. 1999. A novel cis-regulatory element, the PTS, mediates

an anti-insulator activity in the Drosophila embryo. Cell 99, 567-575.

20

Figure legends

Figure 1. A) Scheme of the replacement of a P-lacZ by a P-Gal4 line, indicated for

the Ubx-lacZ (UbxPlac(-31)) line (McCall et al., 1994). For the other Ubx and Abd-B

substitutions we followed a similar procedure. B) Location of the Ubx-Gal4 and Abd-B-Gal4

lines obtained. The transcription units of the Ubx, abdominal-A and Abd-B genes of the

bithorax complex are schematically represented.

Figure 2. Expression driven by the Ubx-Gal4 lines. (A) Haltere of a y mutant fly.

(B) Haltere of the y; Ubx-Gal4LDN/UAS-y+ genotype, strongly pigmented. (C) Haltere disc

of a Ubx-Gal4LDN

/UAS-GFP larva, showing GFP expression in the haltere pouch. (D) In

the third leg disc this line drives expression in groups of cells. (E, H) ß-galactosidase

expression in germ band extended embryos of Ubx-Gal4M1/UAS-lacZ (E) and Ubx-

Gal4M3/UAS-lacZ (H) embryos. The expression pattern is similar to the Ubx expression

pattern in wildtype embryos detected with an anti-Ubx antibody (K). Numbers indicate

parasegments. (F, G, I, J) Haltere (F, I) and third leg (G, J) discs of Ubx-Gal4M1

/UAS-GFP

(F, G) and Ubx-Gal4M3/UAS-GFP (I, J) larvae, showing GFP expression. The Ubx protein

is also expressed in haltere (L) and third leg (M) discs.

Figure 3. Pattern of expression driven by the Abd-B- Gal4 lines. (A) Female

genitalia (g) of a yellow mutant fly. (B) Female genitalia of a y; Abd-B-Gal4LDN/UAS-y+

fly. Note the dark pigmentation of the vaginal teeth (arrow); a, analia. (C, D) ß-galactosidase

21

expression in germ band extended embryos of Abd-B-Gal4LDN/UAS-lacZ (C) and Abd-B-

Gal4199/UAS-lacZ (D) embryos. Both lines reproduce in part the pattern of expression of the

Abd-B m RNA (E), with strong signal in PS13. (F-K). Female (F, H, J) and male (G, I, K)

genital discs showing GFP expression driven by Abd-B-Gal4LDN (F, G) or Abd-B-Gal4199

(H, I), and wildtype Abd-B expression, detected with an anti-Abd-B antibody (J, K). Note

that Abd-B-Gal4LDN drives strong expression in A8 of males and females, but that Abd-B-

Gal4199 also shows weak signal in A9 of both sexes.

Figure 4. Eye color variation of the Abd-B-Gal4LDN line in Polycomb (Pc) or

trithorax (trx) heterozygous mutant backgrounds. A) the Abd-B-Gal4LDN individuals have

pale orange color. In Pc3/Abd-B-Gal4LDN flies this color is darker, while in trx/Abd-B-

Gal4LDN flies (B) the eye color it is slightly lighter.

Figure 5. Phenotypic effects of the functional substitution of a BX-C Hox

gene for another one. A) Wildtype haltere (h). B) Haltere partially transformed to wing in

the abx bx3 pbx/Ubx-Gal4LDN mutant combination. (C, D) Rescue of this phenotype in Ubx-

Gal4LDN/abx bx3 pbx UAS-Ubx (C) and Ubx-Gal4LDN/abx bx3 pbx UAS-abd-A (D) flies.

(E) Wildtype female genitalia (g). (F) Posterior ventral region of an Abd-B-Gal4LDN/Abd-

BM1

adult, showing absence of genitalia. Occasionally there is a small eighth sternite (S8)

with a few bristles, as shown in the figure. (G) Genitalia of an UAS-Abd-B m/+; Abd-B-

Gal4LDN/ Abd-BM1

fly. Note the presence of vulva and some vaginal teeth (arrows). (H) In

Abd-B-Gal4LDN/UAS-abd-A Abd-BM1

flies, by contrast, there is no rescue of the mutant

phenotype (the arrow points to a sternite bristle). S7, seventh sternite. (I) Posterior dorsal

22

abdomen of a wild-type female. Note the small seventh tergite (T7) . (J) In Abd-B-

Gal4LDN/Abd-BM1

females the T7 is bigger than in the wild-type. This phenotype is almost

completely rescued in UAS-Abd-B m/+; Abd-B-Gal4LDN/Abd-BM1

flies (K) but not in Abd-

B-Gal4LDN/UAS-abd-A Abd-BM1

females (L; compare with J); these flies also show a small

eighth tergite pigmented and with bristles (T8) ; a, analia (absent in J and L); T6, sixth

tergite.