QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 November 2005
doi: 10.1242/dev.02173

This Article Summary Figures Only Full Text (PDF) A corrigendum has been published All Versions of this Article: dev.02173v1 133/1/117    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Foronda, D. Articles by Sánchez-Herrero, E. Search for Related Content PubMed PubMed Citation Articles by Foronda, D. Articles by Sánchez-Herrero, E.

Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule

David Foronda^*, Beatriz Estrada^*,, Luis de Navas 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.

Author for correspondence (e-mail: esherrero{at}cbm.uam.es)

Accepted 24 October 2005

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The genitalia of Drosophila derive from the genital disc and require the activity of the Abdominal-B (Abd-B) Hox gene. This gene encodes two different proteins, Abd-B M and Abd-B R. We show here that the embryonic genital disc, like the larval genital disc, is formed by cells from the eighth (A8), ninth (A9) and tenth (A10) abdominal segments, which most likely express the Abd-B M, Abd-B R and Caudal products, respectively. Abd-B m is needed for the development of A8 derivatives such as the external and internal female genitalia, the latter also requiring abdominal-A (abd-A), whereas Abd-B r shapes male genitalia (A9 in males). Although Abd-B r represses Abd-B m in the embryo, in at least part of the male A9 such regulation does not occur. In the male A9, some Abd-B m^–r^– or Abd-B r^– clones activate Distal-less and transform part of the genitalia into leg or antenna. In the female A8, many Abd-B m^–r^– mutant clones produce similar effects, and also downregulate or eliminate abdominal-A expression. By contrast, although Abd-B m is the main or only Abd-B transcript present in the female A8, Abd-B m^– clones induced in this primordium do not alter Distal-less or abd-A expression, and transform the A8 segment into the A4. The relationship between Abd-B and abd-A in the female genital disc is opposite to that of the embryonic epidermis, and contravenes the rule that posteriorly expressed Hox genes downregulate more anterior ones.

Key words: Hox, abdominal-A, Abdominal-B, Distal-less, Genitalia, Imaginal disc

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Hox genes specify the morphological diversity along the anteroposterior axis of most animal species (Mann and Morata, 2000; McGinnis and Krumlauf, 1992). Hox proteins contain a DNA-binding domain, the homeodomain (Gehring et al., 1994), and act as transcription factors, controlling the expression of different downstream genes (Graba et al., 1997). The outcome of Hox gene activity is the formation of different structures. In fact, one remarkable aspect of some Hox mutations is the transformation of one structure into another one (Mann and Morata, 2000; McGinnis and Krumlauf, 1992).

These transformations are particularly striking in Drosophila appendages. For example, the ectopic expression of the Hox gene Antennapedia in the antennal primordium converts the antenna into a leg (Frischer et al., 1986; Jorgensen and Garber, 1987; Schneuwly et al., 1987a; Schneuwly et al., 1987b). In some cases, homeotic transformations have revealed the hidden appendage structure of certain organs. Thus, the genitalia of Drosophila derive from a primordium with appendage-like characteristics (Gorfinkiel et al., 1999), and are indeed transformed into a leg or an antenna in the absence of the Hox gene Abdominal-B (Abd-B) (Estrada and Sánchez-Herrero, 2001).

The genitalia and analia (collectively known as terminalia) are ectodermic structures located at the posterior region of the adult that derive from the genital disc. This disc is the only unpaired disc of Drosophila, and is formed by the fusion of three primordia corresponding to the eighth (A8), ninth (A9) and tenth (A10) abdominal segments. The different development of these primordia in males or females depends on the sex-determination signals. In females, the A8 segment forms the female genitalia and the eighth tergite, the A9 forms the parovaria and part of the uterine wall (both belonging to the internal genitalia), and the A10 forms the female analia. In males, the A8 gives rise to a tiny A8 segment, the A9 to the male genitalia and the A10 to the male analia (Keisman et al., 2001; Nöthiger et al., 1977; Schüpbach et al., 1978). The morphology of these structures, however, also depends on two other groups of genes: one group is formed by genes such as engrailed, hedgehog, decapentaplegic (dpp) and wingless, genes that are involved in signaling pathways (Casares et al., 1997; Chen and Baker, 1997; Emerald and Roy, 1998; Freeland and Kuhn, 1996; Keisman and Baker, 2001; Sánchez et al., 1997; Sánchez et al., 2001); the other group includes the Hox gene Abd-B, which is needed for the formation of the genitalia (Casanova et al., 1986; Celniker et al., 1990; Estrada and Sánchez-Herrero, 2001; Karch et al., 1985; Sánchez-Herrero et al., 1985; Tiong et al., 1985), and the Hox-like gene caudal (cad), which is required for analia development (Moreno and Morata, 1999). It is the combined activity of these three sets of elements (sex-determination genes, signaling pathways and Hox genes) that shapes the terminalia of the adult fly (reviewed by Christiansen et al., 2002; Estrada et al., 2003; Sánchez and Guerrero, 2001).

Homeotic gene function in the genital primordia, however, is more complex than what we have just described. First, the abdominal-A (abd-A) Hox gene, required for the development of A1-A8 in the embryo (Sánchez-Herrero et al., 1985; Tiong et al., 1985), is expressed in the A8 of the female genital disc (Casares et al., 1997; Freeland and Kuhn, 1996). No function has been ascribed yet to such expression. Second, Abd-B is a complex gene: the use of four different promoters and the existence of specific exons give rise to several transcripts that encode two different proteins. The A (m) transcript encodes the Abd-B M (or Abd-B I) protein, and the B, C (r) and RNAs encode the Abd-B R (or Abd-B II) protein (Celniker et al., 1989; DeLorenzi et al., 1988; Kuziora and McGinnis, 1988; Zavortink and Sakonju, 1989). The Abd-B M protein has 221 amino acids more than the Abd-B R product does in its N-terminal domain but both proteins share a common C-terminal region, which includes the homeodomain (Celniker et al., 1989; Zavortink and Sakonju, 1989). In the embryonic epidermis, the Abd-B M transcript and protein are expressed in parasegments (PS) 10-13 (A5-A8 segments), whereas the Abd-B R transcript and protein are present in PS14-PS15 (A9-A10) initially, and in PS14 (A9) at late stages (Boulet et al., 1991; Celniker et al., 1989; DeLorenzi et al., 1988; DeLorenzi and Bienz, 1990; Kuziora and McGinnis, 1988; Sánchez-Herrero and Crosby, 1988). The RNA is transcribed in just a few cells of PS14 or PS15 (Kuziora and McGinnis, 1988).

The role of Abd-B M and Abd-B R products in genital development remains unclear. Abd-B m mutations transform the A5-A8 segments into the A4 segment, both in males and females; the female genitalia are lost whereas male genitalia remain intact (Casanova et al., 1986; Karch et al., 1985; Sánchez-Herrero et al., 1985; Tiong et al., 1985). By contrast, mutations in Abd-B r function eliminate genitalia and analia in both sexes (Casanova et al., 1986). Significantly, the transformations obtained in either Abd-B m or Abd-B r mutants clearly differ from those observed when all Abd-B functions are eliminated: in some of the clones mutant for Abd-B (m and r), part of the male or female genitalia are transformed into leg or antenna (Estrada and Sánchez-Herrero, 2001). Therefore, the precise role of abd-A, Abd-B m and Abd-B r in genitalia development is not well defined.

We have analyzed homeotic expression and requirement in terminalia development. We propose that in the embryonic genital disc, as in the larval discs, Abd-B m, Abd-B r and cad are expressed in the A8, A9 and A10, respectively. We also report that abd-A, Abd-B m and Abd-B r are needed for development of the internal female genitalia, Abd-B m for the development of female external genitalia and Abd-B r for the development of male genitalia. Strikingly, abd-A and Abd-B bear unexpected relationships in mature genital discs. In the A8 of the female genital disc, Abd-B M maintains abd-A expression. In Abd-B m mutant clones, however, another Abd-B protein maintains abd-A expression but does not prevent abd-A function, as these clones transform the A8 segment into the A4. In the male A9, Abd-B r function does not repress the Abd-B m transcript, at least in part of the primordium, and some Abd-B r mutant clones transform male genitalia into leg or antenna. These relationships between Hox genes are different from those reported in the embryonic epidermis and contravene the rule that posteriorly expressed Hox genes repress those expressed more anteriorly.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Genetics
abd-A^M1 is an abd-A null mutation (Sánchez-Herrero et al., 1985); abd-A^iab3-277 is a breakpoint located at +63.0-64.5 kb [coordinates according to Karch et al. (Karch et al., 1985)], which transforms abdominal segments posterior to the A2 into the A2 (Busturia et al., 1989; Karch et al., 1985). The Df109 (or DfUbx¹⁰⁹) deficiency (89D1-89E1-2) eliminates the Ubx and abd-A genes (Karch et al., 1985; Lewis, 1978; Sánchez-Herrero et al., 1985), and the DfR59 deficiency eliminates the bxd regulatory region of Ubx, the abd-A gene and the 3' regulatory region of Abd-B (Gyurkovics et al., 1990). Dpbxd¹¹¹ is a duplication on the X chromosome that includes the abd-A and Abd-B genes (Lewis, 1981). Abd-B^D18, a small deficiency (from +148.5-150.0 to +163.5-166.5 kb) removing the Abd-B gene (Hopmann et al., 1995), and Abd-B^M1 (Casanova et al., 1986; Sánchez-Herrero et al., 1985) are Abd-B (m^– r^–) mutations. Abd-B^M5, Abd-B^M3 (Casanova et al., 1986; Sánchez-Herrero et al., 1985), Abd-B^T2N (Estrada et al., 2002) and Abd-B^D14 (Karch et al., 1985) are Abd-B m^– mutations: Abd-B^M5 is caused by a C to T base substitution that produces a stop codon at amino acid 119, and thus resulting in an Abd-B M protein of 118 amino acids instead of the normal 493 (S. Pelaz and G. Morata, personal communication); Abd-B^T2N is a derivative of a P-element insertion located at 48,957 (Estrada et al., 2002) [coordinates according to Martin et al. (Martin et al., 1995)]; Abd-B^D14 is a 411 bp deletion extending from 66 bp upstream to 345 bp downstream of the transcription initiation site of the Abd-B m RNA (Zavortink and Sakonju, 1989). Abd-B^Uab1 and Abd-B^X23-1 are Abd-B r^– mutations (Casanova et al., 1986): Abd-B^Uab1 is an inversion within the bithorax complex with a breakpoint at +185 kb (Karch et al., 1985) and Abd-B^X23-1 has a breakpoint at, approximately, +189 kb (Mack et al., 1997). The abd-A-lacZ (HC7JA1) (Bender and Hudson, 2000) and hdc-lacZ (B5) (Weaver and White, 1995) lines are P-lacZ insertions that reproduce the expression of the abd-A and headcase genes, respectively. The Gal4/UAS system (Brand and Perrimon, 1993) was used to drive the expression of different genes with the following constructs: UAS-abd-A (Michelson, 1994), UAS-Abd-B m (Castelli-Gair et al., 1994), UAS-Dll (Gorfinkiel et al., 1997), UAS-GFP (Ito et al., 1997), UAS-lacZ (Brand and Perrimon, 1993) and UAS-myc-EGFP^F (Allan et al., 2003). The Abd-B-Gal4^LDN line (L.d.N. and E.S.-H., unpublished) and the cad-Gal4 line (MD509) (Calleja et al., 1996) are insertions in the Abd-B and cad genes, respectively, which reproduce the pattern of expression of the Abd-B m and cad transcripts in the posterior embryonic abdominal segments. The dpp-Gal4 line has been previously described (Staehling-Hampton et al., 1994). The MKRS, abd-A-lacZ (Bender and Hudson, 2000), TM6B, and TM6B, abd-A-lacZ balancers were used to identify mutant larvae and embryos.

In situ hybridization
In situ hybridization was done basically as described (Cubas et al., 1991; Wolff, 2000), with slight modifications. The DNA probe for the Abd-B m (A) transcript is a BamHI genomic probe from 50,702 to 48,864 [coordinates as in Martin et al. (Martin et al., 1995)]. RNA probes for the Abd-B m (A) or Abd-B r transcripts were obtained by the amplification of Abd-B cDNA or genomic DNA with region-specific primers followed by in vitro transcription reactions (Stoflet et al., 1988), using the Roche DIG RNA-labeling mix. The Abd-B m probe includes 671 bp of the Abd-B m-specific exon and was obtained with the following primers: 5'-CAGCAACTACAACAACAGCCGAC-3' and 5'-ACACGCACACTGCCTAAAGAGC-3'. Two `common' probes were used to detect all Abd-B r RNAs. The first one (I) includes 220 bp of two exons common to all the Abd-B r cDNAs and 159 bp of the exon specific for the Abd-B r C cDNA, and was obtained with the following primers: 5'-TGGAAAGATCAGACTTGCAGGTCACG-3' and 5'-TGGGATGGGAACTGACGCTGGA-3'. The second one (II) includes 7 bp of an exon common to all the Abd-B cDNAS, 223 bp of two exons common to all the Abd-B r cDNAs and 66 bp of the exon specific for the Abd-B r cDNA, and was obtained with the following primers: 5'-CGGAAGATTGTATTTGTGCGGTTG-3' and 5'-TTGATGTCTGTGGGATGGGAAC-3'.

Double X-gal staining and in situ hybridization was performed basically as described previously (Wolff, 2000). For double antibody staining and in situ hybridization in imaginal discs, in situ hybridization was carried out first (Wolff, 2000), and this was followed by four washes in PBS for 15 minutes each, incubation with the primary antibody overnight at 4°C, four washes in PBS and incubation with the appropriate biotinylated secondary antibody for two hours at room temperature; the imaginal discs where then washed and stained with the Vectastain ABC kit.

Immunohistochemistry
Immunohistochemistry was carried out as previously described (Sánchez-Herrero, 1991; Estrada and Sánchez-Herrero, 2001), with slight modifications. The antibodies used were mouse and rabbit anti-ß-galactosidase (Cappel), mouse anti-Abd-B lA2E9 (Celniker et al., 1989), rabbit anti-Abd-B (DeLorenzi and Bienz, 1990), rat anti-abd-A (Macías et al., 1990), guinea-pig anti-Hth (Azpiazu and Morata, 2002), guinea-pig anti-Snail (Kosman et al., 1991), rabbit anti-Tsh (Wu and Cohen, 2000), rabbit Anti-Dll (Panganiban et al., 1995), rat anti-Dll (Wu and Cohen, 2000) and mouse anti-Dll (Duncan et al., 1998). Secondary antibodies were coupled to Red-X, Texas Red, FITC and Cy5 fluorochromes (Jackson Immunoresearch). Staining with To-Pro-3 iodide was done as previously described (Baena-López et al., 2003).

Histochemical staining
Internal genitalia were dissected out of late pupae, pharates or adults, and the staining to detect ß-galactosidase expression was carried out as described previously (Ashburner, 1989).

Clonal analysis
Mitotic recombination clones were induced during the larval period by the FLP/FRT system (Xu and Rubin, 1993), with or without using the Minute technique (Morata and Ripoll, 1975). Clones were identified in the adult by the yellow cuticular marker and in the imaginal discs by the loss of the lacZ or GFP markers. The genotypes of the larvae in which the clones were induced are the following (in females the hs-flp is carried as an heterozygous insertion).

abd-A^– clones: y hs-flp122; FRT82B abd-A^M1/FRT82B arm-lacZ.

Abd-B^– (Abd-B m^–r^–) clones: w f^36a hs-flp122; FRT82B Abd-B^D18/FRT82B arm-lacZ Dp(f⁺) M(3)w123, y hs-flp122; FRT82B Abd-B^D18/FRT82B hs-CD2 M(3)w y⁺ and y hs-flp122; FRT82B Abd-B^D18/FRT82B Ubi-GFP M(3)rpS3.

Abd-B m^– clones: y hs-flp122; FRT82B Abd-B^M5 or FRT82B Abd-B^M3/FRT82B arm-lacZ, y hs-flp122; FRT82B Abd-B^M5 or FRT82B Abd-B^M3/FRT82B hs-CD2 M(3)w y⁺, y hs-flp122; FRT82B Abd-B^M5 or FRT82B Abd-B^M3/FRT82B Ubi-GFP M(3)rpS3, w f^36a hs-flp122; FRT82B Abd-B^M5 or FRT82B Abd-B^M3/FRT82B arm-lacZ Dp(f⁺) M(3)w123, and y hs-flp hs-GFP FRT18A/Dpbxd¹¹¹ arm-lacZ FRT18A; Abd-B^D14/Abd-B^D18.

Abd-B r^– clones: y hs-flp122; FRT82B Abd-B^Uab1 or FRT82B Abd-B^X23-1/FRT82B arm-lacZ, w f^36a hs-flp122; FRT82B Abd-B^Uab1 or FRT82B Abd-B^X23-1/FRT82B arm-lacZ Dp(f⁺) M(3)w123 and y hs-flp122; FRT82B Abd-B^X23-1/FRT82B hs-CD2 M(3)w y⁺.

Abd-B^– abd-A⁺ clones: y tub-Gal4 UAS-GFP; FRT82B Gal80/FRT82B Abd-B^D18 UAS-abd-A.

Adult cuticle analysis
Flies were kept in a mixture of ethanol: glycerol (3:1) macerated in 10% KOH at 60°C for 10 minutes, dissected, washed with water, dehydrated with ethanol and mounted in Euparal for inspection under a compound microscope.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression and function of Abdominal-B in the embryonic genital disc
In the third instar genital disc of Drosophila, Abd-B is expressed in the A8 and A9 segments, and cad in the A10 (Casares et al., 1997; Freeland and Kuhn, 1996). To study whether these expression domains are established early in development, we have analyzed Abd-B and cad transcription in the embryonic genital disc. This disc is identified by the expression of genes like snail, escargot or headcase (hdc), and we selected the hdc-lacZ B5 line, which reproduces the pattern of hdc RNA expression (Weaver and White, 1985), to mark the genital disc. At about stage 15 [stages according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1995)], hdc is expressed in three clusters of cells, two anterior ones placed bilaterally, and a third one located in a more posterior and central position (Weaver and White, 1995) (Fig. 1A and Fig. 2A). The three clusters fuse later in development to form the genital disc. At stage 15, we counted six to seven cells at each of the two anterior groups, and two to three cells in the posterior one, making up a total of 14-17 cells (n=12). Double staining with anti-Abd-B and anti-ß-galactosidase antibodies (in hdc-lacZ embryos), or with GFP and anti-ß-galactosidase antibody (in cad-Gal4/UAS-GFP; hdc-lacZ/+ embryos), shows that Abd-B is expressed in the two anterior clusters and cad in the posterior one (Fig. 1A-F).

View larger version (72K): [in this window] [in a new window]   Fig. 1. Expression of Abd-B and cad in the embryonic genital disc. Anterior is to the top. (A-C) Double staining of a late stage 15 hdc-lacZ embryo with anti-ß-galactosidase (green, A, note the three clusters) and anti-Abd-B (red, B) antibodies. A merged image is shown in C. Note that the posterior cluster of genital disc cells is not marked with Abd-B (arrow). (D-F) Double staining of a stage 15 cad-Gal4/UAS-GFP; hdc-lacZ/+ embryo marked with anti-ß-galactosidase (red, D) and GFP (green, E). A merged image is shown in F. See that the posterior cluster (arrow) shows cad expression. (G-L) Embryonic genital disc cells of a UAS-myc-EGFPF/+; Abd-B-Gal4LDN/hdc-lacZ embryo, showing expression corresponding to the Abd-B m transcript (GFP expression, green, I-L) in the anterior lateral cells of the disc primordium, marked with an anti-ß-galactosidase antibody (blue, G,J,L). GFP-marked cells are a subset of those expressing Abd-B (red, H,K,L). Note that about four anterior-lateral cells of the disc primordium express GFP (arrows, J) and that some Abd-B-expressing cells are not marked with GFP (arrows, K). A merged image is shown in L. vc, ventral cord.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Abd-B is needed for the formation of the embryonic genital disc. Anterior is to the top. (A) Late stage 15 hdc-lacZ embryo showing the three clusters of cells that will form the genital disc. (B) Stage 15 Abd-BM5 hdc-lacZ homozygous embryo. The embryonic genital disc is formed by fewer cells in the mutant than in the wild type. (C) Similar stage embryo of the genotype Abd-BUab1/Abd-BM1 hdc-lacZ. The genital disc is disorganized and includes fewer cells than the wild type does. (D) Stage 15 Abd-BM1 hdc-lacZ homozygous embryo. Some ß-galactosidase-expressing cells are scattered (out of focus) and only the posterior cluster remains (arrow). (E,F) In Abd-BM1 hdc-lacZ homozygous embryos, Dll is ectopically activated in some posterior cells (arrows, E; red, F); hdc is marked in green in F. vc, ventral cord.

While this manuscript was under review, Chen and collaborators (Chen et al., 2005) showed that whereas snail (sna) or escargot are expressed in all of the cells from the embryonic genital disc (Chen et al., 2005; Fuse et al., 1996; Hartenstein and Jan, 1992; Whiteley et al., 1992), hdc is transcribed just in a subset of these cells. We have confirmed this result by staining hdc-lacZ embryos with anti-ß-galactosidase and anti-Sna antibodies: three to five cells between the two anterior hdc-expressing groups of cells, and some cells posterior to them are stained with the anti-Sna antibody (data not shown). However, whether or not we mark the disc with either hdc-lacZ or Sna, Abd-B is expressed only in the anterior cells of the disc, and the Abd-B m-Gal4 line drives signal just a subset of these cells (Fig. 1A-C,I-L; data not shown). Chen et al. (Chen et al., 2005) have also analyzed homeotic gene expression and requirement in the genital disc, and conclude, as we do, that the embryonic genital disc is formed by cells that express Abd-B m (plus abd-A), Abd-B r and cad, and which probably correspond to the A8, A9 and A10 segments, respectively.

abdominal-A is required to form the internal female genitalia
To study homeotic gene function in the genital disc after embryonic stages, we analyzed abd-A and Abd-B expression and function in larvae and pupae. The mature female genital disc expresses abd-A (Casares et al., 1997; Freeland and Kuhn, 1996) (Fig. 3A) in a domain within the A8 segment that, according to fate maps, corresponds to the presumptive internal female genitalia (Epper, 1983; Littlefield and Bryant, 1979). We have confirmed this correspondence by studying abd-A expression throughout late larval and pupal development with an abd-A-lacZ line that reproduces abd-A expression in the third instar female genital disc (Bender and Hudson, 2000) (Fig. 3B, compare with 3A). In abd-A-lacZ pupae, the internal female genitalia are intensively stained, including the uterus, seminal receptacle, oviducts and spermatheca, but not the parovaria (Fig. 3C,D). To explore whether abd-A is required for female internal genitalia development, we used the abd-A^iab3-277 allele (see Material and methods). In abd-A^iab3-277/abd-A^– female genital discs, abd-A expression is substantially reduced (Fig. 3E), and in abd-A^iab3-277/abd-A^– females most of the internal genitalia disappear or are highly abnormal (Fig. 3F, compare with wild-type internal genitalia in 3C). As previously reported (Karch et al., 1985) these mutant females lack ovaries.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Expression and requirement of abd-A in the female internal genitalia. (A) Third instar female genital disc, stained with an anti-abd-A antibody. (B) Similar disc of an abd-A-lacZ larva stained with X-Gal. The pattern of expression reproduces that of the anti-abd-A antibody. (C,D) X-Gal staining of internal female genitalia from two late pupae. The seminal receptacle (sr), spermathecae (s), uterus (u) and oviducts (ov) are stained, whereas parovaria (p) are not. o, ovaries. (E) Female genital disc of an abd-Aiab3-277/DfR59 female larva stained with anti-abd-A antibody, showing a reduction in abd-A expression compared with wild type. (F) Female internal genitalia of an abd-Aiab3-277/Df 109 female. See that most elements of the internal genitalia and the ovaries have disappeared (compare with C). vp, vaginal plates (part of the external genitalia).

As Abd-B M is the main or only product expressed in the A8, most Abd-B m mutant clones in this primordium should behave as clones that eliminate both Abd-B functions, that is, they should transform part of the female genitalia into leg or antenna (Estrada and Sánchez-Herrero, 2001). However, Abd-B m mutant clones, similar to Abd-B m escapers (Casanova et al., 1986; Karch et al., 1985; Sánchez-Herrero et al., 1985; Tiong et al., 1985), transform the dorsal eighth tergite into an anterior tergite, and the genitalia into structures sometimes resembling a sternite (Fig. 5A,B). In males, these clones also transform the tiny A8 into an anterior abdominal segment (Fig. 5C).

The difference between Abd-B^– and Abd-B m^– clones is also observed in the A8 of the female genital disc. Although Abd-B^– clones present smooth borders and sometimes activate Dll (Estrada and Sánchez-Herrero, 2001), Abd-B m^– clones are indented and do not express Dll (Fig. 5D-F). We note that most of the Abd-B^– clones that induce Dll transcription are located in the region where abd-A is expressed. Because abd-A represses Dll in the embryo (Cohen et al., 1991; Simcox et al., 1991; Vachon et al., 1992), we decided to examine in more detail the relationship between abd-A, Abd-B and Dll. abd-A mutant clones do not activate Dll (Fig. 5G-I) and do not change Abd-B expression (Fig. 5J-L). Reciprocally, Abd-B m^– clones do not alter abd-A transcription (Fig. 5M-O).

View larger version (43K): [in this window] [in a new window]   Fig. 4. Abd-B m and Abd-B r expression in mature genital discs. (A) Scheme representing the Abd-B transcription unit (not drawn to scale). The RNA is not represented. Red or blue rectangles represent exons of Abd-B m (red) or of Abd-B r (blue) transcripts, and black boxes represent coding regions. (B,C) Drawings of the female (B) and male (C) genital discs (ventral views), indicating the A8 and A9 primordia. The A10 primordium is on the opposite side in both discs. (D,F) Female discs hybridized with probes detecting the Abd-B m transcript (D) or the Abd-B r RNAs (`common' probe; F). (E,G) Male discs hybridized with Abd-B m (E) and Abd-B r (G) probes. Arrowheads in D and F indicate the A9, and in E and G, the A8 segment. Note that the Abd-B m transcript is expressed in the A8 of female and male discs, with some weak expression in the male A9, and that the Abd-B r transcript is present in the male and female A9, with some signal also in the female A8 (arrows; the most central expression in this primordium, out of focus, is the signal from the dorsal A9). Note also the low or absent Abd-B r expression in the anterior female A9.

View larger version (86K): [in this window] [in a new window]   Fig. 5. Abd-B m function in the genitalia and the female genital disc. (A) An Abd-BM5 mutant clone in the female right eighth hemi-tergite, marked with yellow, is converted into an anterior tergite (arrow). Compare with the left, wild-type, hemi-tergite (arrowhead). (B) Similar mutant clone showing transformation of the genitalia into a sternite (arrow). S7, seventh sternite. (C) Abd-BM3 clone, marked with yellow, showing transformation of the male A8 tergite (normally very small) into an anterior one (arrow). T7, seventh tergite; g, genitalia. (D-F) Abd-BM5 clones in the A8 of the female disc, marked by the absence of the lacZ marker (green, D), do not activate Dll (red, E; arrowheads in E and H mark the wild-type Dll expression). a, analia. The merged image is shown in F. (G-L) abd-AM1 mutant clones in the female A8, marked by the absence of the ß-galactosidase marker (G,J, green), do not eliminate Dll (H, red) or Abd-B (K, red) expression. Merged images are shown in I and L. (M-O) Abd-BM5 clones in the A8 of the female genital disc, marked by the absence of the lacZ marker (in green in M), do not change abd-A expression (red, N). A merged image is shown in O. (P-R) Abd-BD18 mutant clones in the A8 segment, marked by the absence of the ß-galactosidase marker (blue, P,R; clones are outlined), eliminate abd-A expression, detected by an anti-abd-A antibody (green, Q,R), and activate Dll protein expression (red, P,R). (S) Abd-BD18 abd-A+ clones, marked with GFP (in green), eliminate Abd-B (blue) and activate Dll (red) expression. The boxed clone is shown in detail in T-W. (X) Similar clones induced in the leg disc do not eliminate Dll, as shown by the co-expression of the GFP marker (green) and Dll (red), giving a yellow color (arrowheads). (Y) dpp-Gal4/UAS-Dll female disc showing elimination of abd-A expression in the dpp domain (arrowheads).

Our phenotypic analysis of Abd-B m function has been confined so far to the external genitalia and the eighth tergite. However, Abd-B m transcripts are also present, although at low levels, in the presumptive internal genitalia of third instar genital discs (Fig. 4D). To follow Abd-B m transcription at later stages, we examined lacZ expression in the internal female genitalia of Abd-BGal4^LDN/UAS-lacZ late pupae and pharate adults. As shown in Fig. 6I,J, and similar to the expression observed in abd-A-lacZ females (Fig. 3C), X-gal staining is detected in the spermathecae, the uterus and, weakly, in the seminal receptacle, but not in the parovaria; however, and in contrast to the results obtained with the abd-A insertion, oviducts were not stained (Fig. 6I). In females mutant for Abd-B m, the internal female genitalia do not develop properly and, in some cases, parovaria are the only structures remaining (Fig. 6K). In males, Abd-B m mutations do not affect the internal genitalia, although the testes are reduced in size and are not connected properly with the internal genitalia (data not shown).

View larger version (68K): [in this window] [in a new window]   Fig. 6. Abd-B expression in Abd-B m mutant embryos and clones, and Abd-B m expression and function in the female internal genitalia. (A) Wild-type stage 11 embryo showing Abd-B expression, detected with the lA2E9 monoclonal antibody, in PS12-PS14 (A7-A9). (B) Abd-BM5 homozygous embryo at a similar stage, stained with the same antibody. The Abd-B expression is restricted to PS14. (C-E) Abd-BM5 clones induced in female A8 do not eliminate Abd-B expression. Clones are marked by the absence of GFP marker (green, C), and Abd-B expression is detected with the anti-rabbit anti-Abd-B (red, D). A merged image is shown in E. (F) Abd-BM5 clones induced in the same primordium, marked by the absence of X-gal staining (light blue), do not ectopically activate Abd-B r expression (purple). (G,H) Detail of the regions boxed in F. Note the Abd-B r wild-type expression in the female A9 (arrowheads) and its absence, or very low expression, in the clones (arrows). (I) Female internal genitalia of an UAS-lacZ/+; Abd-B-Gal4LDN/+ late pupa. The spermathecae (s) and uterus (u) are stained, whereas parovaria (p) and oviducts (ov) are not. o, ovaries; vp, vaginal plates. (J) Detail of I, showing absence of staining in the parovaria. (K) Abd-B m mutant female showing disappearance of all the external and internal genitalia except the parovaria (p). h, hindgut; a, analia.

In Abd-B r mutant flies, the internal genitalia of males is absent (not shown), while that of females is abnormal. A common feature of these females is the absence of parovaria, and in about half of them we observe three and even four spermathecae instead of the normal two (Fig. 7L). Supernumerary spermathecae are also detected in females bearing Abd-B r^– clones (data not shown). In a few cases, duplications of part of the internal genitalia are observed in Abd-B r mutant females.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this work, we describe the expression and function of abd-A and Abd-B in Drosophila genitalia development. As in the embryonic cuticle, abd-A and Abd-B m are needed in the A8 whereas Abd-B r is required in the A9. The relationship between these homeotic products in the mature genital discs, however, clearly differs from what is observed in the embryonic epidermis. In what follows, we discuss several aspects of abd-A and Abd-B expression and function in genital development.

Abdominal-B expression and function in the embryonic genital disc
We report that the embryonic genital disc has three distinct cell populations at stages 15/16: some anterior-lateral cells transcribe Abd-B m, anterior-central and middle cells express Abd-B r and posterior cells transcribe cad, although the expression of these products may overlap. Because the genital disc is formed by the fusion of cells coming from the A8, A9 and A10 segments (Nöthiger et al., 1977; Schüpbach et al., 1978), and by analogy to the expression of these genes in the mature genital discs (Casares et al., 1997; Freeland and Kuhn, 1996) (this report), we conclude that Abd-B m, Abd-B r and cad are probably expressed in the A8, A9 and A10 segments, respectively, of the embryonic genital disc.

Abd-B is not only expressed, but also required in the embryonic genital primordium. In the absence of Abd-B m, the number of hdc-expressing cells in the disc is reduced, most likely because these cells adopt now a more anterior fate, as occurs in the cuticle (Sánchez-Herrero et al., 1985; Tiong et al., 1985). When Abd-B r is absent, the genital primordium lacks some cells and is disorganized, and when both Abd-B products are absent, the primordium is reduced to a few, dispersed cells, some of which express Dll ectopically, suggesting a transformation into a leg primordium.

View larger version (74K): [in this window] [in a new window]   Fig. 7. The Abd-B r function is required for the development of the male genitalia. (A-D) In the male genitalia, some Abd-BUab1 clones transform into leg tissue (A, boxed region is amplified in B; arrows point to bracted bristles) or into antenna (C, boxed region is amplified in D), ar, malformed arista; III, third antennal segment. (E-G) Some Abd-BUab1 clones in the A9 of the male disc, marked by the absence of the lacZ marker (red, E) eliminate Abd-B expression (green, F). Merged image in G. (H-J) Male disc bearing similar clones, marked by the absence of GFP (green, H), eliminate Abd-B expression (I, blue), and in some cells activate Dll (I,J, red, arrow). In some clones there is no Abd-B expression but no Dll activation either (arrowheads, I). (K) Drawing of a male genital disc showing the distribution of Abd-BUab1 clones that eliminate Abd-B expression (pink) and those that do not (green). The region where most `pink' clones accumulate includes, mainly, the presumptive penis apparatus domain. (L) Internal genitalia of an Abd-BUab1/Abd-Bx23-1 female. Note the presence of three spermathecae (s) in the highly abnormal internal genitalia. vp, vaginal plates.

abdominal-A and Abdominal-B m requirement in internal female genitalia development
We have shown that abd-A is expressed in the whole internal female genitalia except for the parovaria, and this is consistent with experiments indicating that parovaria derive from the female A9 segment (Keisman et al., 2001). abd-A has been shown to be required for gonad development (Boyle and Dinardo, 1995; Brookman et al., 1992; Cumberledge et al., 1992; Greig and Akam, 1995; Karch et al., 1985; Karch et al., 1990; Moore et al., 1998; Warrior, 1994), and in the abd-A^iab-3/Df mutant combinations ovaries are also absent. However, the defects we observe in the female internal genitalia are not simply due to an indirect effect of the lack of gonads, as iab-4 mutations prevent the formation of the ovaries but do not alter internal genitalia formation (Cumberledge et al., 1992).

Our results indicate that Abd-B m is required for the development of female external and internal genitalia, both derived from the female A8 (Nöthiger et al., 1977; Schüpbach et al., 1978). The internal genitalia of Abd-B-Gal4^LDN/UAS-lacZ females were stained with X-gal except in two structures, the oviducts and parovaria. The absence of oviduct staining in Abd-B-Gal 4^LDN/UAS-lacZ females is probably due to the particular expression driven by this reporter, and does not imply an absence of Abd-B m transcription in these organs, for two reasons: first, Abd-B m transcripts are present in the whole A8 segment of the female genital disc; and second, oviduct development is affected in Abd-B m mutant females. Parovaria, by contrast, are not stained in Abd-B-Gal 4^LDN/UAS-lacZ or abd-A-lacZ females, and this agrees with their A9 provenance (Keisman et al., 2001). This is supported by the observation that in some Abd-B m mutant females parovaria are the only structures that remain in the internal female genitalia.

abdominal-A, Abdominal-B m and Abdominal-B r cross-regulatory interactions in the genital disc
Abd-B M seems to be the main or only Abd-B product present in the female A8, so it was expected that elimination in this segment of just Abd-B M or of all Abd-B proteins would give similar results. This is not so. Some Abd-B^– clones transform part of the female genitalia into leg or antenna (Estrada and Sánchez-Herrero, 2001), whereas Abd-B m mutant clones convert the eighth tergite, and probably the female genitalia, into an anterior abdominal segment. The differences between Abd-B m^– and AbdB^– clones in the A8 of the female genital disc reveal the existence of unsuspected regulatory interactions between the abd-A and Abd-B genes: whereas Abd-B m^– clones do not affect abd-A, in AbdB^– clones abd-A expression is eliminated. This is a surprising result, because it is contrary to what is observed in the embryo, where Abd-B represses abd-A (Karch et al., 1990; Macías et al., 1990; Sánchez-Herrero, 1991).

Abd-B m^– clones induced in the female A8 do not alter abd-A expression but do not change Abd-B expression levels either. This is observed with mutations that do not make Abd-B M protein, so the Abd-B protein detected is not the Abd-B M product. Surprisingly, although we detect some Abd-B r expression in the female A8, we do not see uniform Abd-B r expression throughout this primordium and Abd-B r transcripts seem not to be derepressed in Abd-B^M5 mutant clones. We have no explanation for this conundrum. Perhaps the probe used, although it includes sequences complementary to all of the Abd-B r cDNA sequences that have been published (Celniker et al., 1989; DeLorenzi et al., 1988; Kuziora and McGinnis, 1988; Zavortink and Sakonju, 1989), does not efficiently detect all of the non-Abd-B m transcripts.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Comparison of genetic interactions in embryonic epidermis and in the mature female genital disc, and organization of the A8 in the female genital disc. (A) Regulatory interactions between abd-A, Abd-B and Dll in the embryonic epidermis and in the mature genital discs. In the embryo, Abd-B represses abd-A in the A8, and abd-A represses Dll in the abdominal segments. In the third instar female genital disc, it seems that Abd-B maintains abd-A transcription, and that ectopic Dll can repress abd-A. (B) In the A8 of the female disc we can distinguish two regions (Estrada and Sánchez-Herrero, 2001) (this report). The anterior region (region I, purple) expresses buttonhead, abd-A and low levels of Abd-B, and can activate Dll in the absence of Abd-B. It represents the `appendage' part of the genitalia and corresponds mainly or exclusively to the internal genitalia. The lower region (region II) does not express abd-A or buttonhead, but expresses high levels of Abd-B. It corresponds to the `trunk' region of the female A8. In this region, Dll is not activated in the absence of Abd-B.

The Abdominal r function and male genitalia development
Abd-B r expression is restricted to the A9 segment in male genital discs, but shows expression in the A9 and in some cells of the A8 in female genital discs. In spite of this, Abd-B r^– clones in the external female genitalia (A8) are phenotypically wild type. In the male A9, some Abd-B r mutant clones eliminate Abd-B, activate Dll and transform part of the genitalia into distal leg or antenna. This is similar to the result obtained in some Abd-B^– clones, and it implies that Abd-B m is not derepressed in these mutant clones. However, Abd-B m is perhaps derepressed in those Abd-B r mutant clones where Abd-B signal (detected by the common antibody) remains.

Although Abd-B r^– clones affect, almost exclusively, male genitalia development, Abd-B r hemizygous or trans-heterozygous flies lack genitalia and analia in both sexes (Casanova et al., 1986). This probably reflects the absence of proper interactions between the different primordia needed for the growth of the genital disc (Gorfinkiel et al., 2003). In Abd-B r mutant females, the internal genitalia are abnormal, and in some of these females we observe an absence of parovaria and the presence of three or four spermathecae. This phenotype is consistent with a segment-autonomous transformation of A9 derivatives (parovaria) into A8 structures (spermathecae), similar to the embryonic cuticular transformation of A9 into A8 observed in Abd-B r mutations (Casanova et al., 1986). A transformation of parovaria into spermathecae has been previously described in Polycomblike mutants (Duncan, 1982), and may also indicate a transformation of A9 to A8.

Genetic organization of the genital disc
Our results illustrate that there are quite different Hox cross-regulatory interactions in the embryo and in the genital disc (Fig. 8A). The effects in the genital discs contradict the general rule that genes transcribed more posteriorly suppress or downregulate the expression of more anterior ones (Hafen et al., 1984; Struhl and White, 1985). This rule has, nevertheless, some exceptions in genes of the Antennapedia complex (Miller et al., 2001). Further, differences in Hox cross-regulation between the embryo and imaginal discs are not unprecedented: the proboscipedia (pb) Hox gene is positively regulated by Sex combs reduced in the embryo (Rusch and Kaufman, 2000), but pb activates Sex combs reduced in the labial imaginal disc (Abzhanov et al., 2001).

It has been proposed that the primordia of female and male genitalia could be subdivided into an `appendage-like' and a `trunk-like' region (Estrada and Sánchez-Herrero, 2001). We can now define these two regions of the female A8 more precisely. The `appendage-like' region would be that expressing abd-A and low levels of Abd-B, and corresponds approximately to the presumptive internal female genitalia (region I in Fig. 8B). This domain is roughly coincident with the region of expression of a reporter insertion in buttonhead, the gene that defines ventral appendage development (C. Estella, PhD Thesis, Universidad Autónoma de Madrid, 2003) (Estella et al., 2003), and this is also, approximately, the domain where Abd-B<sup>–</sup> clones may activate Dll (Estrada and Sánchez-Herrero, 2001). If this subdivision is correct, the `appendage' specification defined by buttonhead would be repressed in the wild type by Abd-B, which both limits Dll expression to a few cells of the A8 primordium and prevents Dll function (Estrada and Sánchez-Herrero, 2001). Abd-B^– clones in this region eliminate abd-A expression and promote leg or antenna development. This subdivision may also apply to the male disc, the penis apparatus presumptive region being the main `appendage' domain. Similar to what we have described, the labial disc possesses a large `appendage' region that is revealed by Dll derepression in pb mutations (Abzhanov et al., 2001). This characteristic, and the changes in Hox gene cross-regulation between the embryo and the imaginal disc, are two features shared by pb/labial disc and Abd-B/genital disc.

	ACKNOWLEDGMENTS

 
We thank G. Morata for support and discussions; N. Azpiazu and D. L. Garaulet for help with the experiments; A. Busturia, I. Guerrero, G. Morata and M. Suzanne for comments on the manuscript; S. Pelaz and G. Morata for communicating unpublished results; and R. González for technical assistance. We also thank M. Akam, W. Bender, S. Celniker, I. Duncan, I. Guerrero, F. Karch, A. Michelson, I. Miguel-Aliaga, R. White and the Bloomington Stock Center for stocks; and N. Azpiazu, M. Bienz, J. Casanova, F. Casares, S. Celniker, S. Cohen, A. Michelson, G. Panganiban and R. Reuter for antibodies. This work has been supported by grants from the Dirección General de Investigación Científica y Técnica (number BMC 2002-00300), the Comunidad Autónoma de Madrid (number 08.1/0031/2001.1) and an Institutional Grant from the Fundación Ramón Areces. D.F. and L.d.N. are supported by fellowships from the Spanish Ministerio de Educación y Ciencia.

	Footnotes

 
^* These authors contributed equally to this work

Present address: Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

	REFERENCES

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

Abzhanov, A., Holtzman, S. and Kaufman, T. C. (2001). The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendages. Development 128,2803 -2814.

Allan, D. W., Pierre, S. E., Miguel-Aliaga, I. and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113,73 -86.[CrossRef][Medline]

Ashburner, M. (1989). Drosophila: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.

Azpiazu, N. and Morata, G. (2002). Distinct functions of homothorax in leg development in Drosophila. Mech. Dev. 119,55 -67.[CrossRef][Medline]

Baena-López, A., Pastor-Pareja, J. C. and Rerino, J. (2003). Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs. Development 130,6497 -6506.[Abstract/Free Full Text]

Bender, W. and Hudson, A. (2000). P element homing to the Drosophila bithorax complex. Development 127,3981 -3982.[Abstract]

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.[Abstract]

Boyle, M. and DiNardo, S. (1995). Specification, migration and assembly of the somatic cells of the Drosophila gonad. Development 121,1815 -1825.[Abstract]

Brand, A. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant pheno