Expressing UAS-bab1 and UAS-bab2:
A Comparative Study of Gain-of-Function Effects and the
Potential to Rescue the bric a` brac Mutant Phenotype
Olivier Bardot,1† Dorothea Godt,2†Frank A. Laski,3and Jean-Louis Couderc1*
1INSERM UMR 384, Laboratoire de Biochimie, Clermont-Ferrand, France 2Department of Zoology, University of Toronto, Toronto, Ontario, Canada
3Department of Molecular, Cell, and Developmental Biology and Molecular Biology Institute, University of California at
Los Angeles, Los Angeles, California, USA Received 3 July 2002; Accepted 9 July 2002
The bric a` brac (bab) locus acts as a homeotic and morphogenetic regulator in the development of adult ovaries (Godt and Laski, 1995; Sahut-Barnola et al.; 1995; Couderc et al., 2002); legs (Godt and Laski, 1995; Chu et
al., 2002; Couderc et al., 2002); antennae (Godt et al., 1993; Chu et al., 2002) and abdomen (Kopp et al., 2000; Couderc et al., 2002) in Drosophila melanogaster. It contains two structurally related genes, bric a` brac1
(bab1) and bric a` brac2(bab2) (Couderc et al., 2002) that encode nuclear proteins sharing two strongly con-served domains, an N-terminal BTB domain and a C-terminal Psq domain (Godt et al., 1993; Zollman et al., 1994; Couderc et al., 2002). Both domains have been found in a number of transcriptional regulators (Collins
et al.; 2001; Lehmann et al.; 1998). The Bab1 and Bab2 proteins interact with BIP2/TAFII155/TAF3, a compo-nent of TFIID (Pointud et al., 2001), suggesting a func-tion in transcripfunc-tional regulafunc-tion by directly interacting with the basal transcriptional machinery.
The analysis of bab mutants suggested a largely redun-dant but also partially differential requirement of bab1 and bab2 for the development of legs, ovaries, and the abdomen, with bab2 apparently playing a predominant role in legs and ovaries (Couderc et al., 2002). However, we found it to be difficult to precisely determine the individual function of bab1 and bab2 for two reasons. First, it is not certain whether any of the isolated bab mutations is a true null for bab1 or bab2. Second, it is unclear whether bab mutations that map to one bab gene affect only the function of this gene, as we have evidence for cross-regulation between the two bab genes (Godt, Laski, and Couderc, unpubl. data). There-fore, to further analyze the respective roles of the two genes we compared the effects produced by mis/over-expression of bab1 and bab2 and their potential to rescue the bab mutant phenotype.
Ectopic Expression of UAS-bab1 and UAS-bab2 Transgenes
A bab1 minigene and a bab2 cDNA, both containing the entire open reading frame, were cloned into the
pUAST vector (Brand and Perrimon, 1993). Five inde-pendent transgenic lines were established for bab1 and more than 20 for bab2 (Couderc et al., 2002; Table 1). Ubiquitous expression of bab transgenes under the con-trol of Hsp70-Gal4 (P{GAL4-Hsp70.PB}2) (Brand et al., 1994) causes semi-lethality at postembryonic stages when flies are raised at a constant temperature of 25°C (Table 1). The bab1 and bab2 transgenes cause similar defects in adult escaper flies. Escapers have a small body and show an overall reduced pigmentation of the cuti-cle, particularly prominent in the posterior most seg-ments of the abdomen and in the pigmentation of most bristles including the sex combs in males (Fig. 2A,B). The macrochaetae appear completely unpigmented and are flaccid and wavy (Fig. 2C). In addition, such flies show incomplete dorsal closure of the tergites, which form the dorsal cuticle of the abdomen, a phenotypic aspect seen in more than 50% of the flies that misexpress
bab1 (Fig. 2D), but not seen in flies that misexpress
bab2at 25°C. After heatshocks at 37°C during the 3rd larval instar, however, split tergites are also frequently observed in bab2 misexpressing flies.
Additional Gal4 driver lines, known to be expressed in imaginal discs, have been used to over/misexpress the UAS-bab genes. These were the Gal4 lines 339 and E132 (Halder et al., 1995), 30A, 32B, and 71B (Brand and Perrimon, 1993), T80 (Wilder and Perrimon, 1995),
ac-tin-Gal4 (a gift from Yash Hiromi), and babPgal4-1 (Ca-brera et al., 2002). UAS-bab1 lines were lethal in com-bination with all tested drivers (339, actin, T80). Some drivers (T80, 32B) also caused lethality when combined
* Correspondence to: J.L. Couderc, INSERM UMR 384, Laboratoire de Biochimie, 28 place Henri Dunant, 63001 Clermont-Ferrand, Cedex, France, or D. Godt, Dept of Zoology, University of Toronto, Toronto, Ontario, Canada.
†Olivier Bardot and Dorothea Godt are co-first authors.
E-mail: J.L. Couderc (jl.couderc@u-clermont1.fr) or D. Godt (dgodt@zoo. utoronto.ca)
Published online 00 Month 2002 in
Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/gene.10124
with UAS-bab2 lines. It is notable that lethality was not observed with any driver prior to the 2nd instar. Al-though the mutant phenotypes induced by different drivers in combination with UAS-bab2 were not identi-cal, several similarities were found. Decreased pigmen-tation in the abdominal cuticle and the claw organs was observed with Gal4 lines 339 and 30A, similar to Hsp70 as described above. Incomplete dorsal fusion of tergites was observed with 71B-Gal4 and babPgal4-1. Shortening, broadening, spreading, and curving/crumpling of the wings was seen with Gal4 lines E132, 30A, 71B, and 339. E132-Gal4 caused a complex phenotype in combina-tion with UAS-bab266/3, characterized by deformed and branching leg segments (Fig. 1A), antenna to leg trans-formation (Fig. 1B), duplicated halteres (Fig. 1C), and deformed wings that show a bubble-like outgrowth in the hinge region (Fig. 1D–F). To gain a better under-standing of the underlying cause of these wing abnor-malities, we analyzed wing imaginal discs at the 3rd larval instar. E132-Gal4 drives strong ectopic expression of Bab2 protein in the presumptive hinge of the wing (Fig. 1 G–J), as detected with the Bab2-R10 antibody (Couderc et al., 2002). Areas of exogenous Bab2 expres-sion are defective, giving the wing disc an abnormal shape (Fig. 1I). Histological sections indicate disruptions of the epithelium in these areas and massive cell degen-eration, which appears to be necrotic, as no apoptotic bodies were detected (Fig. 1J). This and other analyses (not shown) indicate that the over- or ectopic expres-sion of bab1 or bab2 causes pronounced cell death in many imaginal tissues. Such tissue degeneration may stimulate regeneration, which in turn could lead to the formation of outgrowths and secondary axes in fly ap-pendages.
UAS-bab1 and UAS-bab2 Transgenes Rescue bab Mutant Leg and Ovary Phenotypes
The best assay for studying the respective function of the two bab genes is to test each bab gene for its potential to rescue phenotypic defects associated with mutations in the bab locus. We decided against the use of genomic rescue constructs, as both bab genes are fairly large (covering approximately 30 and 60 Kb of genomic DNA, respectively) and as they may share cis-regulatory sequences. Instead, we used the babPGal4-2 driver, which drives expression in a pattern similar to the bab genes (Cabrera et al., 2002, this issue) and UAS-bab transgenes to rescue bab mutants. babPGal4-2is inserted into bab1, but neither causes a bab mutant phenotype (Cabrera et al., 2002, this issue) nor affects
bab1expression as indicated by Northern blot analysis (not shown). One advantage of using this driver is that the bab1 and bab2 transgenes will be expressed in an identical pattern, allowing the detection of potential differences in the function of the two genes.
To obtain flies that contain the babPgal4-2driver and a UAS-bab transgene in a bab mutant background, we constructed a recombinant chromosome containing
babPgal4-2and the babE4allele (a mutation in the coding region of bab2; Couderc et al., 2002). The recombinant chromosome was generated by crossing babE4/babPgal4-2 females to babE4 homozygous males and selecting for
white⫹ eyes and ectopic sex combs indicative of the
babPgal4-2 insertion and the babE4 mutation, respec-tively. This chromosome generates a bab-specific ex-pression pattern when combined with a UAS-GFP re-porter (not shown) and when homozygous produces all phenotypic traits described for babE4mutants (Couderc
et al., 2002). Flies carrying the recombinant chromo-some were then crossed to flies containing a UAS-bab Table 1
UAS– bab1 and UAS– bab2 Transgenic Lines Phenotype of
UAS-bab/⫹, Hsp70-Gal4/⫹
adult flies Wild-type Weak Intermediate Strong Lethal
UAS-bab1 lines — — 1–2 (II)[32%] 61–63 (III)[4%]* 38–41 (X)*
46–49 (III)[18%] 42–45 (III)
UAS-bab2 lines 49/3 (III) 49A2 (II)[39%] 3/2 (II)[81%] 15/3 (III)[3%] 49C3 (III)
51A3 (III)[67%] 16B2 (II)[6%] 44A3 (III)[15%] 56/3 (III)[36%] 44B2 (II)[33%] 66/3 (III)[67%] 72/2 (II)[39%] 51B3 (II)[24%] 83/3 (III)[3%]
75/2 (II)[42%] 91B2 (II)[3%]
UAS-bab lines are classified according to the strength of phenotypic defects observed in combination with a Hsp70-Gal4 driver after raising flies at a constant temperature of 25°C. Flies heterozygous for both UAS-bab and Hsp70-Gal4 were checked for lethality, loss of cuticle pigmentation, and bristle defects. Phenotypes “weak” to “strong” indicate an increase in strength of cuticle and bristle defects and often but not always also an increase in lethality. Indicated are the designation of the UAS-bab transgenic lines, their chromosomal location (in parentheses), and their survival rate (in brackets, shown as percentage of the ratio with which the genotype was expected).
*These lines have been lost.
When 3rd instar larvae harboring any of the UAS-bab1 insertions were heatshocked for 2 h at 37°C, no flies were emerging whereas flies carrying the UAS-bab244B2or UAS-bab266/3insertions were viable under the same conditions, although they showed a more severe mutant phenotype that included split tergites and deformed legs.
insertion on the second chromosome and the babAR07 mutation (a null of both bab1 and bab2; Couderc et al., 2002). We analyzed the phenotype of flies carrying
babPgal4-2 and babE4 in trans to babAR07 and having either the UAS-bab11-2 or the UAS-bab244B2 transgene, which are transgenes of similar strength (Table 1). In this genetic background, the UAS-bab244B2 line produces viable flies at 25°C and 20°C, the UAS-bab11-2line only at 20°C, with many flies showing split tergites (⬎50% of the flies).
One phenotypic characteristic of bab mutants is the transformation of the distal tarsal segments (TS) to more proximal tarsal segments (Godt et al., 1993). This is best shown by the presence of sex combs not only on TS1, as in wild type, but also on more distal segments. Strong
bab mutants (Fig. 2E) show ectopic sex combs on TS2
(7– 8 bristles) and TS3 (3– 4 bristles) and a fusion of TS4 and TS5. In bab mutant flies overexpressing UAS-bab1 under the control of babPgal4-2(Fig. 2F), there is no sex comb on TS3, but there is a small sex comb on TS2 (1–2 bristles), and TS4 and TS5 are separated by a well-defined joint. In bab mutant flies overexpressing UAS-bab2 with the babPgal4-2driver (Fig. 2G), TS2 and TS3 have no sex comb and TS4 and TS5 are always properly separated. Thus, the bab mutant leg phenotype is completely sup-pressed in flies overexpressing UAS-bab2, but only par-tially rescued in flies overexpressing UAS-bab1 using the
babPgal4-2driver.
A second phenotypic trait of bab mutants is the dis-organization and reduced number of ovarioles in the ovary that is primarily due to a defect in terminal fila-ment formation (Godt and Laski, 1995; Sahut-Barnola et
al., 1995; Couderc et al., 2002). The babE4/babAR07
mutant females develop ovaries (Fig. 2H) of reduced size containing only a few follicles with abnormal orienta-tion. These flies are subfertile and lay very few eggs. This phenotype is partially rescued by UAS-bab1, expression of which produces much larger ovaries (Fig. 2I), some with normally formed ovarioles filled with aligned ma-turing follicles, and restores fertility. A UAS-bab2 fully rescues the bab mutant phenotype, producing ovaries as large as wild type (Fig. 2J) with the correct number of well-formed ovarioles and normal egg-laying capacity.
A third phenotypic aspect of bab mutants is an in-crease of the abdominal pigmentation in females. Unex-pectedly, this defect is not rescued by any of the bab transgenes driven by babPgal4-2(Fig. 2K,L). This driver is expressed in the abdomen, since we observed problems with abdominal dorsal closure associated with ectopic
bab1or bab2 expression. However, this driver may not be expressed at the required time or level or in the appropriate tissue to induce suppression of the abnor-mally dark pigmentation in bab mutants. That overex-pression of either bab1 and bab2 can decrease the level of cuticle pigmentation has been demonstrated by driv-ing UAS-bab genes with the Hsp70-Gal4 driver. This suggests that the babPgal4-2 driver does not respond to the complete set of cis-regulatory elements of the bab locus. We are now in the process of testing a second
bab-specific driver, babAgal4-5which expresses Gal4 in a slightly different pattern than babPgal4-2for its ability to restore normal pigmentation in a bab mutant back-ground.
In summary, we first show that ectopic expression of
bab1 and bab2 can result in severe developmental de-fects and tissue degeneration at postembryonic stages. Second, our experiments demonstrate that a bab2 trans-gene is able to fully rescue the ovary and leg defects of a bab2 mutant, and also a bab1 transgene can rescue the
bab2mutant phenotype, although only partially. Expres-sion of UAS-bab1 and UAS-bab2 have similar gain-of-function effects, indicating that they are gain-of-functionally active at a comparative level, so that the difference in the rescuing activity is presumably due to functional differ-ences between the Bab proteins, although insertion-FIG. 1. Misexpression of bab2 causes developmental defects in
imaginal tissues. Flies of the genotype E132-Gal4/⫹; UAS-bab266/3/⫹ show (A) malformed and branching legs, (B) a transformation of basal cylinder and arista into a tarsus, and (C) a duplication of the haltere. In comparison to wild-type (D), this genotype produces shortened and broadened wings (E) and an outgrowth in the hinge of the wing (arrows in F). E132-Gal4 drives expression in the hinge primordium of a 3rd larval instar wing disc (G) and a prepupal extending wing (H) as shown with the help of a lacZ reporter and visualized by X-gal staining. Wing discs of E132-Gal4/⫹; UAS-bab266/3/⫹ 3rd instar larvae that were stained with the Bab2-R10 antibody show deformations (I, whole mount) and tissue de-generation (arrowhead in J, histological section) in areas of exoge-nous Bab2 expression. There is only a very low level of endogeexoge-nous Bab2 expression in the wing disc that is barely detectable in com-parison to the strong ectopic Bab2 expression driven by E132-Gal4.
specific effects cannot be ruled out entirely. Together, these findings support our previous hypothesis of redun-dancy and differential requirement for the two bab genes (Couderc et al., 2002). Finally, we demonstrate that the babPgal4-2 driver sufficiently mimics the bab expression pattern to allow a rescue of the bab mutant phenotype in ovaries and legs, making this driver a valuable tool for future structure–function analysis of
bab1and bab2.
ACKNOWLEDGMENTS
We thank Laury Arthaud for excellent technical assis-tance. We thank the Bloomington, Indiana stock center, Walter Gehring, Yash Hiromi, and Gerd Technau for GAL4 lines, and Ulrich Tepass for comments on the manuscript. Funding was provided by INSERM (to JLC), by the NSERC (to DG), and by the NIH (#GM40451 to FAL).
FIG. 2. Misexpression effects (A–D)
and rescue of bab mutant phenotypic traits with UAS-bab1 and UAS-bab2 (E–L). A: Prothoracic leg of a Hsp70-Gal4/⫹ male showing a wild-type pig-mented sex comb and claw (arrow-heads). B: Prothoracic leg of a Hsp70-Gal4/⫹; UAS-bab266/3/⫹ male showing an unpigmented sex comb (ar-rowhead) and claw (arrow). C: Thorax of an Hsp70-Gal4/⫹; UAS-bab146-49/⫹ fly with unpigmented flaccid and wavy macrochaetae. D: Abdomen of an Hsp70-Gal4/ UAS-bab11-2 female showing incomplete dorsal closure of the tergites. E–G: Prothoracic leg of an adult male with the respective genotypes (E) babE4, babPgal4-2
/ba-bAR07, (F) UAS-bab11-2/⫹; babE4,
babPgal4-2/babAR07, (G)
UAS-bab244B2/⫹; babE4, babPgal4-2
/ba-bAR07. The five tarsal segments (TS1–5) are indicated, TS4/5 indi-cates the fusion between TS4 and TS5, the arrowheads point to the sex combs. H–J: Ovaries from adult fe-males with the respective genotypes (H) babE4, babPgal4-2/babAR07, (I)
UAS-bab11-2/⫹; babE4, babPgal4-2/babAR07, (J) UAS-bab244B2/⫹; babE4, bab Pgal4-2/babAR07. Dissected ovaries were fixed and stained with Hoechst, and observed by epifluorescence. Two complete ovaries are shown in (H), a complete ovary in (I), and only some ovarioles in (J). Scale bar ⫽ 300 m. K–L: Abdomen from adult females with the respective genotypes (K) babE4, babPgal4-2/babAR07, (L)
UAS-bab244B2/⫹; babE4, babPgal4-2/ babAR07. Abdominal segments A5 and A6 are fully pigmented in females of both genotypes.
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