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The bric à brac locus consists of two paralogous genes encoding BTB/POZ domain proteins and acts as a homeotic and morphogenetic regulator of imaginal development in Drosophila

Jean-Louis Couderc^1,*,, Dorothea Godt^2,*,, Susan Zollman^3,*, Jiong Chen³, Michelle Li², Stanley Tiong⁴, Sarah E. Cramton³, Isabelle Sahut-Barnola¹ and Frank A. Laski^3,

¹ INSERM UMR 384, Laboratoire de Biochimie, 28 place Henri Dunant, 63001 Clermont-Ferrand, Cedex, France
² Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada
³ Department of Molecular, Cell, and Developmental Biology and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA
⁴ Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
^* These authors contributed equally to this work

View larger version (14K): [in a new window]   Fig. 1. The bab locus consists of two genes. (A) Molecular map with the center line representing the genomic DNA. NotI (N) and EcoRI sites (vertical lines) and the P-element insertion sites of babA128, babP and babA30 are indicated. The babP insertion point is defined as 0 on the map, the entire locus covers approximately 130 kb of DNA. The positions of the cosmid and lambda fragments of the genomic walk are shown at the top. A 412 transposable element is located in intron 1 of bab1. At the bottom are two overlapping deficiencies that each have one breakpoint within the bab locus. White regions represent deleted sequences, black regions represent DNA still present, and the deletion breakpoints are within the dashed regions. (B) Structure of the bab1 and bab2 transcripts. Untranslated regions (open boxes), exons (black boxes) and positions and sizes of introns are shown. The BTB domain and BabCD-encoding regions are also indicated.

View larger version (54K): [in a new window]   Fig. 2. Bab1 and Bab2 have a BTB domain, a Psq-domain and an AT-hook-like motif. (A) Sequence similarity between Bab1 and Bab2 is restricted to the N-terminal BTB domain and the C-terminal BabCD. Sequence identity is indicated in percent. (B) Alignment of the BTB domains of Bab1 and Bab2. (C) Alignment of the BabCDs of Bab1 and Bab2. A Psq domain (underlined) and an AT-hook-like motif are contained within the BabCD. The BabCDs of Bab1 and Bab2 are encoded by three exons (black triangles indicate exon boundaries). (D) Comparison of the Psq domains of Bab1, Bab2, Tkr and Piefke, and the four Psq domains of the Psq protein. Residues conserved in all the domains are highlighted in black, less conserved residues are highlighted in gray. (E) Glutamine- (Q, gln) and histidine- (H, his) rich domains of Bab1 and Bab2.

View larger version (100K): [in a new window]   Fig. 3. Comparison of the bab1 and bab2 mRNA distribution patterns in ovaries of white prepupae (A-C) and leg imaginal discs of late 3rd instar larvae (D,E). (A) A lateral view (anterior is upwards) showing high levels of bab2 expression in terminal filaments (arrow) and lower levels in the apical cells (large arrowhead) and the swarm cells (small arrowhead). (B) Lateral view, and (C) top view showing the specific expression of bab1 in the terminal filaments. (D) bab2 and (E) bab1 show a similar expression in the primordium of tarsal segments TS1-TS4 in leg imaginal discs. A 4.2 kb genomic fragment of bab1 that includes the first exon and a full-length cDNA of bab2 were used as probes for tissue in situ hybridization.

View larger version (127K): [in a new window]   Fig. 4. Comparison of the Bab1 and Bab2 expression patterns in imaginal primordia. (A) Early-mid and (B) mid-late instar larval ovary (lateral view), showing high levels of Bab2 expression in the developing terminal filaments (arrow) and migrating swarm cells (small arrowhead), and low levels of expression in apical cells (large arrowhead) and other somatic cells. (C) Ovary at puparium formation (frontal view): the level of Bab2 expression remains high in the terminal filaments (arrow), and has increased in the apical cells that migrate posteriorly between the terminal filaments (arrowhead) and decreased in the posteriorly located swarm cells. (D) Ovary at puparium formation: Bab1 is seen only in the nuclei of terminal filament cells. (E) In the prepupal CNS, Bab1-expressing cells are found in a peripheral layer of the central brain hemispheres and the thoracic ganglia. (F) In a late 3rd instar female genital disc, and (G) in a male disc, strongest Bab2 staining is seen in the female genital primordium and the male genital primordium (arrows). (H-J) In leg imaginal discs, both Bab proteins are found in a graded distribution in the tarsal primordium with the highest level of expression seen in TS4 and 3, lower in TS2 and lowest in the distal region of TS1 (numbers indicate tarsal segment primordia). In contrast to Bab1 (J), Bab2 is also found in the proximal region of TS5 (H,I), in the peripodial membrane (arrowhead in I), and in the disc periphery (arrow in I). Anterior is upwards in A-D,F,G; distal is towards the left in H-J. Bab2 is detected with Bab2-R7 (A-C) or Bab2-R10 antibodies (F-I), and Bab1 with the Bab1-r2 antibody (D,E,J).

View larger version (34K): [in a new window]   Fig. 5. Molecular analysis of bab mutations. (A) PolyA+ RNA (10 µg) from 3rd instar larvae that were wild type (+/+), babP/+ or babP/babP were each hybridized with bab1- and bab2-specific riboprobes. Both, bab1 and bab2 encode a 5.4 kb transcript. Detection of the bab1 wild-type signal required 4 days of film exposure, whereas the much stronger bab2 signal was detected after only 12 hours. The babP insertion causes a truncation of the bab1 mRNA, yielding a 2.6 kb transcript, but does not affect the bab2 transcript. (B) Bab2 has a molecular weight of approximately 145 kDa. babE1 causes a strong reduction of Bab2, and babE4 and babE5 cause a truncation of Bab2. (C,D) Bab2 is not detected in Df(3)Fpa1/Df(3)Fpa2 or babAR07/babAR07 mutants, and reduced levels of Bab2 are found in babPR72 homozygotes. babA128 (C) and babE3 mutants (B) produce a Bab2 protein of normal size. Bab2 protein was detected using Bab2-R6 (B,C) or Bab2-R7 antibodies (D), which recognize the N-terminal region of Bab2.

View larger version (86K): [in a new window]   Fig. 6. Phenotypic series of bab mutant adult ovaries. Morphology of (A) a wild-type ovary, and (B-F) bab mutant ovaries of increasing phenotypic strength, showing changes in size and shape of the female reproductive organ because of a decreasing number of ovarioles and developing follicles. Genotypes: (B) babE5/babE6, (C) babE5/babPR72, (D) babE1/babE4, (E) babPR72/babE1 and (F) babPR72/babPR72. (A,B) A single ovary, (C-F) a pair of ovaries of 2- to 3-day-old females. All panels are at the same magnification, and anterior is upwards.

View larger version (49K): [in a new window]   Fig. 7. bab specifies tarsal segment morphology. The tarsal region of the prothoracic leg of a male fly is shown in A,C,E,G, and the metathoracic leg in B,D,F,H. (A) Wild-type prothoracic leg showing a sex comb and transverse bristle rows only on TS1. (B) Wild-type metathoracic leg showing transverse bristle rows on TS1 and TS2. (C,D) babPR72/babPR72 mutants (strong hypomorphic) show sex comb bristles and transverse bristle rows on TS1-TS3, and a fusion between TS5 and TS4. (E-H) Df(3L)Fpa1/Df(3L)Fpa2 mutants show the bab null phenotype. Tarsal segments are strongly shortened and thickened. Although the bristle pattern of TS2-4 is transformed, the ectopic sex combs on TS2 and TS3 are smaller than in a hypomorphic mutant (E) or are missing altogether (G), and the TS1 sex comb is smaller than in wild type (E). Tarsal fusion extends further proximally, often leading to a fusion of TS5 to TS2 (F,G). Kinks and abnormal bristle arrangements (H) or loss of distal segments are frequently seen in metathoracic tarsi. In all panels, distal is towards the left; numbers 1-5 mark TS1-TS5; arrows indicate sex combs; arrowheads indicate transverse bristle rows; an angled line points to tarsal segments that are fused rather than connected by joints.

View larger version (130K): [in a new window]   Fig. 8. bab controls the morphology of abdominal segments. Female tergite pigmentation in wild type (A) and in loss-of-function bab mutants of increasing phenotypic strength: (B) babA128/ babA128 (C) babDl/ babDl and (D) babAR07/Df(3L)Fpa2, a null mutant. Ectopic dark pigmentation in the anterior portion of the tergites is indicated by asterisks. (F) In a Df(3L)Fpa1/Df(3L)Fpa2 female, the two tergite plates of A7 are fused (arrowhead) and enlarged, and contain more bristles in comparison with wild type (E). In addition, the A6 tergite is enlarged (double arrow in E,F). (G) Wild-type and (H) Df(3L)Fpa1/Df(3L)Fpa2 male abdomina, showing ectopic pigmentation in bab mutant tergites. Ventral view of the abdomen of (I) wild-type, (J) Df(3L)Fpa1/Df(3L)Fpa2 and (K) babAR07/Df(3L)Fpa2 females, and a (L) wild-type male showing features of the A6 and A7 sternites, and the female vaginal plates (arrows in I,K). (M-P) Ubiquitous overexpression of bab2 causes a loss of pigmentation. A Hsp70-Gal4/+;UAS-bab266/3/+ female (M) and male fly (P), raised at 25°C, lack dark pigmentation in A6. The loss of pigmentation is stronger in posterior than more anterior abdominal segments. (N) In a Hsp70-Gal4/+;UAS-bab266/3/+ female fly, raised at 18°C until late 3rd instar and at 32°C during the pupal stage, the tergite primordia have not fused. (O) A Hsp70-Gal4/+;UAS-bab251A3/+ male fly, raised at 25°C, shows reduced pigmentation in A5 and A6. In all panels, anterior is upwards and segment identity is indicated by numbers.