Figure 1.
Synopsis of the Abd-B locus of the BX-C and diagram of the mutations created for this study.
A. Synopsis of the Abd-B locus of the BX-C. Diagram of the Abd-B gene and its 3′cis-regulatory region. The horizontal line represents the DNA sequence of the BX-C (see scale on top left). The Abd-B expression pattern in the central nervous system of a 10 hours embryo is shown above the DNA line. In parasegment 10 (PS10) Abd-B is present in a few nuclei at a relatively low level. This PS10-specific expression pattern is controlled by the iab-5 regulatory domain located 55 kb downstream from the Abd-B promoter. In PS11, PS12 and PS13, Abd-B is present in progressively more nuclei and at higher levels. These patterns are controlled by the iab-6, iab-7 and iab-8 regulatory domains, respectively. Each regulatory domain functions autonomously from its neighbors due to the presence of the boundaries that flank them (red ovals). B. Diagram of the mutations created for this study. The top line shows the DNA coordinates of iab-6, according to the Drosophila Genome Project. Below this line, and to approximate scale, are the locations of the various elements isolated from the BX-C including the IAB5 initiator[12], DNase hypersentive site 1 (HS1/Fab-6 including the CTCF binding sites) and 2 (HS2/PRE) [43]–[44], the 2.8 kb iab-6 initiator fragment [22], the minimal initiator fragment and the Fab-7 boundary [14], [30]. Below this line are the DNAs reintegrated to make the mutations. The various iab-6 alleles are indicated as solid bars, with gaps indicating the areas deleted. These bars are color coded such that blue bars indicate mutants that show no cuticle or CNS phenotypes at 25°C, red bars indicate mutants with Fab-6-type phenotypes, turquoise bars indicate mutants with iab-5,6 phenotypes, and green bars indicate mutants with iab-6 phenotypes.
Table 1.
Mutations and phenotypes.
Table 2.
Oligos used to generate the deletions.
Figure 2.
A. Step one: Homologous Recombination. The original “ends-out” donor vector (pW25) was modified to contain an attP insertion site and a removable yellow reporter gene. Using the yellow reporter, homologous recombination events could be identified by screening for flies with yellow expression in the A5 and A6 segments (a consequence of having yellow inserted in the iab-5 domain). The yellow reporter could then be removed to leave only the attP site and a single loxP recombination site (white triangle) in place of iab-6. B. Step two: Reintegration. Plasmids containing a 288 bp attB site, a single loxP site, a yellow reporter and a version of the 19.3 kb fragment were injected into iab-5,6CI embryos expressing a maternally supplied φC31 integrase [36]. Integration events were isolated based on yellow gene expression, then crossed to the Cre recombinase to remove the yellow gene and all vector backbone sequence.
Figure 3.
iab-5,6CI phenotype and rescue.
A. A wild-type adult male cuticle with A4-A6 labeled. Segment A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. For reference, the A6 tergite is indicated by a red arrowhead and the A6 sternite is indicated by a red arrow. B. A wild-type embryonic nerve cord (anterior towards the top) stained with an antibody to Abd-B (brown). Notice the step gradient of Abd-B expression increasing in each parasegment towards the posterior. C. An adult male cuticle of a fly homozygous for the iab-5,6CI chromosome with A5 and A6 transformed towards A4 (notice the A4-like pigmentation on the tergites and the bristled sternites). D. The embryonic nerve cord of homozygous iab-5,6CI embryos shows only a transformation of A6 into A5, as seen by the repetition of PS10/A5-like Abd-B levels in PS11/A6, indicating that the inactivation of iab-5 is incomplete and not seen in the embryo. E. An adult male cuticle from a fly homozygous for the iab-5,6rescue chromosome, where the entire 19.3 kb area deleted in iab-5,6CI is reintegrated into iab-5,6CI, looks completely wild type. F. The complete rescue is confirmed by the wild-type pattern of Abd-B in the embryonic ventral nerve cord.
Figure 4.
Phenotypes from initiator mutants.
Genotypes are as follows: A.–C. iab-61, D.–F. iab-64 and G.–I. iab-68. A., D. and G. show adult male cuticles. B., E. and H. show pseudo-darkfield views of the fifth and sixth tergites to visualize the trichome patterns. C., F. and I. show the Abd-B staining pattern in the embryonic nerve cord. In wild-type flies, A5/PS10 differs from A6/PS11 based on the sternite shape, the bristles present on the A5 sternite, the trichome pattern on the fifth and sixth tergites, and the Abd-B staining pattern in the CNS (see Figure 3 and Figure 4). The iab-61 and iab-64 show transformations of A6 to A5 for all phenotypes monitored. Meanwhile iab-68 shows only a partial transformation of A6 to A5 as seen by the sternite shape and trichome pattern on A6, which remain A6-like.
Figure 5.
Phenotypes from initiator mutants.
Genotypes are as follows: A. and D. iab-64. B. and E. wild type. C. and F. Fab-6IAB5. A.–C. Show the ventral sternite cuticles made from adult males, homozygous for the genotype indicated above. Notice that A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. The opposite homeotic transformations are highlighted by the direction of the arrows on the left and the right of the cuticles. D.–F. Show ventral nerve chords made from homozygous embryos of the genotypes indicated above. Parasegment borders are marked to the left.
Figure 6.
The genotypes of the adult male cuticles of A. Fab-62, and B. Fab-63. C. (wild type) and D. (Fab-63) are embryonic nerve cords stained for Abd-B protein. Notice the increased level of Abd-B in PS10 in mutants (D.) relative to wild-type (C.).