1. Replacement of Fab-7 by the gypsy or scs Insulator Disrupts Long-Distance Regulatory Interactions in the Abd-B Gene of the Bithorax Complex 
Ilham Hogga, Jozsef Mihaly, Stéphane Barges, François Karch 
Molecular Cell 
Volume 8, Issue 5, Pages (November 2001)
DOI: /S (01)00377-X

2. Figure 1 Molecular Map of the Abdominal Region of the BX-C
(A) The genomic DNA is marked off in kilobases. The approximate extent of the iab-2–iab-8 cis-regulatory domains are shown as blocks of different colors. The arrows indicate the target promoter (abd-A or Abd-B) for each cis-regulatory domain and the parasegment/segment in which the interaction occurs. The positions of the Fab-7 and Fab-8 boundaries are indicated.
(B) Magnification of the Fab-7 boundary region drawn at the scale indicated (see Experimental Procedures).
Molecular Cell 2001 8, DOI: ( /S (01)00377-X)

3. Figure 2
Abd-B Expression Pattern in Wild-Type, gypsy, and scs Homozygous Embryos
Abd-B was detected with a mouse monoclonal antibody (Celniker et al., 1990) followed by staining with a peroxidase-coupled secondary antibody.
(A) Embryos were opened along the dorsal midline, flattened, and mounted on a microscope slide. Parasegments 10–13 are indicated. At about 10 hr of development, Abd-B is expressed at its maximum level in the epidermis and appears in some neuroblasts along the midline. In wild-type, the typical Abd-B expression pattern is characterized by an anterior to posterior gradient in the number of positive nuclei per parasegment as well as by the intensity in each nucleus. In homozygous gypsy, there is no detectable signal in PS10 and PS11, indicating that both iab-5 and iab-6 are unable to interact with the Abd-B promoter. In homozygous scsmin embryos, a weak signal is detected in PS10 and PS11.
(B) Central nervous systems (CNS) that were dissected from 12-hr-old embryos are shown. In scsmin embryos, an impediment between iab-5/iab-6 and Abd-B is visible by the weaker signal in PS10 and PS11. In homozygous gypsy, Abd-B appears in the CNS at levels that are almost as intense as in wild-type, contrasting with the complete absence of detectable staining at an earlier stage in the epidermis and CNS of PS10 and PS11 (A). The level and pattern in PS11 resembles that seen in PS12, indicating that iab-7 is ectopically activated in PS11 (the Fab-72 phenotype).
Molecular Cell 2001 8, DOI: ( /S (01)00377-X)

4. Figure 3
Homeotic Transformations Associated with the Swapping of Fab-7 by the gypsy or scs Insulator
Male abdomens were cut along the dorsal midline and flattened on a slide. Only half cuticles are shown in which A4–A6 are well visible. The dorsal surface of each abdominal segment has a rectangular plate of hard cuticle called the tergite (only half of the tergites are visible on the right of each panel, as well as the genitalia at the bottom). Note that in wild-type, the fifth and sixth tergites are pigmented (numbered). The ventral surface of abdominal segments is composed of soft cuticle called the pleura. On the midline of the pleura of A2–A6, there are small plates of harder cuticle called sternites. In wild-type, the sixth sternite (framed by small arrows in all panels) can be easily distinguished from the more anterior sternites by its different shape and by the absence of bristles. In males homozygous for the chromosome carrying the gypsy insulator, the sixth sternite is covered by bristles and has the shape of a fifth sternite, indicating a transformation of A6 into A5. The normal pigmentation of the fifth tergite indicates that A5 has normal identity (see text). In homozygous males in which scsmin replaces Fab-7, the phenotypic effects are the same as in the case of gypsy swapping. In iab-611 homozygous males, a rearrangement break interrupts chromosomal continuity between iab-6 and iab-7. Although iab-5 is unable to regulate Abd-B, A5 identity appears normal (see text).
Molecular Cell 2001 8, DOI: ( /S (01)00377-X)

5. Figure 4
The Su(Hw) but Not Mod Is Required for gypsy Insulation in the Context of Fab-7
A wild-type male has six abdominal segments. The seventh abdominal segment (A7), present in larvae, is suppressed during metamorphosis. In Fab-72, iab-7 is ectopically activated in most cells of A6. As a consequence, A6 assumes A7 identity, and most of the sixth tergite and sternite are absent (data not shown). Fab-72 is dominant, and these transformations can also be seen in heterozygous animals (Fab-72/+). However, the homeotic transformation is weaker because only one dose of iab-7 is ectopically activated in A6. This is revealed by the smaller size of the sixth tergite (shown by two long arrows) and the absence of the sixth sternite (framed by four small arrows). In heterozygous males in which Fab-7 is substituted by the gypsy insulator, the sizes of the sixth tergite and sternite appear normal, indicating that gypsy can revert the Fab-72 phenotype. In the context of Fab-7, we have confirmed that insulation by gypsy requires Su(Hw). The male cuticle shown is heterozygous for the chromosome in which Fab-7 is replaced by the gypsy insulator and transheterozygote for a viable combination of su(Hw)v/su(Hw)f. Note that the phenotype is similar to Fab-72/+. In some but not all cases, the gypsy insulator requires a second protein, Mod(mdg4), for enhancer blocking (Gerasimova et al., 1995). In the homozygous mod(mdg4)u1 (modu1) mutant male shown, both copies of Fab-7 have been replaced by the gypsy insulator. Note that the cuticle phenotype is the same as in Figure 3 (gypsy/gypsy), indicating that Mod is dispensable for insulating activity in this context.
Molecular Cell 2001 8, DOI: ( /S (01)00377-X)