1. Control of Colinearity in AbdB Genes of the Mouse HoxD Complex
Takashi Kondo, József Zákány, Denis Duboule 
Molecular Cell 
Volume 1, Issue 2, Pages (January 1998)
DOI: /S (00)

2. Figure 1
Schematic of the Posterior Part of the HoxD Locus and Constructs Used for Targeting
(A) The starting ES cells had one chromosome in which lacZ reporter sequences had been already introduced by recombination within Hoxd-12 (the Hoxd-12lacGe locus; Kondo et al. 1996).
(B) The resulting targeted allele HoxD[12-13neo]lacGe, i.e., with two lacZ insertions in cis, was obtained before treatment with the Cre recombinase.
(C) Treatment in vitro with the Cre recombinase led to various excision events. A clone that had selectively lost the PGK neo cassette was obtained, thus giving rise to the HoxD[12-13]lacGe locus.
(D) HoxD[12-13]lacGe ES cells were introduced into mice, which were in turn crossed with a Cre-expressor mouse to generate in vivo the deleted HoxD[12Δ13]lacGe locus. Rectangles indicate exons and directions of transcription are shown by arrows. All of these various genetic configurations were precisely monitored by using a battery of probes in Southern blots and through PCR.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 Limb Skeletal Preparations of 8-Week-Old Adult Animals
Comparisons between forelimbs of wild-type animals, a Hoxd-12lac homozygous specimen, a Hoxd-13st homozygous animal, and a HoxD[12-13]lac homozygous double mutant specimen in cis, from left to right, respectively. The phenotypes of limbs from HoxD[12-13]lac HoxD[12Δ13]lac animals were indistinguishable from each other (not shown).
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
Whole-mount β-Galactosidase Stainings of Littermates of Different Genetic Configurations
(A) Control comparison between the staining intensities obtained either from the Hoxd-12 promoter after overnight staining (left; Hoxd-12lac), or from both Hoxd-12 and Hoxd-13 promoters after 4 hr of staining (right; HoxD[12-13]lac) using heterozygous animals. Because of this difference in intensities, lacZ staining observed from the HoxD[12-13]lac locus after short staining periods only reveals the activity of the Hoxd-13 promoter.
(B–F) Comparisons of lacZ staining patterns as controlled by the Hoxd-13 promoter in either nondeleted HoxD[12-13]lac animals (left panels) or in animals with the intergenic region deleted (HoxD[12Δ13]lac; right panels). Comparative panels show littermates derived from the cross of a Cre-expressor heterozygote male with HoxD[12-13]lac homozygous females. (B) In E9.5 animals, HoxD[12-13]lac started to express Hoxd-13 in the proctodeal region, while expression in HoxD[12Δ13]lac animals extended anteriorly and dorsally. (C) E10.5 embryos showed important differences in spinal cord (arrow). Staining in limb buds was much broader in HoxD[12Δ13]lac embryos (arrowheads) resembling that of the endogenous Hoxd-11 gene. (D) At E13.5, the HoxD[12-13]lac locus behaved as Hoxd-13, whereas the HoxD[12Δ13]lac locus had many features of Hoxd-11, some of them being enlarged in (E) and (F). (E) Magnification of the posterior parts of the trunks of the specimen shown under (D) in a rectangle. The HoxD[12Δ13]lac animal (after deletion) showed striking anteriorization of lacZ expression and staining in spinal cord at the level of Hoxd-11 (arrow). Likewise, staining was detected in kidney (arrowhead), in the forearm regions of the limbs (triangles) and in the prevertebral column, whereas HoxD[12-13]lac animal showed no (or weak) expression in these areas, as expected from the Hoxd-13 expression domains. (F) Higher magnification of the trunks (ventral view) of animals from the same genotypes. HoxD[12Δ13]lac animals revealed an anteriorization of lacZ staining in the digestive tract, such that an intestinal loop in the hernia was positive (arrowheads), as for endogenous Hoxd-11. (G) Uterine horns from animals 6 days after birth showing again a remarkable anteriorization of the blue staining in the deleted animals when compared to the nondeleted version (arrows). Similar observations were collected from inspection of male genitalia (data not shown).
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4
Scheme of the Strategy Used for the Production of the Mouse-Fish Exchanged Locus
(A) 5′ parts of the HoxD complex of both mouse and zebrafish. In zebrafish, the Hoxd-12-to-Hoxd-13 intergenic sequence is 3 kb large instead of the 5 kb found in the mouse complex. By contrast, the zebrafish Hoxd-12 intron is 2.5 kb larger than its mouse counterpart.
(B) Targeting vector used for the intergenic exchange. The mouse sequence, from the Hoxd-13 termination codon to the Hoxd-12 initiation, was substituted for the corresponding zebrafish DNA fragment.
(C) The targeted allele was subsequently treated with the Cre recombinase in vivo to excise the PGK neo cassette from the allele, thus generating the HoxD[12-13]FiGe locus.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5
Scheme of the Different Genetic Configurations Drawn at the Same Scale ([B]–[E]) to Allow for a Comparison of Genomic Distances
(A) HoxD complex.
(B) Enlargement of the 5′ posterior part containing genes related to AbdB.
(C) Scheme of the HoxD[12-13]lac locus, with lacZ reporter sequences introduced in both Hoxd-13 and Hoxd-12.
(D) Same chromosome after treatment with the Cre recombinase. The two transcription units are now fused and the Hoxd-13 promoter (arrow) is closer to the Hoxd-11 locus than it was in the (C) configuration.
(E) In the mouse-fish exchange configuration (HoxD[12-13]Fi, the Hoxd-13 promoter is at the same distance from Hoxd-11 than in the hybrid configuration (compare [D] and [E]; see the text).
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6
Expression of the Three Posterior HoxD Genes Surrounding the Exchanged DNA Fragment in Wild-Type and HoxD[12-13]Fi Homozygous Animals
At either E9.5 (top panels) or E11.5 (bottom panels), no significant spatial difference was observed in the expression of either Hoxd-11 (A), Hoxd-12 (B), or Hoxd-13 (C). However, a clear general reduction in the level of Hoxd-12 transcripts was detected in HoxD[12-13]Fi homozygous specimens that correlated with the hypomorphic Hoxd-12 phenotype observed in these animals.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7
Scheme of a Potential Mechanism Underlying Temporal Colinearity
Proteins (circles, triangles, squares) aggregate from a starting point of high affinity (S) located at the 5′ end of the complex and extend in the 3′ direction. This leads to a block in the accessibility of Hox genes (closed rectangles), up to the most 3′ part of the complex. This progression is helped (reinforced) by relay elements (R, R′) scattered in the complex. If the components of this repressive state (the various proteins) are rate-limiting, and in the case where a dilution factor is introduced in the steady-state level of these components, repression cannot be achieved until the 3′ end, and anterior genes become progressively accessible (A). Such a mechanism does not necessarily depend upon a linear spreading of the repressive state. As shown in (B), the S and R, R′ elements may require such rate-limiting proteins to establish or stabilize the formation of interfaces through looping. These two possibilities are conceptually the same, and in both cases, deletion of the R element, as hypothesized in the present study, would accelerate the accessibility of the 5′ (posterior) genes.
Molecular Cell 1998 1, DOI: ( /S (00) )