1. Breaking Colinearity in the Mouse HoxD Complex
Takashi Kondo, Denis Duboule 
Cell 
Volume 97, Issue 3, Pages (April 1999)
DOI: /S (00)

2. Figure 1 Genomic Walk Upstream of the HoxD Complex
(A) Locations of the insertion sites for the Hoxd9/lacZ transgene. Three phages are shown below a scheme of the HoxD complex. Three different insertion sites are depicted on the top with the extent of the various homologous arms used for recombination. One site is located between Hoxd13 and Evx2 (Ge0 or rel 0), one is on the other side of Evx2 (rel I), and the third is further away (rel II). S, SacI; N, NotI.
(B) Targeting strategy to insert the Hoxd9/lacZ transgene. The TgH[d9/lac/neo]GeI construct was electroporated in ES cells (top). After homologous recombination, the TgH[d9/lac/neo]GeI locus was produced (middle). These cells were treated with the Cre recombinase to excise the PGK neo selection cassette, giving rise to the TgH[d9/lac]GeI configuration (also referred to as rel I, for relocation I).
(C) The three different rel loci produced in this work, that is, from top to bottom, rel0, rel I, and rel II. The exact same transgene is inserted at three neighboring positions separated from each other by approximately 12 kb.
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3. Figure 2
lacZ Expression in Animals Carrying Various Relocations of the Hoxd9/lacZ Transgene
(A) Scheme of the upstream part of the HoxD complex with, on the top, the three insertion sites (rel0, relI, and rel II). The corresponding expression profiles at two different stages are shown below (B). At 8.5 dpc, β-gal expression, as measured by whole-mount in situ hybridization using a lacZ RNA probe, was detected only from the rel II locus in the most posterior part of developing embryos (arrowhead). No staining was ever scored in wild-type, rel0, or rel I counterparts. The lower panels show whole-mount enzymatic β-gal activity using X-gal staining. All three animals show signals in the distal parts of their limbs. In addition, strong expression of the reporter transgene was seen in the CNS of both rel I and rel II specimens, whereas it was absent in rel 0 animals. Samples were stained overnight.
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4. Figure 3 Strategy for Targeted Deletions
(A) HoxD is shown (Hoxd9 to Evx2) together with the rel 0 and rel II insertion sites. Each targeted transgene (green) has a loxP site in 3′, as indicated by a red or blue triangle below the line of the cluster. The complex also contains a loxP site downstream of Hoxd11 (black). The deletions depicted under (B) were induced by crossing either rel 0 or rel II mice with a Cre-expressing animal. The resulting loci are shown, with del 0 lacking the blue fragment, whereas del II animals have deleted both red and blue fragments. The difference between these two loci is the presence or absence of the DNA shown in red. They otherwise have the same transgene (green) inserted at the same position, upstream of Hoxd10.
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5. Figure 4 Phenotypes of Heterozygous Deleted Animals
Comparison between wild-type (A) and del II heterozygous (B) fetuses at 8.5 dpc (four to five somites). Somitic condensations are not visible in the del II embryo (arrows), and the posterior neuropore region is ill formed (arrowheads) while the midline has a chaotic aspect. hb, hindbrain.
(C–H) Skeletal alterations in del II mice. del 0 animals display a wild-type morphology and are used as controls. All animals are heterozygous. (C and D) Comparison between del 0 and del II cervical vertebrae. The del II specimen has an extra rib on C7 (asterisk). In addition, the axis (C2) is abnormal. (E and F) Close comparisons between dissected vertebrae show the del II C6 with unfused and truncated anterior tuberculi (asterisk), resembling C7. del II C7 sporadically displayed a moderate phenotype with small rib-like bones on either side (arrowhead in [F]). (G and H) In contrast to the del 0 sternum (six sternebrae), delII animals often had a transformation of the fourth sternebra into the morphology of the fifth, with three pairs of ribs articulating instead of two (H). C1 to C7, cervical vertebrae; T1, first thoracic vertebra; at, anterior tuberculi.
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6. Figure 5 Expression of the Hoxd9 Transgene in del II and del 0 Animals
Schemes of both deletions are shown at the top (colors as in Figure 3). The upper panels show 8.5 dpc del II (left) and del 0 (right) embryos. While no expression was detected in del0 specimens, delII embryos show a conspicuous expression in the caudal part of the body. Panels on the bottom show the same comparison at 11.5 dpc (left older than right). The background pattern of the Hoxd9 transgene is now visible in posterior trunk, up to the level 20–21. In addition, anterior CNS expression was detected for the del II fetus, resembling the rel II or Evx2 pattern. In limbs, expression of the transgene was a mixture of Hoxd9 pattern (strong anterior domains in the right panel) and a distal domain (weak but clearly present in both panels; arrowheads). An enlargement of developing del II limbs at 13.5 dpc shows increased expression in the digit domain (arrowhead). All samples stained overnight.
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7. Figure 6
Expression of Hoxd Genes in 7.25 to 8.5 dpc del II or del 0 Embryos
(A) Schematic of the HoxD complex with both deletions (colors as in Figure 3).
(B) Expression of Hoxd4 and Hoxd10. In the upper panels, two examples are shown of del II embryos that prematurely expressed Hoxd10 posteriorly (arrowheads), whereas del 0 counterparts were negative. At 8.5 dpc, expression was strong in caudal parts of the body, the expected place for a posterior Hoxd gene. All embryos were heterozygous. The bottom left panel shows Hoxd10 staining of a litter of 7.5 dpc embryos derived from a cross between del II and wild-type mice. In such litters, some embryos stained at their most posterior aspects (arrowheads). Staining was never observed in control litters derived from either wild-type mice or del 0 and wild-type parents. The bottom right panel shows the same series of crosses stained with a Hoxd4 probe. Stained embryos (arrow) were found only in those litters deriving from a del II parent. In both cases, expression was clearly premature.
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8. Figure 7
Deletion Upstream of the HoxD Complex through Targeted Meiotic Recombination (TAMERE)
(A) The top two lines represent the homologous chromosomes (Ch. 1 and Ch. 2) used for interallelic recombination. The rel II chromosome (Ch. 1) with the Hoxd9/lacZ transgene (green) and another chromosome carrying a Hoxd11/lacZ transgene (brown) inserted at rel 0 (Ch. 2). Transheterozygous males were produced with, in addition, a Cre transgene expressed in meiotic cells. In germ cells of these males, targeted crossover occurred and produced unequal exchange between chromosomes (light blue or magenta), giving rise to two novel chromosomes shown below (Ch. 3 and Ch. 4). Chromosome 3 had a deletion between rel II and rel 0, with the Hoxd9/lacZ transgene (green) moved upstream of Hoxd13. Chromosome 4 showed a deletion equivalent to del 0 but with a Hoxd11/lacZ transgene (brown) inserted upstream of Hoxd10 instead of Hoxd9/lacZ. Colors as in Figure 3. In Ch. 1, part of the complex is drawn as deleted (dashed red and blue lines) because the chromosome came from the father together with the Cre transgene and thus has undergone cis-recombination (deletion). P1 to P7 indicate the positions and orientations of the primers used under (B).
(B) PCR genotyping of the progeny. Three embryos are shown together (a, b, c or d, e, f). Using the P2:P5 pair, a band was amplified only in sample (a), which identified Ch. 3 (indicated below). Likewise, the double band seen with sample (f) when using the P6:P7 pair revealed the presence of the reciprocal chromosome (Ch. 4). Other lanes verify this typing.
(C) Expression of Hoxd9/lacZ (Ch. 1 and Ch. 3) and Hoxd11/lacZ (Ch. 2 and Ch. 4) transgenes in 11.5 dpc fetuses derived from the trans-loxer male. Expected expression profiles were obtained for Ch. 1, Ch. 2, and Ch. 4. For example, note the slight anteriorization of the pattern in the trunk of Ch. 4 animals versus their Ch. 3 counterparts due to the small deletion in the posterior HoxD complex (Zákány and Duboule 1996). These three latter configurations showed normal morphologies. By contrast, the Ch. 3 littermate was much smaller and displayed severe malformations in the head, the face, and the caudal part. A weak lacZ staining was detected around the proctodeum (pr) and in forelimb buds (flb).
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