(A) The inversion divides the HoxD cluster into two sub-clusters. In the wild type configuration, the cluster is under the mixed regulatory influences coming from either the centromeric (purple), or the telomeric (yellow) sides. After targeted inversion (bottom line), 3 Mb separate the centromeric part of the cluster from the remaining ‘anterior’ part. Hoxd11, Hoxd12 and Hoxd13 are now under the influence of the centromeric neighborhood only (purple), whereas Hoxd1 to Hoxd10 are associated with the telomeric region (yellow). Red triangles depict loxP sites. (B–F) Each panel is accompanied by a scheme of the respective sub-cluster, and the gene analyzed is shown in red. (B–D) Wild type (left) and mutant (Inv; right) embryos hybridized with Hoxd11, Hoxd12 and Hoxd13 probes. In mutant embryos, Hoxd11 expression is completely lost from the trunk (B, upper panel), whereas the developing limb and genitalia show the expected wild type pattern at E12.5 (B, lower panel). Similar effects are scored for both Hoxd12 (C) and Hoxd13 (D) at E11.5, showing expression in both limb and genital buds, but no detectable signal in the primary body axis. (E, F) Wild type (left) and mutant (right) embryos hybridized for Hoxd10 and Hoxd9, which are located in the other sub-cluster. At E8, both Hoxd10 and Hoxd9 show elevated expression in the developing trunk.

Mutant configurations are depicted on the top, with Evx2 in black and the analyzed gene in red. Wild type embryos are always shown on the left of each panel, next to a representative mutant embryo, on the right. (A–I) E8 to E9 embryos hybridized either with a Hoxd13 (A–D), a Hoxd11 (E–H) or a Hoxd10 (I) probe. In some cases, the gene tested is not directly neighboring the breakpoint, but lies further in 5′ (C, D, H and I). For all deletion alleles, expression is up-regulated in the mutant embryos, as compared to age-matched controls. The onset of expression was scored near the most posterior aspect of the embryo (e.g. Hoxd13 in A). (K–M) Analysis of two duplication alleles, Dup(i-10) (J, K) and Dup(i-9) (L). E8.5 to E9 control (left) and mutant (right) embryos were hybridized with either Hoxd13 (K), Hoxd11 (L) or Hoxd10 (M) probe and, in all three cases, signal accumulation was evident in control embryos, whereas undetectable in mutant specimens, indicating a delay in transcriptional activation for those genes that were relocated away from the telomeric end of the cluster.

Hoxd gene expression patterns in a set of nested deletions. Schemes are as for Figure 3. (A–C) E10.5 wild type (A) and Del(i-8) mutant (B, C) embryos hybridized with a Hoxd9 (A, B) or a Hoxd10 (C) probe. Hoxd9, the gene neighboring the breakpoint shows both up-regulation and anteriorization in the neural tube (arrow) and the paraxial mesoderm (arrowhead) (B), whereas Hoxd10, located one transcription unit further away, follows its wild type expression patterns (C), unlike what was detected for the timing of activation. (D–F) E12.5 wild type (D) and mutant (E, F) embryos hybridized with a Hoxd11 probe. Del(8-10) embryos have decreased Hoxd11 expression levels in both the trunk and limbs, yet show largely wild type spatial distribution (E). Del(i-10) embryos display dramatic anterior gains of expression for Hoxd11 in the neural tube (arrow), mesoderm derivatives (arrowhead) and branchial arch derivatives (asterisk) (F), caused by deleting the intergenic region “i” (in blue in schemes D, E). (G–I) E11.5 wild type (G) and mutant (H, I) embryos hybridized with a Hoxd13 probe. While a down-regulation of Hoxd13 is observed in the trunk of Del(9-12) embryos (I), no obvious difference with respect to wild type embryos is scored for Del(10-12) (H).

Distinct mechanisms underlie the two collinear processes in the trunk. (A) In an early phase, a time-sequenced activation occurs, resulting from a balance between a repressive influence coming from the telomeric neighborhood (red), on the one hand, and a positive influence originating from the telomeric side (green), on the other hand. Initial activation starts from the telomeric side to subsequently expand over the entire length of the cluster in a 3′ to 5′ sequence. The gene's relative position within the cluster determines its timing of activation, in a distance-dependent manner. Active genes are shown with a black arrow, whereas silent genes are labeled with a red X. Approximate developmental stages are schematized on the right hand side with expression domains of the last activated genes highlighted in blue. (B) Subsequently (late phase), spatial transcript distribution along the anterior-posterior axis (as well as tissue-specificity) is determined by local regulatory elements, operating once the initial activation has occurred. Activating (green arrows) and repressive (red bars) elements can be shared by neighboring genes in a wild type situation. These elements can ectopically activate genes when relocated adjacent to them. The schematized E12.5 embryo on the right shows anterior (light blue) to posterior (dark blue) gene transcript distribution in both the neural tube and the paraxial mesoderm.

(A–D) Expression patterns of the same Hoxd11/lacZ transgene after deletion of various DNA segments located telomeric from its insertion site. From left to right (schemes on the top): TgH[d11/lac], Del(11-13), Del(4-13) and Del(1-13). At E8.5 (top panels), only Del(4-13) and Del(1-13) (A–D) embryos show beta-gal expression in mesoderm derivatives. At E9.5 (lower panels), however, all configurations show staining in the primary body axis. Similar time dynamics are observed for transgene activation during limb outgrowth, such that only the Del(4-13) and Del(1-13) embryos are expressed in limb buds at E9.5 (C,D).

(A, D, G) Schemes of embryonic stages shown in the right panels. The orientations of section(s) are indicated by dashed line. Progression of the Hoxd11 expression pattern, either in wild type (B, E, H), or in Del(8-10) mutant embryos (C, F, I). After initial activation at the posterior tip, expression is first scored as two lateral lines, within lateral plate mesoderm only, starting at about the dorso-ventral level of the junction between the future splanchnopleure and somatopleure (E, arrowhead). Subsequently, the gene becomes activated more dorsally, in paraxial mesoderm as well as in the neural tube (H). The same temporal sequence of tissue specificity is maintained for Del(8-10) mutant embryos, even though it was advanced in time (C, F, I). At E8.5 expression is indeed already apparent in mutant lateral plate mesoderm, whereas the wild type embryo is still devoid of any Hoxd11 transcripts (compare C to B). Half a day later, expression has expanded into paraxial mesoderm and the neural tube in mutant embryos (F), while in wild type specimen transcripts just appear in lateral plate mesoderm (E). Both wild type and mutant embryos show the same Hoxd11 expression pattern at E9.5, with lateral plate mesoderm, paraxial mesoderm and the neural tube all scoring positive (H, I). Bar is 50 mm. ne, neurectoderm; pm, paraxial mesoderm; lpm, lateral plate mesoderm.

Expression patterns of Hoxd3, Hoxd4 and Hoxd9 in embryos carrying deletions in 5′ of these genes. Schemes and colors are as for Figure 3. (A–H) wild type (left) and mutant (right) embryos at E8–E9. Genes brought closer to the centromeric end of the cluster following a deletion systematically show reduced expression during early embryogenesis. A similar robust down regulation is observed in the mesoderm of Del(8-9) embryos for both Hoxd4 (A) and Hoxd3 (C), whereas expression persists in the anterior neural tube. In Del(i-10) mutant embryos (B, F) the signal for both Hoxd4 (F) and Hoxd3 (B) is decreased in the mesoderm as well as in the neural tube. Expression of the posterior gene Hoxd9 disappears in pre-somitic mesoderm upon relocation closer to the centromeric side of the cluster (H). (I–L) Dorsal views of bisected and reconstituted pictures of wild type (left half) and mutant (right half) embryos at E11.5. Arrowheads demarcate anterior expression limits in the mesoderm. The same mutant alleles as in (E–H) are shown at a later developmental stage. Unlike for early embryos, no coordinated changes are observed. Expression of Hoxd4 is posteriorized in the mesoderm of both Del(i-8) (I) and Del(i-10) (J). Changes in expression levels are observed for Hoxd3 in Del(4-9) (K) and Hoxd9 in Del(10-12) (L), yet both genes retain their wild type anterior expression boundary.