Quantitative collinearity in developing digits. (A, top) Posterior Hoxd genes are expressed in developing digits following a gradient of transcriptional efficiency: Hoxd13 is expressed robustly, whereas more 3′-located genes display progressively weaker expression levels, up to Hoxd9, which is barely expressed in this domain. Only Hoxd13 is detected in presumptive digit I (arrowhead). (Bottom) Genomic organization of the HoxD locus and centromeric sequences. Evx2 and Lnp are coexpressed with Hoxd genes in the developing digits, under the control of two shared enhancer sequences (GCR, Prox). (B) Experimental flowchart using an in situ hybridization showing the wild-type expression of Hoxd12 at E13.5. Digits (top) and genital buds (bottom) were dissected off of either wild-type or mutant fetuses, and expression levels of all posterior Hoxd genes (from Hoxd13 to Hoxd9), Lnp, and Evx2 were quantified by real-time RT–PCR (see Fig. 2). We looked for potential similarities or differences in the regulation of Hoxd genes in digits and genitalia by comparing the detected modification occurring in both developing tissues. (C) Validation of the quantification by comparing the expression levels of Hoxd13 in the digits of either wild-type (wt) embryos or embryos heterozygous and homozygous for a deletion of the Hoxd13 locus [del(13)]. (Left) Amplification curves. (Right) Relative Hoxd13 mRNA levels. Expression levels fit exactly with gene copy number, showing the accuracy and specificity of the approach. (D) Quantitative collinearity. For all genes, transcript copy numbers in wild-type developing digits were measured by external calibration. Expression levels are shown as a proportion of Hoxd13. Hoxd13 shows the highest transcript level, and RNA copy numbers decrease progressively for more 3′-located genes, up to Hoxd9. Hoxd8 is detected near background levels. Lnp and Evx2 levels correspond to ∼10% of that of Hoxd13.

Regulatory reallocations. Real-time PCR comparisons of gene expression levels in presumptive digits of either wild-type embryos (light-blue bars) or embryos homozygous for deletions of Hoxd loci located in 5′ of the cluster. The levels are always shown as a proportion of Hoxd13 wild-type RNA level. To better visualize the results, different scales are used for either Lnp and Evx2, or “posterior” Hoxd genes. (A) Deletions of the Hoxd13 locus, either alone [del(13), red bars] or in combination with either Hoxd12 [del(13–12), yellow bars] or Hoxd12 and Hoxd11 loci [del(13–11), green bars]. The deletions lead to up-regulations of the remaining genes. Regulatory reallocations are more important on the 3′ side (Hoxd genes) than on the 5′ side (Evx2) of the deletion. Lnp shows a significant up-regulation in the largest deletion, exclusively. In all cases, the newly positioned 5′-most Hoxd gene (rank 1) shows the strongest expression. (B) Similar genetic configurations, but combined in cis with a deletion of conserved RXII. Deleting RXII (dark-blue bars) results in both a decrease in Evx2 transcription and an overall higher expression of posterior Hoxd genes. Deleting RXII together with Hoxd13 [del(RXII)–del(13), orange bars], or Hoxd13 and Hoxd12 [del(RXII)–del(13-12), green bars] lead to partial recovery of Evx2 levels, together with an increase of the remaining Hoxd gene expression. However, quantitative collinearity is maintained in all configurations.

Predicted versus observed expression levels. Comparison between expression levels predicted by the model (yellow bars) and observed values in either the wild-type (light-blue bars) or mutant configurations (red bars). The various configurations are depicted below each graph. (A) Wild-type situation. (B–D) Deletions including Hoxd13. The wild-type levels are shown on each panel for comparison. The model accurately reproduces the observed values in wild-type and mutant configurations, all Hoxd gene predictions lying within two standard deviations of the observed values. The up-regulation of Evx2, although predicted, is slightly underestimated by the model. The blue oval between Hoxd13 and Evx2 indicates the presence of RXII.

Deletions excluding Hoxd13. Observed and predicted gene expression levels in five “internal deletions.” Colors and schemes are as in Figure 6. (A) del(12). (B) del(12-11). (C) del(12-10). (D) del(11). (E) del(11-10). All deletions lead to increased expression of 3′-located genes, whereas genes located 5′ of the deletions were less affected. The observed levels are in good agreement with predicted values, with the exception of the strong up-regulation of Hoxd9 in del(11-10), not accounted for by the model. Blue ovals are as for Figure 5.

Stocks of mutant mice used in this study with, at the top, a drawing of the wild-type HoxD cluster. Posterior Hoxd genes are depicted by using a color code, from Hoxd13 in red, to Hoxd9 in blue. The position of conserved RXII is shown by the light-blue oval. The mutant strains used in this work are listed below. Deleted segments are shown by broken lines, whereas duplicated loci are indicated by the same color code. Breakpoints are shown as red triangles.

Modeling quantitative collinearity. (A) Expression levels of Hoxd genes as a function of their relative position with respect to the 5′ extemity of the cluster (their “rank”), in the various genetic configurations. While gene rank has a major impact on expression level, different genes are not expressed at the same absolute level whenever positioned at the same rank. (B) The total transcriptional activity at the locus (i.e., the sum of the expression levels of all Hoxd genes active in developing digits) correlates with the number of transcription units: Deletions or duplications decrease or increase the total transcription, respectively. The wild-type configuration is shown with a light-blue bar. Configurations associated with a deletion of RXII are excluded from this analysis. (C) A two-step model. The digit enhancers (GCR, Prox) first contact the Evx2-Hoxd13 intergenic region. They may subsequently activate the transcription of Lnp, Evx2, or Hoxd genes, with different probabilities. Hoxd genes are activated in a polar fashion, the enhancers scanning (or sensing) the complex in a 5′–3′ direction. This model accounts for most of the observed features and generated several predictions that were subsequently assayed (see the text).

Duplication alleles. Observed (red) and predicted (yellow) gene expression levels in three duplications. Wild-type levels (light blue) are shown on each panel for comparison. (A) dup(13). (B) dup(13-11). (C) dup(12-11). In all cases, the expression of duplicated loci was less than twice the wild-type level, and genes located 3′ of the duplication were down-regulated, highlighting the importance of gene rank. Hoxd13 expression remained unaffected by the duplication of Hoxd12 and Hoxd11, whereas duplications including Hoxd13 lead to a decrease in Evx2 transcription. The predictions of the model matched the observed values, with the exception of Evx2 down-regulation in dup(13) and dup(13-11), underestimated by the model. Blue ovals are as in Figure 5.

Hoxd gene regulation in the developing genital tubercle. The same model structure was applied to investigate Hoxd gene regulation in the genital eminence. The various parameters of the model were optimized using expression values observed in the genital bud in all configurations, except for internal deletions (see the text). (A) In the wild-type situation, quantitative collinearity in the genital bud was similar to what was observed in presumptive digits. (B–E) The 5′ deletions and duplications induced comparable regulatory reallocations in the genital bud, as compared with digits. (B) del(13). (C) del(13-12). (D) dup(13-11). (E) dup(12-11). (F) As in the case of digits, the overall transcription level correlated with the number of transcription units at the locus. In all cases, the observed expression levels were in good agreement with the predictions of the model. (G–I) In contrast, the model could not account for the expression levels observed following internal deletions. For instance, del(12-11) (G) and del(11) (H) lead to a decreased expression of Hoxd10, and del(11-10) leads to a down-regulation of Hoxd12 (I). As in developing digits, del(11-10) elicited a strong increase in Hoxd9 transcription, which was not predicted by the model. Blue ovals are as in Figure 5.