articles Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs Marie Kmita*, Nadine Fraudeau*, Yann He´rault*† & Denis Duboule* * Department of Zoology and Animal Biology, NCCR Frontiers in Genetics, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland ........................................................................................................................................................................................................................... Hox genes, located at one end of the HoxD cluster, are essential for the development of the extremities of our limbs; that is, the digits. This ‘collinear’ correspondence is accompanied by a gradual decrease in the transcriptional efficiency of the genes. To decipher the underlying regulatory mechanisms, and thus to understand better how digits develop, we engineered a series of deletions and duplications in vivo. We find that HoxD genes compete for a remote enhancer that recognizes the locus in a polar fashion, with a preference for the 5 0 extremity. Modifications in either the number or topography of Hoxd loci induced regulatory reallocations affecting both the number and morphology of digits. These results demonstrate why genes located at the extremity of the cluster are expressed at the distal end of the limbs, following a gradual reduction in transcriptional efficiency, and thus highlight the mechanistic nature of collinearity in limbs. During vertebrate evolution, Hox genes were co-opted to achieve a ing that modifications in the presence, the absence, the position or variety of functions in addition to their ancestral role in organizing the number of genes would provide insights into both aspects. We structures along the trunk axis. One example is the limb skeleton, used targeted meiotic recombination (TAMERE19) to produce which is patterned by genes from the HoxA and HoxD clusters1,2. unequal recombination between the Hoxd13, Hoxd12 and Hoxd11 These transcription factors are essential for limb development3,4, loci. Furthermore, some deletions and duplications were engineered and their collinear regulation is similar to that observed in the along with other mutations in cis (Fig. 1b). This allelic series trunk: genes located in the middle of the HoxD complex (for clarified functional issues and provided explanations as to why example, Hoxd9) are expressed in proximal areas of the limb bud genes situated in 5 0 of the HoxD cluster preferentially respond to whereas genes that are located in a more 5 0 direction (for example, this regulation, and why they do it with a decrease in transcriptional Hoxd11) have a more distal expression, in the future forearm region. efficiency; thereby deciphering part of the mechanism underlying Accordingly, the most distal parts of the limb, the future hands and collinearity in limbs. feet, express the four genes situated in the most 5 0 location (Hoxd10 to Hoxd13). Therefore, the skeletal organization of the limb is prefigured in the genomic topology of these genes5–7 (Fig. 1a). Serial deletions Furthermore, a decrease in transcription efficiency correlates with We generated deletions and duplications by crossing mice contain- gene order (quantitative collinearity8; Fig. 1a). ing loxP sites between Evx2 and Hoxd13, between Hoxd13 and The proper regulation and function of these genes is critical, as Hoxd12, between Hoxd12 and Hoxd11, or between Hoxd11 and their inactivation leads to the absence of the corresponding struc- Hoxd10 (Fig. 1b). After recombination, all remaining genes were tures: the loss of Hoxd11 and Hoxa11 function is accompanied by fully functional. This series of deficiencies and duplications involved the loss of forearms3, whereas the absence of both Hoxd13 and from one to three loci, such as deletion (or duplication) of Hoxd13, Hoxa13 prevents the development of hands and feet9–11.These of Hoxd13 and Hoxd12 in cis, or of three loci (Fig. 1b). The phenotypes were difficult to evaluate, owing to redundant and phenotypes of the homozygous animals were examined. Severe compensatory mechanisms, and to a functional hierarchy attribut- skeletal phenotypes were scored only on excision of Hoxd13— ing a prevalent role to the most ‘posterior’ proteins over more deletion of both Hoxd12 and Hoxd11 did not lead to important ‘anterior’ ones12. We reported previously that the element necessary alterations (not shown), in agreement with the functional domi- 20 for digit expression of the four Hoxd genes situated in the most 5 0 nance of Hoxd13 over other posterior Hoxd genes . However, location (Hoxd13 to Hoxd10), and of the neighbouring gene Evx2,is although deletions containing Hoxd13 impinged on digit mor- located outside of the HoxD cluster13–15. Moreover, other Hox16,17 phology, the alterations appeared unexpectedly different from and foreign18 promoters responded to this enhancer whenever those described for the loss of function of the corresponding introduced at the extremity of the locus, and this response was genes generated by disruption of coding sequences. independent of their transcriptional orientation. This raised the This finding was exemplified by deleting the Hoxd13 locus, which question of the specificity of action of this regulatory element; that generated a mild digit alteration in comparison with animals 21–23 is, its potential to discriminate between various, distantly located carrying insertional mutations (Fig. 2a, b) that show a reduction Hox promoters and to lead reproducibly to the observed collinear in digit length, a supernumerary posterior digit and an overall ill- expression patterns, thus producing the correct digit morphology. formed and stiffer aspect of bony elements (Fig. 2a). In contrast, To dissect the function of these genes and to investigate the after deletion of Hoxd13, digit length was no longer affected, apart mechanism underlying collinearity in limbs, we carried out a from a slight reduction of digits II and V, and no supernumerary systematic deletion and duplication approach of Hoxd loci, reason- digit was scored. Altogether, the morphology was close to that of control animals (Fig. 2b, c). Furthermore, mice carrying the † Present address: Molecular and Experimental Genetics, FRE2358, CNRS, Institut de Transge´nose, rue de deletion had normal hindlimbs, without the skeletal defects associ- 21 la Fe´rollerie, 3B, 45071, Orle´ans cedex 2, France. ated with Hoxd13 loss of function . Consequently, whereas Hoxd13 NATURE | VOL 420 | 14 NOVEMBER 2002 | www.nature.com/nature © 2002 Nature Publishing Group 145 articles disruptions induce severe limb alterations, the corresponding To see whether this was specific for Hoxd12 or if it reflected a general deletion has little effect on these structures. This phenomenon versatility of Hoxd promoters, we analysed a deletion whereby illustrates the difficulty over assigning an objective meaning to the Hoxd11 was placed at the position of Hoxd13, and compared it concept of ‘gene function’, as different full loss-of-function alleles with a double disrupted allele, inactivating both Hoxd13 and have distinct consequences on the final morphology. Within such Hoxd12 (ref. 25) (Fig. 1b). Again, limbs of deleted mice appeared gene clusters, the ‘function’ of a given gene cannot be considered on different from those of the double inactivation in cis25. In this case, its own, but instead must be integrated into a larger functional animals carrying the two-gene deletion were affected more severely context, that of the cluster itself. than with the double inactivation in cis (Fig. 2e, f), and up to ten We analysed the expression of neighbouring genes to the dis- supernumerary cartilaginous condensations were scored and the rupted allele and found no difference with control (Fig. 2). In entire autopod (the most distal skeletal limb segment) was markedly contrast, deleting the Hoxd13 locus resulted in a deregulation of the ill formed (Fig. 2). Metacarpal bones appeared chaotic, with partial 3 0 neighbouring gene (Hoxd12; Fig. 2a, b). A robust enhancement in fusions, and phalanges failed to elongate properly. Hoxd12 transcription was observed, and Hoxd12 transcripts were This severe phenotype correlated with both an enhancement of detected in the most anterior aspect of the distal bud, within the Hoxd11 transcription and the anterior expansion of its transcript presumptive domain of digit I, where only Hoxd13 transcripts were domain (Fig. 2f). Again, Hoxd11 behaved like Hoxd13 when located seen in control animals24 (Fig. 2b, c). This gain of expression at the (relative) Hoxd13 position. In contrast, Hoxd11 expression explained why these mice were less affected than mice with dis- was normal in the double inactivation mutant (Fig. 2d, e), indicat- rupted alleles: increased transcription of Hoxd12 partially substi- ing that the severity of the defects was associated with the upregula- tuted for the absence of Hoxd13. tion of Hoxd11. The potential of Hoxd11 to trigger supernumerary Therefore, moving Hoxd12 to the position of Hoxd13, with chondrogenic condensations was verified by using a deletion respect to the extremity of the cluster, led to a regulatory realloca- removing Hoxd13, Hoxd12 and Hoxd11; in this condition, the tion of Hoxd12, which became transcribed like Hoxd13 (Fig. 2b, c). number of digits was back to five and Hoxd10 expression was Figure 1 Collinearity in developing limbs. a, Scheme of a forelimb skeleton (top) and triangles) in between transcription units (red arrows). Brackets