Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice

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1 Proc. Natl. Acad. Sci. USA Vol. 92, pp , January 1995 Developmental Biology Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice BERTRAND FAVIER, MARIANNE LE MEUR, PIERRE CHAMBON, AND PASCAL DOLL1E Institut de G6netique et de Biologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Sante et de la Recherche Medicale/Universite Louis Pasteur/College de France, BP Illkirch-Cedex, C.U. de Strasbourg, France Contributed by Pierre Chambon, September 23, 1994 ABSTRACT The Hoxd-11 gene was disrupted by homologous recombination in embryonic stem cells. We found that Hoxd-11/ mutant mice are viable and display homeotic transformations of their sacral vertebrae, while their forelimbs present abnormalities of some metacarpals and of the first row of carpal bones. These results are discussed in the light of current models of tetrapod axial skeleton and limb patterning. The mouse genome contains 38 Hox genes linked in four complexes (HoxA, -B, -C, and -D) and expressed along the embryo axis in spatially restricted domains colinear with the order of the genes within each Hox complex (reviewed in refs. 1 and 2). Hox gene disruptions have shown that loss of their function can lead to homeosis in the skeleton and to defects of various structures at defined axial levels (reviewed in ref. 1). Five contiguous genes in the 5' region of the HoxD complex (Hoxd-9 to -13) also display specific and colinear expression domains in the mesenchyme of the developing limb buds resulting from the successive activation of these genes in the posterior and distal area of the buds (3, 4). The corresponding set of Hoxa genes also exhibit colinear expression domains in the limb, whose boundaries are slightly distinct from those of their Hoxd paralogs (5). Disruptions of both Hoxd-13 and Hoxa-li affect axial and limb skeletons (6,7), leading to homeosis of sacral vertebrae and to growth and patterning defects in the limb autopod (the most distal "segment" of the limbs). We have now disrupted the Hoxd-11 gene, which is expressed in the trunk up to the lumbosacral transition (4). It is expressed up to the zeugopod (the radius/ulna and tibia/fibula "segments") in the developing forelimbs and hindlimbs (3) and in the developing genital tubercle (8). Hoxd-11-/- mutant mice are viable and display local homeosis of the sacral vertebrae, as well as abnormalities of the forelimb autopod. However, the Hoxd-11 mutation does not appear to affect the hindlimb skeleton and the genital tubercle-derived penian bone. MATERIALS AND METHODS A 129/Sv murine 7.8-kb EcoRI genomic DNA fragment containing both exons of Hoxd-11 was purified from a cosmid containing the 5' part of the HoxD locus. A 633-bp Acc I fragment (extending from anacc I site located in the Hoxd-11 intron 189 bp from the splice donor site to an Acc I site overlapping the 23rd codon of the homeobox sequence) was removed and replaced by a pmclneo (Stratagene) fragment containing the neomycin resistance gene driven by a thymidine kinase promoter, thus yielding the px26 targeting construct in which the Hoxd-11 open reading frame is interrupted at amino acid position 245. Embryonic stem (ES) cell culture, homologous recombination, DNA extraction, and Southern blotting were as described (9). The probe we used for screening ES cell The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. clones and subsequent mice offspring analysis was the 0.3-kb EcoRI-Pvu II genomic fragment located 3' of the targeting construct. This probe recognizes a 17.7-kb Pvu II fragment in the wild-type (WT) and a 3.1-kb fragment in the mutated allele. A 0.9-kb neo fragment and a 0.8-kb EcoRI 5'-flanking genomic fragment were also used as probes on HindIII, Pvu II, or Mlu I digests to confirm the targeting event (details available upon request). Two electroporation experiments, each with 107 D3 ES cells (9), were performed and resulted in one targeted ES cell clone out of 58 and 67 neomycin-resistant clones, respectively. Upon blastocyst injection, one of these clones yielded three germ-line transmitting male chimeras. These chimeras were crossed with C57BL/6 females or with 129/Sv females to produce heterozygous mutants in a pure (129/Sv) background. Mutant mice displayed the same phenotype irrespective of their genetic background. Mutant and littermate control animals were sacrificed at 1, 2, 3, or 6 days or 1, 3 to 4, or 6 months postpartum, and alcian blue/alizarin red skeletal staining was performed as described (6). Fetuses were collected at 15.5 days postcoitum (dpc) and stained as whole mounts with alcian blue (6). The metacarpal and phalanges of 15.5-dpc fetuses and 6-day-old newborns were measured from fixed magnification photographs. For histological examination, organs were fixed in Bouin's fluid and processed according to standard technique. The carpal bone nomenclature of Milaire (10) was used. RESULTS Hoxd-11'/- mice appeared normal and Hoxd-11-/- mutants, which were obtained in a Mendelian ratio (25%) from heterozygote intercrosses, were outwardly indiscernible from their littermates. While Hoxd females were fertile, only 3 out of 13 mutant males presently tested (the oldest one being 8 months old) gave rise to one litter each. One of these males died after a second mating. Autopsy revealed a urethral obstruction by a sperm plug. Thus, the Hoxd-11 mutation clearly resulted in male hypofertility of unknown origin, since dissection and histological analysis of genitourinary organs revealed no abnormalities. The penian bone of Hoxd males was normal. Axial Skeleton Homeosis. The axial skeleton was normal in 47% of the homozygous mutants (Table 1). In 38% of these mutants an "additional" lumbar vertebra (L7) was present, whereas the next four vertebrae had the appearance of four normal sacral vertebrae (Fig. 1B). In these cases, the ilium articulated to one vertebra more posterior than in WT animals (S1*; Fig. 1B). Other Hoxd mutants had an additional vertebra (SO) which appeared "intermediate" between lumbar and sacral, with transverse processes not tightly joined to the Abbreviations: WT, wild type; dpc, days postcoitum; SL, scapholunate; PPSL, palmar process of the SL; PI, pisiform; PY, pyramidal; SU, styloid process of the ulna; SR, styloid process of the radius; Ln, lumbar vertebra number; Sn, sacral vertebra number; ES, embryonic stem. 310

2 Developmental Biology: Favier et al. Table 1. Vertebral and forelimb phenotype of Hoxd-11 mutant mice Proc NatL Acad Sci USA 92 (1995) 311 Hoxd-1 -/- Hoxd-11/- WT (53, 106)* (28, 56)* (11, 22)* Vertebral patternt 6L/4S (WT) L/S0 asym/4s L/SO/4S L/4S Metacarpals II, III, and IV: normal/shortened 10/96 56/0 22/0 SL and PY: normal/fused 82/24 55/1 22/0 PY and PI: normal/fused 11/95 56/0 22/0 Shape of PI head: normal/abnormal 6/100 56/0 22/0 PPSL: normal/separated 37/69 55/1 22/0 SL dorsal aspectt: normal/lunate enlarged 12/46 16/0 16/0 SU: normal/smaller 21/85 56/0 22/0 Palmar side of radial epiphysis : normal/flattened 8/40 40/0 6/0 Gap between radius and ulna : normal/enlarged 9/39 38/2 6/0 Sesamoid: absent/small/big 28/16/4 10/30/0 6/0/0 PI, pisiform; PY, pyramidal; SL, scapholunate; PPSL, palmar process of the SL; SU, styloid process of the ulna; Ln, lumbar vertebra number; Sn, sacral vertebra number. *Number of axial skeletons and forelimbs analyzed. t6l/4s, WT pattern with 6 lumbar and 4 sacral vertebrae and L6 adjacent to S1; SO asym: when an additional asymmetric vertebra was noticed between L6 and Si, it resembled L6 on the left side and Si on the right side; SO, additional sacral-like vertebra anterior to Si but with smaller articular wings for illium; 7L, seven lumbar vertebrae. tnumbers are given for newborn (1-6 days) limbs only. Numbers are given for adult (>1 month) limbs only. ilium and smaller than normal first sacral transverse processes (data not shown, Table 1). In four mutants, this SO vertebra was clearly asymmetric, with a lumbar-like transverse process on the left side and a first sacral-like transverse process on the right side (Fig. 1 C and E), and the ilia were articulated to two distinct vertebrae (SO on the right side and S1* on the left). Note that the S1* was clearly asymmetric, with an S2 morphology on the right side. Interestingly, in all four cases it was the left side of the SO and S1* vertebrae that was anteriorly transformed, indicating that the variability in expressivity of the mutation was not fully stochastic. The occasional presence of such asymmetric SO and S1* vertebrae strongly suggests that the Hoxd-11 vertebral abnormalities correspond to an anterior homeosis of the four sacral and (at least) the first caudal vertebrae, rather than to the addition of one lumbar vertebra. Adult Limb Phenotype. Specific defects were observed in some digits, in the carpus, and in the distal extremities of the radius and ulna of Hoxd forelimbs (Table 1). The severity and the extent of the malformations varied between mutants (incomplete penetrance) and between the forelimbs of a given mutant (variable expressivity). The length of the metacarpal bones of digits II, III, and IV was clearly reduced by --30% in most of the adult mutants, with their distal part appearing often thicker, especially for the second metacarpal (Fig. 2 B and C and data not shown). In contrast, with the exception of the second phalanx of digit II, which was slightly smaller in some mutants (data not shown), other metacarpals and phalanges were not significantly reduced in size. No abnormal bone fusions were ever observed in mutant digits. The bones of the distal row of the carpus were apparently not altered in Hoxd-11-/- mutants, but fusions of proximal carpal bones were almost constant (Figs. 2B and 3). In 90% of the limbs, the PI and PY bones were fused (Fig. 3A). Occasionally, the PY was also fused to the SL (Fig. 2B). In some cases, only the PY and the SL were fused (Fig. 3C). The PI bone, irrespective of its fusion to the PY bone, appeared truncated and thicker, and its head was misshapen (Fig. 3 A-C). In 65% of the mutants, the PPSLwas completely separated from the main bone (Figs. 2 E and F and 3 A and B). The Hoxd-11 mutation also affected the distal extremities of the radius and ulna (see legends to Figs. 2 and 3 and Table 1). The hindlimbs of Hoxd mutants appeared normal in all cases. Developmental Limb Phenotype. The length of the metacarpal cartilaginous anlagen (and of all other digit elements) was not reduced in 15.5 dpc mutant fetuses (Fig. 4 A and B). Three days after birth, the metacarpals of digits II, III, and IV were on average 10% smaller in the mutant than in the WT. This reduction was uniform over the entire length of the metacarpals (Fig. 4 C and D; length data not shown). This size reduction was greatest in adult mutants ('30% on average). Six out of dpc mutant carpi displayed an irregular SL anlage, with an enlarged lunate portion (compare Fig. 4A and B). This feature is typical of younger (14.5 dpc) WT fetuses (not shown). Most of the newborn and 3-day-old mutants (80%) still exhibited a similarly misshapen SL cartilage (Fig. 4 C and D). Exceptionally, one adult mutant SL retained its fetal appearance (compare Fig. 4A and C with Fig. 2C). The PY and PI cartilages were always separated in 15.5-dpc WT and mutant fetuses, while the PPSL could not be discerned at this stage. However, several 1- and 3-day-old mutants displayed fusions of the SL or the PI with the PY cartilages (not shown). The PPSL was clearly seen as a separate cartilage in most newborn and 3-day-old mutants, whereas it was properly fused to the SL cartilage in WT littermates (not shown). DISCUSSION Mice homozygous for a Hoxd-11-targeted mutation have a normal life-span but display anterior homeosis of the sacral (and possibly caudal) vertebrae and alterations of the forelimb skeleton. These phenotypes exhibit both incomplete penetrance and variable expressivity. The anterior limit of the transformed vertebral region (S1) coincides with the rostral boundary of Hoxd-11 transcript expression in the developing prevertebral column (4), whereas the posterior limit cannot be unambiguously determined because of the similarity between caudal vertebrae. The targeted mutation of the paralogous gene Hoxa-il produces similar anterior transformations of sacral vertebrae, suggesting that both of these genes are involved in the patterning of this region (7). The incomplete penetrance and variable expressivity of S1 transformation often results in an "intermediate" lumbar-

3 312 Developmental Biology: Favier et al. i'n I D Proc- NatL Acad ScL USA 92 (1995) The autopod of the forelimb is classically divided into the archipodium (the proximal and central carpal elements, the SR and SU) and the neopodium (the distal carpal arch and digital rays) (13). The Hoxd mutation affects both the archipodium and the neopodium, in agreement with the two domains ofhoxd-11 enhanced expression in dpc limb buds (ref. 14; D. Duboule, D. Decimo, and B.F., unpublished data): one located towards the distal extremity (the presumptive neopodium) and the other along the base of the footplates (the presumptive archipodium). The archipodium abnormalities, which affect the three axes of the limb (proximodistal, anteroposterior, and palmodorsal), can be considered as the result of specific faults in the secondary modifications of the pattern of carpal chondrogenic condensations (including the condensations for the SR and SU). These abnormalities are very similar to those present in Hoxa-1-J/- mutants (7), since the PY/PI bone fusions and the misshapen PI and SU are common to both mutations. The neopodium abnormalities correspond to a size reduction of some metacarpals that is not due to a reduction of their primary cartilaginous models but occurs during their subsequent growth and ossification. These alterations have no counterpart in Hoxa-11-/- mutants, but spatially overlap with similar abnormalities in Hoxd mutants (6). However, the Hoxd- A B C_ E FIG. 1. Anterior homeosis of sacral vertebrae. (A) Ventral view of the lumbosacral skeleton of a 1-month-old WT animal showing the normal pattern of six lumbar (Li to L6) and four sacral (Si to S4) vertebrae. (B) Hoxd mutant displaying an additional L6-like (L7) vertebra. The next posterior vertebrae have normal sacral morphology (S1* to S4*). (C) Hoxd-11-/- mutant with an asymmetrical transformation of S1 (SO). On the right side the transformation is very weak, whereas the left half has a morphology intermediate between L6 and Si. The next posterior vertebrae are apparently all anteriorly transformed as in the previous specimen (S1* to S4*). (D) Laterodorsal view of a WT specimen. (E) Laterodorsal view of the corresponding vertebrae of the mutant displayed in C showing the intermediate morphology of the SO left transverse process, which has a small articular surface for the illium although it is shaped as an L6 process (curved arrow). sacral morphology. This corresponds to an incomplete or asymmetric transformation and reflects the occurrence of genetic and stochastic variations (respectively) in the amounts of other gene products functionally redundant with Hoxd-11-e.g., Hox proteins coexpressed in the same prevertebrae, such as Hoxa-11. The extent of the Hoxd anterior vertebral homeosis, which includes S3 and the more posterior vertebrae, appears to contradict the "posterior prevalence" rule (12), which predicts that vertebrae where the more "posterior" Hox genes are expressed should be normal (S3 corresponds to the anterior limit of Hoxrd-12 expression). This rule may have to be modified to include its temporal dimension (i.e., the colinear temporal activation of Hox genes between gastrulation and prevertebral condensations) in a spatiotemporal "prevalence" model. The morphological identity of a given prevertebra would depend on the extent of activation of the Hox network at the time this prevertebra is determined, rather than at the time it can be seen as a condensation. In this hypothesis, the identity of vertebrae caudal to S3 may also depend on Hoxd-11 expression. The hypofertility of Hoxd-11-/- males may reflect a subtle effect of this mutation in the genital apparatus. The penian bones ofhoxd-11-/- mutants appear normal, whereas those of Hoxd mutants are malformed, even though both of these genes are expressed in the developing genital tubercle (8). This suggests that patterning of the penian bone may require the products of only the most 5'-located gene(s) in the HoxD complex (such as Hoxd-13; ref. 6). D FIG. 2. I E PPSLJ (A-C) Dorsal views of the right forefoot of a WT (A) and two Hoxd mutant 1-month-old mice (B and C). These views show the distal extremities of the radius and ulna (SR, styloid process of the radius and SU), two bones of the proximal row [SL and PY (os triquetrium)], the central carpal bone (C), the distal row of the carpus (only two bones are labeled, D2, distal carpal 2; D4, distal carpals 4 and 5 fused into the os hamatum), and the metacarpal bones (II to V). The defects seen in both mutant forelimbs include the shortening of metacarpal bones II, III, and IV (note also the malformation of the head of metacarpal II in B, black arrow) and the flattening of the SU, which is more severe in C. The forelimb in B displays a fusion between the SL and the PY (arrowheads) whereas C shows the only adult mutant with separated lunate and scaphoid bones (compare with Fig. 4D). (D-F) Palmar views of the right carpus of a WT (D) and of the right (E) and left (F) carpus of the same mutant animal, which are thus mirror images. These views show the PI, adjacent to and partly hiding the PY, as well as the PPSL, which has been outlined in white. Both mutant limbs show a full separation of the PPSL, a truncated medial process of the SR (arrowheads), and a malformed head of the PI. A large supernumerary bone is found medially from the SU (arrow in F). A very small sesamoid-like bone was found more commonly at this location in mutant and heterozygous forelimbs (see Table 1). It is unclear whether these supernumerary bones arise from blastemas that are not properly incorporated into the SU or represent novel sesamoid bones. SL- * ::

4 Developmental Biology: Favier et al. WT...: ~~":-:::~.f"-- ~ -, -/ D FIG. 3. (A and B) Proximal (A) and distal (B) views of the dissected SL, PY, and PI bones from a WT and a mutant forelimb. This mutant shows a grossly misshapen PI bone fused to the PY and a separated PPSL (arrows). (C) Proximal view of the same bones from a different pair of specimens. The PI of this mutant is not fused to the PY, but is severely truncated. SL and PY are fused, and the PPSL is absent (arrow). (D) Palmar views of the distal extremities of the radius (R) and ulna (U) of a WT and a mutant forelimb. In the mutant, the styloid apophysis of the ulna is flattened and more rounded (open arrow), and an excrescence at the palmar and medial edge of the radius styloid apophysis is less protruding (arrow). This alteration may be related to the observation that the distal gap between these two bones usually appears wider in mutant than in control animals, but an abnormal distal curvature of radius and ulna might also existper se (see also Fig. 2E and F). 11-/- phenotype extends more proximally and does not clearly affect the most distal elements altered in Hoxd-13 mutants. (For instance, the second phalanges are normal or very mildly altered in Hoxd-11-/- mutants.) Altogether, these partly overlapping phenotypes fit well with the sequential activation of the 5'-located Hoxd genes during the process of early limb bud formation leading to successive (proximodistal) transcript domains (3) and support the "progress zone" model (15), which postulates that patterning information for proximodistal elements is sequentially provided to mitotic cells of the limb buds. As in the case of the axial skeleton (see above), there is no absolute functional prevalence of a 5'-located gene (e.g., Hoxd-13) in the domain where it is coexpressed with more 3'-located gene(s) (e.g., Hoxd-11), since such a prevalence would exclude any metacarpal alterations in Hoxd-1-1/- mutants. The absence of Hoxd abnormalities in elements distal to the metacarpals (which also derive from Hoxd-11-expressing areas) nevertheless indicates that the products of the most 5'-located Hox genes are sufficient for correct morphogenesis of these elements. In contrast to what is observed in the axial skeleton, the forelimb alterations ofhoxd-11-/- mutants, as well as those of Hoxd-13 (6) and Hoxa-il mutants (7), cannot be explained in terms of homeosis. Thus, the function of Hoxd-11 cannot be readily integrated in a model stating that specific Hox genes would control the morphological identity of individual digits, as it was suggested by the phenotype of retroviral-induced, ectopic expression of Hoxd-11 in the chick hindlimb bud (16). The complex network of Hox gene interactions that we are beginning to unravel seems to control local cell growth, but it does not appear that specific positional information can be assigned to each gene. Similarly, another Hox gene (Hoxa-1) was suggested to control local cell growth in some hindbrain segments (17). All of the 15.5-dpc and most (80%) of the newborn mutants display an abnormal SL cartilage with an enlarged and poorly fused lunate portion, thus resembling the SL of 14.5-dpc WT fetuses. This may correspond to a local heterochronic event, with mutant animals displaying an abnormal feature reminiscent of an earlier normal situation. Compensatory mechanisms Proc. Natl Acad Sci USA 92 (1995) 313 appear, nevertheless, to act before full ossification of the SL blastema, so that the enlargement of the lunate portion persists only occasionally in adult mutants. In Hoxd-11 mutants, the abnormal fusion between the PI and PY cartilages takes place between 15.5 dpc and birth. During the same time interval, the condensation of the PPSL is recognized in Hoxd-11 mutants as a cartilage that remains independent from the main part of the SL. This aspect of the mutant phenotype supports the theory of Holmgren (13) concerning the evolution of tetrapod limb elements from the fish fin. This theory proposes that the SL results from the fusion of four transient blastemas: the radiale (origin of the scaphoid portion), the intermedium (origin of the lunate portion), and two blastemas of the central row (the centrale I and the centrale prepollicis). However, the two latter blastemas have never been observed, which has led to the questioning of this theory (11). The separate PPSL present in Hoxd-1l-J/- mutants may correspond to such an ancestral central carpal cartilage, which would not properly fuse to the other elements of the SL. Our present mutants are very similar to those obtained independently by Davies and Capecchi (14), who disrupted the Hoxd-11 gene at amino acid position 274. The discrepancies appear to be mainly related to the different genetic backgrounds of the parental WT strains. Our WT animals never displayed spontaneous fusions between the second distal carpal bone and the central carpal bone or between the equivalent bones of the hindlimb, whereas such fusions were usually observed in the WT strain analyzed by Davies and Capecchi (14) and were absent from their Hoxd mutants. Differences in genetic backgrounds may also account for the absence of fusions between phalanges in the case of our Hoxd-11 mutants. We also found that the size of the tibial medial sesamoid bone, which is either absent or reduced in Davis and Capecchi's mutants (14), was not significantly affected in our mutants compared with WT animals V I A WT -I-- W T1 lp 1 Xp P2 41 Q- C l D FIG. 4. Hoxd-11-/- forelimb abnormalities during development. (A and B) Dorsal views of the left forefeet of 15.5-dpc WT (A) and mutant (B) fetuses stained with alcian blue. The mutant lunate blastema is thicker than its WT counterpart. This was observed in 6 of the 10 mutant forelimbs examined at this stage. See ref. 11 for details about the delay of chondrification of the lunate vs. the scaphoid blastema. (C and D) Dorsal views of 3-day-old newborn WT (C) and mutant (D) left forefeet. The enlarged lunate portion of the SL strongly alters the overall aspect of the mutant first carpal row. I-V, digit number; L, lunate part of the SL; M, metacarpal; P1, P2, and P3, phalanges; S, scaphoid part of the SL. B W-

5 314 Developmental Biology: Favier et al. of the same age. Overall, the hindlimbs of our Hoxd-11 -/- mice appeared normal. By contrast, the Hoxd (6) and Hoxa- 11-/- (7) mutations affect both hindlimbs and forelimbs. These observations support the idea that the mechanisms involved in fore- and hindlimb patterning are not identical (18). More generally, all of the above variations in penetrance and expressivity of the Hoxd-11 mutation, which are most probably related to functional redundancies, emphasize the complexity of the gene network controlling the morphology of vertebrate skeletal elements. We thank Prof. D. Duboule for the gift of Hoxd genomic material and for discussions, Dr. A. Dierich and her staff for ES cell culture, M. Duval for blastocyst injections, F. Tixier for animal care, and Drs. D. Decimo, E. Barale, and C. Canoun for useful advice and comments. This work was supported by funds from the Institut National de la Sante et de la Recherche M6dicale, the Centre Nationale de la Recherche Scientifique, the Centre Hospitalier Universitaire Regional, the Association pour la Recherche sur le Cancer, the Human Frontier Science Program, and the Fondation pour la Recherche M6dicale. B.F. was supported by a fellowship from the Ligue contre le Cancer. 1. Krumlauf, R. (1994) Cell 78, Doll6, P. & Duboule, D. (1993) Adv. Dev. Biochem. 2, Proc. Natt Acad ScL USA 92 (1995) 3. Dolle, P., Izpisua-Belmonte, J. C., Falkenstein, H., Renucci, A. & Duboule, D. (1989) Nature (London) 342, Izpisua-Belmonte, J. C., Falkenstein, H., Dolle, P., Renucci, A. & Duboule, D. (1991) EMBO J. 10, Haack, H. & Gruss, P. (1993) Dev. Biol. 157, Dolle, P., Dierich, A., LeMeur, M., Schimmang, T., Schuhbaur, B., Chambon, P. & Duboule, D. (1993) Cell 75, Small, K. M. & Potter, S. S. (1993) Genes Dev. 7, Doll6, P., Izpisua-Belmonte, J. C., Tickle, C., Brown, J. & Duboule, D. (1991) Genes Dev. 5, Lufkin, T., Dierich, A., LeMeur, M., Mark, M. & Chambon, P. (1991) Cell 66, Milaire, J. (1978) Arch. Biol. 89, Shubin, N. H. & Alberch, P. (1986) Evol. Biol. 20, Duboule, D. (1992) BioEssays 14, Holmgren, N. (1952) Acta Zool. 33, Davis, A. P. & Capecchi, M. R. (1994) Development (Cambridge, U.K) 120, Summerbell, D., Lewis, J. H. & Wolpert, L. (1973) Nature (London) 224, Morgan, B. A., Izpisua-Belmonte, J. C., Duboule, D. & Tabin, C. J. (1992) Nature (London) 358, Doll6, P., Lufkin, T., Krumlauf, R., Mark, M., Duboule, D. & Chambon, P. (1993) Proc. Natl. Acad. Sci. USA 90, Lohnes, D., Mark, M., Mendelsohn, C., Dolle, P., Dierich, A., Gorry, P., Gansmuller, A. & Chambon, P. (1994) Development (Cambridge, UK) 120,