Proc. Natl. Acad. Sci. USAVol. 92, pp. 310-314, January 1995Developmental Biology

Axial skeleton homeosis and forelimb malformations in Hoxd-11mutant miceBERTRAND FAVIER, MARIANNE LE MEUR, PIERRE CHAMBON, AND PASCAL DOLL1EInstitut de G6netique et de Biologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Sante et de la RechercheMedicale/Universite Louis Pasteur/College de France, BP 163-67404 Illkirch-Cedex, C.U. de Strasbourg, France

Contributed by Pierre Chambon, September 23, 1994

ABSTRACT The Hoxd-11 gene was disrupted by homol-ogous recombination in embryonic stem cells. We found thatHoxd-11/ mutant mice are viable and display homeotictransformations of their sacral vertebrae, while their fore-limbs present abnormalities of some metacarpals and of thefirst row of carpal bones. These results are discussed in thelight of current models of tetrapod axial skeleton and limbpatterning.

The mouse genome contains 38 Hox genes linked in fourcomplexes (HoxA, -B, -C, and -D) and expressed along theembryo axis in spatially restricted domains colinear with theorder of the genes within each Hox complex (reviewed in refs.1 and 2). Hox gene disruptions have shown that loss of theirfunction can lead to homeosis in the skeleton and to defects ofvarious 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 expressiondomains in the mesenchyme of the developing limb budsresulting from the successive activation of these genes in theposterior and distal area of the buds (3, 4). The correspondingset of Hoxa genes also exhibit colinear expression domains inthe limb, whose boundaries are slightly distinct from those oftheir Hoxd paralogs (5). Disruptions of both Hoxd-13 andHoxa-li affect axial and limb skeletons (6,7), leading to homeosisof sacral vertebrae and to growth and patterning defects in thelimb autopod (the most distal "segment" of the limbs).We have now disrupted the Hoxd-11 gene, which is ex-

pressed in the trunk up to the lumbosacral transition (4). It isexpressed up to the zeugopod (the radius/ulna and tibia/fibula"segments") in the developing forelimbs and hindlimbs (3) andin the developing genital tubercle (8). Hoxd-11-/- mutantmice are viable and display local homeosis of the sacral vertebrae,as well as abnormalities of the forelimb autopod. However, theHoxd-11 mutation does not appear to affect the hindlimb skeletonand the genital tubercle-derived penian bone.

MATERIALS AND METHODSA 129/Sv murine 7.8-kb EcoRI genomic DNA fragmentcontaining both exons of Hoxd-11 was purified from a cosmidcontaining the 5' part of the HoxD locus. A 633-bp Acc Ifragment (extending from anAcc I site located in the Hoxd-11intron 189 bp from the splice donor site to an Acc I siteoverlapping the 23rd codon of the homeobox sequence) wasremoved and replaced by a pMClNeo (Stratagene) fragmentcontaining the neomycin resistance gene driven by a thymidinekinase promoter, thus yielding the px26 targeting construct inwhich the Hoxd-11 open reading frame is interrupted at aminoacid position 245. Embryonic stem (ES) cell culture, homol-ogous recombination, DNA extraction, and Southern blottingwere as described (9). The probe we used for screening ES cell

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clones and subsequent mice offspring analysis was the 0.3-kbEcoRI-Pvu II genomic fragment located 3' of the targetingconstruct. This probe recognizes a 17.7-kb Pvu II fragment inthe wild-type (WT) and a 3.1-kb fragment in the mutatedallele. A 0.9-kb neo fragment and a 0.8-kb EcoRI 5'-flankinggenomic fragment were also used as probes on HindIII, Pvu II,or Mlu I digests to confirm the targeting event (details availableupon request).Two electroporation experiments, each with 107 D3 ES cells

(9), were performed and resulted in one targeted ES cell cloneout of 58 and 67 neomycin-resistant clones, respectively. Uponblastocyst injection, one of these clones yielded three germ-linetransmitting male chimeras. These chimeras were crossed withC57BL/6 females or with 129/Sv females to produce heterozy-gous mutants in a pure (129/Sv) background. Mutant micedisplayed the same phenotype irrespective of their geneticbackground.Mutant and littermate control animals were sacrificed at 1,

2, 3, or 6 days or 1, 3 to 4, or 6 months postpartum, and alcianblue/alizarin red skeletal staining was performed as described(6). Fetuses were collected at 15.5 days postcoitum (dpc) andstained as whole mounts with alcian blue (6). The metacarpaland phalanges of 15.5-dpc fetuses and 6-day-old newbornswere measured from fixed magnification photographs. Forhistological examination, organs were fixed in Bouin's fluidand processed according to standard technique. The carpalbone nomenclature of Milaire (10) was used.

RESULTSHoxd-11'/- mice appeared normal and Hoxd-11-/- mutants,which were obtained in a Mendelian ratio (25%) from het-erozygote intercrosses, were outwardly indiscernible fromtheir littermates. While Hoxd-11-1- females were fertile, only3 out of 13 mutant males presently tested (the oldest one being8 months old) gave rise to one litter each. One of these malesdied after a second mating. Autopsy revealed a urethral obstruc-tion by a sperm plug. Thus, the Hoxd-11 mutation clearly resultedin male hypofertility of unknown origin, since dissection andhistological analysis of genitourinary organs revealed no abnor-malities. The penian bone of Hoxd-11-1- males was normal.

Axial Skeleton Homeosis. The axial skeleton was normal in47% of the homozygous mutants (Table 1). In 38% of thesemutants an "additional" lumbar vertebra (L7) was present,whereas the next four vertebrae had the appearance of fournormal sacral vertebrae (Fig. 1B). In these cases, the iliumarticulated to one vertebra more posterior than inWT animals(S1*; Fig. 1B). Other Hoxd-11-1- mutants had an additionalvertebra (SO) which appeared "intermediate" between lumbarand 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, embryonicstem.

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Table 1. Vertebral and forelimb phenotype of Hoxd-11 mutant mice

Hoxd-1 -/- Hoxd-11/- WT(53, 106)* (28, 56)* (11, 22)*

Vertebral patternt6L/4S (WT) 24 28 116L/S0 asym/4S 4 0 06L/SO/4S 5 0 07L/4S 20 0 0

Metacarpals II, III, and IV: normal/shortened 10/96 56/0 22/0SL and PY: normal/fused 82/24 55/1 22/0PY and PI: normal/fused 11/95 56/0 22/0Shape of PI head: normal/abnormal 6/100 56/0 22/0PPSL: normal/separated 37/69 55/1 22/0SL dorsal aspectt: normal/lunate enlarged 12/46 16/0 16/0SU: normal/smaller 21/85 56/0 22/0Palmar side of radial epiphysis§: normal/flattened 8/40 40/0 6/0Gap between radius and ulna§: normal/enlarged 9/39 38/2 6/0Sesamoid: 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 ofthe 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 anadditional asymmetric vertebra was noticed between L6 and Si, it resembled L6 on the left side and Sion the right side; SO, additional sacral-like vertebra anterior to Si but with smaller articular wings forillium; 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 wasclearly asymmetric, with a lumbar-like transverse process onthe left side and a first sacral-like transverse process on theright side (Fig. 1 C and E), and the ilia were articulated to twodistinct vertebrae (SO on the right side and S1* on the left).Note that the S1* was clearly asymmetric, with an S2 mor-phology on the right side. Interestingly, in all four cases it wasthe left side of the SO and S1* vertebrae that was anteriorlytransformed, indicating that the variability in expressivity ofthe mutation was not fully stochastic. The occasional presenceof such asymmetric SO and S1* vertebrae strongly suggests thatthe Hoxd-11 vertebral abnormalities correspond to an anteriorhomeosis of the four sacral and (at least) the first caudalvertebrae, rather than to the addition of one lumbar vertebra.

Adult Limb Phenotype. Specific defects were observed insome digits, in the carpus, and in the distal extremities of theradius and ulna ofHoxd-11-1- forelimbs (Table 1). The severityand the extent of the malformations varied between mutants(incomplete penetrance) and between the forelimbs of a givenmutant (variable expressivity). The length of the metacarpalbones of digits II, III, and IV was clearly reduced by --30% inmost of the adult mutants, with their distal part appearing oftenthicker, especially for the second metacarpal (Fig. 2 B and C anddata not shown). In contrast, with the exception of the secondphalanx of digit II, which was slightly smaller in some mutants(data not shown), other metacarpals and phalanges were notsignificantly reduced in size. No abnormal bone fusions were everobserved in mutant digits.The bones of the distal row of the carpus were apparently

not altered in Hoxd-11-/- mutants, but fusions of proximalcarpal bones were almost constant (Figs. 2B and 3). In 90% ofthe limbs, the PI and PY bones were fused (Fig. 3A). Occa-sionally, the PY was also fused to the SL (Fig. 2B). In somecases, only the PY and the SL were fused (Fig. 3C). The PI bone,irrespective of its fusion to the PY bone, appeared truncated andthicker, and its head was misshapen (Fig. 3 A-C). In 65% of themutants, the PPSLwas completely separated from the main bone(Figs. 2 E and F and 3 A and B). The Hoxd-11 mutation alsoaffected the distal extremities of the radius and ulna (see legendsto Figs. 2 and 3 and Table 1).

The hindlimbs of Hoxd-11-1- mutants appeared normal inall cases.

Developmental Limb Phenotype. The length of the meta-carpal cartilaginous anlagen (and of all other digit elements)was not reduced in 15.5 dpc mutant fetuses (Fig. 4A and B).Three days after birth, the metacarpals of digits II, III, and IVwere on average 10% smaller in the mutant than in the WT.This reduction was uniform over the entire length of the meta-carpals (Fig. 4 C and D; length data not shown). This sizereduction was greatest in adult mutants ('30% on average).

Six out of 10 15.5-dpc mutant carpi displayed an irregular SLanlage, with an enlarged lunate portion (compare Fig. 4A andB). 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. 4C and D). Exceptionally, one adult mutant SL retained its fetalappearance (compare Fig. 4A and C with Fig. 2C). The PY andPI cartilages were always separated in 15.5-dpc WT andmutant fetuses, while the PPSL could not be discerned at thisstage. However, several 1- and 3-day-old mutants displayedfusions of the SL or the PI with the PY cartilages (not shown).The PPSL was clearly seen as a separate cartilage in mostnewborn and 3-day-old mutants, whereas it was properly fusedto the SL cartilage in WT littermates (not shown).

DISCUSSIONMice homozygous for a Hoxd-11-targeted mutation have anormal life-span but display anterior homeosis of the sacral(and possibly caudal) vertebrae and alterations of the forelimbskeleton. These phenotypes exhibit both incomplete penetranceand variable expressivity. The anterior limit of the transformedvertebral region (S1) coincides with the rostral boundary ofHoxd-11 transcript expression in the developing prevertebralcolumn (4), whereas the posterior limit cannot be unambiguouslydetermined because of the similarity between caudal vertebrae.The targeted mutation of the paralogous gene Hoxa-il producessimilar anterior transformations of sacral vertebrae, suggestingthat both of these genes are involved in the patterning of thisregion (7). The incomplete penetrance and variable expressivityof S1 transformation often results in an "intermediate" lumbar-

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312 Developmental Biology: Favier et al.

The autopod of the forelimb is classically divided into thearchipodium (the proximal and central carpal elements, theSR and SU) and the neopodium (the distal carpal arch anddigital rays) (13). The Hoxd-11-1- mutation affects both thearchipodium and the neopodium, in agreement with the twodomains ofHoxd-11 enhanced expression in 11.5-12.5 dpc limbbuds (ref. 14; D. Duboule, D. Decimo, and B.F., unpublisheddata): one located towards the distal extremity (the presump-tive neopodium) and the other along the base of the footplates(the presumptive archipodium). The archipodium abnormal-ities, which affect the three axes of the limb (proximodistal,anteroposterior, and palmodorsal), can be considered as theresult of specific faults in the secondary modifications of thepattern of carpal chondrogenic condensations (including thecondensations for the SR and SU). These abnormalities arevery similar to those present in Hoxa-1-J/- mutants (7), sincethe PY/PI bone fusions and the misshapen PI and SU arecommon to both mutations.The neopodium abnormalities correspond to a size reduc-

tion of some metacarpals that is not due to a reduction of theirprimary cartilaginous models but occurs during their subse-quent growth and ossification. These alterations have no coun-terpart in Hoxa-11-/- mutants, but spatially overlap with similarabnormalities in Hoxd-13-1- mutants (6). However, the Hoxd-

A B C_ E

FIG. 1. Anterior homeosis of sacral vertebrae. (A) Ventral view ofthe lumbosacral skeleton of a 1-month-old WT animal showing thenormal pattern of six lumbar (Li to L6) and four sacral (Si to S4)vertebrae. (B) Hoxd-11-1- mutant displaying an additional L6-like(L7) vertebra. The next posterior vertebrae have normal sacral mor-phology (S1* to S4*). (C) Hoxd-11-/- mutant with an asymmetricaltransformation of S1 (SO). On the right side the transformation is veryweak, whereas the left half has a morphology intermediate between L6and Si. The next posterior vertebrae are apparently all anteriorlytransformed as in the previous specimen (S1* to S4*). (D) Laterodor-sal view of aWT specimen. (E) Laterodorsal view of the correspondingvertebrae of the mutant displayed in C showing the intermediatemorphology of the SO left transverse process, which has a smallarticular surface for the illium although it is shaped as an L6 process(curved arrow).

sacral morphology. This corresponds to an incomplete or asym-metric transformation and reflects the occurrence of genetic andstochastic variations (respectively) in the amounts of other geneproducts functionally redundant with Hoxd-11-e.g., Hox pro-

teins coexpressed in the same prevertebrae, such as Hoxa-11.The extent of the Hoxd-11-1- anterior vertebral homeosis,

which includes S3 and the more posterior vertebrae, appearsto contradict the "posterior prevalence" rule (12), which predictsthat vertebrae where the more "posterior" Hox genes are ex-

pressed should be normal (S3 corresponds to the anterior limit ofHoxrd-12 expression). This rule may have to be modified toinclude its temporal dimension (i.e., the colinear temporal acti-vation of Hox genes between gastrulation and prevertebral con-

densations) in a spatiotemporal "prevalence" model. The mor-

phological identity of a given prevertebra would depend on theextent of activation of the Hox network at the time this prever-tebra is determined, rather than at the time it can be seen as a

condensation. In this hypothesis, the identity of vertebrae caudalto 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 penianbones ofHoxd-11-/- mutants appear normal, whereas those ofHoxd-13-1- mutants are malformed, even though both ofthese genes are expressed in the developing genital tubercle(8). This suggests that patterning of the penian bone mayrequire the products of only the most 5'-located gene(s) in theHoxD complex (such as Hoxd-13; ref. 6).

IPPSLJ

SL-

* ::

D E

FIG. 2. (A-C) Dorsal views of the right forefoot of a WT (A) andtwo Hoxd-11-1- mutant 1-month-old mice (B and C). These viewsshow the distal extremities of the radius and ulna (SR, styloid processof the radius and SU), two bones of the proximal row [SL and PY (ostriquetrium)], the central carpal bone (C), the distal row of the carpus(only two bones are labeled, D2, distal carpal 2; D4, distal carpals 4 and5 fused into the os hamatum), and the metacarpal bones (II to V). Thedefects seen in both mutant forelimbs include the shortening ofmetacarpal bones II, III, and IV (note also the malformation of thehead 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 betweenthe SL and the PY (arrowheads) whereas C shows the only adultmutant with separated lunate and scaphoid bones (compare with Fig.4D). (D-F) Palmar views of the right carpus of a WT (D) and of theright (E) and left (F) carpus of the same mutant animal, which are thusmirror images. These views show the PI, adjacent to and partly hidingthe PY, as well as the PPSL, which has been outlined in white. Bothmutant limbs show a full separation of the PPSL, a truncated medialprocess of the SR (arrowheads), and a malformed head of the PI. Alarge supernumerary bone is found medially from the SU (arrow in F).A very small sesamoid-like bone was found more commonly at thislocation in mutant and heterozygous forelimbs (see Table 1). It isunclear whether these supernumerary bones arise from blastemas thatare not properly incorporated into the SU or represent novel sesamoidbones.

i'N I

D

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Proc. Natl Acad Sci USA 92 (1995) 313

WT ~~":-:::~.f"-- -/ D..............: ~ -,

FIG. 3. (A and B) Proximal (A) and distal (B) views of the dissectedSL, PY, and PI bones from a WT and a mutant forelimb. This mutantshows a grossly misshapen PI bone fused to the PY and a separatedPPSL (arrows). (C) Proximal view of the same bones from a differentpair of specimens. The PI of this mutant is not fused to the PY, butis 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 aWT and a mutant forelimb. In the mutant, the styloidapophysis of the ulna is flattened and more rounded (open arrow), andan excrescence at the palmar and medial edge of the radius styloidapophysis is less protruding (arrow). This alteration may be related tothe observation that the distal gap between these two bones usuallyappears wider in mutant than in control animals, but an abnormaldistal curvature of radius and ulna might also existper se (see also Fig.2E and F).

11-/- phenotype extends more proximally and does not clearlyaffect the most distal elements altered in Hoxd-13 mutants. (Forinstance, the second phalanges are normal or very mildly alteredin Hoxd-11-/- mutants.) Altogether, these partly overlappingphenotypes fit well with the sequential activation of the 5'-locatedHoxd genes during the process of early limb bud formationleading to successive (proximodistal) transcript domains (3) andsupport the "progress zone" model (15), which postulates thatpatterning information for proximodistal elements is sequentiallyprovided to mitotic cells of the limb buds. As in the case of theaxial skeleton (see above), there is no absolute functional prev-alence of a 5'-located gene (e.g., Hoxd-13) in the domain whereit is coexpressed with more 3'-located gene(s) (e.g., Hoxd-11),since such a prevalence would exclude any metacarpal alterationsin Hoxd-1-1/- mutants. The absence of Hoxd-11-1- abnormal-ities in elements distal to the metacarpals (which also derive fromHoxd-11-expressing areas) nevertheless indicates that the prod-ucts of the most 5'-located Hox genes are sufficient for correctmorphogenesis of these elements.

In contrast to what is observed in the axial skeleton, theforelimb alterations ofHoxd-11-/- mutants, as well as those ofHoxd-13 (6) and Hoxa-il mutants (7), cannot be explained interms of homeosis. Thus, the function of Hoxd-11 cannot bereadily integrated in a model stating that specific Hox geneswould 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 arebeginning to unravel seems to control local cell growth, but itdoes not appear that specific positional information can beassigned to each gene. Similarly, another Hox gene (Hoxa-1)was suggested to control local cell growth in some hindbrainsegments (17).

All of the 15.5-dpc and most (80%) of the newborn mutantsdisplay an abnormal SL cartilage with an enlarged and poorlyfused lunate portion, thus resembling the SL of 14.5-dpc WTfetuses. This may correspond to a local heterochronic event,with mutant animals displaying an abnormal feature reminis-cent of an earlier normal situation. Compensatory mechanisms

appear, nevertheless, to act before full ossification of the SLblastema, so that the enlargement of the lunate portion persistsonly occasionally in adult mutants.

In Hoxd-11 mutants, the abnormal fusion between the PIand PY cartilages takes place between 15.5 dpc and birth.During the same time interval, the condensation of the PPSLis recognized in Hoxd-11 mutants as a cartilage that remainsindependent from the main part of the SL. This aspect of themutant phenotype supports the theory of Holmgren (13) con-cerning the evolution of tetrapod limb elements from the fish fin.This theory proposes that the SL results from the fusion of fourtransient blastemas: the radiale (origin of the scaphoid portion),the intermedium (origin of the lunate portion), and two blastemasof 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 separatePPSL present in Hoxd-1l-J/- mutants may correspond to such anancestral central carpal cartilage, which would not properly fuseto the other elements of the SL.Our present mutants are very similar to those obtained

independently by Davies and Capecchi (14), who disrupted theHoxd-11 gene at amino acid position 274. The discrepanciesappear to be mainly related to the different genetic back-grounds of the parental WT strains. Our WT animals neverdisplayed spontaneous fusions between the second distal carpalbone and the central carpal bone or between the equivalent bonesof the hindlimb, whereas such fusions were usually observed inthe WT strain analyzed by Davies and Capecchi (14) and wereabsent from their Hoxd-11-1- mutants. Differences in geneticbackgrounds may also account for the absence of fusions betweenphalanges in the case of ourHoxd-11 mutants. We also found thatthe size of the tibial medial sesamoid bone, which is either absentor reduced in Davis and Capecchi's mutants (14), was notsignificantly affected in our mutants compared withWT animals

WT -I--W T1 lp 1 Xp

P241 Q-

V I

A B

W-

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) andmutant (B) fetuses stained with alcian blue. The mutant lunateblastema is thicker than its WT counterpart. This was observed in 6 ofthe 10 mutant forelimbs examined at this stage. See ref. 11 for detailsabout the delay of chondrification of the lunate vs. the scaphoidblastema. (C and D) Dorsal views of 3-day-old newborn WT (C) andmutant (D) left forefeet. The enlarged lunate portion of the SLstrongly 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.

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of the same age. Overall, the hindlimbs of our Hoxd-11 -/- miceappeared normal. By contrast, the Hoxd-13-1- (6) and Hoxa-11-/- (7) mutations affect both hindlimbs and forelimbs. Theseobservations support the idea that the mechanisms involved infore- and hindlimb patterning are not identical (18). Moregenerally, all of the above variations in penetrance and expres-sivity of the Hoxd-11 mutation, which are most probably relatedto functional redundancies, emphasize the complexity of the genenetwork controlling the morphology of vertebrate skeletal ele-ments.

We thank Prof. D. Duboule for the gift of Hoxd genomic materialand 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 laSante et de la Recherche M6dicale, the Centre Nationale de laRecherche Scientifique, the Centre Hospitalier Universitaire Re-gional, the Association pour la Recherche sur le Cancer, the HumanFrontier Science Program, and the Fondation pour la RechercheM6dicale. B.F. was supported by a fellowship from the Ligue contrele Cancer.

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