Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12644-12648, December 1994 Developmental Biology Homeotic transformation of cervical vertebrae in Hoxa4 mutant mice (pattern formation/skeleton) GERALD S. B. HORAN*, KE WUt, DEBRA J. WOLGEMUTHt, AND RICHARD R. BEHRINGER* *Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; tDepartment of Genetics and Development, The Center for Reproductive Sciences, and Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032 Communicated by R. L. Brinster, September 9, 1994 (receivedfor review August 25, 1994)

ABSTRACT Hoxa-4 (previously known as Hox-1.4) is a gastrulation-stage embryos and, subsequently, in the central mouse -containing that is expressed in the nervous system. A discrete anterior limit of expression can presumptive hindbrain and spinal cord, prevertebrae, and be seen in the central nervous system and in the prevertebrae other tissues during embryogenesis. To understand the role of (6). In situ hybridization studies show that Hoxa4 has an Hoxa-4 during development, we generated Hoxa-4 mutant anterior limit of expression in the hindbrain at the rhom- mice. Homozygous mutants were viable and fertile. Analysis of bomere 6/7 boundary (7). In prevertebrae at 12.5 days of neonatal skeletons revealed the development of ribs on the gestation (E12.5), the anterior limit of Hoxa4 expression is seventh cervical vertebra at variable penetrance and expres- at the second cervical vertebra (C2), although transcripts are sivity. A low frequency of alterations in sternal detected weakly in C2 and more strongly in C3 through C7. was also observed. In addition, we analyzed the skeletons of Expression is also detected in the mesodermal components of transgenic mice that overexpress Hoxa-4 and found that the other organs, including lung, kidney, and portions of the gut. formation of the small rib anlagen that often develop on the In the adult, Hoxa4 expression is restricted to meiotic and seventh cervical vertebra was suppressed. Analysis of adult postmeiotic male germ cells (8, 9). homozyogus mutant skeletons revealed that the dorsal process Loss-of-function studies using homologous recombination normally associated with the second cervical vertebra was also in embryonic stem (ES) cells have begun to reveal the specific found on the third cervical vertebra. These results demonstrate functions of individual Hox in vivo, including neural that Hoxa-4 plays a role in conferring positional information crest specification, limb development, and patterning of the along the anteroposterior axis to specify the identity ofthe third axial skeleton (10). Therefore, to understand the function of and the seventh cervical vertebrae. Hoxa4 during development, we mutated the Hoxa4 locus in ES cells and used the mutated ES cells to generate Hoxa4 The cells of a developing embryo must have positional mutant mice. Hoxa4 homozygous mutant mice are viable information to form secondary structures at the proper lo- and fertile but display homeotic transformations of cervical cations along the embryonic axes. A group ofgenes involved vertebrae; C3 is partially transformed to have characteristics in this process was originally identified in Drosophila, be- ofthe C2 and C7 adopts the identity ofa thoracic vertebra (T). cause mutations at these loci resulted in homeotic transfor- In light of this finding, we examined the skeletons of mice mations (respecification of segments) along the anteroposte- overexpressing Hoxa-4 and found that the formation of rib rior axis (1). These homeotic genes map to two clusters in the anlagen at C7 was suppressed. Our results suggest that Drosophila genome, the Antennapedia complex and the Hoxa4 functions to specify the identity ofC3 and C7, and its bithorax complex. The relative positions of genes within action in determining the identity of C7 is dependent on the these clusters correlate with their functional domains along level of Hoxa-4 expression. We also compare these results the axis: the 5'-most gene is required for the most posterior with the mutant phenotypes of one of its paralogs, Hoxb-4. structures, and genes located toward the 3' end ofthe cluster affect progressively more anterior domains (2, 3). In addition, these genes share a DNA sequence known as the homeobox MATERIALS AND METHODS that encodes the DNA-binding homeodomain, which is struc- Disruption of the Hoxa-4 Locus in Mouse ES Cells. A turally similar to the helix-turn-helix motif of prokaryotic gene-targeting vector was generated from a A phage DNA transcriptional repressors (4). Because the homeotic genes clone containing the Hoxa-4 gene previously isolated from a encode transcription factors, it is thought that they regulate C57BL/6J (B6) mouse genomic library (Fig. 1). The vector specific sets of downstream genes, in a cascade leading to the contains 6.0 kb of with genomic DNA morphogenesis of a given segment (1). and extends from a 5' Kpn I site to an EcoRI site that lies 3' Cross-hybridization with Drosophila homeobox sequences ofthe homeodomain. A PGKneobpA cassette was inserted in facilitated the cloning and identification of similar gene reverse orientation relative to the direction of Hoxa-4 tran- clusters in the mouse. There are 38 murine Hox genes scription into the EcoRI site located in helix 1 of the homeo- localized in four clusters on four different (5). domain (11). An MCltkpA cassette was also added to enrich Many of these genes can be divided into paralogous subfam- for homologous recombinants by using negative selection ilies, each containing up to four members encoding homeo- with 1-(2-deoxy-2-fluoro-,-D-arabinofuranosyl)-5 iodouracil domains that are more similar to each other and to those (FIAU) (12). Ten micrograms of linearized vector was elec- encoded by their Drosophila homologues than to the homeo- troporated into 107 AB-1 ES cells (originally derived from a domains encoded by the other mouse Hox genes. The Hox Black Agouti 129 mouse embryo) and cultured on a mono- genes are usually expressed in the mesoderm of late- layer of SNL 76/7 STO cells in the presence of G418 and FIAU (11, 13). A total of 140 G418/FIAU-resistant ES clones The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: B6, C57BL/6J; Cn, cervical vertebra n; En, gesta- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tional day n; ES, embryonic stem; Tn, thoracic vertebra n.

12644 Downloaded by guest on September 29, 2021 Developmental Biology: Horan et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12645 were screened by Southern analysis by hybridizing EcoRI disruption of the homeobox by the neo cassette. Thus, there digested genomic DNA with a unique 5' probe that lies should be no Hoxa4 transcripts with a continuous coding external to the vector homology. Correctly targeted cell region, and, at most, a truncated lacking DNA- clones were then expanded and reanalyzed by hybridizing binding activity could be produced. Three correctly targeted EcoRV digested DNA with a 3' probe external to the vector ES clones were identified, and two of these clones, 1D3 and homology. 2D4, were found to successfully contribute to the germ line Germ-Line Transmission of the Hoxa-4 Mutant Allele. The of chimeric mice (Fig. 2). Hoxa4 mutant ES clones were microinjected into B6 blas- Male and female Hoxa-4 heterozygotes appeared normal tocysts to generate chimeras that were subsequently bred and were fertile. These heterozygotes were subsequently with B6 mates (14). Tail DNA samples from the agouti pups interbred to generate mice homozygous for the Hoxa4 that resulted from those matings were analyzed by Southern mutation. The genotypes of the offspring from these hetero- blotting with the 5' or 3' probes to identify individuals that zygous crosses (Fig. 2) followed predicted Mendelian fre- were heterozygous for the Hoxa-4 mutation. The chimeras quencies, and the homozygous mutants were externally were also bred with 129/SvEv mates to produce an inbred indistinguishable from their wild-type littermates. All male mouse strain carrying the Hoxa4 mutation. and female homozygous mutants were fertile. Northern Blot Analysis. Total RNA was isolated by lithium Northern Analysis of Hoxa-4 Mutant Embryos. To deter- chloride precipitation (15). Total RNA (25 ,ug per sample) was mine whether the mutation in the Hoxa-4 gene disrupted the analyzed by Northern blotting as described (16). The integrity transcription of wild-type Hoxa-4, RNA isolated from E12.5 of the RNA on the blots was confirmed by the appearance of wild-type, heterozygous, and homozygous mutant embryos the 18S and 28S rRNA bands after ethidium bromide staining. was analyzed by Northern blotting. The blots were hybrid- Two Hoxa-4 probes were used for the analysis. One probe is ized either with a probe containing a portion of the 3'- located 5' of the neo insertion and contains -300 bp of untranslated region of the Hoxa-4 gene located 3' of the neo Hoxa4 cDNA sequence between the Nru I and EcoRl sites. insertion or with a cDNA probe containing portions of exon The other probe is an -680-bp Xba 1-HindIII fragment that 1 and exon 2 that are 5' of the neo insertion (Fig. 3). Both of is located 3' of the neo insertion and that contains 3' un- the Hoxa-4 probes detected the 1.7-kb embryonic transcript translated sequence (17). in the wild-type and heterozygous embryos but not in the Skeleton Preparations. Skeletons were prepared by alkaline homozygous mutant embryos. The intensity of the hybrid- digestion and stained with alizarin red S for ossified bone and ization signal in the heterozygotes was approximately one- alcian blue 8GX for cartilage (18). halfthat ofthe wild-type embryos. Although there were faint Hoxa-4 Overexpressing Transgenic Mice. The transgenic bands at -3.1 and 4.0 kb detected in all samples, it is clear mouse strain Tg(Hox-1.4)69Bri carries multiple copies of a that the major embryonic wild-type transcript has been mouse Hoxa-4 transgene (17). This mouse line was initially abolished by the targeted mutation. generated in a B6 x SJL hybrid genetic background. It has Homeotic Transformations of Cervical Vertebrae in Hoxa-4 subsequently been maintained by crosses with outbred Swiss Mutant Mice. Histological analysis of various organs includ- mice for four generations and for subsequent generations ing liver, lung, kidney, small intestine, colon, brain, testis, with B6 x DBA/2 F1 hybrids. The genotype of mice carrying epididymis, stomach, ovary, thymus, and spleen from adult the transgene was determined with a simian virus 40 probe homozygous mutants revealed no obvious differences in that is specific for the transgene. comparison to wild-type controls. Since targeted mutations in several Hox genes resulted in homeotic transformation of skeletal segments (10), we looked for similar changes in the RESULTS Hoxa-4 mutants from matings between Fl heterozygotes. A Hoxa-4 Mutant Mice Are Viable and Fertile. To disrupt the significant number of skeletons from newborns had an ex- Hoxa-4 gene in mouse ES cells, we generated a vector that tensive ossified rib on C7 (Fig. 4). Since small ossified rib contains a neomycin resistance (neo) expression cassette anlagen at C7 are common in some strains of mice and inserted into the EcoRI site in the first helix of the Hoxa-4 extensive ribs have been reported in 1 of 48 wild-type mice homeodomain (Fig. 1). Correctly targeted clones can be of a hybrid strain (19), we quantitated the cervical-rib effect detected by the presence of an additional 7.8-kb band on a by scoring each skeleton for the presence or absence of a rib Southern blot of genomic DNA digested with EcoRI and anlage and for the presence of an extensive rib (one with a hybridized with a 5' EcoRI-Kpn I fragment (1.1 kb) or by the presence of a 4.8-kb band when genomic DNA is digested 1 2 3 1 2 3 with EcoRV and hybridized with a 3' EcoRI fragment (0.5 .Jl kb). Correct targeting of the Hoxa-4 locus results in the *_ - 6.9 kb _0_ -7.8 kb __ -6.1 kb RI K RV RI RI RI RV ... - I~2 *: 4.8 kb x x 5 probe 3' probe

@3 [ H } ~~~~~~~~~~~~~1 kb .\x\ x\ x\ RI I wt (6.9 kb) RI K RV RI RV W ~- -Mutant (4.8 kb)

5' Probe 3' Probe FIG. 2. Southern blot analysis of targeted Hoxa-4 alleles in ES cells and mice. Upper panels, ES cell lines. Lane 1, 1D3; lane 2, 2D4; FIG. 1. Strategy to mutate the Hoxa-4 gene in ES cells. Top line, lane 3, wild type. The expected diagnostic EcoRI fragments hybrid- Hoxa-4 locus; middle line, targeting vector; bottom line, recombi- izing with the 5' probe are 6.1 kb (wild type) and 7.8 kb (mutant), and nant allele. neo, PGKneobpA; tk, MCltkpA; Rl, EcoRI; RV, the EcoRV fragments hybridizing with the 3' probe are 6.9 kb (wild EcoRV; K, Kpn I. The Hoxa-4 homology in the vector, including type) and 4.8 kb (mutant). Lower panel, progeny of a heterozygote exons 1 and 2 (open boxes), is indicated by the heavy line. The cross. +/+, Wild type; +/-, heterozygote; -/-, homozygous homeobox is indicated as a filled box in exon 2. mutant. Downloaded by guest on September 29, 2021 12646 Developmental Biology: Horan et al. Proc. Natl. Acad. Sci. USA 91 (1994)

+1+ +1- _1- +/+ +/- _/,

......

:: - 28S - ......

.. X * X ....,.

...... *... w...... I . . - 18S - ...... iWS _*'.. _ :::

5' probe 3' probe FIG. 3. Northern blot of total RNA isolated from E12.5 wild- type, heterozygous, and homozygous mutant embryos. Filters were hybridized with Hoxa-4 probes located 5' or 3' of the neo insertion. +/+, Wild-type; +/-, heterozygote; -/-, homozygous mutant. The position of 28S and 18S ribosomal RNA bands is indicated. length approximately 2 or more times its width). These scores were then compared with genotype. An extensive rib was observed in 22 of 46 (48%) homozygous mutants and in 1 of FIG. 5. Homeotic transformation of C3 in Hoxa-4 homozygous 41 (2.4%) wild-type pups of the 2D4 line. Nine (20%) of the mutant mice. Dorsolateral view ofcervical region ofadult skeletons. extensive ribs in the homozygous mutants were long enough (A) Heterozygote. (B) Homozygous mutant. C2 and C3 are num- to fuse to the costal cartilage ofthe first thoracic (Ti) rib (Fig. bered. The arrow points to the dorsocaudally projecting ectopic 4B), but none fused directly to the sternum. Eleven (24%) of dorsal process on C3 in the homozygous mutant skeleton. the extensive ribs were bilateral. Interestingly, 20 of 109 heterozygotes (19%) also had extensive ribs, including two abnormalities, most commonly one or two fused sternebrae. that were fused to the ribs at Ti. Extensive cervical ribs that In two mutant homozygotes (10o), the malformation was fused to the ribs at Ti were never observed in wild-type mice. more severe: there was asymmetric ossification ofthe sterne- To determine if the penetrance of the cervical rib phenotype brae (Fig. 6), similar to that seen in Hoxa-S and Hoxd-3 is higher in an inbred background, we analyzed 47 neonatal mutants (21, 22). Presumably, in the mutants, the offset in skeletons from heterozygote crosses of inbred 129 mice. attachment points ofthe costal cartilages on opposite sides of While some of the mutants had rib anlagen slightly longer the sternal bars results in the direct apposition ofossified and than their widths, there were no extensive ribs in these nonossified segments. In one of these mutants (Fig. 6), the skeletons. Thus, the expressivity of the Hoxa-4 cervical rib costal cartilage of one of the Ti ribs is bifurcated. On the left phenotype decreased in strain 129 mice. side, the two processes from the bifurcation attach to the It was surprising that the more anterior cervical vertebrae sternum at different sites, one at the normal attachment site of neonatal Hoxa-4 mutants were not affected, since the for Ti and one at the normal attachment site for T2. anterior limit of Hoxa-4 expression is at the level of C2. Suppression of Cervical Rib Anlagen in Transgenic Mice Therefore, we also examined the cervical region of skeletons That Overexpress Hoxa-4. We previously generated lines of from adults (animals greater than 8 months of age). In transgenic mice that overexpress Hoxa4 in the tissues in wild-type skeletons, a skeletal element known as the proces- which Hoxa4 is normally expressed (17). Although expres- sus spinosus is found on C2 but not on C3 (20). This dorsal sion ofthe transgene is highest in the developing gut, it is also process is a fin-shaped axial ossification that projects dor- expressed at levels comparable to the endogenous levels of socaudally. In five of seven (71%) Hoxa-4 homozygous Hoxa4 in the prevertebrae. The combined expression from mutants, a dorsal process was also found on C3 (Fig. 5). The both the transgene and the endogenous loci results in over- six heterozygotes that were examined did not possess the expression of Hoxa-4 within its normal domain. Since the ectopic dorsal process on C3. loss of Hoxa4 transcripts in Hoxa-4 mutant mice correlates Sternal Malformations in Hoxa-4 Mutant Mice. In neonatal with more extensive cervical ribs, we asked whether over- skeletons from the products of matings of F2 homozygous mutants to either F1 heterozygotes or F2 homozygous mu- tants, 7 of 21 (33%) Hoxa-4 homozygous mutants had sternal

+/+1

I:.

FIG. 4. Homeotic transformation of C7 in Hoxa-4 homozygous mutant mice. Lateral views of the cervical region of skeletons from a newborn control (A) and a Hoxa-4 homozygous mutant (B). The arrow points to the ossified rib emanating from C7 and fusing with the FIG. 6. Sternum defects in a Hoxa-4 homozygous mutant. Note costal cartilage of the first thoracic rib. The cervical vertebrae are the asymmetric pattern ofossification in the sternebrae and the offset numbered 1 through 7. in the opposing sternocostal junctions on either side of the sternum. Downloaded by guest on September 29, 2021 Developmental Biology: Horan et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12647 expression of Hoxa-4 transcripts could suppress cervical rib the gene with the most posterior limit of expression (26). formation. We analyzed the skeletons of neonates from Thus, a loss-of-function mutation should result in deficien- matings between Hoxa-4 transgenic males and Swiss fe- cies or transformations ofaxial regions near the anterior limit males. In this genetic background, 22 of 31 (71%) wild-type of expression. Although Hoxa-4 is expressed in C2, it is mice had small rib anlagen at C7, while only 6 of 28 (21%) expressed only weakly, and is more strongly expressed in C3 transgenic mice had them. These results suggest that over- through C5. Thus, it is perhaps not surprising for the Hoxa-4 expression of Hoxa-4 suppresses the formation of ribs on C7 mutants to show a transformation at the level of C3. The (P < 0.001, X2 analysis). This is consistent with the inter- incompleteness of the transformation suggests that different pretation that the intermediate phenotype seen in mice het- Hox genes may be responsible for specifying different aspects erozygous for the Hoxa4 mutant allele may be due to loss of ofthe same vertebra. Alternatively, in the absence ofHoxa-4 expression of the wild-type gene from one allele rather than other Hox genes may substitute for Hoxa-4 function. to a dominant action of the mutant allele, particularly since The cervical rib phenotype does not strictly conform to the Northern analysis shows the intensity of the wild-type tran- posterior prevalence model. Perhaps this is because the script from heterozygotes is decreased relative to wild types specification of the identity of C7 is more susceptible to (Fig. 3). perturbations in the expression of Hox genes and to other events that are involved in rib formation. Indeed, the forma- tion of extensive ribs in three different Hox mutants, in DISCUSSION retinoic acid-treated embryos (19), and in a small percentage We generated Hoxa-4 mutant mice to investigate the role of of wild-type animals suggests that this is a process which is Hoxa4 during development. There is no decrease in viability easily perturbable. In the B6 x 129 F2 genetic background, associated with this Hoxa4 mutation. While the embryonic extensive cervical ribs were significantly more frequent in tissues in which Hoxa-4 is expressed are quite diverse, in the homozygous mutants than in heterozygotes, and, in turn, adult, expression is restricted to meiotic and postmeiotic extensive ribs were more frequent in heterozygotes than in male germ cells (9). The highly restricted expression pattern wild-type littermates. Thus, the frequency of extensive cer- in the male gonad suggested that this homeoprotein has a vical ribs increases as the number ofwild-type Hoxa4 alleles function during spermatogenesis. Surprisingly, our Hoxa-4 decreases, probably due to a concomitant decrease in wild- mutant mice were fertile. In addition, mice carrying a partial type transcripts. In an independent experiment, we com- deletion allele of Hoxa-4 were also found to be fertile (23). pared the frequency of small cervical rib anlagen in trans- These results suggest that Hoxa-4 is not essential for sper- genic mice overexpressing Hoxa-4 with that of their wild- matogenesis. Other Hoxa-4 paralogs may provide redundant type littermates. We showed that the frequency offormation or compensatory functions during male germ cell develop- of a small cervical rib anlagen in the Hoxa-4 transgenics was ment. Hoxd-4 is expressed in the somatic cells of the testis significantly reduced compared with controls. Thus, overex- but not in germ cells and is thus an unlikely candidate for pression ofHoxa4 can suppress cervical rib formation, while redundant function. However, Hoxb-4 is transcribed in sper- loss of wild-type Hoxa-4 transcripts correlates with forma- matogenic cells, though at much lower levels than Hoxa4 tion of extensive cervical ribs. It is still possible that these (24). It should be noted that Hoxb4 mutant male mice are effects could be due to altered expression of other Hox genes also fertile (25). If Hoxa-4/Hoxb-4 double-mutant males are in these mutants; however, there is still no report of signif- viable, perhaps they will have defects in spermatogenesis. icant changes in the expression ofother Hox genes in any Hox The formation ofthe C2-type processus spinosus on the C3 mutant. While it is likely that Hoxa-4 is only one of many of homozygous mutants is indicative of a transformation of genes involved in specifying the identity of C7, these results C3 toward a C2 identity. This is a partial anterior transfor- indicate that levels of Hoxa4 expression can influence the mation, since C3 does not assume other characteristics of C2, expressivity and penetrance of the cervical rib phenotype. specifically the dens and widened neural arches. In Hoxa-4 A significant number of the Hoxa-4 homozygous mutant mutants, the presence of extensive ribs at C7 is characteristic mice from matings of F2 homozygotes to either F1 heterozy- of a posterior transformation: a cervical vertebra assuming gotes or F2 homozygotes had sternal abnormalities reminis- the fate of a thoracic vertebra. Intriguingly, mice with mu- cent of those found in Hoxa-S and Hoxd-3 mutants (21, 22). tations of the Hoxa-S gene (21) and the Hoxa-6 gene (23), It was suggested that the low penetrance and variability in which are locatedjust 5' ofHoxa-4, have a similar phenotype. expressivity of this phenotype could be due to the existence These mice have a more complete C7 to thoracic transfor- of an alternate pathway (controlled by one or more genes) mation that is also more penetrant, although different genetic that is stochastically used to compensate for the mutation. backgrounds were analyzed. Nonetheless, this is a clear Indeed, the identification of more and more Hox mutants example of overlapping phenotypes among three different with sternal abnormalities suggests that a number cf genes Hox mutants (Hoxa-4, Hoxa-S, and Hoxa-6), suggesting the could be involved. The expression of Hoxa4 in prevertebra possibility that some phenotypes may be shared between Hox Ti through T7 could be responsible for these minor defects mutants for genes whose anterior limits of expression are in sternum morphogenesis. close. Interestingly, another Hoxa4 mutation that is differ- Finally, the Hoxa4 mutation had no obvious effects in any ent from the one reported here causes a similar phenotype at of the other tissues, not even in the central nervous system, C3, but posterior transformations of C7 were not mentioned where Hoxa-4 is most strongly expressed. The high percent- (23). This mutation differs from the one reported here be- age of amino acid identity among the homeodomains encoded cause it deletes the coding region of the recognition helix, 3' by different Hox genes suggests that different Hox to the EcoRI site in which the disruption reported here was may bind to the same or similar sites in downstream target made. While it is possible that the differences could be due genes. It has therefore been proposed that other Hox genes to different partially functional gene products in the two can compensate for at least some of the functions of the mutants, it is more likely that the cervical rib phenotype and mutated gene. The most obvious candidate genes for func- sternal defects were not reported in the other mutation tional compensation are the Hox paralogs. Like the Hoxa-3 because the low expressivity and penetrance of these phe- and Hoxd-3 mutants (22, 27), which share no common notypes, combined with the influence of genetic background, phenotypes, the Hoxa4 and Hoxb-4 mutants also have no obscured their detection. phenotype in common. Hoxb-4 mutants exhibit partial ante- The posterior prevalence model states that the function of rior transformations of C2, specifically the development of an a given manifests in the axial region in which it is anterior arch of the atlas and widening of the neural arches Downloaded by guest on September 29, 2021 12648 Developmental Biology: Horan et al. Proc. Natl. Acad. Sci. USA 91 (1994) (25), while Hoxa-4 mutants show partial anterior transfor- 8. Wolgemuth, D. J., Engelmyer, E., Duggel, R. N., Gizang- mations of C3 and posterior transformations of C7 to a Ginsberg, E., Mutter, G. L., Ponzetto, C., Viviano, C. & thoracic identity. However, the Hoxa-4, Hoxa-S, and Hoxa-6 Zakeri, Z. (1986) EMBO J. 5, 1229-1235. mutants do share a common 9. Wolgemuth, D. J., Viviano, C. M., Gizang-Ginsberg, E., Froh- phenotype. This apparent over- man, M. A., Joyner, A. L. & Martin, G. R. (1987) Proc. Natl. lap in function suggests that there may be shared functions Acad. Sci. USA 84, 5813-5817. among the Hox genes, particularly among genes whose 10. Krumlauf, R. (1994) Cell 78, 191-201. anterior limits ofexpression are very close, as is the case with 11. Soriano, P., Montgomery, C., Geske, R. & Bradley, A. (1991) Hoxa-4 and Hoxa-S. Now that mutants for the paralogous Cell 64, 693-702. genes Hoxa-4 and Hoxb-4 have been generated, it will be 12. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988) possible through matings to generate double mutants to Nature (London) 336, 348-352. determine the degree of functional redundancy among para- 13. McMahon, A. P. & Bradley, A. (1990) Cell 62, 1073-1085. logs. Likewise, creation of mice that are mutant for both 14. Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, ed. Robertson, E. J. (IRL, Ox- Hoxa4 and Hoxa-5 or Hoxa-6 should better define the degree ford, U.K.), pp. 113-151. offunctional redundancy between nonparalogous Hox genes. 15. 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