Development 125, 2711-2721 (1998) 2711 Printed in Great Britain © The Company of Biologists Limited 1998 DEV2303

Mutations in mouse Aristaless-like4 cause Strong’s luxoid

Shimian Qu1,*, S. Craig Tucker1,*, Jason S. Ehrlich3,*, John M. Levorse3, Lorraine A. Flaherty4, Ron Wisdom1,2,† and Thomas F. Vogt3,† 1Departments of Biochemistry and 2Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA 3Department of Molecular Biology, Washington Road, Princeton University, Princeton, NJ 08544, USA 4Molecular Genetics Program, Wadsworth Center, Albany, NY 12201, USA *These authors contributed equally †Authors for correspondence (e-mail: [email protected])

Accepted 4 May; published on WWW 23 June 1998

SUMMARY

Mutations that affect vertebrate limb development provide encodes a paired-type homeodomain that plays a insight into pattern formation, evolutionary biology and role in limb patterning, as a strong molecular candidate for human birth defects. Patterning of the limb axes depends the Strong’s luxoid . In genetic crosses, the three lst on several interacting signaling centers; one of these, the mutant alleles fail to complement an Alx4 gene-targeted zone of polarizing activity (ZPA), comprises a group of allele. Molecular and biochemical characterization of the mesenchymal cells along the posterior aspect of the limb three lst alleles reveal mutations of the Alx4 gene that result bud that express (Shh) and plays a key role in loss of function. Alx4 haploinsufficiency and the in patterning the anterior-posterior (AP) axis. The importance of strain-specific modifiers leading to mechanisms by which the ZPA and Shh expression are polydactyly are indicative of a critical threshold confined to the posterior aspect of the limb bud requirement for Alx4 in a genetic program operating to mesenchyme are not well understood. The polydactylous restrict polarizing activity and Shh expression in the mouse mutant Strong’s luxoid (lst) exhibits an ectopic anterior mesenchyme of the limb bud, and suggest that anterior ZPA and expression of Shh that results in the mutations in Alx4 may also underlie human polydactyly. formation of extra anterior digits. Here we describe a new Alb chlorambucil-induced deletion allele, lst , that uncovers Key words: Limb bud, Pattern formation, aristaless, Paired-type the lst . Integration of the lst genetic and physical maps homeodomain, Zone of polarizing activity, sonic hedgehog, suggested the mouse Aristaless-like4 (Alx4) gene, which Evolution, Mouse

INTRODUCTION molecular analysis has revealed the existence of positive feedback loops that serve to coordinate the growth and The development of the vertebrate limb serves as an instructive patterning of the limb bud in three dimensions over paradigm for investigations of the genetic and cellular embryological time (Johnson and Tabin, 1997; Martin, 1998). programs that specify the formation of patterned structures The nature of the genetic hierarchy that acts to deploy and/or (Hinchliffe and Johnson, 1980). Classical embryological restrict these signaling centers within the limb bud remains an manipulations in amphibians and avian embryos have important area of inquiry. identified three morphogenetic signaling centers that act to Investigation of existing mutants identified by virtue of coordinate the patterning and outgrowth of the limb along the their phenotypes provides an opportunity to identify dorsal-ventral (DV), proximal-distal (PD) and anterior- that direct limb patterning by combining experimental posterior (AP) axes: respectively, the dorsal ectoderm, the embryology with the power of molecular genetics and gene apical ectodermal ridge (AER), a group of columnar epithelial discovery (Niswander, 1997). There are a large number of cells localized to the dorsal-ventral border of the limb bud spontaneous and induced mouse and chick mutants that distal tip, and the zone of polarizing activity (ZPA), a group of exhibit as part of their phenotype a dysmorphic limb mesenchymal cells localized to the posterior margin of the limb (Johnson, 1986). The study of these phenotypically identified bud mesoderm (Tickle and Eichele, 1994). The patterning mutants complements the approach of gene targeting in mice functions of these signaling centers have been ascribed to their by presenting opportunities to both identify novel genes and expression of specific signaling molecules. Specifically, Wnt- to uncover allelic variation of of a known class, 7a expressed in the dorsal ectoderm, fibroblast growth factors thereby providing insight into structure-function (FGFs) expressed in the AER and Shh expressed in the ZPA relationships (Bedell et al., 1997; Eppig, 1997). The set of have been shown to be major determinants of the patterning existing mutants that exhibit preaxial (anterior) polydactyly functions of these signaling centers. In addition, recent provide opportunities to gain insight into the genetic control 2712 S. Qu and others of AP patterning and the restriction of polarizing activity and Green’s luxoid (lu) and one oligosyndactyly mutant, Shh expression. forminlimb deformity (Fmnld) have been investigated (Forsthoefel, A number of mouse mutants with preaxial polydactyly 1959, 1962; Vogt and Leder, 1996). Analysis of mice exhibit ectopic Shh expression in the anterior mesenchyme of segregating single, double or triple combinations of lst, lu and the limb bud during development; these include Extra toes (Xt), lx led to the interpretation that their interactions result in an Strong’s luxoid (lst), luxate, X-linked polydactyly, Rim4 and additive effect on the phenotype and suggest that the lst, lu and Hemimelic extra toes (Buscher and Ruther, 1998; Chan et al., lx genes may act in independent pathways (Forsthoefel, 1959, 1995; Eppig, 1997; Masuya et al., 1995, 1997). Among these, 1962). In outcrosses, Fmn ld alleles were shown to suppress the only the Xt mutant, which is due to mutational inactivation of lst polydactylous phenotype (Vogt and Leder, 1996). Despite the Gli3 zinc-finger factor gene, has been the detailed developmental and genetic analysis of lst mice, molecularly defined (Hui and Joyner, 1993; Schimmang et al., mechanistic interpretations of the defects in lst mutants and its 1992; Vortkamp et al., 1992). Further evidence for a role for genetic interactions have been limited by both the fact that the Gli3 in patterning the limb comes from the analysis of three lst is semidominant, making it difficult to discern human dysmorphic syndromes, Greig-cephalopolysyndactyly, whether it represents a gain-of-function or loss-of-function Pallister-Hall and postaxial polydactyly type A, which define mutation, and by the lack of knowledge about the molecular an allelic series of mutations in the Gli3 gene and which serve identity of the affected gene (Chan et al., 1995; Vogt and Leder, to illustrate phenotypic heterogeneity associated with the 1996). mutational spectra within a single gene (Biesecker, 1997; Here we describe the identification of a chlorambucil- Vortkamp et al., 1991). The other phenotypic limb mutants induced deletion that uncovers a new lst allele, lstAlb. Genetic define additional loci that may function together with, or and physical mapping of the deleted interval identified Alx4 as independently of, Gli3 to restrict ZPA formation and Shh a candidate gene for the lst mutation. Consistent with the expression. We have focused on an identification of the lst gene possibility of allelism between Alx4 and lst, mice homozygous to gain a better understanding of the molecular basis by which for a targeted mutation of Alx4 (Alx4tm1qw) display preaxial this locus acts to restrict the localization of the ZPA. polydactyly and ectopic Shh expression, as well as craniofacial The Strong’s luxoid mutant, lst, was first noted in 1946 as a and ventral body wall defects (Qu et al., 1997). In addition, we polydactylous mouse (originally called the Springville mouse) provide genetic and molecular evidence that demonstrate that that arose in the course of 3-methylcholanthrene mutagenesis Alx4 is the target of mutation in lst mice. Additional genetic experiments performed by Leonnel Strong in his investigations evidence indicates that other genes interact with Alx4 to repress of chemical induction of mammary tumors (Strong, 1961; ZPA formation in the anterior limb bud mesenchyme in a way Strong and Hardy, 1956). A second allele, designated lstJ, arose that strongly modifies the polydactyly observed in spontaneously at The Jackson Laboratory (P. W. Lane, personal heterozygotes. Molecular and biochemical characterization communication). Both lst alleles exhibit semidominant provide structure-function information that demonstrates that inheritance, such that heterozygotes have preaxial polydactyly the Strong’s luxoid phenotype is the result of loss-of-function that is most often manifest as a broadened hallux or a single mutations in Alx4. additional anterior triphalangeal digit on the hindlimb (Forsthoefel, 1962; P. W. Lane, personal communication). However, selection for modifiers has shown that the penetrance MATERIALS AND METHODS and expressivity of the lst limb phenotype is sensitive to genetic background (Forsthoefel, 1962, 1968; P. W. Lane, personal Mice communication). In contrast, homozygous mutant mice have Procedures for chlorambucil (CHL) mutagenesis have been previously extensive preaxial polydactyly of all four limbs (up to nine described (Flaherty et al., 1992). The mutation was observed in the digits), hemimelia (absence) of the tibia, craniofacial defects, progeny of a CHL-mutagenized C3H/HeJ (C3H) male, which was dorsal alopecia, weakness of the ventral body wall and, in mated to a wild-type C57BL/6 (B6) female. The lstAlb mutation is, males, anomalies of the phallus and cryptorchidism therefore, on a parental C3H . The stock has been (Forsthoefel, 1959, 1962, 1963). The lst limb phenotype is first maintained both by backcrossing to B6 and intercrossing to increase morphologically evident at embryonic day 11 p.c. as an the penetrance of the mutation. C3H/HeJ and A/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and enlarged preaxial extension of the AER and overall broadened C57BL/6 mice were either obtained from The Jackson Laboratory or AP axis (Chan et al., 1995; Forsthoefel, 1963; Masuya et al., bred at the Wadsworth Center (Albany, NY). Mice heterozygous for 1995). Heterotypic transplantation of lst anterior mesenchyme the Alx4 targeted mutation (Alx-4tm1qw/+) maintained on an inbred into the anterior aspect of recipient chick limb buds has 129/SvEvTac background (Qu et al., 1997) were outcrossed to mice demonstrated that the broadening across the AP axis is heterozygous for each of the lst mutations and to C57BL/6J. The lst associated with the presence of an ectopic anterior ZPA (Chan allele maintained on the lstDP/Fo background was originally obtained et al., 1995). Consistent with this observation, molecular from Dr P. Forsthoefel (University of Detroit) (Forsthoefel, 1968) and markers of anterior ZPA formation and downstream events, has been maintained as a closed inbred colony. The lstJ allele is including ectopic Shh, ptc and 5′ Hox-d gene expression in the maintained on a B6C3H background and was purchased from the mesoderm, as well as a concomitant anterior extension of an Jackson Laboratory. Progeny were killed either at the time of birth or following derivation by Cesarean section at day 18.5 and were stained Fgf4 expression domain in the AER, are detected in the limb with alizarin red and alcian blue to visualize bone and cartilage, buds of lst embryos (Chan et al., 1995; Goodrich et al., 1996; respectively (Qu et al., 1997). Masuya et al., 1997; Platt et al., 1997). In addition to phenotypic analysis, genetic interactions of lst SSLP analysis with two preaxial polydactylous mutants, Carter’s luxate (lx), Genomic DNA from tail clippings was extracted using a standard Alx4 mutations causes lst polydactyly 2713 proteinase K/phenol-chloroform/EtOH precipitation procedure (Qu et polyacrylamide gels in 0.5× TBE at 150 volts for 90 minutes. For al., 1997). These were used as PCR templates to amplify simple competition experiments, a 50-fold excess of the appropriate sequence length polymorphisms (SSLPs); primer pairs for specific unlabelled competitor was added during the preincubation. To test for MIT SSLP markers were obtained from Research Genetics the ability of R206Q to inhibit the DNA-binding activity of the wild- (Huntsville, AL). PCR was carried out essentially as described type Alx4 homeodomain, increasing amounts of the R206Q (Dietrich et al., 1996). homeodomain (up to 32 ng per reaction) were mixed with a fixed amount of wild-type homeodomain (3.2 ng) in gel shift reactions DNA analysis using the P3 probe. DNA-binding activity from proteins expressed in For the molecular characterization of the lstAlb allele, DNA was culture used 4 µg of nuclear extract from transiently transfected isolated from newborns from a Alx4tm1qw/+×lstAlb/+ cross and 293 cells (Andrews and Faller, 1991). Southern blotting was performed as described (Qu et al., 1997). For the molecular characterization of the lst and lstJ alleles, the protein- Reporter gene studies coding region of Alx4 was examined by PCR amplification and For transient expression and reporter activation assays, cDNAs sequencing of products from first-strand cDNA and genomic DNA encoding either Alx4 wild-type or R206Q versions were introduced templates. RT-PCR was performed on first-strand cDNA synthesized into the mammalian expression vector pCMX (Umesono et al., 1991). from total RNA isolated from phenotypically homozygous mutant Reporter constructs contained three tandem repeats of either the P3, E11.5 embryos derived from Alx4tm1qw/+×lstJ /+ crosses. The use of P3 mutant or P1/2 sequences upstream of the chloramphenicol acetyl primers that flanked the neo insertion site in the Alx4tm1qw targeted transferase (CAT) gene driven by the basal promoter of the adenovirus allele restricted amplification to the lstJ allele (Qu et al., 1997). To E1b gene. Human 293 cells were transiently transfected using obtain additional Alx4 genomic sequence a 129 genomic library in Lipofectamine (Gibco) with 100 ng of expression construct, 500 ng BACs (Research Genetics) was screened with an Alx4 cDNA probe of the indicated reporter construct and 1.0 µg of a β-galactosidase (Qu et al., 1997). Hybridization-positive BACs were either sequenced expression plasmid (pCH110) that served as a control for transfection directly or following subcloning to obtain intron DNA sequence efficiency. 40 hours after transfection, cell lysates were prepared and (Shizuya et al., 1992). Primers and details on Alx4-positive BACs are CAT activity was determined. For dominant negative experiments, available upon request. PCR was performed on genomic DNA isolated 100 ng of pCMX-Alx4 (wild type) was mixed with increasing from phenotypically homozygous lst/lst and lstJ/lstJ mutants. The amounts, up to 500 ng, of pCMX-Alx4 R206Q. Nuclear extracts following primer pairs were used: prepared from cells transiently transfected with the appropriate Alx4 coding region of exon 1: expression plasmid were analyzed for expression of Alx4 protein by forward primer: 5′ CGTCGCCCGCGCAGGCCA 3′ (exon western blotting with an Alx4-specific antibody (Qu et al., 1997); sequence 5′ of AUG) detection was by enhanced chemiluminescence. reverse primer: 5′ GGTCTCGCACGTGCACTC 3′ (first intron sequence) exon 2: RESULTS forward primer: 5′ GGTTGAGGGAGGGACAGAC 3′ (first intron sequence) A chlorambucil-induced deletion on chromosome 2 reverse primer: 5′ GGTACATTGAGTTGTGCTGTCC 3′ (second uncovers Strong’s luxoid intron sequence) As the chemical and spontaneous origins of the lst and lstJ exon 3: ′ ′ alleles, respectively, suggested they were likely to harbor subtle forward primer: 5 CCTGACTTGTGGTGTCACTGC 3 (second mutations, we sought to identify additional alleles associated intron sequence) reverse primer: 5′ CTGGGGACAGTGAAGGGAGAGA 3′ (third with chromosomal alterations that would facilitate intron sequence) identification of the lst gene. The chemotherapeutic drug exon 4: chlorambucil (CHL) has been found to induce deletions and − forward primer: 5′ TCTCTCCCTTCACTGTCCCCAG 3′ (third complex rearrangements at a high frequency (7.5×10 4 per intron sequence) locus per gamete) in male postmeiotic germ cells (Russell et reverse primer: 5′ GGAGTTCGAGGAGGGGTTCTG 3′ (exon al., 1989; Rinchik et al., 1990). CHL-induced deletions are sequence 3′ of stop codon) useful because they often result in complete loss-of-function RT-PCR: alleles and are effective genetic tools for mapping, forward primer: 5′ CCAGAGTTGCCTCCTGACTCT 3′ (exon 2 ′ complementation analysis and positional cloning (Rinchik et sequence 5 of paired-homeodomain) al., 1993). One of us (L. A. F.) has been conducting a CHL- reverse primer: 5′ GGAGTTCGAGGAGGGGTTCTG 3′ (exon 4 sequence 3′ of stop codon). based mutagenesis screen for developmental mutants (Flaherty et al., 1992). DNA-binding assays Progeny of a CHL-mutagenized C3H male were recovered For in vitro DNA-binding studies, peptides containing the Alx-4 that exhibited preaxial polydactyly of the hindlimb. The mode homeodomain (residues 185-268) or the corresponding R206Q of inheritance of the polydactyly was indicative of a dominant mutant were expressed with a (His)6 tag in E. coli BL21 and mutation with incomplete penetrance. As a starting point for recombinant fusion proteins were purified on Ni+2-NTA agarose. Gel tests of allelism, the CHL-induced polydactylous mutation was shift reactions contained 75 mM NaCl, 15 mM Tris pH 7.5, 1.5 mM first examined for linkage to mouse 2 and 13 EDTA, 1.5 mM DTT, 7.5% glycerol, 0.3% NP-40, 4 mM spermine, µ 32 (locations of the lst and Xt mutations, respectively) by 4 mM sperimidine, 0.8 g poly(dIdC), and the appropriate P- following the segregation of polydactyly with the SSLP labelled probe. For gel shifts using the P3 probe, fourfold increases in protein were used beginning with 0.4 ng of protein. For gel shifts markers D2Mit4 and D13Mit57. These preliminary mapping using the P1/2 probe, tenfold more protein was used for each results excluded linkage to chromosome 13 and were condition. Reactions were incubated on ice without the probe for 5 consistent with linkage to chromosome 2, suggesting possible minutes, the probe was then added and samples were incubated at allelism with lst (n=1/15 animals recombinant with D2Mit4, room temperature for 20 minutes. Samples were then resolved on 5% 7/15 recombinant with D13Mit57). 2714 S. Qu and others

Fig. 1. lstAlb fails to complement lst. (A) 1-month-old lstAlb/lst mouse exhibiting dorsal alopecia and polydactyly. Arrowhead highlights the dorsal-ventral boundary of alopecia (Forsthoefel et al., 1966). (B) Skeleton of an lstAlb/lst neonate stained with alizarin red and alcian blue to visualize bone and cartilage, respectively. (C,D) Close- up of forelimb (C) and hindlimb (D) of an lstAlb/lst neonate shown in B. lst homozygotes and the lstAlb/lst transheterozygotes display preaxial polydactyly of all four limbs and hemimelia of the tibia. (E) Genetic mapping data from the 163-animal backcross segregating lst (Vogt and Leder, 1996). Numbers refer to MIT SSLP markers on chromosome 2 (D2Mit***) (Dietrich et al., 1996). The lstAlb deletion mapping data (see F below) was integrated with this cross. Four of the ten deleted SSLPs (indicated in red) were informative in the lst backcross, and these markers appear in red on the lst genetic map. Of the four deleted markers, three failed to recombine with lst and the fourth, D2MIT161, mapped 0.6 cM distal to the mutation. (F) The minimal CHL-associated lstAlb deletion interval. SSLP markers are indicated beneath physical contigs identified on the MIT Mouse Physical Map (Release 15, October 1997; http://www- genome.wi.mit.edu/cgi-bin/mouse/index). Markers scored in the lst genetic map are in green (centromeric) or yellow (telomeric). Markers indicated in black print are not deleted in lstAlb animals. The SSLPs D2Mit98, D2Mit101, D2Mit102, D2Mit163, D2Mit207, D2Mit442, D2Mit438, D2Mit474 and D2Mit476 were also found to not be deleted in lstAlb, but have not yet been placed on the physical map. SSLP markers indicated in red were found to be deleted in lstAlb (D2Mit221, D2Mit129, D2Mit386, D2Mit351, D2Mit350, D2Mit41, D2Mit253, D2Mit439, D2Mit15 and D2Mit161). The deletion of these markers was confirmed by genotyping the mutant offspring of an LstAlb×A/J cross (data not shown). Nine of these SSLPs fall on a doubly-linked YAC contig; one marker, D2Mit161, was deleted but has not yet been placed on the physical map. One marker, D2MIT130, was not informative in the C3H and B6 backgrounds and therefore could not be scored for deletion. (G) SSLPs from the lst cross were used to define a candidate interval on the Jackson Laboratory’s (B6×SPRET)×SPRET community mapping panel (Rowe et al., 1994). The SSLP D2Mit130 does not recombine with lst. Several potential lst candidates fall within the defined interval: Xrf79, D2Xrf79, EST with homology to yeast CBF5; Wsu101e, D2Wsu101e, anonymous cDNA from ectoplacental cone-specific library; Alx4; Xrf428, D2Xrf428, EST with homology to yeast CDC19; Xrf174, D2Xrf174, EST with homology to yeast YAK1; Xrf212, D2Xrf212, EST with homology to yeast PDA2 (Eppig, 1997). The SSLP D2Mit15, deleted in LstAlb had previously been mapped in the BSS cross to the same location as D2Mit14 (Peichel et al., 1996).

To directly address allelism of the CHL-induced mutation wall defects and dorsal alopecia in progeny of lstAlb/+×lstAlb/+ with lst, we used the difference in the severity of the lst/+ and intercrosses. Since CHL is known to produce deletions, this lst/lst phenotypes as an assay in complementation tests. The suggested that lstAlb might be encompassed by a deletion pleiotropic phenotype observed in lst homozygotes has not sizable enough to uncover multiple loci, at least one of which been observed in either lst/+ or CHL-induced heterozygotes. may be required for viability (Rinchik et al., 1993). To Mice carrying the CHL-induced mutation were mated to lst/+ determine if lstAlb was, indeed, a deletion allele, we took heterozygotes (both parents displaying only hindlimb advantage of the high density of SSLP markers in the lst region. Alb polydactyly). The CHL-induced Albany mutation failed to We examined the mutant F1 progeny of a lst ×B6 cross for complement lst, as indicated by the presence of the severe absence of the C3H allele of SSLPs in the lst region. Two F1 lst/lst phenotype in ~25% of the offspring (Fig. 1A-D). Based mutants from different lstAlb parents were genotyped at each of on this failure to complement and additional data described the 62 SSLPs informative between B6 and C3H in a 3.3 cM Alb below, we refer to the CHL-induced mutation as lst . interval flanking lst on the MIT F2 Intercross Map (Dietrich et al., 1996). By this methodology, 10 SSLP markers were found Deletion analysis and integration of mapping data to be deleted in lstAlb (Fig. 1F). Integration of the genetic data Although the lst homozygous phenotype was observed in with the MIT Mouse Physical Map demonstrated that the transheterozygous animals, we have not observed the deleted markers span a large doubly linked YAC contig (Fig. pleiotropic phenotype of profound skeletal and ventral body 1F). Alx4 mutations causes lst polydactyly 2715

To anchor the lst genetic maps, we examined the SSLP lst, and a fourth, D2Mit161, was placed 0.6 cM distal to lst. markers deleted in lstAlb in a backcross segregating the original (Fig. 1E). We next anchored the lst cross to The Jackson lst allele (Vogt and Leder, 1996) and the Jackson Laboratory Laboratory’s (B6×SPRET)×SPRET (BSS) community Community Interspecific Backcross panel (Rowe et al., 1994) backcross mapping panel (Rowe et al., 1994), by scoring the (Fig. 1E-G). First, the cohort of deleted markers was tested for relevant BSS recombinants at the SSLPs D2Mit14 (proximal polymorphism in the lst cross. Three informative markers to lst), D2Mit130 (non-recombinant with lst) and D2Mit43 (D2Mit386, D2Mit439 and D2Mit253) did not recombine with (distal to lst). This integrated map defines a minimal lst region and suggested Alx4 as a candidate gene (Fig. 1G). lst and Alx4 fail to complement While mapping of the lst mutation was in progress, functional characterization also suggested Alx4 as a candidate gene for the lst mutation. Specifically, animals homozygous for a targeted mutation in the Alx4 gene, on an inbred 129 background, have preaxial polydactyly and ventral body wall defects, reminiscient of the lst homozygous phenotype. However, in contrast to lst mutants, Alx4tm1qw homozygotes have only a single extra preaxial digit, and heterozygotes are unaffected (Qu et al., 1997). To test the possibility of allelism between lst alleles and Alx4, outcrosses between heterozygotes were performed. In each case, non- complementation, scored as the presence of profound polydactyly on all four limbs (Fig. 2) and ventral body wall defects (data not shown), was observed in the progeny. These results also led us to reassess the Alx4tm1qw allele, which unlike the three lst alleles, does not exhibit semidominant hindlimb polydactyly (Qu et al., 1997). The Alx4tm1qw allele has been maintained on an inbred 129 genetic background; however, one generation of outcrossing of Alx4tm1qw heterozygotes to other genetic backgrounds (e.g. C57BL/6J) produced Alx4tm1qw heterozygous progeny with hindlimb preaxial polydactyly similar to that previously observed for lst heterozygotes (Fig. 3, compare to Fig. 2). Thus, as previously documented for the lst alleles (Forsthoefel, 1968), the penetrance of the Alx4tm1qw heterozygous hindlimb polydactyly phenotype is sensitive to

Fig. 2. Genetic interaction between lst alleles and Alx4tm1qw. Skeletal stains of forelimbs (fore) and hindlimbs (hind) of progeny are shown with the genotypes Fig. 3. Genetic background modifies the Alx4tm1qw indicated to the left. The appearance of fore- and hindlimb preaxial polydactyly heterozygous phenotype. (A,B) Skeletal stains of the and variably expressive radial and/or tibial hemimelia is indicative of the polydactylous hindlimbs of a (C57BL6/J +/+ × tm1qw tm1qw phenotype seen in homozygous lst and Alx4 mice (Chan et al., 1995; 129/SvEvTac Alx4 /+) F1 progeny displaying Forsthoefel, 1962, 1968; Qu et al., 1997). preaxial polydactyly. 2716 S. Qu and others strain-specific modifying loci. The linkage of lst and Alx4 and a monomer with lower affinity. In contrast to the Alx4 wild- their failure to complement provide strong genetic evidence type homeodomain, the R206Q homeodomain fails to bind that the lst alleles harbor loss-of-function mutations in Alx4 and DNA as either a dimer or monomer (Fig. 5A). Consistent with prompted molecular analysis of the Alx4 gene in lst mice. our in vitro analysis of DNA binding, transient expression assays in human 293 cells show that wild-type Alx4 protein Alx4 is mutated in all lst alleles efficiently activates transcription of reporter constructs in a We looked for molecular alterations of Alx4 in the three lst manner that is dependent on the presence of high-affinity P3- alleles (Fig. 4). In the 3-methylcholanthrene-associated lst binding sites, while the Alx4lst R206Q protein does not (Fig. allele, the coding sequence of Alx4 has a single base change in 5B). Nuclear extracts prepared from transfected cells show codon 206 (CGA>CAA, R206Q), resulting in the substitution that, although the R206Q mutant protein is expressed at levels of glutamine for arginine at the fifth residue of the similar to wild-type Alx4 protein, the mutant protein has homeodomain (Fig. 4B,C). Sequencing of the Alx4-coding markedly reduced DNA-binding activity (Fig. 5C,D). The region in the spontaneously arising lstJ allele revealed a 16 base Alx4lst R206Q protein failed to compete with wild-type Alx4 pair deletion within the paired-type homeodomain. This for DNA binding in vitro (Fig. 5E) and to inhibit deletion disrupts the DNA recognition helix and results in a transactivation in co-transfection experiments (Fig. 5F). By frame-shift of the downstream protein-coding sequence (Fig. these criteria, Alx4lst R206Q does not act in a dominant 4B). Genomic Southern analysis of the CHL-associated lstAlb negative manner. This interpretation is consistent with prior allele revealed that the Alx4 gene is deleted (Fig. 4D). Thus, data demonstrating that dimerization of paired-type each of the three independently generated lst alleles have homeodomain proteins is a consequence of cooperative DNA mutations in Alx4. Hereafter, we refer to the lst alleles as binding, and does not occur in solution (Wilson et al., 1993, Alx4lst, Alx4lstJ and Alx4lstAlb. 1995). Therefore, we conclude that the Alx4lst R206Q mutation represents a severe loss-of-function or null allele. The lst alleles represent Alx4 loss-of-function mutations Previous models to explain the abnormal limb patterning in lst DISCUSSION mice could not distinguish whether the lst mutations represented gain- or loss-of-function alleles (Chan et al., 1995; Strong’s luxoid mice exhibit preaxial polydactyly, which is Vogt and Leder, 1996). The Alx4lstAlb gene deletion indicates preceded during development by the formation of an ectopic that a loss of Alx4 function can underlie the lst-associated ZPA and expression of Shh in the anterior mesenchyme of the polydactylous phenotype. Because of their large effects on limb bud. Our genetic, molecular and functional analyses protein structure, the Alx4lstJ and the Alx4tm1qw alleles are also demonstrate that loss-of-function mutations in Alx4 cause the likely to result in a loss of function (Qu et al., 1997). In phenotype of Strong’s luxoid polydactylous mutants. contrast, the Alx4lst R206Q mutation could represent either a loss-of-function or a dominant negative allele, and its further Chlorambucil-induced chromosomal deletion characterization would provide additional insight into uncovers lst structure-function relationships of the paired-type Comparative genetic mapping and the identification of a homeodomain. RNA expression of Alx4lst in mouse embryos chromosomal deletion that uncovered lst provided a genetic at day 11 of gestation is indistinguishable from wild type, and physical framework to assess candidate genes and served suggesting that the effect of the Alx4lst mutation would be post- to focus attention on Alx4. The deletion uncovering Alx4lstAlb transcriptional (B. Prabhakaran and T. F. V., data not shown; is certain to uncover additional loci, one or more of which is Qu et al., 1997). Therefore, we examined the effect of the likely to account for our failure to observe viable progeny R206Q mutation on the biochemical function of Alx4 in more homozygous for the deletion. The deletion spans a genetic detail. distance of 2-3 cM, removes 10 SSLPs and is spread across Both biochemical and crystallographic analyses have shown multiple YACs on the MIT Physical Map, indicating that its that the paired-type homeodomain binds cooperatively as a size may be measured in the hundreds of kilobases. At this homodimer to specific palindromic DNA sequences (Wilson et level of inspection, the CHL-induced deletion appears to be an al., 1993, 1995). The crystal structure of the paired-type interstitial deletion and not a complex rearrangement. The size homeodomain dimer bound to DNA shows that arginine 5 of of the deletion falls within the range (0.5 cM-15 cM) the homeodomain (the residue altered in the R206Q mutant) previously indicated by analysis of CHL-induced mutations makes specific DNA contacts in the minor groove (Fig. 4C) centered around the albino locus on mouse chromosome 7 and (Wilson et al., 1995). Thus, the substitution of the neutral serves to highlight the utility of integrating CHL-mutagenesis glutamine for the basic arginine 206 could adversely affect with the increasingly dense mouse genetic and physical maps DNA binding. Furthermore, amino acid sequence alignment (Bedell et al., 1997; Dietrich et al., 1996; Rinchik et al., 1993). reveals that the arginine mutated in Alx4lst is highly conserved in the paired-homeodomain and, therefore, is likely to be Polydactyly results from Alx4 loss of function important for function (Burglin, 1994). Prior studies of lst have been unable to distinguish whether the Functional analysis of Alx4lst R206Q confirms that it mutations represented a loss or gain of function. In conjunction represents a loss-of-function allele. The wild-type Alx4 paired- with the gene-targeted disruption of the Alx4 homeodomain, homeodomain binds the palindromic P3 sequence (5′ the present identification of the Alx4lstAlb chromosomal TAATCTGATTA3′) cooperatively as a dimer with high deletion and the Alx4lstJ allele’s 16 bp deletion of the affinity, and the corresponding P1/2 half-site (5′TAATC3′) as homeodomain demonstrate that the phenotypes associated with Alx4 mutations causes lst polydactyly 2717 lst can be caused by loss-of-function mutations. The Alx4lst beads soaked in RA antagonists to the posterior aspect of the single amino acid R206Q substitution is also consistent with a limb bud results in decreased Shh expression, and engraftment loss of function. A number of lines of evidence lead us to of RA-soaked beads to the anterior limb bud results in an conclude the R206Q mutation results in loss of function. ectopic anterior domain of Shh expression in the limb bud and Arginine 5 of the amino terminal arm of the homeodomain is the formation of an ectopic anterior ZPA (Lu et al., 1997). one of the most conserved residues in the homeodomain (98% Although the mechanism by which retinoids exert this effect is of 346 homeodomains surveyed, an exception is the divergent unclear, it may relate to the known ability of RA to regulate AP PrH/Hex proteins which have a glutamine at amino terminal positional specification by controlling axial Hox gene position 5 and an arginine at position 7) (Burglin, 1994). A expression. Hoxb-8 expression in chick limb buds is altered in high resolution crystallographic analysis of the paired-type response to RA bead grafts (Stratford et al., 1997), and homeodomain bound as a dimer to the palindromic P3 element misexpression of Hoxb-8 in the anterior mouse forelimb bud shows that arginine 5 is insinuated into the minor groove of the results in anterior Shh expression and polydactyly (Charité et DNA, where it physically interacts with both strands (Wilson al., 1994). Ectopic forelimb expression of Hoxb-8 is not et al., 1993, 1995). Consistent with the evolutionary invariance detected in either Alx4lst or Alx4tm1qw limb buds, suggesting that and the structural analysis, our biochemical evidence Alx4 is not required for restriction of Hoxb-8 expression (Chan demonstrates that the R206Q mutant behaves as a functional et al., 1995; Qu et al., 1997). Whether Alx4 is a target of null in terms of DNA binding and transcriptional regulation. patterning by retinoids or Hox genes remains to be determined. Furthermore, our observation that the R206Q homeodomain Second, the continued expression of Shh in the posterior does not interact with DNA as a monomer corroborates the mesoderm is contingent on the expression of Wnt-7a in the importance of arginine 5 in DNA binding and predicts that dorsal ectoderm and FGFs in the AER; this regulation mutation of arginine in this position would result in a loss of presumably serves to coordinate patterning along the three axes function in many different classes of homeodomain proteins. (Johnson and Tabin, 1997). Prior genetic studies of the Consistent with our assignment of R206Q as a loss-of- interaction of lst with recessive forminlimb deformity mutant function mutation, we were unable to find any evidence that alleles have indirectly suggested a dependence of the ectopic the R206Q protein could inhibit either the DNA-binding or ZPA on a preaxial extension of the AER and its reciprocal transcriptional properties of the wild-type protein. Although all signaling for Shh maintenance (Vogt and Leder, 1996). Formin biochemical properties of Alx4 that are dependent on DNA mutations cause an inability to form or maintain a proper apical binding are absent in the Alx4 R206Q mutant, it remains ectodermal ridge that subsequently leads to a failure to possible the R206Q protein may exhibit a non-DNA-binding- maintain Shh expression, resulting in oligosyndactyly (Zeller dependent activity in some contexts (Benassayag et al., 1997). et al., 1989). When Alx4lst is placed in trans to mutant formin The biochemical profile of Alx4lst as a loss-of-function alleles, the semidominant polydactyly of Alx4lst heterozygotes mutation is consistent with our genetic evidence that the in vivo is suppressed and a wild-type limb results (Vogt and Leder, behavior of R206Q as assessed by phenotype is largely 1996). The genetic evidence that the Alx4 alleles are likely to indistinguishable from the null alleles. This interpretation is represent severe hypomorphic or null alleles suggests that lst consistent with prior genetic evidence from Drosophila that the and formin are operating on distinct developmental pathways. interaction of the paired homeodomain with palindromic Presumably, Alx4 loss leads to ectopic Shh expression in the elements is critical for biological activity (Bertuccioli et al., anterior limb bud mesenchyme that is dependent on formin 1996). In summary, both biochemical and genetic analysis of function for the anterior expansion of the AER that may be Alx4lst demonstrate a critical role for arginine 5 in required to support ectopic Shh expression. homeodomain function. Third, the large number of mouse and human polydactylous conditions suggests that the genetic mechanisms that restrict Developmental and evolutionary control of ZPA Shh from the anterior mesoderm of the limb bud are complex. formation Prior studies examining genetic interactions between lst and Proper functioning of the ZPA demands that it be precisely the polydactylous mutants luxate and luxoid led to a view that localized in time and space (Johnson and Tabin, 1997). The the phenotypic consequences were additive (Forsthoefel, identification of loss-of-function mutations in Alx4 mice, 1962). With respect to Gli3Xt and Alx4, it is not clear whether together with the expression of Alx4 in the anterior limb bud, they participate in hierarchical or parallel pathways. At the demonstrates that Alx4 function is not required for the level of RNA in situ expression analysis, neither Alx4 nor Gli3 formation of the normal posterior ZPA; in contrast, Alx4 is expression are significantly altered in the respective mutant required to repress both ZPA formation and Shh expression in backgrounds, suggesting they function in separate biochemical the anterior aspect of the limb bud. This requirement for Alx4 pathways to repress Shh expression in the anterior limb bud function is similar to the requirement for Gli3 gene activity to mesenchyme (Qu et al., 1997). Whether Alx4 expression is repress ectopic Shh expression and ZPA formation (Buscher et altered in the limb buds of other preaxial polydactylous al., 1997; Buscher and Ruther, 1998; Hui and Joyner, 1993). mutants remains to be examined. Based on molecular marker and genetic analysis, Alx4 During vertebrate evolution the transition of fins into limbs currently cannot be positioned in a genetic pathway restricting has been proposed to involve a developmental elaboration of anterior Shh expression (Dunn et al., 1997; Qu et al., 1997). digits as novel distal autopod elements (Daeschler and Shubin, Three different lines of evidence provide insight into the 1998; Sordino et al., 1995). The molecular changes underlying refinement of ZPA activity and subsequently Shh expression this evolutionary process are not clear, but may reflect distal and serve to provide a framework for viewing Alx4 function. in response to distal Hox gene expression (Sordino First, signaling by retinoids is implicated, as the engraftment of et al., 1995; Zakany et al., 1997). Regardless of mechanism, 2718 S. Qu and others

Fig. 4. lst alleles have mutations in Alx4. (A) Schematic representation of the Alx4 open reading frame with the paired homeodomain indicated as a blue box and a 22 amino acid region conserved amongst aristaless-related family members termed the paired-tail indicated as a yellow box (Mathers et al., 1997). (B) The nucleotide and amino sequence of the Alx4 homeodomain, showing the location of the mutations present in the lst (R206Q) base transition and lstJ (16 deletion) alleles outlined in red. The inverted arrowhead indicates the position of intron 2. (C) The crystal structure of a paired-type homeodomain (Wilson et al., 1995) showing the position of the homeodomain R5 residue that is altered in the Alxlst R206Q protein. The DNA is in space-filling representation, the paired-type homeodomain monomer is represented as a blue ribbon trace of the polypeptide backbone and the side chain of Arg5 is indicated in red depicting its contact with the DNA backbone in the minor groove (Wilson et al., 1995). (D) Genomic Southern analysis of Alx4 in lstAlb. Genomic DNA Alb tm1qw prepared from the parental strains, lst /+ and Alx4 /+, and F1 progeny was digested with ApaI, transferred to a nylon membrane and hybridized with an Alx4 genomic probe (Qu et al., 1997). Deletion of Alx4 sequences is observed in F1 progeny no. 5 as an absence of the upper wild-type Alx4 hybridizing band. In all cases the observed phenotype of the F1 progeny was consistent with their genotype at Alx4.

co-opted from a role in axial patterning as part of a genetic program to repress anterior ZPA formation. Alternatively, as may be argued from the restriction of Shh to the proximal posterior region of the fin bud in the teleost zebrafish (Krauss et al., 1993), the polydactylous specimens in the fossil record may represent the ‘capture’ of an evolutionary offshoot and Alx4 expression in the anterior limb bud may instead be ancestral. An ancestral role for Alx4 is also suggested by the expression of Drosophila relatives of Alx4 and Gli3 (aristaless and ci, respectively) in the anterior compartment of the wing imaginal disc (Campbell et al., 1993; Schneitz et al., 1993; Shubin et al., 1997). Whereas in AP patterning of the fly wing ci appears to act downstream of hedgehog (Dominguez et al., 1996), the function of aristaless and its relationship to hedgehog and AP patterning is less well defined (Campbell et al., 1993; Schneitz et al., 1993). Analysis of molecular markers such as Shh, Gli3 and Alx4 expression in zebrafish and in more primitive species such as lungfish and coelacanths should provide information by which to evaluate these speculations. Modifiers of Alx4 polydactyly and implications for human polydactyly Previous analysis of the Alx4 targeted mutation, which was carried out in 129/Sv inbred mice, failed to reveal hindlimb polydactyly in heterozygous animals as had been observed in the fossil record suggests that the ancestral Devonian tetrapods Strong’s luxoid heterozygous animals (Qu et al., 1997). Several had limbs with up to eight digits (Coates, 1994; Coates and lines of genetic evidence suggest that the differences in Clack, 1990). The digits of polydactylous limbs of heterozygous phenotype are the result of strain-specific genetic Acanthostega and Ichthyostega appear at some level to changes and are not the result of different Alx4 alleles. First, it resemble the mirror-image polydactyly seen in Alx4 alleles. has previously been established that the lst allele is subject to This resemblance suggests that during the fin-to-limb strain-specific modifying loci (Forsthoefel, 1962, 1968). Second, transition, a branch of ancestral tetrapod limbs were in fact the nature of the Alx4lstAlb, Alx4lstJ and Alx4tm1qw alleles are all polydactylous due to the presence of symmetric anterior and indicative of protein nulls. Third, although the Alx4tm1qw targeted posterior ZPAs (Raff, 1996). Subsequently, during evolution of allele does not generate a heterozygous phenotype on the 129/Sv the pentadactyl limb, the presence of an anterior ZPA would background, outcrossing the mutation to C57BL/6J mice or to have become actively repressed. Based on this notion, the Alx4 the lstDP background results in the appearance of heterozygous mutant alleles would represent atavistic mutations, suggesting polydactyly in the first generation. Fourth, when the strain lst tm1qw that during tetrapod evolution Alx4 may have been recruited or background is the same (F1 progeny of an Alx4 ×Alx4 Alx4 mutations causes lst polydactyly 2719

Fig. 5. Functional characterization of the Alx4lst R206Q mutant protein reveals defective DNA binding. (A) DNA binding by the Alx4 homeodomain. Peptides containing either the wild-type Alx4 homeodomain, with flanking sequences, or the corresponding R206Q mutant protein, were expressed in E. coli and purified to homogeneity. The purified proteins were used in equal amounts in gel shift assays with either the P3 palindromic probe, which Alx4 binds as a homodimer, or the corresponding half-site probe (P1/2), which Alx4 binds as a monomer. Lane 1, probe only; lanes 2-4, increasing amounts of Alx4 wild-type homeodomain; lane 5, 50-fold excess of unlabelled P3 DNA as competitor; lane 6, 50- fold excess of unlabelled P3 mutant DNA as competitor; lane 7, probe only; lanes 8-10, increasing amounts of Alx4 R206Q homeodomain. D designates the migration of dimeric Alx4 bound to probe; M designates the migration of Alx4 monomer bound to probe. Tenfold more protein was used to examine monomeric DNA binding to the P1/2 probe than to assay dimeric binding to the P3 palindromic probe (see Materials and Methods). (B) Transcriptional activation by Alx4. Plasmids directing the expression of either wild-type Alx4 or Alx4 R206Q were co- transfected into human 293 cells along with the indicated reporter construct. Lysates were prepared 40 hours later and analyzed for CAT activity. Schematic diagrams of the reporter constructs are shown. Activity of wild-type Alx4 on the P3 reporter was assigned as 100%; values represent the average of four independent experiments. (C) Expression of Alx4 proteins in transiently transfected cells. 40 hours after transfection of 293 cells with either wild-type or Alx4 R206Q expression constructs, nuclear extracts were prepared and analyzed by Western blotting with an Alx4- specific antibody. (D) Nuclear extracts prepared from human 293 cells transiently transfected with either wild-type Alx4 or Alx4 R206Q were assessed for binding to a P3 DNA probe by gel shift assay. Lane 1, probe alone; lane 2, vector transfected cells; lane 3, wild- type Alx4 extracts; lane 4, Alx4 R206Q extracts. (E) DNA binding by Alx4 is not inhibited by Alx4 R206Q. Recombinant Alx4 homeodomain (3.2 ng) was used in gel shift assays with the P3 probe as above; increasing concentrations of Alx4 R206Q homeodomain were added into the reaction. The amount of R206Q in highest concentration (32 ng) was tenfold more than wild-type Alx4 (3.2 ng). (F) Transcriptional activation by Alx4 is not inhibited by Alx4 R206Q. Expression plasmids directing the expression of wild-type Alx4 and increasing amounts of Alx4 R206Q were co-transfected into human 293 cells along with P3 reporter construct. Lysates were prepared 40 hours later and analyzed for CAT activity. Activity of wild-type Alx4 on the P3 reporter was assigned as 100%; values represent the average of four independent experiments. cross), animals heterozygous for the Alx4lst and Alx4tm1qw alleles The strain-specific changes in phenotype make it possible to exhibit a similar degree of polydactyly. Taken together, these genetically define other genes that play a role in AP patterning data indicate that strain-specific genetic changes strongly modify in the limb bud by genetic mapping. Among the candidate Alx4-dependent polydactyly in heterozygotes. By extension, it modifiers are genes that encode proteins that are highly related seems reasonable to infer that differences in the homozygous to, and possibly functionally overlapping with, Alx4. These phenotypes, including the polydactyly, hemimelia, ventral body include other aristaless-related genes (Cart-1 and Alx3) and wall defects and alopecia are also related to strain-specific rather members of the paired-type homeodomain gene class. In this than allele-specific effects. context, the Cart-1 gene functions in a partially redundant 2720 S. Qu and others manner to suppress ZPA formation in the anterior aspect of the Bruford, E. A., Riise, R., Teague, P. W., Porter, K., Thomson, K. L., Moore, limb (Zhao et al., 1996) (S. Q., R. W., and B. deCrombrugghe, A. T., Jay, M., Warburg, M., Schinzel, A., Tommerup, N., Tornqvist, K., unpublished data). In addition, the possible interaction of Alx4 Rosenberg, T., Patton, M., Mansfield, D. C. and Wright, A. F. (1997). Linkage-Mapping in 29 Bardet-Biedl Syndrome Families Confirms Loci in with transcriptional co-activators may serve to modify Chromosomal Regions 11q13, 15q22.3-q23, and 16q21. Genomics 41, 93-99. functional activity and therefore patterning. The finding that Burglin, T.R. (1994). A comprehensive classification of genes. In mutations in the transcriptional coactivator CBP underlie the Guidebook to the Homeobox Genes (ed. D. Duboule), pp. 27-71. New York: human Rubinstein-Tabyi syndrome and its associated preaxial Oxford University Press. limb phenotype of a broadened digit 1 (Petrij et al., 1995) are Buscher, D., Bosse, B., Heymer, J. and Ruther, U. (1997). Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb bud development. consistent with a model in which limb patterning is very Mech. Dev. 62, 175-182. sensitive to alterations in gene dosage or stoichiometric Buscher, D. and Ruther, U. (1998). Expression profile of Gli family members relationships of transcriptional regulators. and Shh in normal and mutant mouse limb development. Dev Dyn. 211, 88- The demonstration that mouse Alx4 mutant alleles are 96. Campbell, G., Weaver, T. and Tomlinson, A. (1993). Axis Specification in haploinsufficient and dissociate polydactyly from the ventral the Developing Drosophila Appendage: The Role of wingless, body wall defects suggests that human dysmorphic syndromes decapentalegic, and the Homeobox Gene aristaless. Cell 74, 1113-1123. that include polydactyly, with or without abdominal wall and Chan, D. C., Laufer, E., Tabin, C. J. and Leder, P. (1995). Polydactylous craniofacial defects, could be due to mutations in human ALX4 limbs in Strong’s Luxoid mice result from ectopic polarizing activity. (McKusick, 1997). The map position of Alx4 on mouse Development 121, 1971-78. Charité, J., Graaff, W. d., Shen, S. and Deschamps, J. (1994). Ectopic chromosome 2 is contained in a region of conserved synteny expression of Hoxb-8 causes duplication of the ZPA in the forelimb and that predicts that human ALX4 and any associated syndromes homeotic transformation of axial structures. Cell 78, 589-601. would most likely map to chromosome 11p12-q12 (Eppig, Coates, M. (1994). The Origin of Vertebrate Limbs. Development 1994 1997). A search of the OMIM database did not reveal any Supplement, 169-180. human syndromes mapping to this region that display the full Coates, M. and Clack, J. (1990). Polydactyly in the earliest tetrapod limbs. Nature 347, 66-69. range of lst phenotypes. However, one mapped disorder, Daeschler, E. B. and Shubin, N. (1998). Fish with fingers? Nature 391, 133. Bardet-Biedl syndrome type I, localizes to human chromosome Dietrich, W. M., Miller, J., Steen, R., Merchant, M. A., Damron-Boles, D., 11q13 and displays postaxial polydactyly (Bruford et al., Husain, Z., Dredge, R., Daly, M. J., Ingalls, K. A., O’Connor, T. J., 1997). This is in contrast to the preaxial polydactyly observed Evans, C. A., DeAngelis, M. M., Levinson, D. M., Kruglyak, L., Goodman, N., Copeland, N. G., Jenkins, N. A., Hawkins, T. L., Stein, in lst; however, as noted above, the allelic series of mutations L., Page, D. C. and Lander, E. S. (1996). A comprehensive genetic map in human GLI3 can result in either preaxial or postaxial of the mouse genome. Nature 380, 149-152. polydactyly (Biesecker, 1997). Because the vast majority of Dominguez, M., Brunner, M., Hafen, E. and Basler, K. (1996). Sending and human polydactyly mutations have not yet been mapped, there receiving the hedgehog signal: control by the Drosophila Gli protein cubitus exists a large set of candidates for identity with Alx4 or genes interruptus. Science 272, 1621-1625. Dunn, N., Winnier, G., Hargett, L., Schrick, J., Fogo, A. and Hogan, B. acting within an Alx4-dependent pathway. (1997). Haploinsufficient Phenotypes in Bmp4 Heterozygous Null Mice and Modification by Mutations in Gli3 and Alx4. Dev. Biol. 188, 235-247. We thank V. Ko, B. Prabhakaran, S. Ray, and S. Kyin and the Eppig, J. (1997). Mouse Genome Database (MGD): Mouse Genomics Princeton Syn/Seq facility for technical assistance, F. Hughson and S. Informatics Project. URL: http://www.informatics.jax.org. Johnston for assistance with the graphics, and G. Barsh, M. Cleary, Flaherty, L., Messer, A., Russell, L. B. and Rinchik, E. M. (1992). N. Garvey, J. Moran, C. Peichel, L. Silver, S. Tilghman, and E. Chlorambucil-induced mutations in mice recovered in homozygotes. Proc. Wieschaus for comments on the manuscript. S. Q. was supported by Natl. Acad. Sci. USA 89, 2859-2863. a Hematology Training Grant from the NIH and S. C. T. was Forsthoefel, P. F. (1968). Responses to selection for plus and minus modifiers supported by NIH Training Grant T32 CA09582. J. E. was supported of some effects of Strong’s luxoid gene on the mouse skeleton. Teratology 1, 339-351. in part by an undergraduate fellowship from the Josephine de Karman Forsthoefel, P. F. (1963). The embryological development of the effects of Fellowship Trust. 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