Reciprocal Mouse and Human Limb Phenotypes Caused by Gain- and Loss-Of-Function Mutations Affecting Lmbr1

Reciprocal Mouse and Human Limb Phenotypes Caused by Gain- and Loss-of-Function Mutations Affecting Lmbr1

Richard M. Clark,* Paul C. Marker,*,1 Erich Roessler,† Amalia Dutra,‡ John C. Schimenti,§ Maximilian Muenke† and David M. Kingsley*,** *Department of Developmental Biology, Stanford University, Stanford, California 94305-5327, †Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-1852, ‡Cytogenetic and Confocal Microscopy Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, §The Jackson Laboratory, Bar Harbor, Maine 04609 and **Howard Hughes Medical Institute, Stanford University, Stanford, California 94305-5327 Manuscript received March 20, 2001 Accepted for publication June 1, 2001

ABSTRACT The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently deﬁned for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may underlie preaxial polydactyly in both mice and humans. We have used deletion chromosomes to demonstrate that the dominant mouse and human limb defects arise from gain-of-function mutations and not from haploinsufﬁciency. Furthermore, we created a loss-of-function mutation in the mouse Lmbr1 gene that causes digit number reduction (oligodactyly) on its own and in trans to a deletion chromosome. The loss of digits that we observed in mice with reduced Lmbr1 activity is in contrast to the gain of digits observed in Hx mice and human polydactyly patients. Our results suggest that the Lmbr1 gene is required for limb formation and that reciprocal changes in levels of Lmbr1 activity can lead to either increases or decreases in the number of digits in the vertebrate limb.

ERTEBRATE limb malformations that cause al. 1994; Tsukurov et al. 1994; Vargas et al. 1995; Zgur- V changes in digit number are relatively common and icas et al. 1999). Despite this phenotypic variation, all include polydactyly (extra digits) and oligodactyly (too these mutations are dominantly inherited and highly few digits). Of these defects, preaxial polydactyly is the penetrant, and recent mapping studies have localized most common in humans and includes forms of thumb many of the mutations to the same 7q36 region (Heu- duplications, triphalangeal thumb, and index ﬁnger du- tink et al. 1994; Tsukurov et al. 1994; Hing et al. 1995; plications on the anterior (preaxial) side of the limb Radhakrishna et al. 1996; Vargas et al. 1998; Zguricas (Temtamy and McKusick 1978). While preaxial poly- et al. 1999; Dobbs et al. 2000). Therefore, a major lo- dactyly is frequently associated with additional defects at cus for triphalangeal thumb-polysyndactyly syndrome sites outside the limbs, a subset of families with inherited (TPTPS; OMIM 190605) at 7q36 is responsible for al- preaxial polydactyly have limb-speciﬁc defects (Zguri- most all dominant preaxial polydactylies and polysyn- cas et al. 1999). Limbs from patients harboring these dactylies with defects restricted to the limbs. preaxial polydactyly mutations typically present with re- In the mouse, the dominant Hemimelic extra toes (Hx) placement of the thumb with one or more triphalangeal and Hammertoe (Hm) limb mutations are thought to elements (Canun et al. 1984; Cordeiro et al. 1986; Heu- be analogous to the human TPTPS mutations at 7q36 tink et al. 1994; Tsukurov et al. 1994; Hing et al. 1995; (Heutink et al. 1994; Tsukurov et al. 1994), and both Vargas et al. 1995; Radhakrishna et al. 1996; Zguricas map to a mouse chromosome region homologous to et al. 1999; Dobbs et al. 2000). Many of these polydactyly 7q36 (Clark et al. 2000). Hx mice have limb defects mutations are also associated with additional distal limb that include preaxial polydactyly and radial and tibial defects that include soft tissue fusions (syndactyly) of hemimelia (Knudsen and Kochhar 1981; Masuya et adjacent digits and/or radial or tibial dysplasia/aplasia al. 1995) that closely resemble the human limb pheno- (Canun et al. 1984; Cordeiro et al. 1986; Heutink et types. Hm mice do not have changes in digit number but have highly penetrant webbing between digits (Green 1964) similar to that observed in some polysyndactyly Corresponding author: David M. Kingsley, Howard Hughes Medical mutations that have been mapped to 7q36 (Tsukurov Institute, Stanford University, Beckman Ctr., B300, 279 Campus Dr., Stanford, CA 94305-5327. E-mail: [email protected]. et al. 1994). In crosses segregating Hx and Hm only 1 Present address: Department of Anatomy, University of California, a single recombination was observed in 3664 meioses San Francisco, CA 94143-0452. (Sweet 1982). This extremely tight linkage suggests

Genetics 159: 715–726 (October 2001) 716 R. M. Clark et al. that the mouse mutations affect neighboring genes or that amplified the expected size fragments from commercial alternatively may be different alleles of the same gene genomic DNA (Clontech, Palo Alto, CA). Following sequence verification of test amplicons, two primer pairs [HxF1b (5Ј-acc with the observed recombination arising from an intra- tgttccaacacggctcgc-3Ј) and HxR1 (5Ј-actcccgcacttggctgtgg-3Ј) genic crossover. and the nested pair HxF1b (above) and HxR1b (5Ј-acacct kb re- cgtcctgcccttcc-3Ј)] were used by the Physical Mapping Core-450ف Recently, Heus et al. (1999) defined an gion on 7q36 that contains dominant polydactyly muta- (National Human Genome Research Institute/National Insti- tions and identified a small set of genes that are con- tutes of Health) to screen a human bacterial artificial chromosome (BAC) library (Incyte Genomics, Palo Alto, CA), re- tained within the TPTPS critical region. In a parallel sulting in the identification of clone address 575h20. Direct study in the mouse, Clark et al. (2000) defined an sequencing of this clone as well as Southern blot hybridization kb interval for the mouse Hm and Hx mutations experiments verified that it contains exon 1 and 5Ј flanking-450ف and identified several genes within this critical region. sequences of LMBR1 (data not shown). The mouse and human candidate genes are ortholo- Fluorescent in situ hybridization analysis: Slides with chromosome metaphase spreads were incubated for 1 hr at 37Њ in gous, confirming previous conjecture that the human 2ϫ SSC (0.3 m NaCl and 0.3 m sodium citrate) and then and mouse phenotypes arise by defects in similar genes dehydrated sequentially in 70, 80, and 90% ethanol. Chromo- (Clark et al. 2000). However, extensive mutational anal- some DNA was denatured in 70% formamide, 2ϫ SSC for 2 ysis in both human and mouse has not identified lesions min at 72Њ followed by dehydration in ethanol washes of 70, in the coding sequences of any candidate genes (Heus 80, 90, and 100%. Fluorescent in situ hybridization (FISH) was performed with probes labeled with spectrum orange- et al. 1999; Clark et al. 2000). dUTP (Vysis, Downers Grove, IL), essentially as described pre- The absence of coding region mutations raises the viously (Pinkel et al. 1986; Lichter et al. 1988). On each possibility that the dominant mouse and human muta- slide, 100 ng of labeled DNA was applied. Nonunique and tions are regulatory alleles that disrupt expression of a nonspecific DNA hybridization was blocked by preannealing gene or genes in the interval. Clark et al. (2000) showed the probe with a 10-fold excess of human Cot1 DNA. Labeled and blocking DNAs were denatured at 75Њ for 10 min and that one of the genes within the mouse critical region, then preannealed at 37Њ for 15 min. The hybridization mixture called Limb region 1 (Lmbr1), is normally expressed in contained labeled DNA in 10 ml of 50% formamide, 2ϫ SSC, developing limbs at the times that both the Hx and Hm and 10% dextran sulfate at pH 7.0. Slides were hybridized phenotypes arise. More importantly, Lmbr1 was dynami- overnight at 37Њ. Post-hybridization washes were performed Њ ϫ cally misexpressed in Hx limbs at the exact time that at 45 as follows: (1) 50% formamide, 2 SSC, 20 min; (2) 1ϫ SSC, 10 min; and (3) 0.1ϫ SSC, 10 min. Slides were coun- Hx limb morphology first appears. Expression changes terstained with propidium iodide-Antifade (Intergen, Pur- included a possible overexpression of the gene followed chase, NY) or 250 ng/ml 4Ј,6-diamidino-2-phenylindole (Boeh- by a dramatic decrease in Lmbr1 transcript levels at later ringer Mannheim, Indianapolis) with Antifade. stages (Clark et al. 2000). The human Lmbr1 ortholog Design of Lmbr1 targeting vector and homologous recombi- Ј (LMBR1) is contained entirely within the human critical nation: The 5 end of the Lmbr1 gene is present on mouse region for TPTPS mutations at 7q36 (Heus et al. 1999), BAC clone 136E36 from the 129 strain CITB mouse BAC II library (Research Genetics, Huntsville, AL; Clark et al. 2000, and the striking correlation between the appearance of and our unpublished data). DNA from this BAC was digested defects in Hx mice and Lmbr1 misregulation suggests with restriction enzymes and subcloned into a plasmid vector, that the mouse and human limb mutations may be al- and an 8.3-kb BamHI fragment harboring the exon that con- leles of the Lmbr1 gene (Clark et al. 2000). tains the start site of the Lmbr1 open reading frame was isolated Here we use deletion chromosomes in both mice and by hybridization with Lmbr1 sequences. A 1.1-kb MluI/KpnI fragment that contains this exon (see results) was replaced humans to show that the dominant mouse and human by a PGKneo-positive selection cassette by cloning sequences limb phenotypes are likely to arise by gain-of-function flanking the 1.1-kb fragment into the pPNT vector. The pPNT mechanisms. In addition, we created a loss-of-function vector contains the herpes thymidine kinase gene for negative allele of the Lmbr1 gene to test whether this gene is selection. required for normal limb development. Mice with re- Targeting of the endogenous Lmbr1 locus was performed in R1 embryonic stem (ES) cells (kindly provided by Janet duced Lmbr1 function show distal limb reductions in- Rossant) as described previously (Joyner 1993). Construct cluding oligodactyly. These phenotypes are reciprocal linearized with NotI was electroporated into ES cells and posi- to those caused by the classical dominant mutations. tive selection was performed using G418 while Gancyclovir The complementary defects suggest that levels of ex- was used to select against nonhomologous integration events. pression of the Lmbr1 gene play a key role in controlling Cells were grown on G418-resistant irradiated mouse embryonic fibroblasts. ES cell colonies with targeted integrations the development of skeletal structures in the vertebrate were detected by nested PCR amplification of a 3.1-kb junction limb. fragment created by homologous recombination between the 3Ј arm of the replacement construct and the endogenous Lmbr1 locus. Primers used for amplification were neo1 (5Ј- Ј Ј MATERIALS AND METHODS gcagcctctgttccacataca-3 ) and LK1 (5 -tgagggagccagaggagtca- 3Ј) for primary PCR and neo2 (5Ј-gccaagttctaattccatcagaa-3Ј) Isolation of human genomic bacterial artificial chromosome and LK2 (5Ј-aaaatacaagaaaacctacagaatc-3Ј) for secondary PCR. clones: Primers were prepared on the basis of partial human Amplifications were performed with ampliTaq (Applied Bio- expressed sequence tag (EST) sequences predicted to corre- systems, Foster City, CA) with cycle conditions of 94Њ (3 min); spond to the 5Ј untranslated region and first exon of LMBR1 24 cycles of 94Њ (30 sec), 59Њ (1 min), 72Њ (4 min); followed Lmbr1 and Reciprocal Limb Phenotypes 717 by 6 cycles of 94Њ (30 sec), 59Њ (1 min), and 72Њ (5 min); Hm limb phenotypes. Where necessary, inheritance of the followed by 72Њ (15 min). Homologous recombinants were Lmbr1ATG allele was determined by typing with primers N1F and verified by additional PCR with primers LK3 (5Ј-ggtaggggttatt N1R. Hdhdf4J/ϩ males were also crossed to Lmbr1ATG/Lmbr1ATG ggtacagactt-3Ј) and neo3 (5Ј-gcctcccctacccggtagaatt-3Ј) that females. Deletion progeny from this cross were identified by amplify a junction fragment created by homologous recombi- typing animals with primers ATGF and ATGR. The Hdhdf4J nation between the 5Ј arm of the targeting construct and the deletion includes Lmbr1 exon 1, and in Lmbr1ATG/Hdhdf4J mice endogenous Lmbr1 locus. PCR with primers LK3 and neo3 the exon 1 product amplified by ATGF and ATGR is not was performed using the expand long template PCR system present (see results). (Hoffmann La Roche, Basel, Switzerland) with cycle conditions Skeletal preparations: Alizarian red-stained skeletons were of 94Њ (2 min); 10 cycles of 94Њ (10 sec), 65Њ (30 sec), 68Њ (8 prepared as previously described (Green 1952) from weaning min); followed by 25 cycles of 94Њ (10 sec), 65Њ (30 sec), 68Њ age or adult mice. (8 min with an additional 20-sec/cycle); followed by 68Њ (10 min). We refer to the allele created by targeting as Lmbr1ATG. Generation and typing of Lmbr1ATG mutant mice: Two inde- pendently selected ES cell clones were injected into C57BL/ RESULTS 6J host blastocysts, and chimeric animals (agouti coat color) The Hx and Hm mutations are gain of function: While were crossed to B6D2F1/J animals (The Jackson Laboratory). Germline transmission was determined by coat color, and the phenotypes and mode of inheritance of the mouse heterozygous progeny were identified. Homozygous animals Hx and Hm mutations are described in detail (Green were generated by intercrossing heterozygotes, and pheno- 1964; Knudsen and Kochhar 1981; Zakeri et al. 1994; typic analysis of Lmbr1ATG/ϩ and Lmbr1ATG/Lmbr1ATG animals Masuya et al. 1995), it has not been clear whether these ϫ as well as wild-type controls was performed on the 129SV/J dominant mutations produce limb abnormalities by B6D2F1/J mixed genetic background. Initially, heterozygous animals were typed with primers that were used to identify gain-of-function or loss-of-function mechanisms. A dele- targeted ES clones (primers neo1, LK1, neo2, and LK2; see tion of proximal mouse chromosome 5 (Hdhdf4J) that above). Afterward, progeny were typed with duplexed primer does not produce obvious webbing or gross polydactyly pairs that allowed all genotypes to be distinguished in single was previously reported (Schimenti et al. 2000). The PCR reactions. One primer set amplifies a 174-bp fragment Hx and Hm mutations were previously mapped between of Lmbr1 exon 1 that is deleted by the targeted mutation [primers ATGF (5Ј-tcttgaaccgcttctccctgag-3Ј) and ATGR (5Ј- the Shh and Il6 loci on proximal chromosome 5 (Mar- cccttccatcctcctttcatacc-3Ј)], and another pair amplifies a 268- tin et al. 1990; Robert et al. 1994; Marigo et al. 1995; bp fragment from the neo resistance cassette inserted into the Clark et al. 2000) in a region likely to be included locus by targeting [primers N1F (5Ј-acagacaatcggctgctctgatg- in the Hdhdf4J deletion. PCR typing confirmed that this Ј Ј Ј 3 ) and N1R (5 -gatggatactttctcggcaggag-3 )]. PCR amplifica- deletion removes Lmbr1 coding sequences (see materi- tion conditions were 94Њ (2 min); 30 cycles of 94Њ (30 sec), 63Њ (1 min), 72Њ (30 sec); followed by 72Њ (10 min). als and methods and Figure 3F), demonstrating that Northern blot analysis with wild-type and mutant RNA sam- Hdhdf4J/ϩ mice carry a single copy of the Lmbr1 candi- ples: Total brain RNA from wild-type and mutant mice was date region. prepared using TRIzol (Invitrogen, Carlsbad, CA) according We examined limbs of Hdhdf4J/ϩ mice to test whether to the manufacturer’s instructions. Messenger RNA was iso- deletion of the candidate interval produces limb pheno- lated from total RNA using oligo(T) cellulose (FastTrack 2.0 ϩ ϩ kit; Invitrogen). For Northern blot analysis, 2 ␮g of poly(A) types typical of those observed in either Hm/ or Hx/ RNA per lane was loaded and separated on 1% agarose/1.5% mice. No webbing was seen between digits, and Alizarin formaldehyde gels by electrophoresis. RNA was then blotted red-stained skeletal preparations did not show extra to Hybond Nϩ membrane (Amersham, Arlington Heights, skeletal elements characteristic of Hx/ϩ mice, sug- 32 IL). Blots were probed with a P-labeled Lmbr1 cDNA probe. gesting that the classical Hm and Hx phenotypes arise This probe contains the open reading frame (ORF) of the Lmbr1 gene that encodes LMBR1L 3Ј of the sequence for exon by a gain-of-function mechanism rather than from loss- 1. Blots were scanned on a model 425 E phosphor imager of-function mutations in a gene in the interval (Table (Molecular Dynamics, Sunnyvale, CA) and relative expression 1 and Figure 1). Although Hdhdf4J/ϩ mice do not have levels were calculated using a Gapdh probe to control for limb defects that resemble those in Hx and Hm mice, loading differences. df4J minor coalitions of distal wrist bones were observed in Hdh mice, crosses, and typing: A male mouse carrying df4J ϩ the proximal chromosome 5 deficiency Hdhdf4J (Schimenti et 25% of wrists in Hdh / animals and included fusions al. 2000) in trans to a Mus mus castaneous chromosome of the central to either distal carpals 2 or 3 (dc2 and (C57BL/6J ϫ 129/Jae ϫ M. mus castaneous mixed-strain back- dc3, respectively; Table 2). Animals carrying the Hdhdf4J ground) was crossed to either Hm/Hm (C3HeB/FeJLe strain) deletion were also smaller than wild-type mice as has or wild-type (B10.D2/nSn strain) females. To determine in- been observed for several other chromosomal deletions. heritance of the deletion, DNAs from progeny animals were typed with primers that amplify the microsatellite locus Dominant preaxial polydactyly mutations that map to D5Mit148 that is predicted to be removed by the Hdhdf4J defi- 7q36 are gain of function: The critical region for human ciency. This locus is polymorphic between M. mus castaneous TPTPS mutations at 7q36 is homologous to the critical and C3HeB/FeJLe and B10.D2/nSn strain DNA (data not region for the mouse Hm and Hx mutations and con- shown), and progeny that inherited the deficiency that lacked tains the human ortholog of the mouse Lmbr1 gene the M. mus castaneous allele were identified. Hm/Hdhdf4J male progeny were crossed to wild-type B6D2F1/J females or to (LMBR1; Heus et al. 1999; Clark et al. 2000). To deter- Lmbr1ATG/ϩ or Lmbr1ATG/Lmbr1ATG females and progeny that mine whether the dominant human limb mutations inherited the deletion were distinguished by the absence of arise by a gain-of-function mechanism, we used FISH 718 R. M. Clark et al.

TABLE 1 Frequency of defects affecting the digits

Genotype ϩ/ϩϩ/Lmbr1ATG Lmbr1ATG/Lmbr1ATG ϩ/Hdhdf4J Lmbr1ATG/Hdhdf4J Total fore- and hindlimbs examined (each)a 28 24 36b 20 38 Soft tissue syndactylies in forelimbs (%) 0 0 2 (6) 0 14 (37) Soft tissue syndactylies in hindlimbs (%) 0 0 1 (3) 0 6 (16) Forelimbs with reduced digit no. (%)c 0 0 1 (3) 0 23 (61) Four digits/limb 0 0 1 (3) 0 16 (42) Three digits/limb 0 0 0 0 7 (18) Hindlimbs with reduced digit no. (%) 0 0 1 (3) 0 19 (50) Four digits/limb 0 0 1 (3) 0 19 (50) Phalangeal length or no. reductions in forelimbs (%) 0 0 3 (8)d 0 11 (29)e a All limbs examined for both soft tissue and skeletal defects. b Two of 128 Lmbr1ATG homozygous animals (Ͻ1%) had obvious limb defects (syndactyly and/or oligodactyly) that were detected as live mice. Limbs from these mice were prepared as skeletons and are included in this data set along with all other homozygotes that were also prepared as skeletons. c Digit number reduction refers to instances of lateral digit fusions that produce a single distal-most phalanx (bony syndactylism) or to cases where one or more digits are completely absent (oligodactyly). d In each case the most posterior digit (digit V) was affected. In one instance the second phalanx was reduced in size, while in the other two affected digits a single bone replaced the first and second phalanges. e In each of the affected limbs the most posterior digit was affected, and in one limb a second digit was also affected. In 9 of the 12 affected digits the first and second phalanges were replaced by a single bone. In the remaining affected digits the second phalanx was grossly reduced in length. analysis to map the region surrounding the LMBR1 in- gene (Roessler et al. 1997). None of these patients terval in human patients with deletions or translocations have limb defects that include preaxial polydactyly or in the 7q36 region (Roessler et al. 1997 and references polysyndactyly (Roessler et al. 1997). Therefore, the therein). A 135-kb human BAC clone that contains exon entire region containing the LMBR1 critical area can 1 of the LMBR1 gene was isolated. This BAC produces be deleted without producing the TPTPS characteristics two chromosome 7q36-specific hybridization signals in of the dominant limb mutations that were indepen- FISH analysis of control human cells (Figure 2A). FISH dently mapped to 7q36 (Figure 2E). These data suggest analysis of cells from a patient with a t(7;17) transloca- that, like the mouse Hx and Hm mutations, the domi- tion (T4) shows two chromosome 7 LMBR1 hybridiza- nant human limb mutations that map to 7q36 are likely tion signals [7 and der(7), Figure 2B], indicating that to act by a gain-of-function rather than a loss-of-function LMBR1 is proximal to the T4 translocation breakpoint. mechanism. FISH analysis of cells harboring a t(7;9) translocation Generation of a loss-of-function allele of the Lmbr1 (T1) shows that one LMBR1 hybridization signal is asso- gene: The location of the Lmbr1 gene in the critical ciated with the intact copy of chromosome 7 while the interval for both the mouse and human limb mutations other is on the chromosome 9 derivative [7 and der(9), and the altered expression of this gene in Hx mice Figure 2C], indicating that LMBR1 is distal to the T1 suggest that regulatory mutations in Lmbr1 may be re- translocation breakpoint. Only a single chromosome 7 sponsible for the dominant mouse and human limb hybridization signal was seen in cells from a patient with defects. To examine the role of this gene during normal a de novo 7q36 deletion (Figure 2D). These data suggest limb development, we created a loss-of-function muta- that the LMBR1 BAC maps precisely between the T1 tion in the Lmbr1 gene using embryonic stem cell tar- and T4 translocations in a region completely covered geting. by the 7q36 deletion (Figure 2E). Physical estimates The Lmbr1 gene encodes a highly conserved product based on assembly of genomic clones in the 7q36 region of 490 amino acids (LMBR1L) as well as a smaller prod- suggest that the T1 and T4 translocations are located uct of 32 amino acids (LMBR1S) produced by an alter- .(and 335 kb distal to the SHH gene (Roessler et natively spliced Lmbr1 transcript (Clark et al. 2000 265ف al. 1997). The present Lmbr1 mapping results, combined The first 22 amino acids of both proteins are identical. with the earlier cytogenetic data, suggest that as many To create an allele of Lmbr1, we subcloned genomic as 32 unrelated patients with cytogenetic deletions in DNA containing the 5Ј-most coding exon of the Lmbr1 the 7q36 region may also lack one copy of the LMBR1 gene. Sequence analysis revealed that this exon encodes Lmbr1 and Reciprocal Limb Phenotypes 719

tion cassette (Figure 3, A–C). The deleted fragment contains 365 bp 5Ј of the predicted translational start site of exon 1 and 696 bp 3Ј of the exon 1 splice donor site. The targeted mutation therefore deletes the first known exon of the Lmbr1 gene that contains the translational start site as well as coding sequence for both known Lmbr1 products. The targeted mutation may also remove the endogenous Lmbr1 transcriptional start site. Two independent ES cell clones transmitted the Lmbr1 mutation (Lmbr1ATG) through the germline. Mice heterozygous for the targeted mutation were inter- crossed to produce Lmbr1ATG/Lmbr1ATG mice, and PCR analysis confirmed that the coding exon containing the translational start site of both predicted Lmbr1 products was completely missing in DNA from Lmbr1ATG homozygotes (Figure 3D). Homozygous mice were present at normal Mendelian ratios (36 ϩ/ϩ;91Lmbr1ATG/ϩ;44 Lmbr1ATG/Lmbr1ATG; P ϭ 0.20, chi-square test). Both male and female Lmbr1ATG homozygous mice are fertile and we have been able to maintain the Lmbr1ATG allele by intercrossing homozygotes. We also examined a variety of tissues from Lmbr1ATG homozygotes by histology (including liver, kidney, spleen, testis, epididymus, and seminal vesicle) and did not detect significant abnormalities when compared to wild-type controls (data not shown). To assess whether the targeted mutation created a null allele of the Lmbr1 gene, we probed Northern blots ATG ATG Figure 1.—Mice hemizygous for the Hx-Hm interval do not of wild-type and Lmbr1 /Lmbr1 adult brain poly(A) have classical Hx or Hm limb phenotypes. For A–D, distal is RNA with a Lmbr1 cDNA probe. The normal 3- and 5-kb up, proximal is down, anterior is left, and posterior is right. messages that contain the first exon of the Lmbr1 gene Digit number is indicated by Roman numerals. (A and B) (Clark et al. 2000) were greatly reduced or undetect- ϩ Ventral views of adult forefeet showing soft tissues. In Hm/ able in Lmbr1ATG homozygotes (Figure 3E). However, limbs (A) digits II–V are connected to each other by lateral soft tissue fusions (asterisks) as well as to the ventral surface long exposures of Northern blots showed multiple tran- of the limb, and digits are flexed ventrally toward the viewer scripts in brain RNA from homozygotes (Figure 3E). that of %7ف the “hammertoe” phenotype). In contrast, no soft tissue webs These transcripts were present at levels of) are observed in Hdhdf4J/ϩ limbs that lack one copy of the wild-type Lmbr1 messages in brain. These molecular data region that contains the Hm-Hx interval (B), and digits from suggest that the targeted mutation reduced Lmbr1 func- these mice are fully extended (compare to Figure 4A). (C and D) Dorsal views of cleared forelimbs stained with alizarin red tion but that the mutation may not have created a null to identify skeletal elements. Hx/ϩ limbs (C) have preaxial allele (see discussion). polydactyly in which digit I, which normally has two small Lmbr1ATG homozygous mice have low incidences of phalanges (see Figure 4D), is replaced by extra triphalangeal limb defects: As expected from our studies of the chro- df4J ϩ digits (asterisks). In contrast, limbs from Hdh / mice (D) mosome 5 deletion mice, the loss-of-function mutation have normal digit morphology. These results demonstrate that the classical webbing and polydactyly phenotypes result from in Lmbr1 did not produce a phenocopy of either the gain-of-function mechanisms and not from haploinsufficiency. Hx or Hm mutations. We did, however, detect a very low incidence of limb abnormalities in Lmbr1ATG homozygous animals (Table 1). These phenotypes presented the 22 amino acids that are common to the N termini as digit loss or reduction rather than the gain-of-digit of both the LMBR1L and LMBR1S proteins (Clark et al. number typically seen in Hx animals. One mouse ap- 2000). Genomic sequence upstream of the translational peared to have only four digits on its right forefoot, start site for Lmbr1 products is contiguous with sequence while a second homozygous mouse had only four digits of Lmbr1 5Ј rapid amplification of cDNA ends (RACE) on a hindfoot. The latter mouse also had mild soft tissue products for 166 bp, at which point RACE products end syndactylies between central digits on its other three (our unpublished data). This point may represent the limbs (Table 1 and Figure 4B). Skeletal preparation transcriptional start site for the Lmbr1 gene, and we revealed that reduction of digit number in the affected refer to the exon we isolated as Lmbr1 exon 1. We used Lmbr1ATG/Lmbr1ATG forelimb arose from loss of the meta- homologous recombination in ES cells to replace a 1.1- carpal and phalanges of a single digit (Figure 4E). The kb fragment that contains exon 1 with a PGKneo selec- morphology of the four remaining digits suggested that 720 R. M. Clark et al.

TABLE 2 Frequency of wrist and ankle abnormalities in mice of different genotypes

Genotype ϩ/ϩϩ/Lmbr1ATG Lmbr1ATG/Lmbr1ATG ϩ/Hdhdf4J Lmbr1ATG/Hdhdf4J Total wrists examined 28 24 36 20 38 Wrists with distal coalitions (%)a 9 (32) 2 (8) 14 (39) 5 (25) 38 (100) Distal defects dc2-c fusion (only) 9 (32) 2 (8) 0 3 (15) 0 c-dc3 fusion (only) 0 0 14 (39) 2 (10) 0 More severe fusions/reductions 0 0 0 0 38 (100) Wrists with affected proximal structures (%)b 0 0 1 (3) 0 36 (95) Total ankles examined 28 24 36 20 38 Ankles with distal coalitions (%)c 8 (29) 6 (25) 2 (6) 0 33 (87) Distal defects dt2, c, and/or dt3 fused (only) 8 (29) 6 (25) 1 (3) 0 0 More severe fusions/reductions 0 0 1 (3) 0 33 (87) a dc2, dc3, and “c” are distal carpals 2 and 3 and the central, respectively. “More severe fusions/reductions” refers to fusions that may involve dc2, dc3, and the central but also distal carpals 1 and 4/5 as well as reduced size of remaining elements. b Refers to fusions involving the radiale, ulnar, and/or pisiforme and that were frequently accompanied by reduction in size of remaining fused elements. c dt2, dt3, and “c” are distal tarsals 2 and 3 and the central, respectively. “More severe fusions/reductions” refers to fusions that may involve dt2, dt3, and the central but also dt4/5 and that were frequently accompanied by reduced size of remaining elements.

the missing digit is digit V (compare Figure 4E to 4D). ure 4M). However, the number of distal wrist bones has A small posterior element in this limb may be a rudiment been observed to vary depending on strain background of the fifth metacarpal (see arrow in Figure 4E). In (Davis and Capecchi 1994). In wild-type control mice contrast, in the affected hindlimb of the homozygous from our cross, we observed a high frequency (32%) of mutant animal, reduction in digit number was caused forelimbs in which dc2 was partially or completely fused by fusion of digits III and IV at the level of the phalanges to the central (Table 2). In Lmbr1ATG heterozygous mice, to produce a single digit distally that ended in a single we observed a lower frequency (8%) of distal wrist coali- third phalanx (Table 1 and Figure 4H). tions that involved fusion of the central to dc2 (Table Further examination of homozygous Lmbr1ATG ani- 2). In contrast, in Lmbr1ATG/Lmbr1ATG wrists we observed mals revealed low incidences of additional digit defects a high frequency (39%) of distal coalitions that included that included reduction in phalange length or number only fusions between the central and dc3 (Table 2 and that were never observed in wild-type or heterozygous Figure 4N). While no wild-type or heterozygous wrists control limbs (Table 1). On both fore- and hindlimbs that we examined had defects in proximal carpal bones, digit I normally has two phalanges (P1 and P2, both of in 1 of 36 homozygous wrists the ulnare and pisiforme which are very small in forelimbs), while digits II–V have were partially fused (Table 2). three phalanges each (P1, P2, and P3; Figure 4, D, G, We also observed coalitions of anklebones in wild- and J). While the fifth digits of most Lmbr1ATG/Lmbr1ATG type and heterozygous and homozygous Lmbr1ATG mice forelimbs had three phalanges of normal relative (Table 2). Normally, six bones comprise the distal ankle lengths, one forelimb had a fifth digit in which the size [distal tarsals 1 (dt1), 2 (dt2), 3 (dt3), and 4/5 (dt4/ of P2 was greatly reduced (Table 1 and data not shown). 5); the pisiforme; and the central] while the talus and In two other limbs, the P1 and P2 elements were re- the calcaneus comprise the proximal ankle. While most placed by a single element (Table 1 and Figure 4K). No ankles of each genotype that we examined had the ca- Lmbr1ATG/Lmbr1ATG hindlimbs had obvious reductions in nonical ankle organization, we observed coalitions of the length of phalanges or any reductions in phalangeal distal anklebones in wild-type and Lmbr1ATG/ϩ mice that number (data not shown). included dt2, dt3, and/or the central (Table 2). In We also examined the organization of the wrists and Lmbr1ATG homozygous ankles we observed distal ankle- ankles in wild-type, heterozygous, and homozygous bone fusions that included coalitions involving dc2, dc3, mice. In general, five bones comprise the distal wrist dc4/5, and/or the central (Table 2). We did not observe [distal carpals 1 (dc1), 2 (dc2), 3 (dc3), and 4/5 (dc4/ any defects in the proximal anklebones of wild-type or 5); and the central], while three bones comprise the Lmbr1ATG heterozygous or homozygous mice (data not proximal wrist (the radiale, ulnare, and pisiforme; Fig- shown). Lmbr1 and Reciprocal Limb Phenotypes 721

Defects in Lmbr1ATG mice appear to be confined to Hdhdf4J mice were also produced by crossing Hdhdf4J/Hm the wrist, ankle, and footplate regions, with no obvious mice to Lmbr1ATG/ϩ animals (see materials and meth- defects detected in more proximal limb structures or ods). As with Hdhdf4J/ϩ mice, Lmbr1ATG/Hdhdf4J mice were structures outside the limb. typically smaller than littermates (either Lmbr1ATG/ϩ or Lmbr1ATG/Hdhdf4J mice have severe distal limb defects: Lmbr1ATG/Hm mice; data not shown). Whether the ob- To further test the role of Lmbr1 in development, we served decrease in survival of Lmbr1ATG/Hdhdf4J relative generated mice trans-heterozygous for the Lmbr1ATG al- to Hdhdf4J/ϩ mice results from a specific effect of Lmbr1 lele and the Hdhdf4J deletion that removes the region or rather from differences in the interaction of the containing the Lmbr1 locus. If residual Lmbr1 function deletion with different stain backgrounds used in our is present in Lmbr1ATG homozygotes, we reasoned that crosses is not known. Lmbr1ATG/Hdhdf4J mice should have an even greater re- Limbs from Lmbr1ATG/Hdhdf4J mice showed dramatic duction in Lmbr1 activity and therefore may display digit defects that include soft tissue webbing (Table 1 more severe phenotypes. Most Lmbr1ATG/Hdhdf4J mice and Figure 4C), reductions in the number of digits were produced by crossing either Hdhdf4J/ϩ or Hdhdf4J/ (Table 1 and Figure 4, C, F, and I), and reductions in Hm mice to Lmbr1ATG/Lmbr1ATG animals. In these crosses, the number or length of phalanges in digits (Table 1 progeny that inherited the Hdhdf4J deletion (Lmbr1ATG/ and Figure 4L). These defects were more severe and Hdhdf4J mice) were recovered at less than the expected occurred at much greater frequencies than those in frequency of 50% at weaning (40 Lmbr1ATG/ϩ or Lmbr1ATG/Lmbr1ATG mice. For example, 55% of all Lmbr1ATG/Hm progeny; 16 Lmbr1ATG/Hdhdf4J progeny; P ϭ Lmbr1ATG/Hdhdf4J limbs had fewer than five digits, com- 0.001, chi-square test). A small number of Lmbr1ATG/ pared to Ͻ1% in Lmbr1ATG homozygotes (Table 1). The digit reduction was also more severe in Lmbr1ATG/Hdhdf4J animals, with one-third of the affected forelimbs showing only three remaining digits (Table 1 and Figure 4F). The reduction of digits appeared to be restricted to central or posterior digits on the basis of morphological criteria (compare Figure 4F and 4I to 4D and 4G). Lmbr1ATG/Hdhdf4J mice also had high incidences of wrist and ankle defects (Table 2). While we observed minor distal wrist bone coalitions restricted to dc2, dc3, and/or the central in Lmbr1ATG homozygous mice and control mice, we observed dramatic coalitions/reductions of distal wrist bones that involved dc2, dc3, and/or

Figure 2.—Dominant TPTPS limb mutations at 7q36 are gain of function. (A–D) FISH analysis to chromosome metaphase spreads of cells from patients with 7q36 rearrangements previously described by Roessler et al. (1997). In all cases, a BAC clone probe that contains exon 1 of the LMBR1 gene was used for hybridization. Hybridization signal is red, and chromosome 7 and derivatives are as labeled (arrows). In normal cells (A) and in cells harboring the T4 (B) or the T1 (C) translocations the LMBR1 probe recognizes two chromosome 7 signals on either normal or translocated 7q36 segments as indicated. In contrast, in cells from a patient with a de novo 7q36 deletion (patient 30, Roessler et al. 1997) (D), only a single chromosome 7-specific hybridization signal was observed. These results localize a portion of the LMBR1 gene between the T1 and T4 breakpoints in a region that was previously shown by Roessler et al. (1997) to be distal to the SHH locus on 7q36 (E). The LMBR1 gene, which is within the TPTPS critical interval defined by Heus et al. (1999), is contained within the 7q36 deletion that also removes the SHH locus. None of the rearrangements cause TPTPS phenotypes, although patients with the deletion and T1 translocation have holoprosencephaly (HPE) phenotypes resembling those that result from haploinsufficiency for the SHH gene (Belloni et al. 1996; Roessler et al. 1997; see discussion). These results demonstrate that TPTPS phenotypes are unlikely to arise from haploinsufficiency. 722 R. M. Clark et al.

Figure 3.—Generation, typing, and characterization of animals harboring a targeted mutation in the Lmbr1 gene. (A) Genomic locus and replacement construct are shown. B, M, and K denote sites recognized by BamHI, MluI, and KpnI, respectively. A 1.1-kb MluI- KpnI fragment including the exon that contains the putative translational start site of the Lmbr1 gene (ATG) was replaced by a PGKneo selection cassette by homologous recombination in ES cells (dashed lines). Orientations of the Lmbr1 and neo genes are as indicated (arrows). Primer sites used to detect homologous recombination or for subsequent typing experiments are indicated (split arrows). (B) Primers LK3 and neo3 amplify a 4.2-kb fragment generated by homologous recombination between the 5Ј arm of the replacement construct and the Lmbr1 genomic locus. (C) Primary PCR (1Њ PCR) with primers neo1 and LK1 and secondary PCR (2Њ PCR) with primers neo2 and LK2 amplify a 3.1-kb fragment generated by homologous recombination between the 3Ј arm of the replacement construct and the endogenous Lmbr1 locus. (D) Genotyping by duplexed PCR with primer pairs that amplify (1) a 174-bp fragment (primers ATGF and ATGR) that is deleted from the Lmbr1 locus by homologous recombination and (2) a 268-bp fragment from the neo gene that was inserted into the Lmbr1 locus by targeting (primers N1F and N1R). (E) Northern blot analysis of poly(A) brain RNA prepared from wild-type and Lmbr1ATG/ Lmbr1ATG mice as indicated. A Lmbr1 cDNA probe recognizes major /and 5 kb in wild-type RNA (these data and Clark et al. 2000). Lmbr1-specific signal is still observed in Lmbr1ATG 3ف transcripts of and 5.5 kb along with additional 3.5ف Lmbr1ATG RNA after long exposures (right), with most prominent transcripts (asterisks) of of wild-type levels %7ف less abundant species (arrows). Remaining transcripts were present in homozygous mutant brain RNA at as determined by quantification using a Gapdh control probe (bottom). (F) Typing of DNA from progeny from a Hm/Hdhdf4J ϫ Lmbr1ATG/Lmbr1ATG cross by PCR as indicated. While Lmbr1 exon 1 sequence was amplified from DNA of phenotypically Hm progeny (left lane, bottom band), amplification was not observed from DNA from non-Hm (Hdhdf4J/Lmbr1ATG) progeny (right lane, bottom band absent). This result demonstrates that Lmbr1 is absent from Hdhdf4J chromosomes. Amplification of the neo product (top band) served as a positive control for PCR. the central as well as other distal carpals in all Lmbr1ATG/ and the ulna were sometimes narrow and the junction Hdhdf4J wrists that we examined (Table 2, compare Figure between the radius and ulna and abnormal proximal 4O to 4N). Furthermore, 36 of 38 Lmbr1ATG/Hdhdf4J limbs wrist bones was disorganized (data not shown). How- that we examined had coalitions/reductions of proxi- ever, lengths of the long bones of both the fore- and mal wrist bones (Figure 4O) in contrast to only one hindlimbs were approximately identical to those from observed proximal wrist bone coalition in Lmbr1ATG ho- control wild-type and Lmbr1ATG/ϩ animals, and obvious mozygous mice (Table 2). skeletal defects outside limbs were not apparent. The limb defects that we observed in Lmbr1ATG/Hdhdf4J The abnormalities seen in both Lmbr1ATG/Lmbr1ATG mice are primarily restricted to the most distal structures and Lmbr1ATG/Hdhdf4J mice suggest that the Lmbr1 gene of the limb (wrist/ankle and digits). In forelimbs with is required for normal development of the distal struc- severely affected wrists, the distal ends of the radius tures of the mouse limb skeleton. Lmbr1 and Reciprocal Limb Phenotypes 723

DISCUSSION at 7q36 do not produce dominant limb phenotypes. Several dominant limb mutations that cause polydac- The dominant limb phenotypes previously associated tyly were recently shown to arise by haploinsufficiency. with these regions thus must occur by gain-of-function For example, mice heterozygous for a loss-of-function mechanisms (see also Schimenti et al. 2000). As no mutation in Alx4 have preaxial polydactyly (Qu et al. coding region mutations were found in any of the candi- 1998; Takahashi et al. 1998), and loss of a single copy of date genes within the mouse Hx/Hm or human TPTPS the Gli3 gene in both mice and humans causes preaxial critical regions (Heus et al. 1999; Clark et al. 2000), we polydactyly and syndactyly (Vortkamp et al. 1991; Hui think it likely that the dominant limb phenotypes arise and Joyner 1993). In contrast, our genetic data here by gain-of-function regulatory mutations that alter the show that chromosome deletions covering either the expression of one or more genes in the region. mouse Hm/Hx interval or the human TPTPS interval The most obvious candidate for the Hx and Hm mutations is the novel Lmbr1 gene. Lmbr1 is one of only two known genes that are located entirely within the critical intervals for both the mouse and human limb mutations, and levels of Lmbr1 transcripts are dramatically misregulated in Hx limbs at the exact time that the Hx phenotype is first morphologically apparent (Clark et al. 2000). To test the requirement for the Lmbr1 gene during development, we created a mutation in the Lmbr1 gene that deletes the first known exon that contains the pre-

Figure 4.—Mice with reduced Lmbr1 function have dramatic reductions in distal limb structures. Genotypes are indicated at the top of each column, and digit number is indicated by Roman numerals. Question marks denote instances where digit identity could not be assigned with certainty. For A–O, distal is up, proximal is down, anterior is left, and posterior is right. Soft tissues (A–C) and cleared skeletal preparations that were stained with alizarin red (D–O) are shown. (A–C) Ventral views of adult forefeet. (A) Lmbr1ATG heterozygous forefoot with wild-type digit separations. (B) Lmbr1ATG/ Lmbr1ATG forefoot with mild soft tissue webbing between central digits (asterisk). (C) Four-digit Lmbr1ATG/Hdhdf4J forefoot displaying dramatic syndactyly of remaining central digits (asterisk). (D–F) Dorsal views of forefeet. (D) Lmbr1ATG/ϩ heterozygotes have normal digit number and skeletal morphology. (E) A Lmbr1ATG homozygous limb with reduced digit number that appeared to result from reduction of digit V (an arrow denotes what may be a rudimentary ﬁfth metacarpal). (F) Severely affected Lmbr1ATG/Hdhdf4J forelimb with only three remaining digits. (G–I) Dorsal views of hindfeet. (G) Lmbr1ATG/ϩ hindlimb of normal morphology. (H) Homozy- gous Lmbr1ATG hindlimb with reduced digit number resulting from bony syndactyly of digits III and IV to produce a single digit distal to the site of fusion (asterisk). (I) Lmbr1ATG/Hdhdf4J hindlimb with only four digits. (J–L) Dorsal/lateral views high- lighting posteriormost digits of forelimbs. (J) Lmbr1ATG/ϩ limbs have normal posterior digits consisting of metacarpals (m) and three phalanges (P1, P2, and P3). (K) Lmbr1ATG/ Lmbr1ATG limb in which P1 and P2 elements of digit V have been replaced by a single element. (L) Lmbr1ATG/Hdhdf4J limb in which the most posterior digit (asterisk) is reduced in size and P1 and P2 are replaced by a single element. The base of posteriormost metacarpal was also fused to its nearest neigh- bor (arrow). (M–O) Dorsal or dorsal/lateral views of wrists. (M) Lmbr1ATG/ϩ wrist with canonical organization of distal carpals 1, 2, 3, 4/5, and the central (c) in the distal wrist and the radiale (r), ulnare (u), and pisiforme (p) in the proximal wrist. (N) Homozygous Lmbr1ATG wrist with minor fusion of the central to distal carpal 3. (O) Severely affected Lmbr1ATG/ Hdhdf4J wrist in which only a small single distal element (asterisk) and a small single proximal element (arrow) remain. 724 R. M. Clark et al.

Figure 5.—Mutational and phenotypic summary and model for role of Lmbr1 in causing limb phenotypes. (A) The human Lmbr1 ortholog consists of 17 coding exons spread over 212 kb as determined by analysis of human genomic sequences (see also Ianakiev et al. 2001). While the exact size of Lmbr1 in mouse has not been determined, the mouse ortholog is at least 100 kb in size (Clark et al. 2000). The targeted mutation that we created in the mouse deletes the first exon of Lmbr1 and causes loss of distal limb structures. Ianakiev et al. (2001) showed that a small deletion including exon 4 of the human LMBR1 gene causes ACHP. Patients with this dis- order have distal limb truncations that are more severe than those observed in mice. In humans the distance between exons 1 (deleted in mice) and 4 (deleted in ACHP patients) is 66 kb. The location of mutations at different sites in the Lmbr1 gene, each of which causes distal limb reductions, suggests a specific requirement for Lmbr1 during normal limb growth and patterning. (B) We pro- pose a model whereby differences in Lmbr1 activity lead to reciprocal limb phenotypes. In gain-of-function (GOF) mutations, additional skeletal elements are formed. In contrast, loss-of-function (LOF) Lmbr1 mutations cause reductions of the distal limb. dicted site of translational initiation for both known of hands and feet) suggest that the human LMBR1 gene Lmbr1 products. Mice carrying this allele show greatly is also required for formation of distal limb structures. reduced expression of Lmbr1 transcripts but still express Acheiropodia (ACHP; OMIM 200500) is an autosomal low levels of novel transcripts. These transcripts may recessive condition that has been mapped to a small initiate from an alternative promoter in the region or region on 7q36 that overlaps the region Heus et al. from within the PGKneo selection cassette that was in- (1999) defined as containing TPTPS mutations (Esca- serted into the gene to create the Lmbr1ATG allele. On milla et al. 2000). Unlike the dominant TPTPS pheno- the basis of the residual expression seen in Northern types, ACHP patients present with loss or truncation of blots and the genetic behavior of the mutation, we be- hands and feet, although more proximal limb skeletal lieve that the Lmbr1ATG mutation is a hypomorphic rather elements are relatively spared (Toledo and Saldanha than a null mutation in the Lmbr1 gene. 1969; Toledo et al. 1972; Grimaldi et al. 1983). The Mice heterozygous or homozygous for the Lmbr1ATG molecular lesion for ACHP was recently identified as a mutation are viable and fertile and do not show the small 4- to 6-kb deletion that eliminates exon 4 of the typical limb defects that are induced by the classical LMBR1 gene (see Figure 5A; Ianakiev et al. 2001). Loss dominant gain-of-function mutations Hx and Hm. In- of this exon causes premature truncation of the LMBR1 stead, homozygous mutant mice show a very low inci- open reading frame and likely generates a null allele dence of limb defects, including oligodactyly, reduction of the gene (Ianakiev et al. 2001). The phenotypes in length or number of phalanges, and soft tissue or observed in ACHP patients clearly show a key role for bony syndactyly. The severity and penetrance of digit Lmbr1/LMBR1 during human limb development. Al- defects is markedly increased when the Lmbr1ATG allele though the human hand and foot truncations are more is placed over the deficiency mutation, and severe carpal severe than those seen in Lmbr1ATG homozygous and and tarsal coalitions are also observed. The striking dis- LmbrATG/Hdhdf4J mice, the human and mouse phenotypes tal limb phenotypes seen in mice carrying the Lmbr1ATG both involve distal limb truncations, loss of digits, and allele strongly argue that the Lmbr1 gene is required for relative sparing of proximal skeletal structures. The dif- normal formation of distal limb structures in the mouse. ferences in the severity of the mouse and human limb Recent studies of patients with acheiropodia (absence phenotypes could be the result of residual Lmbr1 activity Lmbr1 and Reciprocal Limb Phenotypes 725 in affected mice. This possibility can be tested by gener- ing limb development are currently unknown. It is ating additional Lmbr1 alleles. formally possible that the Lmbr1ATG mutation and the The loss-of-function mouse and human phenotypes human ACHP mutation also disrupt distant cis-regula- do not directly address whether the dominant limb mu- tory interactions with the Shh gene. Since the mouse and tations are also due to mutations in the Lmbr1/LMBR1 human mutations cause small disruptions that remove gene. However, the types of defects caused by loss-of- single coding exons of Lmbr1/LMBR1 and are located function Lmbr1 mutations (reductions of distal limb at two distinct locations within the gene, we think it is structures including digits) are reciprocal to those much more likely that they disrupt the function of the caused by the gain-of-function mutations (extra distal Lmbr1/LMBR1 gene itself and cause the limb defects. limb structures including digits). Combined with previ- The predicted protein product of the Lmbr1/LMBR1 ous expression data showing that Lmbr1 is misregulated gene is a novel multipass transmembrane protein that in polydactylous limbs of Hx animals (Clark et al. 2000), does not fall into any known functional class but has it is likely that the reciprocal phenotypes are due to been highly conserved in different organisms (Clark altered levels of expression of the Lmbr1 gene (see Fig- et al. 2000). Its structure suggests that it may encode a ure 5B). An important goal for future experiments will membrane anchoring protein, adhesion molecule, be to identify the nature of the DNA alterations that transporter, or cell surface receptor. An important goal generate the dominant limb phenotypes in both mice for future studies will be to determine how this gene and humans. interacts with other pathways and how it acts to control The reciprocal phenotypes seen in the current studies the development of distal skeletal structures in the verte- resemble the reciprocal effects of transplantation or brate limb. ablation of zone of polarizing activity (ZPA) cells during normal limb patterning. The presence of an additional source of ZPA activity on the anterior side of a devel- LITERATURE CITED oping limb induces preaxial polydactyly (Saunders Belloni, E., M. Muenke, E. Roessler, G. Traverso, J. Siegel-Bar- 1948; Honig and Summerbell 1985), while ablation of telt et al., 1996 Identification of Sonic hedgehog as a candidate the ZPA region from the posterior of the limb causes gene responsible for holoprosencephaly. Nat. Genet. 14: 353– loss of distal limb structures (Pagan et al. 1996). Shh is 356. Canun, S., R. M. Lomeli, R. Martinez and A. Canevale, 1984 Ab- normally expressed along the posterior limb margin and sent tibiae, triphalangeal thumbs and polydactyly: description of is thought to mediate the proliferative and patterning a family and prenatal diagnosis. Clin. Genet. 25: 182–186. activity of the ZPA (Riddle et al. 1993). Previous studies Chang, D. T., A. Lopez, D. P. von Kessler, C. Chiang, B. K. Simandl et al., 1994 Products, genetic linkage and limb patterning activity showed that the Shh gene is ectopically expressed along of a murine hedgehog gene. Development 120: 3339–3353. the anterior of the limb margin of Hx mice in the region Clark, R. M., P. C. Marker and D. M. Kingsley, 2000 A novel where extra digits form in affected limbs (Masuya et al. candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice. 1995). 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