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

Corresponding author: David M. Kingsley, Howard Hughes Medical Institute, Stanford University, Beckman Ctr., B300, 279 Campus Dr., Stanford, CA 94305-5327., kingsley{at}cmgm.stanford.edu (E-mail)

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

VERTEBRATE limb malformations that cause changes in digit number are relatively common and include polydactyly (extra digits) and oligodactyly (too few digits). Of these defects, preaxial polydactyly is the most common in humans and includes forms of thumb duplications, triphalangeal thumb, and index finger duplications on the anterior (preaxial) side of the limb (TEMTAMY and MCKUSICK 1978 ). While preaxial polydactyly is frequently associated with additional defects at sites outside the limbs, a subset of families with inherited preaxial polydactyly have limb-specific defects (ZGURICAS et al. 1999 ). Limbs from patients harboring these preaxial polydactyly mutations typically present with replacement of the thumb with one or more triphalangeal elements (CANUN et al. 1984 ; CORDEIRO et al. 1986 ; HEUTINK et al. 1994 ; TSUKUROV et al. 1994 ; HING et al. 1995 ; VARGAS et al. 1995 ; RADHAKRISHNA et al. 1996 ; ZGURICAS et al. 1999 ; DOBBS et al. 2000 ). Many of these polydactyly mutations are also associated with additional distal limb defects that include soft tissue fusions (syndactyly) of adjacent digits and/or radial or tibial dysplasia/aplasia (CANUN et al. 1984 ; CORDEIRO et al. 1986 ; HEUTINK et al. 1994 ; TSUKUROV et al. 1994 ; VARGAS et al. 1995 ; ZGURICAS et al. 1999 ). Despite this phenotypic variation, all these mutations are dominantly inherited and highly penetrant, and recent mapping studies have localized many of the mutations to the same 7q36 region (HEUTINK et al. 1994 ; TSUKUROV et al. 1994 ; HING et al. 1995 ; RADHAKRISHNA et al. 1996 ; VARGAS et al. 1998 ; ZGURICAS et al. 1999 ; DOBBS et al. 2000 ). Therefore, a major locus for triphalangeal thumb-polysyndactyly syndrome (TPTPS; OMIM 190605) at 7q36 is responsible for almost all dominant preaxial polydactylies and polysyndactylies with defects restricted to the limbs.

In the mouse, the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) limb mutations are thought to be analogous to the human TPTPS mutations at 7q36 (HEUTINK et al. 1994 ; TSUKUROV et al. 1994 ), and both map to a mouse chromosome region homologous to 7q36 (CLARK et al. 2000 ). Hx mice have limb defects that include preaxial polydactyly and radial and tibial hemimelia (KNUDSEN and KOCHHAR 1981 ; MASUYA et al. 1995 ) that closely resemble the human limb phenotypes. 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 mutations that have been mapped to 7q36 (TSUKUROV et al. 1994 ). In crosses segregating Hx and Hm only a single recombination was observed in 3664 meioses (SWEET 1982 ). This extremely tight linkage suggests that the mouse mutations affect neighboring genes or alternatively may be different alleles of the same gene with the observed recombination arising from an intragenic crossover.

Recently, HEUS et al. 1999 defined an 450-kb region on 7q36 that contains dominant polydactyly mutations and identified a small set of genes that are contained within the TPTPS critical region. In a parallel study in the mouse, CLARK et al. 2000 defined an 450-kb interval for the mouse Hm and Hx mutations and identified several genes within this critical region. The mouse and human candidate genes are orthologous, confirming previous conjecture that the human and mouse phenotypes arise by defects in similar genes (CLARK et al. 2000 ). However, extensive mutational analysis in both human and mouse has not identified lesions in the coding sequences of any candidate genes (HEUS et al. 1999 ; CLARK et al. 2000 ).

The absence of coding region mutations raises the possibility that the dominant mouse and human mutations are regulatory alleles that disrupt expression of a gene or genes in the interval. CLARK et al. 2000 showed that one of the genes within the mouse critical region, called Limb region 1 (Lmbr1), is normally expressed in developing limbs at the times that both the Hx and Hm phenotypes arise. More importantly, Lmbr1 was dynamically misexpressed in Hx limbs at the exact time that Hx limb morphology first appears. Expression changes included a possible overexpression of the gene followed by a dramatic decrease in Lmbr1 transcript levels at later stages (CLARK et al. 2000 ). The human Lmbr1 ortholog (LMBR1) is contained entirely within the human critical region for TPTPS mutations at 7q36 (HEUS et al. 1999 ), and the striking correlation between the appearance of defects in Hx mice and Lmbr1 misregulation suggests that the mouse and human limb mutations may be alleles of the Lmbr1 gene (CLARK et al. 2000 ).

Here we use deletion chromosomes in both mice and humans to show that the dominant mouse and human limb phenotypes are likely to arise by gain-of-function mechanisms. In addition, we created a loss-of-function allele of the Lmbr1 gene to test whether this gene is required for normal limb development. Mice with reduced Lmbr1 function show distal limb reductions including oligodactyly. These phenotypes are reciprocal to those caused by the classical dominant mutations. The complementary defects suggest that levels of expression of the Lmbr1 gene play a key role in controlling the development of skeletal structures in the vertebrate limb.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of human genomic bacterial artificial chromosome clones:
Primers were prepared on the basis of partial human expressed sequence tag (EST) sequences predicted to correspond to the 5' untranslated region and first exon of LMBR1 that amplified the expected size fragments from commercial genomic DNA (Clontech, Palo Alto, CA). Following sequence verification of test amplicons, two primer pairs [HxF1b (5'-acctgttccaacacggctcgc-3') and HxR1 (5'-actcccgcacttggctgtgg-3') and the nested pair HxF1b (above) and HxR1b (5'-acacctcgtcctgcccttcc-3')] were used by the Physical Mapping Core (National Human Genome Research Institute/National Institutes of Health) to screen a human bacterial artificial chromosome (BAC) library (Incyte Genomics, Palo Alto, CA), resulting in the identification of clone address 575h20. Direct sequencing of this clone as well as Southern blot hybridization experiments verified that it contains exon 1 and 5' flanking sequences of LMBR1 (data not shown).

Fluorescent in situ hybridization analysis:
Slides with chromosome metaphase spreads were incubated for 1 hr at 37° in 2x SSC (0.3 M NaCl and 0.3 M sodium citrate) and then dehydrated sequentially in 70, 80, and 90% ethanol. Chromosome DNA was denatured in 70% formamide, 2x SSC for 2 min at 72° followed by dehydration in ethanol washes of 70, 80, 90, and 100%. Fluorescent in situ hybridization (FISH) was performed with probes labeled with spectrum orange-dUTP (Vysis, Downers Grove, IL), essentially as described previously (PINKEL et al. 1986 ; LICHTER et al. 1988 ). On each slide, 100 ng of labeled DNA was applied. Nonunique and nonspecific DNA hybridization was blocked by preannealing the probe with a 10-fold excess of human Cot1 DNA. Labeled and blocking DNAs were denatured at 75° for 10 min and then preannealed at 37° for 15 min. The hybridization mixture contained labeled DNA in 10 ml of 50% formamide, 2x SSC, and 10% dextran sulfate at pH 7.0. Slides were hybridized overnight at 37°. Post-hybridization washes were performed at 45° as follows: (1) 50% formamide, 2x SSC, 20 min; (2) 1x SSC, 10 min; and (3) 0.1x SSC, 10 min. Slides were counterstained with propidium iodide-Antifade (Intergen, Purchase, NY) or 250 ng/ml 4',6-diamidino-2-phenylindole (Boehringer Mannheim, Indianapolis) with Antifade.

Design of Lmbr1 targeting vector and homologous recombination:
The 5' end of the Lmbr1 gene is present on mouse BAC clone 136E36 from the 129 strain CITB mouse BAC II library (Research Genetics, Huntsville, AL; CLARK et al. 2000 , and our unpublished data). DNA from this BAC was digested with restriction enzymes and subcloned into a plasmid vector, and an 8.3-kb BamHI fragment harboring the exon that contains the start site of the Lmbr1 open reading frame was isolated by hybridization with Lmbr1 sequences. A 1.1-kb MluI/KpnI fragment that contains this exon (see RESULTS) was replaced by a PGKneo-positive selection cassette by cloning sequences flanking the 1.1-kb fragment into the pPNT vector. The pPNT vector contains the herpes thymidine kinase gene for negative selection.

Targeting of the endogenous Lmbr1 locus was performed in R1 embryonic stem (ES) cells (kindly provided by Janet Rossant) as described previously (JOYNER 1993 ). Construct linearized with NotI was electroporated into ES cells and positive selection was performed using G418 while Gancyclovir was used to select against nonhomologous integration events. Cells were grown on G418-resistant irradiated mouse embryonic fibroblasts. ES cell colonies with targeted integrations were detected by nested PCR amplification of a 3.1-kb junction fragment created by homologous recombination between the 3' arm of the replacement construct and the endogenous Lmbr1 locus. Primers used for amplification were neo1 (5'-gcagcctctgttccacataca-3') and LK1 (5'-tgagggagccagaggagtca-3') for primary PCR and neo2 (5'-gccaagttctaattccatcagaa-3') and LK2 (5'-aaaatacaagaaaacctacagaatc-3') for secondary PCR. Amplifications were performed with ampliTaq (Applied Biosystems, Foster City, CA) with cycle conditions of 94° (3 min); 24 cycles of 94° (30 sec), 59° (1 min), 72° (4 min); followed by 6 cycles of 94° (30 sec), 59° (1 min), and 72° (5 min); followed by 72° (15 min). Homologous recombinants were verified by additional PCR with primers LK3 (5'-ggtaggggttattggtacagactt-3') and neo3 (5'-gcctcccctacccggtagaatt-3') that amplify a junction fragment created by homologous recombination between the 5' arm of the targeting construct and the endogenous Lmbr1 locus. PCR with primers LK3 and neo3 was performed using the expand long template PCR system (Hoffmann La Roche, Basel, Switzerland) with cycle conditions of 94° (2 min); 10 cycles of 94° (10 sec), 65° (30 sec), 68° (8 min); followed by 25 cycles of 94° (10 sec), 65° (30 sec), 68° (8 min with an additional 20-sec/cycle); followed by 68° (10 min). We refer to the allele created by targeting as Lmbr1^ATG.

Generation and typing of Lmbr1^ATG mutant mice:
Two independently selected ES cell clones were injected into C57BL/6J host blastocysts, and chimeric animals (agouti coat color) were crossed to B6D2F1/J animals (The Jackson Laboratory). Germline transmission was determined by coat color, and heterozygous progeny were identified. Homozygous animals were generated by intercrossing heterozygotes, and phenotypic analysis of Lmbr1^ATG/+ and Lmbr1^ATG/Lmbr1^ATG animals as well as wild-type controls was performed on the 129SV/J x B6D2F1/J mixed genetic background. Initially, heterozygous animals were typed with primers that were used to identify targeted ES clones (primers neo1, LK1, neo2, and LK2; see above). Afterward, progeny were typed with duplexed primer pairs that allowed all genotypes to be distinguished in single PCR reactions. One primer set amplifies a 174-bp fragment of Lmbr1 exon 1 that is deleted by the targeted mutation [primers ATGF (5'-tcttgaaccgcttctccctgag-3') and ATGR (5'-cccttccatcctcctttcatacc-3')], and another pair amplifies a 268-bp fragment from the neo resistance cassette inserted into the locus by targeting [primers N1F (5'-acagacaatcggctgctctgatg-3') and N1R (5'-gatggatactttctcggcaggag-3')]. PCR amplification conditions were 94° (2 min); 30 cycles of 94° (30 sec), 63° (1 min), 72° (30 sec); followed by 72° (10 min).

Northern blot analysis with wild-type and mutant RNA samples:
Total brain RNA from wild-type and mutant mice was prepared using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Messenger RNA was isolated from total RNA using oligo(T) cellulose (FastTrack 2.0 kit; Invitrogen). For Northern blot analysis, 2 µg of poly(A) RNA per lane was loaded and separated on 1% agarose/1.5% formaldehyde gels by electrophoresis. RNA was then blotted to Hybond N+ membrane (Amersham, Arlington Heights, IL). Blots were probed with a ³²P-labeled Lmbr1 cDNA probe. This probe contains the open reading frame (ORF) of the Lmbr1 gene that encodes LMBR1L 3' of the sequence for exon 1. Blots were scanned on a model 425 E phosphor imager (Molecular Dynamics, Sunnyvale, CA) and relative expression levels were calculated using a Gapdh probe to control for loading differences.

Hdh^df4J mice, crosses, and typing:
A male mouse carrying the proximal chromosome 5 deficiency Hdh^df4J (SCHIMENTI et al. 2000 ) in trans to a Mus mus castaneous chromosome (C57BL/6J x 129/Jae x M. mus castaneous mixed-strain background) was crossed to either Hm/Hm (C3HeB/FeJLe strain) or wild-type (B10.D2/nSn strain) females. To determine inheritance of the deletion, DNAs from progeny animals were typed with primers that amplify the microsatellite locus D5Mit148 that is predicted to be removed by the Hdh^df4J deficiency. This locus is polymorphic between M. mus castaneous and C3HeB/FeJLe and B10.D2/nSn strain DNA (data not shown), and progeny that inherited the deficiency that lacked the M. mus castaneous allele were identified. Hm/Hdh^df4J male progeny were crossed to wild-type B6D2F1/J females or to Lmbr1^ATG/+ or Lmbr1^ATG/Lmbr1^ATG females and progeny that inherited the deletion were distinguished by the absence of Hm limb phenotypes. Where necessary, inheritance of the Lmbr1^ATG allele was determined by typing with primers N1F and N1R. Hdh^df4J/+ males were also crossed to Lmbr1^ATG/Lmbr1^ATG females. Deletion progeny from this cross were identified by typing animals with primers ATGF and ATGR. The Hdh^df4J deletion includes Lmbr1 exon 1, and in Lmbr1^ATG/Hdh^df4J mice the exon 1 product amplified by ATGF and ATGR is not present (see RESULTS).

Skeletal preparations:
Alizarian red-stained skeletons were prepared as previously described (GREEN 1952 ) from weaning age or adult mice.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Hx and Hm mutations are gain of function:
While the phenotypes and mode of inheritance of the mouse Hx and Hm mutations are described in detail (GREEN 1964 ; KNUDSEN and KOCHHAR 1981 ; ZAKERI et al. 1994 ; MASUYA et al. 1995 ), it has not been clear whether these dominant mutations produce limb abnormalities by gain-of-function or loss-of-function mechanisms. A deletion of proximal mouse chromosome 5 (Hdh^df4J) that does not produce obvious webbing or gross polydactyly was previously reported (SCHIMENTI et al. 2000 ). The Hx and Hm mutations were previously mapped between the Shh and Il6 loci on proximal chromosome 5 (MARTIN et al. 1990 ; ROBERT et al. 1994 ; MARIGO et al. 1995 ; CLARK et al. 2000 ) in a region likely to be included in the Hdh^df4J deletion. PCR typing confirmed that this deletion removes Lmbr1 coding sequences (see MATERIALS AND METHODS and Fig 3F), demonstrating that Hdh^df4J/+ mice carry a single copy of the Lmbr1 candidate region.

View larger version (67K): In this window In a new window Download PPT slide   Figure 1. Mice hemizygous for the Hx-Hm interval do not have classical Hx or Hm limb phenotypes. For A–D, distal is up, proximal is down, anterior is left, and posterior is right. Digit number is indicated by Roman numerals. (A and B) Ventral views of adult forefeet showing soft tissues. In Hm/+ limbs (A) digits II–V are connected to each other by lateral soft tissue fusions (asterisks) as well as to the ventral surface of the limb, and digits are flexed ventrally toward the viewer (the "hammertoe" phenotype). In contrast, no soft tissue webs are observed in Hdhdf4J/+ limbs that lack one copy of the region that contains the Hm-Hx interval (B), and digits from these mice are fully extended (compare to Fig 4A). (C and D) Dorsal views of cleared forelimbs stained with alizarin red to identify skeletal elements. Hx/+ limbs (C) have preaxial polydactyly in which digit I, which normally has two small phalanges (see Fig 4D), is replaced by extra triphalangeal digits (asterisks). In contrast, limbs from Hdhdf4J/+ mice (D) have normal digit morphology. These results demonstrate that the classical webbing and polydactyly phenotypes result from gain-of-function mechanisms and not from haploinsufficiency.

View larger version (34K): In this window In a new window Download PPT slide   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.

View larger version (41K): In this window In a new window Download PPT slide   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 5' arm of the replacement construct and the Lmbr1 genomic locus. (C) Primary PCR (1° PCR) with primers neo1 and LK1 and secondary PCR (2° PCR) with primers neo2 and LK2 amplify a 3.1-kb fragment generated by homologous recombination between the 3' 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 transcripts of 3 and 5 kb in wild-type RNA (these data and CLARK et al. 2000 ). Lmbr1-specific signal is still observed in Lmbr1ATG/ Lmbr1ATG RNA after long exposures (right), with most prominent transcripts (asterisks) of 3.5 and 5.5 kb along with additional less abundant species (arrows). Remaining transcripts were present in homozygous mutant brain RNA at 7% of wild-type levels as determined by quantification using a Gapdh control probe (bottom). (F) Typing of DNA from progeny from a Hm/Hdhdf4J x 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.

We examined limbs of Hdh^df4J/+ mice to test whether deletion of the candidate interval produces limb phenotypes typical of those observed in either Hm/+ or Hx/+ mice. No webbing was seen between digits, and Alizarin red-stained skeletal preparations did not show extra skeletal elements characteristic of Hx/+ mice, suggesting that the classical Hm and Hx phenotypes arise by a gain-of-function mechanism rather than from loss-of-function mutations in a gene in the interval (Table 1 and Fig 1). Although Hdh^df4J/+ mice do not have limb defects that resemble those in Hx and Hm mice, minor coalitions of distal wrist bones were observed in 25% of wrists in Hdh^df4J/+ animals and included fusions of the central to either distal carpals 2 or 3 (dc2 and dc3, respectively; Table 2). Animals carrying the Hdh^df4J deletion were also smaller than wild-type mice as has been observed for several other chromosomal deletions.

Dominant preaxial polydactyly mutations that map to 7q36 are gain of function:
The critical region for human TPTPS mutations at 7q36 is homologous to the critical region for the mouse Hm and Hx mutations and contains the human ortholog of the mouse Lmbr1 gene (LMBR1; HEUS et al. 1999 ; CLARK et al. 2000 ). To determine whether the dominant human limb mutations arise by a gain-of-function mechanism, we used FISH analysis to map the region surrounding the LMBR1 interval in human patients with deletions or translocations in the 7q36 region (ROESSLER et al. 1997 and references therein). A 135-kb human BAC clone that contains exon 1 of the LMBR1 gene was isolated. This BAC produces two chromosome 7q36-specific hybridization signals in FISH analysis of control human cells (Fig 2A). FISH analysis of cells from a patient with a t(7;17) translocation (T4) shows two chromosome 7 LMBR1 hybridization signals [7 and der(7), Fig 2B], indicating that LMBR1 is proximal to the T4 translocation breakpoint. FISH analysis of cells harboring a t(7;9) translocation (T1) shows that one LMBR1 hybridization signal is associated with the intact copy of chromosome 7 while the other is on the chromosome 9 derivative [7 and der(9), Fig 2C], indicating that LMBR1 is distal to the T1 translocation breakpoint. Only a single chromosome 7 hybridization signal was seen in cells from a patient with a de novo 7q36 deletion (Fig 2D). These data suggest that the LMBR1 BAC maps precisely between the T1 and T4 translocations in a region completely covered by the 7q36 deletion (Fig 2E). Physical estimates based on assembly of genomic clones in the 7q36 region suggest that the T1 and T4 translocations are located 265 and 335 kb distal to the SHH gene (ROESSLER et al. 1997 ). The present Lmbr1 mapping results, combined with the earlier cytogenetic data, suggest that as many as 32 unrelated patients with cytogenetic deletions in the 7q36 region may also lack one copy of the LMBR1 gene (ROESSLER et al. 1997 ). None of these patients have limb defects that include preaxial polydactyly or polysyndactyly (ROESSLER et al. 1997 ). Therefore, the entire region containing the LMBR1 critical area can be deleted without producing the TPTPS characteristics of the dominant limb mutations that were independently mapped to 7q36 (Fig 2E). These data suggest that, like the mouse Hx and Hm mutations, the dominant human limb mutations that map to 7q36 are likely to act by a gain-of-function rather than a loss-of-function mechanism.

Generation of a loss-of-function allele of the Lmbr1 gene:
The location of the Lmbr1 gene in the critical interval for both the mouse and human limb mutations and the altered expression of this gene in Hx mice suggest that regulatory mutations in Lmbr1 may be responsible for the dominant mouse and human limb defects. To examine the role of this gene during normal limb development, we created a loss-of-function mutation in the Lmbr1 gene using embryonic stem cell targeting.

The Lmbr1 gene encodes a highly conserved product of 490 amino acids (LMBR1L) as well as a smaller product of 32 amino acids (LMBR1S) produced by an alternatively spliced Lmbr1 transcript (CLARK et al. 2000 ). The first 22 amino acids of both proteins are identical. To create an allele of Lmbr1, we subcloned genomic DNA containing the 5'-most coding exon of the Lmbr1 gene. Sequence analysis revealed that this exon encodes the 22 amino acids that are common to the N termini of both the LMBR1L and LMBR1S proteins (CLARK et al. 2000 ). Genomic sequence upstream of the translational start site for Lmbr1 products is contiguous with sequence of Lmbr1 5' rapid amplification of cDNA ends (RACE) products for 166 bp, at which point RACE products end (our unpublished data). This point may represent the transcriptional start site for the Lmbr1 gene, and we refer to the exon we isolated as Lmbr1 exon 1. We used homologous recombination in ES cells to replace a 1.1-kb fragment that contains exon 1 with a PGKneo selection cassette (Fig 3, A–C). The deleted fragment contains 365 bp 5' of the predicted translational start site of exon 1 and 696 bp 3' 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 (Lmbr1^ATG) through the germline. Mice heterozygous for the targeted mutation were intercrossed to produce Lmbr1^ATG/Lmbr1^ATG 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 Lmbr1^ATG homozygotes (Fig 3D). Homozygous mice were present at normal Mendelian ratios (36 +/+; 91 Lmbr1^ATG/+; 44 Lmbr1^ATG/Lmbr1^ATG; P = 0.20, chi-square test). Both male and female Lmbr1^ATG homozygous mice are fertile and we have been able to maintain the Lmbr1^ATG allele by intercrossing homozygotes. We also examined a variety of tissues from Lmbr1^ATG 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 of wild-type and Lmbr1^ATG/Lmbr1^ATG adult brain poly(A) RNA with a Lmbr1 cDNA probe. The normal 3- and 5-kb messages that contain the first exon of the Lmbr1 gene (CLARK et al. 2000 ) were greatly reduced or undetectable in Lmbr1^ATG homozygotes (Fig 3E). However, long exposures of Northern blots showed multiple transcripts in brain RNA from homozygotes (Fig 3E). These transcripts were present at levels of 7% that of wild-type Lmbr1 messages in brain. These molecular data suggest that the targeted mutation reduced Lmbr1 function but that the mutation may not have created a null allele (see DISCUSSION).

Lmbr1^ATG homozygous mice have low incidences of limb defects:
As expected from our studies of the chromosome 5 deletion mice, the loss-of-function mutation in Lmbr1 did not produce a phenocopy of either the Hx or Hm mutations. We did, however, detect a very low incidence of limb abnormalities in Lmbr1^ATG homozygous animals (Table 1). These phenotypes presented as digit loss or reduction rather than the gain-of-digit number typically seen in Hx animals. One mouse appeared to have only four digits on its right forefoot, while a second homozygous mouse had only four digits on a hindfoot. The latter mouse also had mild soft tissue syndactylies between central digits on its other three limbs (Table 1 and Fig 4B). Skeletal preparation revealed that reduction of digit number in the affected Lmbr1^ATG/Lmbr1^ATG forelimb arose from loss of the metacarpal and phalanges of a single digit (Fig 4E). The morphology of the four remaining digits suggested that the missing digit is digit V (compare Fig 4E). A small posterior element in this limb may be a rudiment of the fifth metacarpal (see arrow in Fig 4E). In contrast, in the affected hindlimb of the homozygous mutant animal, reduction in digit number was caused by fusion of digits III and IV at the level of the phalanges to produce a single digit distally that ended in a single third phalanx (Table 1 and Fig 4H).

View larger version (53K): In this window In a new window Download PPT slide   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/+ 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/+ hindlimb of normal morphology. (H) Homozygous 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 highlighting posteriormost digits of forelimbs. (J) Lmbr1ATG/+ 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 neighbor (arrow). (M–O) Dorsal or dorsal/lateral views of wrists. (M) Lmbr1ATG/+ 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.

Further examination of homozygous Lmbr1^ATG animals revealed low incidences of additional digit defects that included reduction in phalange length or number that were never observed in wild-type or heterozygous control limbs (Table 1). On both fore- and hindlimbs digit I normally has two phalanges (P1 and P2, both of which are very small in forelimbs), while digits II–V have three phalanges each (P1, P2, and P3; Fig 4D, Fig G, and Fig J). While the fifth digits of most Lmbr1^ATG/Lmbr1^ATG forelimbs had three phalanges of normal relative lengths, one forelimb had a fifth digit in which the size of P2 was greatly reduced (Table 1 and data not shown). In two other limbs, the P1 and P2 elements were replaced by a single element (Table 1 and Fig 4K). No Lmbr1^ATG/Lmbr1^ATG hindlimbs had obvious reductions in the length of phalanges or any reductions in phalangeal number (data not shown).

We also examined the organization of the wrists and ankles in wild-type, heterozygous, and homozygous mice. In general, five bones comprise the distal wrist [distal carpals 1 (dc1), 2 (dc2), 3 (dc3), and 4/5 (dc4/5); and the central], while three bones comprise the proximal wrist (the radiale, ulnare, and pisiforme; Fig 4M). However, the number of distal wrist bones has been observed to vary depending on strain background (DAVIS and CAPECCHI 1994 ). In wild-type control mice from our cross, we observed a high frequency (32%) of forelimbs in which dc2 was partially or completely fused to the central (Table 2). In Lmbr1^ATG heterozygous mice, we observed a lower frequency (8%) of distal wrist coalitions that involved fusion of the central to dc2 (Table 2). In contrast, in Lmbr1^ATG/Lmbr1^ATG wrists we observed a high frequency (39%) of distal coalitions that included only fusions between the central and dc3 (Table 2 and Fig 4N). While no wild-type or heterozygous wrists that we examined had defects in proximal carpal bones, in 1 of 36 homozygous wrists the ulnare and pisiforme were partially fused (Table 2).

We also observed coalitions of anklebones in wild-type and heterozygous and homozygous Lmbr1^ATG mice (Table 2). Normally, six bones comprise the distal ankle [distal tarsals 1 (dt1), 2 (dt2), 3 (dt3), and 4/5 (dt4/5); the pisiforme; and the central] while the talus and the calcaneus comprise the proximal ankle. While most ankles of each genotype that we examined had the canonical ankle organization, we observed coalitions of distal anklebones in wild-type and Lmbr1^ATG/+ mice that included dt2, dt3, and/or the central (Table 2). In Lmbr1^ATG homozygous ankles we observed distal anklebone fusions that included coalitions involving dc2, dc3, dc4/5, and/or the central (Table 2). We did not observe any defects in the proximal anklebones of wild-type or Lmbr1^ATG heterozygous or homozygous mice (data not shown).

Defects in Lmbr1^ATG mice appear to be confined to the wrist, ankle, and footplate regions, with no obvious defects detected in more proximal limb structures or structures outside the limb.

Lmbr1^ATG/Hdh^df4J mice have severe distal limb defects:
To further test the role of Lmbr1 in development, we generated mice trans-heterozygous for the Lmbr1^ATG allele and the Hdh^df4J deletion that removes the region containing the Lmbr1 locus. If residual Lmbr1 function is present in Lmbr1^ATG homozygotes, we reasoned that Lmbr1^ATG/Hdh^df4J mice should have an even greater reduction in Lmbr1 activity and therefore may display more severe phenotypes. Most Lmbr1^ATG/Hdh^df4J mice were produced by crossing either Hdh^df4J/+ or Hdh^df4J/Hm mice to Lmbr1^ATG/Lmbr1^ATG animals. In these crosses, progeny that inherited the Hdh^df4J deletion (Lmbr1^ATG/Hdh^df4J mice) were recovered at less than the expected frequency of 50% at weaning (40 Lmbr1^ATG/+ or Lmbr1^ATG/Hm progeny; 16 Lmbr1^ATG/Hdh^df4J progeny; P = 0.001, chi-square test). A small number of Lmbr1^ATG/Hdh^df4J mice were also produced by crossing Hdh^df4J/Hm mice to Lmbr1^ATG/+ animals (see MATERIALS AND METHODS). As with Hdh^df4J/+ mice, Lmbr1^ATG/Hdh^df4J mice were typically smaller than littermates (either Lmbr1^ATG/+ or Lmbr1^ATG/Hm mice; data not shown). Whether the observed decrease in survival of Lmbr1^ATG/Hdh^df4J relative to Hdh^df4J/+ mice results from a specific effect of Lmbr1 or rather from differences in the interaction of the deletion with different stain backgrounds used in our crosses is not known.

Limbs from Lmbr1^ATG/Hdh^df4J mice showed dramatic digit defects that include soft tissue webbing (Table 1 and Fig 4C), reductions in the number of digits (Table 1 and Fig 4C, Fig F, and Fig I), and reductions in the number or length of phalanges in digits (Table 1 and Fig 4L). These defects were more severe and occurred at much greater frequencies than those in Lmbr1^ATG/Lmbr1^ATG mice. For example, 55% of all Lmbr1^ATG/Hdh^df4J limbs had fewer than five digits, compared to <1% in Lmbr1^ATG homozygotes (Table 1). The digit reduction was also more severe in Lmbr1^ATG/Hdh^df4J animals, with one-third of the affected forelimbs showing only three remaining digits (Table 1 and Fig 4F). The reduction of digits appeared to be restricted to central or posterior digits on the basis of morphological criteria (compare Fig 4F and Fig 4I and Fig 4G).

Lmbr1^ATG/Hdh^df4J 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 Lmbr1^ATG homozygous mice and control mice, we observed dramatic coalitions/reductions of distal wrist bones that involved dc2, dc3, and/or the central as well as other distal carpals in all Lmbr1^ATG/Hdh^df4J wrists that we examined (Table 2, compare Fig 4O). Furthermore, 36 of 38 Lmbr1^ATG/Hdh^df4J limbs that we examined had coalitions/reductions of proximal wrist bones (Fig 4O) in contrast to only one observed proximal wrist bone coalition in Lmbr1^ATG homozygous mice (Table 2).

The limb defects that we observed in Lmbr1^ATG/Hdh^df4J mice are primarily restricted to the most distal structures of the limb (wrist/ankle and digits). In forelimbs with severely affected wrists, the distal ends of the radius and the ulna were sometimes narrow and the junction between the radius and ulna and abnormal proximal wrist bones was disorganized (data not shown). However, lengths of the long bones of both the fore- and hindlimbs were approximately identical to those from control wild-type and Lmbr1^ATG/+ animals, and obvious skeletal defects outside limbs were not apparent.

The abnormalities seen in both Lmbr1^ATG/Lmbr1^ATG and Lmbr1^ATG/Hdh^df4J mice suggest that the Lmbr1 gene is required for normal development of the distal structures of the mouse limb skeleton.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Several dominant limb mutations that cause polydactyly were recently shown to arise by haploinsufficiency. For example, mice heterozygous for a loss-of-function mutation in Alx4 have preaxial polydactyly (QU et al. 1998 ; TAKAHASHI et al. 1998 ), and loss of a single copy of the Gli3 gene in both mice and humans causes preaxial polydactyly and syndactyly (VORTKAMP et al. 1991 ; HUI and JOYNER 1993 ). In contrast, our genetic data here show that chromosome deletions covering either the mouse Hm/Hx interval or the human TPTPS interval at 7q36 do not produce dominant limb phenotypes. The dominant limb phenotypes previously associated with these regions thus must occur by gain-of-function mechanisms (see also SCHIMENTI et al. 2000 ). As no coding region mutations were found in any of the candidate genes within the mouse Hx/Hm or human TPTPS critical regions (HEUS et al. 1999 ; CLARK et al. 2000 ), we think it likely that the dominant limb phenotypes arise by gain-of-function regulatory mutations that alter the expression of one or more genes in the region.

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 predicted site of translational initiation for both known Lmbr1 products. Mice carrying this allele show greatly reduced expression of Lmbr1 transcripts but still express low levels of novel transcripts. These transcripts may initiate from an alternative promoter in the region or from within the PGKneo selection cassette that was inserted into the gene to create the Lmbr1^ATG allele. On the basis of the residual expression seen in Northern blots and the genetic behavior of the mutation, we believe that the Lmbr1^ATG mutation is a hypomorphic rather than a null mutation in the Lmbr1 gene.

Mice heterozygous or homozygous for the Lmbr1^ATG mutation are viable and fertile and do not show the typical limb defects that are induced by the classical dominant gain-of-function mutations Hx and Hm. Instead, homozygous mutant mice show a very low incidence of limb defects, including oligodactyly, reduction in length or number of phalanges, and soft tissue or bony syndactyly. The severity and penetrance of digit defects is markedly increased when the Lmbr1^ATG allele is placed over the deficiency mutation, and severe carpal and tarsal coalitions are also observed. The striking distal limb phenotypes seen in mice carrying the Lmbr1^ATG allele strongly argue that the Lmbr1 gene is required for normal formation of distal limb structures in the mouse.

Recent studies of patients with acheiropodia (absence of hands and feet) suggest that the human LMBR1 gene is also required for formation of distal limb structures. Acheiropodia (ACHP; OMIM 200500) is an autosomal recessive condition that has been mapped to a small region on 7q36 that overlaps the region HEUS et al. 1999 defined as containing TPTPS mutations (ESCAMILLA et al. 2000 ). Unlike the dominant TPTPS phenotypes, ACHP patients present with loss or truncation of hands and feet, although more proximal limb skeletal elements are relatively spared (TOLEDO and SALDANHA 1969 ; TOLEDO et al. 1972 ; GRIMALDI et al. 1983 ). The molecular lesion for ACHP was recently identified as a small 4- to 6-kb deletion that eliminates exon 4 of the LMBR1 gene (see Fig 5A; IANAKIEV et al. 2001 ). Loss of this exon causes premature truncation of the LMBR1 open reading frame and likely generates a null allele of the gene (IANAKIEV et al. 2001 ). The phenotypes observed in ACHP patients clearly show a key role for Lmbr1/LMBR1 during human limb development. Although the human hand and foot truncations are more severe than those seen in Lmbr1^ATG homozygous and Lmbr^ATG/Hdh^df4J mice, the human and mouse phenotypes both involve distal limb truncations, loss of digits, and relative sparing of proximal skeletal structures. The differences in the severity of the mouse and human limb phenotypes could be the result of residual Lmbr1 activity in affected mice. This possibility can be tested by generating additional Lmbr1 alleles.

View larger version (23K): In this window In a new window Download PPT slide   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 disorder 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 propose 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.

The loss-of-function mouse and human phenotypes do not directly address whether the dominant limb mutations are also due to mutations in the Lmbr1/LMBR1 gene. However, the types of defects caused by loss-of-function Lmbr1 mutations (reductions of distal limb structures including digits) are reciprocal to those caused by the gain-of-function mutations (extra distal limb structures including digits). Combined with previous expression data showing that Lmbr1 is misregulated in polydactylous limbs of Hx animals (CLARK et al. 2000 ), it is likely that the reciprocal phenotypes are due to altered levels of expression of the Lmbr1 gene (see Fig 5B). An important goal for future experiments will be to identify the nature of the DNA alterations that generate the dominant limb phenotypes in both mice and humans.

The reciprocal phenotypes seen in the current studies resemble the reciprocal effects of transplantation or 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 developing limb induces preaxial polydactyly (SAUNDERS 1948 ; HONIG and SUMMERBELL 1985 ), while ablation of the ZPA region from the posterior of the limb causes loss of distal limb structures (PAGAN et al. 1996 ). Shh is normally expressed along the posterior limb margin and is thought to mediate the proliferative and patterning activity of the ZPA (RIDDLE et al. 1993 ). Previous studies showed that the Shh gene is ectopically expressed along the anterior of the limb margin of Hx mice in the region where extra digits form in affected limbs (MASUYA et al. 1995 ). These phenotypes and the phenotypes seen in the current studies could be explained if the Lmbr1 gene normally acts as a positive trans-acting regulator of Shh activity.

The Shh gene is also physically linked to the Hx/Hm and 7q36 critical regions, and it was previously proposed that dominant human and mouse limb mutations may be due to defects in cis-acting regulatory elements of the Shh gene (CHANG et al. 1994 ; SHARPE et al. 1999 ). The data reported here show that the human TPTPS critical region containing the LMBR1 gene is located 300 kb distal to SHH in the region between the T1 and T4 breakpoints. Patients carrying the T1 but not the T4 translocation show mild holoprosencephaly phenotypes thought to be caused by loss of SHH in cranial midline structures (see Fig 2E; ROESSLER et al. 1997 ). These data suggest that control elements required for normal expression of Shh during craniofacial development may be located far from the coding regions of SHH (ROESSLER et al. 1997 ). However, patients with the T1 and T4 translocations and numerous other patients with LMBR1 deletions do not have TPTPS limb phenotypes (ROESSLER et al. 1997 ), and the locations of regulatory sequences required for normal Shh regulation during limb development are currently unknown. It is formally possible that the Lmbr1^ATG mutation and the human ACHP mutation also disrupt distant cis-regulatory interactions with the Shh gene. Since the mouse and human mutations cause small disruptions that remove single coding exons of Lmbr1/LMBR1 and are located at two distinct locations within the gene, we think it is much more likely that they disrupt the function of the Lmbr1/LMBR1 gene itself and cause the limb defects.

The predicted protein product of the Lmbr1/LMBR1 gene is a novel multipass transmembrane protein that does not fall into any known functional class but has been highly conserved in different organisms (CLARK et al. 2000 ). Its structure suggests that it may encode a membrane anchoring protein, adhesion molecule, transporter, or cell surface receptor. An important goal for future studies will be to determine how this gene interacts with other pathways and how it acts to control the development of distal skeletal structures in the vertebrate limb.

	FOOTNOTES

¹ Present address: Department of Anatomy, University of California, San Francisco, CA 94143-0452.

Manuscript received March 20, 2001; Accepted for publication June 1, 2001.

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