Copyright 2001 by the Genetics Society of America 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 chromo- some (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 chro- mosome 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.