ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

Background

Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan''s syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.

Methodology/Principal Findings

As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2^fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.

Conclusions

These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.     

Chromosomal locations of the mouse fatty acid oxidation genes Cpt1a, Cpt1b, Cpt2, Acadvl, and metabolically related Crat gene

Mitochondrial β-oxidation of long-chain fatty acids (LCFA) is essential for mammalian life. Because portions of this metabolic pathway are composed of enzymes that are coordinately regulated and share structural and functional similarities, we evaluated five of these enzyme genes for possible chromosomal linkages. Regulation of LCFA catabolism influences cell signal pathways and apoptosis, as well as energy production from LCFA. Partial cDNA fragments of the mouse mitochondrial proteins carnitine acetyltransferase (Crat), very-long-chain acyl coenzyme A dehydrogenase (Acadvl), the liver and muscle isoforms of carnitine acyltransferase I (Cpt1a and Cpt1b respectively), and a genomic PCR product of mitochondrial protein carnitine acyltransferase II (Cpt2) were used in a previously established mapping panel to determine their chromosomal locations. No pseudogenes were detected for any of the genes in Mus musculus, and all of the genes mapped to different chromosome locations, including the tissue-specific isoforms of carnitine palmitoyltransferase. Crat mapped to Chromosome (Chr) 2, at a position approximately 18 cM from the centromere and 2 cM proximal to the gene Ass1. Acadvl mapped to the middle of Chr 11, 8.3 cM distal to Il4 and 2.8 cM proximal to Mpmv2. Cpt1a mapped to the centromeric region of Chr 19, 8.7 cM proximal to Pomc-ps1. Cpt1b mapped to Chr 15, 4.9 distal to Gpt1 and 3.5 cM proximal to Wnt1. Cpt2 mapped to Chr 4 near the locus Pmv19. Received: 29 January 1998 / Accepted: 25 March 1998     

Scraggly, a new hair loss mutation on mouse Chromosome 19

By use of chlorambucil, we have generated a mouse mutation called scraggly (sgl) that exhibits skin and hair defects. Homozygous mutant mice exhibit hair loss, skin defects, and abnormalities in sebaceous lipid composition. We have constructed a high-resolution genetic map of mouse Chromosome (Chr) 19 that links this mutation to the anonymous DNA marker D19Umi1. An additional cross, (BALB/c × CAST/Ei) F₁× BALB/c, was used to map markers around this mutation as well as to map the potential candidate genes, Fgf8 and Cyp17. Allelism tests between sgl and asebia (ab), another hair loss mutation on mouse Chr 19, showed that these genes were separate and distinct. Received: 8 December 1998 / Accepted: 10 May 1999     

Linkage of phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, on the mouse X Chromosome

The X Chromosome (Chr) genes for phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, were mapped on the mouse X Chr with interspecific backcrosses between C57BL/6 (B6) and M. spretus (S). Southern analysis showed that Prps1 mapped between Plp and DXWas31, a mouse X Chr region that is homologous to Xq21-24 on the human X Chr while Prps2 mapped between DXWas31 and Amg, a region that is homologous to the map position of PRPS2 on Xp22 of the human X Chr. Additionally, other restriction fragments highlighted by PRS II showed autosomal segregation. In situ hybridization and FISH analysis of metaphase chromosome spreads prepared from lymphocytes of B6 or S male mice confirmed that there were in fact two different locations on the X Chr, X F1-2 and X F2-3 for Prps1 and 2 respectively, as well as two autosomal sites for Prps-like genes.     

Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

The mouse homolog of FRG1, a candidate gene for FSHD, maps proximal to the myodystrophy mutation on chromosome 8

The human autosomal dominant neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) is associated with deletions within a complex tandem DNA repeat (D4Z4) on Chromosome (Chr) 4q35. The molecular mechanism underlying this association of FSHD with DNA rearrangements is unknown, and, thus far, no gene has been identified within the repeat. We isolated a gene mapping 100 kb proximal to D4Z4 (FSHD Region Gene 1:FRG1), but were unable to detect any alterations in total or allele-specific mRNA levels of FRG1 in FSHD patients. Human Chr 4q35 exhibits synteny homology with the region of mouse Chr 8 containing the gene for the myodystrophy mutation (myd), a possible mouse homolog of FSHD. We report the cloning of the mouse gene (Frg1) and show that it maps to mouse Chr 8. Using a cross segregating the myd mutation and the European Collaborative Interspecific Backcross, we showed that Frg1 maps proximal to the myd locus and to the Clc3 and Ant1 genes. Received: 24 September 1996 / Accepted: 7 February 1997     

A new mouse model for marfan syndrome presents phenotypic variability associated with the genetic background and overall levels of Fbn1 expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19-24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.     

Genetic mapping near the myd locus on mouse Chromosome 8

Myodystrophy (myd), an autosomal recessive mutation of the mouse characterized by progressive weakness and dystrophic muscle histology, maps to the central portion of Chromosome (Chr) 8 (Lane et al. J. Hered 67, 135, 1976). This portion of Chr 8 contains the genes for a mitochondrial uncoupling protein (Ucp) and kallikrein (Kal3), which map to distal 4q in the human, providing evidence for a segment of homology. Characteristics of the myd phenotype coupled with this homology suggest that myd may be a mouse homolog of facioscapulohumeral muscular dystrophy (FSHD), which maps to human 4q35. We have confirmed and expanded the region of mouse 8-human 4 homology by generating a map of Chr 8 in an interspecific backcross of C57BL/6J and a partially inbred strain derived from M. spretus. The map is comprised of the genes for Ucp, coagulation factor XI (Cf11), and chloride channel 5 (Clc5), all of which have homologs on distal human 4q, 15 microsatellite loci, and the membrane cofactor protein pseudogene (Mcp-ps). To place myd in the genetic map, 75 affected progeny from an intersubspecific backcross of animals heterozygous for myd with Mus musculus castaneus were genotyped with Chr 8 microsatellite loci. The mutation maps between D8Mit30 and D8Mit75, an interval that is flanked by genes with human homologs at distal 4q. These results are consistent with the possibility that myd is the mouse homolog of FSHD.     

Localization of the IGHG,PRKACB, and TNP2 genes in pigs by in situ hybridization

The porcine genes encoding the immunoglobulin gamma heavy chain (IGHG), cAMP-dependent protein kinase catalytic beta subunit (PRKACB), and transition protein 2 (TNP2) were mapped to Chromosomes (Chrs) 7 q25–q26, 6q31–q33, and 3p13-cent, respectively, by in situ hybridization. Localization of the IGHG gene confirms the assignment of linkage group III to Chr 7. Our results show that the IGHG locus in pigs, similar to the situation in other mammalian species, viz. humans, mouse, cattle, and river buffaloes, is located on the terminal region of the chromosome. The assignment of the PRKACB gene extends the homology observed between porcine Chr 6q and human Chr 1p. Mapping of the TNP2 gene provides the first marker assigned to the p arm of Chr 3 in pigs. The present study contributes to the development of the physical gene map in pigs and also bears significance in terms of comparative gene mapping.     

Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO,ACY, NP,CKBB, ADH2, and ME1 loci in mink (Mustela vison)

To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.     

A new mutation Rim3 resembling Re den is mapped close to retinoic acid receptor alpha (Rara) gene on mouse Chromosome 11

A new mouse mutation, recombination-induced mutation 3 (Rim3), arose spontaneously in our mouse facility. This mutation exhibits corneal opacity as well as abnormal skin and hair development resembling rex denuded (Re ^den ) and bareskin (Bsk). Large-scale linkage analysis with two kinds of intersubspecific backcrosses revealed that Rim3 is mapped to the distal portion of Chromosome (Chr) 11, in which Re ^den and Bsk have been located, and is very close to the retinoic acid receptor, alpha (Rara). The genes, keratin gene complex-1, acidic, gene 10, 12 (Krt1-10, 12), granulin (Grn), junctional plakoglobin (Jup) and Rara, all of which regulate growth and differentiation of epithelial cells, are genetically excluded as candidate genes for Rim3, but are clustered in the short segment on mouse Chr 11. Received: 3 July 1997 / Accepted: 26 August 1997     

Assignment of the rat genes coding for DOPA decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2)

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5-phosphate (PLP)-dependent decarboxylases—namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)—were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)-Tpl2-Gad2 on the rat Chr 17.     

Genetic mapping of the human and mouse phospholipase C genes

To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human × rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8. Received: 24 January 1996 / Accepted: 2 April 1996     

Thirteen loci physically assigned to sheep Chromosome 2 by cell hybrid analysis and in situ hybridization

Sheep x hamster cell hybrids containing sheep metacentric Chromosome (Chr) 2 were produced by fusing blood leukocytes from normal sheep with hamster auxotrophic Ade F-minus mutants. Cell clones that were isocitrate dehydrogenase 1 (IDH1) positive were cytogenetically characterized, confirming that they contained sheep Chr 2. The following loci were newly assigned by Southern hybridization to sheep Chr 2: lipoprotein lipase (LPL), glycoprotein-4-beta galactosyltransferase 2 (GGTB2), neurofilament light polypeptide (68 kDa; NEFL), surfactant-associated protein 2 (SFTP2), lymphocyte-specific protein tyrosine kinase (LCK), and nebulin (NEB). These new assignments and the in situ localization of gelsolin (GSN) to sheep Chr 2pter-p24 are consistent with the predicted homology of cattle Chr 8 (U18) with sheep Chr 2p, and of cattle Chr 2 (U17) with sheep 2q. In addition, the assignment by cell hybrid analysis of loci previously mapped to Chr 2 in sheep, viz., cholinergic receptor, nicotinic, delta polypeptide (CHRND), collagen type III alpha 1 (COL3A1), fibronectin 1 (FN1), isocitrate dehydrogenase (IDH1), and villin 1 (VIL1), confirmed the localization of sheep syntenic group U11 to this chromosome. By nutritional selection and complementation of the hamster auxotrophic Ade F mutation, the multifunctional enzyme locus phosphoribosylaminoimidazolecarboxamide formyltransferase (AICAR transformylase)/IMP cyclohydrolase (inosinicase) (provisionally given the symbol PRACFT) has also been newly assigned to sheep Chr 2. This report significantly extends the number of loci physically mapped to sheep Chr 2 and confirms its close homology with cattle Chrs 2 and 8.     

Localization of growth arrest-specific genes on mouse Chromosomes 1, 7, 8, 11, 13, and 16

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.     

Chromosomal mapping in the mouse of eight K-channel-genes representing the four Shaker-like subfamilies Shaker, Shab, Shaw, and Shal

The four Shaker-like subfamilies of Shaker-, Shab-,Shaw-, and Shal-related K⁺ channels in mammals have been defined on the basis of their sequence homologies to the corresponding Drosophila genes. Using interspecific backcrosses between Mus musculus and Mus spretus, we have chromosomally mapped in the mouse the Shaker-related K⁺-channel genes Kcna1, Kcna2, Kcna4, Kcna5, and Kcna6; the Shab-related gene Kcnb1; the Shaw-related gene Kcnc4; and the Shal-related gene Kcnd2. The following localizations were determined: Chr 2, cen-Acra-Kcna4-Pax-6-a-Pck-1-Kras-3-Kcnb1 (corresponding human Chrs 11p and 20q, respectively); Chr 3, cen-Hao-2-(Kcna2, Kcnc4)-Amy-1 (human Chr 1); and Chr 6, cen-Cola-2-Met-Kcnd2-Cpa-Tcrb-adr/Clc-1-Hox-1.1-Myk-103-Raf-1-(Tpi-1, Kcna1, Kcna5, Kcna6) (human Chrs 7q and 12p, respectively). Thus, there is a cluster of at least three Shaker-related K⁺-channel genes on distal mouse Chr 6 and a cluster on Chr 2 that at least consists of one Shaker-related and one Shaw-related gene. The three other K⁺-channel genes are not linked to each other. The map positions of the different types of K⁺-channel genes in the mouse are discussed in relation to those of their homologs in man and to hereditary diseases of mouse and man that might involve K⁺ channels.     

A new spontaneous mouse mutation in the Kcne1 gene

A new mouse mutant, punk rocker (allele symbol Kcne1 ^pkr ), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1 ^pkr mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21. Received: 20 April 2000 / Accepted: 2 June 2000     

Chromosomal and regional localization of the loci for IGKC,IGGC, ALDB,HOXB, GPT,and PRNP in the American mink (Mustela vison): comparisons with human and mouse

Chromosomal localization of the genes for gamma- and kappa-immunoglobulins (IGGC and IGKC, respectively), aldolase B (ALDB), prion protein (PRNP), homeo box B (HOXB), and glutamate pyruvate transaminase (GPT) were determined with the use of mink-rodent hybrid cells. Analysis of segregation of the mink markers and chromosomes in these hybrid cells allowed us to assign the gene for HOXE to Chromosome (Chr) 8, IGGC to Chr 10, PRNP and IGKC to Chr 11, ALDB to Chr 12, and GPT to Chr 14 in mink. Furthermore, using a set of mink-mouse hybrid cells carrying fragments of mink Chr 8 of different sizes, we assigned the gene for HOXB to the pter-p26 region of the short arm of Chr 8. Comparative mapping of the genes of mink, human, and mouse, as well as other mammalian species, demonstrated that the mink genes HOXB, PRNP, ALDB, and IGGC are members of a conserved region shared by many mammalian species in common; the IGKC gene is a member of a conserved region common to carnivores and primates, not rodents; the GPT gene is a member of a syntenic gene group probably unique to the Mustelidae family or carnivores.     

Rat Chromosome 1: Regional localization of seven genes (Slc9a3, Srd5a1, Esr, Tcp1, Grik5, Tnnt3, Jak2) and anchoring of the genetic linkage map to the cytogenetic map

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na⁺/H⁺ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes. Received: 21 March 1997 / Accepted: 3 May 1997