The Journal of Heredity 2002:93(3)
© 2002 The American Genetic Association 93:210-213
Brief Communication |
The Charles River "Hairless" Rat Mutation Maps to Chromosome 1: Allelic With Fuzzy and a Likely Orthologue of Mouse Frizzy
From the Department of Biological Sciences, Central Connecticut State University, 1615 Stanley Street, New Britain, CT 06050.
Address correspondence to T. R. King at the above address.
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Abstract |
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Recent evidence has indicated that the recessive mutation affecting hypotrichosis in the Charles River (CR) "hairless" rat does not involve the hairless gene (hr) on rat chromosome 15. To determine if this mutation might be allelic (or orthologous) with any other previously mapped hypotrichosis-generating mutation in mammals, we have produced a panel of backcross rats segregating for the CR hairless rat mutation as well as numerous other markers from throughout the rat genome. Analysis of this panel has located the CR hairless rat's hypotrichosis-generating mutation on chromosome 1, near Myl2, where only the fuzzy mutation in rat (fz) and the frizzy mutation in mouse (fr) have been previously localized. Intercrossing fz/fz and CR hairless rats produced hybrid offspring with abnormal hair, showing that these two rat mutations are allelic. We suggest that the CR hairless rat mutation and fuzzy be renamed frizzy-Charles River (frCR) and frizzy-Harlan (frH), respectively, to reflect their likely orthology with the mouse fr mutation.
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Introduction |
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More than a dozen mutations have been reported that generate recessive hypotrichosis in rats (reviewed by Ferguson et al. 1979; Hanada et al. 1988; Hedrich 1990; Moemeka et al. 1998; Robinson 1979). Among these, a molecular defect has been identified only for the Rowett and New Zealand nude mutations, which have been found to be the result of a defect in the winged-helix protein-encoding gene, whn (Nehls et al. 1994). Several other hypotrichosis-generating rat mutations have been genetically mapped (e.g., Greaves and Ayres 1985; Hall et al. 2000) or histologically characterized (e.g., Ferguson et al. 1979; Hanada et al. 1988; Palm and Ferguson 1976), but for most, genetic and histologic data are limited or absent. Indeed, several named hypotrichotic rat mutants are likely to be extinct. Thus it has been difficult to determine for many of the named hypotrichosis-generating rat mutations if any might be alleles of one another, or whether any are potential orthologues of hypotrichotic mutations identified in other mammals.
One of the more widely used hypotrichotic rat models is the Charles River (CR) "hairless" rat, which is commercially available from Charles River Laboratories (Wilmington, MA). While the skin morphologic features of mutant CR hairless rats have recently been described (Panteleyev and Christiano 2001), the genetic basis of this variant remains unknown. Indeed, recent molecular analyses (Panteleyev and Christiano 2001) have shown definitively that the CR hairless rat mutation, in spite of its designation in the literature as hr, is not an allele of the cloned and characterized hairless gene (hr; Thompson 1996; GenBank accession no. U71293). While disruption of the hr gene in both mice and humans results in hypotrichosis (Cachon-Gonzalez et al. 1999; Panteleyev et al. 1998), no mutation in this gene on rat chromosome 15 has yet been reported.
To determine if the CR hairless mutation might identify a novel genetic locus with a critical function in the normal development of a hairy mammalian coat, we have produced a panel of 86 backcross rats segregating for the CR hairless mutation as well as numerous polymorphic microsatellite markers from throughout the rat genome (Brown et al. 1998; Jacob et al. 1995; Steen et al. 1999). Analysis of this backcross panel has allowed us to genetically map the CR hairless mutation to rat chromosome 1, in the region where fuzzy has been previously mapped in the Norway rat (Ferguson et al. 1979; Palm and Ferguson 1976). Complementation testing has further revealed that the fuzzy and CR hairless mutations do not complement, but are instead allelic.
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Materials and Methods |
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Outbred CR hairless rats [Crl:CD(SD)-hrBR, designated herein as CR] were obtained from Charles River Laboratories (Wilmington, MA). Inbred Brown Norway (BN/SsNHsd, designated herein as BN) and outbred Fuzzy rats (Hsd:FUZZY-fz) were obtained from Harlan (Indianapolis, IN).
For outcrossing, a single, pigmented BN (C/C) male was crossed with a single, albino CR hairless rat female (c/c). Three hybrid (CR x BN) F1 females were then backcrossed to three CR hairless males to produce 31, 24, and 10 offspring. In addition, two hybrid (CR x BN) F1 males were backcrossed to two CR hairless females to produce 11 and 10 offspring. Backcross rats were scored visually for sex, pigmentation, and hair morphology. All parents and progeny were then killed, and organs (spleen, liver, and kidney) were harvested and stored at -70°C.
Genomic DNA was extracted from the frozen spleen of individual rats according to the method of Jenkins et al. (1982). Dinucleotide repeat DNA markers were typed in 13 µl amplification reactions using 75 ng genomic DNA as a template, 0.2 µM forward and reverse primers (MapPairs from Research Genetics, Huntsville, AL), 0.2 µM dNTPs, Titanium Taq DNA polymerase, and reaction buffer as supplied by the enzyme manufacturer (Clontech Laboratories, Palo Alto, CA). Parameters for amplification reactions were 95°C for 25 s, 60–68°C for 20 s, 68°C for 10 s for 35 cycles, followed by 68°C for 3 min. To visualize products, 10–13 µl aliquots were electrophoresed through 3–4% NuSieve 3:1 agarose gel (BioWhittaker Molecular Applications; Rockland, ME) in TBE buffer with 0.1 µg/ml ethidium bromide. Genetic distances based on backcross data are presented as percentage recombination ± 1 standard error.
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Results |
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Backcrossing (CR x BN) F1 rats to CR hairless rats produced 86 progeny; 33 were hypotrichotic and 53 with normal hairy coats (deviation from the expected 1 bald:1 hairy ratio is significant;
2 = 4.65; P 
0.03). A similar non-Mendelian ratio has also been reported by Panteleyev and Christiano (2001) in a CR hairless rat testcross (which produced only 39% mutants), and apparently reflects the poor viability of the mutant class. In addition, mutant mothers—while initially producing litters of normal size (9.1 ± 4.6 born versus 12.2 ± 2.1 born from F1 mothers)—raised only small numbers of progeny to weaning (2.0 ± 2.4 weaned versus 10.8 ± 1.8 weaned from F1 mothers; based on 10 and 6 litters, respectively).
Immediate scoring of visible markers in this 86-member backcross panel showed that inheritance patterns for the CR hairless mutation and albino—73 parentals (hairy and pigmented, or hypotrichotic and albino) and 13 recombinants (hairy and albino, or hypotrichotic and pigmented)—deviated dramatically from the 1 parental:1 recombinant ratio predicted by independent assortment (2 = 41.9; P < .0001). This suggested a location for the CR hairless mutation on chromosome 1, 15.1 ± 3.9% recombination from the albino locus.
To refine the map location for the CR hairless mutation, DNA was isolated from these 86 backcross rats and typed for polymerase chain reaction (PCR)-scorable DNA markers known to reside on rat chromosome 1. (Numerous microsatellite markers known to map in our region of interest, including all 35 microsatellite markers listed between D1Rat360 and D1Arb22 in the [SHRSP x BN] F2 panel [Rat Genome Data 2001] were tested for polymorphism, but the majority were found to be monomorphic in our cross.) Segregation patterns for all polymorphic markers typed among the 86-member backcross panel are shown in Figure 1. This analysis placed the CR hairless mutation between D1Rat285, -361 (on the centromeric flank) and D1Rat440 (on the telomeric flank). The CR hairless mutation was not separated from D1Arb29, D1Mit13 (alias for myosin, light polypeptide 2, alkali; Myl2), D1Rat219, -287, or -438 in this backcross panel.
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This location suggested possible allelism of the CR hairless mutation and the rat fuzzy mutation (fz; Ferguson et al. 1979; Palm and Ferguson 1976), since fz has been mapped close to warfarin resistance (Rw) in rats (0.0% recombination; Greaves and Ayres 1985), and warfarin resistance in mice (War) has not been separated from sialophorin (Spn; Mouse Genome Database 2001), which has not been separated from Myl2 in rat (Jacob et al. 1995). Therefore crosses between fuzzy females and CR hairless males were conducted to assess complementation. The 27 resulting hybrid progeny (from two litters) displayed curly whiskers at birth and failed to develop normal hair (see Figure 2), indicating that these mutations do not complement. Thus the CR hairless mutation is allelic with the rat fuzzy mutation. It is perhaps noteworthy that these two fuzzy females successfully reared 25 of their 27 hybrid (mutant) progeny, in contrast with the routinely poor survival seen among litters raised by mutant CR hairless mothers.
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Discussion |
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The CR hairless mutation maps to rat chromosome 1 near D1Arb29, Myl2, D1Rat219, -287, and -438 (see Figure 3), and is an allele of rat fz. It would seem that mutations at this site have been identified numerous times in the rat, yielding recessive hypotrichosis of differing degrees (Ferguson et al. 1979; Palm and Ferguson 1976; Panteleyev and Christiano 2001). This region of rat chromosome 1 shares homology with chromosome 7 in mouse (Mouse Genome Database 2001; see Figure 3), where the recessive coat-morphology mutation frizzy (fr; Snell 1951) has previously been mapped (Falconer and Snell 1952; MacSwiney and Wallace 1978; Wallace and MacSwiney 1976). Thus rat fz and mouse fr are likely orthologues, as has already been suggested by Greaves and Ayres (1985). Unfortunately another—clearly distinct—hypotrichosis-generating mutation mapping to mouse chromosome 1 is also named fuzzy (fz). For the sake of clarity, we suggest that both the CR hairless and the rat fz mutations be renamed frizzy-Charles River (frCR) and frizzy-Harlan (frH), respectively, to reflect their likely orthology with (and priority of) the mouse fr mutation, as well as to distinguish this locus from that identified by mouse fz. We recommend distinct allele designations for these commercially available rat fr mutations, especially because they control very distinct phenotypes (in terms of coat morphology, postnatal mutant viability, and mutant maternal success) and are therefore likely to have distinct mutational bases. Nomenclature revision for other rat "fuzzy" alleles (see, e.g., Ferguson et al. 1979), if such lines remain extant, is similarly advised.
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A molecular assignment for fr is now needed to allow a detailed functional analysis of its role in both the normal and disrupted development of the mammalian integument. The map location for fr in both rat and mouse suggests a number of potential candidates for the gene affected by these mutations (see Mouse Genome Database 2001). Perhaps the most compelling among these candidates is the fibroblast growth factor receptor 2 gene (Fgfr2), which has been shown to be expressed in hair follicles (see Figure 4F in Makarenkova et al. 2000). Evaluation of this and any other colocalizing fr candidates should be facilitated by the availability of at least three mutant alleles of the fr gene in rodents.
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Acknowledgments |
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The authors thank Drs. K. A. Martin-Troy and J. P. Mulrooney for critical review of the manuscript. This work was supported by a research grant from Connecticut State University.
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Footnotes |
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Corresponding Editor: Muriel T. Davisson
Received January 28, 2002
Accepted March 29, 2002
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References |
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