| First Author | Liddell RA | Year | 1994 |
| Journal | Mouse Genome | Volume | 92 |
| Issue | 4 | Pages | 688-690 |
| Mgi Jnum | J:22094 | Mgi Id | MGI:69985 |
| Citation | Liddell RA (1994) Molecular genetic mapping of the lethal spotting (ls) mutation in the mouse. Mouse Genome 92(4):688-690 |
| abstractText | Full text of Mouse Genome contribution: Molecular Genetic Mapping of the Lethal Spotting (ls) Mutation in the Mouse. Rebecca A. Liddell(1), Giselle Thibaudeau(2), Paul Maddison(2) Craig Goldstein(1), Kirk M. McHugh(3), Linda D. Siracusa(1) and William J. Pavan(4). 1Department of Microbiology and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107 2Department of Molecular Biology, Princeton University, Princeton, NJ 08544. 3Department of Anatomy, Pathology, and Cell Biology, Jefferson Medical College, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. 4Laboratory for Genetic Disease Research, National Center for Human Genome Research, N.I.H., 9000 Rockville Pike, Bethesda, MD 20892. INTRODUCTION The embryonic neural crest begins as a ridge of cells on the dorsal surface of the neural tube early in vertebrate development. Once these cells become detached from the neural tube, they migrate extensively and differentiate into numerous cell types including pigmented melanocytes in the skin and the neurons and glial cells of the peripheral nervous system (4). Mouse spotting mutants serve as model systems to study the molecular mechanisms involved in neural crest development. Mice homozygous for the lethal spotting (ls) mutation exhibit a white spotted coat and aganglionic megacolon due to a lack of neural crest derived melanocytes and enteric ganglia in the skin and terminal bowel, respectively (3, 8, 10). Hirschsprung's disease in humans is also characterized as an absence of neural crest derived enteric ganglia in the distal colon (7, 11). Identification of the gene responsible for the ls mutation will be useful for understanding its role in normal neural crest cell development as well as for understanding its role in the disease process. We have established a molecular genetic linkage map of the ls region on mouse Chr 2 using intersubspecific backcross analyses and determined the map position of ls with respect to molecular markers. MATERIALS AND METHODS Mice: Mus castaneus (CAST/Ei) and LS/Le -at ls/at ls mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The three intersubspecific backcrosses used for these studies are: Cross 1 [LS/Le Ðat ls/at ls X (LS/Le -at ls/at ls x CAST/Ei)F1], Cross 2 [(LS/Le -at ls/at ls x CAST/Ei)F1 X LS/Le -at ls/at ls], and Cross 3 [(CAST/Ei x LS/Le - at ls/at ls)Fl X LS/Le - at ls/at ls]. In each cross, the female parent is listed first. Crosses 1 and 2 were performed at Princeton University and the NIH-NCHGR; Cross 3 was performed at the JCI. PCR and Southern blot analyses: Genomic DNA isolations, restriction endonuclease digestions, and Southern transfers were as described (16). Primers and fragment sizes for the D2Mit25, D2Mit52, and D2Mit200 microsatellite markers were as described (5, 6). PCR conditions were as described (9, 13). The resulting PCR fragments were visualized by ethidium bromide staining on 10% polyacrylamide or 3% agarose gels (9, 13). The probe, hybridization and washing conditions for the Plcgl (phospholipase C-gamma 1) gene were as described (12). The BglI restriction fragments identified after Southern blot analysis were 9.2 and 7.9 kb in LS/Le - at ls/at ls and 9.2 and 5.5 kb in CAST/Ei. The presence or absence of the 5.5 kb fragment was used to determine the segregation pattern of Plcg1 alleles. (Both strains also exhibited nonvariant, light fragments of 4.0, 2.3 and 2.1 kb). RESULTS Mapping studies performed previously by Phillips (1966) first showed that the ls mutation resided between the visible agouti (a) and ragged (Ra) mutations on mouse Chr 2. The genetic distances reported were 25.7 cM between a and ls, and 3.1 cM between ls and Ra, with the most likely position for ls being between a and Ra (14). Further studies by Beechey (1989) and Beechey, Peters and Ball (1992) confirmed the placement of ls distal to a with distances of 43.6 +/- 6.7 cM, 20.0 +/- 6.8 cM and 23.3 +/- 3.3 cM between the two loci. The results suggested that the ls mutation most likely resides distal to the T1Go and T2Wa translocations, based on cytogenetic localization of the T1Go and T2Wa G-band breakpoints to 2H3. Interval mapping was used to limit and define the region containing the ls mutation. The region distal to a was divided into five intervals to determine the specific location of the ls mutation. The intervals and markers chosen were: 1) a -Plcgl, 2) Plcgl - D2Mit52, 3) D2Mit52 - D2Mit25, 4) D2Mit25 - D2Mit200, and 5) D2Mit200 - telomere. TABLE 1. Linkage of the 1s mutation to molecular markers on mouse Chr 2. Crosses 1 and 2; Pairwise loci combinations(a): a Ð D2Mit52; Recombinants/Total: 7/100; Distance (cM +/- s.e.): 7.0 +/- 2.5. Cross 3; Pairwise loci combinations(a): a Ð Plcg1; Recombinants/Total: 4/100; Distance (cM +/- s.e.): 4.0 +/- 2.0; Cross 3; Pairwise loci combinations(a): Plcg1 Ð D2Mit52; Recombinants/Total: 2/100; Distance (cM +/- s.e.): 2.0 +/- 1.4. Crosses 1 and 2; Pairwise loci combinations(a): D2Mit52 Ð D2Mit25; Recombinants/Total: 6/100; Distance (cM +/- s.e.): 6.0 +/- 2.4. Cross 3; Pairwise loci combinations(a): D2Mit52 Ð D2Mit25; Recombinants/Total: 4/100; Distance (cM +/- s.e.): 4.0 +/- 2.0. Crosses 1 and 2; Pairwise loci combinations(a): D2Mit25 Ð ls; Recombinants/Total: 2/100; Distance (cM +/- s.e.): 2.0 +/- 1.4. Cross 3; Pairwise loci combinations(a): D2Mit25 Ð ls; Recombinants/Total: 3/100; Distance (cM +/- s.e.): 3.0 +/- 1.7. Crosses 1 and 2; Pairwise loci combinations(a): ls Ð D2Mit200; Recombinants/Total: 3/100; Distance (cM +/- s.e.): 3.0 +/- 1.7. Cross 3; Pairwise loci combinations(a): ls Ð D2Mit200; Recombinants/Total: 1/100; Distance (cM +/- s.e.): 1.0 +/- 1.0. aThe most proximal locus is listed first. Loci were ordered by arranging to obtain the least number of multiple crossovers; no double crossovers were found with the loci order shown. A molecular probe for Plcgl was used in Southern blot analyses to determine the segregation pattern of this locus. The segregation patterns of the D2Mit25, D2Mit52, and D2Mit200 loci were determined by PCR analyses. The a locus was typed by observation of coat color. The segregation pattern of the ls mutation was determined by examining the coat color of the same N2 offspring. N2 progeny were typed as homozygous for ls if they had white spots on their ventrums as well as their dorsums. One hundred N2 progeny from each intersubspecific backcross were typed for these loci. The order of the loci, the number of recombinants between the loci, and the genetic distances between the loci are given in Table 1. The data from all three crosses were combined to provide a single linkage map (Figure 1). DISCUSSION The results provide the first determination of the order and genetic distance of the ls mutation with respect to molecular markers on mouse Chr 2. The data demonstrate that the ls mutation resides within a -4.5 cM interval bounded on the proximal side by the D2Mit25 locus and on the distal side by the D2Mit200 locus (Table 1 and Figure 1). The overall genetic distance between the a and ls loci is consistent with previously published reports (2, 14). In addition, the distances between marker loci are comparable to those from other crosses and from the consensus linkage map (15). Therefore, the ls mutation does not appear to result from a major genomic rearrangement, such as an inversion, which would significantly alter recombination distances in crosses containing the mutation. The collective mapping data place ls in the Chr 2H3-4 region, which is within the imprinted region of mouse Chr 2 and is consistent with prior localization using cytogenetically visible translocations (1, 2). The position of the ls mutation places it in a region syntenic with human Chr 20. Therefore, the human homolog of the ls mutation most likely resides on human Chr 20q12-13. This region of the human genome may thus harbor one of the genes responsible for the development of Hirschsprung's disease. FIGURE 1. (Legend). Consensus linkage map of mouse Chr 2 compared to the molecular genetic linkage map surrounding the ls mutation. The maps above are A) a partial consensus linkage map of mouse Chr 2 (15) and B) a combined linkage map of the results of the three intersubspecific backcrosses used for these studies (see Materials and Methods) with distances between the loci given in centiMorgans. Underlined loci have been localized in the human genome and their positions on human chromosomes are shown in C). Genes in boxes were also localized on the cytogenetic map (shown to the far left) by in situ hybridization. Their cytogenetic locations along with selected translocations and inversions are indicated by dotted lines and brackets. Maps A) and B) were aligned at the Picgl locus. The ls mutation has been circled for clarity. ACKNOWLEDGEMENTS We thank Dr. Shirley M. Tilghman for advice and support. Research supported in part by NIH grants HD27252 to KMM and DK45717 to LDS. WJP and LDS are grateful for continued support from the ACS. REFERENCES 1. Beechey, C.V. (1989) Mouse News Letter 84: 85-86. 2. Beechey, C.V., J. Peters and S.T. Ball (1992) Mouse Genome 90: 423-424. 3. Bolande, R.P. and W.F. Towler (1972) Am. J. Pathol. 69:139-162. 4. Bronner-Fraser, M. (1993) Bioessays 15: 221-230. 5. Dietrich, W. et al. (1992) Genetics 131: 423-447. 6. Dietrich, W. et al. (1993) Genetic maps: S.J. O'Brien, ed. CSHLP, NY. 7. Kapur, R.P. (1993) Pediatric Pathology 13: 83-100. 8. Lane, P.W. (1966) J. Hered. 57: 29-31. 9. Ma, Q. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6350-6354. 10. Mayer, T.C. and E.L. Maltby (1964) Dev. Biology 9: 269-286. 11. Meier-Ruge, W. (1974) Curr. Top. Pathol. 79: 131-179. 12. Nelson, K.K., J.L. Knopf and L.D. Siracusa (1992) Mammalian Genome 3: 597-600. 13. Pavan, W.J. and S.M. Tilghman (1994) Proc. Natl. Acad. Sci. USA 91: 7159-7163. 14. Phillips, R.J.S. (1966) Mouse News Letter 34: 27. 15. Siracusa. L.D. and C.M. Abbott (1993) Mammalian Genome 4: S31-S46. 16. Siracusa, L.D. et al. (1989) Genetics 122: 669-679. |
