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BXA recombinant inbred strains of mouse
Mechanisms of Development 91 (2000) 305±309
Short communication
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Mammary gland patterning in the AXB/BXA recombinant inbred strains of mouse Beatrice A. Howard*, Barry A. Gusterson Institute of Cancer Research, The Breakthrough Toby Robins Breast Cancer Research Centre, Section of Cell Biology and Experimental Pathology, 237 Fulham Road, London SW3 6JB, UK Received 16 August 1999; received in revised form 1 October 1999; accepted 5 October 1999
1. Results and discussion In mice, ®ve pairs of mammary glands are distributed in a cranial to caudal sequence on the ventral body wall starting slightly anterior to the forelimbs and extending to the hindlimbs. Little is known about the inductive signals that initiate mammary gland formation and patterning. Mammary glands develop during embryogenesis as a result of reciprocal epithelial±mesenchymal interactions (Cunha et al., 1992; Cunha, 1994; Cunha and Hom, 1996). The mammary glands are arranged as bilaterally symmetric series of organs with no obvious relationship to the basic vertebrate segmentation that occurs earlier during embryogenesis (Bateson, 1894). How the number and spacing of mammary glands is achieved is unknown. De®ciencies and supernumerary mammary glands in inbred strains of mice have been reported and were postulated to have a genetic basis (Gardner and Strong, 1935; Little and McDonald, 1965). We have shown that one major autosomal recessive trait is responsible for the scaramanga (ska) mutation, which is involved in the determination of pattern formation of the mammary gland in mouse (Howard and Gusterson, 2000). The ska mutation was characterized in an inbred strain of mice (A/J) that displays supernumerary mammary glands and nipples, misplaced nipples, and mammary gland de®ciencies. We report here the results of genetic studies in this system using recombinant inbred (RI) strains produced between the A/J and C57BL/6 (B6) strains. 16 AXB RI and 14 BXA RI strains were obtained from The Jackson Laboratory strains and the mammary gland patterning phenotypes were determined (The Jackson Laboratory, Bar Harbor, ME, USA) (Marshall et al., * Corresponding author. Tel.: 144-171-352-8133; fax: 144-171-3525241. E-mail address: [email protected] (B.A. Howard)
1992). We have characterized the phenotypic variation of the ska mammary gland pattern mutant phenotype within the A/J, B6, and 30 AXB/BXA RI strains of mice (Table 1). ska mammary gland pattern abnormalities include absent #3 glands, misplaced #3 glands, and supernumerary nipples connected to a functional, milk-producing ductal system that is distinct from that of the normally occurring gland (Fig. 1). All 75 B6 mice displayed a normal mammary gland pattern, consisting of 5 pairs of glands spaced linearly and bilaterally symmetrically along the body's ventral surface. 95.2 percent of A/J mice (40/42) displayed abnormal mammary gland patterns. Penetrance levels of the total ska mammary gland pattern mutant phenotypes were similar to those of the parental strains, except in the BXA-17 strain where it displayed an intermediate value of 45.5 percent. This suggests the presence of modi®er loci that affect the probability of whether abnormal mammary gland patterns will form or not. The distribution of mammary gland pattern phenotypes varies greatly throughout the AXB/BXA RI set which strongly suggests that modi®er genes also in¯uence the type of phenotype that is likely to arise in that genetic background. It should be possible to map such factors to better understand the nature of the process of mammary gland development. The variability of the ska mammary gland mutant phenotypes within genetically identical backgrounds suggest that genes affecting mammary gland development may be especially prone to modi®cation by other genes and environmental factors. A human syndrome with similar variable mammary phenotype exists. Mutations in the human TBX3 gene (a member of the T-box gene family) cause ulnarmammary syndrome, a pleiotropic disorder (Bamshad et al., 1997). Women and men with ulnar-mammary syndrome may have hypoplastic, absent or extra mammary structures as well as abnormal limb, apocrine gland, tooth, and genital development (Bamshad et al., 1997). Another human disorder with pleiotropic genetic features is limb mammary
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00268-3
306
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
Table 1 Mammary patterning phenotypes of A/J, C57BL/6 and 30 AXB/BXA recombinant inbred strains a Strain
No. of females observed
No. of ska MG pattern phenotypes (percent of total)
No. of super-numerary nipple or MG (percent of total)
No. of misplaced MG (percent of total)
No. of absent #3 MG (percent of total)
No. of normal MG pattern phenotype (percent of total)
A/J C57BL/6 AXB-1 AXB-2 AXB-4 AXB-5 AXB-6 AXB-8 AXB-10 AXB-12 AXB-13 AXB-14 AXB-15 AXB-18 AXB-19 AXB-20 AXB-23 AXB-24 BXA-1 BXA-2 BXA-4 BXA-7 BXA-8 BXA-11 BXA-12 BXA-13 BXA-14 BXA-16 BXA-17 BXA-24 BXA-25 BXA-26
42 75 9 10 9 5 21 24 3 6 4 3 13 25 14 17 4 10 32 14 19 8 1 29 4 11 17 14 11 34 7 2
40 (0.95) 0 (0.00) 8 (0.89) 10 (1.00) 9 (1.00) 0 (0.00) 17 (0.81) 0 (0.00) 3 (1.00) 0 (0.00) 4 (1.00) 3 (1.00) 0 (0.00) 22 (0.88) 14 (1.00) 11 (0.65) 4 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 19 (1.00) 6 (0.75) 1 (1.00) 0 (0.00) 0 (0.00) 11 (1.00) 17 (1.00) 14 (1.00) 5 (0.45) 34 (1.00) 7 (1.00) 2 (1.00)
13 (0.31) 0 (0.00) 1 (0.11) 3 (0.30) 3 (0.33) 0 (0.00) 6 (0.29) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.04) 1 (0.07) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 6 (0.32) 2 (0.25) 0 (0.00) 0 (0.00) 0 (0.00) 2 (0.18) 10 (0.59) 7 (0.50) 0 (0.00) 10 (0.29) 1 (0.14) 1 (0.50)
12 0 7 10 9 0 15 0 1 0 4 3 0 20 14 13 3 0 0 0 15 6 1 0 0 8 14 13 5 29 7 1
23 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 1 0 0 0 2 0 0 0 0 3 2 0 0 0 0 0
2 (0.05) 75 (1.00) 1 (0.11) 0 (0.00) 0 (0.00) 5 (1.00) 4 (0.19) 24 (1.00) 0 (0.00) 6 (1.00) 0 (0.00) 0 (0.00) 13 (1.00) 3 (0.12) 0 (0.00) 6 (0.35) 0 (0.00) 10 (1.00) 32 (1.00) 14 (1.00) 0 (0.00) 2 (0.25) 0 (0.00) 29 (1.00) 4 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 6 (0.55) 4 (0.12) 0 (0.00) 0 (0.00)
(0.29) (0.00) (0.78) (1.00) (1.00) (0.00) (0.71) (0.00) (0.33) (0.00) (1.00) (1.00) (0.00) (0.80) (1.00) (0.76) (0.75) (0.00) (0.00) (0.00) (0.79) (0.75) (1.00) (0.00) (0.00) (0.73) (0.82) (0.93) (0.45) (0.85) (1.00) (0.50)
(0.55) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.67) (0.00) (0.00) (0.00) (0.00) (0.04) (0.00) (0.00) (0.25) (0.00) (0.00) (0.00) (0.11) (0.00) (0.00) (0.00) (0.00) (0.27) (0.12) (0.00) (0.00) (0.00) (0.00) (0.00)
a Results are expressed as the number of female mice observed with mammary gland patterning abnormalities and the percent of each class compared to the total number of mice observed. MG, mammary gland.
syndrome (LMS) (van Bokhoven et al., 1999). LMS patients may have hypoplasia or aplasia of the mammary gland and nipple and severe hand and/or foot anomalies. We have not observed any other developmental defects linked to the ska mutant phenotype. The mammary gland pattern was examined in several mice from each of the 30 available AXB/BXA series of RI strains to obtain a strain distribution pattern (SDP) for the proposed ska locus (Table 2). Consistent with the single locus hypothesis, abnormal mammary gland patterning was found in 21 of the 30 RI strains (17 out of 26 of the independent lines). The bimodal SDP of presence or absence of abnormal mammary gland patterns among the RI strains suggest the presence of a major locus controlling the patterning of mammary glands, with allelic differences presumably resulting in normal or abnormal mammary gland patterns. Linkage to several markers on chromosome 14 was detected using the MapManager program (Manly, 1993). Strongest linkage (LOD 3.0) was to D14Mit80 at the 99.9% signi®cance level (Chr 14). The SDP for markers on
mouse Chr 14 is displayed according to their most likely order in Table 2 with the best placement for ska between markers D14Mit14 and D14Mit80. Comparison of the SDP of the marker loci, D14Mit14 and D14Mit80, that putatively ¯ank the ska locus, provide further evidence for linkage since mostly single recombination events are seen among the three loci within individual RI strains. Two double recombination event are required (in the AXB-18 and BXA-4 strains) between the three markers. There are no other known microsatellite markers that are polymorphic between A/J and B6 strains that map between this interval. When ska is placed between D14Mit14 and D14Mit80, four crossovers exist between ska and D14Mit80 and seven recombination events are required between ska and D14Mit14. These crossovers will be useful for ®ne-scale mapping when more markers are obtained The ska locus maps to mouse Chr 14 to an interval between the microsatellite markers D14Mit14 and D14Mit80 which are located at 10.0 and 13.5 cM, respectively on the 1999 Chromosomal Committee maps. Our
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
307
Table 2 Strain distribution pattern of ska mammary gland phenotype of 30 AXB/BXA recombinant inbred strains and localization of ska candidate locus on Chr14 a,d,e AXB strain 1 D14Mit207
2
4
BXA strain 5
6
8
10 12 13 14 15 18 19 20 23 24 1 B
B
D14Mit201
A A A B A B B x A A A B A B A
B
B
D14Mit202
A A A B A B A
B
B
D14Mit14 b
A A A B A B A
B
ska
A A A B A B A x x B A A B A B B
B B
B x A x B
B
B
D14Mit80
D14Mit129 c B
A A B A B B
B x A x B
2
4
7
8
11 12 13 14 16 17 24 25 26 Ref. B
A
B
A
B
B
A
B
*
B
A
B
A
A
B
A
B
*
B
B
B
B
B
B
B B B
B
B
B
B
B
B
B
B B B
B
B B x A B
B
B
B
B
B
B B B
B
A B
B
A
B
A
A
B
A
B
*
B x A
B
B x A x B
B
A
A
A
A
A
A
B x A
²
B
B x A
A
B
B x A
A
A
*
A
B
B B B x B B A x B B B
B A B x A A B
B
B x A x B
B
A x B
B x A x B
B x A
A A B
B
A
A
A
A
A
A
A
*
B
B
B
B
A
B
B B B
A A B
B
A
A
A
A
A
A
A
*²
B
a Markers are listed from the centromere. Strains displaying abnormal mammary gland pattern (misplaced nipples, absent glands, supernumerary nipples) were scored as having the ska mutation or A allele. Strains displaying normal mammary gland pattern (ten nipples and glands, bilaterally symmetric) were scored as having the wild-type ska allele or B allele. A denotes the A/J allele. B denotes the B6 allele. x denotes a recombinant chromosome. b D14Mit14, D14Mit44*, D14Mit55*, D14Mit133*, D14Mit173*, D14Mit174*, and D14Mit186* display concordant SDPs. c D14Mit129*²³, Mmp-14*, and Np-2*³, display concordant SDPs. ³Our SDP for D14Mit129 differs from that reported by (Sampson et al., 1998). Heterozygous alleles were observed in genotyping reactions with strains AXB-10 and BXA-26 in their analysis. Our genotype of the BXA-4 strain for Np-2 differs from that reported by (Sampson et al., 1998). *This study. ² Sampson et al. (1998). d SDP for markers typed in the AXB/BXA RI set were obtained from published reports (Higgins and Paigen, 1997; Naggert et al., 1997; Prows and Leikauf, 1998; Sampson et al., 1998) and from the World Wide Web (WWW) (http://mcbio.med.buffalo.ed/mapmgr.html) and (http://www.informatics.jax.org/ riset.html). Polymerase chain reaction (PCR) was performed as described (Dietrich et al., 1994). Primers were obtained from Research Genetics, Inc. (Huntsville, AL, USA). e Genetic linkage was determined by segregation analysis. The mapping data were analyzed with Map Manager Version 2.6.6 (Manly, 1993). Genetic contamination of some AXB/BXA RI lines has been reported so only 27 independent lines now exist (Taylor, 1996). AXB-14, AXB-18 and BXA-17 were used for the genetic analysis while AXB-19, AXB-20 and BXA-8 were omitted to avoid false linkage detection.
analysis has found larger recombination frequencies in the AXB and BXA crosses relative to the standard genetic maps which describe a recombination distance between D14Mit14 and D14Mit80 that is approximately one sixth of what we observed in this study. Different mouse lines are known to have line-speci®c rates of recombination and our data may simply be a re¯ection of this or of errors present in consensus maps, which are not always recombination-based due to the cumulative nature in which they are constructed. ska maps to a region of the genome containing a known mouse mutation, pugnose (pn) which is now extinct (Doolittle et al., 1996). pn mutant mice were reported to have skeletal abnormalities including craniofacial defects and mapped to the same region as Bmp-4 and the Bmpr genes (Doolittle et al., 1996). Bmp4 was excluded as a candidate gene for ska based on observed genetic recombination between D14Mit129 (a microsatellite sequence within the Bmp4 gene) and ska (Kurihara et al., 1993). The Mmp-14 gene was also excluded as a candidate for ska based on observed genetic recombination between a microsatellite contained within this gene and ska (Kurihara et al., 1993). The ska gene maps near a proximal region of mouse Chr 14 which has conserved linkage relationships to that of human Chr 10q. Further genetic characterization of this region will
be needed to determine whether this region of conserved linkage extends to and includes the ska locus. Of the many genes that map to the interval within which the ska locus should lie, the Bmpr is the most intriguing candidate gene for ska since Bmp-2 and Bmp-4 are both expressed during mouse mammary gland development in temporal and spatial patterns that suggest that they may play roles in the control of epithelial- mesenchymal interactions in the mouse mammary gland (Phippard et al., 1996). The Bmpr gene encodes a BMP-2/4 type I serine±threonine kinase transmembrane receptor. The Gdf10 gene is a reasonable candidate since it is a member of the TGFb superfamily and is highly related to Bmp-3 and displays expression patterns that suggest multiple roles in regulating cell differentiation events (Cunningham et al., 1995). BMPs are members of the TGFb superfamily and play key roles in developmental processes such dorsal-ventral axis formation, left-right symmetry, epithelial±mesenchymal interactions, as well as growth, death and differentiation of speci®c tissues (Hogan, 1996; Lemaire and Yasuo, 1998). Knockouts of both the Bmp-4 and Bmpr genes in mice exist and are embryonic lethals and die between embryonic day 6.5 and 9.5, before mammary glands have developed (at day E11.5) (Kratochwil, 1987; Mishina et al., 1995; Winnier et al., 1995). We have localized the ska mutation to mouse Chr 14 by
308
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
analyzing the AXB and BXA recombinant inbred strains of mice. Analysis of genetic variation in mammary gland patterning may provide a means to study mechanisms of mammary gland development and identify the products and modes of action of some of the genes that in¯uence the formation and placement of mammary glands. The
results reported here set the groundwork for understanding the molecular genetics of patterning of the embryonic mammary gland, and also how variable phenotypes can be produced in identical genetic backgrounds. Acknowledgements We thank Philip Gladwell, Jayne Hacker, Jenny Teague, and Mabel Iwobi for their excellent technical assistance. This work was supported by the Cancer Research Campaign. References Bamshad, M., Lin, R.C., Jaw, D.J., Watkins, W.S., Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel, A., Seidman, C.E., Jorde, L.B., 1997. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311±315. Bateson, W., 1894. Materials for the Study of Variation, Macmillan, London. Cunha, G., 1994. Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate. Cancer 74, 1030±1044. Cunha, G.R., Hom, Y.K., 1996. Role of mesenchymal-epithelial interactions in mammary gland development. J. Mamm. Gland Biol. Neoplasia 35, 21±35. Cunha, G.R., Young, P., Hamamoto, S., Guzman, R., Nandi, S., 1992. Developmental response of adult mammary epithelial cells to various fetal and neonatal mesenchymes. Epithelial Cell Biol. 1, 105±118. Cunningham, N.S., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., Reddi, A.H., Lee, S.J., 1995. Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3. Growth Fact. 12, 99±109. Dietrich, W.F., Miller, J.C., Steen, R.G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D.C., Wessel, M., Dredge, R.D., et al., 1994. A genetic map of the mouse with 4006 simple sequence length polymorphisms. Nat. Genet. 7, 220±245. Doolittle, D., Davisson, M.T., Guidi, J.N., Green, M.C., 1996. Catalog of mutant genes and polymorphic loci. In: Lyon, M.F. (Ed.). Genetic Variants and Strains of the Laboratory Mouse, vol. 1. Oxford University Press, Oxford, pp. 17±854. Gardner, W.U., Strong, L.C., 1935. The normal development of the mammary glands of virgin female mice of ten strains varying in susceptibility to spontaneous neoplasms. Am. J. Cancer 25, 282±290. Higgins, D.C., Paigen, B., 1997. An additional 150 SSLP markers typed for Fig. 1. Mammary gland pattern phenotypes observed in AXB/BXA RI strains of mice. (A) Normal pattern of ®ve pairs of mammary glands which are numbered 1±5, anterior to posterior of a lactating adult BXA-7 mouse. (B) ska mutant phenotype of absent right #3 gland of an adult BXA13 mouse. (C) ska mutant phenotype of misplaced #3 nipple in a 7-day-old AXB-2 pup. The right nipple is lower than the left nipple. (D) ska mutant phenotype of bilateral supernumerary #4 nipples in a 10-day-old BXA-14 pup. These nipples are connected to ductal systems which are distinct from the adjacent #4 gland and lactate and are suckled by pups. (E) Supernumerary right #4 nipple adjacent to the #4 nipple in a lactating AXB-4 mouse. Both are distended by suckling by pups. (F) Wholemount analysis of #4 gland from mouse displaying a supernumerary right #4 nipple and ductal system. The normal #4 nipple is labeled N and the supernumerary nipple is indicated by an arrow.
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309 the AXB and BXA recombinant inbred mouse strains. Mamm. Genome 8, 846±849. Hogan, B.L.M., 1996. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580±1594. Howard, B.A., Gusterson, B.A., 2000. The characterization of a mouse mutant that displays abnormal mammary gland development. Mamm. Genome. in press. Kratochwil, K., 1987. Tissue combination and organ culture studies in the development of the embryonic mammary gland. In: Gwatkin, R.B.L. (Ed.). Developmental Biology: A Comprehensive Synthesis, Plenum Press, New York, pp. 315±334. Kurihara, T., Kitamura, K., Takaoka, K., Nakazato, H., 1993. Murine bone morphogenetic protein-4 gene: existence of multiple promoters and exons for the 5 0 -untranslated region. Biochem. Biophys. Res. Commun. 192, 1049±1056. Lemaire, P., Yasuo, H., 1998. Developmental signalling: a careful balancing act. Curr. Biol. 8, 228±231. Little, C.C., McDonald, H., 1965. Abnormalities of the mammae in the house mouse. J. Hered. 36, 285±288. Manly, K.F., 1993. A Macintosh program for storage and analysis of experimental genetic mapping data. Mamm. Genome 4, 303±313. Marshall, J.D., Mu, J.-L., Cheah, Y.-C., Nesbitt, M.N., Frankel, W.N., Paigen, B., 1992. The AXB and BXA set of recombinant inbred mouse strains. Mamm. Genome 3, 669±680. Mishina, Y., Suzuki, A., Ueno, N., Behringer, R.R., 1995. Bmpr encodes a type I bone morphogenetic receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 3027±3037.
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Naggert, J.K., Svenson, K.L., Lin, L., Cheah, Y.-C., Nishina, P.M., Mu, J.L., Devereux, T.R., You, M., Paigen, B., 1997. An additional 136 SSLP markers typed for the AXB and BXA recombinant inbred mouse strains. Mamm. Genome 8, 209±211. Phippard, D., Weber-Hall, S., Sharpe, P.T., Naylor, M.S., Jayatalake, H., Maas, R., Woo, I., Roberts-Clark, D., Francis-West, P.H., Liug, Y.H., Maxsong, R., Hill, R.E., Dale, T.C., 1996. Regulation of Msx-1. Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development. Development 122, 2729±2737. Prows, D.R., Leikauf, G.D., 1998. Genotypes for 66 microsatellite markers in the AXB and BXA recombinant inbred mouse strains. Mamm. Genome 9, 160±161. Sampson, S.B., Higgins, D.C., Elliot, R.W., Taylor, B.A., Lueders, K.K., Koza, R.A., Paigen, B., 1998. An edited linkage map for the AXB and BXA recombinant inbred mouse strains. Mamm. Genome 9, 688± 694. Taylor, B., 1996. AXB/BXA recombinant inbred strains. JAX Notes 465, 3. van Bokhoven, H., Jung, M., Smits, A.P., van Beersum, S., van Steensel, M., Veenstra, M., Tuerlings, J.H., Mariman, E.C., Brunner, H.G., Wienker, T.F., Reis, A., Ropers, H.H., Hamel, B.C., 1999. Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other hand/foot anomalies maps to human chromosome 3q27. Am. J. Hum. Genet. 64, 538±546. Winnier, G., Blessing, M., Labosky, P.A., Hogan, B.L., 1995. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105±2116.
Short communication
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Mammary gland patterning in the AXB/BXA recombinant inbred strains of mouse Beatrice A. Howard*, Barry A. Gusterson Institute of Cancer Research, The Breakthrough Toby Robins Breast Cancer Research Centre, Section of Cell Biology and Experimental Pathology, 237 Fulham Road, London SW3 6JB, UK Received 16 August 1999; received in revised form 1 October 1999; accepted 5 October 1999
1. Results and discussion In mice, ®ve pairs of mammary glands are distributed in a cranial to caudal sequence on the ventral body wall starting slightly anterior to the forelimbs and extending to the hindlimbs. Little is known about the inductive signals that initiate mammary gland formation and patterning. Mammary glands develop during embryogenesis as a result of reciprocal epithelial±mesenchymal interactions (Cunha et al., 1992; Cunha, 1994; Cunha and Hom, 1996). The mammary glands are arranged as bilaterally symmetric series of organs with no obvious relationship to the basic vertebrate segmentation that occurs earlier during embryogenesis (Bateson, 1894). How the number and spacing of mammary glands is achieved is unknown. De®ciencies and supernumerary mammary glands in inbred strains of mice have been reported and were postulated to have a genetic basis (Gardner and Strong, 1935; Little and McDonald, 1965). We have shown that one major autosomal recessive trait is responsible for the scaramanga (ska) mutation, which is involved in the determination of pattern formation of the mammary gland in mouse (Howard and Gusterson, 2000). The ska mutation was characterized in an inbred strain of mice (A/J) that displays supernumerary mammary glands and nipples, misplaced nipples, and mammary gland de®ciencies. We report here the results of genetic studies in this system using recombinant inbred (RI) strains produced between the A/J and C57BL/6 (B6) strains. 16 AXB RI and 14 BXA RI strains were obtained from The Jackson Laboratory strains and the mammary gland patterning phenotypes were determined (The Jackson Laboratory, Bar Harbor, ME, USA) (Marshall et al., * Corresponding author. Tel.: 144-171-352-8133; fax: 144-171-3525241. E-mail address: [email protected] (B.A. Howard)
1992). We have characterized the phenotypic variation of the ska mammary gland pattern mutant phenotype within the A/J, B6, and 30 AXB/BXA RI strains of mice (Table 1). ska mammary gland pattern abnormalities include absent #3 glands, misplaced #3 glands, and supernumerary nipples connected to a functional, milk-producing ductal system that is distinct from that of the normally occurring gland (Fig. 1). All 75 B6 mice displayed a normal mammary gland pattern, consisting of 5 pairs of glands spaced linearly and bilaterally symmetrically along the body's ventral surface. 95.2 percent of A/J mice (40/42) displayed abnormal mammary gland patterns. Penetrance levels of the total ska mammary gland pattern mutant phenotypes were similar to those of the parental strains, except in the BXA-17 strain where it displayed an intermediate value of 45.5 percent. This suggests the presence of modi®er loci that affect the probability of whether abnormal mammary gland patterns will form or not. The distribution of mammary gland pattern phenotypes varies greatly throughout the AXB/BXA RI set which strongly suggests that modi®er genes also in¯uence the type of phenotype that is likely to arise in that genetic background. It should be possible to map such factors to better understand the nature of the process of mammary gland development. The variability of the ska mammary gland mutant phenotypes within genetically identical backgrounds suggest that genes affecting mammary gland development may be especially prone to modi®cation by other genes and environmental factors. A human syndrome with similar variable mammary phenotype exists. Mutations in the human TBX3 gene (a member of the T-box gene family) cause ulnarmammary syndrome, a pleiotropic disorder (Bamshad et al., 1997). Women and men with ulnar-mammary syndrome may have hypoplastic, absent or extra mammary structures as well as abnormal limb, apocrine gland, tooth, and genital development (Bamshad et al., 1997). Another human disorder with pleiotropic genetic features is limb mammary
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00268-3
306
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
Table 1 Mammary patterning phenotypes of A/J, C57BL/6 and 30 AXB/BXA recombinant inbred strains a Strain
No. of females observed
No. of ska MG pattern phenotypes (percent of total)
No. of super-numerary nipple or MG (percent of total)
No. of misplaced MG (percent of total)
No. of absent #3 MG (percent of total)
No. of normal MG pattern phenotype (percent of total)
A/J C57BL/6 AXB-1 AXB-2 AXB-4 AXB-5 AXB-6 AXB-8 AXB-10 AXB-12 AXB-13 AXB-14 AXB-15 AXB-18 AXB-19 AXB-20 AXB-23 AXB-24 BXA-1 BXA-2 BXA-4 BXA-7 BXA-8 BXA-11 BXA-12 BXA-13 BXA-14 BXA-16 BXA-17 BXA-24 BXA-25 BXA-26
42 75 9 10 9 5 21 24 3 6 4 3 13 25 14 17 4 10 32 14 19 8 1 29 4 11 17 14 11 34 7 2
40 (0.95) 0 (0.00) 8 (0.89) 10 (1.00) 9 (1.00) 0 (0.00) 17 (0.81) 0 (0.00) 3 (1.00) 0 (0.00) 4 (1.00) 3 (1.00) 0 (0.00) 22 (0.88) 14 (1.00) 11 (0.65) 4 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 19 (1.00) 6 (0.75) 1 (1.00) 0 (0.00) 0 (0.00) 11 (1.00) 17 (1.00) 14 (1.00) 5 (0.45) 34 (1.00) 7 (1.00) 2 (1.00)
13 (0.31) 0 (0.00) 1 (0.11) 3 (0.30) 3 (0.33) 0 (0.00) 6 (0.29) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.04) 1 (0.07) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 6 (0.32) 2 (0.25) 0 (0.00) 0 (0.00) 0 (0.00) 2 (0.18) 10 (0.59) 7 (0.50) 0 (0.00) 10 (0.29) 1 (0.14) 1 (0.50)
12 0 7 10 9 0 15 0 1 0 4 3 0 20 14 13 3 0 0 0 15 6 1 0 0 8 14 13 5 29 7 1
23 0 0 0 0 0 0 0 2 0 0 0 0 1 0 0 1 0 0 0 2 0 0 0 0 3 2 0 0 0 0 0
2 (0.05) 75 (1.00) 1 (0.11) 0 (0.00) 0 (0.00) 5 (1.00) 4 (0.19) 24 (1.00) 0 (0.00) 6 (1.00) 0 (0.00) 0 (0.00) 13 (1.00) 3 (0.12) 0 (0.00) 6 (0.35) 0 (0.00) 10 (1.00) 32 (1.00) 14 (1.00) 0 (0.00) 2 (0.25) 0 (0.00) 29 (1.00) 4 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 6 (0.55) 4 (0.12) 0 (0.00) 0 (0.00)
(0.29) (0.00) (0.78) (1.00) (1.00) (0.00) (0.71) (0.00) (0.33) (0.00) (1.00) (1.00) (0.00) (0.80) (1.00) (0.76) (0.75) (0.00) (0.00) (0.00) (0.79) (0.75) (1.00) (0.00) (0.00) (0.73) (0.82) (0.93) (0.45) (0.85) (1.00) (0.50)
(0.55) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.67) (0.00) (0.00) (0.00) (0.00) (0.04) (0.00) (0.00) (0.25) (0.00) (0.00) (0.00) (0.11) (0.00) (0.00) (0.00) (0.00) (0.27) (0.12) (0.00) (0.00) (0.00) (0.00) (0.00)
a Results are expressed as the number of female mice observed with mammary gland patterning abnormalities and the percent of each class compared to the total number of mice observed. MG, mammary gland.
syndrome (LMS) (van Bokhoven et al., 1999). LMS patients may have hypoplasia or aplasia of the mammary gland and nipple and severe hand and/or foot anomalies. We have not observed any other developmental defects linked to the ska mutant phenotype. The mammary gland pattern was examined in several mice from each of the 30 available AXB/BXA series of RI strains to obtain a strain distribution pattern (SDP) for the proposed ska locus (Table 2). Consistent with the single locus hypothesis, abnormal mammary gland patterning was found in 21 of the 30 RI strains (17 out of 26 of the independent lines). The bimodal SDP of presence or absence of abnormal mammary gland patterns among the RI strains suggest the presence of a major locus controlling the patterning of mammary glands, with allelic differences presumably resulting in normal or abnormal mammary gland patterns. Linkage to several markers on chromosome 14 was detected using the MapManager program (Manly, 1993). Strongest linkage (LOD 3.0) was to D14Mit80 at the 99.9% signi®cance level (Chr 14). The SDP for markers on
mouse Chr 14 is displayed according to their most likely order in Table 2 with the best placement for ska between markers D14Mit14 and D14Mit80. Comparison of the SDP of the marker loci, D14Mit14 and D14Mit80, that putatively ¯ank the ska locus, provide further evidence for linkage since mostly single recombination events are seen among the three loci within individual RI strains. Two double recombination event are required (in the AXB-18 and BXA-4 strains) between the three markers. There are no other known microsatellite markers that are polymorphic between A/J and B6 strains that map between this interval. When ska is placed between D14Mit14 and D14Mit80, four crossovers exist between ska and D14Mit80 and seven recombination events are required between ska and D14Mit14. These crossovers will be useful for ®ne-scale mapping when more markers are obtained The ska locus maps to mouse Chr 14 to an interval between the microsatellite markers D14Mit14 and D14Mit80 which are located at 10.0 and 13.5 cM, respectively on the 1999 Chromosomal Committee maps. Our
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
307
Table 2 Strain distribution pattern of ska mammary gland phenotype of 30 AXB/BXA recombinant inbred strains and localization of ska candidate locus on Chr14 a,d,e AXB strain 1 D14Mit207
2
4
BXA strain 5
6
8
10 12 13 14 15 18 19 20 23 24 1 B
B
D14Mit201
A A A B A B B x A A A B A B A
B
B
D14Mit202
A A A B A B A
B
B
D14Mit14 b
A A A B A B A
B
ska
A A A B A B A x x B A A B A B B
B B
B x A x B
B
B
D14Mit80
D14Mit129 c B
A A B A B B
B x A x B
2
4
7
8
11 12 13 14 16 17 24 25 26 Ref. B
A
B
A
B
B
A
B
*
B
A
B
A
A
B
A
B
*
B
B
B
B
B
B
B B B
B
B
B
B
B
B
B
B B B
B
B B x A B
B
B
B
B
B
B B B
B
A B
B
A
B
A
A
B
A
B
*
B x A
B
B x A x B
B
A
A
A
A
A
A
B x A
²
B
B x A
A
B
B x A
A
A
*
A
B
B B B x B B A x B B B
B A B x A A B
B
B x A x B
B
A x B
B x A x B
B x A
A A B
B
A
A
A
A
A
A
A
*
B
B
B
B
A
B
B B B
A A B
B
A
A
A
A
A
A
A
*²
B
a Markers are listed from the centromere. Strains displaying abnormal mammary gland pattern (misplaced nipples, absent glands, supernumerary nipples) were scored as having the ska mutation or A allele. Strains displaying normal mammary gland pattern (ten nipples and glands, bilaterally symmetric) were scored as having the wild-type ska allele or B allele. A denotes the A/J allele. B denotes the B6 allele. x denotes a recombinant chromosome. b D14Mit14, D14Mit44*, D14Mit55*, D14Mit133*, D14Mit173*, D14Mit174*, and D14Mit186* display concordant SDPs. c D14Mit129*²³, Mmp-14*, and Np-2*³, display concordant SDPs. ³Our SDP for D14Mit129 differs from that reported by (Sampson et al., 1998). Heterozygous alleles were observed in genotyping reactions with strains AXB-10 and BXA-26 in their analysis. Our genotype of the BXA-4 strain for Np-2 differs from that reported by (Sampson et al., 1998). *This study. ² Sampson et al. (1998). d SDP for markers typed in the AXB/BXA RI set were obtained from published reports (Higgins and Paigen, 1997; Naggert et al., 1997; Prows and Leikauf, 1998; Sampson et al., 1998) and from the World Wide Web (WWW) (http://mcbio.med.buffalo.ed/mapmgr.html) and (http://www.informatics.jax.org/ riset.html). Polymerase chain reaction (PCR) was performed as described (Dietrich et al., 1994). Primers were obtained from Research Genetics, Inc. (Huntsville, AL, USA). e Genetic linkage was determined by segregation analysis. The mapping data were analyzed with Map Manager Version 2.6.6 (Manly, 1993). Genetic contamination of some AXB/BXA RI lines has been reported so only 27 independent lines now exist (Taylor, 1996). AXB-14, AXB-18 and BXA-17 were used for the genetic analysis while AXB-19, AXB-20 and BXA-8 were omitted to avoid false linkage detection.
analysis has found larger recombination frequencies in the AXB and BXA crosses relative to the standard genetic maps which describe a recombination distance between D14Mit14 and D14Mit80 that is approximately one sixth of what we observed in this study. Different mouse lines are known to have line-speci®c rates of recombination and our data may simply be a re¯ection of this or of errors present in consensus maps, which are not always recombination-based due to the cumulative nature in which they are constructed. ska maps to a region of the genome containing a known mouse mutation, pugnose (pn) which is now extinct (Doolittle et al., 1996). pn mutant mice were reported to have skeletal abnormalities including craniofacial defects and mapped to the same region as Bmp-4 and the Bmpr genes (Doolittle et al., 1996). Bmp4 was excluded as a candidate gene for ska based on observed genetic recombination between D14Mit129 (a microsatellite sequence within the Bmp4 gene) and ska (Kurihara et al., 1993). The Mmp-14 gene was also excluded as a candidate for ska based on observed genetic recombination between a microsatellite contained within this gene and ska (Kurihara et al., 1993). The ska gene maps near a proximal region of mouse Chr 14 which has conserved linkage relationships to that of human Chr 10q. Further genetic characterization of this region will
be needed to determine whether this region of conserved linkage extends to and includes the ska locus. Of the many genes that map to the interval within which the ska locus should lie, the Bmpr is the most intriguing candidate gene for ska since Bmp-2 and Bmp-4 are both expressed during mouse mammary gland development in temporal and spatial patterns that suggest that they may play roles in the control of epithelial- mesenchymal interactions in the mouse mammary gland (Phippard et al., 1996). The Bmpr gene encodes a BMP-2/4 type I serine±threonine kinase transmembrane receptor. The Gdf10 gene is a reasonable candidate since it is a member of the TGFb superfamily and is highly related to Bmp-3 and displays expression patterns that suggest multiple roles in regulating cell differentiation events (Cunningham et al., 1995). BMPs are members of the TGFb superfamily and play key roles in developmental processes such dorsal-ventral axis formation, left-right symmetry, epithelial±mesenchymal interactions, as well as growth, death and differentiation of speci®c tissues (Hogan, 1996; Lemaire and Yasuo, 1998). Knockouts of both the Bmp-4 and Bmpr genes in mice exist and are embryonic lethals and die between embryonic day 6.5 and 9.5, before mammary glands have developed (at day E11.5) (Kratochwil, 1987; Mishina et al., 1995; Winnier et al., 1995). We have localized the ska mutation to mouse Chr 14 by
308
B.A. Howard, B.A. Gusterson / Mechanisms of Development 91 (2000) 305±309
analyzing the AXB and BXA recombinant inbred strains of mice. Analysis of genetic variation in mammary gland patterning may provide a means to study mechanisms of mammary gland development and identify the products and modes of action of some of the genes that in¯uence the formation and placement of mammary glands. The
results reported here set the groundwork for understanding the molecular genetics of patterning of the embryonic mammary gland, and also how variable phenotypes can be produced in identical genetic backgrounds. Acknowledgements We thank Philip Gladwell, Jayne Hacker, Jenny Teague, and Mabel Iwobi for their excellent technical assistance. This work was supported by the Cancer Research Campaign. References Bamshad, M., Lin, R.C., Jaw, D.J., Watkins, W.S., Krakowiak, P.A., Moore, M.E., Franceschini, P., Lala, R., Holmes, L.B., Gebuhr, T.C., Bruneau, B.G., Schinzel, A., Seidman, C.E., Jorde, L.B., 1997. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16, 311±315. Bateson, W., 1894. Materials for the Study of Variation, Macmillan, London. Cunha, G., 1994. Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate. Cancer 74, 1030±1044. Cunha, G.R., Hom, Y.K., 1996. Role of mesenchymal-epithelial interactions in mammary gland development. J. Mamm. Gland Biol. Neoplasia 35, 21±35. Cunha, G.R., Young, P., Hamamoto, S., Guzman, R., Nandi, S., 1992. Developmental response of adult mammary epithelial cells to various fetal and neonatal mesenchymes. Epithelial Cell Biol. 1, 105±118. Cunningham, N.S., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., Reddi, A.H., Lee, S.J., 1995. Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3. Growth Fact. 12, 99±109. Dietrich, W.F., Miller, J.C., Steen, R.G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D.C., Wessel, M., Dredge, R.D., et al., 1994. A genetic map of the mouse with 4006 simple sequence length polymorphisms. Nat. Genet. 7, 220±245. Doolittle, D., Davisson, M.T., Guidi, J.N., Green, M.C., 1996. Catalog of mutant genes and polymorphic loci. In: Lyon, M.F. (Ed.). Genetic Variants and Strains of the Laboratory Mouse, vol. 1. Oxford University Press, Oxford, pp. 17±854. Gardner, W.U., Strong, L.C., 1935. The normal development of the mammary glands of virgin female mice of ten strains varying in susceptibility to spontaneous neoplasms. Am. J. Cancer 25, 282±290. Higgins, D.C., Paigen, B., 1997. An additional 150 SSLP markers typed for Fig. 1. Mammary gland pattern phenotypes observed in AXB/BXA RI strains of mice. (A) Normal pattern of ®ve pairs of mammary glands which are numbered 1±5, anterior to posterior of a lactating adult BXA-7 mouse. (B) ska mutant phenotype of absent right #3 gland of an adult BXA13 mouse. (C) ska mutant phenotype of misplaced #3 nipple in a 7-day-old AXB-2 pup. The right nipple is lower than the left nipple. (D) ska mutant phenotype of bilateral supernumerary #4 nipples in a 10-day-old BXA-14 pup. These nipples are connected to ductal systems which are distinct from the adjacent #4 gland and lactate and are suckled by pups. (E) Supernumerary right #4 nipple adjacent to the #4 nipple in a lactating AXB-4 mouse. Both are distended by suckling by pups. (F) Wholemount analysis of #4 gland from mouse displaying a supernumerary right #4 nipple and ductal system. The normal #4 nipple is labeled N and the supernumerary nipple is indicated by an arrow.
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