A Novel Mouse Chromosome 17 Hybrid Sterility Locus - Genetics

Copyright 0 1991 by the Genetics Society of America

A Novel Mouse Chromosome 17 Hybrid Sterility Locus: Implications for the Origin of t Haplotypes

Stephen H. Pilder, Michael F. Hammer' and Lee M. Silver Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

Manuscript received January 2 1, 199 1 Accepted for publication May 1 1, 199 1

ABSTRACT The effects of heterospecific combinations of mouse chromosome 17 on male fertility and trans-

mission ratio were investigated through a series of breeding studies. Animals were bred to carry complete chromosome 17 homologs, or portions thereof, from three different sources-Mus domesticus, Mus spretus and t haplotypes. These chromosome 17 combinations were analyzed for fertility within the context of a M. domesticus or M. spretus genetic background. Two new forms of hybrid sterility were identified. First, the heterospecific combination of M. spretus and t haplotype homologs leads to complete male sterility on both M. spretus and M. domesticus genetic backgrounds. This is an example of symmetrical hybrid sterility. Second, the presence of a single M. domesticus chromosome 17 homolog within a M. spretus background causes sterility, however, the same combination of chromosome 17 homologs does not cause sterility within the M. domesticus background. This is a case of asymmetrical hybrid sterility. Through an analysis of recombinant chromosomes, it was possible to map the M. domesticus, M. spretus and t haplotype alleles responsible for these two hybrid sterility phenotypes to the same novel locus (Hybrid sterility-4). Previous structural studies had led to the hypothesis that the ancestral t haplotype originated through an introgression event from M. spretus or a related species. If this were true, one might expect that (1) M. spretus homologs would be transmitted at a non- Mendelian ratio within the M. domesticus background, and (2) t haplotypes would be transmitted at a ratio closer to Mendelian within the M. spretus background. The functional data generated in the current study indicate that neither of these predictions is fulfilled, and thus, the M. spretus introgression hypothesis appears to be unlikely.

M OST naturally occurring populations of the house mouse (which include the sibling species

Mus musculus, Mus domesticus, as well as others) are polymorphic for a selfish chromosomal entity known as a t haplotype. A t haplotype occupies a 20 cM region at the proximal end of chromosome 17 and it maintains its structural integrity through a series of four inversions that block recombination with the wild-type homolog (Committee for Mouse Chromo- some 17 199 1 ; Figure 1A). t Haplotypes are maintained at relatively high levels in natural populations through the expression of a male-specific phenotype of transmission ratio distortion (TRD) by which 95% or more of the offspring from heterozygous +/t males receive the t-bearing chromosome (SILVER 1985). Al- though TRD provides a powerful selective advantage, t haplotypes have not become fixed in the mouse genome because males homozygous for the t-form of the chromosome are completely sterile. From mapping studies, it appears likely that this recessive sterility phenotype is a consequence of homozygosity for the same t genes that are involved in the dominant TRD phenotype (LYON 1986). In addition, most (but

' Current address: Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts 021 38.

Genetics 149: 237-246 (September, 1991)

not all) naturally occurring t haplotypes carry recessive embryonic lethal mutations that also counteract TRD (BENNETT 1975). These three selective forces-TRD, sterility and lethality-balance each other out giving rise to equilibrium allele frequencies of 5-1 5% in most populations that have been studied (FICUEROA et al. 1988a).

Since their discovery over 60 years ago, the origin and evolution of these unusual genetic entities has intrigued many investigators. Until the last decade, the question of t haplotype derivation had been a complete enigma. However, with the ability to use DNA probes to compare t haplotypes with other forms of chromosome 17, it became possible to begin to unravel this puzzle in the context of a model shown in Figure 1B. The crux of this model is as follows: first, all current-day t haplotypes derive from a single ancestor; second, t haplotypes diverged apart from the line leading to the M. domesticus form of chromosome 17 between 1 and 6 million years ago, prior to the divergence of the various strongly commensal mouse species from each other, but not prior to the divergence of the M . domesticus line from the M. spretus line (DELARBRE et al. 1988; HAMMER, SCHI- MENTI and SILVER 1989). Finally, once the primordial

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238 S. H. Pilder, M. F. Hammer and L. M. Silver

A 48 119 66 Tcpl Sai2 66 Tu50122 54 Him Piml C y 89

M. domesticus 0 : : : ; b f ; I b j

Tai-1 Tal4 T~Y Tai-3 Tai-5

t Haplotype! 1. I I 7 1 7

B fixation of rima1 inversion p"

FIGURE 1 .-(A) Diagram of three forms of the t region from mouse chromosome 17. The top line represents the M. domesticus t region, the middle line, the t haplotype form, and the bottom line, the M. spretus form. Boxes represent t region-associated inversions, in(17)I through in(l7)4, while arrows within the boxes illustrate their relative orientations. Circles at the far left of each line denote the positions of the centromeres. The 13 loci displayed above the topmost chromosomal segment have been placed in the order in which they would be found in the M. domesticus t region. The loci indicated with numbers alone (48, 1 19, 66, 122, 54 and 89) are abbreviations for Dl 7Leh48, Dl7Lehl19, D17Leh66, D l 7Leh122, D17Leh54 and D17Leh89, respectively. Markers for all loci except D17Tu50 have been used in this study. The position of DI7Tu5O has been shown to denote the distal boundary of the Hst-I hybrid sterility locus. Loci involved in t haplotype- associated spermatogenic phenotypes (Tcd-I through Tcd-5 and Tcr) are placed above the middle chromosomal segment and ordered as they would be found in t haplotypes. (B) An evolutionary tree depicting the divergence of different forms of chromosome 17 carried by several modernday species of strongly and weakly commensal mice. The complete range of possible ancestral origins for t haplotypes is denoted by the shaded box and the question mark.

t chromosome had appeared with a slight TRD advantage, it is possible to explain its evolution into current- day t haplotypes by invoking selective forces that acted to increase the absolute level of TRD through (1) internal single gene mutations and (2) a series of inversions that locked together all of the genes responsible for this phenotype (CHARLESWORTH and HARTL 1978).

One critical question that remains unanswered con- cerns the nature of the initial set of events that led to the origin of the primordial t chromosome (repre- sented within the "shaded box" in Figure 1B). In a previous attempt to better understand the parameters involved in this problem, Hammer and co-workers compared the structure of the t region of chromosome 17 present in three different species of mice ( M . domesticus, Mus macedonicus (previously Mus abbotti) and M. spretus with that of t haplotypes (HAMMER, SCHIMENTI and SILVER 1989). The results demonstrated that the oldest of the four inversion polymorphisms that distinguishes t haplotypes from M. domesticus forms of chromosome 17 [in(l7)2] actually originated within the M . domesticus line and not on the

line leading to t haplotypes (Figure 1 A). Furthermore, the M. spretus form of the chromosome, like t haplotypes, does not carry this inversion. This finding led to the proposal of two alternative models to explain the origin oft haplotypes.

According to the first model, the in(l7)2 inversion became fixed within the M. domesticus ancestral line, and subsequently, a noninverted chromosome intro- gressed into this population from M . spretus (or a related species in existence at that time). The basic tenet of this model is that at the time of this event, the M. spretus-like species carried a normal form of chromosome 17 that could express meiotic drive upon transfer to the M. domesticus genetic environment (HAMMER, SCHIMENTI and SILVER 1989; SILVER 1982, 1985). Precedents have been described for this type of cryptic male-specific meiotic drive system (CAM- ERON and MOAV 1957; LOEGERINC and SEARS 1963).

According to the second model, the primordial t haplotype originated entirely within the M . domesticus line through the stepwise accumulation of mutations on a normal noninverted form of the chromosome that acquired an initial TRD advantage. Subsequently,

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Chromosome I 7 Hybrid Sterility 239

the in(l7)Z inversion arose on a wild-type chromosome within the same population, and was selected for in + / t heterozygotes because it reduced recombination between two or more alleles required for the TRD phenotype (CHARLESWORTH and HARTL 1978).

In order to distinguish between these models, it was essential to extend our studies beyond that of chromosomal organization to an analysis of the functional relationships that exist between the different forms of chromosome 17. The original rationale of the studies reported herein was to test certain predictions made by the M . spretus-origin model. In particular, one might expect current-day t haplotypes to exhibit a reduced meiotic drive phenotype when placed within a putative "species of origin." In addition, we reasoned that it might be possible to unmask a cryptic meiotic drive system within the M. spe tus form of chromosome 17 by placing it experimentally into the M. domesticus genome. Here we report the effects on fertility and transmission ratio of various heterospecific chromosome I7 combinations within the genetic backgrounds of M. spretus and M. domesticus.

To perform this analysis, it was necessary to inter- cross the two species that normally carried the three forms of chromosome 17 analyzed. However, crosses between M. spretus and M. domesticus result in a classic case of hybrid sterility at the first generation, which is defined as a situation where two parental forms, each of which is fertile inter se, produce a hybrid which is sterile (BONHOMME, MARTIN and THALER 1972; HALDANE 1922). The hybrid sterility phenotype is often confined to the "heterogametic" sex, as is the case here. Two major loci, Hst-2 and Hst-3, appear to be involved in this example of hybrid sterility, and after three generations of backcrossing to M. domesticus (through females), most males express full fertility (BONHOMME, GUENET and CATALAN 1982; GUENET et al. 1990). Thus, to view the possible effects on sperm function of heterospecific forms of chromosome I7 in isolation from other genic effects, it was necessary to backcross the heterospecific chromosome into each genetic background for at least four generations.

The results obtained indicate that present-day t haplotypes are not functionally related to present-day chromosome I7 representatives from the M. spretus species. Thus, the M . spretus origin model for t haplotypes is rendered unlikely. However, these studies have led to the identification and genetic characterization of a novel hybrid sterility locus with a complex phenotypic expression.

MATERIALS AND METHODS

Nomenclature: The species of mice used in this study are named according to standard practice (AUFFRAY et al . 199 1 ; SAGE 198 1). The sibling species M . domesticus and M. musculus are considered subspecies (M. m. domesticus and M. m.

musculus respectively) by some investigators (AUFFRAY et al. 1991). The genotypes of all hybrid mice used in this study are described by a notation which takes the following form: X. [Y/Z]. The first letter, outside the brackets, is indicative of the genetic background of the animal. Letters inside the brackets, separated by a "/," symbolize the chromosome I7 homologs. D represents M. domesticus, S represents M. spretus, and t represents a t-form of chromosome 17. For example, D[S/t] represents an animal with a M. domesticus genetic background harboring a M . spretus form of chromosome 17 and a complete t haplotype. The notation X - Y represents a recombinant chromosome I7 with a proximal X-derived region and a distal Yderived region. For example, t t, D denotes a proximal partial t haplotype that originated within a M . domesticus mouse, D c-, t denotes a distal partial t haplotype, and S c* D and D c-, S denote structurally reciprocal forms of recombinant M. spretus-M. domesticus chromosome 17. Each unique class of recombinant M. spretus-M. domesticus chromosomes used in this study is named according to the alleles present at each of six DNA loci (DI7Leh66, DI7Leh54, Hba-ps4, Pim-I, Crya-I and DI7Leh89) that were typed as shown in Figure 2. For example, SSDDDD denotes a recombinant chromosome I 7 with M. spretus alleles at DI7Leh66 and DI7Leh54, and M. domesticus alleles at Hba-ps4, Pim-I, Crya-I and D17Leh89. When independent recombinant chromosomes were recovered with the same set of typed alleles, they were distin- guished by numbers as indicated in Table 1.

Mice and crosses: All breeding experiments were con- ducted at Princeton University in accordance with NIH regulations and guidelines. All t haplotypes have been maintained in the colony of L. SILVER at Princeton University. Outbred CD1 mice were obtained from Charles River (Wil- mington, Massachusetts). M. spretus animals originally col- lected from Cadiz, Spain, were obtained from M. POTTER (Bethesda, Maryland).

M. domesticus females heterozygous for the complete t haplotypes P5, Pb7, tTUwz4 were crossed to M . spretus males to produce two types of F1 hybrids for chromosome I7 (D/S and t/S). t/S F1 females were crossed to M. spretus males while D/S FI females were crossed to either M. spretus or M. domesticus males to produce N2 backcross generation offspring. Further backcrossing of the same type was carried out through the N4 generation. N5 and successive backcross generation D[S/D] offspring were derived by crossing to either sex. Further backcrossing to produce S[S/D] offspring was accomplished only through the maternal line. N5 and N6 D[S/t] males were produced by crossing N4 and N5 D[S/D] males to homozygous D[t"'/t"'] females maintained routinely in our colony. N5, N6 and N7 generation D[S/t c-, D] and D[S/D t, t] males were produced in the first instance by crossing D[S/D] males and females of the appro- priate generation to animals that carried the desired partial t haplotype. These same genotypes were also produced by intercrossing. Males harboring recombinant S c-, D or D c-, S chromosomes in conjunction with a variety of other chromosome 17 homologs were produced as described above for all genotypes.

Definition of sterility: Males to be tested for sterility were placed with at least four females of breeding age for at least one month per pair of females. To be classified as sterile, a male had to produce vaginal plugs in at least two different females without giving rise to any progeny. This definition is absolute-the birth of a single offspring indicates that the male parent is nonsterile.

Assay for chromosome 27 genotype: All experimental animals were tested with eleven informative markers that identify restriction fragment length polymorphisms (RFLPs)

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D S t D S t D S t D S t D S t D S t D S t D S t D S t D S t D S t

7 -

48 119 Tcpl Sod2 66 122 54 Hba Piml Cryal 89 FIGURE 2.-Southern analysis of RFLPs between different forms of chromosome 17. Each panel of three lanes represents a Southern

hybridization with one of eleven molecular probes to 10 gm per lane of blotted high molecular weight DNA digested with the restriction endonuclease, Ta91. The letters D, S and t above each lane represent domesticus, spretus and t haplotype samples. Hybridiiration panels are presented from left to right according to the order of corresponding loci along the chromosome. The loci are indicated below each panel and are abbreviated as indicated in the legend to Figure 1. Clones used to identify RFLPs at these loci are indicated in the same order: Tu48 (FOX et al. 1985), Tu1 19 (HERRMANN et al. 1986). 29x (WILLISON, DUDLEY and POTTER 1986), Sod2 (FIGUEROA et al. 1988b). Cg3-100 (SCHIMENTI et al. 1987). T u 1 22 (Fox et al. 1985), 54M ( B ~ C A N et al. 1987), Hba4 (Fox, SILVER and MARTIN 1984), Pim 1A (NADEAU and PHILLIPS 1987), Crya-10 (KING, SHINOHARA and PIATICORSKY 1982), and Tu89 ( B ~ C A N et al. 1987).

TABLE 1

Summary of breeding results

17-1 17-2 Background Backcross generation tested Total

No. Fertile

fmpl'u S S N4 (8) 8 0 S S S N4 ( 2 ) sibling control 2 2 tyz S D N5 (1 2). N6 (2) 14 0 1-2 D D N5 (3) sibling control 3 3 t6 S D N6 (4) 4 0 T f 6 S D N5 (6), N6 (1) 7 Tr' S D N5 (2), N6 (4), N7 (7) 13 0 t r ~ 3 z S D N5 (1). N6 (7) 8 5 P S D N5 (I), N6 (4). N7 (3) 8 5 r' S D N7 (6) 6 5 P SSSDDD #1 D N5 (1) 1 0 r5 SSSDDD #1 D N5 (1) 1 0 T P SSSDDD # I D N5 (2) 2 0 PZ SSSDDD #2 D N5 (1) 1 0

0

T P SSSDDD #3 D N5 (1) 1 0 T P SSSDDD #4 D N6 (1) 1 0 T P SSSDDD #5 D N8 (1) 1 1 rZ SSDDDD D N5 (1) 1 1 D S D N4 (7), N5 (3). N6 (3). N7 (3) 16 12 D S S N4 (1 6), N5 (1) 17 0 S S S N4 (6) sibling control 6 6 S SSSDDD #6 S N4 (1) 1 1 S SSSSDD S N4 (1) 1 1 S DDSSSS S N4 (1) 1 S

1 DSSSSS S N4 (1) 1 1

Columns 17-1 and 17-2 indicate the two forms of chromosome 17 present in a particular genotype within the background indicated in the third column. S represents M . spretus, and D represents M . domesticus. Partial and complete t haplotypes are discussed in MATERIALS AND METHODS. Classes of M . spretus-M.domesticus recombinant chromosomes are named as described i n MATERIALS AND METHODS. The number of mice tested at each backcross generation is indicated in parentheses.

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Chromosome 17 Hybrid Sterility 24 1

among the three forms of chromosome 17 under analysis in this report". spretus, M. domesticus and t haplotypes. Mark- ers and corresponding RFLPs are shown in Figure 2. High molecular weight DNA, prepared from tail clippings ( H e GAN, COSTANTINI and LACY 1986), was cut to completion with TaqI restriction endonuclease (New England Biolabs, Beverly, Massachusetts), electrophoresed and blotted onto nylon membranes (Genescreen, New England Nuclear, Bos- ton, Massachusetts) according to the manufacturer's instruc- tions. DNA was bound to the membrane by UV light and hybridized according to the procedure of CHURCH and GILBERT (1984). Radioactive probes were produced by po- lymerization from a mixture of random oligonucleotides on templates of denatured DNA (FEINBERG and VOGEISTEIN 1984). Membranes were routinely stripped and reprobed multiple times according to the procedure described by the manufacturer.

RESULTS

t Haplotypes cause male sterility when placed into the M. spretus genome: One of the predictions of the M. spretus-origin model of t haplotypes is that these chromosomes might not distort transmission ratios as drastically in a heterozygous combination with the M. spretus form of the chromosome as they do normally with the M. domesticus chromosome, since they would retain residual functional homology with a putative chromosome of origin. To examine this prediction, we generated males that were heterozygous for a complete t haplotype within a M. spretus background (S. [S/t]) at the N4 generation of backcrossing, and assessed their fertility according to the protocols described in the MATERIALS AND METHODS. All eight males tested proved to be sterile, whereas the two backcross sibling males tested without a t haplotype were found to be fertile (Table 1). Thus, it appears that one or more genes within or closely linked to t haplotypes is causing male sterility within M. spretus animals. The prediction of an altered transmission ratio obviously cannot be tested within the context of this genotype.

The M. spretus form of chromosome 17 does not distort transmission ratios when placed into the M. domesticus genome: The second prediction of the M. spretus-origin model for t haplotypes is that the normal M . spretus form of chromosome 17 might act to distort transmission ratios when it is placed experimentally into the M. domesticus genome. T o test this prediction, we backcrossed the M. spretus form of chromosome 17 into the M. domesticus genome. At each generation, offspring with an intact M. spretus chromosome across the complete length of the t region (from D17Leh48 to Dl7Leh89) were identified for further breeding (Figure 1A). Of the 16 males of the D - [D/S] type that were analyzed from the N4 through N7 generations, 12 were found to be fertile. When eight of these fertile males were bred to determine the ratio of transmission to their offspring of each chromosome 17 homolog, no significant distortion from 50% was

TABLE 2

M. dontesticus/M. spretus chromosome 17 transmission ratios for D . [S/D] males

generation Male No. offspring offspring Backcross domesticus spretus domesfinrsjspretus

ratio

N4 1 10 9 1.11 N4 2 48 47 1.02 N4 3 22 18 1.22 N4 4 44 36 1.22 N5 5 38 48 0.79 N5 6 26 52 0.5 N6 7 15 14 1.07 N6 8 49 27 1.81

Total 1-8 252 253 0.996

observed (Table 2). This result implies that the present-day M . spretus chromosome 17 is functionally un- related to the primordial t haplotype. A caveat to this interpretation is that a small distortion of transmission ratio could go undetected in laboratory studies, but nevertheless, have a profound effect in natural populations.

The M. spretus/t haplotype chromosome 17 combination causes hybrid sterility irrespective of genetic background: Two general genetic models can be proposed to account for the sterility phenotype caused by t haplotypes in a M. spretus background. The first model posits a direct incompatibility between the M . spretus and t alleles at a particular chromosome 17 locus that results in sterility irrespective of the genetic background of the animal. According to this model, the chromosome 17 gene(s) involved would represent a classic symmetrical hybrid sterility locus. The second model posits an incompatibility between one or more t haplotype gene(s) and other genes present in the M. spretus genetic background. Accord- ing to this model, the guilty t haplotype gene(s) would be acting as a true dominant mutation only within the context of the M. spretus genetic background, giving rise to an asymmetrical hybrid sterility phenotype.

In order to distinguish between these two models, we generated animals that carried the same chromosome 17 genotype as that described above (one homolog from M . spretus and a t haplotype) but within the M. domesticus genetic background (D.[S/t]). If the dominant mutation explanation of the original sterility phenotype were correct, these new males would be fertile. If this were the case, it would be possible to investigate whether t haplotypes could continue to distort transmission ratios against non-M. domesticus forms of chromosome 17. On the other hand, if the symmetrical hybrid sterility explanation were correct, these new males would be sterile, and once again, it would not be possible to obtain a value for TRD.

Fourteen D-[S/t] males were tested for fertility. All 14 proved to be sterile, whereas all three sibling males

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tested with a D. [D/t] genotype showed fertility (Table 1). Thus, the M . spretuslt haplotype hybrid sterility phenotype is independent of genetic background.

One or more M. domesticus chromosome 17 loci acts as a dominant male sterile mutation within the M. spretus genome: To further investigate the nature of chromosome 17 effects on fertility in animals with hybrid genotypes, we backcrossed the M . domesticus form of chromosome 17 into the M . spretus genome in order to obtain S . [ D/S] males. At each generation, offspring with an intact M . domesticus chromosome across the complete length of the t region (from D17Leh48 to Dl 7Leh89) were identified for further breeding. Unexpectedly, all 17 males of the S. [D/S] type proved to be sterile, whereas all six sibling males not carrying a M. domesticus chromosome 17 (S - [S/S] ) were found to be fertile (Table 1). When these data are viewed in conjunction with the observation described above that D - [ D/S] males are fertile, it is clear that the hybrid chromosome I7 combination of M . domesticus and M. spretus is not in itself causing sterility. Rather, it appears that one or more loci on the M . domesticus form of chromosome I7 can act as a dominant male sterile mutation within the context of the M. spretus genome. This hybrid sterility phenotype is termed asymmetrical in contrast to the symmetrical hybrid sterility result obtained with the M. spretuslt haplotype chromosome 17 combination.

Mapping of a t haplotype locus responsible for hybrid sterility: We reasoned that since D. [D/S] males are fertile, it should be possible to map the t allele responsible for the sterility of D.[t/S] males by replacing defined portions of complete t haplotypes with M . domesticus DNA to produce D. [D c, t/S] and D. [t c* D/S] animals. In practice, this can be accomplished through breeding protocols that make use of existing stocks of mice carrying different recombinant [ t c, M . domesticus] chromosomes known as partial t haplotypes that have resulted from rare crossover events in De [D/t] animals. Partial t haplotypes are classified as "proximal" if they retain a proximal portion of the complete t haplotype from which they are derived with a M. domesticus distal region [t c, Dl. "Distal" partial t haplotypes retain only a distal portion of a complete t haplotype with a proximal region from M . domesticus [D c, t].

Six different partial t haplotypes were used in this experiment. The extent of t-DNA present in each is shown in Figure 3. Fertility was observed in D. [t c, D/S] males that carried any one of three different proximal partial t haplotypes (tTuu32, th2, t') (Table 1). No significant difference was observed between the fraction of these males that are fertile and the fraction of males with a control D-[S/D] genotype that are fertile. In contrast, all of the D.[D c, t/S] males that carried any one of three distal partial t haplotypes (t6,

c"---f Region - 48SodTcp1196612251Hb.PhnDY.89~DNAy.rLnr

spretus

7- ? S l h v w . l w rhdhwMh

5/8

518

48SodTcp1196612251HbaplmcrY.89 f 3 516

f @ 014 4 6 6 1 1 9 T ~ ~ s o d ~ 1 ~ ~ ~ , , ~ ~ 8 , ~

Oi7

48661l#T~Sod6612289crY.p imHb.Y 0113

FIGURE 3.-Mapping the Hst-4' allele. Male mice with a M. domesticus background having one chromosome 17 homolog from M. spretus (black box at top) and the other with one of six partial 1 haplotypes (mosaic boxes below the line) were tested for fertility as indicated in the text. The shaded areas represent the extent of t chromatin; the remaining portion of these chromosomes is derived from M. domesticus. The small boxes below the M. spretus chromosome represent inverted regions in(17)J through in(17)4, and the marker loci employed in the genetic analysis are shown above each chromosome. The fertility of each genotypic class is presented as a fraction of the males tested. The Hst-4' map position is indicated by the solid bar at the bottom of the figure.

f6, th") were completely sterile (Table 1). These data allow the localization of the t haplotype gene($ responsible for sterility to the distal in(l7)4 region defined by the markers D l 7Leh54 and DI7Leh89.

Mapping of the M. spretus hybrid sterility locus: To map the M . spretus allele responsible for the sterility of D - [t/S] males, we took advantage of the obvious fact that D - [t/D] males are fertile. Thus, it is possible to replace portions of the M. spretus chromosome with M . domesticus DNA in D - [t/D c* SI and D - [t/S c* Dl males and test for the restoration of fertility. Recom- binant forms of chromosome 17 containing M. spretus- and M . domesticus-derived portions occurred in the offspring from a number of crosses described in this report. These chromosomes were characterized at 11 molecular loci that span the t region (Figure 4). Fur- ther crosses were performed to obtain animals that carried each recombinant D c, S or S t, D chromosome in conjunction with a t haplotype on a M. domesticus background. Since distal partial t haplotypes appear indistinguishable from complete t haplotypes in the expression of the D.[t/S] sterility phenotype, we used them interchangeably with complete t haplotypes in this experiment (data obtained with each are reported separately in Table 1).

Fertility was observed in a male that carried a recombinant chromosome with a breakpoint between Dl7Leh54 and Hba-ps4 (D-[t/SSDDDD]) (Figure 4). Since the sterility phenotype expressed by D-[t/S] animals is absolute, the recovery of single fertile De [t/D c, SI or D.[t/S t, Dl male of a particular genotype

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Chromosome 17 Hybrid Sterility 243

- t Region -

No. of Fertlle Males With

96 hlt 48 Tc 1 1 9 6 6 1 2 2 Y H b a P l m C m 8 9

t i t SSSDDD#l- 1 012 0l2 n.d.

SSSDDD#2 - SSSDDD#3 I)

48Sod Tc 11966 12254Hba PImcN.89 011 n,d, n.d.

48SodTc 1196612254HbsPlmQywBB n.d. n.d. 0,1

48SodTc 1196612254HbsPlm9y~89 SSSDDDM - j n.d. n.d. 011

SSSDDD#5

SSDDDD

48SodTc 1196612254HbaPlmCm89 n.d. n.d.

48SodTc 1196612254HbsPlmuY.89 1111 n.d. n.d. -

FIGURE 4.-Mapping the Hst-4' allele. Male mice with a M. domesticus background. carrying either a complete t haplotype or one of two disral partial t haplotypes (indicated at the top of the figure with mosaic boxes) and one of six M. sprefus-M. domesticus recombinant t regions (indicated below with mosaic boxes) were tested for fertility as indicated in the text. Black portions of mosaic boxes represent M. spretus DNA, white portions represent M . domesticus DNA, and shaded portions represent t haplotype DNA. Marker loci used in the analysis of each male are shown above each chromosome. Fertility values are indicated for each combination of M . spretus-M. domesficus and f haplotype homologs. n.d. indicates not done. The large solid bar at the bottom of the figure represents the Hst-4' map position from Figure 3, while the small solid bar indicates the Hst-4' map position.

is a significant outcome and can be used to indicate the presence of the M. domesticus allele rather than the M . spretus allele at the hybrid sterility locus. Thus, the M . spretus allele responsible for the sterility of De [t/S] males must map distal to DI7Leh54.

Five independent SSSDDD recombinant chromosomes were bred into a D.[t/SSSDDD] genotype. In total, eight males were analyzed that carried one of these five chromosomes. Seven males representing four of the breakpoints proved to be sterile, whereas one male representing the fifth breakpoint was fertile (Figure 4). This result further defines the proximal boundary of the sterility locus distal to the recombination breakpoint present in the one fertile D. [t/ SSSDDD] male (distal to Hba-ps4).

Although the fertility of a particular genotype can be demonstrated with the birth of a single offspring, the sterility of a particular genotype cannot be demonstrated unless data from enough animals are accumulated to be significantly different from the expected fraction of fertile males observed with control genotypes. Consequently, the data from this class of recombinants do not allow the determination of a definitive distal boundary for this hybrid sterility lo-

SSSSDD 4 8 S o d T c p 1 1 9 6 6 1 2 2 5 4 H b s P I m C ~

1 I1

DDSSSS - 4866119TcpSod6612254HbaPlm BB 1 I1

1 I1 - FIGURE 5.-Mapping the Hst-4' ;~lIc'Ic. \l;lle mice with a M .

sprefus background h;uhoring one complete M. spretus chromosome I7 homolog in conjunction with one of four M. spretus-M. domesficus recombinant t regions were tested for fertility as indicated in the text. The black boxes represent M. spretus DNA. Fertility values are presented a s a fraction of the total number of males tested. Marker loci employed i n the genetic analysis of each male are shown above each chromosome. The Hst-4' map position is indicated by the largest of the three solid bars at the bottom of the figure, the Hst-4' map position by the smallest of the three bars. and the Ifst- 4d map position by the middle sized bar.

cus. However, the results just described can be used to circumscribe the most probable region harboring the locus. First, only one of the eight males from the entire D.[t/SSSDDD] class was fertile. This fraction (1/8) is significantly different from the lowest fertility value obtained for any other fertile genome analyzed (5/8) when the chi-square test, corrected for small sample size, is employed ( P < 0.0 15). Second, when the fertile recombinant chromosome is removed from the remaining chromosome members of this class, the difference from other fertile genotypes is significant with a P value of less than 0.005. Finally, four males that carry one particular recombinant chromosome from this class (SSSDDD#l) were all determined to be sterile, yielding a P value of less than 0.05. Taken together, these results strongly suggest that the M . spretus sterility locus lies between Hba-ps4 and Pim-1.

Mapping of the M. domesticus hybrid sterility locus: The M . domesticus allele responsible for the sterility of S.[D/S] males was mapped according to the same general protocol used in the mapping studies described above. T w o recombinant [D * SI chromosomes (DSSSSS and DDSSSS) were recovered and bred into the M . spretus genetic background to produce S. [D * S/S] males that were tested and found to be fertile (Figure 5). This result indicates a proximal boundary for the M. domesticus sterility locus distal to Dl7Leh54. Recombinant chromosomes of the [S t, Dl type with breakpoints between Hba-ps4 and Pim-1 in one case, and between Pim-I and Crya-I in the second case, were also found to produce fertile S-[S * D/S] males. Thus the M. domesticus sterility locus must map between D l 7Leh54 and Pim-I.

DISCUSSION

Present-day samples of M. spretus chromosome I7 do not express functional properties characteristic

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of presentday t haplotypes: Comparative studies of chromosome structure have led to the formulation of two models for the origin of t haplotypes (HAMMER, SCHIMENTI and SILVER 1989). According to one model, t haplotypes originated through the introgression of a normal M. spretus (or M. spretus-like) chromosome I7 into an ancestral M. domesticus population. The major assumption underlying this model is that although the M. spretus chromosome I7 would have functioned normally within its host species, it might have carried unique alleles that allowed a preferential transmission relative to the normal M. domesticus chromosome in heterozygous animals. A precedent of this type has been described in tobacco (CAMERON and MOAV 1957), and in further support of this model, the current M. spretus range overlaps that of M. domesticus, and occasional formation of hybrids between these two species has been observed in the wild (F. BONHOMME, personal communication).

To determine whether present-day t haplotypes and M. spretus forms of chromosome I7 might continue to have the original functional relationship predicted by this model, a series of genetic studies were carried out. In the first experiment, a M. spretus chromosome I 7 was bred into a M. domesticus background and males heterozygous for this chromosome were tested for transmission ratios. The results demonstrate the equal transmission of the two chromosome I7 homologs, ruling out the possibility that the present-day wild-type M. spretus chromosome carries a cryptic meiotic drive system.

The second experiment was designed to determine whether t haplotypes would lose their meiotic drive activity when they were placed "back" into a M. spretus genome, as might be expected if these two forms of the chromosome were closely related. With the dem- onstration that t haplotype/M. spretus combinations of chromosome 17 caused hybrid sterility, it was not possible to test this hypothesis directly. However, one conclusion can be drawn from this result-it is ex- tremely unlikely that complete t haplotypes would be found as a polymorphism within present day populations of M. spretus because chromosomes carrying sterility factors tend to be lost from populations.

Finally, the transmission results obtained with a male that carries a third heterospecific combination of chromosome I7 homologs sheds further light on the relative functional properties of M. spretus alleles at four of the genes involved in the M. domesticuslt haplotype TRD phenotype. The informative male carries a complete t haplotype (t"") and a recombinant [S c* Dl homolog (SSDDDD in Table 1) with M. spretus alleles at the Tcr, Tcd-I, Tcd-3 and Tcd-4 loci involved in the expression of the TRD phenotype by t haplotypes. This male is fertile and expresses a transmission ratio of 100% in favor of the t-carrying

chromosome. If the M. spretus alleles at the three Tcd loci functioned like present-day t haplotype alleles, one would expect male sterility (LYON 1986). If the M. spretus allele at Tcr functioned like a t allele, then this genotype would be effectively homozygous at the Tcr locus and meiotic drive activity would be sup- pressed (LYON and MASON 1977).

This last result provides further evidence for the lack of a functional relationship between present-day M. spretus and t haplotype alleles at loci involved in the TRD phenotype. Rather, the M. spretus alleles at Tcr, Tcd-I, Tcd-3 and Tcd-4 appear to be functioning in a manner expected of M. domesticus alleles at these loci. In conclusion, the accumulated results described in this report argue against the likelihood that t haplotypes were derived from a M. spretus form of chromosome 17. Nevertheless, this possibility cannot be ruled out completely because all traces of a relationship between the two chromosomes could have been eliminated through a long period of divergence. Fur- thermore, as discussed earlier, a small distortion of transmission ratio may have gone undetected in our studies.

The hybrid sterility-1 (Hst-I) locus is not responsible for the M. spretuslM. domesticuslt haplotype hybrid sterility phenotype: T o date, three other hybrid sterility loci (Hst-I , Hst-2, Hst-3) have been described in the mouse genome (BONHOMME, GUENET and CATALAN 1982; FOREJT and IVANYI 1975; GUE- NET et al. 1990). One of these loci-Hst-l-maps to chromosome 17 and is responsible for a classic hybrid sterility phenotype in males that carry certain heterospecific combinations of chromosome 17 from the sibling species M. domesticus and M. musculus (FOREJT and IVANYI 1975). Recently obtained genetic data have allowed the high resolution mapping of Hst-1 between Sod-2 and Dl 7Tu50 proximal to the in(l7)4 inversion (FOREJT et al. 1991) (Figure 1A).

Since the hybrid sterility phenotypes described in this report are also caused by a gene(s) mapping to chromosome 17, it was important to determine whether the Hst-I locus was involved as well. TWO independent lines of evidence indicate that this is not the case. First, the loci involved in the M. spretus/M. domesticuslt haplotype phenotypes have been mapped definitively to a region distal to the D17Tu50 IOCUS. Second, preliminary studies indicate that the physiological basis for the sterility phenotypes described here is different from that caused by Hst-I (S. H. PILDER and P. OLDS-CLARKE, unpublished data). The Hst-1 phenotype is associated with a significant reduction in testes weight and the absence of epididymal sperm (FOREJT and IVANYI 1975). In contrast, the sterile males reported in our study have normal testes weights and display no abnormalities in sperm count. Thus, we have identified a novel locus, Hybrid sterility-

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Chromosome 17 Hybrid Sterility 245

4 (Hst-4), which is responsible for the sterility of males that carry heterospecific combinations of chromosome 17 with one M . spretus homolog and either a M . domesticus or t haplotype homolog.

The M. spretus, M. domesticus and t haplotype genes responsible for hybrid sterility are likely to represent alternate alleles at the same locus: In three separate sets of experiments, the hybrid sterility genes of M. spretus, M . domesticus, and t haplotypes were mapped to overlapping intervals in the distal portion of the t complex on chromosome 17. The highest resolution mapping was accomplished with M. spretus to the 3-cM region between Hba-ps4 and Pim-1. The next highest resolution was achieved with M. domesticus to the 5-cM region between D l 7Leh.54 and Pim-1. Finally, the least resolution was obtained with t haplotypes to the 9-cM region between D17Leh122 and DI7Leh89. The simplest interpretation of these data is that a single hybrid sterility locus (Hst-4) is entirely responsible for all forms of hybrid sterility described in this report. This locus would map between Hba-ps4 and Pim-1, and would have three alternate alleles, Hst- 4d in M. domesticus, Hst-4" in M. spretus, and Hst-4' in t haplotypes. More complex scenarios, however, cannot be ruled out at the present time and it is possible that the sterility phenotypes result from dominant interactions between closely linked but multiple genes present within or among the three different chromosome types (DUTCHER and LUX 1989; SANO 1990). Notwithstanding, until proven to the contrary, we will assume that a single Hst-4 locus is responsible for all of the sterility phenotypes described herein.

Speculation on the functional basis for the Hst-4 hybrid sterility phenotypes: Two types of Hst-4-associated hybrid sterility phenotypes have been described in this report. In the first, the Hst-4*/Hst-4" genotype causes sterility in a background-independent fashion. This result can be explained most simply by an incompatibility between the M. spretus and t haplotype products of the Hst-4 gene. In the second form of hybrid sterility, the H~t-4~/Hst-4" genotype causes sterility only in the M . spretus genetic background, and not in the M. domesticus background. This result cannot be explained by allelic incompatibility. Rather, it would appear that the product of the H ~ t - 4 ~ allele must be incompatible with one or more products expressed by unlinked M . spretus-specific genes.

A biochemical explanation of the first phenotype would hold that the product of the Hst-4 gene normally forms homodimers or higher order multimeric proteins, and Hst-4'/Hst-4" hybrid animals form non- functional dimers (or multimers) that interfere with some aspect of normal sperm cell differentiation. The second phenotype can be explained biochemically through the formation of heterodimeric proteins that

include the Hst-4 product as well as non-chromosome 17-encoded polypeptides.

An advantage of these two biochemical explanations is that they are unified through the proposition that Hst-4 products function within the context of larger multimeric units. Nevertheless, it is certainly possible to formulate more complex models, and it is only with the cloning of the Hst-4 gene or the biochemical and physiological characterization of its product that any model will be confirmed.

Finally, it is intriguing that the Hst-4 locus is not separable genetically from the t complex distorter-2 (Tcd-2) locus involved in t haplotype effects on male transmission ratio distortion and sterility. Further- more, it has been demonstrated that Tcd-2 must also function within the context of interacting protein products (LYON 1984, 1986). Nevertheless, it is not possible to further resolve the mapping of Tcd-2 within a 7-cM region because of an associated inversion (in(l7)4). Therefore, it is likely that Hst-4 will be cloned and characterized first, at which point, it will be possible to test its functional identity with Tcd-2.

This research was supported by grants from the National Insti- tutes of Health to L.M.S., and by postdoctoral fellowships from the National Institutes of Health to S.H.P. and M.F.H. We thank CHRISTINE BUCK for technical assistance, andJuov CEBRA-THOMAS and JEN-YUE TSAI for animal assistance. S.H.P. thanks JOANNA WIUON for technical advice and comments on the manuscript.

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Communicating editor: R. E. GANSCHOW

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A Novel Mouse Chromosome 17 Hybrid Sterility Locus - Genetics