Download Genotype, Phenotype, and Karyotype Correlation in the XO Mouse

Journal of Heredity 2008:99(5):512–517
doi:10.1093/jhered/esn027
Advance Access publication May 21, 2008
Ó The American Genetic Association. 2008. All rights reserved.
For permissions, please email: [email protected].
Genotype, Phenotype, and Karyotype
Correlation in the XO Mouse Model of
Turner Syndrome
FRANK J. PROBST, MITCHELL LANCE COOPER, SAU WAI CHEUNG,
AND
MONICA J. JUSTICE
From the Department of Molecular and Human Genetics, Baylor College of Medicine, Room R804, One Baylor Plaza,
Houston, TX 77030.
Address correspondence to Monica J. Justice at the address above, or e-mail: [email protected].
Abstract
The murine model for Turner Syndrome is the XO mouse. Unlike their human counterparts, XO mice are typically fertile,
and their lack of a second sex chromosome can be transmitted from one generation to the next as an X-linked dominant
trait with male lethality. The introduction of an X-linked coat-color marker (tabby) has greatly facilitated the maintenance of
this useful mouse strain. XO mice can be produced in large numbers, generation after generation, and rapidly identified on
the basis of their sex and coat color. Although this breeding scheme appears to be effective at the phenotype level, its utility
has never been conclusively proved at the molecular or cytogenetic levels. Here, we clone and sequence the tabby deletion
break point and present a multiplex polymerase chain reaction–based assay for the tabby mutation. By combining the results
of this assay with whole-chromosome painting data, we demonstrate that genotype, phenotype, and karyotype all show
perfect correlation in the publicly available XO breeding stock. This work lays the foundation for the use of this strain to
study Turner Syndrome in particular and the X chromosome in general.
Women with Turner Syndrome typically have short stature,
webbing of the posterior neck, an increased ‘‘carrying angle’’
at the elbows (cubitus valgus), and delayed or absent puberty
(Turner 1938). Gonadal dysgenesis is seen in the vast
majority of cases, and almost all affected individuals are
infertile (Sybert and McCauley 2004). The classic cause of
the disease is the complete absence of a second sex chromosome, leading to an abnormal 45,X karyotype, as
opposed to the normal 46,XX or 46,XY karyotypes (Ford
et al. 1959).
The murine model for Turner Syndrome is the XO
mouse. Like their human counterparts, XO mice have
a single X chromosome and no second sex chromosome.
Note that the nomenclature differs between human and
mouse. The International Standing Committee on Human
Cytogenetic Nomenclature defines the human karyotype as
‘‘45,X,’’ whereas the International Committee on Standardized Genetic Nomenclature for Mice defines the mouse
karyotype as ‘‘39,XO’’ or simply ‘‘XO,’’ where the letter ‘‘O’’
is a placeholder indicating the absence of a second sex
chromosome; there is no ‘‘O’’ chromosome.
The existence of XO mice was first suspected after the
discovery of mice that appeared to be homozygous for
various X-linked traits but failed to transmit an allele of the
trait to some of their female offspring. This finding was
512
explained by the hypothesis that these unusual animals were
not homozygous XX females but hemizygous XO females,
and their female progenies who did not inherit an allele of
the X-linked trait were also XO animals that failed to inherit
an entire X chromosome from their mothers (Russell et al.
1959; Welshons and Russell 1959). Chromosome counts on
a subset of these putative XO animals revealed 39
chromosomes in each case, as opposed to the normal 40,
providing strong support for this hypothesis (Welshons and
Russell 1959).
XO mouse stocks were subsequently developed using
the tabby mutation as a coat-color marker. The tabby
mutation (Ta) was the one of the first reported mouse traits
to show a clear X-linked pattern of inheritance. On an
agouti background, mice that are hemizygous or homozygous for the mutation have tan-colored fur and bald patches
behind the ears, whereas heterozygous females have stripes
of agouti fur and stripes of tan-colored fur, similar to that of
a tabby cat (Falconer 1952). The mutation has been shown
to be an ;2-kb deletion at the 5#-end of the ectodysplasinA gene (Eda) and has therefore been renamed EdaTa
(Ferguson et al. 1997; Srivastava et al. 1997).
The first XO mouse stock was created by breeding
female mice that were believed to be hemizygous for the
wild-type ectodysplasin-A allele (Edaþ/O) to male mice that
Probst et al. XO Mice
Figure 1. Phenotypes of animals in the XO breeding stock. To maintain the XO breeding stock, an agouti male from an outside
colony (I-1; Edaþ/Y, XY) is bred to a presumed XO female (with tan-colored fur and bald spots behind the ears) from the XO
breeding stock (I-2; presumed EdaTa/O, XO). Three types of offspring are seen: female animals with stripes of agouti fur and
stripes of tan-colored fur (II-1; presumed Edaþ/EdaTa, XX), male animals with tan-colored fur and bald spots behind the ears (II2; presumed EdaTa/Y, XY), and female animals with agouti fur (II-3; presumed Edaþ/O, XO). Animals II-2 and II-3 are interbred,
and there are again 3 different kinds of progeny: female animals with stripes of agouti fur and stripes of tan-colored fur (III-1;
presumed Edaþ/EdaTa, XX), male animals with agouti fur (III-2; presumed Edaþ/Y, XY), and female animals with tan-colored fur
and bald spots behind the ears (III-3; presumed EdaTa/O, XO). Animal III-3 can then be bred to another agouti male from an
outside colony to propagate the stock indefinitely. The sex of each animal is shown at the bottom right of each photograph (# 5
male, $ 5 female). All phenotypings (both sex and coat color) are done by simple visual inspection. The photographs also
demonstrate that the hemizygous EdaTa mutants (animals I-2, II-2, and III-3) have a tendency to squint, which is likely secondary to
well-documented deficiencies in the glands surrounding the eyes in mutant animals (Grüneberg 1971). Note that EdaTa/O, XO
females (I-2 and III-3) are bred to Edaþ/Y, XY males from an outside colony (I-1; a male from JAX stock number 001201) as
opposed to Edaþ/Y, XY males from within the XO breeding colony (III-2). This is done to promote fertility within the colony as
the stock is difficult to maintain if brother–sister matings are used exclusively (Deckers and van der Kroon 1981).
were hemizygous for the EdaTa mutation (EdaTa/Y). Three
kinds of offspring resulted: females with striped fur, who
were presumably XX and heterozygous for the EdaTa
mutation (Edaþ/EdaTa); males with agouti fur, who were
presumably XY and hemizygous for the wild-type Eda allele
(Edaþ/Y); and females with tan-colored fur and bald
patches behind the ears, who were presumably XO and
hemizygous for the EdaTa mutation (EdaTa/O). These
presumed XO (EdaTa/O) females were then bred to their
XY (Edaþ/Y) brothers, and again 3 kinds of offspring
resulted: females with striped fur (presumably XX, Edaþ/
EdaTa); males with tan-colored fur and bald spots behind the
ears (presumably XY, EdaTa/Y); and females with agouti fur
(presumably XO, Edaþ/O). Brother–sister matings between
XY and XO individuals could then be used to propagate the
stock indefinitely (Cattanach 1962). The expected YO
chromosome complement has been seen in 2-celled embryos but not thereafter, suggesting that it results in early
embryonic lethality; YO mice are never seen in the live
offspring (Morris 1968; Luthardt 1976).
Analysis of trypsin-Giemsa–banded chromosomes of these
presumed XO mice has yielded 39 XO karyotypes at the gross
cytogenetic level (Luthardt 1976), but more sophisticated
chromosomal analysis, such as whole-chromosome painting,
has never been reported. These studies would rule out
other anomalies, such as cryptic translocations, which can
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Journal of Heredity 2008:99(5)
be missed on routine karyotyping. Furthermore, although
the genotype at the Eda locus is believed to correlate with
the expected phenotype and karyotype of mice in an XO
breeding stock, this has never been demonstrated. The
genotype at the linked DXMit25 locus has previously been
shown to correlate with the coat-color phenotype in one
XO breeding stock, but this finding was not, in turn,
shown to correlate with the animals’ karyotypes (Valdivia
et al. 1996).
We describe here the cloning and sequencing of the
EdaTa mutation and the development of a multiplex polymerase chain reaction (PCR)–based assay for genotyping at
the Eda locus. We then use this assay to show that, for each
kind of mouse seen in the publicly available XO mouse
colony, an animal’s genotype at the Eda locus correlates
with its coat-color phenotype. Finally, we demonstrate that
each mouse’s genotype and phenotype also correlate with its
karyotype, based on whole-chromosome painting of bone
marrow cells. These results lay the groundwork for the use
of this XO strain both as a murine model for Turner
Syndrome and as a tool for the comprehensive functional
analysis of the X chromosome.
Materials and Methods
Mice
The XO mouse breeding stock was obtained from The
Jackson Laboratory (JAX, Bar Harbor, ME; JAX stock
number 000314). The scheme for maintaining this stock has
been described (Jackson Laboratory Staff 1990) and is
explained in Figure 1. The animals were housed and cared
for in the Transgenic Mouse Facility at Baylor College of
Medicine (BCM). This is a specific pathogen-free mouse
facility that is accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care (AAALAC).
Animals were provided food and water ad libitum.
The EdaTa mutation is known to cause tooth defects in
hemizygous and homozygous animals, which appears to
lead to impaired feeding and poor weight gain (Grüneberg
1965). In our hands, this effect is more pronounced in
hemizygous female animals than in hemizygous males. Thus,
cages containing EdaTa/O female mice were provided highfat mouse chow (PicoLab Rodent Diet 20, #5058; LabDiet,
Richmond, IN) as this chow is softer than regular mouse
chow and has a higher caloric content. In addition, a quarter
cup of powdered high-fat mouse chow (ground in a household kitchen blender) was added to these animals’ cages
once a week. The rest of the animals were fed regular mouse
chow (PicoLab Rodent Diet 20, #5053).
This work was approved by the Institutional Animal
Care and Use Committee at BCM (Protocol #AN-1757).
Genomic DNA Preparation
Mouse genomic DNA was prepared from tail biopsies as
described (Nagy et al. 2003), with minor modifications. The
DNA was quantified on an ultraviolet (UV)
514
Figure 2. Eda genotyping. (a) The EdaTa mutation was cloned
and sequenced and found to be a pure 1073-bp deletion that removes
the entire first exon of the Eda gene (GenBank accession number
EU178100). In a multiplex PCR reaction, primers A and B amplify
a 216-bp product from wild-type DNA, whereas primers C and D
amplify a 162-bp product. The EdaTa deletion removes the annealing
sites for primers B and C and brings together the annealing sites for
primers A and D, yielding a single 168-bp product. Thus, DNA
from a homozygous or hemizygous wild-type animal yields 216-bp
and 162-bp PCR products, DNA from a homozygous or
hemizygous mutant animal yields a single band of 168 bp, and
DNA from a heterozygote yields all 3 bands. The 3 different PCR
products were all sequenced to confirm their identities (data not
shown). (b) The pedigree shows a graphic representation of the
breeding scheme shown in Figure 1. Males are shown as squares,
and females are shown as circles. Dark brown shading indicates an
agouti coat color, tan shading indicates a tan-colored coat color
(and bald spots behind the ears), and stripes indicate a striped coat.
The electrophoresis gel shows the results of the Eda multiplex PCR
reaction for each of the respective animals in the pedigree from
Figure 1. All animals have the expected genotypes, and there is
perfect genotype–phenotype correlation. DNA from agouti
animals yields 216-bp and 162-bp bands (Edaþ), DNA from tancolored animals (with bald spots behind the ears) yields a single
band at 168 bp (EdaTa), and DNA from striped animals yields all 3
bands (Edaþ/EdaTa). The assay is not quantitative and does not
directly distinguish homozygotes (Edaþ/Edaþ and EdaTa/EdaTa)
from hemizygotes (Edaþ/Y, Edaþ/O, EdaTa/Y, and EdaTa/O).
However, it demonstrates that animal I-2 fails to transmit her
EdaTa allele to her female agouti offspring (II-3), and animal II-3 in
turn fails to transmit her Edaþ allele to her offspring with tancolored fur and bald spots behind the ears (III-3), which is
consistent with all 3 animals having the XO karyotype.
Probst et al. XO Mice
Eda Multiplex PCR
Figure 3. Whole-chromosome painting. Wholechromosome painting of the X and Y chromosomes was
performed on chromosome spreads from bone marrow cells of
the animals from generations II and III in Figures 1 and 2. The
DAPI counterstain gives all chromosomes a blue fluorescent
background color. The cyanine 3–labeled X chromosome
probe produces a red fluorescent signal, and the fluorescein
isothiocyanate-labeled Y chromosome probe produces a green
fluorescent signal. Cells from animals II-1 and III-1 have 2
chromosomes with red signals (XX), and cells from animals II2 and III-2 have 1 chromosome with a red signal and 1 with
a green signal (XY), whereas cells from animals II-3 and III-3
have 1 chromosome with a red signal and no second sex
chromosome signal (XO).
spectrophotometer and diluted to a concentration of 25 ng/
ll with Tris EDTA (TE) buffer (Sambrook and Russell
2001).
Amplification and Sequencing of the Eda
Ta
Mutation
Southern blotting has previously shown that the EdaTa
mutation is an ;2-kb deletion at the 5#-end of the Eda gene
(Ferguson et al. 1997; Srivastava et al. 1997). The deletion break
point was amplified with the following primers: 5#-GAG GAC
CTC CTC CCT CAT TC-3# and 5#-CCA AAA ATC CTT
GAC CAC CA-3#. This PCR product was then sequenced via
standard protocols (Sambrook and Russell 2001) (GenBank
accession number EU178100). Comparison of the break point
sequence to the wild-type sequence of the mouse X
chromosome (Kent et al. 2002) revealed the lesion to be a pure
1073-bp deletion that removes the entire first exon of the Eda
gene (based on GenBank accession number AF016628).
Table 1.
Based on the sequence of the Edaþ and EdaTa alleles, we
devised and optimized the following multiplex PCR reaction
to amplify both the Edaþ and EdaTa alleles from genomic
DNA. Primers were designed using the Primer3 algorithm
(Rozen and Skaletsky 2000). Each 25-ll reaction consisted
of 22-pmol primer A (5#-GTG AGG ACC TCC TCC CTC
AT-3#), 10-pmol primer B (5#-CCT CTG CCT CAG TCC
TTC AC-3#), 2.15-pmol primer C (5#-CAG GCT CCT CTT
TGG AGT TG-3#), 13.4-pmol primer D (5#-CGC AGA
AGA GAA TCC GTC TC-3#), 5-nm dNTPs, 1 standard
Taq reaction buffer, 2.5 U Taq polymerase (New England
Biolabs; Ipswich, MA), and 25 ng of genomic DNA. The
thermocycler profile used was 94 °C for 3 min; 30 cycles of
94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; 72 °C for
10 min; and 4 °C until the PCR products were ready to be
analyzed. The PCR products were then run on a 4%
MetaPhor agarose (Cambrex Bio Science; Rockland, ME),
0.5 Tris-borate-EDTA (TBE) gel at 100 V and visualized
via UV transillumination (Sambrook and Russell 2001).
Chromosome Preparation
Chromosome preparations were performed on bone
marrow cells via a slightly modified version of the protocol
found at The Jackson Laboratory’s Web site (Available
at: http://www.jax.org/cyto/marrow_preps_alt.html). The
final cell pellet was resuspended in 0.5–1 ml of fixative.
Approximately 20 ll of each cell suspension was placed
on a standard microscope slide for whole-chromosome
painting.
Whole-Chromosome Painting
The above slides were baked at 45 °C for 10 min, rinsed
with acetic acid, and then washed for 2 min each in 75%,
85%, and 95% ethanol. A premade mixture of the following
was added to each slide: 12-ll cyanine 3–labeled chromosome X probe, 12-ll fluorescein isothiocyanate (FITC)labeled chromosome Y probe, and 36-ll hybridization
solution (Open Biosystems, Huntsville, AL; Catalog numbers SFP2281 and SFP2241). The slides were heated to 73
°C for 3 min to denature the probes and then hybridized at
37 °C overnight. The following morning, the slides were
rinsed twice in 0.4 standard saline citrate (SSC), 0.3% NP40 at 73 °C for 2 min and then rinsed once in 2 standard
saline citrate, 0.1% Igepal for 1 min. Finally, the slides were
counterstained with 4#,6-diamidino-2-phenylindole (DAPI)
and examined via standard fluorescence microscopy.
Progeny counts: Edaþ/Y EdaTa/O
Laboratory
Number of
litters
Average litter
size at weaning
Striped females
(Edaþ/EdaTa, XX)
Tan males
(EdaTa/Y, XY)
Agouti females
(Edaþ/O, XO)
BCM
JAX
36
—
4.5
—
68 (42%)
272 (46%)
65 (40%)
249 (42%)
30 (18%)
76 (13%)
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Journal of Heredity 2008:99(5)
Table 2.
Progeny counts: EdaTa/Y Edaþ/O
Laboratory
Number of
litters
Average litter
size at weaning
Striped females
(Edaþ/EdaTa, XX)
Agouti males
(Edaþ/Y, XY)
Tan females
(EdaTa/O, XO)
BCM
JAX
133
—
4.3
—
267 (47%)
257 (41.5%)
236 (42%)
239 (39%)
65 (11%)
121 (19.5%)
Results and Discussion
Like humans with the classic 45,X Turner Syndrome
karyotype, XO mice have only a single-sex chromosome
in all their cells. Unlike their human counterparts, however,
affected mice are usually fertile. The introduction of the
tabby (EdaTa) mutation as an X-linked coat-color marker has
allowed XO animals to be maintained for decades at The
Jackson Laboratory by selecting for animals with the
appropriate sex and coat color (Figure 1).
Genotyping by Eda multiplex PCR was performed on
each kind of animal observed in this XO breeding stock and
confirms that each animal has the expected genotype
predicted by its observed coat-color phenotype (Figure 2).
This assay conclusively demonstrates that XO females fail to
transmit a copy of their Eda gene to their XO offspring, and
it excludes the possibility that the predicted XO animals are
Edaþ/EdaTa, XX females with skewed X inactivation.
In order to confirm that the predicted XO animals have
a pure XO karyotype and not a cryptic translocation or
other subtle chromosomal anomaly, whole-chromosome
painting was performed on the animals from generations II
and III of the pedigree shown in Figures 1 and 2. These
results show that animals II-3 and III-3 inherited a single X
chromosome and no second sex chromosome. Animals II-1
and III-1 have the expected XX karyotype, and animals II-2
and III-2 have the expected XY karyotype. No evidence of
a complex rearrangement involving the X or Y chromosomes was seen in any of the cells examined (Figure 3). This
confirms that this stock is truly segregating the absence of
a second sex chromosome as an X-linked dominant trait
with male lethality. Taken together, these results show that
this breeding stock permits the maintenance of XO mice
with perfect genotype, phenotype, and karyotype correlation.
Progeny counts on animals in the stock from both our
laboratory at Baylor College of Medicine (BCM) and from
The Jackson Laboratory (JAX) show a clear deviation from
the expected 1:1:1 ratio of XX, XY, and XO animals (Tables
1 and 2). This likely occurs for several reasons. First, it has
been previously demonstrated that there is nonrandom
segregation of the single X chromosome during oogenesis in
XO mice, with the X chromosome being transmitted to the
oocyte about two-thirds of the time (as opposed to the
predicted one-half) (Kaufman 1972), so XO embryos are
expected to occur only half as frequently as XX and XY
embryos (i.e., the expected XX:XY:XO ratio immediately
after fertilization is 2:2:1). Second, some XO embryos that
implant near the cervix develop extremely poorly. It is not
clear why this phenomenon occurs, but it could presumably
reduce the number of XO progenies that are born
516
(Burgoyne et al. 1983). Third, the EdaTa mutation is not
simply a coat-color mutation. It is a pleiotropic mutation
that affects a number of organ systems and has a significant
deleterious effect on homozygous and hemizygous mutant
animals (Grüneberg 1965, 1971).
Two of the most clinically important features of Turner
Syndrome are cardiac disease and renal malformations
(Sybert and McCauley 2004). There are no published reports
of studies on XO mice for either of these conditions. Our
multiplex PCR assay allows the rapid and easy identification
of XO mice at any stage of development, so studies of the
hearts and kidneys of these animals can now be performed
to determine if cardiac or renal disease is a common finding
in XO mice.
Finally, the XO mouse can be a powerful tool for the
functional analysis of the mouse X chromosome. Previously,
phenotype-driven mutagenesis screens have focused on
genome-wide autosomal dominant mutations (Hrabe de
Angelis et al. 2000; Nolan et al. 2000) or region-specific
autosomal recessive mutations (Kile et al. 2003).
A phenotype-driven mutagenesis screen for X-linked recessive mutations has never been reported. The XO mouse
provides an obvious avenue to accomplish this. ENUmutagenized males (Edaþ/Y, XY) can be bred to EdaTa/O,
XO females, and the Edaþ/O animals that result from such
a cross can be identified via our multiplex PCR assay. These
mice will be hemizygous for an N-ethyl-N-nitrosourea
(ENU)-mutagenized X chromosome and will therefore
express any X-linked recessive traits that were induced by
the mutagen. Thus, the use of the XO mouse allows for
a one-generation screen for X-linked recessive mouse
mutants. Furthermore, the Edaþ/EdaTa, XX females that
result from such a cross can also be examined for any
deviation from the expected striped-fur phenotype as these
animals may represent mutations that cause skewing of X
inactivation. Clearly, the XO mouse is valuable not only just
as a murine model for Turner Syndrome but also as a tool
for the study of all the genes on the X chromosome.
Funding
Burroughs Wellcome Fund (Career Award for Medical
Scientists #1006860 to F.J.P.); National Institutes of Health
(F32HG003942 to F.J.P. and R01CA115503 to M.J.J.).
Acknowledgments
We thank Curtis Clark of California State Polytechnic University for
designing the male and female symbols used in the text and figures, Julie
Probst et al. XO Mice
Soukup and Muriel Davisson of The Jackson Laboratory for providing
progeny counts from their XO colony, and Muriel Davisson of The Jackson
Laboratory for her suggestions for improving the manuscript.
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Received September 25, 2007
Accepted March 10, 2008
Corresponding Editor: Roger Reeves
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