J. Embryol. exp. Morph. 78, 1-22 (1983)
Printed in Great Britain (E) The Company of Biologists Limited 1983
Non-random X-chromosome inactivation in mouse
X-autosome translocation embryos—location of the
inactivation centre
By SOHAILA RASTAN 1
From the Division of Comparative Medicine, Clinical Research Centre,
Harrow
SUMMARY
X-chromosome inactivation was investigated cytologically using the modified Katida
method which differentially stains inactive X-chromosome material at metaphase in balanced
13|-day female embryos heterozygous for four X-autosome rearrangements, reciprocal translocations T(X;4)37H, T(X;11)38H and T(X;16)16H (Searle's translocation) and the insertion
translocation Is(7;X)lCt (Cattanach's translocation). In all cases non-random inactivation
was found. In the reciprocal translocation heterozygotes only one translocation product ever
showed Kanda staining. In addition in a proportion of cells from T(X;4)37H, T(X;11)38H &nd
Is(7;X)lCt the Kanda staining revealed differential staining of X-chromosome material and
attached autosomal material within the translocation product.
In a study of 8£-day female embryos doubly heterozygous for Searle's translocation and
Cattanach's translocation two unbalanced types of embryo were found. In one type of
unbalanced female embryo of the karyotype 40(X(7)/X16;16/16) no inactivated Xchromosomal material is found. A second unbalanced type of female embryo, of the presumptive karyotype 40(X(7)/XN;16x/l6) was found in which two inactivated chromosomes were
present in the majority of metaphase spreads. A simple model for the initiation of Xchromosome inactivation based on the presence of a single inactivation centre distal to the
breakpoint in Searle's translocation explains these findings.
INTRODUCTION
Chromosomal rearrangements between the X chromosome and the autosomes interrupt the physical continuity of the X chromosome and are thus of use
in the study of both the randomness and mechanisms of X-inactivation. One
property of X-autosome translocations is the spread of inactivation into
autosomal loci attached to the X chromosome analogous to position-effect
variegation in Drosophila (Baker, 1971; Cattanach, 1974; Russell & Montgomery, 1970). Female mice which are heterozygous for X-autosomal translocations and carry marker genes which are recessive in the relevant autosomes
exhibit a variegated phenotype due to spreading of inactivation into attached
autosomal material in cells where the translocated X chromosome is inactive.
1
Author's address: Division of Comparative Medicine, Clinical Research Centre, Watford
Road, Harrow, Middlesex HA1 3UJ, U.K.
2
S. RASTAN
To date 14 X-autosome translocations have been reported in the mouse
(reviewed by Eicher, 1970 and Searle, 1981). X-autosome rearrangements are
often characterized by non-random X-inactivation. This could be the result of
initial random X-inactivation followed by selection for cells with the maximum
genetic balance or the result of primary non-random X-inactivation caused by a
disturbance in the process of X-inactivation due to rearranged control centres.
There is ample evidence that cell selection does operate in the two cell populations generated by random X-inactivation, for example in the female mule and
hinny (Giannelli & Hamerton, 1971; Hamerton et al. 1971; Hook & Brustman,
1971; Cohen & Rattazzi, 1972), in heterozygotes for certain X-autosome aberrations in mouse and man (Cattanach, 1975; Disteche, Eicher & Latt, 1979;
Russell & Cacheiro, 1978) and in blood cell populations of women heterozygous
for X-linked hypoxanthine phosphoribosyl transferase deficiency (Nyhan et al.
1970). The data on inactivation centres are less clear cut. Various models for the
initiation of X-inactivation have been proposed based on either the concept of
a single inactivation centre on the X chromosome (e.g. Russell & Cacheiro,
1978) or more than one inactivation centre (e.g. Eicher, 1970; Disteche, Eicher
& Latt, 1981). Previous work on balanced carriers of various reciprocal Xautosome translocations in the mouse suggest that only one translocation
product is ever inactivated in cells in which the normal X is active (Russell &
Montgomery, 1965, 1970). In particular in the autoradiographic studies of
Russell & Cacheiro (1978) in which late replication was used as evidence of
inactivation in cells from adults and 18-day embryos heterozygous for six
independent translocations T(X;7)2R1, T(X;7)3R1, T(X;7)5R1, T(X;7)6R1,
T(X;4)1R1 and T(X;4)7R1 it was found that in each case the shorter translocation product was never late-labelling in any cell. These results support the concept of a single inactivation centre from which inactivation is able to spread in
both directions. However the inactivation of both parts of an X chromosome
divided by an autosomal insertion such as in Is(X;7)lCt (Cattanach's translocation) has been used to support the concept of at least two inactivation centres
(Eicher, 1970).
Searle's translocation (T(X;16)16H) is of particular interest in the study of the
mechanism of X-inactivation as it is the only X-autosome translocation in the
mouse described to date that has the breakpoint in the X in a more or less central
position. It is also characterized by marked nonrrandom inactivation.
Heterozygous females behave as if the normal X is completely inactive in all
cells. All X-linked mutant genes located on either part of the translocated X
behave as if dominant, without variegation in the heterozygote (Lyon, 1966;
Lyon, Searle, Ford & Ohno, 1964). In a cytogenetic study using Budr (5-Bromo2'-deoxyuridine) labelling Takagi (1980) reported that in balanced Searle's
translocation heterozygotes at 6-5 days most cells had the normal X chromosome
inactivated. He also found an unbalanced carrier embryo of the karyotype
40(XN/X16;16/16) which did not show any asynchronously replicating
Non-random X-chromosome inactivation in mouse embryos
3
16
chromosome in any cell, despite the fact that inactivation of the X translocation
product (containing the centromeric portion of the X) would have restored
genetic balance. He concluded that the X16 product was incapable of being
inactivated due to lack of an -inactivation centre and also" suggested that the
concurrence of at least two X-chromosome loci separated by the breakpoint in
Searle's translocation was necessary for the homologous X to be inactivated. In
conflict with these results is the report by Disteche et al. (1981) using Budr
labelling in balanced female Searle's translocation heterozygotes that in adult
bone marrow and in 9-day embryos respectively, 7 % and 1 % of cells had the
X16 product late replicating. This has been used to support the concept of at least
two inactivation centres on the X chromosome.
The studies reported here were designed to obtain further data on this
controversy using the modified Kanda method (1973), (Rastan, Kaufman,
Handyside & Lyon, 1980; Rastan, 1981) for differential dark staining of the
inactive X chromosome, on female embryos heterozygous for the reciprocal
translocations T(X;4)37H, T(X;11)38H and T(X;16)16H (Searle's translocation) and the insertion Is(7;X)lCt (Cattanach's translocation).
MATERIALS AND METHODS
X-autosomal translocations (for review see Searle, 1981)
T(X;4)37H (Fig. 1A). A reciprocal translocation between the X and
chromosome 4 to give long and short somatic marker chromosomes. The translocation will be hereafter abbreviated to T37H.
T(X;11)38H (Fig. IB). A reciprocal translocation between the X and
chromosome 11 which gives long and short somatic markers. The translocation
will be hereafter abbreviated to T38H.
Is(7;X)lCt (see Fig. 1C). This translocation involves an inverted piece of
chromosome 7 inserted into the X to produce a long somatic marker. The translocation occurs as two types, Type I, balanced, with the inserted X and a deleted
chromosome 7, and Type II with the inserted piece of chromosome 7 present as
a duplication (Cattanach, 1974). Unlike carriers of the other X-autosome translocations males of both types can be fertile. In this study only Type II (i.e.
unbalanced duplication form) females and males were used. The translocation
will be hereafter abbreviated to IslCt.
T(X;16)16H (see Fig. ID). A reciprocal translocation between the X and
chromosome 16. The longer translocation product with the centromeric segment
of the X (X16) corresponds roughly in length to the intact X, and the shorter
translocation product with the centromeric segment of chromosome 16 (16X), to
the intact chromosome 16 (Eicher, Nesbitt & Francke, 1972). The translocation
will be hereafter abbreviated to T16H.
S. RASTAN
T38H
T37H
ft wa-2
spf
spf
vt
wi
b
m
Ta
Ta
Gy
Gy
TICt
T16H
D
X 16
spf
spf
Ta
Mo
Bn
Gy
c
P
ru-2
Gy
Fig. 1. Relative cytological lengths of the various translocations: (A) T(X;4)37H,
(B) T(X;11)38H, (C) Is(7;X)lCt (Cattanach's translocation) and (D) T(X,16)16H
(Searle's translocation).
Embryos
Embryos heterozygous for the various X-autosome translocations were
produced by mating spontaneously ovulating females heterozygous for T37H,
Non-random X-chromosome inactivation in mouse embryos
5
T38H and IslCt to normal Fi males of the 3H1 strain (Fi between two inbred
strains 101/H and C3H/HeH). The Xce status of the translocated Xes have not
been characterized; however the X chromosome of the 3HI males is known to
carry Xce?.
For T16H/+ heterozygous embryos, as the translocation products are not
sufficiently different in size from a normal X chromosome or a normal
chromosome 16 to be morphologically distinguishable, embryos doubly
heterozygous for T16H and IslCt were produced to facilitate distinguishing
between X chromosomes. This was achieved by mating spontaneously ovulating
T16H/+ females to fertile Type II IslCt males. The validity of this approach has
been demonstrated by Takagi (1980) who produced T16H/+ and T16H/IslCt
heterozygous embryos and confirmed cytologically that there was no behavioural
difference between the XN and X ^ in the presence of T16H. This was also
demonstrated genetically by Cattanach (1974).
The day of finding the copulation plug was designated day \ of pregnancy.
Pregnant females were killed by cervical dislocation on day 13! of pregnancy, the
embryos dissected out of the uterus and the extraembryonic membranes discarded. The embryos were sexed by dissection of the gonad, male embryos were
discarded and female embryos were kept for chromosome preparations. A number of pregnant T16H/+ females which had been mated to IslCt males were
killed on day %\ of pregnancy in order to recover unbalanced embryos which
would otherwise be resorbed later in pregnancy.
Chromosome preparations
The 13^-day female embryos were treated individually by the modified Kanda
(1973) method for revealing the inactive X chromosome at metaphase, as
previously described (Rastan etal. 1980). Briefly the embryos were incubated in
medium 199 containing colchicine (4/igml" 1 final cone.) to accumulate
metaphases, then placed for 15min in hypotonic 0-5 % KC1 (w/v) at 50 °C and
fixed in 3:1 absolute alcohol/glacial acetic acid fixative. The embryos were then
disaggregated in 60 % acetic acid for 5 min and slides made on a hotplate at 40 9 C.
After staining in 2 % Giemsa (buffered at pH6-8) for 20 min the inactive Xchromosome material stains much darker than any of the other chromosomes.
The 8£-day Searle's translocation embryos were dissected into two parts. One
part was treated by the modified Kanda technique for revealing the inactive X
chromosome and the second part was used for conventional chromosome
preparation for G-banding for karyotype analysis (Wurster, 1972).
Analysis
Translocation carriers were recognized by the presence of marker
chromosome(s) in the metaphase cells. In the case of T37H and T38H both long
and short marker chromosomes are present. For IslCt, because it is an insertion,
a long marker chromosome only is observed. In the case of T16H as no long or
6
S. RASTAN
short markers are present, it is not possible to screen directly for translocation
heterozygosity. However to distinguish T16H heterozygotes at 13£ days a genetic
approach was adopted based on the premise that T16H causes extreme nonrandom inactivation. Female embryos from the cross IslCt/Y males by
T16H/+ females, will be of the type IslCt/+ or IslCt/T16H. Since it is known
that T16H causes extreme preferential inactivation of the X not involved in the
reciprocal translocation, embryos in which virtually all the cells showed inactivation of only the marker IslCt chromosome were deduced to be double heterozygotes of the type IslCt/T16H. Embryos which had two types of cells (i) with
the long marker IslCt inactive or (ii) with a chromosome of approximately the
size of a normal X inactive, were deduced to be heterozygous for IslCt only, i.e.
of type IslCt/+, in which random X-inactivation had occurred.
Metaphase cells from female embryos which had been established to be translocation heterozygotes were scored in the following way:- (1) for the presence
of a dark staining normal X chromosome or a dark staining translocated X
chromosome; (2) for whether both translocation products were dark staining or
whether only one translocation product was dark staining in cells in which the
normal X was the active chromosome, to distinguish between inactivation of one
or both translocation products; and (3) for whether or not differential staining
could be seen in an inactive translocation product(s).
The 8i-day embryos were sexed cytologically by determining the presence or
absence of a Y chromosome. This task was facilitated by the fact that the Y
chromosome is the darkest staining element in over 70 % of cells from postimplantation male mouse embryos treated by the modified Kanda method (Rastan, 1981). Slides of embryos without a Y chromosome were assumed to be of
female embryos and were scored for the presence or absence of a dark staining
inactive X chromosome(s).
RESULTS
T37H/+ and T38H/+ embryos
Four adult T37H/+ heterozygous females produced a total of 19 embryos at
13| days of which 11 were female and 3 proved to be heterozygous for T37H on
chromosomal analysis. All three embryos were balanced carriers of the translocation. Four adult T38H/+ heterozygous females produced a total of 21 embryos, of which 8 were female and 3 proved to be heterozygous on chromosomal
analysis. Again, all three embryos were balanced carriers of the translocation.
In both T37H heterozygotes and T38H heterozygotes the short translocation
product was never dark staining in any cell. The dark X was either the normal
X or the long translocated X (see Table 1 and Figs 2A and 2B). No cell in which
both translocation products were dark staining was ever seen for either translocation. In both T38H and T37H heterozygotes the proportion of cells with one or
the other X dark staining differed significantly from the 1:1 ratio expected for
3
3
7
T37H/+
T38H/+
IslCt/+
230
196
297
Total
191
(70-5 ± 2-8)
113
(63-5 ± 3-6)
84
(39-1 ±3-3)
Dark
(%±S.E.M.)
XN
20
6
Diff (%)
60
(75-0)
59
(90-8)
73
(55-7)
58
Unif.
A
LTX dark
80
(29-0 ± 2-8)
65
(36-5 ± 3-6)
131
(60-9 ±3-3)
(%±S.E.M.)
Total
0
0
NA
0
NA
Both
TX dark
0
STX
dark
15
18
26
No dark
X
8-s
3
chromosi
Key TX = Translocated X
LTX = Long translocated X
STX = Short translocated X
Diff. = Differential staining
Unif. = Uniform staining
No. of
embryos
Genotype of
embryo
No. of Metaphases
Table 1.
3
3
Non
nactivation
8
S. RASTAN
random X-inactivation (f = 45-46 for T37H, P < 0-001 and / = 12-94 for T38H,
P< 0-001).
For both T37H and T38H heterozygotes when the long translocation product
X
(4 or ll x ) was the dark-staining chromosome in the cell differential staining
within the long translocated X could frequently be seen (Figs 2A and 2B). For
both translocations dark staining is clearly seen in the distal part of the
translocated chromosome, which corresponds roughly to the region of the Xchromosomal material, whereas the proximal part of the translocation, corresponding roughly to the autosomal part of the translocation is pale staining. Table
1 shows that for T37H on average 75 % of cells in which 4X was the inactive
chromosome showed differential staining within the translocated chromosome,
and for T38H on average 90-8 % of cells in which the l l x was the inactive
chromosome showed differential staining within the translocated chromosome.
IslCt/+ heterozygous embryos
Four adult IslCt/+ females produced a total of 30 embryos at 13 days of which
16 were female and 7 proved to be heterozygous for IslCt on chromosomal
analysis. All 7 embryos were unbalanced Type II carriers. The inserted X
chromosome (X7) was the dark-staining chromosome in 60-9 % of these cells
compared with the normal X as the dark-staining chromosome in 39-1 % of cells
(Table 1). This represents a departure from the 1:1 ratio expected from random
inactivation which is significant at P = 0-01 level. A proportion of cells (55 %)
also showed differential staining within the X ^ chromosome when inactive
(Table 1). Fig. 2C shows a cell in which X(7) is the inactive X chromosome, where
a pale-staining region corresponding to the region of the autosomal insertion can
be seen.
T16H/IslCt doubly heterozygous embryos
a) 131-day embryos
Four adult T16H/+ females crossed to Type II IslCtY males produced a total
of 19 embryos at 13| days, 8 of which were female. Five of these embryos were
deduced to be of the type IslCt/+, as discussed in the Analysis (Materials and
Methods). The three remaining embryos were deduced to be double heterozygotes of the type T16H/IslCt, with the karyotype 40(X(7>/X16;16x/16), as
Fig. 2. (A) Metaphase spread from a female embryo heterozygous for T37H with the
4X chromosome, the long translocation product (arrow), inactive and showing a differential pale-staining region corresponding to the autosomal part of the translocation. Arrowhead indicates short translocation product. (B) Metaphase spread from a
female embryo heterozygous for T38H with the ll x chromosome, the long translocation product (arrow), inactive and showing a differential pale-staining region corresponding to the autosomal part of the translocation. Arrowhead indicates short translocation product. (C) Metaphase spread from a female embryo heterozygous for IslCt
with the translocated X (X^7)) inactive (arrow) showing a pale-staining region corresponding to the region of the autosomal insertion.
Non-random X-chromosome inactivation in mouse embryos
2A
B
:•. r
9
10
S. RASTAN
previously discussed. In these three embryos the long marker IslCt chromosome
was the dark-staining chromosome in 100 out of 102 cells that showed a darkstaining chromosome, i.e. 98-0 % which is in accord with the evidence previously
discussed of the extreme non-random inactivation caused by T16H. The two cells
that did not have the long IslCt marker dark staining both showed an exceptionally short dark-staining chromosome, deduced to be the 16X, the shorter translocation product of T16H with the centromeric segment of chromosome 16, on
the basis of size. No cell with both translocation products dark staining was ever
seen.
b) 8j-day embryos
19 embryos were recovered from six pregnant T16H females. Fig. 3 shows the
possible outcome of balanced and unbalanced zygotes produced from the cross
T16H female and IslCt Y male. Five of these embryos were male, as ascertained
by the presence of a dark-staining Y chromosome. As described in Analysis
(Materials and Methods) for the 13|-day embryos two types of balanced female
embryos were found (i) four embryos in which virtually all the cells showed
inactivation of only the marker IslCt chromosome and (ii) seven embryos which
had two types of cells, with the longer marker IslCt inactive or with a
chromosome of approximately the size of a normal X-inactive. These proved on
karyotype analysis to represent females of the type IslCt/T16H and IslCt/+
respectively, as deduced for the 13^-day embryos. Two female embryos which
showed no dark-staining X-chromosomal material proved on karyotype analysis
to be unbalanced Type (a) i.e. X (7) /X 16 ;16/16 thus confirming Takagi's observation that no inactive X is present in unbalanced female embryos of this type
(Type (a), Fig. 3) (Takagi, 1980).
In addition one exceptional female embryo was recovered in which two darkstaining chromosomes, one longer and one shorter, could be seen in the majority
of cells. The 42 cells (out of the 44 analysed) from this embryo with two darkstaining chromosomes could be divided into two types, (i) 18 cells in which the
ratio of the length of the long dark-staining chromosome to the short darkstaining chromosome was about 2-5 (Figs 4A, B) and (ii) 24 cells in which the
ratio of the long to the short dark-staining chromosome was about 1-75 (Figs 4C,
D). One presumptive tetraploid cell was also found which showed four darkstaining chromosomes, two longer and two shorter (Fig. 4E). Of the two cells
(out of the 44 analysed) which did not have two dark-staining chromosomes, one
had a long dark-staining chromosome and the other had a short dark-staining
chromosome. Unfortunately this embryo was small and retarded so no
karyotype analysis was performed on it due to the small amount of material
available. However, it was deduced to be a chromosomally unbalanced Type (b)
(Fig. 3) with the karyotype 40(X(7)/XN;16x/16) for the following reasons:
(1) The two classes of cells with the ratio of the longer to the shorter darkstaining chromosome of 2-5 and 1-75 are thought to represent cells in which
o
s
W
H
/[ALE
V
£
Balanced $ carrier for T16H
also carrying TlCt
*
16 j
#
fx 17 j
#
Sterile cf carrier
A
lY
t Ix I
4
(x
J
16
X
X
(
JY
|x
J16 [l« {X
Normal cf
Tl6 Tl6
116
leterozygous for TlCt
(
X
<
16]
1
X
• \ y iv
I
16
lx
27
J16 Tx
Unbalanced $
(Type a)
|16
Cf nullisomic for
distal segment of X
16;
X
4
16]
X
I"
[ I[X16 |16
I h'
<
X
1
Unbalanced $
(Type b)
Jx
(x i
j
Cf monosomic for
distal segment of 16
X
X
UNBALANCED
Fig. 3. Diagram to show the possible balanced and unbalanced zygotes produced from the cross T16H/+ female x IslCt Y male.
xf
1%
<16
X
16
X
16
X
BALANCED
FEMALE GAMETES
12
S. RASTAN
*•*»
LU
-* »
1+'
>
/
*
A
V
CQ
Non-random X-chromosome inactivation in mouse embryos 13
plus 16X and XN plus 16X respectively are inactive. This surmise is supported by
the fact that the long chromosome in cells with the ratio of long: short of 2-5 often
showed a pale-staining region corresponding to the region of the autosomal
insertion of chromosome 7 into the X in IslCt (see Fig. 4B) whereas the longer
chromosome in cells with a ratio of long: short of 1-75 never showed such a palestaining region;
(2) The shorter, dark-staining chromosome is thought to be 16X, the shorter
translocation product of T16H, as it appears to be shorter than a normal X
chromosome appears after the heat/hypotonic treatment;
(3) The Kanda method occasionally produces G-banding (Rastan,
unpublished observations). In Fig. 4D where G bands are visible, the G band
pattern is compatible with the dark-staining chromosome being 16X, the shorter
translocation product;
(4) In Fig. 4E, the tetraploid cell in which four dark-staining chromosomes can
be seen, a pale-staining area near the centromere can be seen in both the shorter
chromosomes, compatible with these chromosomes being the 16X translocation
products and spread of heterochromatinization from the X into the autosome
being incomplete.
DISCUSSION
Three inferences may be drawn from the results presented in this paper; (l)
non-random inactivation is seen in embryos heterozygous for the X-autosome
translocations T37H and T38H as well as for T16H and IslCt; (2) when an X
chromosome is divided into two parts by a reciprocal translocation, only one part
is capable of Kanda staining (3) spread of genetic inactivation from the X
chromosome into translocated autosomal material, which results in positioneffect variegation, can be reflected by heterochromatinization at the
chromosomal level, and the spread of heterochromatinization appears to be
variable.
Inactivation of only one translocation product
The fact that only one translocation product in the reciprocal translocations
T37H and T38H was ever seen to be dark staining by the modified Kanda method
Fig. 4. Metaphase chromosomes from an exceptional unbalanced female embryo
carrier of Searle's translocation in which there are two dark-staining inactive
chromosomes per cell, one longer and one shorter (arrows). (A) and (B) cells in
which the ratio of the long: short dark-staining chromosome is 2-5. Note palestaining region corresponding to the autosomal insertion in IslCt in longer darkstaining chromosome in (B). (C) and (D) cells in which the ratio of the long: short
dark-staining chromosome is 1-75. (E) A tetraploid cell with four inactive darkstaining chromosomes, two longer and two shorter (arrows). Note pale-staining
region on the proximal part of the two shorter dark-staining chromosomes.
14
S. RASTAN
in 13|-day embryos supports the concept of a single inactivation centre located
distal to the breakpoints on the X in T37H and T38H, resulting in inability of the
short translocation product in each case to become inactivated. An alternative
explanation is that lack of dark staining of the small translocation product could
be an artefact. As discussed elsewhere (Rastan, 1981) the modified Kanda
method does not reveal an inactive X in 100 % of cells, and it could be that the
small translocation products in these cases are more prone to failure of the
differential staining perhaps by virtue of their small size. This explanation is
considered unlikely, however, as the Kanda method reveals a dark-staining Y
chromosome in over 70% of cells from 13^-day male embryos (Rastan, 1981)
and therefore small size per se is unlikely to preclude or interfere with the
differential staining produced by the modified Kanda method. In addition, the
fact that for T37H and T38H not a single metaphase was found with the short
translocation product dark staining constitutes compelling evidence that the
observed results represent failure of the short translocation products to become
inactivated, and are not artefactual. Unfortunately genetic evidence is somewhat
lacking due to a shortage of suitable markers near the centromere of the X
chromosome.
The data presented here supporting the concept of a single inactivation centre
on the X chromosome are in agreement with the autoradiographic studies of
Russell & Cacheiro (1978) on six of Russell's X-autosome translocations in which
the short translocation product was never late-labelling in any cell. There is also
some genetic evidence from Russell's translocations which suggests that only one
part of an X divided by a reciprocal translocation is capable of inactivating
attached autosomal loci (Russell & Montgomery, 1965, 1970). In particular in
the R2 translocation (T(X;7)2R1), a reciprocal translocation between the X
chromosome and chromosome 7, in which the breakpoint on chromosome 7 is
between c and p (c being distal to p) and the breakpoint in the X is 23 units on
the proximal side of Ta, the c locus is never inactivated, even though this locus
lies close to the breakpoint and is readily inactivated in the three other reciprocal
translocations involving chromosome 7, namely, T(X;7)3R1 T(X;7)5R1 and
T(X;7)6R1.
The inactivation of both parts of an X chromosome divided by an autosomal
insertion as in IslCt has been used to support the concept of at least two inactivation centres. However, there is no a priori reason why one inactivation centre
should not be capable of inactivating both parts of the X separated by an
autosomal insertion if the spread of inactivation were not interrupted in the
autosomal material. At first sight the differential pale staining of a region corresponding to the autosomal insertion found in IslCt when treated by the modified
Kanda method may appear incompatible with this last stipulation. However,
Cattanach (1974) has shown that subsequent reactivation of initially inactivated
autosomal loci is possible in IslCt. This could explain the pale-staining region
seen in about 55 % of cells in which the X ^ was the inactivated dark-staining
Non-random X-chromosome inactivation in mouse embryos 15
chromosome in 13^-day embryos, and also the observation that the autosomal
insertion was early replicating in about 50 % of cells from 9-day and 13-day
IslCt/+ embryos labelled with Budr in which the X(7) was the late-replicating
chromosome(Disteche et al. 1979).
Differential staining within translocated chromosomes
The differential staining seen in a proportion of cells within the translocation
products when inactive could represent variable and limited spread of heterochromatinization from the inactive X portion into the attached autosomal
material, or it could be the result of initial complete heterochromatinization of
attached autosomal material followed by subsequent retreat of heterochromatinization in some cells during development of the embryo (cf. Cattanach,
1974). In spite of differential pale staining of what appears to be all, or most of,
the autosomal region in the 4X chromosome in the majority of T37H/+ cells
which have the long-translocation product inactive, it is known that X inactivation can spread at least 25 map units, the distance between b and the Xchromosomal breakpoint in T37H (Beechey & Searle, 1977; Searle & Beechey,
1977). Unfortunately, no inferences may be drawn from this as the precise
relationship between map distance and cytological distance remains unknown.
In addition, measurement of the relative lengths of the dark- and pale-staining
regions in the translocated chromosome in different cells from an embryo, while
giving an estimate of variability of extent of spread of heterochromatinization,
is of limited value as length relationship will be complicated by the extension of
the chromatin of the non-heterochromatinized portion due to heat denaturation.
Non-randomness of X-inactivation
In all the translocation heterozygotes used in the present study a departure
from the 1:1 ratio expected from random inactivation is seen. Extreme nonrandom inactivation of the X chromosome not involved in the translocation has
already been established for T16H/+ heterozygotes (Ohno & Lyon, 1965), and
for IslCt/+ heterozygous embryos Disteche et al. (1979) have shown that cell
selection causes a departure from the 1:1 ratio expected from random inactivation, the direction of which depends on whether the embryo is the balanced Type
I carrier or the unbalanced Type II carrier. This rationale explains the nonrandom inactivation in Type II unbalanced IslCt/+ heterozygous embryos
found in this study.
Cell selection arguments can be convincingly used to explain the non-random
inactivation seen in both T37H and T38H heterozygotes as well as in IslCt
heterozygotes. Spread of inactivation into attached autosomal loci would result
in partial monosomy for the loci in question in cells in which the translocated X
was inactivated, and would thus be selected against to a greater or lesser degree
and might even result in cell death. This is in accord with the results presented
here for T37H and T38H. The fact that size and viability are reduced in females
16
S. RASTAN
heterozygous for T37H supports the idea of selective death of some cells with the
translocated chromosome inactive in the developing embryo.
However, for the reciprocal translocations another possibility must be considered. Since the results presented here and by Russell & Cacheiro (1978)
indicate that only one translocation product is capable of being inactivated in the
case of reciprocal translocations, one might also expect selection against cells in
which the translocated X is inactive due to the remaining active segment of the
X resulting in lack of dosage compensation ('functional disomy') for some Xlinked loci. The larger the non-inactivated piece of X, the greater one would
expect selection against such cells to be. T37H and T38H both have very small
non-inactivated segments of X, but T16H, the only X-autosome translocation in
the mouse in which the X is divided into two more or less equal-sized parts, is
characterized by extreme non-random inactivation.
T16H/+ heterozygotes
Takagi (1980) suggested that the extreme non-random inactivation seen in
T16H heterozygotes was the result of (1) inability of the X16 product to become
inactive, (2) inactivation in favour of XN and (3) rapid elimination of 16X inactive
cells by cell selection. He further suggested that there was no X-inactivation at
all in cells of embryos of the type (XN/X16;16/16) or (X^/X 1 6 ; 16/16) due to
inability of the X16 product to be inactivated and the necessity for the concurrence of at least two chromosomal loci separated by the T16H breakpoint for
the homologous X chromosome to become inactivated. An alternative interpretation of the results of Takagi and this paper is presented here in a simple
model (Fig. 5) for the initiation of X-inactivation which explains the following
facts:
(1) inactivation of only one part of an X chromosome separated by a reciprocal
translocation (Russell & Cacheiro, 1978; present data);
(2) non-inactivation of the X16 product as found by Takagi (1980);
(3) lack of inactivation of any kind in unbalanced embryos of the type
40(XN/X16;16/16) or 40(X(?VX16;16/16);
(4) inactivation of two chromosomes in the exceptional unbalanced embryo
deduced to be of the karyotype 40(X(7)/XN;16x/16).
Details of the model (Fig. 5)
This model is an extension of one of the hypotheses suggested by Russell &
Cacheiro (1979). It deals with only the initiation of X-inactivation, not the spread
Fig. 5. Model for initiation of X-chromosome inactivation based on a single inactivation centre on the X chromosome distal to the breakpoint in T16H. Only one inactivation centre per cell may be blocked; physical linkage to an empty inactivation
centre guarantees inactivation. Any other X-chromosomal material remains active.
(For further explanation see text.)
Non-random X-chromosome inactivation in mouse embryos 17
X n /X 16 . 16X/16
BALANCED T16H/+HETEROZYGOTE
Alternative 1
Alternative 2
X 16
Xn
16X
X 16
xn
16
lfi
X n OFF
X n ON
X ON
X 16 ON
16X OFF
16XON
16
UNBALANCED 9 TYPE a
X (7) /X 16 . 16/16
Alternative 2
Alternative 1
-16
lgX
16
16
same as alternative 1
X 16 ON
X(7) ON
UNBALANCED $ TYPE b
Alternative 1
X
16
Alternative 2
n
X™ X
16 X ON
X (7) /X N . 16X/16
16
X
16
X
(7)
16X OFF
7)
Alternative 3
n
X
16
16X
X (7)
16X OFF
X(7) OFF
X< ON
X (7) OFF
X n OFF
X n OFF
X"ON
• FULL INACTIVATION CENTRE
D EMPTY INACTIVATION CENTRE
Fig. 5
Xn
16
18
S. RASTAN
of inactivation along the X chromosome nor the maintenance of inactivation.
The following conditions are stipulated:
(1) the presence of a single inactivation centre on the X chromosome located
distal to the breakpoint in T37H, T38H and T16H;
(2) the inactivation centre may be in one of two states, 'free' or 'blocked'. The
possible ways in which the inactivation centre could be blocked include by
attachment of an episome (Grumbach, Horishima & Taylor, 1963), attachment
of protein products of a particular pair of autosomes or one set of autosomes etc.
The details are not important to the general scheme of the model;
(3) only one inactivation centre may be blocked in any cell, i.e. there is only
one episome, or sufficient autosomal product etc., to block one inactivation
centre;
(4) if X-chromosomal material is physically linked to a free inactivation centre,
inactivation is guaranteed. If, on the other hand X-chromosomal material is
physically linked to a blocked inactivation centre, or no inactivation centre is
present, it remains active;
(5) initially any inactivation centre present in a cell may be blocked at random.
For balanced carriers of T16H there are two alternative patterns of inactivation as two inactivation centres are present in each cell. Inactivation pattern
alternative 1 produces cells which are not dosage compensated ('functionally
disomic') for the proximal part of the X chromosome and partially monosomic
for the proximal part of chromosome 16 to a greater or lesser extent, depending
on the extent of spread of inactivation from the X into chromosome 16. Inactivation pattern alternative 2, on the other hand, results in a cell which is balanced
for both the X chromosome and chromosome 16. Although Takagi (1980) reported that in balanced Searle's translocation heterozygotes at 6-5 days most cells
inactivated the normal X chromosome, McMahon & Monk (1982) showed that
in T16H/+ female embryos heterozygous for isozymes of the X-linked
phosphoglycerate kinase (PGK-1) X-chromosome inactivation (thought to be
complete in the embryo by 5-5 days (Rastan, 1982a)) is initially random (with
respect to the Pgk-1 locus) and followed by rapid selection against those cells
having inactivated the Pgk-1 locus carried on T16H. Even if this meant losing
half the cells of the early embryo full compensatory growth is known to be able
to occur (Snow & Tarn, 1979). In this paper non-random inactivation in T16H
heterozygotes is postulated to be the result of a combination of primary disturbance of inactivation centres, resulting in inactivation of only one translocation
product, and secondary cell selection.
For unbalanced carriers of Type (a) i.e. X (7) /X 16 ;16/16, as shown in Fig. 5,
only one inactivation centre is present, so there is only one possible alternative.
X16 can never be inactivated because there is no inactivation centre present on
this chromosome. In every cell the single inactivation centre present on X ^ will
be necessarily blocked and thus incapable of being inactivated and therefore
there will be no X-inactivation at all in such embryos. This model explains the
Non-random X-chromosome inactivation in mouse embryos 19
result found by Takagi (1980) and confirmed in the present study for such
unbalanced embryos.
For unbalanced carriers of Type (b), i.e. X(7)/XN;16X/16, Fig. 5 shows that
tljere are three alternative patterns of inactivation, as three inactivation centres
are present in each cell, each resulting in two inactivated chromosomes per cell.
It must be remembered that even before inactivation all cells of such an embryo
are monosomic for the distal half of chromosome 16. Cells with inactivation
pattern alternative 1 will be, in addition, functionally nullisomic for the proximal
part of the X chromosome and would thus be rapidly eliminated by cell selection.
Disregarding for the time being the possible selective effects of having the X ^
chromosome switched on or off in a cell, cells with inactivation pattern alternatives 2 or 3 will both be partially monosomic for part of the proximal part of
chromosome 16 to the same degree, which will depend on the extent of spread
of inactivation from the X chromosome into chromosome 16. The two cell types,
with either XN and 16X inactivated or X(7) and 16* inactivated, could thus be
expected to be represented in equal proportions. However, as the embryo is also
an unbalanced Type II carrier for IslCt, cell selection considerations for
maximum genetic balance predict that inactivation pattern alternative 3, with
X ^ and 16X inactivated, would be the one most likely to restore maximum
genetic balance. The single unbalanced embryo found of this putative type had,
in fact, a slight preponderance of cells with XN plus 16X inactive, contrary to
expectations. This difference is not statistically significant, however, and may be
an artefact caused by low cell numbers.
The finding of such an unbalanced embryo at 8£ days is probably a very rare
event. Monosomies are known generally to be more deleterious to embryonic
development than trisomies (reviewed by Searle, 1981) and are likely to be
eliminated early in development. Takagi (1980) found no embryos of this type
in a total of 88 embryos at 6£ to 8£ days from T16H/+ mothers. It is possible that
the presumed karyotype of the unbalanced embryo is incorrect. In Fig. 3 showing
the balanced and unbalanced gametes from T16H/+ heterozygous females the
possibility of so-called 'adjacent-2' disjunction, in which homologous
centromeres proceed to the same pole (McClintock, 1945) is not included. If
adjacent-2 disjunction had occurred in this case an alternative karyotype for the
unbalanced female embryo could be 40(X (7) /X N /X 16 ;16). Such an embryo would
still be monosomic, but for the proximal part of chromosome 16 rather than the
distal part. If the exceptional unbalanced embryo were of this karyotype it would
place the postulated inactivation centre in the model proximal to the breakpoint
in T16H rather than distal. However, as an inactivation centre proximal to the
breakpoint in T16H is incompatible with the evidence that the X16 product is
incapable of being inactivated, the possibility of the unbalanced embryo having
such a karyotype is considered to be unlikely.
The report by Disteche et al. (1981) that the X16 product may be late replicating in a small proportion of cells has been used to support the concept of at least
20
S. RASTAN
two inactivation centres on the X chromosome. However, according to Takagi
(1980) and as reported here, no cell in which the X16 product was inactive was
ever seen, even in unbalanced embryos where inactivation of the X16 product
would have restored genetic balance. Non-inactivation of the X16 product due to
absence of an inactivation centre is thus a basic tenet of the model proposed here.
It is also known that the inactive X, although allocyclic, is not necessarily always
late replicating. There is evidence that the allocyclic X chromosome may be
either late replicating or early replicating in extraembryonic tissues of the early
postimplantation mouse embryo (Takagi, Sugawara & Sasaki, 1982). In
addition, Takagi (personal communication) has recently analysed replication
patterns in bone marrow cells from adult female T16H/+ heterozygotes using
Budr labelling and has found that the allocyclic X chromosome may be early
replicating in a considerable proportion of cells, ranging from 30 % to 67 % of
cells depending on the individual. In view of this variability in replication pattern
of the allocyclic X it is possible therefore that the supposed presence of the X16
chromosome late replicating in a small minority of cells in the 1981 study of
Disteche et al. was due to a misinterpretation.
The model of initiation of X-inactivation presented here is compatible with
the data from all Russell's X-autosome translocations especially the genetic
evidence of non-inactivation of the c locus in the R2 translocation (Russell &
Montgomery, 1965, 1970). Russell's data have placed the postulated inactivation centre between the breakpoints in R2 and R6. The model presented in this
paper considerably further localizes the postulated inactivation centre to between the breakpoints of T16H and R6. It is of interest to note that this location
of the inactivation centre, distal to the breakpoint in T16H and proximal to the
breakpoint in R6 is compatible with it mapping in the same region as the Xce
locus (Cattanach & Papworth, 1981). Although there is no direct proof that the
Xce locus is the inactivation centre there is both biochemical evidence (Johnston & Cattanach, 1981) and cytogenetic evidence (Rastan, 1982b) that alleles
at the Xce locus do, indeed, cause primary non-randomness of X-inactivation.
Different alleles of the Xce locus could specify differences at the inactivation
centre and thus affect the probability of an X chromosome carrying them
remaining active or becoming inactivated. As only one inactivation centre per
cell may be blocked off the model also explains the inactivation of all but one
X in individuals with supernumerary X chromosomes. The model is at present
being tested using different pluripotential stem cell lines derived from
parthenogenetic embryos (E.K. lines) (Evans & Kaufman, 1981) containing
various deletions of the second X chromosome (Robertson, Evans & Kaufman,
1983).
I would like to thank Sheila Brown for technical assistance and Mary Lyon for critically
reading the manuscript and for much valuable discussion. Part of this work was supported
by an MRC Studentship.
Non-random X-chromosome inactivation in mouse embryos 21
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(Accepted 10 August 1983)