/. Embryol. exp. Morph., Vol. 16, 3, pp. 559-568, December 1966
Printed in Great Britain
559
The role of the genome in the development
of the haploid syndrome in Anura
By LOUIE HAMILTON
From the Department of Biology as Applied to Medicine,
Middlesex Hospital Medical School, London
INTRODUCTION
The problem of the factors involved in the development of the haploid
syndrome in anuran embryos is as yet unsolved. It is known that about 90 % of
all haploid frog embryos develop the haploid syndrome, which is characterized
by the presence of oedema and sluggishness, by reduction in pigmentation and
in the efficiency of the heart and circulation, and by a partial failure of the gut to
coil and of muscle to differentiate. The two most favoured explanations of the
development of the haploid syndrome have been nucleocytoplasmic imbalance,
since a haploid nucleus is only half the size of a diploid nucleus in the same-sized
egg, and unmasked recessive lethal genes.
There is good evidence that an abnormal nucleocytoplasmic ratio is an
important contributory factor in the development of the haploid syndrome.
Briggs (1949) compared populations of haploids developing from large and
small eggs and Subtelny (1958) compared the development of haploids and
homozygous diploids which possessed a reduplicated set of haploid chromosomes. Both these workers found that the proportion of embryos developing the
haploid syndrome was smaller when the nucleocytoplasmic ratio approached
normal (i.e. in the small-egged haploids and the homozygous diploids) but was
not reduced to such an extent that a genetic influence could be ruled out.
Other evidence from grafting experiments (Hadorn, 1936; Baltzer, 1941) has
shown that although lethal genes may be unmasked in haploids, they are not
necessarily cell-lethal, and Hamilton (1963) also observed that haploid organs
may become viable and functional in a diploid environment. The demonstration
that the haploid syndrome can be mimicked by treatment of heterozygous
diploids with lithium chloride and sodium thiocyanate (Hamilton, 1965), and
that similar abnormalities are found in magnesium-starved and nucleolarless
Xenopus (Brown & Gurdon, 1964), led to the conclusion that developmental
stresses could give rise to the haploid syndrome in both haploid and diploid
embryos but that the threshold was very much higher in heterozygous animals.
1
Author's address: Department of Biology as Applied to Medicine, Middlesex Hospital
Medical School, London, W.I, England.
35-2
560
L. HAMILTON
This phenomenon of heterosis or the stability of heterozygotes has been
studied extensively and mention of some of the work is pertinent here. Robertson
& Reeve (1952) found that the variance in wing length of hybrid Drosophila
melanogaster was less than that of inbred individuals, and they suggested that
the greater number of alleles possessed by heterozygotes would allow a greater
selection of responses to environmental changes and thus minimize the effect of
such changes. Inbred mice are also more variable than heterozygotes in their
development (Griineberg, 1954) and in their response to narcotics (McLaren &
Michie, 1954).
The problem of the haploid syndrome can be attacked by producing clones
of haploids or homozygous diploids by the method of nuclear transplantation.
If recessive lethal alleles are responsible for the haploid syndrome, isogenic
haploids or homozygous diploids should be no more variable than isogenic
heterozygous diploids, whereas if these two classes of embryos differ appreciably
in the variability of their development, then this is best attributed to the degree
of heterozygosity, the only respect in which they are thought to differ.
METHOD
The experiments were performed with eggs and embryos of Rana pipiens and
Xenopus laevis.
Rana pipiens females were induced to ovulate by injection of fresh pituitaries.
Eggs were artificially fertilized and enucleated in order to make androgenetic
haploids by the method of Porter (1939). Moore's modification (1958) of the
nuclear transplantation procedure of Briggs & King (1953) was followed and
the injected cells were always taken from the animal pole of blastulae. Donor
embryos were one of five types: diploid or haploid developing from fertilized
eggs, diploid transfer, haploid transfer or homozygous diploid. The latter arose
when the recipient of a haploid transfer nucleus was delayed in first cleavage.
Since reduplication of any injected nucleus is fairly common it was important
to notice whether the time of first cleavage was delayed or not (Subtelny, 1958).
Similar nuclear transfer experiments were performed with Xenopus laevis but
in this case the females were induced to spawn by injection of gonadotrophin
(Pregnyl, Organon), and fertilization of the eggs, when required, was natural
since the male Xenopus could be induced to clasp by a similar injection. The
female pronucleus was inactivated in eggs by UV-irradiation (Gurdon, 1960).
Both methods of enucleation were 100 % successful.
When normal heterozygous controls reached early feeding stage all animals
which had appeared normal at the tail-bud stage were classified into those which
were normal and those which were not ('haploid syndrome'). Many embryos
survived to this age but were not scored because they had been abnormal before
reaching tail bud.
561
Genome and haploid syndrome
RESULTS
1. Rana pipiens
The overall results of transplanting the five types of nuclei are summarized
in Table 1, and so far as haploid and diploid nuclei are concerned agree very
closely with the results of Subtelny's (1958) experiments on haploid and diploid
transplantability. Unfortunately the poor transplantability of haploid nuclei,
together with the fact that not every embryo was suitable for scoring, made the
Table 1. Development of embryos obtained by nuclear transfer in Rana pipiens:
a comparison of the transplantability of different types of nuclei
Donor nuclei
No. of
transfers
No. of
blastulae(100%)
Diploid
Haploid
Diploid serial
Haploid serial
Homozygous diploid
65
663
54
326
262
54(100%)
443 (100%)
27 (100%)
207(100%)
185(100%)
Gastrulae Hatching
(%)
larvae (%)
65
48
52
61
66
42
18
32
16
35
Table 2. Details of largest Rana pipiens homozygous diploid clone
Survival of nuclear transplant embryos
eggs
injected
Blastulae
(%)
Day 2: haploid
29
22(100%)
17(77%)
Day 3: homoz. dip.
(delayed cleavage)
Day 4: homoz. dip.
(no delay)
Day 5: homoz. dip.
(no delay)
32
25 (100%)
24(96%)
29
22(100%)
20 (91 %)
38
27(100%)
13(48%)
f2 haploid) 1 4 0 /
11 diploid J 4 / o
/14 oedematous 1 ,. 0/
\ 2 normal
J b 4 /o
/ 5 oedematousl - n o /
12 deformed J JU / o
(2 normal
\
\ 4 oedematous V 33 %
(1 normal \
14 deformed/
Donor nuclei
Gastrulae
Young post-hatching
larvae (%)
[3 deformed J
Day 6: homoz. dip.
(no delay) new
recipient
Day 7: homoz. dip.
(no delay)
Total: homozygous
diploid
39
32(100%)
22(69%)
31
28(100%)
21(75%)
169
134(100%)
100(75%)
.
l b /o
(2 normal
}
h oedematous \ 36%
[l deformed J
47(35%)
production of sufficiently large haploid clones a more difficult task than initially
envisaged. The only embryos that could usefully be scored for degree of normal
development were those that had appeared to be perfect at the tail-bud stage,
for abnormalities at this stage can lead to the development of the 'haploid
562
L. HAMILTON
syndrome' in heterozygous nuclear-transplant diploids and it was essential to
compare uniformly normal heterozygous embryos with the homozygous clones.
On the basis of Subtelny's (1958) experiments showing good survival of
homozygous diploids, it was hoped that homozygous diploid clones might be
easier to produce than haploid clones and that the results might be easier to
assess since there was no nucleocytoplasmic imbalance to consider. Table 1 also
includes the results of transplanting homozygous diploid nuclei and shows that
55?
1 mm
Diploid control
Homozygous diploid
Haploid control
Fig. 1. Camera lucida drawings of Ranapipiens larvae from day 4 of the experiment
at the time of scoring. Haploids and heterozygous diploids have developed from
fertilized eggs, homozygous diploids by nuclear transfer. Notice that five of the
homozygous diploids are oedematous and two display spina bifida.
they survive up to hatching about as well as heterozygous diploids; yet, despite
this, only one clone with more than twenty scorable members was produced,
and this at the expense of nuclear transfer controls. Unfortunately the production of a large clone of homozygous diploid embryos by nuclear transfer together
with the production of isogenic heterozygous diploids in the same experiment
proved impossible. The single large homozygous diploid clone will now be
described in more detail. The experiment lasted 7 days and was planned as
follows (Table 2). On the first day haploid embryos were made by mechanical
enucleation of fertilized eggs when extrusion of the second polar body was
visible (Porter, 1939). On the second day one of these haploids became the donor
of nuclei for transplantation and those embryos in which first cleavage was
delayed were set aside as possible donors of homozygous diploid nuclei for the
Genome and haploid syndrome
563
following day. Only two sibs of this first homozygous diploid donor in the
series were haploid at scoring, and a third was diploid (homozygous). On all
subsequent days the donor embryos were selected from those that cleaved on
time so that further generations were also homozygous diploid.
Out of the 169 eggs injected with homozygous diploid nuclei in this experiment, 47 (27 %) reached the age at which they should be scored. Unfortunately
many transfer embryos had tended to exogastrulate and because a persistent
yolk plug was present at the neurula stage spina bifida developed. This type of
1 mm
Diploid control
Homozygous diploid
Haploid control
Fig. 2. Camera lucida drawings of Rana pipiens larvae from the 5th day of the experiment at the time of scoring. The homozygous diploids produced by nuclear transfer
were isogenic with those shown in Fig. 1. In this case two were indistinguishable from
heterozygous diploids, four were oedematous, and three display spina bifida.
defect even developed in heterozygous diploid transfers, no doubt from faulty
transfer technique, and in order to avoid complications in scoring all such
abnormal embryos were discounted. This meant that a further ten embryos had
to be eliminated from the experiment under discussion so that eventually only
37 were scored. Of these 37, 30 had developed the 'haploid syndrome' and 7 were
indistinguishable from normal heterozygous diploids. The original 169 eggs
injected may be subdivided into 99 of one host female on days 2-5 and 70 eggs
of a second female on days 6 and 7. The results in this case were 23 oedematous,
4 normal from the first host eggs; and 7 oedematous, 3 normal from the second.
There were 5 spinae bifidae or otherwise seriously malformed embryos in each
group that were not scored. All the embryos that were scored were genetically
564
L. HAMILTON
identical and yet were not phenotypically identical. Heterozygous diploid
controls for this experiment had not been obtained by nuclear transfer, but in
all other experiments where heterozygous nuclear transfer embryos were normal
at tail bud their subsequent development was normal.
Tail-tip preparations of the embryos were made which confirmed that all
supposedly diploid animals were in fact diploid. Figs. 1 and 2 show the experimental animals from days 4 and 5 of the experiment which has been described,
together with control haploids and heterozygous diploids.
Other clones of homozygous diploids were produced in the same way and
showed similar variation but were composed of fewer individuals; their heterozygous nuclear transfer controls were uniformly normal.
Table 3. Development of embryos obtained by nuclear transfer in Xenopus laevis:
a comparison of the transplantability of different types of nuclei
Survival of nuclear transplant embryos
Donor nuclei
Blastulae
Diploid
Haploid
Haploid serial
Homozygous diploid
60(100%)
115(100%)
109(100%)
31 (100%)
Gastrulae Hatching
(%)
larvae (%)
73
44
57
50
30
11
17
26
Doubling of the transferred nucleus is a fairly common occurrence (Briggs &
King, 1957) and many of the homozygous diploid transfers were delayed in
entering first cleavage. However, none of the homozygous tetraploids so
produced survived to scoring.
2. Xenopus laevis
Essentially the same results were obtained in the nuclear transfer experiments
using Xenopus. The overall results are set out in Table 3. The greatest handicap
in clone production in Xenopus is the need to use a different host every day and
the rarity of Xenopus which lay good recipient eggs. No clones of sufficient size
were produced but even so there was variation within the small clones. Fig. 3
shows the development of the 'haploid' syndrome in some members of a homozygous diploid clone while one remains free from it; the heterozygous clone is
uniformly normal. In Xenopus again, none of the eggs with delayed first cleavage
which should be homozygous tetraploids survived to scoring.
DISCUSSION
Subtelny (1958) pointed out in his discussion of the development of homozygous diploids that although they possessed a duplicate set of haploid chromosomes and displayed normal early development they all had deficiencies in later
Genome and haploid syndrome
565
embryonic or larval development. Two-thirds of them developed the 'haploid
syndrome', but if recessive lethals were its cause the proportion should be the
same as in haploids (90-95 %). Subtelny was thus reluctant to support the idea
of * genetic influence on the expression of these deficiencies' but at the same time
6 days
8 days
13 days
1 mm
Heterozygous diploid
Homozygous diploid
Fig. 3. Camera lucida drawings of one group of nuclear transplant homozygous and
heterozygous diploid Xenopus to show their development. The 'haploid syndrome'
develops in two out of the three scorable homozygous diploids.
he said that the differences between homozygous and heterozygous diploid
development 'must be due somehow to the nuclear condition of the homozygous
diploids'. This should also be a nuclear condition shared by haploids and I
suggest it is lack of heterozygosity.
The work of Robertson & Reeve (1952), McLaren & Michie (1954) and
Griineberg (1954) already referred to presents general evidence not only for the
566
L. HAMILTON
phenomenon of heterosis but also for greater variation within populations of
homozygotes than heterozygotes. These may be two sides of the same coin, for
if we assume that a species is fitted to its environment its individuals must
respond to changes in the environment by deviating as little as possible for
the norm. In any case highly inbred mice and fruit-flies and homozygous
diploid amphibians cannot be considered to be genetically normal members
of their species, so there is no reason for expecting them to be phenotypically
normal.
My suggestion is that the haploid syndrome is not specific but may develop
in any embryo in response to conditions to which it cannot adapt. For instance,
heterozygous diploid Xenopus develop the 'haploid syndrome' if they are
treated with sodium thiocyanate or lithium chloride (Hamilton, 1965), are
reared in magnesium-free medium, or are nucleolarless (Brown & Gurdon,
1964); under ordinary conditions, however, the tadpoles are uniformly normal
because of the developmental stability afforded by heterosis. Homozygous
diploids, on the other hand, are developmentally less stable and their responses
to stress less predictable, so that under normal laboratory conditions some may
develop the' haploid syndrome'. One also finds, as one might expect, that haploids
are more sensitive to lithium chloride and similar treatments than heterozygous
diploids (Hamilton, 1965).
The extreme sensitivity of homozygous embryos can explain why no homozygous tetraploids and so few haploids survived nuclear transplantation in the
present experiments. The possibility that chromosome damage or loss during
manipulation of nuclei causes the development of the haploid syndrome is
unlikely for two reasons. First, members of heterozygous diploid clones do not
develop the haploid syndrome despite the fact that they are produced by the
same technique that is used for producing homozygous diploid clones; and,
secondly, some normal embryos did develop from each day of serial transplantation and the variability of transfer clones was as great at the end of an
experiment as at the beginning.
The real crux of the matter is that clones of genetically identical homozygous
diploids do not develop uniformly, and those individuals that are abnormal
develop the 'haploid syndrome'. Lack of heterozygosity is the nuclear condition
that haploids and homozygous diploids have in common. By rendering them
less stable genetically this makes them more susceptible to stresses, such as unmasked deleterious genes and nucleocytoplasmic imbalance, and thus reduces
their chances of developing normally.
SUMMARY
1. Homozygous diploid embryos were obtained by using Subtelny's method
of nuclear transplantation of haploid nuclei and selection of the eggs which were
delayed in first cleavage.
Genome and haploid syndrome
567
2. Genetically uniform clones of homozygous diploids were produced by
serial transplantation and their uniformity assessed as tadpoles.
3. Homozygous diploid nuclear transplant embryos are quite variable within
clones, about 20% normal and 80% with the 'haploid syndrome'. Heterozygous diploid transplant embryos are uniform and normal.
4. It is concluded that lack of heterozygosity leads to developmental instability of which the 'haploid syndrome' is an expression.
ZUSAMMENFASSUNG
Die Rolle des Genoms bei der Entwicklung des Haploid-Syndroms in Anura
1. Man erhielt homozygote diploide Embryonen durch Kerntransplantation
auf haploide Nuclei und durch Auslese der Eier, welche in der ersten Teilungsphase zuruckgeblieben waren.
2. Genetisch einheitliche Klone der homozygoten Diploiden wurden an hand
von Serientransplantation erzielt, und ihre Identitat an Kaulquappen gemessen.
3. Homozygote Diploide sind recht variabel innerhalb der Klone: circa 20 %
sind normal und 80% zeigen das 'Haploid-Syndrom'. Heterozygote Diploide
sind einheitlich und normal.
4. Es wird gefolgert, dass ein Mangel an Heterozygozitat zu Entwicklungslabilitat fiihrt, die seinen Ausdruck im ' Haploid-Syndrom' findet.
I wish to thank Dr J. A. Moore for guidance and hospitality at Columbia University,
New York, during the work on Rana pipiens; and the National Science Foundation for
grant G 9001. I am also grateful to the Wellcome Trust for a travel grant and the Nuffield
Foundation for support in London.
REFERENCES
BALTZER, F. (1941). Ober die Pigmentierung merogonische-haploider Bastarde zwischen der
schwarzen und weissen Axolotlrasse. Verh. schweiz. naturf. Ges. 121, 169-71.
BRIGGS, R. W. (1949). The influence of egg volume on the development of haploid and diploid
embryos of the frog Rana pipiens. J. exp. Zool. Ill, 255-94.
BRIGGS, R. W. & KING, T. J. (1953). Factors affecting transplantability of nuclei of frog
embryonic cells. /. exp. Zool. 122, 485-506.
BRIGGS, R. W. & KING, T. J. (1957). Changes in the nuclei of differentiating endoderm cells
as revealed by nuclear transplantation. /. Morph. 100, 269-312.
BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139—46.
GRUNEBERG, H. (1954). Variation within inbred strains of mice. Nature, Lond. 173, 674.
GURDON, J. B. (1960). The effects of ultraviolet irradiation on uncleaved eggs of Xenopus
laevis. Quart. J. micr. Sci. 101, 299-311.
HADORN, E. (1936). Ubertragung von Artmerkmalen durch das entkernte Eiplasma beim
merogonischen Tritonbastard, palmatus Plasma x cristatus Kern. Verh. dt. zool. Ges.,
Freiburg, pp. 97-104.
HAMILTON, L. (1963). An experimental analysis of the development of the haploid syndrome
in embryos of Xenopus laevis. J. Embryol. exp. Morph. 11, 267-78.
HAMILTON, L. (1965). The development of haploid and diploid embryos of Xenopus laevis after
treatment with sodium thiocyanate and lithium chloride. Expl Cell Res. 38, 684-5.
568
L. HAMILTON
A. & MICHIE, D. (1954). Are inbred strains suitable for bioassay? Nature, Lond.
173, 686.
MOORE, J. A. (1958). The transfer of haploid nuclei between Rana pipiens and Rana sylvatica.
Expl Cell Res. Suppl. 6, 179-91.
PORTER, K. R. (1939). Androgenetic development of the egg of Rana pipiens. Biol. Bull. 77,
233-57.
ROBERTSON, F. W. & REEVE, E. C. R. (1952). Heterozygosity, environmental variation and
heterosis. Nature, Lond. 170, 286.
SUBTELNY, S. (1958). The development of haploid and homozygous diploid frog embryos
obtained from transplantation of haploid nuclei. /. exp. Zool. 139, 263-305.
MCLAREN,
{Manuscript received 29 June 1966)
/ . Embryol. exp. Morph., Vol. 16, 3, pp. 569-590, December 1966
With 1 plate
Printed in Great Britain
569
More about the. tabby mouse and about
the Lyon hypothesis
By HANS GRUNEBERG 1
From the Department of Animal Genetics, University College London
INTRODUCTION
Autosomal genes are present in duplicate in the body cells of both sexes.
Genes carried in the X-chromosome are present in double dose in the mammalian female, but only in single dose in the mammalian male. Despite this
disparity in gene dosage, the phenotypic effects of such genes are generally the
same in homozygous and in hemizygous condition. To bring about this situation, some kind of 'dosage compensation' is required. A possible mechanism of
dosage compensation in mammals which has been widely discussed in recent
years is the 'inactive-Z-chromosome' or 'single-active X-chromosome' hypothesis. As originally put forward by Lyon (1961, 1962), this postulates that
during embryonic development, either the maternal or the paternal Z-chromosome of the female is inactivated. Inactivation happens at random and is
irreversible; it thus persists in the descendants of the cell in which it has occurred.
The mammalian female, according to this hypothesis, is thus a patchwork mosaic
so far as its Z-borne genes are concerned, and as in any one cell only one of the
two Z-chromosomes is active, the effective dosage is the same in both sexes.
The observational starting-point of the hypothesis was the fact that heterozygotes for several sex-linked genes in the mouse and other mammals show what
appears to be, or could be interpreted as, a patchwork phenotype, the tortoiseshell cat being the most striking and widely known example. However, a prima
facie case in favour of a hypothesis must not be mistaken for a proof. A test of
validity will have to establish whether the facts claimed to support the hypothesis are indeed in quantitative agreement with its consequences. Methodologically it is obvious that in the first instance this will have to be done in the
simplest genetic situations, taking one gene at a time and studying its behaviour
in a structurally normal X-chromosome. Only on the basis of such studies can
genetically more complex situations (double heterozygotes, and genes carried in
structurally abnormal chromosomes) be critically evaluated.
It is surprising that, whereas there are many data involving complex situations,
there is very little information as to whether the individual genes conform to the
1
Author's address: Department of Animal Genetics, University College London, W.C. 1,
U.K.
570
H. GRUNEBERG
Lyon hypothesis (or L.H. for short). The gene for tabby in the mouse (Falconer,
1953) affects the fur and the teeth (Griineberg, 1965), and it has been shown
recently (Griineberg, 19666) that the dental syndrome of Ta/+ ?? cannot be
accounted for in terms of the L.H. In this paper, evidence will be presented that
this is equally true of the effects of tabby on fur and skin. A survey of the other
sex-linked genes in the mouse and other mammals (other than man) will show
that, in so far as they provide evidence at all, this evidence is uniformly against
the L.H.
No attempt will be made in this paper to deal with complex situations involving the simultaneous segregation of several sex-linked genes or the behaviour
of genes carried in structurally abnormal chromosomes; with data from human
genetics; or with the cytological aspects of the hypothesis. The possibility thus
remains that valid evidence in favour of the hypothesis may come from sources
of information not discussed in this paper. It is hoped to come back to an
assessment of the remainder of the evidence on a later occasion.
CRITERIA FOR TESTING THE VALIDITY OF THE LYON HYPOTHESIS
A test of the L.H. will, in the first instance, have to establish whether individual
sex-linked genes, in structurally normal chromosomes, behave according to
expectation in heterozygous females. Some of the criteria for this simple
situation have been discussed in a previous paper (Griineberg, 19666); others
will be added here. No doubt the list is still not complete.
(1) To establish critically whether the phenotype of a heterozygote is in
agreement with the L.H., the phenotype of both hemizygotes must be known, as,
for example, in tabby. In the mottled series of (probable) alleles, the fur of
brindled (Mobr) <$£ is affected uniformly all over, and it may reasonably be
supposed that this would also be true for other alleles like mottled (Mo) or
dappled (Modp) if the $$ lived to grow fur. On the other hand, the phenotype
of the lethal striated {Str) $$ is of necessity conjectural.
(2) Where an organ or structure is affected in its entirety in the hemizygote
(such as fur and molars in Ta or the fur in Mobr <£<£), the patches of normal and
mutant phenotype in the heterozygote, on the L.H., must be arranged at random.
The existence of an orderly arrangement (pattern) is quite incompatible with
that hypothesis, as random inactivation of chromosomes cannot generate an
orderly (i.e. non-random) pattern. Patterns may be recognizable by inspection,
or demonstrated statistically by unequal involvement of, or correlations between,
different parts of the body.
(3) Where the phenotype of the hemizygote is itself a pattern (as in bent-tail
(Bn) #(£), the emergence of a similar pattern in the heterozygote is equally
compatible with the L.H. and conventional semi-dominance and thus does not
discriminate between these alternatives.
(4) Clearly demarcated patches of normal and mutant phenotype in heterozy-
Tabby mouse and Lyon hypothesis
571
gotes should show the respective phenotypes pure and uncontaminated, and no
intermediacy. This, of course, depends on autonomous development (without
which clearly defined patches could not be formed), and on the absence or nearabsence of mingling of cells of contrasted type. The latter should be the case
near the middle of large patches.
(5) As a corollary to (4), the regular occurrence of large areas of uniformly
intermediate phenotype in heterozygotes is incompatible with the L.H., as in
such areas both alleles must be at work together, as in ordinary (autosomal)
heterozygotes.
(6) The contrasted patches, to fit the L.H., should on an average cover equal
areas in the aggregate. This again depends on autonomous development and on
equal growth of the respective sectors following the inactivation of the paternal
and maternal Z-chromosomes respectively. As I have pointed out elsewhere
(Griineberg, 19666), 'in a genetically heterogeneous strain [differential growth],
if it should occur, would also be influenced by other sex-linked genes which, in
relation to A/a, would be in coupling and repulsion at random and thus act as a
buffer'. Moreover, as any appreciable differential growth of sectors would
inevitably lead to gross malformations, it is improbable that any major disparity
in the aggregate area of contrasted sectors can be explained in terms of differential
growth.
(7) According to the L.H., paternal and maternal X-chromosomes are inactivated at random, i.e. regardless of the genes they carry. If so, the manifestation
of contrasted alleles in heterozygotes should be refractory to selection. Successful
selection for or against the manifestation of alleles in heterozygotes would
demonstrate genetic (Fisherian) control of dominance at the level of the gene.
Selection for a gene, of course, always involves adjacent regions of the chromosome and sometimes the whole of it, but not the propensity of the opposite
chromosome to be inactivated (if, for example, in a heterozygote Aja, one can
select for increased manifestation of A9 this would, in terms of the L.H., involve
increased inactivation of the chromosome carrying a, and vice versa). Random
chromosome inactivation, ex hypothesi, involves absence of any control and
hence failure to respond to selection.
(8) If the heterozygote for an autosomal gene shows 'mosaic' manifestation,
this must be interpreted in terms of ordinary semi-dominance in conjunction
with a threshold mechanism. If a sex-linked mimic of such a gene behaves in the
same way, the parsimony principle militates against invoking a totally different
interpretation for the same phenomenon.
Throughout, the above criteria either contradict the L.H., or they fail to
discriminate. Thus, existence of a pattern is incompatible with the hypothesis,
but absence of a pattern is equally compatible with the L.H. and with ordinary
semi-dominance, and similarly for all the other criteria.
572
H. GRUNEBERG
THE COAT OF THE NORMAL MOUSE
The coat of the normal mouse has been studied by Dry (1926), Fraser (1951)
and Slee (1957). In the mid-dorsal region, Dry distinguishes four types of hairs,
with very few intermediates. The overhair includes three coarse types of fibres
(guard-hairs, awls and auchenes, respectively) which together in Dry's mice
accounted for about 16 % and in Fraser and Slee's animals for about 28 % of all
the hairs of the baby coat. The remainder consists of fine fibres (zigzag hairs)
which form the underfur. Dry (1926) found that 1627/2000 hair follicles produced
the same kind of fibre in the first and second hair generation; in 362 follicles
(18-1 %), a finer hair was succeeded by a coarser one (mainly auchenes by awls
(137) and zigzags by auchenes (174 cases)), and only in 11 instances was a
stronger hair replaced by a thinner one. Dry used mainly three criteria for the
classification of the four hair types. Guard-hairs (which are the longest fibres)
and awls lack constrictions, auchenes have one constriction and zigzags from
3 to 5, with angulations at each (hence the name). Medullary pigment is present
in the transverse septa (complete partitions) or in columns of septules (incomplete
partitions) the latter of which only occur in the overhairs. The internal structure
of the medulla is a function of hair calibre. Thus the fine zigzag hairs (with
maximum diameters of 14-15 ju) are septate throughout except at the constrictions where the medulla may be interrupted. The overhairs, in the thicker
middle regions, have rows of septules (2 in guard-hairs, and up to 5 in the
strongest awls of adult mice); but near the tip and near the base where they are
thin, the overhairs are also septate. The change of calibre is steady, and the
transition from septate to septulate and back again to septate near the base
happens within a few partitions and generally without uncertainty and hesitation.
Guard-hairs have a longer solid tip than the other hair types and lack the
yellow agouti band which is present in nearly all the other hairs; they are
roughly circular in cross-section whereas awls are bean-shaped.
In the normal mouse, the four hair types distinguished by Dry can easily be
sorted out under the dissecting microscope, mainly on the basis of the constrictions. But this method breaks down completely in a coat which includes
anomalous hair types. Here it becomes necessary to scan each hair from one end
to the other under the medium or high power of the microscope, a procedure as
tedious as it is essential for the discovery of smaller deviations from normality.
As this inevitably restricts the size of the hair sample which can be examined,
some authors have preferred to classify a large sample of hairs superficially
under the dissecting microscope rather than to scrutinize a small sample in
detail under the microscope. This has led to important features in several
mutants being overlooked and severely restricts the critical value of these
earlier enumerations.
Tabby mouse and Lyon hypothesis
573
THE COAT OF THE TABBY MOUSE
Historically, the structure and development of the coat of the crinkled (crjcr)
mouse was described first (Falconer, Fraser & King, 1951). The structure of the
tabby coat was found to be indistinguishable from that of crinkled, and its
development was presumed to be the same (Falconer, 1953). Tabby hemizygotes
and homozygotes have a darker coloration along the mid-dorsum than normal
mice. By contrast, the remainder of the hairs has a wider band of phaeomelanin
so that the fur looks yellower than that of a normal mouse. Typical guard-hairs
are absent in the tabby mouse whose coat is short and rather thin. There are
no hairs with constrictions. The hairs of tabbies are on an average considerably
finer, and the strongest fibres do not exceed 22 /i in diameter. The stronger
fibres in the tabby coat have an abnormal internal structure, with irregular
sequences of septa and septules, i.e. complete and incomplete partitions. There
is a conspicuous bald patch behind the ear, and the tail is usually completely
naked though occasionally a few hairs are present.
This situation can be accounted for in two different ways. Either the abnormality of the tabby coat is a failure of differentiation; i.e. the fur corresponds
to the whole of the normal pelage but lacks its differentiation into guard-hairs,
awls, auchenes and zigzags; or certain hair types are absent in the tabbies, namely
the long guard-hairs and the hairs with constrictions (zigzags and auchenes),
with the result that the tabby fur corresponds to the awls only. These, in the
normal baby coat, are about 15-20 % of all hairs. Falconer et al. (1951) did not
succeed in getting a direct estimate of hair density, but decided in favour of the
'awls-only' hypothesis in view of the obvious thinness of the coat together with
embryological evidence for which the reader may be referred to the original
paper.
The present author has succeeded in getting direct estimates of follicle
density in normal and tabby mice. To avoid shrinkage and distortion, the
dorsal skin was fixed in situ by subcutaneous injection of Bouin's fluid; during
the process of fixation, the mouse was put on its back and weighted down
slightly to flatten the dorsal skin. The rectangular piece of skin subsequently
removed was then dehydrated and cleared in methyl salicylate. The anagen
phase of the first and second hair generation was studied. The formation of
follicles, in the normal mouse, ceases towards the end of the first week after
birth. Thereafter, the follicles spread out as the mouse grows. To make animals
of different size comparable with each other, a correction is necessary; in Table 1,
the original counts (covering an area of 5-12 mm2) are thus given along with
adjusted values of hair density which would have been found if the mouse had
reached a weight of 20 g; i.e. the original count of each mouse has been multiplied by (W/IO)^ where W\s the weight of the animal. If in the baby coat of the
normal mouse (14 days) the proportion of awls were as high as 25 % (which is
certainly an overestimate), the (adjusted) number of hairs expected in tabbies
36
JEEM 16
574
H. GRUNEBERG
on the 'awls-only' hypothesis would be 97 whereas 246 were actually observed.
The 'awls-only' hypothesis is thus untenable. As, in the second hair generation
(31 days), the hair density of the tabby is not far from normal, the reduced hair
count of tabbies in the first hair generation is probably largely due to retardation,
not all hair follicles being active or perhaps even formed. In the light of these
data a detailed study of the development of the tabby fur is desirable. In any
case, it is obvious that the fur anomaly of tabbies is mainly a failure of differentiation with reduction in hair calibre rather than of hair number. Microscopic
Table 1. Mid-dorsal hair counts in tabbies and normal litter mates
Tac )O
Weight Hair
count
(g)
Age
14
4-91
4-94
571
683
Mean .
31
12-12
+ <?
Hair count
(adjusted)
223-9
268-9
Weight Hair
count
(g)
5-58
7-04
926
763
246-4
453
324-4
19-67
347
^
Hair count
(adjusted)
Ta<J<J
+ 66
395-4
380-4
387-9
0-635
343-1
0-945
examination indeed suggests that tabby hairs are not a homogeneous population
as one should expect if they were all (atypical) awls. In the absence of constrictions, a sharp distinction between overhairs and underfur is not possible. Hair
calibre in general is much reduced. The finer fibres (believed to be the equivalents
of zigzags) usually have a maximum diameter of 10-11 p (normal 14-15 /*); they
are essentially septate, but often have a few short and irregular septulate regions.
The absence of constrictions may be a consequence of their reduced calibre.
The stronger fibres are usually septate for long stretches at both ends, only the
middle region being irregularly septulate; evidently, even the thicker parts of
the fibre only just reach the threshold at which septation turns into septulation.
A minority of the stronger hairs corresponds to fine (2-septulate) awls.
As Falconer et al. (1951) have pointed out, in the normal mouse the tuft of
hair behind the ear consists entirely of zigzags. However, these are finer than
zigzags elsewhere, have a longer solid tip and generally a long non-medullated
base. I am inclined to think that they are absent in tabbies not because they are
zigzags, but because they are the finest hairs and thus the first to be pushed over
the brink in the general reduction of hair calibre. A similar but diffuse reduction
of the finer zigzags is probably present elsewhere in the coat. Incidentally, the
bald patch behind the ear in tabbies is a good deal larger than the area occupied
by the tuft of zigzags in normal mice.
/. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 1
Ta
H. GRUNEBERG
facing p. 575
Tabby mouse and Lyon hypothesis
575
THE COAT OF TABBY HETEROZYGOTES
The coat of Taj + $$ has characteristic transverse stripes of black hair which
lack the agouti band. They are most obvious in the baby coat and tend to
become less marked in the adult animal. Somewhat surprisingly, Lyon and her
collaborators have referred to the tabby markings as 'variegation'. That term
is defined in the Concise Oxford Dictionary (4th ed., 1950) as 'being marked with
irregular patches of different colours' and thus clearly cannot be used legitimately
to describe the regular transverse pattern of the Taj + mouse. The arrangement
of the stripes is unmistakably orderly, and orderly patterns are not the result
of random events. Moreover, the change of the tabby markings with age is
quite alien to the L.H., and the average area covered by the black stripes is far
less than one half of the whole coat which would be expected on that hypothesis.
Whereas orderly stripes cannot conceivably be generated by random events,
they can easily be accounted for by transverse structures in the developing skin.
A slight transverse wrinkling of the skin on the flanks first appears in 15-day
embryos (Griineberg, 1943) and soon becomes very marked (Plate 1, fig. 1).
During the growth of the baby fur and again, to a lesser extent, during that of
the second hair generation, the skin of the mouse shows marked transverse folds
(David, 1934) which are hidden under the fur and which follow approximately
the transverse structure of the skin which arises in the 15- to 16-day embryo.
Position and size of these folds and their relation to the hair growth cycles
suggest that they are the structural features which underlie the transverse
markings of Ta/+ ?$ (and perhaps similarly in other mammals). The coarser
pattern of skin folds during the growth of the first coat explains why the tabby
markings are most striking in the baby coat. It also accounts for the fact that
the face of Ta\ + $? does not show stripes; according to the L.H. there is no
reason why it should be immune.
Kindred (1967) rightly remarks that 'Tabby does not fit into this scheme
[the L.H.] very well even on a superficial examination since the dark stripes are
equated to the mutant which is, in most parts, more intensely agouti [i.e. has
wider yellow bands] than normal'. In fact, the fur composition of the black
stripes differs from that of Ta by the presence of guard-hairs (Falconer, 1953;
Lyon, 1963; Kindred, 1967) and of some zigzags (Lyon, 1963; Kindred, 1967),
PLATE 1
Fig. 1. Normal mouse embryo, 16 days old, showing the transverse wrinkling of the skin
which is believed to underlie the transverse stripes in Ta/+ and probably Str/+ 9$.
Figs. 2-4. Tail skin of a normal <J, a.Ta/+ $ and a Ta <$ (49, 65 and 47 days old; tail lengths
91, 91 and 16 mm respectively). The central 3 cm of each tail are shown. This represents the
middle third in the first two mice; the corresponding region in the Ta S is indicated by the
arrows. Approximate tail-ring number in the middle third 54, 65 and 70 respectively. The
tail skin of the first two mice is shown from the inside as the scale pattern on the outside is
partly obscured by hairs.
36-2
576
H. GRUNEBERG
neither of which should be present. On the other hand, the intervening agouti
areas differ from the normal fur by a reduction in number of (segmented)
zigzags (Lyon, 1963; Kindred, 1967); the latter author found 50-9 % where some
75-80 % would have been expected. Of course one can try to save the L.H. by
making ad hoc assumptions such as that the development of guard-hairs (unlike that of other hair types) is non-autonomous, or that the areas sampled were
not, in fact, pure (though Kindred, and no doubt Lyon also, took pains to
avoid this source of error). Past enumerations of hair types suffer from the fact
that under the dissecting microscope little can be seen beyond the presence or
absence of constrictions and of the yellow subterminal band. Microscopic
examination shows that some of the hairs in agouti areas of Taf + $$ correspond
to the finer fibres of Ta 3$ which are probably unsegmented zigzags; these
would be misclassified at low magnifications as awls. Pending a more detailed
investigation, the most probable interpretation of the Taj 4- coat is that stripes
and agouti areas differ in degree rather than in kind, and that, as in the tail
rings (see below), the effects of both alleles are detectable over the whole area of
the coat.
The orderly arrangement of the stripes in Taj + $$ is obvious to the eye.
The existence of a pattern is further demonstrated by a negative correlation
between striping and vibrissa number (see next section); the more nearly
normal the total number of vibrissae, the less intense the striping, and vice versa
(Dun, 1959). The manifestation of the gene in the face (vibrissae) is thus not
independent of that on the back (stripes) and hence involves a pattern.
As shown by Fraser & Kindred (1962, and earlier papers), the vibrissa number
in Tal + $$ responds to selection (a behaviour which in itself is quite incompatible with the L.H.). Dun & Fraser (1959) also found that selection for vibrissa
number indirectly affects the degree of striping. Thus in the upwards selection
line in which vibrissa number approached normal the stripes tended to disappear,
whereas in the downwards selection line they became strikingly more marked—
again clear evidence for the existence of a pattern. The genetic background of
these selection lines acted specifically on Ta. Kindred (1967) crossed dappled
(Modp) and brindled (Mobr) on to the background of the high vibrissa selection
line, but observed no effect on the phenotype of these genes.
THE SINUS HAIRS (VIBRISSAE)
The sensory or sinus hairs of the face of the mouse include the mystacial
vibrissae (whiskers) which are not appreciably affected by Ta, and certain groups
of secondary vibrissae. In the normal mouse, these are almost completely
invariant in number. On each side, there are two supraorbital, one postorbital
and two postoral vibrissae, and near the midline under the chin there is a group
of three inter-ramals. The total number is thus 13. Ta reduces some of these
sinus hairs slightly and others strikingly (Table 2). For the group of secondary
Tabby mouse and Lyon hypothesis
577
vibrissae as a whole, Ta\ + is about intermediate. But the situation is different for
individual locations. The gene is dominant for the supraorbitals which are only
slightly affected, but almost recessive for the postorbitals which are considerably
reduced. In the two remaining groups, the gene is more nearly dominant.
Contrary to what one should expect on the L.H., the heterozygous manifestation
of tabby as regards the secondary vibrissae thus again shows a pattern, favouring
some bristles and avoiding others. The situation is thus similar to that in the
molars (Griineberg, \966b), where heterozygous manifestation favours m2 as
compared with mx.
Table 2. Secondary vibrissae in normal and tabby mice (after Fraser,
Nay & Kindred, 1959)
Genotype
+ <?<?, + / + 99
Ta/+ 99
Ta/Ta 99
Supraorbitals
Postorbitals
Postorals
Interramals
Total
4
3-7
3-7
3-8
2
1-8
0-3
0-4
4
2-2
1-5
1-5
3
1-6
1-2
1-2
13
9-4
6-7
6-8
TAIL RINGS
The tail of the mouse is covered with scales which are arranged in fairly
regular rings, with rows of stiff hairs in the intervals between the rings. The
tail of crinkled mice lacks hairs and is thus usually entirely naked, and tail rings
are absent from most crinkled mice (Falconer et al 1951). The same is the case
in tabby where the tail is 'nearly always quite devoid of hair and tail-rings'
(Falconer, 1953).
A closer study of the flat tail skin mounted between glass plates shows that our
tabby mice usually have some scales; the amount is variable and the distribution
sometimes patchy, and particularly the ventral surface of the tail is often smooth.
The scales of tabby mice are much smaller than those of normals (Plate 1,fig.4),
and their arrangement in rings is less regular. The large scales of normal mice
are roughly rectangular in outline, and the interval between adjacent scales in
the row is narrower than that between successive rows. The scales of tabbies
are separated from their neighbours to the right and left by wider intervals, and
the whole arrangement of the scales is distinctly hexagonal as in a honeycomb.
The number of scales in a row is about the same in normals and tabbies. The
scales of Taj + $$ are uniformly intermediate in size between those of + $$ and
Ta <&? (Plate 1); their arrangement in rings is about as regular as in a normal
mouse, but as the rings are narrower, there are more of them per unit length of
tail. This is detectable by inspection (Plate 1) where there are about 54, 65 and
70 rings in the middle third of the tail in a + £, a Taj + ? and a Ta $ respectively.
Counts of tail rings are approximate only on account of irregularities; to
578
H. GRUNEBERG
minimize the error, the mean of three counts (one near the midline and two
near the sides) was taken and recorded to the nearest integer. The counts may
be regarded as accurate to within ± 1 ring. The sample examined included 20
normal mice (19 $$, 1 ?) and 23 Ta/+ $$ from the same matings; the mean
tail length of the normals was 9005 mm and that of the Ta/+ ?? 89-00 mm,
with almost identical ranges around the mean. The rings have been counted in
each case in the 4th and 6th cm, counting from the base of the tail (Table 3). In
both segments, the Taj + $$ have significantly more (i.e. narrower) rings than
the normal mice; the ratio of tail ring numbers (Ta/+ divided by normal) is
about the same in both segments (1-160 and 1-127, respectively). No attempt
has been made to obtain similar counts for tabbies, as the distribution of scales
is too irregular; it is, however, obvious by inspection that their scales are much
smaller than those of normals.
Table 3. Number of tail rings in the 4th and 6th cm of the tail (upper
and lower half of the table, respectively) in normal andTa/ + mice
17 18 19 20 21 22 23 24 25
Normal
Ta/+ $$
Normal
Ta/ + 9?
2 8 7
3
— — 4 2
— 1 6 5
— — 1 1
— — — — —
4 5 7 1 —
4 3 1 —
—
2 5 6 4 4
Total
Mean
20
23
20
23
18-55
21-52
20-25
22-83
The comparison between normal and Taj + mice is complicated by the fact
that nearly all the normals are <$<$ whereas the Taj + mice are all $$. A comparison between 10 £$ and 10 $$ from a normal stock showed no sign of a sex
difference in either segment of the tail, in conformity with the findings of
Fortuyn (1939 and earlier papers).
The scale size of Taj + $$ is uniformly intermediate over the whole area of
the tail, without any sign of patchiness such as would be expected on the L.H;
and individual Taj + $$ are similar in this respect. Evidently, both alleles are
interacting with each other as in an ordinary autosomal heterozygote. The
situation is probably the same as in the coat where the influence of both alleles
was detectable both in the stripes and in the intervening agouti areas (though in
that case the possibility of a mingling of contrasted types of hairs has not yet
been excluded critically).
There are several probable recurrences of Ta and at least one distinguishable
new allele {Ta1; Stevens, 1963) whose tail is not naked: 'They have fewer hairs
on the tail than normal, and the hairs are curved. TaTa1 $$ have hairless areas
and areas of curved hairs as would be expected on the basis of the inactive-X
theory of Lyon.' In the absence of detailed critical data, I am inclined to treat this
declaration of faith in the L.H. with some reserve.
We mention here another sex-linked gene, greasy (Gs; Larsen, 1964) which
has not yet been described in detail. It is very closely linked to, but not allelic
Tabby mouse and Lyon hypothesis
579
with, Ta. 'G5/+ females superficially resemble Ta/ + females. GsjY males and
GsjGs females have uniformly shiny fur. They lack the following features of
Ta/Y: sticky [i.e. hairless] tail, bare patches behind ears, decrease in yellow
pigment along mid-dorsal.'
STRIATED
This gene was probably induced by X-rays (Phillips, 1963). Str/ + $$ superficially resemble Taj + ?$ in having dark transverse stripes; however, the stripes
are often not distinguishable clearly until the animals are 16-18 days old, and
there is some normal overlapping. Str <$g are not viable (death between \\\ and
13 days of gestation). Striated is about 8 units from Ta (Lyon, 1966b) and has a
different pathology (Lyon, 1963): the black stripes are due to a local shortening
of hairs which exposes the dark base of the hairs behind; there is no absence of
(segmented) zigzags as in Ta. Str thus affects hair structure rather than pigmentation. According to Lyon, smaller patches of short hair are sometimes not
visible unless the fur is brushed back, and the regular transverse stripes are only
part of the story.
As the (potential) phenotype of Str $$ is unknown, that of Strj + $? cannot
give critical evidence in favour of the L.H. However, as will be shown presently, it
can quite critically rule it out. As seen in dry preparations at medium microscopic magnifications, all the hairs from short patches are grossly abnormal. The
hairs as a whole appear irregularly wavy somewhat like the 'de-kinked' hairs
of some Negro women. The hair calibre varies considerably along the length of
the hair and, as a consequence, repeated short sections of septate and septulate
structure may follow each other. Many hairs, and particularly the zigzags, are
flat ribbons which tend to be axially twisted. Details will be reported on a later
occasion. Now, according to the L.H., the long hairs of these mice should be
normal. This, however, is far from the truth. In virtually every hair, slight
irregularities of septation and minor changes of calibre are clearly detectable
which do not occur in the normal coat, and it is difficult, if indeed possible, to
find a hair which is normal throughout its length. Clearly, the action of the
Str gene is not confined to the short patches, but affects the coat as a whole, and
the difference between short patches and the remainder of the coat is one of
degree and not of kind. This is only possible if both alleles are active throughout
the coat of Str I + $$, and this, for the gene in question, critically rules out the
mechanism postulated by the L.H.
MOTTLED, BRINDLED, DAPPLED AND TORTOISESHELL
The heterozygotes for all these genes (Falconer, 1953; Fraser, Sobey &
Spicer, 1953; Dickie, 1954; Lyon, 1960; Phillips, 1961) show a similar type of
mottled fur. As the hemizygotes perish before birth or, in the case of brindled
(Mobr), in the nest, direct tests for allelism cannot generally be carried out; how-
580
H. GRUNEBERG
ever, by the use of an exceptional Mobr $ which survived and was fertile (a
'Durchbrenner', to use Hadorn's expression), the allelism of Mobr and dappled
(Modp) has been virtually proved: the putative Mohr\Modv $$ had the near-white
phenotype of Mobr $$ and died at about the same age. Allelism is, in any case,
likely on account of the similarity of phenotype of the heterozygotes and similar
linkage relations with Ta which is about 4 units away (though the latter is not
yet known for tortoiseshell).
The original description of mottled {Mo) by Fraser et al. (1953) speaks of
' many regions of light-coloured (off-white) hair scattered without pattern over
the body' and adds that 'Mottled and Brindled females are similar both in the
colour of the off-white regions, and in random scattering of them over the body'.
This may be accepted as a first approximation. However, Falconer (1953)
observed that the diffuse areas of very lightly pigmented hairs are ' sometimes
arranged in an irregular pattern of transverse bars reminiscent of the markings
of Tabby heterozygotes', an observation which I can confirm. It thus seems
that the markings of Mo/ + and Mohr\ + $$ are not strictly random though
the transverse pattern is less obvious and regular than that of Ta/+ and
Strl+ $$.
All four heterozygotes show some waving or curling of the whiskers, and for
tortoiseshell, at any rate, a silkier hair texture has also been reported (Dickie,
1954). The vibrissae of Mobr $$ are strikingly curly, and the hairs of the coat are
also affected (Falconer, 1953). Recent observations by the present author show
that the fur consists of the usual hair types, but the structure of the hairs is
grossly abnormal. Details (also on Str and other genes affecting hair structure in
the mouse) will be published in due course. As seen in dry preparations, the
transverse structure of the hairs is interrupted irregularly over shorter or longer
distances, perhaps due to absence of septa, more probably due to the presence
of liquid in the hair. Often there are fluctuations in hair calibre not found in
normal hairs and various other abnormalities. The picture as a whole is striking
and recognizable at a glance. As one might expect, a similar but less extreme
situation is encountered in the light-coloured patches of Mobr/+ $$ which,
incidentally, include many hairs which are partly pigmented. However, structural abnormalities are by no means confined to the light-coloured areas.
Almost without exception, pigmented hairs have milder but easily detectable
irregularities in septation and often fluctuations in hair calibre. It is obvious
that the whole of the fur of Mobrj + ?? is involved, and not only the lightcoloured patches. Where the structural abnormalities are slight, pigment is
formed; where they are severe, pigment is reduced or absent. Evidently, Mobr
(and presumably the other mottled alleles) affects hair structure in the first
instance, and pigmentation is only secondarily involved where the structural
anomalies are severe. Beyond any doubt, the light-coloured areas are due to a
threshold mechanism, and in the fur as a whole, both alleles are active as in an
ordinary autosomal heterozygote.
Tabby mouse and Lyon hypothesis
581
This is in agreement with the findings of Phillips (1961) in the dappled
(Modpl + ) heterozygote where the amount of curling of the whiskers is correlated
with the degree of lightness of the coat at weaning: 'the greater the curling, the
lighter the coat', i.e. the more severe the structural abnormalities, the fewer
hairs are able to form pigment. On the other hand, Phillips (1961) did not
succeed in selection experiments for lighter and darker strains; this negative
finding, of course, does not discriminate between the hypotheses.
We mention here briefly a similar sex-linked gene, blotchy (Russell, 19606;
Russell & Saylors, 1962; Lyon, 19666), also about 4 units from Ta and possibly
(probably) an allele of mottled. 'Heterozygous females have irregular patches of
more diluted fur. Expression is occasionally poor at weaning age... but is complete at adulthood. Hemizygous males, hemizygous females (XO), and homozygous females are light all over (no blotching)... Their whiskers are kinked at birth
but straight by the time of weaning...'
The fact that, like Ta and Str, the mottled alleles affect hair structure in the
first instance will prove of importance in the interpretation of experiments
(Lyon, 1963) to be discussed on a subsequent occasion. In that paper, Lyon
treated the mottled series as colour genes like albinism (c) or pink-eyed dilution
(p). In the light of the facts presented in this paper, this obviously requires
modification and reassessment of the situation.
BENT-TAIL AND OTHER SEX-LINKED GENES IN THE MOUSE
The semi-dominant gene for bent-tail (Bn; Garber, 1952) has been claimed
to provide supporting evidence for the L.H. (Lyon, 1966 a). The only published
data on its anatomy and development are my own (Griineberg, 1955). Whereas
penetrance is complete in Bn 3$, there are some normal overlaps in Bn / + $$.
Normal overlapping is found in countless autosomal genes and thus cannot be
used to discriminate in favour of one hypothesis rather than the other. In Bn <$$,
the anomalies start with the 6th caudal vertebra and steadily increase in frequency
to a plateau which is maintained in the distal parts of the tail. In Bnj + $$, the
general pattern is the same, but expressivity is lower. In 14 Bn <$$, there was an
average of 5-07 severely affected vertebrae (with double centres) and of 8-07
mildly affected vertebrae per mouse. If the vertebral anomalies in Bn\ + ?? were
due to inactivation of the normal allele in the sense of Lyon, they should show
a similar ratio. In actual fact, 27 manifesting Bnj+ °.°. had an average of 0-70
severely and 6-96 mildly affected tail vertebrae. Evidently, in the manifesting
region, both alleles are active to bring about this lowered expressivity. The
evidence provided by the Bn gene is thus against the L.H.
Several of the remaining sex-linked genes in the mouse have not yet been
described in any detail, and none of them provides any critical information for
or against the L.H. Jimpy (Phillips, 1954) is a sex-linked recessive gene which in
SS causes intention tremor and later convulsions, with death round about
582
H. GRUNEBERG
28 days. A single manifesting jimpy female (which was Ta + / + jp and hence
not an XO ?) has been explained as probably due to somatic crossing over, a
sector including part of the CNS becoming homozygousy/?///? (Griineberg, 1966 a).
Gyro, according to preliminary reports by Lyon (1960, 1961), in $$ shows
circling behaviour with deafness, abnormalities of the long bones and ribs and
sterility; heterozygous ?$ show incomplete penetrance of the circling behaviour
and apparently no skeletal abnormalities. Scurfy (Russell, Russell & Gower,
1959), sparse fur (Russell, 1960 a), and sex-linked anaemia (Grewal, 1962) are
all recessive without any known manifestations in heterozygous $$. The same
applies to a sex-linked lethal (Hauschka, Goodwin & Brown, 1951), if the
existence of such a gene was the correct interpretation of the anomalous segregations observed by these authors. Finally, there is a sex-linked histocompatibility
gene (Bailey, 1962) so far only reported in an abstract which contains no
relevant information.
MOTTLED-WHITE IN THE GOLDEN HAMSTER
This condition (Magalhaes, 1954) is lethal in <$<$ whose potential phenotype
is thus conjectural. Heterozygous $? 'have a thinner than normal coat with
normal colored fur intermingled with white. Some animals are almost entirely
white while others are only slightly grayer than normal.' Hair structure as well
as colour is thus involved as in the mottled alleles in the mouse, and pending a
microscopic study of both white and pigmented regions of the fur, the 'mosaic'
nature of these heterozygotes is clearly problematical.
STREAKED HAIRLESSNESS IN CATTLE
This sex-linked condition in Holstein-Friesian cattle (Eldridge & Atkeson,
1953) has been claimed in support of the L.H. (Lyon, 1966 a). As affected &J are
not viable, their (potential) phenotype is unknown. Supposing they were, in fact,
completely hairless, heterozygous $$ should, on an average, be half-hairless. This
is very far from the truth, a s ' It was roughly estimated that not more than 5 %
of the hide was lacking in hair in the most severely affected animal'. Moreover,
the arrangement of the hairless areas is not random. 'The affected cattle were
found to have areas devoid of hair on various parts of the body, the hairless
areas occurring in more or less consistent patterns. On all affected animals there
were approximately perpendicular hairless streaks over the thurls, some more
extensive than others, with considerable variation between the two sides of the
same animal...' Clearly, this case does not support the L.H.
THE TORTOISESHELL CAT
Unlike the sex-linked genes in mouse and hamster which affect hair structure
(with or without involvement of pigmentation), in the tortoiseshell cat pigmentation (eumelanin versus phaeomelanin) seems to be affected in the first instance.
Tabby mouse and Lyon hypothesis
583
These two pigments generally occupy separate sites such as in the banded agouti
hairs, or on the ventral surface of d mice, etc. Several species of animals have
series of multiple alleles which go from one extreme to the other. Thus all fur
pigment in the lethal yellow mouse (Av) is phaeomelanin and all fur pigment in
extreme non-agouti (ae) is eumelanin. Some alleles seem to be teetering on the
brink; in viable yellow (Avv; Dickie, 1962), whereas the baby coat is usually a
clear yellow, many animals subsequently become irregularly mottled with black
patches intermixed with yellow (and sometimes with agouti hairs), a pattern
very much like that of the tortoiseshell cat. A similar situation occurs in the
Japanese rabbit (ej), a member of the extension series which also spans the
whole range from eumelanin to phaeomelanin, and in the tortoiseshell (ep)
guinea-pig. In the latter animal, a remarkable case of factor interaction has been
known for a long time; it is described by Sewall Wright (1963) as follows:
'Tortoise shells of genotype SSepep are predominantly eumelanic but usually
show scattered yellow hairs and less frequently more or less yellow in blotches.
With ssepep or even Ssepep, the amount of yellow is increased and there is a
strong tendency to segregation of yellow and eumelanin into a few large areas
each often with scattering admixture of the other color. These areas are often
separated in whole or in part by white streaks. Sometimes a streak between
eumelanic areas is white at one end, yellow at the other, indicating that the
determination of yellow is related to the process that leads in more extreme
cases to white by absence of the pigment cells.'
As every observer of cats knows, the same interaction is strikingly present in
the tortoiseshell cat. To quote an early source (Whiting, 1919), '"Self" tortoiseshells have yellow hair closely intermixed with non-yellow... Tortoiseshells with
restricted white-spotting tend to have yellow separated into patches, while
further extension of white separates yellow and non-yellow areas still more.
Separation of yellow into patches appears not to be correlated with amount of
yellow.'
Avy in the mouse, e> in the rabbit and ep in the guinea-pig are all autosomal
genes, and it is obvious that in all of them, the eumelanin-phaeomelanin dichotomy is the result of a threshold mechanism and not the result of chromosome
inactivation. The physiological process in the tortoiseshall cat is obviously the
same, notwithstanding the fact that it happens in a heterozygote rather than in
homozygotes as in the other animals. To invoke a threshold for the autosomals
and chromosome inactivation for the sex-linked gene would be an arbitrary
procedure which could not strengthen the L.H. Similarly, in the guinea-pig, the
influence of spotting genes on the manifestation of ev is clearly a case of factor
interaction, and there is no justification for a totally different interpretation in
the analogous case in the cat. Indeed, as has been pointed out above, according
to the L.H. chromosome inactivation must be refractory to the effects of other
genes, and the tortoiseshell phenotype plainly is not. So far from supporting the
L.H., the tortoiseshell cat. increases its difficulties.
584
H. GRUNEBERG
DISCUSSION
Of all the sex-linked genes in the mouse, tabby affords the best opportunities
for a critical test of the L.H. Six different test criteria are applicable, and in every
single instance the evidence goes clearly against the L.H.
(1) There is a clear-cut pattern (fur, vibrissae, molars).
(2) Contrasted areas differ from the hemizygous phenotypes (fur).
(3) Intermediate areas are regularly present (tail rings).
(4) Contrasted areas are unequal in the aggregate (fur, molars).
(5) The heterozygous phenotype responds to selection (vibrissae, stripes).
(6) Resemblance of heterozygous manifestation to that of autosomal mimic
(molars).
With everything against and nothing in favour of the hypothesis, attempts to
explain away by ad hoc assumptions one or the other of these items (perhaps
no. 2) will not carry conviction: the rest of the evidence, so far as I can see,
cannot be shaken without abandoning essential parts of the hypothesis in the
process. In conformity with previous findings in the molars (Griineberg, 19666),
we must conclude that the known phenotype of Ta\ + $$ cannot be explained in
terms of the Lyon hypothesis. Its whole behaviour is in agreement with the
conclusion that both alleles act together in the same way as in heterozygotes for
autosomal genes.
Decisive evidence against the L.H. also comes from the mottled series and
from striated. Disregarding lesser points, the structural abnormalities of the
coat are not confined to the unpigmented or short-haired areas respectively, but
occur in a milder form throughout the pelage. The fur of the heterozygotes is
thus clearly not a patchwork of normal and mutant areas: both alleles act
together over the whole area of the coat, with some regions more severely
affected than others and hence unpigmented or short-haired, respectively. In
Bnj + $$ the pattern of tail abnormalities is similar to that in Bn #<£, but its
lower expressivity shows that both normal and mutant allele are active. In the
latter case, ad hoc assumptions (non-autonomous development, etc.) would
remove the gene from the category of contradictors to that of non-discriminators,
but no more.
The mottled-white gene in the golden hamster is very similar to the mottled
series in the mouse and may well be homologous to it. In the absence of evidence
to the contrary, there is no reason to suppose that its heterozygous phenotype is
a patchwork mosaic in the sense of the L.H. any more than that of Mobrl + in
the mouse. The heterozygous phenotype of streaked hairlessness in cattle
disagrees with the consequences of the L.H. in every respect, quite apart from the
fact that the phenotype of the hemizygote is unknown. The eumelanin-phaeomelanin dichotomy of the tortoiseshell cat has autosomal counterparts in
mouse, rabbit and guinea-pig, and in the tortoiseshell cat and in the tortoiseshell
guinea-pig there is also the same interaction with spotting genes which separate
Tabby mouse and Lyon hypothesis
585
the two kinds of pigment into distinct areas. Clearly, none of these genes in
hamster, cattle and cat gives any support to the L.H., and the tortoiseshell cat,
at any rate, raises considerable difficulties for the hypothesis.
Taken one at a time, and in a structurally normal Z-chromosome, none of the
sex-linked genes in the mouse thus agrees with the L.H. Returning to the starting
point of the hypothesis, the peculiar heterozygotes for Ta, Str and Mo turn out
to be the result of threshold mechanisms like countless others in the autosomes,
and not to be explicable by 'phenotypic segregation' of chromosomes which is
another way of expressing the L.H. Evidently, in mouse females, there is no
inactivation of whole Z-chromosomes, and the data discussed in this paper do
not suggest that there is any inactivation at all. This, of course, does not rule
out the possibility that instances of inactivation of individual sex-linked genes
may yet be discovered, but this is a concept totally different from the L.H.
Henceforth, as a minimum requirement, claims that the phenotype of a
heterozygote conforms to the L.H. will have to be based on the criteria discussed
in this paper: mere assertions will not do.
The question arises of whether the behaviour of the sex-linked genes in the
mouse calls for any special explanation. The present author feels that it would be
rash to answer this question in the negative. It may be peculiar that there are
six semi-dominant genes (Ta, Str, Mo, Gs, Bn, Gy) and only four recessives
(jimpy, scurfy, sparse fur and anaemia), disregarding the histocompatibility gene
and the lethal which belong to a different category. It is difficult to obtain an
estimate of the ratio of 'dominant' to 'recessive' autosomal genes in the mouse,
if only because these are not distinct categories, and small effects in the heterozygote can probably be discovered in the majority of cases commonly referred to
as recessives. However, if we omit histocompatibility genes, 'biochemical'
mutants and the like, and if we count as semi-dominant any locus which has
given rise to at least one such allele, it seems that autosomal' recessives' exceed
genes with easily detectable effects in the heterozygote by roughly 3:1. Hence the
^-chromosome of the mouse (disregarding the possibility of a bias in their
discovery) probably has more than its fair share of such genes. Amongst these,
the group of hair structure genes (Ta, Str, Mo and probably Gs) stands out. The
question therefore arises of how common are autosomal genes of this kind, and
do they include instances where the heterozygote is poised as closely to the
threshold of manifestation as in the sex-linked trio (or quartet) of genes. Excluding colour and spotting genes, the Mouse News Letter no. 34 (1966) lists
some 30 loci which clearly affect skin and/or pelage of the mouse; many of them
have not yet been published, and some may ultimately turn out to be allelic to
each other. They include about nine semi-dominants or dominants, none of them
with a variegated or striped heterozygous phenotype comparable to the sexlinked ones. On this basis it would appear that the sex-linked genes do in fact
differ from the autosomal genes affecting hair structure.
However, the case of the mottled series may not be unique, and there may be
586
H. GRUNEBERG
more structure genes masquerading as colour genes, etc. The study of hair
structure in the mouse is a badly neglected field. Even grossly abnormal coats
have in many instances never been investigated, and nobody, apparently, has
thought of examining the coat of 'colour mutants' for the possible existence of
structural anomalies of the fur. Following the study of the coats of the sexlinked genes, the present author has started to make a survey of various autosomal
conditions not hitherto suspected of affecting hair structure. The result has been
quite surprising, and the full exploitation of the large field opened up will take
time. Here, two examples may suffice. The clumping of eumelanin pigment in
dilute (d/d) has been known for a long time, and it is perhaps not surprising that
clumping of phaeomelanin in these animals has now also been found: but indeed
the hair structure of these animals is characteristically abnormal, and the suspicion arises that this may be more basic than the colour effect by which the gene
has been discovered in the first instance. The second example concerns the
grey-lethal gene which I described more than 30 years ago: absence of yellow
pigment seemed obvious from inspection of the coat which was pure grey and
lacked the yellow colour in the agouti bands. Microscopic examination now
shows that phaeomelanin is, in fact, present in the form of a few massive clumps
(much like the eumelanin clumps in dilute mice); but the hairs themselves are
structurally abnormal, and this may well be behind the clumping of the yellow
pigment. In grey-lethal and brindled alike, the assessment of the mutants by
visual inspection has misled the respective authors!
The most striking case of autosomal mottling is the varitint-waddler mouse
(Cloudman & Bunker, 1945); Va/+ mice show irregular and finely interspersed
areas of white, dilute and normally pigmented hairs; the dilute areas gradually
tend to become white with increasing age. Like the mottled genes which it
resembles phenotypically, varitint-waddler is now also unmasked as a gene
affecting both hair structure and melanin formation. The white hairs are grossly
abnormal in structure, the grey ones rather less so, whereas the normally
pigmented hairs are structurally nearly, but apparently not quite, normal.
Evidently defective pigmentation is secondary to abnormal hair structure, and
the colour changes with age reflect a gradual deterioration of hair structure.
Some of the ordinary autosomal spotting genes are remarkably similar to the
sex-linked mottled series. Thus, the homozygotes of dominant spotting (WjW,
WvjWvi etc.) are uniformly white, the respective heterozygotes, on certain
genetic backgrounds, variegated (and Wvl + also dilute). Similarly, the homozygotes for microphthalmia and for white (mi/mi and MiwhIMiwh) are white, the
+ lmi and Miwh/+ heterozygotes, on certain genetic backgrounds, spotted
(though not variegated; the latter also with some dilution of fur colour). Other
spotting genes have more regular and more sharply defined patterns of black
and white, and the respective homozygotes are usually not completely white. An
increasing body of information shows that in several of these genes spotting is
not merely a matter of pigmentation, but also involves hair structure, and this
Tabby mouse and Lyon hypothesis
587
may well turn out to be true for most if not all of them. As critical and quantitative studies will take time, we shall here confine ourselves to pointing out the
essential similarity of autosomal and sex-linked genes whether their phenotype
is compared by inspection or by microscopic examination.
Manifestly, sex-linked and autosomal heterozygotes in the mouse do not
differ in kind. In both groups, cases occur in which the physiological interaction
between the two alleles brings about a situation in which a developmental
threshold is crossed in some parts of the body, but not in others. The suspicion
remains, however, that this kind of situation may be commoner in sex-linked
than in autosomal heterozygotes, and that something in the Z-chromosome may
tend to keep alleles in a state of semi-dominance rather than to allow one allele
to gain complete ascendancy over the other. This is also suggested by the behaviour of genes in structural heterozygotes, with which I hope to deal on a
later occasion.
The relationship between hair structure and pigmentation discussed above
is not peculiar to the mouse. Mudge (1908) described a transitory ghost pattern
in albino rats (Rattus norvegicus) homozygous for the hooded gene, and
Goldschmidt (1927) mentioned a tiger embryo in which the future transverse
stripes were clearly visible long before pigment formation in the shape of
thickenings of the skin. Many people will also be familiar with the ghost tabby
pattern in certain black cats, and with the ghost rosettes in black panthers.
[Note added September 16,1966, Since this paper was written, a scrutiny of the sex-linked
genes in man indicates that they provide as little evidence in favour of the Lyon hypothesis
as their counterparts in the mouse and other mammals. The evidence will be published
shortly (' Sex-linked genes in man and the Lyon hypothesis', Ann. Hum. Genet., in the press).]
SUMMARY
The Lyon hypothesis
(L.H.)
of dosage compensation of sex-linked genes in
mammals postulates that in the female, during embryonic development and at
the cellular level, either the paternal or the maternal X-chromosome is inactivated; that this inactivation happens at random; and that it persists in the
descendants of the cell in which it has taken place. The genetic evidence hitherto
adduced as favouring the L.H. has mainly involved complex situations such as
the simultaneous segregation of two sex-linked genes and/or the behaviour of
genes in structurally abnormal chromosomes.
By contrast, this paper examines the behaviour of sex-linked genes in
mammals (other than man) taken one at a time and in structurally normal
Z-chromosomes. Criteria are discussed by means of which the validity of the
L.H. in such genetically simple situations can be tested. These tests reveal that
the behaviour of heterozygotes for three sex-linked genes in the mouse (tabby,
striated, brindled) is decisively at variance with the L.H., and the same is probably true for a fourth gene, bent-tail. The remaining sex-linked genes in the
mouse give no evidence for or against the L.H. AS there is thus clear evidence
588
H. GRUNEBERG
that in three, and probably in all four instances, both alleles are active as in
ordinary autosomal heterozygotes, it is evident that in the mouse there is no
inactivation of a whole X-chromosome. Indeed, the facts discussed in this
paper (including those of one instance each in hamster, cattle and cat) do not
suggest that there is any inactivation at all.
Nonetheless there is a suspicion that heterozygotes for sex-linked genes, taken
as a group, may include more cases of semi-dominance than occur in autosomal
heterozygotes. If this should be substantiated, it would require an explanation.
ZUSAMMENFASSUNG
Weitere Mitteilungen tiber die tabby Maus und die Lyon-Hypothese
Die Hypothese von Lyon iiber die Dosis-Kompensation geschlechtsgebundener Gene von Saugern fordert, dass in der Embryonalentwicklung des
Weibchens auf zellularer Gmndlage eine Inaktivierung entweder des vaterlichen
oder des nriitterlichen X-Chromosomes stattfindet; dass die Inaktivierung
zufallsgemass erfolgt; und dass sie erhaltenbleibtinden Nachkommen der Zelle,
in der sie stattgefunden hat. Die genetischen Tatsachen, die bisher als Stiitze
dieser Hypothese angefiihrt worden sind, sind iiberwiegend komplizierter Natur,
wie z.B. die gleichzeitige Spaltung zweier geschlechtsgebundener Gene oder das
Verhalten von Genen in strukturell abnormen Chromosomen.
Demgegeniiber beschaftigt sich diese Arbeit mit geschlechtsgebundenen
Genen bei Saugern (ausser denen des Menschen) derart, dass jedes Gen fiir sich
allein betrachtet wird, und zwar in strukturell normalem X-Chromosom.
Zunachst werden Kriterien erortert, mit denen die Gultigkeit der Hypothese in
derartigen genetisch einfachen Situationen gepriift werden kann. Dabei stellt
sich heraus, dass das Verhalten der Heterozygoten fiir drei geschlechtsgebundene
Gene bei der Maus {tabby, striated, brindled) sich durchaus nicht mit der
Hypothese von Lyon in Ubereinstimmung bringen lasst, and das gleiche trifft
wahrscheinlich auch fiir ein viertes Gen {bent-tail) zu. Die iibrigen geschlechtsgebundenen Gene der Maus sprechen weder fiir noch gegen die Hypothese. Da
demgemass bei drei und wahrscheinlich bei alien vier Genen beide Allele ganz
wie bei autosomalen Heterozygoten wirksam sind, ist es oflfenkundig, dass bei
der Maus nicht das ganze Geschlechtschromosom inaktiviert wird. Die in dieser
Arbeit erorterten Tatsachen (einschliesslich je eines Falles beim Hamster, beim
Rind und bei der Katze) geben keinerlei Hinweise dafiir, dass Inaktivierung
iiberhaupt vorkommt.
Andererseits besteht ein Verdacht, dass Heterozygoten fiir geschlechtsgebundene Gene ofter als solche fiir autosomale Gene sich intermediar verhalten.
Sollte sich dies bestatigen, wiirde eine besondere Erklarung erforderlich sein.
The author wishes to thank his colleagues, Dr Gillian M. Truslove and Miss Jean M. Gray,
for assistance in various ways. He also wishes to thank Dr D. S. Falconer (Edinburgh) and
Dr Mary F. Lyon (Harwell) for the gift of some of the mutants discussed in this paper.
Tabby mouse and Lyon hypothesis
589
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(Manuscript received 30 June 1966)