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J. Embryol.exp. Morph. Vol. 22, 2, pp. 145-79, September 1969
145
Printed in Great Britain
Threshold phenomena
versus cell heredity in the manifestation of
sex-linked genes in mammals
By HANS GRUNEBERG 1
Department of Animal Genetics, University College London
The mode of action of sex-linked genes in mammals has been the subject of
much discussion in recent years. This has centred round the concept that in the
mammalian female, either the maternal or the paternal ^-chromosome is
randomly and irreversibly inactivated during development, with the result
that in the female, as in the male, one X-chromosome only is active in a given
cell (Lyon, 1966, and earlier papers). The Lyon Hypothesis (L.H.) is based, in
part, on the mottled or striped phenotypes of heterozygotes for certain sexlinked genes affecting the coat of the mouse and other mammals: these phenotypes are regarded as a mixture of clones descended from cells in which either
one or the other ^-chromosome has been inactivated. As Baker (1967) rightly
insists, the interpretation of a pattern as clonal must be based on known cell
lineages, as without such a basis one simply postulates what should be put to the
test.
The only multi-clonal mosaics so far known in mammals are mouse chimeras
(Mintz, 1967; Mystkowska & Tarkowski, 1968), which are the result of aggregation of genetically different embryos in early cleavage (about 8-cell) stages. The
data so far published include typical colour genes like that of albinism (c), but
no genes affecting coat structure. According to Mystkowska & Tarkowski, the
chimeras are finely mottled animals with darker and lighter areas which differ
mainly quantitatively from each other. Mintz has put stress on a transverse
pattern of bands of dark and light hairs which, in certain animals, more or less
alternate with each other; sometimes they are mismatched on the dorsal midline so that a dark band faces a light one. The transverse pattern has led Mintz
to postulate the existence of 17 pairs of primordial melanoblasts.
In view of the unique origin of these chimeras, one may perhaps hesitate to
accept them as close models of normal development. For instance, blastocysts
derived from the aggregation of two morulae regulate down their size to normal;
cells lost during this process presumably account for the fact that, in the
1
Author's address: Department of Animal Genetics, University College London,
4 Stephenson Way, London, N.W. 1, England.
10
JEEM 22
146
H. GRUNEBERG
experiments of Mintz, as many as 68/109 surviving mice, or 62%, exhibited
the coat colour of one of the parent strains only.
As judged by eye, there is some resemblance between the chimeras and some
of the phenotypes discussed in this paper (brindled, flecked), but closer inspection immediately reveals consistent differences. For instance, according to both
Mintz (1967) and Mystkowska & Tarkowski (1968), the tails of chimeras show a
sequence of pigmented and unpigmented bands; the tails of brindled $$ are
solidly coloured whereas those of flecked $$ are irregularly mottled. At the
level of individual hairs, no comparison is possible yet as those of the chimeras
have not so far been studied. The phenotypes of other sex-linked heterozygotes
discussed here (tabby, striated) are quite unlike those of the chimeras for colour
genes, but no adequate descriptions of chimeras for genes for coat structure are
yet available for comparison.
On the other hand, the phenotype of the chimeras is also similar to that of
certain autosomal genes for which a clonal basis cannot be invoked. Under these
circumstances the only way open is to scrutinize the phenotypes of heterozygotes
for sex-linked genes for internal evidence concerning their origin. This paper
deals with the genes of the house mouse for tabby {Ta; Falconer, 1953), brindled
(Mobr; Fraser, Sobey & Spicer, 1953) and striated (Str; Phillips, 1963). The test
for the validity of the L.H. is essentially a comparison between the heterozygote
and the two hemizygotes, normal and mutant, which is possible for Ta and Mohr.
No such comparison is possible in the third case, as Str <$<$ perish as early
embryos; as it is thus unknown what kind of coat (if any) they would have had,
the phenotype of Str/ + $? (and of Ta + / + Str $$) cannot be interpreted in
terms of the L.H. without begging the question (Griineberg, 19666). For factual
reasons which will be explained below, the phenotypes of Ta MobT\ + + and of
Ta +1 + Mobr $£ are also irrelevant as tests of the L.H. All of them are, however,
important in their own right, as their phenotype will have to be accounted for
by any hypothesis of dosage compensation which may emerge. The phenotype
of an insertional translocation (Cattanach, 1961) which brings the autosomal
locus of albinism under the influence of the X-chromosome is also included.
Preliminary descriptions have already been given (Griineberg, 19666, 19676).
The more complete study presented here differs in some respects from the preliminary reports, but leads to the same conclusion, namely that the phenotypes
in question cannot be accounted for on a clonal basis. An alternative hypothesis
of dosage compensation in mammals is therefore formulated which is in agreement not only with the genetical evidence, but also with the body of cytological
facts which has hitherto been regarded as favouring the L.H.
Sex-linked mammalian genes
147
MATERIAL AND METHODS
This paper is based essentially on some 430 preparations made in 1968,
earlier material being regarded as preliminary studies. In the case of striated, a
stock recently obtained from Miss Rita J. S. Phillips (Harwell) has been used, as
a stock previously supplied by Dr Mary F. Lyon proved to be contaminated
with the gene for tabby; this has been the reason for the one major discrepancy
between a preliminary report (Griineberg, 1967 b) and the present study. Other
differences are mainly due to the fact that certain minor anomalies at first
believed to be the effects of mutant genes are now known to be due to the genetic
background.
Hair samples are plucked from the back of the mouse (mid-dorsal) by means
of flat-tipped forceps. The baby coat was usually sampled at about 3 weeks
(earlier in brindled SS) and the adult coat at about 8 weeks or later. A small
bunch of hairs is spread and gently separated on a 1 x 3 in. slide flooded with
absolute alcohol which is then allowed to evaporate on a hotplate. A coverslip
is gently put down on top and secured with narrow adhesive strips all round. A
preparation should include about 150 hairs. To remove the air from the hairs,
the slide is first smeared with albumen-glycerine before being flooded with
absolute alcohol; it is left on a hotplate overnight before being transferred to
absolute alcohol, alcohol-xylene and xylene in a vacuum embedding oven
(2 days, 1 day and 1 day respectively) followed by embedding in Canada balsam.
THE COAT OF THE NORMAL MOUSE
As described by Dry (1926), the dorsal fur of the mouse consists of four
distinct types of hairs, with very few intermediates. Hair follicles are thus
'canalized', and as they tend to form the same kind of hair in successive hair
generations, determination evidently has some degree of stability.
The coat (Fig. 1) consists of three types of overhairs (together about 16-28 %
of all the fibres) and the finer underfur which makes up the rest. The overhairs
include the guard hairs or monotrichs (a) and the awls (b) which have no constrictions, and the 'auchenes' of Dry (c) with a single constriction which separates a shorter distal blade from a much longer stalk. The name 'auchene' is derived from the Greek word avxqv (the neck, throat, metaph. a narrow passage, a
neck of land, isthmus; also a narrow sea, strait; the narrow bed of a river; a defile)
which, even if familiar to biologists, would not convey the appearance of this
type of hair at all graphically. I venture to suggest the name of 'flails' for these
hairs, which both on linguistic and descriptive grounds appears rather more
appropriate. The wool hairs are known as zigzags (Fig. 1 d) on account of the
flat constrictions, usually three in number, at which successive segments are
angulated against each other.
The hairs consist of the very thin cuticle on the outside, the cortex and the
148
H. GRUNEBERG
medulla. The tips and bases of all hairs are solid and lack medullary cells, and the
medulla may also be interrupted at the flattened constrictions of zigzags. The
medullary cells are arranged transversely and separated from each other by air
spaces. In thin hairs like zigzags (k), the medullary cells form a single row of
'septa'. The same is true near the tapering tip and base of overhairs. In thicker
hairs there are rows of two, three or more 'septules', depending on hair calibre.
In Fig. 1 e the transitional region of an awl is shown where septa just turn into
septules; nearer the middle (/, g) there are three or four rows of septules, or even
2 mm
50
I
I
I
100//
I
2
te
*5
(g)
h
I
I
I
(n)
0)
CO
(0
Fig. 1. Hairs from the baby coat of normal (<$) mice, (a) Guard hair, (b) awl, (c) flail,
and {d) zigzag, (e-n) Enlarged regions of the hairs shown in (Jb-d). (ij). Constrictions
of zigzags seen edge-on and from the fiat surface, respectively. Further explanation
in the text.
(a)
(b)
(c)
(d)
Sex-linked mammalian genes
149
five in the strongest awls of adults. The transitional zone of awls is generally
fairly short. The stalk of flails (/?) tapers so gradually that irregular sequences
of septa and septules may alternate with each other many times, and the same
is common in guard hairs. Occasionally zigzags reach the critical diameter where
some septules may make their appearance, and sometimes whole sequences, as
in Fig. 1/. This occurs in some otherwise normal mice, may persist from the
baby to the adult coat, and is possibly in part under genetic control.
Pigmentation is present both in the cortex and in the medulla. In guard hairs cortical pigmentation is heavy enough to obscure the pattern of septa and septules
over much of the hair. In the other hair types, it is mainly found near the tip. Most
of the pigment occurs in the medullary cells, which it may fill almost completely
(e.g. Fig. 1 k). Lesser quantities tend to be aggregated in the distal (apical) region
of the cell, leaving the proximal (basal) region empty (Fig. 1 e-h). In the region
of the yellow band, awls and particularly flails are occasionally compressed
longitudinally in the middle. In the baby fur (but apparently not in the adult
coat) of normal mice, an occasional hair may be collapsed and form a ribbon,
sometimes twisted round its axis. Such hairs are common in certain mutants.
We mention here an artifact (mostly in zigzags) which can easily be produced by
pressure (Fig. Ira): successive septa appear to run together, sometimes only a
few, sometimes long sequences. Whether some hairs are more prone to this
kind of artifact than others is a moot question.
Roughly 1 % of the dorsal hairs of mice are guard hairs. Among the remaining overhairs, awls usually predominate and flails are rare. But sometimes,
for reasons unknown, the situation is reversed. As will be described below, the
same can happen in tabby mice.
TABBY
The coat of the tabby mouse (Ta <$<$, TajTa ??) lacks guard hairs and zigzags;
the fibres which are present have been regarded as awls by Falconer (1953)
though many of them are atypical. The strongest awls (Fig. 2b) are rarely more
than 2-septulate. They differ little from normal awls of similar calibre, except
that often they have a long and thin (and consequently septate) base. Most
hairs are finer in calibre and typically show irregular sequences of septa and
septules (Fig. 2 a), often associated with irregular changes in calibre (Fig. 2d).
Twisting of hairs is common: in the darker-appearing parts of such hairs, two
septules are seen end-on and can be confused with septa. The finest hairs
(particularly in the baby fur and most strikingly in tabby runts), are essentially
or completely septate (e). The suggestion (Griineberg, 1966/?) that the hair
population of tabbies might be a mixture of several hair types is not borne out
by subsequent and more detailed studies. Flattened and sometimes twisted
fibres are common in baby and occasionally still in adult tabbies.
Most tabbies have no hairs with constrictions, but in two out of 16 7a SS
examined there were numerous flails both in the baby and in the adult coat and
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H. GRtJNEBERG
over the whole of the dorsal surface. The situation persisted unchanged up to
3 months of age, and flails formed about one-half or more of all the fibres; the
blade was mainly septulate, the stalk more variable, being either partly septulate
•
50
^
,
m
Bii
mm
*4
m
(a)
(b)
(c)
(d)
(e)
Fig. 2. Hairs from the baby coat of Ta <$<$. Explanation in the text.
or essentially septate; but at 6^ and 8 months, respectively, the number of
flails had greatly decreased. Numerous flails were also encountered in three
litter-mates of one of these males ( + <?; + / + $; and Ta/+ ?), those of the
Ta/+ $ being mostly of tabby type. Evidently, as in the normal mouse, a switch
Sex-linked mammalian genes
151
from awls to flails is possible in tabbies, and as far as the evidence goes, the
change seems to have some stability.
As the total number of hair follicles is not far from normal (Griineberg,
19666), the coat anomaly is one of differentiation. The normal mouse turns
about three-quarters of its follicles into underfur; tabby uses all its follicles for
overhairs (usually awls) and thus makes a coat comparable to the hairs of the
human scalp. Hair calibre is reduced, guard hairs and the coarser awls being
absent, and many of the finer hairs are thinner than normal zigzags. Presumably,
all this is somehow connected with the delay in follicle formation characteristic
of tabby.
Table 1. Percentage of hair types in the dark stripes and the intervening agouti
areas of Ta/ + $$ as classified under the dissecting microscope
Average of five animals. N = 'normal', A = 'abnormal'.
Based on data of Kindred (1967).
Zigzags
Flails
Awls
Area sampled
Guard
hairs
N
A
N
A
N
A
Agouti
Dark stripes
0-8
1-2
34-8
63-2
7-8
22- 4
5-3
1-2
0-4
0-4
SO- 1
1
0-9
0-5
The transverse stripes of Taj + ?? have been interpreted in terms of the L.H., it
being claimed that the dark stripes correspond to areas in which Ta is active,
with the normal allele active in the intervening agouti areas. The regularity of
the stripes is, of course, not easy to explain as the result of a random process
(Gr uneberg, 1966 b, 1968), and the fact that the intensity of stripingcan be increased
or decreased by selection (with correlated orderly changes in the sensory hairs) is
equally at variance with the L.H. Quite recently (Bangham, 1968), the same
phenotype has also been described for an autosomal gene (autosomal striping,
Sta) in which both male and female heterozygotes resemble Ta\ + , the homozygote probably being lethal. As the phenotype of Sta/+ clearly cannot be
interpreted in terms of the L.H., it is logically inadmissible to invoke a different
interpretation for the same phenotype when it is caused by a sex-linked gene.
The coat of Taj + $$ regularly includes some typical tabby hairs with irregular
sequences of septa and septules; more seem to be present in the baby than in the
adult coat, but an accurate enumeration is difficult as there are intergrades
between tabby and near-normal and normal awls. The recent data of Kindred
(1967; Table 1 above) are particularly informative, as in her selected lines she
was able to obtain uncontaminated samples from the stripes and the intervening agouti areas. In the dark stripes, guard hairs and zigzags are present;
if the dark stripes really correspond to tabby, they should be absent. Awls are
greatly increased and zigzags decreased in the dark stripes. But the same is also
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H. GRLINEBERG
true, though to a lesser extent, for the agouti areas which should be normal.
The agouti areas thus differ from the normal, and the stripes from the tabby
phenotype. One Taj + $ (not included in Table 1) in which both the stripes and
the agouti areas consisted entirely of tabby hairs, can clearly not be explained
in terms of the L.H. : this being so, there is no reason to invoke that concept for
the rest of the Taj + $$. The abnormal flails of Table 1 presumably correspond
to those in tabbies described above. As abnormal zigzags with septulate regions
are fairly common in certain 'normal' mice (Fig. 1/), it must remain problematical whether their occurrence in Taj + ?$ may be regarded as a heterozygous
effect of tabby. For embryological evidence also at variance with the L.H. the
reader may be referred to Kindred's (1967) paper.
The data on coat, vibrissae and molars (Griineberg, 1966«; Sofaer, 1969) of
tabby are now quite extensive. They all agree in showing that all along the line
the phenotype of Taj+ ?? cannot be accounted for in terms of the L.H.
Tabby has two autosomal mimic genes, crinkled (cr; Falconer, Fraser &
King, 1951) and downless (dl; Phillips, 1960), both of which are indistinguishable
from it as regards coat and molars. A prolonged search in + jcr mice for hairs
corresponding to the crjcr type has been completely negative. Crinkled is thus
recessive as regards the coat, but not as regards the morphology of its molars
(Griineberg, 1966 a).
BRINDLED
The gene for brindled is lethal in hemizygous condition. As originally described (Fraser, Sobey & Spicer, 1953), Mobr $<$ died between 10 and 14 days;
at present they not rarely live up to 3 weeks. One which lived for 5 weeks was by
far the darkest of the series, another which lived to 40 days had normal light fur.
The cause of their death is still unknown. Usually, the coat of Mobr SS is almost
white, with slightly sooty hair tips, but sometimes it is very light grey in colour.
The eyes are dark and skin pigmentation (ears, tail, scrotum) is about normal,
as best seen in the naked tails of Ta Mobr $<$. The resemblance to Himalayan
albinism is, however, only superficial as pigmentation resides in the skin rather
than in the hairs. The whiskers are extremely curly and irregular; the coat as a
whole is short, and many hairs are finely curved or undulated. The halo of
guard hairs which normally projects above the rest of the coat is absent; the tail
hairs are reduced and less regular in arrangement than in a normal animal.
The coat of Mo brj+ $$ is irregularly mottled or brindled; ill-defined lighter and
darker areas tend to grade imperceptibly into each other. At the extremes, some
animals are phenotypically almost normal, others very light. The hair bases
are much lighter than the tips, as easily seen when the hairs are blown back. As in
dappled (Modpj + ; Phillips, 1961), an allele of brindled, the whiskers are normal
in dark, but a little curled in light individuals (Fig. 3). The tail hairs are reduced
and irregular in a somewhat patchy fashion like the brindling of the fur. There is
no colour mottling on ears or tail.
Sex-linked mammalian genes
hr
153
br
Mo l+ $$ sometimes change colour with age. Unselected groups of Mo l +
$$, Ta Mobr/+ + ?? and Ta + I + Mobr $$ were graded at fortnightly intervals
by reference to series of skins covering the whole range of variation for each
genotype in five or six steps. Grading was done by two observers without
knowledge of the grade accorded to a given mouse on previous occasions
(Table 2). All three genotypes tend to get darker with age. This is significant at
the 001 level for both Mobr/ + and TaMobr/+ + $$; the slight changes in
Ta + / + Mobr $$ when considered in isolation are not significant in the present
sample.
Fig. 3. Brindled (Mobr/ + ) $ with dorsal midline effect, aged 47 days. Most midline
effects in brindled ?? are much less marked.
In addition to mottling, some Mobr\ + $$ have certain more regular patterns.
As already pointed out by Falconer (1953), the light hairs sometimes form 'an
irregular pattern of transverse bars reminiscent of the markings of Tabby
heterozygotes'. As in Taf + ?$, the transverse pattern is most marked in the
baby coat and tends to decrease with age. The transverse patterns in both types
of heterozygotes may reflect the transverse wrinkling of the embryonic skin
(Griineberg, 19666). The second regular pattern is a 'midline effect' found
occasionally in Mobrl+ $$ in which dark and light areas meet sharply along the
midline; the animal shown in Fig. 3 is an extreme case. Midline effects are
154
H. GRUNEBERG
common in Cattanach's translocation where they will be discussed in more
detail; they also occur occasionally in certain human skin conditions.
The coat of Mobr S<$ (Fig. 4) includes both overhairs (awls, flails and possibly
guard hairs) and underfur (zigzags), all of them reduced in calibre and strucTable 2. Average degree ofbrindlmg in ' young" animals (3-5 weeks) and as' adults'
{4\-6\ months): the higher the grade the lighter the mouse
Mice
Genotype
br
Mo /+ 99
TaMobr/+ + 9?
Ta + / + Mobr 99
n
19
20
11
Young Adult Change
(A)
(A/Y) Unchanged
(50
2-82
3
2-24
0-79
2-65
2
1-65
0-62
3-55
3
0-88
314
Darker
Lighter
14
18
6
2
0
2
500//
to
(a)
(b)
(<0
(d)
(e)
(f)
(g)
(h)
Fig. 4. Brindled hairs, (a) Awl from a Mobr/ + 9 (19 days), (6) awl from Mobr 6 (littermate, 12 days), (c-f) other hairs from the same animal at lower magnification (all
completely devoid of pigment in the segments shown), (g, h) Normal 3 (19 days).
(a, b, d, e and h) awls, (d a distal tip), (c, g) zigzags, (/) presumed guard hair with
long non-medullated tip.
Sex-linked mammalian genes
155
turally abnormal. Most hairs are unpigmented, but some have a little pigment
in the tip which is mostly cortical though in the first few septa some yellowishbrownish eumelanin pigment is also sometimes present. Due to the presence of
liquid (absence of air) between the medullary cells coupled with absence of
1 mm
1
(b)
a
- <3
(0)
(i)
(f)
Fig. 5. Brindled (Mobr/ + ) ?, 19 days (same as in Fig. 4). (a) A zigzag, (/) aflail,both
with polarized pigmentation as shown in the enlarged regions (b-e) and (g-j),
respectively.
(a)
pigment, little internal structure can be made out in most of the hairs. Some are
flatter than normal, and the stronger awls in particular tend to have a groove
along the centre.
The baby coat of Mobr\ + $$ includes some completely pigmented and structurally normal hairs and some with as little pigment as in Mobr &?; however, they
are usually structurally more nearly normal (Fig. 4a) and thus cannot simply be
156
H. GRUNEBERG
equated to the hemizygous phenotype. Most hairs are intermediate between these
extremes (Fig. 5); typically they start normal both as regards structure and pigmentation. Gradually pigmentation decreases and eventually stops altogether,
and hair structure becomes increasingly abnormal; deterioration may start
early or late and some hairs escape altogether and appear normal. Occasionally,
there is some recovery leading to an interstitial abnormal segment. But there are
no hairs which start abnormal and gradually become normal. There is thus the
same polarization of pigment as in Mobr SS except that much more of the hair is
pigmented and pigmentation is much heavier. Polarization is visible at a glance
in the living mouse when, on blowing back the hairs, the light bases are spectacularly revealed.
In the coat of adult Mohr\ + $$, hairs as extreme as are found in the baby
coat are all but absent. Changes in degree of brindling (Table 2) are evidently
largely due to differences in the extent of polarization in different hair generations.
Clearly, the coat of Mobr/ + ?$ is not simply a mixture of Mobr and + hairs,
respectively. The lightest hairs are structurally less abnormal than those of
Mobr <$$\ in the polarized hairs, contrary to the L.H., both alleles manifest
themselves seriatim, + distally and Mobr proximally, with a gradual transition
from one to the other. If all these hair follicles had mixed populations of
cells (with either Mobr or + active), pigmentation in individual hairs should be
randomly distributed. The polarization decisively excludes a random process.
As the same type of polarity is typical of the Mobr $, it is difficult to imagine a
more complete demonstration of allelic interaction with intermediate result. As
in tabby, the evidence is thus unambiguously against the L.H.
The relationship between hair structure and pigmentation in brindled is not
yet clear. Either the effect on hair structure is primary, and melanocytes cannot
function properly in follicles which give rise to abnormal hairs, or the effect on
the melanocytes is primary, it being assumed that these cells do more in hair
development than to produce pigment. The fact that skin pigmentation is not
affected would favour the view that the primary effect is on hair structure. In the
context of the present discussion, the mode of action of Mobr is of no immediate
importance, as on neither interpretation can the phenotype of Mobrj + $? be
accounted for in terms of the L.H.
As discussed in the next section, mottled alleles have been used in double
heterozygotes in attempts to test the L.H., but the results have been rather
contradictory. Without going into details, the observations fit the hypothesis best
on the assumption (which has been seriously put forward) that Modp acts
primarily in melanocytes, but Mobr in follicle cells. The argument is so naively
circular that it is astonishing that it has gone uncontradicted, quite apart from
the inherent improbability of the concept. As will be discussed below, the various
double heterozygotes are in any case irrelevant to the L.H. and thus do not
require any subsidiary assumptions as props for that hypothesis.
The brindled mouse has several autosomal counterparts of which the well-
Sex-linked mammalian genes
157
known roan gene in cattle is probably the most striking. The homozygote is
white or nearly so, that of the normal allele, in the absence of other spotting
genes, is solidly coloured. Roan heterozygotes range from a slight degree of
silvering to heavy preponderance of white over pigmentation. As in brindled
mice, random mottling is commonly associated with orderly systematized
patterns, including transverse bars like those in Mobr/+ °.$. The similarity in
phenotype is so unmistakable and striking that, if two such genes were found in
the same species, one would immediately suspect allelism. Yet the bovine gene is
autosomal and hence its phenotype must be due to some threshold phenomenon.
That being so, on what evidence, or by what logical process can one invoke a
clonal mechanism when the same phenotype occurs in a heterozygote for a sexlinked gene?
By way of example from other mammals, let us consider the extension series
in the guinea-pig (Chase, 1939), which is autosomal; i.e. E (self black), ep
(brindled or tortoiseshell) and e (self yellow). E is dominant over ep and e, but
e1' is not completely dominant over e. In the absence of spotting, ev\ev guineapigs show a brindling of yellow hairs on a black background; in the presence of
spotting, black and yellow areas become increasingly segregated from each
other and brindling is correspondingly reduced; in such tortoiseshell (tricolour)
guinea-pigs, yellow tends to occur in areas which are most frequently white, and
where spots remain brindled, the black hairs tend to be central and the yellow
ones peripheral. The formation of eumelanin versus phaeomelanin is evidently
dependent on a threshold mechanism, with E on one and e\e on the other side of
the divide; ep/ep guinea-pigs teeter on the brink, and the decision one way or the
other is made locally and influenced by genetic factors like spotting and, to a
lesser extent, by sex; ev\e animals are nearer e/e and thus have less black and
more yellow.
The e"[e" pattern of the guinea-pig is virtually indistinguishable from that of the
tortoiseshell cat and subject to the same interaction with spotting. But in the
guinea-pig it is produced by a mechanism which clearly cannot involve chromosome inactivation. The same happens in the extension series of other mammals
(rabbit, pig), and there are other autosomal colour genes in the mouse like
viable yellow (Arv) and mottled agouti (am) as well as two alleles of p (Russell,
1964) which give rise to mottled phenotypes in heterozygotes. Searle (1968)
admits the close phenotypic resemblance between the tortoiseshell cat and e1 in
the rabbit and el) in the guinea-pig, but opines that 'it seems clear that the last
two patterns are produced by an entirely different mechanism. They are due to
homozygosity for an allele of the autosomal extension locus, while the tortoiseshell pattern in cats is due to heterozygosity for a sex-linked gene.' No matter
whether the emphasis is on 'heterozygosity' or on 'sex-linked gene', the argument begs the question because it gives as evidence for the supposed difference
in mechanism the very concepts which are at issue. Moreover, the statement is
not even completely true as to facts. Sewall Wright (unpublished, quoted by
158
H. GRUNEBERG
Chase, 1939), in a sample of about 900 E/e guinea-pigs, observed one individual
which was black with the right hind leg red and hence in the phenotypic range of
ep/ep, but in an Eje heterozygote! A second point is only a suggestion. The rare
male tortoiseshell cats are usually sterile and apparently XXY in constitution
(Thuline & Norby, 1961; Chu, Thuline & Norby, 1964). There are, however,
sometimes fertile tortoiseshell <$$, and two such animals examined by Komai &
Ishihara (1956) had a normal 38 chromosome complement 'including a distinctly unequal X-Y pair'. Such animals cannot be heterozygous for a sexlinked gene. I venture to suggest that there may exist, in the cat, a rare allele
similar to ev in the guinea-pig which produces the tortoiseshell pattern by itself
in the hemizygote and presumably in the homozygote.
TABBY-BRINDLED DOUBLE HETEROZYGOTES
Lyon (1963) and Cattanach (19666) have attempted to test the validity of the
L.H. by investigating the phenotype of double heterozygotes for genes (or structural rearrangements) affecting the coat. Provided both act through the same cells
and provided each of them individually behaves in accordance with the L.H.,
coupling double heterozygotes (AB/ + +) should have areas manifesting either
both A and B, or their two normal alleles, but not A or B alone; whereas
repulsion double heterozygotes (A + / + B) should in some areas manifest A,
in others B, but never both or neither. As neither Ta nor Mobr individually
behaves in accordance with the L.H., it cannot be expected that the double
heterozygotes will, and in fact they do not. However, they show a marked
cis-trans position effect which is interesting in its own right, particularly as it
can be studied at the level of individual hairs or parts of hairs. This position
effect is, of course, a fact which will have to be explained by any hypothesis on
the action of sex-linked genes in mammals.
Ta Mobr/+ + ?$ differ markedly from Ta + I + Mobr $$ in phenotype. In the
former, the tabby stripes are fine but remarkably regular and rather more
conspicuous than those in ordinary Taj + ??. The manifestation of Mobr varies
from virtual absence to animals in which light hairs definitely predominate. The
over-all impression is one of light on a dark background, with the tabby striping
often seen as if through a thin veil of light hairs. By contrast, most Ta + I + Mobr
$? give the impression of a dark pattern on a light background. Usually there
are no real tabby stripes though often some transverse banding as in M.obr\ + $$.
Only when brindling is very slight are tabby stripes sometimes visible. As in
Ta Mobr/+ + $?, brindling varies from an almost imperceptible admixture of
light hairs to their definite preponderance. Mid-line effects sometimes occur in
either phase.
Ta Mobr/+ + differ from Ta + / + Mobr $$ in an orderly way at the level of
individual hairs (Table 3). The two halves of Table 3 are essentially mirror
images of each other. In coupling, hairs tend to be structurally normal and fully
pigmented or tabby with little or no pigment. Conversely, in repulsion, non-
Sex-linked mammalian genes
159
tabby hairs tend to be light and tabby hairs to be fully pigmented. But in either
phase there are many hairs with polarization as to Mobr which do not conform
to the L.H. as mutant and normal allele are acting side by side. They were
present in numbers in every specimen examined and often dominate the picture;
in general, polarization is commoner in normal than in tabby-type hairs, and
commoner in coupling than in repulsion. In hairs of tabby structure, polarization is sometimes as in normal hairs; more often, it leads to a considerable
dilution of pigment without much of a gradient so that the base of the hair does
not become colourless. Whether present in coupling or in repulsion, in structurally normal or in tabby-type hairs, polarized brindling is contrary to the
L.H. Thus, in coupling, in a polarized zigzag, Mobr is 'active', but not Ta;
similarly, in a polarized tabby-type hair, the normal allele of Mohr is 'active',
but not that of Ta; etc., etc.
Table 3. Hair types encountered in coupling and repulsion double heterozygotes
involving tabby and brindled {baby coat only)
Note that the tabby coat includes some near-normal awls and sometimes also flails.
TaMobrl++
^pigmentation
Ta + l + Mo
i br $$ pigmentation
A
Haii- type
Guard hair
Awl
Flail
Zigzag
Greatly
reduced* Polarizedf
Normal
Greatly
reduced* Polarizedf
Normal
I
+
+
+
(+ )
Tabby
+
+
.
+
+
* As in Mobr $o, i.e. unpigmented hairs and hairs with a little apical pigment.
t As typical for the Mobr/+ $ and including hairs with interstitial segments with defective
pigmentation.
One rarer type of hair not included in Table 3 may be mentioned. Such hairs
start as zigzags, but have only a single constriction; the remainder of the hair is
an unsegmented twisted ribbon with pigment reduced or absent. A length of
tabby-like structure but with full pigmentation is sometimes interposed between
the two. In such hairs, both alleles at both loci are manifest, tabby and brindled
proximally and their normal alleles distally. Such 'fourfold' hairs have been
found repeatedly both in coupling and in repulsion. Other rarer types of mixed
hairs will not be described in detail.
In Ta Mobr <$<$, the combined effect of these genes leads to highly abnormal,
virtually colourless twisted ribbons. In TaMobr/+ + $$, most of the corresponding hairs with little or no pigment are structurally far more normal as
already described for the light hairs of Mobr S$ and Mobr/+ $$ respectively in a
previous section.
160
H. GRUNEBERG
STRIATED
As Str S<$ die early in embryonic life, the phenotype of Str/+ $$ cannot be
interpreted in terms of the L.H. without the circular argument that the abnormal
hairs of such animals are like those which the Str <$S would have had if they
Fig. 6. (a) Zigzags from a short-haired region of a StrI + $ (20 days old),
(b) zigzags from a + / + (litter-mate) $.
lived to grow a coat. The same uncertainty eliminates double heterozygotes
(Ta + I + Str or TaStrj + + $$) as a source of information concerning the L.H.
Str I + ?? superficially resemble Taj + $$ in that they are striped transversely;
however, the stripes are caused by regions of shortened hairs (Lyon, 1963)
which expose the dark bases of the hairs behind to view. An admixture of light
Sex-linked
mammalian genes
161
hairs to the coat is commonly present, sometimes in little groups. Phillips
(1963) suspected some normal overlapping (though this was not directly demonstrated); in the present stock, penetrance seems to be complete.
- o
(0
- 100
(g)
L- 200//
50
100//
(a)
(c)
Fig. 7. Strj+ $ {a-e, g) and + / + ? (/); same animals as in Fig. 6. (a) Awl, transition to narrow base of hair; (6) awl, basal region; (c) awl (same hair as in a); (d) awl,
virtually pigment-free region; (e) zigzag, distal part of 2nd segment; (g) zigzag,
widest part of 2nd segment (to be compared with (/), the corresponding region of a
normal zigzag).
The coat of Strj + $$ includes all four hair types. The most abnormal hairs
are found in the short regions of the coat. Zigzags (except the smallest ones)
usually have normal or nearly normal numbers of constrictions (Fig. 6), but
tend to be undulated rather than angulated. Hair calibre is greatly reduced, and
particularly the zigzags are often whip-like and with irregularly wobbly outlines
(Fig. le). Awls commonly have very long and thin septate bases (la, b); the
internal structure is often irregular (b, d) and twisting of hairs is common (c).
Pigmentation of many hairs is greatly reduced (d), particularly basally where
II
JEEM 22
162
H. GRUNEBERG
many hairs are completely unpigmented. Even in the lightest hairs some phaeomelanin is usually present, but sometimes clumped. Abnormal hairs are also
found in regions of the coat which are not shortened, but their number is much
smaller and the abnormalities are less extreme; a gradual transition leads from
definitely abnormal hairs to fibres of normal or near-normal structure and
dimensions. Whether they are in fact completely normal is a question which
cannot be decided in the absence of detailed measurements. The above descriptions are based on the baby coat; there are some indications that the coat of
Str/ + $$ tends to become more normal later in life.
Str/+ ?$ mated to Ta $<$ produced 41 striped daughters and 25 normal
sons; the first 23 striped daughters included 11 Ta + I + Str $? (recognizable by
the presence of short-haired areas) and 12 Ta +1 + + $$; they were even more
easily distinguishable from each other in hair preparations. The coat of Ta + /
+ Str $$ consists essentially of Str-type hairs, 7«-type hairs and normal hairs;
the latter include all four hair types, but apparently few strong awls, perhaps
due to the interaction of the two mutant genes. Hairs showing the effects of both
Str and Ta are certainly not common and may not occur at all; the few which
could be so interpreted are somewhat problematical. Str may be epistatic over
Ta, as the inner structure of Str-type awls is so irregular that the simultaneous
expression of Ta would scarcely be detectable.
If, for the sake of argument, the circular reasoning concerning the potential
phenotype of the Str $ were accepted, the L.H. would require the coat of
Ta + I + Str $? to consist of hairs manifesting either one mutant or the other: it
cannot account for the occurrence of numerous normal hairs in which neither of
them is 'active'.
As mentioned earlier on, the first observations on Str were carried out with a
stock subsequently proved to have been contaminated with Ta. This has been
the common cause for two erroneous statements. Ta + / + Str $$ do not have bare
patches behind the ears and naked tails (Griineberg, 19676); the animals in
question were in fact Ta + jTa Str and Ta + jTa + in genotype. Secondly, due to
the presence of Ta in the Str stock, some features of Ta have at first been
ascribed to Str (Griineberg, 1966/?), and the statement has been made that
Str affects the coat as a whole; it is now clear that the visual evidence was
misleading (though the statement might still be vindicated by measurements).
Obviously, these corrections make no difference to the argument as a whole.
The phenotype of Str/+ $$ is irrelevant to the L.H., regardless of whether the
Str gene affects the coat as a whole or not. Similarly, whereas it was erroneously
thought that in Ta + / + Str $$ both mutant alleles were simultaneously 'active'
it is now the simultaneous manifestation of the two normal alleles which
contradicts the L.H.
Sex-linked mammalian genes
163
C A T T A N A C H ' S TRANSLOCATION
In this translocation (Cattanach, 1961, 1966a), a segment of linkage group I
which includes the albino locus is inserted into the continuity of the Z-chromosome. A male which is homozygous for albinism (c/c) but which carries the
normal allele for that gene in the X-chromosome with the translocation (XT) is
fully pigmented, but an XTX female is flecked with a mixture of dark and light
hairs which give a mottled effect to the coat. This situation has been described
Fig. 8. Flecked $ (Cattanach's translocation), 61 days old. Crossed midline effects on
the venter in the absence of similar dorsal effects.
in terms of the L.H. on the assumption that X-chromosome inactivation spreads
into the inserted segment of autosome. Depending on whether X or X 2 'has been
inactivated, clonal areas with pigmented or unpigmented hairs will be formed.
The coat of flecked $$ consists mainly of finely interspersed groups of dark
and light hairs; the appearance is brindled or mottled, as on the back of the
animal in Fig. 8. Often, a characteristic pattern is superimposed on this mottled
background, i.e. large dark or light areas with a striking tendency to end sharply
at the midline of the body without crossing it. This is particularly common and
conspicuous ventrally where it sometimes leads to crossed configurations
(Fig. 8). Except for the face, dorsal midline effects are rarer and usually less
conspicuous. Ventrally, dark and light areas tend to confront each other;
164
H. GRUNEBERG
dorsalJy, dark or light areas often border on mottled fur. The segregation into
large areas of dark and light fur somewhat resembles that into large orange and
black patches which, in the presence of spotting, tends to happen in tortoiseshell
cats and guinea-pigs (though without relation to the midline). It is unknown
whether, in flecked $?, the tendency to form large patches would be enhanced by
spotting genes. Unlike brindled, marked mottling is present on ears and tail of
flecked ?$.
The coat of flecked mice includes all intergrades from fully pigmented to
albino hairs (Griineberg, 19676). This makes questionable the method adopted
by Cattanach & Isaacson (1967) for the quantitative estimation of the degree of
flecking, which is based on an attempt to enumerate two distinct kinds of hairs,
pigmented and unpigmented. In many hairs, the granule count per medullary
cell is greatly reduced compared with its normal value of about 90 (Russell,
1946), and many cells lack pigment altogether; the granules also are smaller and
consequently round rather than oval. Pigment reduction may be uniform, but
many hairs are strikingly polarized with pigmentation near the tip gradually
fading out as hair growth proceeds. In the large light areas the few hairs with
pigment usually show a reversed polarity (unpigmented tips with pigment in
the proximal parts of the hair only).
To account for the hairs with intermediate degrees of pigmentation, the L.H.
would have to postulate that a large proportion of hair follicles have mixed
populations of cells with either XT or X inactivated. However, this would not
account for the polarization of pigmentation which is a regular pattern and thus
contrary to the L.H. which requires a random process. Moreover, the same hair
types occur in silver (si I si) mice (Dunn & Thigpen, 1930), an autosomal condition which thus cannot be interpreted in terms of the L.H.
In ageing necked $?, Cattanach & Isaacson (1965) observed that 'pigmented
hairs began to appear in the regions that had earlier been clear white', and as
this happened uniformly over the white areas, it cannot be accounted for by
spread of adjoining pigmented areas or by enzyme diffusion. The observation
does not fit a clonal explanation of necking, but presents no difficulties to a
threshold concept.
Brown (chocolate) mice show significantly more intense flecking than black
flecked mice (Cattanach & Isaacson, 1967). On the L.H. (i.e. two distinct hair
populations) such an interaction would indeed not be easy to understand: how
can an allelic difference in linkage group VIII have an influence on whether XT
or X is to be inactivated, or to what extent inactivation will spread into the
inserted segment of linkage group I ? As the gene for brown (b) considerably
reduces pigment as compared with black (Dunn & Einsele, 1938; Russell,
1946, 1948), it increases the proportion of hairs which appear colourless to the
naked eye. In the presence of silver (Dunn & Thigpen, 1930), b is strikingly
semi-dominant which may be relevant in view of resemblances between silver
and the flecked condition; other instances of semi-dominance of b have been
Sex-linked mammalian genes
165
described by Wallace (1953) and by Tatchell (1963). As b was segregating in the
experiments of Cattanach & Isaacson (1965, 1967) its presence may have contributed to the effects of selection for increased mottling.
Whereas many Mobr\ + $$ come close to normal overlapping and others show
a decided preponderance of light hairs, the range of variation in flecked $$ is
much narrower. If both genotypes have a clonal background, and if Mobr
(like c) acts in the melanocytes, it is difficult to see why there should be this
difference. But, if Mobr acts in the hair follicles proper, the same question must
be asked concerning the dissimilar phenotypes of Mobr/ + and Taj+ $$.
Midline effects are very common in flecked mice and occasionally occur in
brindled $$ (and presumably other alleles of that series); they do not seem to
have been observed in other sex-linked genes, but occur in autosomal conditions, like the fully pigmented coat areas of varitint-waddler (Va/+) mice
(Cloudman & Bunker, 1945). They also occur in capillary angiomata (Port
Wine marks) in man and sporadically in other skin diseases such as acanthosis
nigricans (Dr A. Jarrett, personal communication). Midline effects are thus
neither found in all heterozygotes for sex-linked genes, nor are they confined to
them.
If the midline effects of flecked mice are clonal in origin, they must represent
migration patterns of melanocytes originating in the neural crest. Now, if
Mobr acts in the melanocytes, why are mid-line effects so much rarer than in
flecked mice? Conversely, if Mobr acts in the hair follicles, why are there any
midline effects at all? The clonal interpretation thus runs into difficulties. These
do not arise if the midline acts as a physiological barrier between the two sides.
This is not difficult to imagine considering that, except for the tail, both the
dorsal and the ventral midline of the skin constitutes a raphe; the midline is not
crossed by blood vessels or by cutaneous nerves.
In mosaics of undoubted clonal origin (Wright & Eaton, 1926; Dunn, 1934;
Bhat, 1949; Hoecker, 1950) there is no indication of a midline effect, the midline
being crossed by mutant territories both dorsally and ventrally. In Carter's
(1952) case, whose clonal origin is rather less certain, the midline was crossed in
some places but formed the boundary in others, including a ventral area. In
mouse chimeras, dorsal midline effects have been mentioned by Mintz (1967),
but neither she nor Mystkowska & Tarkowski (1968) refer to ventral midline
effects which are so characteristic of flecked mice. Ventral midline effects can
scarcely be interpreted on a clonal basis, particularly when present in animals
without a trace of dorsal midline effect, as in Fig. 8.
As discussed for ordinary sex-linked genes, complex situations in double
heterozygotes cannot give any information concerning the L.H. unless the single
entities individually behave in accordance with that hypothesis. The same applies
to chromosomal rearrangements like Cattanach's or Searle's translocations. For
that reason, two experiments (Cattanach, 1966b) which, on certain assumptions,
are formally at variance with the L.H., but which, on other assumptions, can
166
H. GRUNEBERG
formally be interpreted in terms of the 'spreading' concept, in reality do not give
any information concerning the L.H. one way or the other.
Ohno & Cattanach (1962) have shown that in metaphase plates the XT of
flecked ?? is distinguishable from the normal A'by its greater length. It has also
been claimed that the same is possible in the single heteropyknotic X-chromosome of somatic prophase nuclei of flecked $$; that in light fur areas the long
XT is heteropyknotic (and hence inactive); and that in dark areas the short X\s
heteropyknotic. This remarkable finding has been claimed as cytological proof
of the L.H. However, as noticed by several authors (e.g. Mukherjee & Sinha,
1964; Baker, 1968), the claim is rather problematical. On the technical side, as
in any prophase nucleus, a single element only is heteropyknotic, it cannot be
compared with its partner in the same cell. The difference between XT and Zis
not so great that the claim can be credibly established without the use of quantitative methods and coded preparations (and, indeed, by independent observers).
However, accepting the claim for sake of argument, its weakness is that it refers
to follicular cells (which do not form pigment) rather than to the melanocytes in
which c is acting. Hence, in any case, the claim would constitute a phenomenon
sui generis without obvious relation to the L.H.
DISCUSSION
The facts related in this paper and earlier studies show that the phenotypes of
heterozygotes for sex-linked genes in mammals cannot be accounted for in terms
of clonal territories. They must thus be due to threshold mechanisms. Contrary
to the L.H., both alleles thus interact with each other as in the case of autosomal
genes. At the level of individual genes, this of course removes all the difficulties
which arise out of the postulates of that hypothesis. There are, however, enough
indications that the phenomena are chromosomal rather than genie to require
discussion in this wider frame of reference.
(1) The cytological behaviour of the mammalian ^-chromosome sets it apart
from the autosomes and thus suggests a corresponding functional difference.
The cytological facts have from the beginning been deemed to support the L.H.
I have surmised (1968) that perhaps these facts might fit a different conceptual
framework equally well, a supposition which will now have to be examined.
(2) The Jf-chromosome of the mouse, at any rate, seems to have more than
its fair share of semi-dominant genes (7a, Str, Mo, Gs, Bn, Gy and spf, assuming
that Bio and To belong to the mottled series; the only' recessives' beingy/?, sf and
sla); by contrast, about three-quarters of autosomal genes are 'recessive'.
(3) Double heterozygotes for sex-linked genes tend to show a cis-trans
position effect, the most spectacular example being that of 71a and Mobr
(Griineberg, 19676, and this paper). Though similar effects also occur in autosomal genes (Phillips, 1966) their apparent prevalence in sex-linked genes may
pose a problem.
Sex-linked mammalian genes
167
(4) In ^-autosome translocations the action of sex-linked genes removed from
the ^-chromosome is stabilized, whereas that of autosomal genes brought under
the influence of the ^-chromosome becomes unstable (Russell, 1964). This is
particularly striking in Cattanach's translocation, where it applies to c, p, ru-2
(Eicher, 1968) and sh-\ (Deol & Green, unpublished) and is evidently a chromosomal rather than a genie phenomenon. Though mottling has not so far been
found in translocations between two autosomes, it does not follow that it is due
to the ^-chromosome per .ye; it could be due to proximity of heterochromatin and
might thus also occasionally be found in interchanges between autosomes as in
Drosophila (Baker, 1968).
Cytology
The cytological basis of the L.H. has been formulated succinctly by Hamerton
(1968) as follows. 'This inactivation is effected by facultative heterochromatinization of all Jf-chromosomes in excess of one in somatic cells and can be demonstrated cytologically by precocious condensation during prophase, late replication
and sex chromatin formation during interphase. In diploid cells the number of
sex chromatin bodies correlates exactly with the number of late replicating
Jf-chromosomes and is one less than the total number of X-chromosomes
present in the cell.' Hamerton's statement does not offer cytological support
for one essential ingredient of the L.H., namely that inactivation once effected
is irreversible. This omission is not due to an oversight. There is, of course,
no means of knowing whether a heterochromatic chromosome is the same
which behaved similarly in the preceding mitotic cycle, or whether it was its
homologue. Where abnormal ^-chromosomes are non-randomly heterochromatinized, their abnormal structure is a sufficient explanation for their different
behaviour. There is thus no cytological evidence for irreversibility of inactivation
as postulated by the L.H.
Cytologists seem to be agreed that heterochromatinization in some way
reflects the state of gene activity. The argument is necessarily indirect, but for
the purposes of this discussion it may be accepted. Let us then pose a question.
The L.H. posutlates that gene activity in the heterochromatic Z-chromosome is
0 whereas that in its non-heterochromatic partner is 100. Supposing both
Z-chromosomes were active with a joint activity of 100, would the cytological
appearances exclude relative activities of, say, 10:90, or perhaps 20:80? It is
not easy to see how, with present methods, cytology could discriminate between
complete and relative inactivity.
As implied in Hamerton's formulation and summarized by Mittwoch (1967),
^-chromosome heterochromatinization is facultative. Thus, Barr bodies are not
present in all cells, tissues or organs of XX$$; and, somewhat ironically, in the
mouse none have been demonstrated at all. In human XXX $$ cells with two
Barr bodies form a small minority, and similarly in XXXX $$, etc. The same
applies to late-labelling ^-chromosomes. In vivo studies in the mouse either
have shown that only a small percentage of labelled cells has a 'hot' X-chromo-
168
H. GRUNEBERG
some (Evans, Ford, Lyon & Gray, 1965; Chandley, 1969), or have given no
evidence for asynchrony of X-chromosome replication at all (Tiepolo et al.
1967), in agreement, perhaps, with the failure to demonstrate sex-chromatin in
that animal. For the mouse, at any rate, the cytological support of the L.H.
cannot be said to be impressive. Even if we ignore this fact, another difficulty
remains. If heterochromatinization is the cytological expression of chromosome inactivation, the n— 1 rule should be obeyed in every cell and not merely as
a limiting case. We shall show below that for the alternative hypothesis to be
discussed presently, this difficulty does not arise.
The complemental-X hypothesis
The question of whether the XY & XX $ mechanism of sex determination
requires a special mechanism of dosage compensation is controversial; Goldschmidt (1954) argued that it does not. However that may be, dosage compensation may be brought about in at least three different ways. The first is that the
single Xin the $ works twice as hard as the individual Xs in the $; the second is
inactivation of one X'm the $; and the third is complementarity, i.e. the two Xs in
the $ jointly do as much work as the single X in the <$. A complemental-X
hypothesis was first proposed (Griineberg, 19676) to account for the fact that in
the mammalian ? the two Xs interact with each other like autosomal genes; it
was further suggested that the two alleles are active at about half-strength each.
That restriction of the hypothesis (sub-equal contributions) should not have
been introduced without good reason—which there was not (actually, I fell a
victim to my own attempt at visualization—that of a cart drawn by two horses).
Abandoning the restriction, the generalized complemental-X hypothesis postulates that, within individual cells or groups of cells, there may be all intergrades
from predominance of the paternal to predominance of the maternal X, with 100:0
or 0:100 situations not necessarily excluded (though perhaps rare, and of course
not irreversible). There is no reason to suppose that a 50:50 situation is favoured,
and considerable deviations either way may be the rule rather than the exception. It is further suggested that when the relative activity of an X-chromosome
falls below a critical value, this becomes detectable cytologically by heterochromatinization. Absence of heterochromatinization may either mean that
the two Xs share their function more evenly, or that in a given cell or tissue, the
activity of the Xs is not complemental (being, perhaps, 70:70 or 100:100, as
with autosomal genes). Unlike the L.H. which requires that the n-\ rule be
obeyed in every cell, the complemental-X hypothesis is consistent with facultative heterochromatinization. Where the two Xs are cytologically distinguishable, as in the Indian gerbil (Rao, Shah & Seshadri, 1968) or in the mule
(Mukherjee & Sinha, 1964), it can be seen that they are involved randomly, as
also applies to some structurally abnormal chromosomes (e.g. Cattanach's
translocation; Evans et al. 1965) though usually structurally abnormal Xchromosomes are heterochromatinized selectively.
Sex-linked mammalian genes
169
Evidently, the cytological observations which have been regarded as such
strong support for the L.H. fit the complemental-X hypothesis at least as well.
Unlike heterozygotes for autosomal genes, those for sex-linked genes will
have cells in all complemental states of activity in mixture. Such a mixture may
reasonably be expected to be developmentally less stable than cells of uniform
physiological state. This presumably accounts both for mottled phenotypes (as
in brindled or flecked $?) and for more orderly patterns (such as the stripes and
dental anomalies of Ta/+ ?$ which probably reflect transverse wrinkles in the
embryonic skin and physiological conditions in the dental laminae, respectively).
The developmental instability inherent in the complemental-AT mechanism will
also lead to the manifestation of genes which, under the more stable conditions
of autosomes, would behave as recessives. This may account for the high proportion of sex-linked genes with heterozygous manifestation.
In an unstable situation a gene will manifest itself when certain critical conditions reach a threshold level; a swing in the opposite direction will, however,
be without effect on the phenotype. In conformity with observation, mutants
will thus tend to manifest themselves in less than a half of the total area. The
L.H. has to invoke ad hoc hypotheses to account for this fact, such as differential
growth following inactivation, etc.
There is no reason to suppose that the complemental state of the AT-chromosomes in a cell is fixed. Unlike the L.H., the complemental-A" hypothesis thus
has no difficulties in accounting for changes in phenotype, such as those with age.
As the complemental state of activity affects the chromosome as a whole,
genes carried in the same chromosome will tend to behave similarly. The complemental-X hypothesis thus immediately accounts for the cis-trans position
effect in Ta Mobr\ + + and Ta + / + Mobr ??. Indeed, similar cases which have
been claimed to support the L.H. (such as the phenotype of Ta + j + Str $$) in
reality support the complemental-A' hypothesis. Both hypotheses require that,
in the coat of repulsion double heterozygotes, the mutant phenotypes should
repel each other; but whereas the L.H. requires that such repulsion should be
complete, the complemental-^ hypothesis is compatible with the occurrence of
hairs showing neither mutant (which are indeed plentiful).
Equally important, whereas in the case of the Ta + j + Str$ the L.H. has to use
a circular argument concerning the potential phenotype of the lethal Str <J, the
complemental-A'hypothesis avoids that logical trap because it has to make no
such assumptions. Whereas, under the L.H., cis-trans position effects should
only occur if the two genes act through the same cells, there is no such restriction under the complemental-^ hypothesis as questions of cell lineage do not
enter into the argument.
In agreement with concepts developed by Russell (1964) from her studies of
yV-autosome translocations, the instability of gene manifestation spreads from the
point of interchange into attached segments of autosomes and peters out with
distance. Russell thus regards inactivation as incomplete, as a gradient rather
170
H. GRUNEBERG
than an all-or-none affair, a view which is much closer to the complemental-X
hypothesis than to the L.H.
Presumably, a state of complementarity as envisaged by the present hypothesis
involves some kind of feedback mechanism. It would scarcely be profitable to
speculate about its possible nature at this stage.
It appears, then, that all the known facts concerning the phenotypes of heterozygotes for sex-linked genes, the behaviour of double heterozygotes and of
Z-autosome translocations and the body of cytological observations concerning
the mammalian Z-chromosome can find a common explanation in the complemental-X hypothesis without introducing ad hoc assumptions to account for
discrepant facts.
Hemizygous manifestation in single-cell clones
There remains one group of phenomena which, on the face of it, does not
easily fit into the complemental-X hypothesis. In human glucose-6-phosphate
dehydrogenase (G-6-PD) deficiency (which is sex-linked), single-cell clones
obtained from heterozygotes manifest either one allele or the other, but not
both or neither (for a fuller discussion see Griineberg, 1967a), and two further
similar examples have recently been published. These facts have been widely
regarded as strong evidence in favour of the L.H.
The observations on the macroscopic and those on the cellular level will have
to be reconciled with each other. Thus the demonstration that the macroscopic
phenotypes cannot be accounted for in terms of clonal territories is no more
invalidated by observations on clones from heterozygotes than vice versa. It
will be shown that the facts can be reconciled in two different ways between which
a final decision does not appear possible at present. It will thus be necessary to
examine the consequences of both alternatives, and to suggest the types of
observations which may ultimately help to resolve the situation.
The question which has to be answered is whether or not the observations on
single-cell clones are relevant to the present inquiry. So far as I can see, there are
at least two conditions which must be met if the facts are to be regarded as
relevant. These are that the phenomenon is specific to the X-chromosome, and
that it is demonstrable in vivo (i.e. that it is not an in vitro artifact). It will be
shown that there is some doubt on both counts. A further desideratum is
evidence as to whether the clones fall sharply into two classes as required by the
L.H., or whether there are some intergrades, as required by the complemental-X
hypothesis. Here again, the evidence is not quite unambiguous.
Specificity for the J\f-chromosome requires that, on the cellular level, hemizygous manifestation should be demonstrable in heterozygotes for all sex-linked,
but not for autosomal genes. Despite claims to the contrary (see below), there is
so far no evidence for the clonal nature of hemizygous manifestation in the case
of the sex-linked Xga blood group system in man. Hemizygous manifestation in
individual lymphocytes certainly occurs in the autosomal gamma globulin
Sex-linked mammalian genes
171
genes in both man and the rabbit (Pernis, Chiappino, Kelus & Gell, 1965;
Oudin, 1966), but in the absence of clonal data, it cannot be decided whether
this represents merely cellular phenotypes or cell heredity (see below).
The case of Hurler's syndrome in man (Danes & Beam, 1967) is particularly
interesting because both autosomal and sex-linked forms of this mucopolysaccharidosis are available for comparison. After the lapse of about 15 cell
generations, fibroblast cultures from skin biopsies of heterozygotes (both autosomal and sex-linked) come to consist of two types of cells, normal ones and
cells which stain metachromatically with certain dyes and contain mucopolysaccharides like those from clinically affected children. Now, single-cell clones
derived from fibroblast cultures of autosomal heterozygotes always give rise
to mixed cell populations: evidently, the two cell types represent reversible
functional states. By contrast, some 90 % of clones derived from sex-linked
heterozygotes are either normal or abnormal (the remaining mixed colonies of
cells, according to the authors, are due to coalescence of neighbouring clones);
there are, however, consistently far too many abnormal and correspondingly too
few normal clones; this cannot be explained by cell selection, as in individual
mixed fibroblast cultures, the proportion remained constant over some 9 months
of cultivation.
The behaviour of the autosomal heterozygotes shows that two discrete cell
phenotypes do not necessarily represent separate clones. Hence all such claims
based on cell phenotypes alone are inconclusive: clonal continuity can only be
demonstrated directly by in vitro methods. (A recent example is a pedigree (Lee,
MacDiarmid, Cartwright & Wintrobe, 1968) with segregation both for the
sex-linked blood group gene Xga and a certain anaemia, said also to be JT-borne
on rather unconvincing evidence. In two double heterozygotes, normal and
anaemic cells could be separated by centrifugation and were found to differ also
as regards the blood-group antigen in question. Claimed as support for the
L.H., the published data do not prove the existence of two distinct clones of
erythroblasts: factor interaction is not ruled out, regardless of whether the
anaemia is sex-linked or autosomal.)
In neither form of Hurler's syndrome are two histologically distinct cell
populations demonstrable in vivo. They appear only after rather prolonged
cultivation in vitro. Does in vitro culture simply make a pre-existing difference
between cells manifest, or is the in vivo fibroblast population perhaps still
homogeneous? In the latter case, the in vitro happenings would not tell us much
about cell differentiation in vivo.
Clonal studies have also recently been carried out in A'-borne hypoxanthineguanine phosphoribosyltransferase (HGPRT) deficiency in man (Rosenbloom,
Kelley, Henderson & Seegmiller, 1967; Migeon et al. 1968; Salzmann, DeMars
& Benke, 1968). The last-named authors counted in fibroblast cultures from
heterozygotes 94-5 % + , 2-8 % - and 2-6 % ' ± ' cells. The latter have a moderate
enzyme activity; they occur regularly in fibroblast cultures from heterozygotes
172
H. GRUNEBERG
and in negative clones derived from such cultures, but not in either kind of
hemizygote. Single-cell clones from fibroblast cultures of heterozygotes included
43 % negatives (still significantly below 50 %). Perhaps many of the + cells in
fibroblast cultures are in reality — which have been cross-fed by + cells; but, as
they carry the normal allele of HGPRT deficiency, that might be an alternative
source of the enzyme activity. As the authors themselves realize, the' ± ' cells do
not properly fit into the L.H. : they present, of course, no difficulty to the complemental-X hypothesis. One starts to wonder, in this and similar cases, whether,
with more refined techniques, the present dichotomous classification into
'normal' and 'abnormal' clones might not prove to have been an undue degree
of simplification.
Ideally, proof of the orthotopic existence of clones in vivo is required. For
instance, whereas clones from cells in the amniotic fluid may be of diagnostic
value (Fujimoto, Seegmiller, Uhlendorf & Jacobson, 1968), they represent, in
essence, in vitro situations and hence they do not give critical evidence concerning the state of affairs in the foetus itself. Similarly, evidence from tumours
arising in heterozygotes (Linder & Gartler, 1965), while suggestive, cannot be
regarded as critical because a 100:0 or 0:100 situation may itself predispose a
cell to become the focus of a neoplasm. As most of the work on clones has of
necessity started from in vitro cultures, the claim that the findings represent the
in vivo situation is, perhaps, not quite as firmly established as has sometimes
been assumed.
The alternatives
More data on sex-linked and particularly on autosomal heterozygotes will be
required before one can decide whether hemizygous manifestation at the cellular level is specific for the Z-chromosome. Doubts as to whether the in vitro
behaviour of cells can be accepted to represent their in vivo behaviour can also
not be wholly dismissed. With present techniques, it is also by no means certain
whether clones from heterozygotes are in fact of two discrete types only.
Clearly, it would be unwise to accept everything at its face value because it
would fit the L.H. so well. Equally clearly, the facts available constitute a prima
facie case which cannot be dismissed out of hand.
Supposing that the facts on clones turn out to be irrelevant, or, if relevant,
that some of the clones turn out to be intermediate between the respective
hemizygotes: the complemental-Z hypothesis would then be consistent with
all the known facts. In particular,rthere would be no reason to invoke irreversibility of the physiological state of the Z-chromosomes in heterozygotes, as the
macroscopic facts speak against that concept and the cytological facts do not
discriminate for or against.
On the other hand, if the facts on clones turn out to be relevant and there are
no intermediate clones, an alternative hypothesis would have to be considered.
It has been postulated above that an intimate mixture of cells in all complemental
Sex-linked mammalian genes
173
states from 100:0 to 0:100 would be developmentally unstable, and that this
instability would account for the macroscopic behaviour both of the heterozygotes and of the X-autosome translocations. Perhaps a similar instability might
also be the result of an intimate mixture of 100:0 and 0:100 cells, but without
intermediates. This modification of the L.H. differs from the original version in
one important respect. Whereas the L.H. regards the macroscopic features of
heterozygotes as representing equally macroscopic clonal territories, the clonal
mixture here envisaged is of cellular dimensions and thus only indirectly (through
the ensuing instability) responsible for the macroscopic features. I have drawn
attention to the fact that the small biopsy specimens used to establish fibroblast
cultures generally include members of both types of clone (Griineberg, 1967 a);
this agrees well with the present hypothesis, but not with the L.H. in its original
form. The mode of origin of such a fine-grained mixture is not quite clear.
Klinger & Schwarzacher (1962) found that in an XYjXXY mosaic embryo
(obviously the result of a single event in an early cleavage division), abnormal
cells were widely distributed, but still tended to form fairly well-defined areas;
multiple clones of early origin would presumably lead to a finer intermixture of
cells. A very intimate dispersion would result if cellular dichotomy originates late
and perhaps continues to arise in adult tissues.
Pending more detailed and more critical information on clones, autosomal
and sex-linked, I am inclined to favour the complemental-X hypothesis. There is
one other major gap in our knowledge. It is clear that the macroscopic phenotype of sex-linked heterozygotes is not the expression of corresponding macroscopic clonal territories. But the concept that an intimate mixture of cells in
different physiological states is developmentally unstable, though plausible, so
far lacks an observational basis. Information may come from the study of
chimeras, whether produced by aggregation of whole embryos or by other
means (Gardner, 1968). It should not necessarily be taken for granted that the
phenotype of a chimera is wholly determined by cell lineage. Gene differences
which behave autonomously in larger aggregates of cells may not do so when
cells are intimately interspersed—a situation which can perhaps be brought
about in chimeras.
SUMMARY
A detailed study of the sex-linked genes for tabby (Ta), striated (Str) and
brindled (Mobr) and of an Z-autosome translocation in the mouse has shown
that in heterozygotes, both alleles interact with each other as in autosomal
genes. The phenotypes are thus due to threshold mechanisms, and not a patchwork of clones in which either one or the other Z-chromosome has been irreversibly inactivated in the sense of the Lyon Hypothesis (L.H.).
A complemental-^T hypothesis is put forward which assumes that, in the
mammalian female, the two Z-chromosomes jointly do the same work as the
single X in the male. There is no reason to suppose that a 50:50 situation is
174
H. GRUNEBERG
favoured, and wide deviations either way may be the rule rather than the exception, with 100:0 or 0:100 not necessarily excluded. As in heterozygotes cells in
all states of complemental action are present in mixture, they may be expected
to be developmentally unstable as compared with autosomal heterozygotes.
This is held to account for the high proportion of sex-linked genes with heterozygous manifestation, as well as for the mottled phenotypes common among
such genes. As the complemental state of activity affects the chromosome as a
whole, linked genes will tend to behave similarly and cis-trans position effects
in double heterozygotes may be expected, in conformity with observation. On
the assumption that relative inactivity of an Z-chromosome beyond a critical
point results (or may result) in visible heterochromatinization, the cytological
facts hitherto regarded as supporting the L.H. fit the complemental-X hypothesis
at least as well.
Data on single-cell clones from heterozygotes for certain sex-linked enzyme
defects in man (hitherto regarded as strong evidence for the L.H.) show certain
features which require more critical information. If this should go against the
complemental-Z hypothesis, a modification of the L.H. would have to be considered such that a microscopic rather than a macroscopic mosaic of two
distinct cell clones leads to a developmentally unstable situation similar to that
postulated for the complemental-X hypothesis.
ZUSAMMENFASSUNG
Schwellen-Mechanismen als Erklarung der Manifestation
geschlechtsgebundener Gene bei Sdugern
Eine eingehende Untersuchung der geschlechtsgebundenen Gene fur tabby
(Ta), striated (Str) und brindled (Mobr) und einer X-Autosom-Translokation
bei der Maus zeigt, dass bei Heterozygoten beide Allele gleichzeitig wirksam
sind. Wie auch bei autosomalen Genen sind also die Phanotypen das Resultat
von Schwellen-Mechanismen: sie sind nicht ein Nebeneinander von Klonen, in
denen das eine oder das andere der beiden X-Chromosomen im Sinne der
Lyon-Hypothese (L.H.) irreversibel ausser Funktion gesetzt sind.
Zur Erklarung des Verhaltens geschlechtsgebundener Gene bei Saugern wird
eine Hypothese komplementarer Wirkung der Z-Chromosomen vorgeschlagen,
nach der beim Weibchen die beiden X-Chromosomen zusammen die gleiche
Wirkung wie das alleinige X-Chromosom beim Mannchen entfalten. Es besteht
kein Grund zu der Annahme, dass eine 50:50 Situation bevorzugt ist: erhebliche
Abweichungen in beiden Richtungen sind vermutlich haufig, und 100:0 oder
0:100 ist nicht unbedingt ausgeschlossen. Da bei Heterozygoten Zellen in alien
komplementaren Funktionszustanden in Mischung vorhanden sind, muss man
erwarten, dass sie in der Entwicklung verglichen mit autosomalen Genen sich
unstabil verhalten werden. Dies ist vermutlich verantwortlich fur die vielen
geschlechtsgebundenen Genemit Manifestation imheterozygoten Zustand, wie
Sex-linked mammalian genes
175
auch fur die unregelmassig gefleckten Phanotypen, die unter solchen Heterozygoten hauftg sind. Da der komplementare Funktionszustand das Chromosom
als ganzes betrifft, werden gekoppelte Gene dazu neigen, sich ahnlich zu
verhalten, und bei Doppel-Heterozygoten sind m-frTms-Positionseffekte zu
erwarten, was ja auch der Fall ist. Wenn man annimmt, dass bei relativer
Unterfunktion jenseits eines kritischen Schwellenwertes das X-Chromosom
heterochromatinisiert wird, so passen die bisher im Sinne der L.H. gedeuteten
cytologischen Befunde mindestens so gut in den Rahmen der komplementarenA'-Hypothese.
Befunde an Ein-Zell-Klonen von Heterozygoten fiir gewisse geschlechtsgebundene Enzymdefekte beim Menschen sprechen weniger eindeutig fur die
L.H. als bisher angenommen worden ist. Falls die erforderlichen kritischen
Befunde gegen die komplementare-Z-Hypothese sprechen sollten, muss eine
Modifikation der L.H. erwogen werden, nach der ein zellulares Mosaik von
Klonen eine unstabile Situation bedingt ahnlich der, die fiir die andere Hypothese angenommen werden muss.
The author is indebted to Miss Rita J. S. Phillips (Harwell) for the gift of the striated stock
used in this paper, to Miss Beryl F. Fannon for technical assistance, to Mr A. J. Lee for the
illustrations, to Mrs G. Javeri for secretarial assistance, and for aid in various ways to
Dr Gillian M. Truslove.
POSTSCRIPT (9 JANUARY 1969)
Since this paper was written, Lyon (1968) has replied to some of the criticisms of the L.H. made by the present author (19666, 1967a, b). We are told
that inactivation leads to two types of cells about equal in number and random
in distribution, but that this original situation tends to get distorted later on
as the result of unequal cell multiplication, cell mingling, regularities of cell
lineage, non-autonomous or not fully autonomous development and other causes.
Is this not an attempt to have it both ways ? If the heterozygotes conform to the
original situation, this would undoubtedly be regarded as evidence in favour of
the L.H.; if not, the discrepancies can be 'explained' by various ad hoc assumptions which are introduced, tailor-made, as and when required. However, to
quote (in translation) from a more recent paper (G., 1968), 'every subsidiary
hypothesis is in reality an admission that the observed facts do not agree with
the main hypothesis. Each subsidiary hypothesis is thus in a sense a promissory
note to the effect that, on closer study, the discrepancy will be found to be
apparent rather than real. But, like monetary debts, subsidiary hypotheses must
ultimately be redeemed: one cannot go on making use of them without ultimately putting them to the test. For the above-mentioned subsidiary hypotheses
this has so far not been done in any single case.'
Lyon maintains that regularities of cell lineage following random inactivation could lead to some degree of orderliness of pattern. That concept was
discussed in detail in connexion with the behaviour of the molars in Taj + ??
176
H. GRUNEBERG
(G., 1966a), and it was shown that correlations between upper and lower jaw
and between right and left could not be so explained. Lyon neither discusses the
facts nor does she attempt to refute the argument. To account for the orderly
transverse stripes of Taj + °.°., she appeals to the metameric arrangement of the
dermatomes and postulates that these form transverse stripes of similarly
metameric hair follicles. There is no evidence whatsoever for this concept; on the
other hand, the transverse structure of the embryonic skin (G., 19666) is not
mentioned though it is there for everybody to see. The bearing of large uniformly
intermediate areas (like the tail rings in Taj + $$; G. 19666) is also ignored, as is
that of polarized hairs (in the flecked mice of Cattanach's translocation, G.,
19676; and in Mobrl+ $$, this paper). I had argued (G., 19666, 1967a) that a
pattern due to inactivation should be refractory to selection; Lyon counters this
with the argument that the intensity of manifestation (expressivity) of sexlinked genes may be under the influence of modifiers like that of autosomal
genes: this is true, but the same does not apply to the relative size of the contrasted areas (penetrance), and it is the effect of selection on the latter which
contradicts the L.H. (such as on the width of the stripes in Ta\ + $$, etc.). It is
altogether irrelevant for the present discussion that, as usual, penetrance and
expressivity tend to go together.
Turning to questions of pure logic, Lyon completely ignores my argument
that, unless both hemizygotes are known, a heterozygote cannot legitimately be
interpreted in terms of the L.H. None the less, the Str/ + ? again figures as supporting her hypothesis. Lyon incorrectly tries to saddle me with the statement
that 'autosomal genes and sex-linked genes should not show similar variegated
effects, since X-inactivation was postulated to be a dosage compensation
mechanism, and such a thing would not be expected in autosomes'. My very
point was that autosomal and sex-linked genes do give rise to similar phenotypes, but that they cannot legitimately be explained by different mechanisms on
that account alone.
To say, as Lyon does, that' Griineberg's criticisms of the genetic evidence for
X-inactivation can be answered' is one thing; to do so is quite another: she
herself has certainly failed in this all along the line.
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Genetics 11, 333-51.
(Manuscript received 13 January 1969)