/ . Embryol. exp. Morph. Vol. 36, 1, pp. 151-161, 1976
\$\
Printed in Great Britain
Patches in the livers of chimaeric mice
By J. D. WEST1
Department of Genetics, University of Edinburgh, Scotland
SUMMARY
Sections of adult chimaeric livers have been histochemically stained for /?-glucuronidase
activity and patches of two cell populations visualized. A one-dimensional clonal analysis has
been used to estimate the number of coherent clones in the adult liver. The data are consistent
with a total of 9-22 million regular, coherent clones, comprising 10-34 nuclei, or a smaller
number of irregular, branched coherent clones. Both of these alternatives suggest considerable cell mixing during liver morphogenesis.
INTRODUCTION
The presence of two genetically marked cell populations in a single mouse
aggregation chimaera has been used to investigate clonal development in several
different tissues (for review see Mintz, 1971). From these studies and from work
with human X-inactivation mosaics (Gartler et al. 1971), the concept of limited
coherent clonal growth has emerged (Nesbitt, 1974).
If cell mixing is very extensive, growth will be non-clonal whereas, if no cell
mixing takes place at all, strict coherent clonal growth will occur and chimaeric
tissues will comprise large continuous clones with smooth outlines as reported
for a number of insect systems (Schneiderman & Bryant, 1971; Garcia-Bellido,
Ripoll & Morata, 1973; Lawrence, 1973). If cell mixing takes place but is
insufficient to randomize the distribution of the two marked cell populations
completely, limited coherent clonal growth will occur and result in the formation of coherent clones which will be either relatively small or very irregular in
shape.
Nesbitt (1974) has defined a coherent clone as 'a group of clonally related
cells which have remained contiguous throughout the history of the embryo'
and the same author defines a patch a s ' a group of cells of like genotype which
are contiguous at the moment of consideration'. A patch, therefore, may comprise several adjacent coherent clones.
The two cell populations in the livers of chimaeric mice can be identified using
biochemical markers. Two studies on the distributions of the two cell types in
chimaeric livers have been reported and both make use of a low activity mutant
(Gush) of the enzyme /?-glucuronidase.
1
Present address: Department of Molecular Biology, Roswell Park Memorial Institute,
Buffalo, New York, 14263, U.S.A.
152
J. D. WEST
Wegmann (1970) investigated the distribution of the two cell populations in
C57BL/10SnJ<-> C3H/HeJ chimaeras, using a biochemical assay for /?-glucuronidase activity. He found considerable variation in specific activity between
small samples of the same liver, which suggested a patchy arrangement of the
two cell types. Wegmann used a statistical analysis based on the variance between samples to estimate a mean of 17-2 'clones' per sample. As each sample
was approximately one-twentieth of the liver, this represents an estimate of
about 344 ' clones' per liver.
A more direct histochemical approach was used by Condamine, Custer &
Mintz (1971) in their analysis of C3H/HeNIcr •-> BALB/cAnNIcr and
C3H/HeNIcr <-> C57BL/6JNIcr chimaeras. This work was primarily concerned
with chimaeric liver tumours, but a number of basic observations were made on
normal chimaeric liver tissue. Patches of each cell population were visualized
using /?-glucuronidase histochemistry and each lobe often contained cells from
both populations. The authors speculated that the liver clonal number may be
'rather large', but a systematic analysis of liver patches was not reported.
The work presented below represents an attempt to investigate the degree of
cell mixing during the development of the mouse liver by analysing the distribution of two cell populations in mouse aggregation chimaeras, using the
/?-glucuronidase marker and histochemical techniques.
MATERIALS AND METHODS
(a) Mice
Chimaeric mice were produced by aggregating two eight-cell morulae
(McLaren & Bowman, 1969). Mice of the C3H/BiMcL strain were used as the
source of the low activity Gush allele and either C57BL/McL or a multiple
recessive stock designated 'Recessive' (see McLaren & Bowman, 1969) were
used as a source of normal /?-glucuronidase activity. (The allele responsible
for normal activity will be referred to as Gush for convenience, although one or
both stocks may carry the alternative, normal-activity allele Gwsa, which can
only be distinguished from Gus^ by electrophoresis.)
(b) Histochemical methods
Small pieces of fixed tissue were sectioned on a freezing microtome and the
sections were stained for /?-glucuronidase for up to three hours as described by
Hayashi, Nakajima & Fishman (1964). The sections were then rinsed in water,
counterstained for l-|min in methyl green (0-5% in 0-1 M veronal acetate
buffer), rapidly dehydrated in graded alcohols, cleared in xylene and mounted
in DPX. Initially the sections were cut at 7-5 /mi, but this was later increased to
10 jam to improve the contrast in staining intensity.
Liver patches in chimaeric mice
153
(c) Liver analysis
The liver of the mouse most commonly consists of four primary divisions or
lobes: a left lateral, a medial, a right lateral and a posterior - and apart from the
left lateral, each is in turn subdivided into two main lobes. (See, for example,
Danforth & Center, 1953.) For the purposes of this study the four major lobes
will be designated lobes 1, 2, 3 and 4 in the above order. These four lobes
normally comprise 34-40 %, 31-37 %, 17-25 % and 5-10 % of the total liver
weight, respectively.
Each chimaeric liver was divided into its four major lobes and two samples
taken from widely separated regions of each lobe. Several sections were cut and
two slides prepared from each sample. One section per slide was selected for
examination. Thus, normally 16 sections were examined from each chimaeric
liver. The only exception to this was chimaera XQ3, where lobe 4 was missing
and so only 12 sections were studied. Control slides were prepared from livers of
inbred C3H/BiMcL and C57BL/McL mice.
Sections of chimaeric liver were analysed in an attempt to determine the mean
size and number of coherent clones in the adult tissue. Two types of clonal
analyses were attempted using histochemically stained sections of chimaeric
liver. Both analyses remove any variation in patch size due to differences in
proportions of the two cell populations. First, a one-dimensional analysis was
used, similar to that described for the retinal epithelium (West, 1976). The patch
lengths were measured along a random line across the section, using a microscope fitted with a calibrated eyepiece micrometer. These data were used to
calculate the mean patch length and the proportion (p) of each cell type, from
which the mean coherent clone length was estimated, as described below. Second
a two-dimensional analysis was attempted using a modified Chalkley grid (see
Curtis, 1960) to measure the areas of patches and the proportion of each cell
type (p) over the entire section. This data was used to estimate the coherent clone
area.
In the one-dimensional analysis the mean coherent clone length was estimated
by dividing the measured mean patch length by 1/(1 — p) as described previously
(West, 1975, 1976). In the two-dimensional analysis the mean length of the
coherent clones was taken to be the square root of the mean area. This mean
coherent clone area was calculated as the mean patch area divided by the antilog10 of (l-48p + 0-3708)3, which is the empirical relationship found for a model
100x100 array (West, 1975). It proved difficult to measure discrete patches
unless p was less than 0-2 and so the two-dimensional analysis was only applied
to fifteen of the 76 sections examined.
The mean coherent-clone size estimated here by these two methods is a
statistical estimate and represents the mean size of groups of cells which would
produce the observed mean patch size if distributed at random. Both of these
analyses make the rather unrealistic assumption that coherent clones are regular
154
J. D. WEST
Liver patches in chimaeric mice
155
in shape, and the two-dimensional relationship was derived specifically for
hexagonal coherent clones (West, 1975). The derived mean size of the coherent
clones will therefore be a minimum estimate, as the size of irregular, highly
branched coherent clones will be underestimated. However, this estimate still
yields information about the amount of cell mixing: a small estimate of coherent
clone size suggests either small regular coherent clones or larger, highly branched
ones. Both of these possibilities imply considerable cell mixing during liver
morphogenesis.
(d) Liver growth and properties
Several parameters were measured in non-chimaeric livers for use in the clonal
analysis. The use of data from non-chimaeric mice in the analysis introduces a
small error but it was impracticable to use chimaeric tissues for these preliminary
studies. Growth curves were constructed for (C57BL x C3H)FX embryos from
\2\ days post coitum and for (C57BLxC3H)F 1 males after birth. Two litters
were examined separately for each embryonic age considered and the embryos
within a litter were pooled to determine the number of nuclei per liver. Six male
mice were used for each age considered after birth and, apart from the 210-day
values, the six mice were taken from at least two different litters in each case.
The liver weight and the number of nuclei per liver were measured and, for postnatal stages, determinations were made on individual mice. The number of
nuclei was estimated by homogenizing the liver in a known volume of 0-01 N-HC1,
in a glass homogenizing tube using a Teflon TRI-R electric homogenizer, and
counting nuclei in a haemocytometer as described by Zumoff & Pachter (1964).
The specific gravity of fresh liver, and the percentage shrinkage after fixation
in formal-calcium, were estimated using a specific-gravity bottle.
RESULTS
After staining for /?-glucuronidase activity, and counter-staining with methyl
green, all the liver cells of adult C57BL, Recessive and other Gw5b-carrying strains
of mice showed an intense red/brown cytoplasmic staining, as did Gus*>lh heterozygotes. Similarly treated adult C3H livers, homozygous for Gus]\ showed no
red/brown staining for /?-glucuronidase in the liver cells, although this was
detectable in blood cells present in the livers of these animals. Although the
liver is a complex organ of several different cell types (Elias & Sherrick, 1969),
with the conditions used the staining intensity appeared similar in hepatocytes
throughout non-chimaeric livers. This is important for the study of chimaeric
Fig. 1. Sections of liver, histochemically stained for /?-glucuronidase activity and
counterstained with methyl green. (a)C3H/BiMcL; 7-5 /*m section, (b) C57BL/McL;
7-5/Am section, (c) Chimaera X34 (C3H/BiMcL <-> Recessive), lobe 2; .10 /*m
section, (d) Chimaera X34 (C3H/BiMcL <-» Recessive), lobe 4; 10 /*m section. In
each case the horizontal bar represents 50/tm.
156
J. D. WEST
Fig. 2. Ten /<m section of lobe 3 of X35 (C3H/BiMcL <-> Recessive) chimaeric liver,
histochemically stained for /?-glucuronidase activity. The horizontal bar represents
100 jim. The dark areas were predominantly red/brown and the light areas were
mainly green in the original section.
Liver patches in chimaeric mice
157
livers using this histochemical technique, and is in marked contrast to kidneys
which show an uneven distribution of /?-glucuronidase activity, even in sections
of C3H kidneys which have a low overall enzyme activity.
Chimaeric livers have a patchy distribution of two cell types, as shown in Figs.
1 and 2, and although individual cells sometimes appear to be isolated from
Table 1. Results from one-dimensional clonal analysis of liver, with figures from
two-dimensional analysis shown in parentheses
Chimaera
C3H <-> C57BL
XQ3
XQ9
XQ12
XQ18
C3H <-> Recessive
X34
X35
Age
(days)
Liver
wt(g)
499
3-33
495
478
203
1-95
521
—
331
1-53
274
1-25
Estimated no. Mean
Mean
coherent
of coherent of nuclei
per
clone length clones per
Mean
proportion (Gus* & Gus* )
liver
coherent
(/tm)
ofC3H(p)
(x!0 G )
clone
0-84
(0-80)
0-71
(0-69)
0-23
(0-25)
100
(100)
0-40
(0-40)
0-36
(0-40)
14
34
19
15
9
31
—
—
—
39
(-)
43
(-)
22
10
13
14
58
(52)
44
(30)
57
(96)
others of like phenotype, this could be an artifact of sectioned material. The
identification of isolated cells of both phenotypes in the sections strongly suggests
that diffusion of enzyme or product is negligible under the conditions employed.
This conclusion is reinforced by another observation: when C57BL and C3H
liver sections are thawed on to the same slide, so that one partially overlaps the
other, and then stained for /?-glucuronidase activity, the red/brown cytoplasmic
staining is restricted to the C57BL section. Therefore, /?-glucuronidase provides a
suitable cell-autonomous marker for investigating the mean size of coherent clones
in the adult liver, and the results of the clonal analyses are presented in Table 1.
Five of the six chimaeras studied proved to have two cell populations in their
livers and in each case both cell populations were present in all four lobes.
Comparisons of the two types of clonal analysis showed a strong positive
correlation for both the proportion oftheC3H component (r = 0-93, P < 0-001,
n = 76) and the coherent clone length (r = 0-78, P < 0-01, n = 15). However,
although a t test on the paired comparisons showed no significant difference in
the data obtained by the two methods for relative proportions (t = 0-34,
P > 0-05, n = 76), the two-dimensional analysis gave a significantly higher value
(t = 2-48, P < 0-05, n = 15) for the mean coherent clone length (81 ± 13 /an)
158
J. D. WEST
0
12 5 19-5
Days p.c.
0
20
40
60
SO
100
210
Days after birth
Fig. 3. Plot of number of nuclei per liver with age for (C57BL/McL x C3H/BiMcL)F1
mice. (The peak 3 days before birth represents maximum haematopoietic activity
of the embryonic liver.) The embryonic data is taken from two litters for each age
group and the post-natal data represents means and standard errors for six males in
each group, (p.c. = post coitum.)
20
40
60
Davs after birth
Fig. 4. Plot of weight of liver per nucleus with age after birth. Each point represents
the mean of six male (C57BL/McL x C3H/BiMcL)F! mice and the vertical bar
represents the standard error of the mean.
Liver patches in chimaeric mice
159
than did the one-dimensional analysis (59 ± 7 ^m) for the fifteen pairs of data.
Since the two-dimensional analysis was only possible on a small proportion of the
material, subsequent analysis is based on data derived from the one-dimensional
method.
No significant difference in mean one-dimensional coherent clone length was
found between the four liver lobes, as tested by an analysis of variance CF3)7o =
0-89, P > 0-05), so in the analysis of the data the lobes are pooled and mean
coherent clone sizes for each liver are used.
Table 1 shows the mean proportion of C3H (p) and the mean coherent clone
length determined by the one-dimensional analysis, with the corresponding
figures from the two-dimensional analysis given in parentheses below. The threedimensional coherent clone volume is estimated as the cube of the coherent clone
length. The number of nuclei per coherent clone and number of coherent clones
per liver is estimated from the liver weights and data from non-chimaeric livers.
The volume of the fixed liver is calculated taking into account both the specific
gravity (1-08) and the expected shrinkage (9 %). Figs. 3 and 4 show that the
weight of liver per nucleus remains fairly constant at about 7 ng per nucleus
after 60 days from birth, although the liver weight and the number of nuclei may
continue to increase.
Using this information it is estimated that the adult liver contains a maximum
of 9-22 million regular coherent clones each comprising about 10-34 nuclei (see
Table 1), or a smaller number of highly branched coherent clones. The number of
cells per coherent clone will be smaller than the number of nuclei, as the adult
liver contains about 55 % binucleate cells (Epstein, 1967; Wheatley, 1972).
DISCUSSION
The results from the chimaeras studied suggest that the adult mouse liver
comprises either an extremely large number (several million) of very small,
regular coherent clones, or a smaller number of irregular, coherent clones. Both
of these alternatives imply considerable cell movement during liver morphogenesis.
Some limitations of this type of analysis, based on non-serial sections, have
already been discussed. Other possible problems include the possibility of differences in clonal growth between chimaeras of different strain combinations,
due perhaps to differences in liver growth, differences in the adhesiveness
between cells of different genotypes, or to cell selection.
It is difficult to compare the results from the present analysis with those of
Wegmann (1970). The numerical validity of both analyses assumes a fairly
regular shape of either the coherent clones or the patches. The patches seen in
histochemically-stained sections (Fig. 2) are clearly highly branched and irregular and this may also be true for the coherent clones. Clearly there is a marked
difference between the present estimate of several million coherent clones per
160
J. D. WEST
adult liver and the estimate of 344 clones implied from Wegmann's experiments.
Wegmann's estimate is based on variation of relative proportions of the two
cell types in small three-dimensional samples taken from three chimaeric livers.
Such variation may be due to the grouping of the two cell populations into
patches as illustrated in Fig. 2 and the data probably refer to patches and not to
coherent clones. Wegmann's estimates probably depend both on patch size and
patch shape. As previously shown (West, 1975), the patch size depends partly on
the relative proportions of the two cell types and so has little general significance
and cannot easily be used to assess the degree of cell mixing.
The present results suggest that considerable cell mixing occurs during liver
morphogenesis. The pattern of patches seen in the chimaeric livers may have
resulted from strict coherent clonal growth following a period of non-clonal
growth earlier in development, but it seems more likely that limited coherent
clonal growth occurs during liver morphogenesis. If so it is likely that limited
coherent clonal growth begins before the liver comprises 9-22 million nuclei,
assuming cell death does not significantly reduce the number of coherent clones
during development. The data shown in Fig. 3 and the data of Silini, Pozzi &
Pons (1967) and Bateman, Cole, Regan & Tarbutt (1972) thus argue for the
onset of limited coherent clonal growth before 13-14 days post coitum.
The coherent clone size has been estimated in two other mammalian tissues,
and in each case the coherent clones comprise surprisingly few cells. Gartler
et al. (1971) estimated 12-60 cells per coherent clone in the scalp epidermis of
foetal human X-inactivation mosaics, and West (1976) estimated 5-9 nuclei per
coherent clone in the retinal epithelium in adult mouse X-inactivation mosaics
and chimaeras. This evidence for so much cell mixing in mammalian development
is in complete contrast to the situation in insect mosaics where the adult clones
are large, continuous and have smooth outlines which suggests 'that there
is little individual cell movement in these systems' (Schneiderman & Bryant,
1971).
The evidence for cell mixing quite late in mouse development appears to be in
conflict with the phenomenon of contact inhibition of movement seen in monolayer cultures (Abercrombie & Heaysman, 1954). However, evidence from threedimensional tissue explants shows that individual cells can migrate through a
solid tissue mass (Wiseman & Steinberg, 1973; Armstrong & Armstrong, 1973).
I wish to thank Dr Anne McLaren for her help, supervision and encouragement. I am also
indebted to Drs Anne McLaren, Patricia Bowman and Mrs Janet Carter for providing the
chimaeras used in this study, to Mr B. Doyle for reliable technical help and to Dr W. K.
Whitten of the Jackson Laboratory, Bar Harbor, for helpful discussion. I am grateful to the
Ford Foundation and to the Science Research Council for financial support for this work.
Liver patches in chimaeric mice
161
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