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J. Embryol. exp. Morph. 73, 179-191, 1983
Printed in Great Britain © The Company of Biologists Limited 1983
279
Identification of embryonic cell lineages in
histological sections of M. musculus ** M. caroli
chimaeras
By J. ROSSANT 1 *, M. VIJH 1 , L. D. SIRACUSA 2 , AND
V. M. CHAPMAN2
From the Department of Biological Sciences, Brock University, St. Catharines,
and the Department of Molecular Biology, Roswell Park Memorial Institute,
Buffalo
SUMMARY
An in situ cell marker system has been developed which allows identification of Mus caroli
and Mus musculus cells in interspecific chimaeras. A radioactively labelled, cloned DNA
probe to M. musculus satellite DNA was hybridized in situ to sections of M. musculus and M.
caroli adult tissues. Autoradiography revealed high levels of hybridization to the nuclei of M.
musculus cells, but little or no label bound to M. caroli cells. The DNA probe could also
distinguish M. musculus and M. caroli cells in the same tissue section. Patches of labelled and
unlabelled cells were clearly identified in sections of adult chimaeric tissues and also in the
embryonic ectoderm of 6-5-day embryonic chimaeras.
The ability to recognize M. musculus and M. caroli cells in sections of chimaeras should
provide a powerful new tool in analyses of cell lineages in both embryonic and adult mouse
chimaeras. The marker system has several advantages over other marker systems so far
developed, the most important of which is its ubiquity. Since it is a nuclear marker, only cells
without nuclei should be unsuited to its use. The potential of the marker system has been
shown by its use in demonstrating directly for the first time the postimplantation derivatives
of inner cell mass and trophectoderm in blastocysts 'reconstituted' with M. musculus trophectoderm and M. caroli inner cell mass.
INTRODUCTION
The use of mammalian chimaeras for detailed cell lineage analysis in development has been limited by the lack of a suitable ubiquitous in situ cell marker
system (McLaren, 1976; Gearhart & Oster-Granite, 1978). In the preceding
paper (Siracusa et al., 1982), it has been shown that a marker system exists which
may allow identification of the two component cell types in interspecific
chimaeras between Mus musculus and Mus caroli. A cloned DNA probe to M.
1
Authors' address: Dept. of Biological Sciences, Brock University, St. Catharines, Ontario,
Canada.
2
Authors' address: Dept. of Molecular Biology, Roswell Park Memorial Institute, Buffalo,
N.Y., U.S.A.
* To whom reprint requests should be sent.
180
J. ROSSANT, M. VIJH, L. D. SIRACUSA & V. M. CHAPMAN
musculus satellite DNA was hybridized in situ to M. musculus and M. caroli cell
spreads and hybridization was detected by autoradiography. This particular
satellite DNA sequence showed little cross hybridization with M. caroli
repetitive DNA sequences so that M. caroli cells appeared virtually unlabelled
after in situ hybridization. The probe was able to distinguish M. caroli and M.
musculus cells in bone marrow preparations from interspecific chimaeras. However, to be truly useful as a marker system, it must be able to distinguish M. caroli
and M. musculus cells in histological sections of chimaeric tissue. In this study
we describe extension of the technique of in situ DNA-DNA hybridization to
sectioned material and demonstrate that the cloned probe to M. musculus
satellite DNA can be used to distinguish M. musculus and M. caroli cells in
sections of adult and embryonic chimaeric tissue. The marker system was also
used experimentally to demonstrate directly for the first time the postimplantation derivatives of the inner cell mass (ICM) and trophectoderm in blastocysts
'reconstituted' with M. musculus trophectoderm and M. caroli ICM.
MATERIALS AND METHODS
Embryo collection and manipulation
Mus musculus blastocysts were obtained by flushing the uteri of Ha/ICR
female mice on the afternoon of the fourth day after natural mating. Mus caroli
blastocysts were obtained following hormonal induction of ovulation and natural
mating as described previously (Rossant & Frels, 1980). Embryos were placed
in PB1 medium (Whittingham & Wales, 1969) plus 10 % foetal calf serum for
manipulation and transfer. M. caroli blastocysts were subjected to immunosurgery (Solter & Knowles, 1975) and the resulting ICMs were injected
microsurgically into either M. musculus blastocysts or M. musculus trophectoderm vesicles. Trophectoderm vesicles were obtained by slitting the zona
pellucida overlying the ICM and allowing the ICM plus its overlying polar
trophectoderm to extrude during a period of culture at 37 °C (Papaioannou,
1981). When the host ICM was clearly outside the zona, a M. caroli ICM was
injected into the trophectoderm vesicle still enclosed by the zona. The last stage
of the procedure, which involved cutting off the extruded ICM, was performed
by hand using glass microneedles under the dissecting microscope and not by
micromanipulation as described previously (Papaioannou, 1981). Both 'reconstituted' blastocysts and injected blastocysts were allowed to recover for an hour
before transfer to pseudopregnant recipients.
Embryo transfer and development
M. caroli blastocysts, M. musculus blastocysts injected with M. caroli ICMs,
and blastocysts reconstituted with M. musculus trophectoderm and M. caroli
ICM were transferred to the uteri of Ha/ICR females on the third day of
Embryonic cell lineages in Mus chimaeras
181
pseudopregnancy. Some females carrying injected blastocysts were allowed to
go through to term. Live interspecific chimaeric offspring were recognized by
coat colour and glucose phosphate isomerase (GPI) mosaicism (Rossant & Frels,
1980). Other females carrying all three types of embryo were killed at 6-5 days
of pregnancy. Uteri containing decidua were removed and fixed in glacial acetic
acid: absolute ethanol (1: 3) at 4°C overnight.
Processing of tissues for hybridization
Various small pieces of tissue from adult M. musculus, M. caroli and chimaeric
animals were fixed as described for embryonic tissue. All tissues were then rinsed
in two 30 min changes of absolute ethanol at 4 °C, transferred to a 50:50 mixture
of ethanol and ester wax (BDH1960) in a 50 °C water bath for 1 h, infiltrated with
two 1 h changes of ester wax and embedded in the wax at 50 °C. Blocks were
cooled and sectioned at 7/im. A few serial sections were mounted on chromicacid-cleaned glass slides. Sections were heated to 40 °C for the minimum time
required for section spreading and then allowed to dry for a few hours at room
temperature. Care was taken throughout to ensure that slides remained clean
and free of dust.
In situ DNA-DNA hybridization
Conditions for in situ DNA-DNA hybridization on sectioned material were
essentially similar to those described for cell spreads (Siracusa et al., 1982),
except that only heat denaturation was used as alkali denaturation usually
removed the sections from the slides. Slides were dewaxed in xylene, washed in
ethanol and air dried briefly before heat denaturation and hybridization overnight. The slides were then washed in 2xSSC to remove non-specifically bound
DNA, dehydrated, air dried and dipped in Kodak NTB-3 emulsion diluted 1:1
with water. Autoradiographs were exposed for five days and then developed for
1-5 min in Kodak D-19 developer, rinsed in distilled water and fixed for 5 min in
Kodak fixer. After washing with distilled water, slides were stained with
haematoxylin and eosin.
RESULTS
In situ hybridization ofM. musculus satellite DNA sequence to histological
sections
In situ hybridization of the cloned M. musculus satellite sequence to histological sections of M. musculus adult tissues was successfully achieved. The
procedure outlined resulted in specific binding of the probe to the nuclei of cells
from a variety of adult tissues, including kidney, liver, uterus, and brain (Fig. 1).
There was always some background labelling in the cytoplasm, which varied
between experiments. However, most label was clearly localized to the nucleus
182
J. ROSSANT, M. VIJH, L. D. SIRACUSA & V. M. CHAPMAN
and often showed patches of intense labelling as observed previously in interphase
nuclei hybridized with satellite DNA in cell spreads (Pardue & Gall, 1970; Singh,
Purdom & Jones, 1977; Siracusa et al., 1982). The amount of label bound was
visually similar to the level observed when the same probe was hybridized to cell
spreads (Siracusa et al., 1982), but there was more variation between nuclei
because of the varying amounts of DNA exposed in the plane of the section. When
the probe was hybridized to adult M. caroli tissue, only background labelling was
observed; no nuclear localization of label was apparent (Fig. IB).
^^1^1-
Fig. 1. Autoradiographs of sections of adult tissues from M. musculus and M. caroli
after hybridization with pMR196. (A) M. musculus uterus, showing nuclear localization of label; (B) M. caroli kidney, snowing only background labelling. Grid bar on
this and succeeding figures = 50fim.
Embryonic cell lineages in Mus chimaeras
183
The low level of binding to M. caroli cells suggested that misidentification of
M. caroli as M. musculus in chimaeric material would be unlikely. However,
occasional classification of a M. musculus cell as unlabelled and hence M. caroli
might occur. Several counts of labelled versus unlabelled nuclei were made from
photographs of M. musculus tissues after in situ hybridization. Nuclei were
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Fig. 2. Autoradiographs of sections of adult chimaeric tissues after in situ hybridization. Patches of labelled and unlabelled cells clearly visible. Diagrams illustrate
patchiness by scoring all labelled nuclei with a solid dot (•) and all unlabelled nuclei
with open dot (O). (A) chimaeric liver; (B) chimaeric brain.
184
J. ROSSANT, M. VIJH, L. D. SIRACUSA & V. M. CHAPMAN
scored as unlabelled if there were fewer than five grains over the nucleus. This
represents a very conservative estimate since M. caroli tissues never showed this
high a level of labelling. An average of 8 % unlabelled cells (range 3-14 %) was
observed in all tissues analysed. In most cases, nuclei scored as unlabelled were
clearly sectioned tangentially and might have appeared labelled in succeeding
sections.
Identification ofM. musculus and M. caroli cells in adult chimaeric tissue
Tissues from adult interspecific chimaeras that showed fairly balanced GPI
mosaicism were used for in situ hybridization. A mixture of labelled and unlabelled nuclei was evident in sections from liver and cerebral hemispheres (Fig.
2). In both tissues, the patch sizes of the two species types were small. The
distinction between labelled and unlabelled cells was particularly evident in the
brain tissue, where nuclei were widely spaced (Fig. 2B). Counts of labelled
versus unlabelled cells in random samples of the tissues gave M. caroli : M.
musculus ratios similar to those calculated from quantitative GPI analysis of
other samples of the same tissues from the same chimaera (Table 1).
In situ hybridization on embryonic cells
An initial experiment revealed that the M. musculus cloned sequence could be
used to distinguish M. musculus and M. caroli blastomeres in cell spreads from
aggregation chimaeras showing that, as expected, hybridization was equally
effective on embryonic and adult tissue. Hybridization of the probe to sectioned
Table 1. Comparison of mosaicism estimated by GPI analysis or by in situ
DNA-DNA hybridization in adult interspecific chimaeric tissues
Mean % unlabelled
M. caroli nuclei
% M. caroli
Tissue
± S.E.
by GPI
Brain
Liver
Kidney
62 ± 2-8
34 ±3-4
36 ± 2-2
58
21
34
M. musculus embryos at 6-5 days was also successful (Fig. 3A). In fact, the large
size of the nuclei and the distinctness of their boundaries, as well as the epithelial
arrangement of many cell types, resulted in clearer localization of label than in
many adult tissues. The mean percentage of cells that would be scored as unlabelled was 7 % (range 5-9 %). Again, sections of M. caroli embryos at similar
stages showed no nuclear localization of label, even when enclosed in the M.
musculus uterus. In such cases, M. musculus decidual cells were clearly labelled
but all M. caroli embryonic cells were unlabelled (Fig. 3B).
Embryonic cell lineages in Mus chimaeras
185
Identification ofM. musculus and M. caroli cells in embryonic chimaeras
Three 6-5-day potential interspecific chimaeras were sectioned and subjected
to in situ DNA-DNA hybridization. In one embryo, no patches of unlabelled
cells could be detected but the remaining two showed patches of unlabelled,
presumably M. caroli, cells in the embryonic region. Two successive sections of
one of these chimaeras are shown in Fig. 4. All cells of the extraembryonic
ectoderm, trophoblast and endoderm were labelled, but patches of unlabelled
M. caroli cells were apparent in the embryonic ectoderm. Complete serial reconstruction of the embryo was not possible since some sections were lost, but
limited analysis suggested that the M. caroli cells formed a fairly contiguous
group of cells showing little mixing with M. musculus cells.
Analysis of reconstituted blastocysts
Five out of eleven reconstituted blastocysts had formed decidua when
analysed at 6-5 days p. c. and three implants contained embryos when sectioned.
Two of the three embryos were analysed using in situ hybridization with the M.
Fig. 3. (A) Autoradiograph of section of ectoplacental cone and extraembryonic
ectoderm from 6-5 day M. musculus embryo, showing virtually all nuclei labelled.
(B) Autoradiograph of M. caroli egg cylinder in M. musculus uterus, showing labelling of nuclei of uterine tissue and only background labelling (fairly high!) over M.
caroli embryo. Note unlabelled trophoblast giant cells (arrows) around periphery of
M. caroli embryo, surrounded by labelled M. musculus nuclei.
186
J. ROSSANT, M. VIJH, L. D . SIRACUSA & V. M. CHAPMAN
Fig. 4. Autoradiographs of sections of 6-5-day M. musculus <-> M. caroli chimaera.
(A) Micrograph and diagram of embryonic region, revealing group of unlabelled M.
caroli cells in embryonic ectoderm. Hatched areas represent unlabelled cells. (B)
Micrograph and diagram of succeeding section showing continuity of unlabelled area
through the embryo.
musculus probe. This analysis revealed that all cells of the ectoplacental cone,
extraembryonic ectoderm and trophoblast giant cell layer were labelled, whereas
the embryonic ectoderm, and visceral and parietal endoderm were unlabelled in
both cases (Fig. 5). There was no evidence for cross contamination of tissue
types. Boundaries between extraembryonic ectoderm and embryonic ectoderm
Embryonic cell lineages in Mus chimaeras
187
Fig. 5. Autoradiograph of section of 6-5 day reconstituted M. caroli/M. musculus
embryo, showing labelled (M. musculus) extraembryonic ectoderm (arrow) and
unlabelled (M. caroli) proximal endoderm.
were clear and the hybridization patterns corresponded exactly to the tissue
boundaries.
DISCUSSION
A cloned probe to M. musculus satellite DNA has been used to distinguish M.
musculus and M. caroli cells in histological sections. Heavy labelling, localized
to the cell nucleus, was observed when the probe was hybridized to M. musculus
tissues but no such labelling was observed in M. caroli cells. Patches of labelled
and unlabelled cells were observed in all three adult chimaeric tissues examined
and the proportions of unlabelled to labelled cells were similar to the proportions
of M. caroli and M. musculus cells assessed by GPI analysis. This suggested that
the patches were not an artifact but truly represented differential binding of the
probe to M. musculus and M. caroli cells. The small size of the patches was also
consistent with the pattern of mosaicism predicted by previous reports of finegrained mosaicism in adult chimaeric tissues, using other in situ markers (Dewey,
Gervais & Mintz, 1976; West, 1976; Oster-Granite & Gearhart, 1981). Confirmation of the ability of the system to distinguish M. musculus and M. caroli
cells in the same section was provided by sections in which unlabelled M. caroli
embryo cells were clearly distinguishable from surrounding labelled M. musculus
uterine cells. The marker system also revealed patches of unlabelled M. caroli
188
J. ROSSANT, M. VIJH, L. D. SIRACUSA & V. M. CHAPMAN
cells in the embryonic regions of 6-5-day interspecific chimaeras. The
distribution of the M. caroli cells suggested that growth of the injected M. caroli
ICM cells was fairly coherent and that little cell mixing had yet occurred. Similar
results have been reported previously in rat <-» mouse chimaeras (Gardner &
Johnson, 1973, 1975).
The most powerful test of the marker system was its use in following the fate
of M. caroli ICM and M. musculus trophectoderm in reconstituted blastocysts.
There have been many previous experimental studies aimed at delineating the
fate of these two cell types (Gardner, Papaioannou & Barton, 1973; Rossant &
Lis, 1979; Papaioannou, 1982) and it has generally been agreed that the ICM
gives rise to the embryonic ectoderm and endoderm, while the trophectoderm
produces the extraembryonic ectoderm, ectoplacental cone and giant cells
(reviewed by Rossant & Papaioannou, 1977). However, most studies have relied
on electrophoretic analysis of GPI isozymes in tissue homogenates of later embryos, where cross contamination of tissues is possible and a minor contribution
to a given tissue may go unnoticed. Direct analysis of the development of reconstituted blastocysts using the marker system has confirmed all previous indirect
studies of ICM and trophectoderm cell fate and simultaneously provided powerful confirmation that in situ labelling with the recombinant DNA probe was
specific to M. musculus cells.
The genetic marker system described here has several advantages over
previous marker systems used for chimaera analysis. First, the marker can be
detected in histological sections and does not require destruction of the spatial
integrity of the chimaeric tissue being analysed. The wax embedding method
used in association with the in situ DNA-DNA hybridization should allow serial
reconstruction of the clonal distribution of M. musculus and Mus caroli cells in
chimaeras. Second, the marker is cell autonomous; no invasive method of cell
marking is required. Exogenous marker systems, such as [3H]thymidine (Kelly
& Rossant, 1976) or horse-radish peroxidase (Balakier & Pedersen, 1982), may
alter cell behaviour and make interpretation of cell relationships difficult. Third,
the marker is confined to the cell nucleus, since the probe hybridizes to a nontranscribed satellite DNA sequence (Flamm, Walker & McCallum, 1969;
Pietras, 1981). This nuclear localization was clearly preserved in histological
sections. Some other marker systems, such as j3-glucuronidase activity variants,
suffer from problems of enzyme transfer between cells (Feder, 1976).
The most important advantage of this marker system over any other in situ
marker system used experimentally in mammals is its ubiquitous nature. The
marker system has already been used to distinguish M. caroli and M. musculus
cells in a variety of chimaeric tissues from embryo to adult and should be applicable to any nucleated tissue, since satellite DNA is present in all nuclei. A variety
of histologically detectable marker systems has been developed, including activity variants of /3-glucuronidase (Mullen & Herrup, 1979) and /3-galactosidase
(Dewey etal., 1976), the ichthyosis nuclear marker (Goldowitz & Mullen, 1982)
Embryonic cell lineages in Mus chimaeras
189
and GPI immunocytochemistry (Gearhart & Oster-Granite, 1978; OsterGranite & Gearhart, 1981), but none has so far proved applicable to more than
a limited selection of chimaeric tissues.
This in situ cell marker system thus possesses many of the properties of an ideal
cell marker (McLaren, 1976) and, although it utilizes interspecific rather than
intraspecific chimaeras, we have no evidence to suggest that this should impose
any serious limitations on its use. This question is considered in more depth in
the succeeding paper (Rossant & Chapman, 1982). The in situ DNA-DNA
hybridization marker system does, however, have some minor technical limitations, which it should be possible to eliminate with further refinement of the
system. The extent of hybridization and of background labelling was somewhat
variable. However, it was always easy to distinguish evenly distributed background labelling over M. caroli cells from high-density nuclear-localized labelling over M. musculus cells. Improvements in hybridization conditions will continue to be sought in order to reduce this background labelling. The marker also
cannot unequivocally identify every single cell in a chimaeric section as M.
musculus or M. caroli. M. musculus cells will occasionally be scored as unlabelled and therefore M. caroli, because of the varying depth of sectioning of
the nuclei. A single diploid M. caroli cell in the middle of a mass of M. musculus
cells would, therefore, be unlikely to be recognized. However, this is common
to most cell marker systems and is unlikely to be a major problem since contiguous growth of clones of cells is more likely to be observed in embryos than
migration of isolated cells. Patches of M. musculus and M. caroli were readily
identified in adult chimaeric tissue and the resolution in earlier embryonic stages
was even greater. Another limitation is that the marker scores M. caroli cells as
unlabelled and M. musculus cells as labelled. For technical reasons, interspecific
chimaeras are usually made by injecting M. caroli cells in M. musculus blastocysts and transferring to the M. musculus uterus. This means that the injected
M. caroli cells will usually be the minority population, especially if single cell
injections are performed. Clearly it would be preferable, although not essential,
to arrange the marker system so that M. caroli cells are recognized as labelled
and M. musculus cells as unlabelled. Preliminary observations using M. caroli
repetitive DNA as a probe for in situ hybridization suggest that this should be
possible (Siracusa et al., 1982). We are currently attempting to clone sequences
from M. caroli repetitive DNA in order to obtain the reverse marker system.
CONCLUSIONS
The use of in situ DNA-DNA hybridization of a M. musculus satellite DNA
probe to interspecific M. musculus ±* M. caroli chimaeras should provide a
powerful new tool in analyses of cell lineages in both embryonic and adult mouse
chimaeras. The system has several advantages over other marker systems so far
developed, the most important of which is its ubiquity. We have not yet tested
EMB73
190
J. ROSSANT, M. VIJH, L. D. SIRACUSA & V. M. CHAPMAN
it on all tissues, but, in theory, only cells without nuclei, like erythrocytes, should
be unsuited to its use. Its utility has already been demonstrated by analysis of
reconstituted M. caroli/M. musculus blastocysts.
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SOLTER, D.
(Accepted 25 July 1982)