Download An in situ transgenic enzyme marker for the

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Development 106, 37-46 (1989)
Printed in Great Britain (S) The Company of Biologists Limited 1989
An in situ transgenic enzyme marker for the midgestation mouse embryo
and the visualization of inner cell mass clones during early organogenesis
ROSA S. P. BEDDINGTON1, JAY MORGERNSTERN2, HARTMUT LAND2 and AILEEN HOGAN1
'ICRF Developmental Biology Unit, Department of Zoology, South Parks Road, Oxford OXl 3PS, UK
ICRF, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK
2
Summary
In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an
in situ cell marker which can be used to follow cell fate.
To create such a marker a transgenic mouse strain,
designated Tg(Act-lac Z)-l , which carries 6 copies of the
Escherichia coli lac Z gene under the control of the rat /Sactin promoter, was made by pronuclear injection of
DNA. Staining early postimplantation hemizygous
mouse concept uses, during gastrulation and early organogenesis, for /i-galactosidase activity shows that lac Z
expression is ubiquitous and constitutive in all epiblast
derivatives of the 10th day conceptus. No activity is seen
in trophectoderm and primitive endoderm derivatives.
Postimplantation grafts of [3H]thymidine-labelled
transgenic cells establish the cell autonomy of this
transgenic marker. Preliminary observations on the
distribution of inner cell mass (ICM) descendant clones,
identified in situ in midgestation concept uses, confirm
the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal
primordia are discussed.
Introduction
at least in the central nervous system, that cells from
different species mix less freely than intraspecific combinations, resulting in unusually large patch sizes in the
cerebellum and elsewhere (Goldwitz, 1986). The
second nuclear marker is intraspecific and makes use of
a transgenic mouse strain containing approximately
1000 copies of an exogenous /3-globin gene integrated as
a concatamer (Lo, 1986; Varmuza et al. 1988). Analyses
of chimaeras using this marker have been reported
(Thomson & Solter, 1988; Clarke et al. 1988) but
reliable detection of a single concatemer in every
nucleus is affected by nuclear size and cell packing
density and, therefore, requires different sectioning
conditions., for different tissues. Furthermore, as
emphasized by Gardner (1985b), resolution of cell type
and position in complex organs necessitates a cytoplasmic marker to define the perimeter of cells. In addition,
cytoplasmic markers which can be used on wholemount material can greatly facilitate the analysis of
chimaeras since this will permit the observation of
clones both in the intact conceptus and in subsequently
sectioned material. Unfortunately, all cytoplasmic
markers so far described, which are usually based on
null or thermolabile variants of known enzymes, have
proved to be applicable only to a limited repertoire of
tissue types where wild-type enzyme activity is high
(West, 1984; Ponder, 1987). For this reason, none are
No descendant preimplantation clone has ever been
visualized in the embryonic portion of the early mammalian conceptus. Consequently, our appreciation of
the pluripotency of individual inner cell mass (ICM)
cells or epiblast cells rests on electrophoretic analysis of
homogenized tissues (Gardner, 1985a). The inability to
follow the fate of single cells in situ and to observe the
distribution of their progeny in the embryo is a serious
deficit to our understanding of cell deployment during
gastrulation and early organogenesis. While the widespread tissue colonization reported for single epiblast
cells is indicative of extensive cell mixing during early
postimplantation development (Beddington, 1983), it is
not clear whether mixing is a transient episode preceding allocation of cells to different primordia or if it is
sustained within nascent definitive tissues. Only the
analysis of descendant clones in the intact conceptus, or
in histological preparations, will resolve this question.
Over the last ten years new cell markers have been
developed for chimaera studies in the mouse (Ponder,
1987). However, all of these have certain shortcomings.
Two nuclear markers now exist. One exploits differences between two species of mouse in highly repetitive
DNA sequences which can be visualized using in situ
hybridization (Rossant et al. 1983; 1986). However,
there is some evidence in these interspecific chimaeras,
Key words: mouse embryo, transgenic, cell marker, lac Z.
38
7?. S. P. Beddington and others
adequate for marking all cell types during the early
stages of fetal development.
This paper describes a cytoplasmic marker suitable
for studying cell deployment during early organogenesis
in the mouse. The marker was produced by making a
transgenic strain of mouse (designated Tg(Act-lac Z)-l )
carrying the E. coli lac Z gene under the control of the
rat /S-actin promoter. Single ICM cells from this strain
were introduced into blastocysts and preliminary observations on their descendant clones in midgestation
(10th day) chimaeric embryos are presented.
pIRV-Neo-Act-Lac
(9-6 kb)
Materials and methods
Media
The recovery and manipulation of all embryos was carried out
in PB1 medium (Whittingham & Wales, 1969) containing
10 % (w/v) fetal calf serum (FCS) instead of bovine serum
albumin. For DNA injections, this medium contained
SjUgml"1 of Cytochalasin B (Sigma). Eggs were incubated
before and after injection in microdrops of standard ovum
culture medium (Biggers et al. 1971) pre-equilibrated under
011 at 37°C in an atmosphere of 5 % CO2. Blastocysts before
and after injection were incubated under similar conditions
but in a medium (Stanners et al. 1971) modified as described
previously (Gardner et al. 1985).
Production of transgenics
Outbred PO (Pathology, Oxford) mice were used in all
experiments. Fertilized eggs were obtained from females
maintained on a light regime of 14 h light/10 h dark, the
midpoint of the dark period being 19.00 h. Pseudopregnant
recipient females were maintained on a similar light regime
except that the midpoint of the dark period was midnight. The
recovery and injection of eggs was essentially identical to that
described elsewhere (Hogan et al. 1986) except that the DNA
injection pipette was attached to tubing filled with paraffin oil
(Boots, UK, Ltd) and controlled by a de Fonbrune suction
and force pump. Injections were carried out in hanging drops
in a Puliv chamber using a fixed-stage microscope (Ergoval,
Zeiss-Jena).
Preparation of DNA for injection
A 4-3 kb BgKl/Scal fragment, containing the E. coli lac Z
gene fused to the rat /3-actin promoter (350bp), was isolated
from pIRV-Neo-Act-lac Z as illustrated in Fig. 1. This was
extracted from a 0-5 % agarose gel, purified using a silica
matrix (Geneclean) and redissolved in 5mM-Tris, 0-5 minEDTA (pH8-0) to a final concentration of approximately
Identification of transgenics
High molecular weight DNA was isolated from tail biopsies of
putative transgenic offspring (Hogan et al. 1986), digested
with various restriction enzymes (Fig. 2) and run on a 0-5 %
agarose gel. DNA was transferred over 4h to nylon filters
(Hybond, Amersham) and covalently linked to the filter with
ultraviolet light (5 min). The filters were prehybridized for 2 h
at 65°C with 5 x SSC, 5 x Denhardfs solution, 0-5% SDS
and 100/igmP 1 herring sperm DNA. Subsequently, they
were incubated overnight at 65°C in the same mix containing
a nick-translated 3 1 kb ^P-labelled BamHl fragment of
pIRV-Neo-Act-lac Z (Fig. 1). Southern blots were incubated
at the same time with a nick-translated mouse oglobin
Fig. 1. Diagram of pIRV-Neo-Act-lac Z. The /3-actin
promoter was a 350 bp Hinfl fragment of p. Ac. 4.1 (Nudel
et al. 1983). Lac Z sequences came from BAG virus (Price
et al. 1987) and the Moloney retroviral sequences from
p.Zi'p.MoMulV (Hoffman et al. 1982). The 4-3 kb
BgRl/Scal fragment was injected into eggs. The 3-1 kb
BamHl fragment was used to probe Southern blots.
(Nishioka & Leder, 1979) probe (2-2kb Hinfl-Sacl fragment). For the nick translation mixture 2-5ngml~1 DNase I,
50^Ci [o-32P]dCTP, 10 ^M each of dGTP, dATP and dTTP,
50mM-Tris-HCl (pH7-4), 10mM-MgSO4, lmM-DTT, and
50/igml"1 BSA were added together for 3min at room
temperature before adding 5 units of E. coli DNA polymerase
I. After incubation for 2h at 14°C the nick-translated DNA
was de-salted on a Sephadex G-50 spun column and added to
the hybridization mixture. The filters were washed twice
following hybridization in 2 x SSC (15 min each at 65°C), in
2 x SSC + 0-1 % SDS (for 20 min at 65°C), and the final high
stringency wash in 0 1 x SSC (for 10min at 65°C). The filters
were exposed to preflashed Kodak XAR film or Fuji X-ray
film in cassettes, containing two intensifier screens, at —70°C.
The number of copies of transgene integrated was determined
on developed films by scanning densitometry using a Gelman
DCD-16 computing densitometer with automatic integration.
Histochemistry
Ear punches from putative transgenic mice and their offspring
were routinely stained for E. coli /J-galactosidase activity. The
tissue was washed in 0-lM-phosphate buffer (pH7-4) and
fixed for 15 min in 0-2 % glutaraldehyde (Gurr) in the same
buffer containing 2mM-MgCl2 and 5mM-EGTA. It was
washed for l h in three changes of 0-lM-phosphate buffer
(pH 7-4) containing 2 mM-MgCl2, 001 % (w/v) sodium desoxycholate and 0-02 % (w/v) Nonidet P-40. Staining was carried
out at 37 °C in a solution of the above buffer containing X-Gal
at a final concentration of 1 mgml"1. The X-Gal was dissolved
in dimethylformamide (40mgml"1) and to this was added
buffer, which in addition to MgCl2 and detergent also
contained 5 mM-K3Fe(CN)6, 5 mM-K4Fe(CN)6. 6H2O and
1 mM-spermidine hydrochloride. Ear tissue was left to stain
for up to 48h. All reagents, unless specified, were obtained
from Sigma.
ICM clones and a transgenic cell marker
Control, hemizygous Tg(Act-lac Z)-l and chimaeric conceptuses were stained intact as whole mounts using identical
conditions. Controls and hemizygotes were recovered and
stained on the 8th, 9th and 10th days of gestation whereas only
9th and 10th day potential chimaeras were stained.
After staining, embryos were briefly fixed in Carnoys' fluid
(15min), dehydrated and embedded in paraffin wax (m.p.
56 °C). They were serially sectioned at 8/an and some were
counterstained with eosin before mounting in DPX and
viewed in a light microscope (Dialux 20EB; Leitz).
Assessment of cell autonomy of X-Gal staining
Ninth day conceptuses, derived from Tg(Act-lac Z)-l males
mated to PO females, were dissected from the decidua into
PB1 + 10% FCS. Reichert's membrane was removed. In all
but one experiment the embryos were subsequently divided
into anterior and posterior halves using fine glass needles
(Beddington, 1987). The anterior halves were stained for /Sgalactosidase activity as described above. The posterior
halves were labelled with [3H]thymidine according to a
procedure described elsewhere (Tam & Beddington, 1987).
After 3h in labelling medium, small clumps of posterior
ectoderm were prepared from those embryos that had been
identified as Tg(Act-lac Z)-l/+ from X-Gal staining. In one
experiment, clumps were prepared from all embryos because
no parallel X-Gal staining was undertaken. These clumps of
labelled ectoderm cells were injected into host unlabelled PO
9th day embryos (Tam & Beddington, 1987). Injections were
made either into the primitive streak region or immediately
anterior to the heart. Embryos that had received grafts were
cultured for 24 h in roller bottles in 50 % rat serum diluted in
Dulbecco's modification of Eagle's medium (Flow Laboratories; Beddington, 1987) before being fixed and stained for /Jgalactosidase activity (see above).
After histochemical staining, embryos were refixed in
Carnoy's and processed for routine histology, sectioned at
6/mi, and subjected to autoradiography as described by
Beddington (1981), except that the trichloroacetic acid treatment was omitted. Following development, after 4 weeks
exposure at 4°C, the slides were scanned and labelled nuclei
counted in every second section. The coincidence, or otherwise, of blue cytoplasm and nuclear silver grains was recorded
for each section.
Recovery and blastocyst injection of single transgenic
ICM cells
Blastocysts were recovered from matings between hemizygous transgenic mice and PO females, on the 4th day of
gestation. The zona pellucida was removed in acidified
Tyrode's solution (Nicolson et al. 1975) and, because blastocysts do not appear to express lac Z and cannot be preselected
(R. Beddington, unpublished observations), all the blastocysts were subjected to routine immunosurgery (see Nichols
& Gardner, 1984). Isolated ICMs were disaggregated into
single cells by the method described by Gardner et al. (1985)
and the isolated cells cultured in <* medium before injection.
Host blastocysts were obtained from PO females on the 4th
day of gestation.
Blastocyst injections were carried out using the two-instrument method described by Babinet (1980). Manipulations
were performed in hanging drops in a Puliv chamber using an
Ergoval microscope. Injected blastocysts were cultured for up
to 1 h before being transferred to recipients that were on the
third day of pseudopregnancy.
39
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4-3 kb
Fig. 2. Southern blot showing the two positive founder
transgenics (R10 and R12) and a plRV-Neo-Act-lac Z
control (cut with BgHI/Scal). The transgenic DNA was cut
with EcoBl and the blot probed with a 3-lkb fragment of
the lac Z gene. There is an £coRI site at the 3' end of the
lac Z gene.
Results
Production and characterization of founder transgenics
201 PO eggs, in three separate experiments, were
injected with the 4-3 kb fragment containing the rat /Sactin promoter fused to the lac Z gene. Of these 99 eggs
survived and 68 were transferred to three recipients
which subsequently became pregnant. 16 mice were
born but 4 were eaten by their mother before DNA
could be extracted for analysis. The remaining 12
offspring were tested for bacterial /3-galactosidase activity in ear punches and by Southern blot analysis of
total genomic DNA. One neonate (R10) had large
patches of blue cells in ear tissue stained with X-Gal.
This animal and one other (R12) contained integrated
transgenes (Fig. 2).
From densitometry measurements, R12 contained
two copies of the transgene but there was evidence (not
shown) of rearrangement of the injected DNA. Neither
this animal nor its offspring, either during fetal life or
after birth, showed any exogenous /3-galactosidase activity that could be detected histochemically. R12 will
not be considered further in this paper.
R10 contained six copies of the transgene integrated
in the usual head-to-tail concatamer formation found in
many transgenic strains (Palmiter & Brinster, 1986).
The transgene was transmitted to F t progeny at a
frequency of approximately 11-5% (Table 1). If this low
frequency of transmission were due to lethal effects of
40
R. S. P. Beddington and others
Table 1. Litter size and transgene transmission frequency o/Tg(Act-lac Z)-l hemizygotes and controls
Mating
POxPO
RIO x PO
Preimplantation litter size
9-3 ±1-1
(168)*
9-1 ±1-7
(218)
-
-
Litter size at birthf
Transmission frequency of transgene
Transmission frequency of lac Z
expressiont
-
8-5 ±1-6
(298)
11-5%
(26)
11-5%
(26)
Tg(Act-lac Z)-l x
PO
9-2 ±0-98
(55)
9-3 ±1-6
(546)
44-4%
(27)
40-7%
(27)
Tg(Act-lac Z)-l x
Tg(Act-lac Z)-l
90 ±0
(45)
8-5 ±1-9
(324)
69-4%
(62)
54-7%
(62)
* Figures in brackets denote the total number of offspring.
t Litter sizes at birth of Tg(Act-lac Z)-l x PO are significantly different from those of Tg(Act-lac Z)-l x Tg(Act-lac Z)-l ((=215),
P<005.
$The transmission frequency of lac Z expression is based on histochemical staining of the same individuals from which total genomic
DNA was extracted for Southern blot analysis.
the transgene one would have expected a smaller litter
size than that recorded (Table 1). It is more likely that
the low transmission frequency stemmed from mosaicism in R10, which was consistent with the patchy
histochemical staining observed in ear tissue.
Expression and transmission of the transgene in
subsequent generations
15 Fx progeny from R10 mated to PO females were
examined for the coincident expression of /3-galactosidase in ear tissue and the presence of the transgene in
genomic DNA. Three Tg(Act-lac Z)-l hemizygotes
were identified histochemically and only these three
mice contained the transgene by Southern blot analysis.
Subsequently, among offspring from Fx matings (both
intercrossed and outcrossed) 10 animals out of 89 tested
(11-2%) have failed to show staining despite the
presence of the transgene (Table 1). Within the limits of
Southern blot analysis, there is no evidence for rearrangement of the transgene and methylation differences are not pronounced (unpublished observations).
There has been no instance of finding apparent enzyme
activity in animals that do not carry the lac Z gene.
No homozygous F2 progeny have been obtained from
intercrossing F] hemizygous mice. Densitometry
measurements on Southern blots, comparing the mouse
a'-globin and transgenic copy numbers in F 2 progeny,
identified likely homozygous offspring although there
was not a clear-cut bimodal distribution. However, test
breeding all transgenic F2 progeny from 2 litters, both
putative hemizygotes and homozygotes identified by
Southern blot analysis, by outcrossing to PO animals
(16 F2 transgenics tested; 370 offspring) did not produce
any litters in which all offspring expressed lac Z and,
therefore, failed to confirm the presence of homozygotes. Consequently, we assume that homozygotes die
in utero, although the stage at which they are dying is
not known. Litter size is within the normal range at
least up to the 14th day of gestation but is somewhat
reduced by birth (Table 1).
In addition, the transmission frequency of the transgene is less than expected. Screening for enzyme
activity within the first week after birth showed that in
Tg(Act-lac Z)-l x PO matings 154 out of 546 (28-2%)
offspring inherited E. coli /S-galactosidase activity.
Even if this percentage is corrected for the known
discrepancy between presence of the transgene and
detectable enzyme activity (11-2 %; see above), this still
leaves a transmission frequency of only 39-4%, as
opposed to the expected 50%. The corrected value
obtained from 291 offspring from hemizygous intercrosses is 49-1%, as opposed to the expected 66-7%
(accepting that there will be no liveborn homozygotes).
Both males and females appear to transmit lac Z at a
reduced rate. This suggests that the insertion of the
transgene may not be neutral either during gametogenesis or in mature gametes.
Expression of lac Z in postimplantation conceptuses
Hemizygous conceptuses from the 8th, 9th and 10th
days of gestation have been analysed for expression of
lac Z in embryonic and extraembryonic tissues. Conceptuses were derived from matings between hemizygous males and PO females, except for one 10th day
litter where the transgene was inherited from the
mother.
Late primitive streak stage (8th day)
Staining in the four embryos analysed is restricted to the
epiblast, embryonic and extraembryonic mesoderm,
cells in the embryonic endoderm layer lying along the
midline in the region of the head process and the
occasional cell overlying the primitive streak (Fig. 3A).
Staining in the epiblast is punctate, as if the enzyme is
localized to intracellular vesicles. Mitotic cells show a
more generalized cytoplasmic staining. Mesoderm and
presumptive definitive endoderm cells appear more
strongly stained than the epiblast. The punctate staining
makes it difficult to evaluate the precise percentage of
positively stained cells in these tissues but within any
one section at least 80 % of the cells contain a blue spot.
Primitive endoderm and trophectoderm derivatives are
uniformly negative and indistinguishable from controls.
Control embryos have no blue staining in any tissue.
Early somite stage (9th day)
The general distribution of lac Z expression remains the
<t
Fig. 3. Hemizygous embryos stained with X-Gal. (A) Longitudinal section of an 8th day embryo. Cells in the ectoderm and
mesoderm show punctate staining. Bar, 100^m; ps, primitive streak; ve, visceral endoderm. (B) Whole mount of a 9th day
embryo during staining. The notochord (n) and archenteron (a) show the strongest staining and stain before other tissues.
The constituents of the parietal yolk sac (pys) and the visceral endoderm of the visceral yolk sac (vys) are unstained. Bar,
100^m. (C) Transverse section through a 9th day embryo. All the epiblast derivatives of the conceptus exhibit /3galactosidase activity, whereas the primitive endoderm and trophectoderm derivatives are unstained. Much of the staining is
punctate although now the cytoplasm is also pale blue (obscured by eosin counterstaining which renders the cells brownish
purple), s, somite; nt, neural tube; n, notochord; t, trophoblast; pe, parietal endoderm. Bar, 20^m. (D) Whole mount of a
10th day embryo. Bar, 100^m. (E) Longitudinal section of a 10th day embryo. Every cell in the fetus shows generalized
staining in the cytoplasm, the nuclei appearing pale. Bar, 100^m. (F) Transverse section through three axial levels of a 10th
day embryo. The endoderm of the visceral yolk sac (vys) is unstained. Bar, 100 jxm.
42
R. S. P. Beddington and others
Table 3. Frequency of chimaeras resulting from single
ICM cell injections into blastocysts
No blastocysts
injected
45
No. transferred
to pregnant
females
45
No. normal
conceptuses
No. chimaeric
conceptuses
40
(88-9%)
3
(7-5%)
were stained blue (Table 2). However, the high density
of grains over 11 unstained cells not only masked much
of the cytoplasm but also indicated that they were
probably dead cells. The failure to detect blue staining
in the other 3 cells may be an extreme example of the
fading of staining during autoradiography. The intensity of blue staining in all donor cells was much reduced
compared to sections which had not been subjected to
autoradiography. Only 2 cells were found, both in the
same embryo, that were stained blue but did not have
grains over their nuclei. These cells may not have been
labelled with sufficient [3H]thymidine before grafting
(Tarn & Beddington, 1987).
Distribution of ICM clones in chimaeras
The injection of a single ICM cell into a blastocyst, and
its subsequent participation in development, permits
not only an analysis of the potential of individual cells
but also serves as a very sensitive means of assessing
whether or not constitutive expression of lac Z affects
development. In these experiments, it was not possible
to distinguish between hemizygous and wild-type ICM
cells before injection. Therefore, only some blastocysts
must have been injected with a transgenic cell. The
frequency of normal development and formation of
chimaeras, detected histochemically, is shown in
Table 3. One of these chimaeras was recovered on the
9th day (early-somite-stage) and the other two on the
10th day. More extensive analysis of clonal chimaeras
will be presented elsewhere. The object of this paper is
to demonstrate the potential use of this transgenic
marker, not to supply a detailed analysis of cell mixing
and growth patterns in nascent primordia. Therefore,
only unequivocal observations, which do not require
substantiation by elaborate serial reconstruction, are
presented below.
The 9th day, early-somite-stage chimaera (CI) contained only relatively few Tg(Act-lac Z)-l cells (466
cells; counted in alternate sections) in the embryo and
extraembryonic mesoderm (Fig. 4A). It is possible that
not all donor descendants were stained in these tissues
at this stage (see above), and, of course, there may have
been more descendants in the primitive endoderm and
trophectoderm lineages but these would have gone
undetected. However, positively staining transgenic
cells were distributed along the entire axis of the
embryos, including representatives in the cranial neurectoderm and cells in the caudal and allantoic mesoderm. 88 cells lay anterior to the first somite and 130
cells were found posterior to the last (7th) somite. The
distribution of the Tg(Act-lac Z)-l cells in the embryo
(Table 4) demonstrates the pluripotency of individual
Table 4. Tissue distribution of donor transgenic cells
in chimaera 1
Tissue
Neurectoderm
Surface ectoderm
Cranial mesoderm
Lateral mesoderm
Caudal mesoderm
Cardiac mesoderm
Extraembryonic mesoderm
Somitic mesoderm
Notochord
Gut endoderm
Number of
donor cells
73
12
32
102
54
6
68
46
25
48
466
ICM cells. Furthermore, the spatial distribution of the
donor cells indicates that cell mixing continues within
nascent primordia such as the notochord (Fig. 4D).
The older chimaeras (C2 and C3) are more informative because the /3-galactosidase activity is higher and
detectable throughout the cytoplasm of individual cells
(Fig. 4B,C). However, the extensive contribution of
donor cells, particularly to the trunk region, precluded
an accurate count of total donor cell contribution. In C2
approximately 50% of cells were of donor origin
whereas in C3 the contribution was nearer to 20%
(Fig. 4B,C).
Examination of serial sections revealed extensive
chimaerism in all regions, although the heart, cranial
neurectoderm and splanchnopleure were less extensively colonized than other tissues. Some degree of cell
mixing was apparent in all tissues, darkly staining cells
often being intermixed with wild-type unstained cells.
However, in certain primordia there was evidence for
limited coherent growth. For example, small patches
were common in somites (Fig. 4E), neural tube, surface
ectoderm and quite large coherent patches were observed in the gut, invariably seen as elongated collections of cells orientated along the long axis of the gut
(Fig. 4F).
Discussion
The objective of this work was to produce an ubiquitous, cytoplasmic, in situ marker, which did not
compromise development and which could be used to
distinguish donor and host mouse cells in the intact
embryo and in histological sections. The transgenic
strain produced may not wholly satisfy these aims but it
does provide, for the first time, a means of studying the
deployment of cells in any fetal primordium during
early organogenesis.
Constitutive, ubiquitous expression
The rat /3-actin promoter was chosen because it is
thought to be constitutively active in all cells, /J-actin
being an integral component of the cytoskeleton and
cell motility (Uyemura & Spudich, 1980). The actin
genes are a highly conserved gene family and, therefore, there was every reason to believe that the rat
ICM clones and a transgenic cell marker
promoter (Nudel et al. 1983) would be effective within
the mouse genome. With the proviso that only enzyme
activity, and not mRNA synthesis, was measured in
hemizygous embryos, it appears that the transgene first
becomes active after implantation (no staining has been
seen in putative hemizygous blastocysts; R. Beddington, unpublished observations). Initially the staining
is punctate, as if the enzyme is localized to vesicles, but
by the 10th day the entire cytoplasm stains blue.
Punctate staining may be a feature of cells expressing
the enzyme at low levels. Cleavage-stage blastomeres,
with low bacterial /3-galactosidase activity, show a
similar punctate pattern (Ueno et al. 1987), but there
was no evidence for segregation of enzyme activity with
particular subcellular fractions. Therefore, the blue
spots were considered to represent cytoplasmic oxidation centres which facilitate the deposition of insoluble pigment (Ueno et al. 1987).
At all developmental stages examined, staining is
restricted to epiblast derivatives. The lack of activity
earlier in development, and its absence in trophectoderm and primitive endoderm derivatives, is unlikely to
be a function of the promoter. A larger fragment of the
/3-actin promoter is active in 2-cell embryos (Bonnerot
et al. 1987), and the pIRV-Neo-Act-lac Z construct,
introduced into blastocysts via retroviral infection, can
produce positive clones in trophectoderm and endoderm cells (P. Savatier & R. Beddington, unpublished
observations). Therefore, although expression in only a
single transgenic strain has been studied, it seems likely
that it is the integration site of the transgene that is
responsible for this observed lineage restriction in lac Z
expression (Palmiter & Brinster, 1986; Allen et al.
1988). If this is the case, the sequences flanking the
integration site may prove informative with respect to
differential gene activity in these primary tissue lineages.
Interestingly, during the latter part of gestation lac Z
expression, judged by enzyme activity, is dramatically
down-regulated, leading in some cases to failure to
detect staining in ear punches (Table 1), and during the
first few weeks after birth the number of positive cells
decreases rapidly and extensively (R. Beddington,
unpublished observations). However, with the exception of a few organs (such as liver) that are entirely
negative, the loss of enzyme activity does not appear to
correspond to any obvious pattern with respect to tissue
or cell type. Inexplicable variation between animals of
the same transgenic strain, in the synthesis of transgene
mRNA and its pattern of inheritance, has been
reported before (Palmiter et al. 1982, 1984). In the case
of Tg(Act-lac Z)-l, it is not clear whether this progressive extinction of activity is a feature peculiar to an
exogenous promoter, a position effect or a reflection of
normal regulatory mechanisms employed to reduce
levels of nonessential proteins. Clearly, an in situ
marker provides a very sensitive method for monitoring
this inactivation.
Cell autonomy of lac Z expression
The apparent ubiquitous expression of lac Z in 10th day
43
hemizygous embryos and the complete absence of
staining in wild-type embryos is not sufficient grounds
for assuming /3-galactosidase to be a reliable cell autonomous marker. It is necessary to demonstrate that
ubiquitous staining is not the product of diffusion, and
that every transgenic cell expresses the marker. On a
gross level, tissues such as the heart, which show
weaker staining, show the same blue profile whether
stained in the intact fetus or isolated by dissection
before histochemistry. However, the grafts of [3H]thymidine-labelled transgenic epiblast provide the most
compelling evidence for cell autonomy since over 94 %
of cells showed unequivocal coincidence of /3-galactosidase activity and nuclear silver grains (Table 2). Allowing for negative staining in putative dead cells (11 cells),
the parallel marking occurs in 98% of cells. It is true
that these grafts all contributed predominantly to mesenchymal tissues and that diffusion may be greater in
tightly packed epithelial structures. However, inspection of epithelial tissues in the clonal ICM chimaeras
reveals frequent instances of negative cells surrounded
by strongly staining transgenic tissue (e.g. Fig. 4E). In
addition, studies using retroviruses containing the lac Z
gene, producing positively staining clones either in vitro
or in vivo, have found little evidence for diffusion of
staining product (Price et al. 1987; Sanes et al. 1986;
Turner & Cepko, 1987; P. Savatier & R. Beddington,
unpublished observations). Therefore, one may be
confident that every blue cell is indeed of transgenic
genotype.
Neutrality in development
The introduction of a foreign gene which produces
constitutive enzyme expression, albeit in the absence of
substrate at the optimal pH, may be expected to
compromise development. Clearly, hemizygous
Tg(Act-lac Z)-l embryos develop normally and are
wholly viable and fertile when adult (Table 1). However, from monitoring the inheritance of lac Z expression and taking into account the percentage of
transgenics that do not express active enzyme, it appears that both hemizygous males and females may
transmit the transgene at a reduced frequency. The
normal litter size in such matings (Table 1) argues
against this reduced transmission frequency being
explained by embryo loss in utero. Therefore, as has
been reported for other transgenic strains (Palmiter et
al. 1984), the transgene may be deleterious to haploid
cells, compromising gametogenesis or fertilization. The
failure to produce homozygous individuals is most
likely due to an insertional mutation, a relatively
common occurrence in DNA injection transgenics (Palmiter & Brinster, 1986), rather than to increased
production of bacterial /3-galactosidase. Certainly, quite
widespread expression of lac Z in other transgenic
strains, which can be bred to homozygosity, does not
appear to impair development (Allen et al. 1988; S.
Darling, personal communication).
The most rigorous test for neutrality of a marker is to
examine the behaviour of cells in competition with wildtype ones. For example, [3H]thymidine was shown to
44
R. S. P. Beddington and others
put labelled preimplantation cells at a selective disadvantage in chimaeras (Kelly & Rossant, 1976). The
inability to identify transgenic ICM donor cells before
injection, in the chimaeras produced here, makes the
interpretation of the frequency of clonal ICM chimaeras (Table 4) complicated. The frequency of chimaeras from injection of single wild-type ICM cells
ranges from 17 to 26% (Gardner & Lyon, 1971;
Gardner & Rossant, 1979; Gardner et al. 1985). The
frequency of chimaeras (7-5%; Table 3) appears low
but considering that donor cells were obtained from
outcross matings, to avoid injecting possibly deleterious
homozygous transgenic cells, and that the transgene is
transmitted at a reduced frequency the number of
chimaeras identified by X-Gal staining is quite acceptable. More importantly the high contribution of transgenic cells seen in the 10th day chimaeras (Fig. 4B,C)
strongly suggests that they are at no particular disadvantage in competition with wild-type cells. However,
in both chimaeras, the contribution to the head and the
heart is less than elsewhere in the fetus and, therefore,
one cannot rule out that wild-type cells have some
advantage in these regions.
Distribution of donor cells in chimaeras
Previous analysis of the embryo, as opposed to extraembryonic membranes (Gardner, 1985; Cockcroft &
Gardner, 1987), in chimaeras at midgestation has relied
on separation of electrophoretic variants of isoenzymes.
The resolution of such an analysis is entirely dependent
on the precision of dissection into component tissues.
Therefore, with the exception of certain specific tissues
such as gut derivatives (Gardner & Rossant, 1979),
somites (Gearhart & Mintz, 1972) and selected organs
later in development where pigment or enzyme variants
can be distinguished in situ (see West, 1984), the
embryo has been treated as a single entity. Consequently, the pluripotency of single ICM cells has been
inferred from the analysis of a few dissected tissues in a
small number of adult clonal chimaeras (Gardner et al.
1985).
The distribution of stained cells in the clonal transgenic chimaeras demonstrates for the first time that the
progeny of a single ICM cell are found in all histologically recognizable tissue primordia, extending from
head to tail, in the midgestation embryo. Although not
specifically identified by alkaline phosphatase staining,
some large cells in the hindgut region, which could be
primordial germ cells, were also stained in both 10th
day chimaeras. In previous studies, test breeding has
confirmed coincident germline chimaerism in adult coat
colour chimaeras derived from single ICM cell injections (Gardner et al. 1985). This makes it likely that the
germ line was, indeed, also colonized in the two 10th
day transgenic chimaeras.
The analysis of growth patterns in nascent primordia
requires careful reconstruction to assess the extent of
cell mixing. However, certain features are evident in
these chimaeras from studying both whole mounts and
individual histological sections. The distribution of
transgenic cells along the entire axial length of the 9th
and 10th day chimaeras, which includes cells in derivatives of all three germ layers, strongly suggests that
there must be extensive mixing of epiblast cells during
development of the egg cylinder, prior to gastrulation
(Beddington, 1983). This is reminiscent of the observed
intermingling seen in descendant clones prior to gastrulation in zebrafish (Kimmel & Warga, 1986). There is
clearly cell mixing in the emergent notochord, the most
strongly staining tissue at the early somite stage
(Fig. 3B,C), of the 9th day chimaera (Cl; Fig. 4D).
This is consistent with the intercalation of notochordal
cells viewed in teleost fishes (Thorogood & Wood,
1987) and supports models of notochord growth invoking elongation by a combination of cell recruitment, cell
rearrangement and shearing forces (Jurand, 1962; Bancroft & Bellairs, 1976; Jacobson & Gordon, 1976; Woo
Younef al. 1980).
In the 10th day chimaeras, the first signs of obvious
coherent growth are apparent. Small patches are evident in individual somites, neural tube, surface ectoderm, endothelium and gut endoderm in individual
histological sections. Such patches are not seen, for
example, in cranial mesenchyme or the lateral mesoderm. However, serial reconstruction is required to
establish the extent of contiguity both in patches and
ostensibly mixed populations. As shown before (Gearhart & Mintz, 1972), somites are clearly not clonal in
origin, nor is there any indication that the most recently
formed somites arise from large coherent patches of
presomitic mesoderm. This corresponds to the notion
that there is cell mixing within the presomitic mesoderm
(Tarn & Beddington, 1987; Keynes & Stern, 1988).
With respect to the gut, the impression of elongated
clones in the midgut suggests that the growth is longitudinal not radial. In the neural tube, patches are
distributed radially in transverse section indicative of
radial growth at this stage. In the future, it is hoped that
analysis of chimaeras, in which the donor cell contributes rather a small proportion of progeny to the
chimaera, will prove very informative regarding patterns of growth during early organogenesis.
In summary, the staining in epiblast derivatives of
hemizygous embryos, particularly on the 10th day,
appears to be ubiquitous and cell autonomous. This
offers the first opportunity for analysing in situ the
developmental potential of individual pre- and postimplantation transplanted cells, and for examining the
patterns of growth in chimaeric organ primordia. In
addition, the migratory pathways of cells emanating
from structures such as somites or neural crest can be
charted. Heterotopic transplantation of marked tissue
will also make it possible to assess the autonomy or
otherwise of certain patterns of gene expression, for
example Hox genes, implicated in pattern formation.
We would like to thank Drs P. Ingham, P. Savatier and A.
J. Copp for useful criticism of the manuscript. This work was
supported by the Imperial Cancer Research Fund.
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