J. Embryol. exp. Morph. 82, 253-266 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
253
The histogenetic capacity of tissues in the caudal
end of the embryonic axis of the mouse
By P. P. L. TAM
Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, N.T., Hong Kong
SUMMARY
The caudal end of the embryonic axis consists of the primitive streak and the tail bud. Small
fragments of this caudal tissue were transplanted from mouse embryos of various developmental stages to the kidney capsule in order to test their histogenetic capacity. The variety of
mature tissues obtained from these small fragments was similar to that obtained by grafting
a larger caudal portion of the embryo. Initially, the grafted tissue broke up into loose masses
of embryonic mesenchyme and this was later re-organized into more compact tissues and into
cysts that were lined with various types of epithelia. After 14 days in the ectopic site, grafted
tissues coming from embryos of the primitive-streak, the early-somite and the forelimb-bud
stages differentiated into structures that has presumably originated from the three embryonic
germ layers. Many of these structures were related to the caudal region of the adult body, such
as the mid- and hindgut segments and urogenital derivatives. The histogenetic capacity for
endodermal tissues and urogenital organs was lost when the grafted tissue consisted entirely
of the tail bud of the hindlimb-bud-stage embryos. The behaviour of the caudal tissues suggested that (1) the primordia for the various parts of embryonic body were derived from a small
progenitor population in the primitive streak and the tail bud, and (2) the histogenetic capacity
of this population changed during development.
INTRODUCTION
The embryonic body pattern of the mouse is laid down in a rostrocaudal
direction by the continuous addition of tissues at the caudal end of the embryo.
This caudal tissue of the embryo is made up successively of the primitive streak
and the tail bud. The primitive streak is formed in the egg cylinder and it persists
until the embryo has made 26-29 somites and the posterior neuropore had closed
(Tarn, Meier & Jacobson, 1982). The primordium of the tail bud appears early
in the forelimb-bud-stage embryo (13-20 somites) at the posterior end of the
primitive streak (Rugh, 1968). A definitive tail bud is formed and replaces the
primitive streak when the embryo has reached the hindlimb-bud stage (Theiler,
1972).
The developmental potential of the caudal tissue has been studied in the early
mouse embryo by following its development in vitro, in ectopic sites and after
grafting to another embryo. When the isolated embryonic fragment containing
the primitive streak of the 7-5-day egg cylinder was cultured in vitro, it formed
rudimentary tail, hindgut, primordial germ cells and allantois (Snow, 1981).
254
P. P. L. TAM
When the cells in the primitive streak area were transferred microsurgically to
the homotypic site in another embryo, their progenies mainly contributed to the
tissues in the caudal region of the chimaeric embryo (Beddington, 1982). When
the primitive streak tissue was grown in an ectopic site, a whole array of tissues
that was presumably derived from all three embryonic germ layers was found in
the teratoma. There was no indication of any regional restriction in the histogenetic capacity since tissues belonging to both cranial and caudal body levels
were formed from the anatomically posterior embryonic fragment (Beddington,
1983). A similar absence of regional differences in histogenetic capacity was also
found in the primitive-streak-stage rat embryo (Svajger, Levak-Svajger,
Kostovic-Knezevic & Bradamante, 1981). A more distinctive regionalization in
histogenetic capacity was seen in mouse embryos by the early-somite stages.
When the caudal portion was grafted to ectopic sites, it gave rise to all tissue types
except respiratory epithelium which was typically found in grafts of the cranial
portion (Fujimoto & Yanagisawa, 1979). At the head-fold stage, the posterior
(streak) fragment of the rat embryo gave rise to, amongst other tissue types,
structures specific to the mid- and hindgut (Svajger & Levak-Svajger, 1974;
Levak-Svajger & Svajger, 1974). These observations thus suggested a
progressive restriction in the histogenetic capacity of the posterior region of the
developing embryo. It is not known, however, whether such a change was due
to the presence of tissues of a more committed fate or to the temporal change in
the capacity of the progenitor tissue in the primitive streak. The histogenetic
behaviour of the caudal tissue in more advanced embryos has not been specifically studied. Large fragments of the caudal region of the forelimb-bud-stage (9-5day) mouse embryo which included axial structures, presomitic mesoderm and
other caudal tissues had been grafted to ectopic site. In comparison to the graft
of the more rostral portion, the resultant teratoma showed a more extensive
development of the cutaneous tissues and the alimentary tube (Bennett, Artzt,
Magnuson & Spiegelman, 1977; Fujimoto & Yanagisawa, 1979). In the present
study, small fragments of the caudal tissue which included the primitive streak
and/or the tail bud were taken from mouse embryos of different developmental
stages. The differentiation of these fragments under the kidney capsule was then
followed to study their histogenetic capacity and the morphogenetic behaviour
of the caudal tissues.
MATERIALS AND METHODS
Two strains of mice were used: C3H/He was from the MRC Mammalian
Development Unit, University College London and ICR was kept in the Chinese
University of Hong Kong. Female mice were paired with males of the same
strain. Embryonic age was estimated by taking the afternoon on the day of plug
as 0-5 dayp.c. Embryos of matching developmental stages were recovered from
the two strains of mice at 7-5,8-5,9-5 and 10-5 daysp.c. The ectoplacental tissue
Differentiation of caudal tissues
255
and extraembryonic membrane were removed with electrolytically polished
tungsten or alloy needles. The remaining embryonic portions were further
dissected for transfer to the kidney capsule.
Primitive-streak-stage (7-5-day) embryos were first bisected into an anterior
portion containing the presumptive cranial region and a posterior portion
(= large fragment) containing the primitive streak (see Snow, 1981). A smaller
fragment (= primitive streak fragment) was obtained by trimming the large
fragment to remove as much of the tissue lateral to the streak as possible (Fig.
1) and the distal third of the streak.
Early-somite-stage (8-5-day) embryos (somite no. = 6-8 ± 0-3 (n = 12)) were
first bisected into a cranial half and a caudal half. The caudal half was trimmed
to leave behind the region posterior to the last formed somite (large caudal
fragment). A small portion of the caudal area was excised by making a transverse
cut at about 500-600 /im from the tip of the embryonic axis to give the streak +
bud fragment (Fig. 2).
Forelimb-bud-stage (9-5-day) embryos (somite no. = 24-0 ± 0-6 (n = 25))
were transected at the level of the last formed somite to give the large caudal
fragments. A further dissection was made by cutting the tail region (at
approximately 600 jum from the tip of embryonic axis) just posterior to the caudal
neuropore to produce the streak + bud fragment (Fig. 3).
The growth of the large caudal fragments of hindlimb-bud-stage (10-5-day)
embryo (somite no. = 41-0 ± 0-9 (n = 23)) was not studied because of the technical difficulty involved in the grafting of such large fragments under the kidney
capsule. Only the tail bud containing a mass of mesenchyme and the encapsulating epidermis was excised for transfer (Fig. 4).
Several small fragments of the primitive streak and tail bud from each dissection were fixed in Karnovsky (1965) fixative and embedded in Spurr resin. Thick
(1-2 /-on) sections were cut on a Reichert-Jung Ultracut microtome and were
stained with 1 % toluidine blue for light microscopy.
The embryonic fragments were transferred individually to the space under the
kidney capsule of syngenic males under Nembutal anaesthesia using fine glass
micropipettes. Recipients were killed by cervical dislocation at 3, 7, 14 and 30
days after the transfer. The teratoma derived from the graft wasfixedin Sanfelice
fluid and embedded in wax for routine histology. Serial 7jum sections were
obtained from most specimens and they were stained with haematoxylin and
eosin, Masson's trichrome stain or PAS stain. Some sections were stained with
silver method for nerve fibres and cresyl violet for ganglionic cell bodies (Bancroft, Stevens & Dawson, 1977). Some specimens were fixed in Karnovsky
fixative, sliced in cacodylate buffer, post-fixed in osmium tetroxide (Kelley,
Dekker & Bluemink, 1973) and critical-point dried for examination in a JEOL
JSM-35CF scanning electron microscope. The presence of various types of tissue
in the teratoma was recorded and the relative amounts of the different types of
tissue were estimated semiquantitatively on a three-point scale (Table 2).
256
P. P. L. TAM
#% %
*# f
Differentiation of caudal tissues
257
Table 1. Development of embryonic fragments under the kidney capsule
No. recovered at
Embryonic tissue
No. transplanted 3 days
7 days 14 days 30 days Total (%)
7-5-day embryo
Large posterior fragments
Primitive streak
22
22
—
5
—
8
—
8
12
—
12 (57)
21 (95)
8-5-day embryo
Large caudal fragments
Streak + bud
5
38
—
9
—
11
—
11
5
—
5 (100)
31(82)
9-5-day embryo
Large caudal fragments
Streak + bud
5
42
—
14
—
11
—
10
5
—
5 (100)
35(83)
10-5-day embryo
Tail bud
24
6
6
7
—
19(79)
RESULTS
Twelve out of the twenty-one large posterior fragments of the primitivestreak-stage embryos formed a teratoma when transferred to the kidney capsule
and all the large caudal portions of early-somite and forelimb-bud-stage embryos
formed teratomas (Table 1). A variety of mature tissues which had presumably
originated from the three embryonic germ layers was formed. Some embryonal
tissues such as the mitotically active pseudostratified epithelium and undifferentiated mesenchyme resembling embryonal carcinoma cells were also found,
particularly in teratomas derived from primitive-streak-stage embryos. The most
prevalent tissues formed were those of the skin, neural tissue, cartilage and
ossifying bones (Fig. 5), smooth and striated muscle (Fig. 6), epithelial cysts
(Fig. 7) and segments of the alimentary tract. Cutaneous tissues were often
organized in the proper topographical relationship to one another (Fig. 8). The
composition of tissues in the teratomas derived from the large posterior/caudal
Fig. 1. A transverse section of the primitive streak fragment of 7-5-day egg cylinder.
p = primitive streak, m = mesoderm, ec = ectoderm, en = endoderm.
Fig. 2. A sagittal section of the streak + bud fragment of 8-5-day embryo, which is
composed of the caudal end of the primitive streak (p), some lateral surface
ectoderm and embryonic mesenchyme (m). ca = caudal artery.
Fig. 3. An oblique section of the streak + bud fragment of 9-5-day embryo, containing the primitive streak (p), the tail-bud mesenchyme (m) and some presumptive
hindgut endoderm (en).
Fig. 4. A horizontal section of the tail-bud fragment of 10-5-day embryo,
m = mesenchyme, nc = condensed mesenchyme of the presumptive notochord.
Scale bar equals 100 jum for Figs 1-4.
258
P. P. L. TAM
Differentiation of caudal tissues
259
Table 2. The tissue composition ofteratomas derived from large posterior/caudal
embryonic fragments
7-5-day
8-5-day
9-5-day
12
5
5
8+
9+ + +
1+
10+ +
0
8+
5+ + +
5+ + +
5+ +
4+
0
4+
5+ +
4+ +
5+ +
5+
0
4+
Striated muscle
Smooth muscle
Bone
Cartilage
Adipose tissue
10+ + +
9+ + +
8+ +
8+ + +
6+ +
5+ + +
5+ + +
5+ + +
3+ +
4+ + +
5+ + +
5+ + +
5+ +
5+ +
5+ + +
Ciliated epithelium
Gut epithelium
Glands
11 + + +
9+ +
5+
3+
5+ + +
4+ + +
4+
5+ +
5+ +
3+ +
5+ + +
4+ 5+ +
No. of teratomas examined
No. containing:
Stratified epithelium
Karatinized epithelium
Hair, sebaceous gland
Neural tissue
Lens
Pigment epithelium
Epithelial cysts
Intestine
4+
3+
fragments of the 7-5-day, 8-5-day and 9-5-day embryos was generally similar.
However, the large 7-5-day fragments tended more frequently to form ciliated
epithelium of the respiratory tract and alimentary tube of both the pharyngeal
and intestinal types (Table 2). The teratomas derived from the large caudal
fragments of 8-5-day and 9-5-day embryos showed much more prolific differentiation of skin structures and the intestine (Table 2). Mesodermal derivatives
such as muscle, adipose and skeletal elements were the most predominant
tissues. Tubular and cystic structures that were lined with cuboidal glandular
epithelium or intestinal epithelium were commonly found.
Figs 5-8. Some common types of tissues found in the teratoma derived from
embryonic fragments as seen under the SEM. Scale bar equals 100 jum for Figs 5, 7
&8; lOjumforFig. 6.
Fig. 5. Cartilage (c) and ossifying bone (b) with marrow.
Fig. 6. Striated muscle fibres organized into bundles.
Fig. 7. Epithelial cyst lined with stratified squammous epithelium showing early
signs of desquamation.
Fig. 8. Skin-like structure with keratinized epithelium (k), hairs with follicles
(arrow head), layers of epidermis (e) and loose connective tissues and adipose
tissue of the dermis (d).
260
P. P. L. TAM
At the time of grafting, the caudal fragment of the 7-5-day embryo consisted
of the primitive streak and some adjacent ectoderm, mesoderm and endoderm
(Fig. 1). The caudal fragment of the 8-5- and 9-5-day embryo was made up of
primitive streak and a mass of mesenchymal cells which was found ventrolateral
to the primitive streak (Figs 2 & 3). These mesenchymal cells were polygonal to
stellate in shape, loosely packed together and made focal junctional contacts
with neighbouring cells. Occasionally, in the 8-5-day and 9-5-day caudal fragments, a short segment of the hindgut was present. The caudal fragments of 10-5day embryo consisted mainly of undifferentiated mesenchymal cells. The cells
were more closely packed in regions close to the caudal end of the neural tube
and notochord (Fig. 4).
Three days after grafting to the kidney capsule, the epithelial tissues in the 7-5day primitive streak fragment formed an admixture of embryonic cells, invading
Table 3. The types of tissues formed by the primitive streak and tail bud grown
under the kidney capsule
7•5 day
Age of embryos
3 7 14
9 11 9
7 14
9 9
Time after grafting (days)
No. of specimens
3
5
7 14
8 8
Tissues
Stratified epithelium
Keratinized epithelium
Hair, sebaceous gland
Neuroepithelium
Mature neural tissue
Peripheral ganglia
1
0
0
2
0
0
8
0
0
5
8
5
3
7
6
3
6
3
2 10
0 3
0 1
1 4
0 8
0 1*
6
8
6
0
4
2*
3
0
0
8
4
0
7
5
2
2
7*
2*
Embryonic mesenchyme
Connective tissues
Differentiating myoblast
Striated muscle
Smooth muscle
Cartilage
Bone
Adipose tissue
5
0
0
0
0
0
0
0
4
8
6
0
0
7
1
0
0
5
1
6
7
7
7
7
9 10
3 9
0 9
0 2
0 8
0 6
0 2
0 3
0
2
0
7
6
7
4
6
10
0
1
0
0
1
0
0
Epithelial cyst
Ciliated epithelium
Gut epithelium
Intestine
Glands
0
2
1
0
0
7
0
6
0
4
7
4
7
7
6
2
0
1
0
0
8
0
8
2
5
6
0
5
4
6
Urogenital structures
Gonad, phallus,
bladder, kidney
0
0
4
0
2
3
* Present in trace amounts.
10•5 day
9-5 day
8•5 day
3
5
7 14
6 6
6
8
9
1
5*
0
0
0
0
0
0
0
3
6
0
0
0
0
6
6
6
0
0
1*
3
6
5
3
6
5
2
2
0
5
1
9
7
7
7
9
5
0
0
6
0
0
0
0
1
0
0
0
0
6
0
0
0
6
3
4
0
6
6
6
7
1
1
0
2
3
1
2
2
3
3
0
1
5
6
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
2
4
0
0
0
3
10
Differentiation of caudal tissues
261
blood vessels and fibrous connective tissue. Occasionally, a small portion of the
graft retained an epithelial appearance. The streak -I- bud fragments of 8-5- and
9-5-day embryo similarly dissociated into masses of loosely packed mesenchymal
cells. However, in addition there were epithelial cysts with either a single layer
of round to cuboidal epithelium or a more elaborate layer of stratified to
pseudostratified epithelium. The tail bud fragment of 10-5-day embryo typically
formed a mesenchyme-like mass of loosely packed cells, with isolated compact
clusters of cells bound peripherally by a basal lamina.
The embryonic tissue underwent further differentiation during the first week
of growth (Table 3). Cystic structures lined by stratified epithelium were formed.
The primitive neuroepithelium differentiated into the more mature neural tissue
(cresyl-violet-stained cell bodies and silver-positive neuronal processes) and a
mantle layer was formed on the periphery of the ependyma. The mesenchyme
was gradually replaced by the mature mesodermal derivatives. The myoblasts
were organized into myotubes and finally into striated muscles and smooth
muscles. The chondrocytes laid down extracellular matrix of the cartilage and
small areas of the cartilage model began to ossify into bones. The gut epithelium
containing the mucus-goblet cells folded extensively and acquired a smooth
muscle coat and glandular tissue of the tubular and acinar type was present. In
those cysts with stratified epithelium, some keratinization were seen in the luminal
stratum and hair follicles and sebaceous glands were developing in the dermis.
After growing for two weeks under the kidney capsule, the caudal fragments
of 7-5-, 8-5- and 9-5-day embryos developed into teratomas containing a multitude of tissue types (Table 3). Most teratomas derived from the 7-5-day primitive
streak contained skin structures, neural tissues, muscle, bone and cartilage.
Large numbers of epithelial cysts and glandular tissues were present. There was
organotypic differentiation of the intestine (Fig. 11) and, in four cases,
urogenital structures such as the spongy tissue of the phallus (Fig. 9), the foetal
kidney (Fig. 10) and the urogenital sinus (Fig. 12) were formed. Similar intestinal
and urogenital tissues were found in teratomas derived from 8-5- and 9-5-day
small caudal fragments. However, there was discernibly less mature neural tissue
in 8-5-day grafts and it was further reduced to a trace quantity in 9-5-day grafts.
Epithelial cysts were less frequently encountered. Ciliated epithelium presumably of the respiratory type was absent but intestinal and urogenital derivatives
were present and were morphologically more differentiated. Differentiated
tissues usually appeared earlier in the developing teratomas of 8-5- and 9-5-day
grafts than those of 7-5-day graft. By 14 days, embryonic cells were absent in the
teratoma of older grafts and only some differentiating neural ependyma
remained in the 7-5-day grafts, suggesting that further tissue differentiation was
unlikely to occur. The tissue composition of the teratomas derived from the small
fragments was generally similar both in types and relative abundance to those
derived from a more substantial caudal portion of the same stage embryo. The
10-5-day tail bud grew less extensively than all other groups and they formed
262
•11
P. P. L. TAM
Differentiation of caudal tissues
263
smaller teratomas. During the early stages of growth, the bud differentiated into
an aggregated mass of embryonic mesenchyme with an occasional cartilage
nodule, several islets of chondrocytes and some glandular cells surrounding a
small cyst. In the final teratoma, only cutaneous tissues and mesodermal tissues
were found. The neural and endodermal tissue were both absent and urogenital
organs were never observed.
DISCUSSION
The present study on the differentiation of small fragments of caudal tissues
has shown that such fragments were capable of extensive histogenesis in the
ectopic site. Furthermore, the composition of tissues formed by these fragments
was qualitatively similar to that produced by the grafting of a much larger caudal
portion of the embryo. These findings suggest that tissues associated with the
caudal fragments were developmentally pleuripotential in nature. Morphologically, these fragments were mainly made up of the primitive streak and/or the tail
bud. The ultrastructural appearance of mesenchymal cells derived from these
two sources was very similar (Tarn, unpublished observation) and showed no
morphological characteristic that indicated any future direction of differentiation. It has been proposed that normal formation of the somitic pattern of the
embryonic body depends on the active generation of primordial tissues in the
primitive streak and the tail bud (Tarn, 1981; Tarn et al. 1982). The results of the
present study suggest that the primordia of other components of the body pattern
may also be similarly generated from the same progenitor population of cells in
the caudal area.
A comparison of the types of tissues formed by caudal fragments of four
developmental stages further indicates a progressive restriction of the histogenetic potential of the progenitor population during development. The younger
caudal fragments generated adult tissues belonging to both the cranial and caudal
body levels (see also Svajger etal. 1981; Beddington, 1983) but older fragments
tended to give tissues that were predominantly found in the caudal level of the
Figs 9-12. Some structures which were derived from grafts of caudal fragments.
Scale bar equals 100/im.
Fig. 9. A structure resembling the developing phallus of the foetus, with a central
core of erectile spongy tissue (sp) similar to that of corpus spongiosum penis (insert)
and superficial layer of skin (k). H&E.
Fig. 10. Clusters of convoluted tubules (t) and collecting ducts (d) in a foetal kidney.
Masson's trichrome.
Fig. 11. A segment of the intestine with mucosal foldings, the epithelium contained
mucus-secreting goblet cells (arrow). Layers of smooth muscle (sm) were formed
around the intestinal segment. Masson's trichrome.
Fig. 12. An urogenital sinus lined with layers of transitional epithelium (arrow) and
connective tissue coat. Masson's trichrome.
264
P. P. L. TAM
adult body (see Fujimoto & Yanagisawa, 1979). The histogenetic capacity was
greatly reduced when the caudal fragment was made up entirely of the tail bud
of more advanced embryos and in such case it differentiated primarily to tissues
found in the adult tail. This progressive change in histogenetic capacity may
indeed be a genuine reflection of the functional competency of the streak and the
bud in normal rostrocaudal development of the embryonic body pattern. The
primordia of the various body parts may be generated according to a sequence
at which the various body levels are being laid down. The caudal tissue thus
behaved very similarly to that of the apical mesenchyme of the chick limb bud
(Summerbell & Lewis, 1975) and to the differentiating cells in the tail bud of the
Xenopus embryo (Elsdale & Davidson, 1983).
A common feature of the initial stage of differentiation of the caudal tissues
in the ectopic site was the dissociation of epithelial tissues and the intermingling
of the released cells with the pre-existing mesenchymal cells. The deranged
tissues found at later stages may therefore be the result of an enhanced mixing
of cells which was then followed by the re-organization of cells either into clusters
from which structures like the neural tissues, adipose, muscle and cartilage were
formed or into epithelia that became the gut, skin, urogenital sinus, glands and
other unidentified epithelial cysts. A similar atypical morphogenetic behaviour
was also observed in the differentiation of embryonic ectoderm of rat embryos
under the kidney capsule (Svajger et al. 1981). The mixing of cells may have
created a much greater variety of tissue interaction and environmental cues
normally not experienced by the embryonic tissues had the normal relationship
been maintained as in the embryo. This may be the major reason for the apparent
discrepancy found in the extent of expression of developmental potential by
embryonic tissues in studies where they were allowed to differentiate in normal
embryonic surroundings on the one hand, and where they developed in ectopic
sites on the other hand (Beddington, 1983). The extent of tissue differentiation
in the teratomas thus reflects the maximum histogenetic capacity possessed by
the caudal fragments.
The contribution of the caudal tissue to the various body structures has been
studied more directly by observing the nature of deficiency in the embryo with
an extirpated caudal area and the autonomous development of the isolated
pieces. In the primitive-streak-stage mouse embryo, extirpation of the posterior
end of the primitive streak resulted in a truncated body axis ending in a rudimentary tail. The embryo lacked the hindgut, allantois and primordial germ cells
(Snow, 1981). In the early-somite-stage embryo, the removal of the primitive
streak and the tail bud resulted in a cessation of the somite formation (Tarn et al.
1982) and the truncation of the axis (Smith, 1964). In early chick, quail and
snapping turtle embryos the extirpation of the whole node and streak area always
resulted in the termination of axis formation and prevented any further production of the-somitomeric pattern (Packard, 1978,1980; Packard & Meier, 1983).
In the chick embryo, transferring the tail bud to another embryo resulted in the
Differentiation of caudal tissues
265
colonization of tissues in the host tail and very seldom in other parts of the body
(Schoenwolf, 1977). The isolated tail bud, however, differentiated poorly when
grafted to the chorioallantoic membrane (Schoenwolf, 1978). Extirpation of only
the tail bud area leaving the streak left most of the trunk structures undisturbed
even though the extirpation was done on early embryos. This suggested that the
major portion of the trunk was derived from the streak and the tail bud
contributes only to tail structures (Schoenwolf, 1978) and that the intact streak
could still maintain some degrees of normal morphogenesis up to the stage when
tail bud contribution is required. It is believed that the transition from a streak
contribution to a purely bud contribution occurred at the lumbosacral level of the
embryo (Criley, 1969; Jelinek & Rychter, 1979; Schoenwolf, 1978, 1979). The
cellular activity in the caudal tissues is therefore crucial to the formation of the
embryonic axis and other related body patterns and such activity is likely to be
limited to a very small population of cells.
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{Accepted 21 March 1984)