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/. Embryol. exp. Morph. Vol. 22, 2, pp. 295-304, September 1969
Printed in Great Britain
295
Effects of a mesoderm-inducing factor
on early chick embryos
By E. M. DEUCHAR 1
From the Anatomy Department, University of Bristol
During recent years Tiedemann and his co-workers have developed methods
for extracting and purifying neural and mesoderm-inducing factors from
homogenates of 9-day-old chick embryos (see Tiedemann, 1966). One type of
extract, obtained initially by phenol treatment of material precipitated with
ammonium sulphate from a pyrophosphate extract of chick embryos (Tiedemann,
1959) appeared to induce mesoderm in a high proportion of cases, when
implanted into Triturus gastrulae. It has been purified by chromatography on
CM-cellulose (Tiedemann, 1959; Tiedemann, Kesselring, Becker & Tiedemann,
1961) and by electrophoresis on Dextran gel (Kocher-Becker, Tiedemann &
Tiedemann, 1965). Triturus larvae which have received implants of mesoderminducing factor at the gastrula stage show extra notochord, muscle and kidney
within their ventrolateral mesoderm. From the illustrations in these authors'
papers, there can be no doubt at all as to the identity of these induced tissues.
Since, however, they develop within areas that would normally have formed
mesoderm of another kind, one may infer that in these cases there has been
no transformation of cells other than mesoderm: rather, the type of mesoderm
has been altered to somite myoblasts, chorda and kidney cells instead of lateral
mesothelium. In tests by the 'sandwich' technique (Holtfreter, 1933) however,
which were evidently also used but not reported in detail (Tiedemann, 1959)
any mesoderm that formed would have to be by transformation of ectoderm
cells. In earlier experiments on the effects of a crude protein extract from chick
embryos on isolated amphibian ectoderm in tissue culture (Becker, Tiedemann &
Tiedemann, 1959) transformations into mesenchyme, myoblasts and chorda
cells were certainly shown to occur. It seems then that a 'mesoderm inductor'
may have two kinds of effect: an enhancement of the growth of mesoderm,
transforming it also into more dorsal, axial tissues; and a transformation of
ectoderm into cells of all mesoderm types. Whether or not endoderm cells can
be transformed by the inductor is not fully clear: Kocher-Becker et ah (1965)
described an apparent migration of endoderm cells outwards over the surface
of Triturus gastrulae which contained implants of highly purified mesoderm
1
Author's address: Anatomy Department, The Medical School, University Walk, Bristol,
BS8 1TD.
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E. M. DEUCHAR
inductor, but they interpreted this as due to ectoderm transformation (with
a resultant increase in affinity between ectoderm and endoderm) rather than
suggesting that the endoderm itself had acquired any mesoderm-like properties.
It would seem desirable to extend tests of the action of Tiedemann's inducing
factors to other kinds of cells in culture. Most workers judge the amphibian
embryo to be an ideal test system for biochemically-prepared inductors. Yet
one could argue that this judgement is rash and that the 'idealness' of this
embryo is due to its cells being peculiarly capable of giving dramatic, varied
responses to foreign implants. This was in fact shown to be the case, during
the 'gold-rush' of tests for neural inductors in the 1930's, when it was found
that numerous materials ranging from Invertebrate tissues to pieces of silica
could produce a neural response in amphibian ectoderm (Needham, 1942).
Eventually it was realized that such 'inductions' were a property of the ectoderm
rather than of the implant and bore little relation to events in normal development.
When dealing with inducing agents extracted from chick embryos, one possible
criterion that could help in deciding whether similar factors operate in normal
development would be a test of these inductor's effects in embryos of their
own species. So far, no successful attempts appear to have been made to carry
out such tests. Grafting experiments are not easy to do in large numbers on
early chick embryos, and from preliminary trials McCallion (see Tiedemann,
1966) concluded that many regions of the early chick blastoderm had already
lost any competence to form mesoderm. Since considerable areas of the surface
layer of the blastoderm at primitive streak stages are, in fact, destined to form
mesoderm (Pasteels, 1940) it is not clear to which regions McCallion's remarks
apply. Rawles's (1936) first detailed mapping experiments indicated that there
were still wide ranges of competence in blastoderm areas at head process
stages.
With the above considerations in mind, a series of experiments has been
carried out in which samples of Tiedemann's mesoderm-inducing factor have
been inserted as grafts between endoderm and ectoderm layers of explanted
early chick embryos, in order to test the effects on ectoderm, mesoderm and
endoderm of the host. These experiments are described below.
MATERIAL AND METHODS
Chick embryos of 16 hours' incubation (primitive streak stages 3-4 of
Hamburger & Hamilton, 1951) were set out in watch-glass cultures according
to New's (1955) method. Tiedemann's inductors 538/2 (phenol extract) and
538/12 (CM-chromatography extract) were stored at - 25 °C before use. A small
piece of the solid inductor (ca. 2 mm diameter) was soaked for a few seconds in
a drop of sterile 1% aqueous Nile blue solution, then a graft piece, not more
than 0-2 mm diameter was cut off it with iris knives. The graft was inserted
Mesoderm-inducing factor
297
under the endoderm of an explanted embryo, either in a region lateral to the
node or as near the anterior end of the area pellucida as possible (Fig. 1).
Embryos were left to incubate for a further 20-24 h, by which time they had
reached stages 8-11 (Hamburger & Hamilton, 1951) with from 4-10 pairs of
somites. Those embryos in which the position of the graft (identifiable by its
blue colour) was clear were then fixed in Bouin's fixative, dehydrated in alcohol,
cleared in methyl benzoate and embedded in paraffin wax. 10/* transverse
sections were stained with Weigert's haematoxylin and eosin. A total of 111
experimental embryos was examined. 34 controls received grafts of human yglobulin.
Primitive
streak
Area opaca
Fig. 1. Diagram showing the two alternative sites in which grafts were placed
(1 and 2) between upper and lower blastoderm layers, at the primitive streak
stage.
RESULTS
1. Phenol extract (538/2)
Of the 60 embryos examined histologically, 21 had graft material within
mesoderm tissue: in six of these, it lay within mesoderm of the heart wall and
in the other 15, within lateral mesoderm. In 12 embryos the graft lay in contact
with endoderm: in one of these it was in the fore gut. Finally, there were 23
embryos with grafts in contact with the neural tube, and seven in which the
graft was in contact with epidermis.
(a) Effects on mesoderm. In all 21 cases where the graft was in contact with
mesoderm, there was a marked thickening of this tissue, mainly due to proliferation of cells, which surrounded the graft. These cells were rounded and
mitotic figures could be seen in several of them. In addition, some fibroblast-like
cells were present and in six of the 21 embryos examined, these cells had
invaded the graft. It was not clear whether they represented true fibroblasts, or
possibly myoblasts (since the cells of the somite tissue in these embryos were
similar in shape to fibroblasts at this stage) (Fig. 2 A.). The effects on mesoderm
are summarized in Table 1.
(b) Effects on endoderm. Six of the embryos of this series showed grafts
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E. M. DEUCHAR
Fig. 2 (A). Graft (pale areas, marked '#') surrounded and invaded by mesoderm
cells (m), somefibroblast-like(/). Transverse section, x 325. (B) Thickened, irregular
epidermis (<?) overlying graft material (g). T.S., x 640. (C) Detached cells of brain wall
(b). Some cells fibroblast-like (/) and wandering either outwards into surrounding
tissue space, or inwards into neural canal. Other cells necrotic (n). T.S., x 325.
(D). Neural tube with enlarged neural crest (nc) on side of graft (g). T.S., x 325.
Mesoderm-inducing factor
299
within endoderm of the head fold (pro-amnion) and 5 others had a graft within
lateral extra-embryonic endoderm. In only one case was the graft in contact
with endoderm within the embryo: this was the one where it lay in the fore gut.
Adjacent to it, the wall of the fore gut was slightly thickened in comparison
with the wall on the side furthest from the graft (Fig. 3B). There appeared,
however, to be no change in the histological characteristics of these endoderm
cells, other than proliferation.
Table 1. Reactions of mesoderm to grafted inductors
Effect of inducer
1. Phenol extract
Embryos examined
Cells proliferated round graft
Fibroblast-like cells invaded graft
2. CM-chromatography extract
Embryos examined
Cells proliferated round graft
Cells invaded graft
Extra blood-island cells
Total cases
21
21
6
13
13
1
1
In two of the six cases where the graft lay in head fold endoderm, some cells
of this layer were fibroblast-like and had penetrated the graft. In the other
four cases, the endoderm cells had simply proliferated to surround the graft.
In one of the five embryos with grafts in lateral endoderm, there were also
fibroblast-like cells which had penetrated the graft: the other four cases showed
just a proliferation of cells to enclose the graft. Table 2 summarizes these
effects on endoderm.
(c) Effects on ectoderm. The seven embryos in which graft material lay in
contact with epidermis showed no effect other than a very slight thickening or
irregularity of this layer, overlying the graft. (Cf. Fig. 2B.)
Among the 23 embryos in which grafts lay in contact with neural tissue,
however, there was evidence of more radical effects. Thirteen of these embryos
showed patches of cells in the neural tube wall that were detached and had
spread either inwards into the neural canal or outwards into the surrounding
tissue spaces (Fig. 2C). Some of these cells were elongated in shape, more like
fibroblasts than neural cells. There were also some necrotic cells in these
regions.
Three other embryos of this group are of interest: in them the neural crest
appeared enlarged on the side next to the graft (e.g. Fig. 2D). The remaining
seven embryos of this series showed no evidence of transformation of neural
cells. The only effects of the graft in these were purely mechanical: wounding,
breakage and displacements of cells. Table 3 summarizes the effects on ectoderm.
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E. M. DEUCHAR
Fig. 3 (A). Graft (g) invaded by lateral plate mesoderm cells (m). T.S., x 780.
(B) Thickened endodermal wall of fore gut (/) on side next to graft (g), as compared
with normal thickness on side remote from graft (c). T.S., x 150. (C) Thickened
lateral endoderm (e) enclosing graft (g). T.S., x 500. (D) Detached, fibroblast-like
cells if) in neural tube wall (/?). Compare Fig. 2C. T.S., x 640.
Mesoderm-inducing factor
301
Table 2. Reactions of endoderm to grafted inductors
Effect of inducer
Total cases
1. Phenol extract
Embryos examined
Thickened epithelium next to graft
Cells proliferated round graft
Fibroblast-like cells invaded graft
2. C M-chromatography extract
Embryos examined
Thickened epithelium next to graft
Cells proliferated round graft
A few cells de-epithelized
12
1
8
3
13
10
2
1
Table 3. Reactions of ectoderm to implanted inductors
Number of cases
r
Effect of inducer
1. Phenol extract
Total embryos examined
Thickened epithelium next to graft
Cells detached and fibroblast-like
Neural crest enlarged
2. CM-chromatography extract
Total embryos examined
Proliferated epithelium next to graft
Cells detached and fibroblast-like
Neural crest enlarged
No effect other than wounding
Epidermis
Neural
crest
Total
7
6
—
—
23
—
13
3
30
6
13
3
4
4
—
—
—
12
—
6
1
5
16
4
6
1
5
2. CM-Chromatography extract (538/12)
Fifty-one embryos that had received grafts of this material were examined.
Of these, 13 had grafts lying within mesoderm: four within heart mesoderm
and nine within lateral mesoderm. A larger number (19) had grafts within
endoderm: in nine of these, the graft lay in the fore gut, in two others it was
in the head fold, and in the remaining eight the graft lay in lateral extraembryonic endoderm. Thirteen specimens showed graft material in contact
with the neural tube and a further four specimens had grafts in contact with
epidermis.
(a) Effects on mesoderm. On the whole there were less marked reactions of
the mesoderm to this chromatography extract than to the phenol extract,
described above. In all 13 cases, mesoderm cells had proliferated to surround
the graft, but as the grafts were much smaller than the phenol extract grafts
(perhaps because some of this crystalline chromatographed product had been
dissolved away by enzymes of the host) little proliferation was needed in
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E. M. DEUCHAR
order to engulf the graft. There was only one case where mesoderm cells had
invaded the graft (Fig. 3 A). In one other case an extra blood island appeared
to have been formed as a result of mesoderm proliferation near the graft. The
effects on mesoderm are summarized in Table 1.
(b) Effects on endoderm. In five out of the nine embryos with grafts in the
fore gut, there was a thickening of the fore-gut wall near the site of the graft
(Fig. 3B). In one other case, endoderm cells had proliferated to surround the
graft. The remaining three embryos showed no effect and in them the graft
was very small (appearing in only two successive 10/* sections). Among the
eight embryos with grafts in contact with lateral endoderm, five showed thickening of this endoderm layer (cf. Fig. 3C). Again, in only one specimen was the
graft surrounded by proliferated endoderm cells. Another case showed apparent
de-epithelization of the endoderm near it. The remaining one embryo showed
no effect, but here it -was found that the graft had become detached and lay at
some distance from the endoderm (i.e. outside the embryo). Table 2 summarizes
the effects of this extract on endoderm.
(c) Effects on ectoderm. Of the very few (four) cases where the graft lay in
contact with epidermis, three showed a proliferation of this layer. In two of
them, it was thrown into folds and in the third, epidermal cells enclosed the graft.
The remaining specimen which showed no effect had an extremely small graft.
Grafts lay in contact with neural tissue in 12 embryos of this series. Five of
these showed changes in cell shape and detachment of cells from the neural
wall (Fig. 3D) as described in section l(c). One embryo had an enlarged
neural fold on the side nearest the graft: another showed irregularities in the
brain wall as if a few cells were beginning to detach. The remaining five
embryos showed no sign of any effects, other than mechanical damage by the
graft. Results of this series are summarized in Table 3.
The 34 controls, given grafts of y-globulin, were histologically normal.
DISCUSSION
Taken as a whole, these results show relatively small effects with these
mesoderm inductors, compared with their effects on Amphibian embryos
described by Tiedemann and his co-workers (loc. cit.). It is questionable whether
the reactions described here in the mesoderm (i.e. proliferation, invasion and
enclosure of grafts) should be regarded as in any sense 'inductions'. They are
more like the normal defence reactions of tissues to foreign bodies: such
bodies are usually encapsulated and, if possible, invaded by mesenchyme cells
as a preliminary to their destruction. The much reduced final size of the chromatography extract grafts (series 2) suggests that they had undergone some
dissolution by host tissue enzymes.
The reactions of endoderm are even less like any kind of 'induction'. Like
the mesoderm, these cells tended only to undergo such changes as proliferation
Mesoderm-inducing factor
303
or thickening, which enabled them to surround the graft or at least to resist
any deep penetration by it. In the extremely few cases where endoderm cells
had invaded a graft, these cells, although described as 'fibroblast-like', were
within the range of forms characteristic of migrating extra-embryonic endoderm:
they did not represent transformations into mesoderm.
The abnormalities observed in neural tissue that was in contact with grafts
of either mesoderm inductor (Series l(c), 2(c)) do seem, however, to represent
some kind of cell transformation. There were very marked changes in shape,
arrangement and staining properties of these cells (e.g. Fig. 2C) which were
detached from the neural wall at the site near the graft, in thirteen of the
embryos in series 1 and five of those of series 2. The changed cells bore a much
closer resemblance to fibroblasts than to any type of ectodermal cell. One may
also wonder whether the enlarged neural crest observed in four other cases
represented a transformation of some cells that would normally have contributed
to the wall of the neural tube, into migratory neural crest cells. It might be
argued that this is a transformation towards a more mesoderm-like cell type.
In conclusion, it can be said that part of the range of effects produced in
amphibian embryos by Tiedemann's extracts which contain mesoderm-inducing
factors, may be evoked in the early chick embryo also. But before it can
be known how characteristic these effects are, further purified extracts of other
kinds of inducing factors need to be tested out on chick embryos by the same
method. New's (1955) culture technique offers good possibilities for tests of
this kind on reasonably large numbers of explants. The chick embryo also
has the advantage over the amphibian as a test system in that there is a wide
choice of sites for grafts. In sites near to the primitive streak, possibilities of
induction of tissues that normally form part of the embryonic axis may be
tested, while in extraembryonic sites the possibility of more subtle transformations of epithelial, mesenchymal and blood cells may be tested.
SUMMARY
Two mesoderm-inducing factors derived from 9-day chick embryos (Tiedemann's phenol extract, 538/2 and his CM-chromatography extract, 538/12)
have been grafted into explanted chick embryos at primitive streak stages, to
test their effects on chick tissues.
Mesoderm of the host showed proliferation and a tendency to surround
and invade the graft. Endoderm showed some proliferation, but rarely any
invasion of grafts. There was virtually no effect on epidermis. In nearly half
the embryos in which the graft was in contact with neural tissue, however,
groups of cells had detached from the neural tube wall and were fibroblast-like
in appearance. It is argued that this may represent transformation into mesoderm.
There was no significant difference between the effects of the two types of
inducing extract.
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E. M. DEUCHAR
RESUME
Ejfets d'un facteur inducteur de mesoderme sur le tres jeune
embryon de Poulet
Deux facteurs inducteurs de mesoderme extraits de Fembryon de Poulet de
9-jours (extrait au phenol, 538/2, de Tiedemann, ainsi que l'extrait chromatographique CM, 538/12, du meme auteur) sont graffes dans des blastodermes
d'embryons de Poulet cultives in vitro. Les greffes sont effectuees au stade de
la ligne primitive. Le but de ces experiences est d'eprouver les effets de ces
facteurs sur les tissus de l'embryon.
Le mesoderme de l'hote presente une nette proliferation et une tendance a
entourer et a envahir le greffon. L'endoderme prolifere egalement, quoique
plus faiblement, mais il est tres rare qu'il envahisse le greffon. II n'y a pratiquement aucun effet sur repiderme. Cependant, dans presque la moitie des
embryons ou le greffon est en contact avec le tube neural, des groupes de
cellules se sont detaches des parois de ce tube et ont pris une apparence fibroblastique. Ce changement de structure pourrait representer une transformation
du tissu neural en mesoderme.
11 n'y a aucune difference significative entre les effets des deux types d'extraits
inducteurs eprouves.
I am very grateful to Professor H. Tiedemann for supplying dried samples of the mesoderm inductor and to Mrs. J. Look and Miss S. Pillinger for technical assistance.
REFERENCES
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(Manuscript received 13 December 1968)