/. Embryol exp. Morph. Vol. 27, 1, pp. 229-234, 1972
Printed in Great Britain
229
Chondrogenesis in chick embryo somites grafted
with adjacent and heterologous tissues
By M. J. O'HARE 1
From the Chester Beatty Research Institute, Institute of Cancer Research:
Royal Cancer Hospital
SUMMARY
A variety of heterologous tissues have been tested for the ability to promote cartilage
differentiation in isolated chick-embryo somites, using a modified chorioallantoic grafting
technique. Of the 12 tissues tested only 3- and 4-day embryonic ectoderm promoted somite
chondrogenesis in somites that fail to chondrify when grafted in isolation. This activity of
ectoderm was evident in grafts of somites isolated with adjacent ectoderm, and in grafts of
somites recombined with ectoderm derived from several sources. Four-day embryonic limbbud ectoderm, including the apical ridge, was capable of promoting somite chondrogenesis,
but to no greater extent than dorsal trunk ectoderm of the same age. It is suggested that the
ability of embryonic ectoderm to promote cartilage differentiation in isolated somites is
associated with its ability to synthesize basement membrane material (sulphated glycosaminoglycans and collagen), in association with adjacent somite mesoderm.
INTRODUCTION
A problem of importance in any proposed inductive interaction is the specificity of that interaction. The existence of other tissues capable of promoting the
same differentiation casts doubt on a unique tissue-specific activity of the
inducer, but may also reveal more general mechanisms whereby the phenotypic
differentiation of a particular tissue type is favoured.
Recent experiments on the organ culture of isolated chick-embryo somites
have revealed that in the in vitro environment factors unrelated to the presence
of inducing tissues may promote the differentiation of cartilage (Ellison, Ambrose
& Easty, 1969). It may be that nutritional supplementation of the organ culture
medium, as employed by Ellison et ah (1969) is merely making good deficiencies
that artificially depress the intrinsic chondrogenic potential of the somites.
Alternatively, it is possible that the 'stabilization' of phenotypic differentiation
observed in these cultures does reflect a response of the somites that may have
a definite morphogenetic role in vivo.
Previous experiments on the specificity of the response of somites to spinal
cord and/or notochord induction are inconclusive. On the one hand, no activity
1
Author's address: Chester Beatty Research Institute, Institute of Cancer Research:
Royal Cancer Hospital. Fulham Road, London SW3 6JB, U.K.
230
M. J. O ' H A R E
of tissues other than spinal cord and notochord has been demonstrated in organ
culture (Avery, Chow & Holtzer, 1956; Stockdale, Holtzer & Lash, 1961). On
the other hand, chorioallantoic cultures of somites indicated that adjacent
tissues other than spinal cord and notochord might promote somite chondrogenesis (Seno & Biiyukozer, 1958). Against the organ culture experiments may
be adduced the fact that under the conditions employed in these experiments,
somites that are now known to possess considerable intrinsic chondrogenic
potential failed to differentiate cartilage. These conditions were thus manifestly
suboptimal for demonstrating somite differentiation, and may have failed to
reveal low levels of cartilage promoting activity in other tissues. The chorioallantoic culture experiments have, however, been subjected to alternative
explanations (Stockdale et al. 1961), and being isolation rather than recombination experiments do not afford a critical analysis of the effect.
This controversial aspect of somite chondrogenesis has been examined using
the modified chorioallantoic grafting technique reported previously (O'Hare,
1972). In this study both isolation and recombination experiments have been
carried out on a more extensive scale than previously attempted. The modified
chorioallantoic grafting technique, using a Millipore filter as graft vehicle, has
been shown to be at least as sensitive in demonstrating somite chondrogenesis
as the best organ culture techniques and superior to them in some respects, and
thus affords a good method for testing the specificity of the somite response.
METHODS
Preparation of somites and other tissues for grafting were as detailed in the
previous paper (O'Hare, 1972), solutions of 3 % trypsin being used to dissociate
the tissue.
Grafts were assembled on pieces of HA-grade Millipore filter prior to grafting.
The slightly adhesive nature of the Millipore surface permitted the tissues to
remain in close proximity during and after the grafting procedure. Grafts were
transferred to the chorioallantoic membrane of 9- to 10-day host embryos, with
the grafted tissues under the Millipore filter in direct contact with the chorioallantoic epithelium.
After 9 days' culture, graft sites were identified by the presence of the Millipore
filter, excised, fixed, embedded and serially sectioned. Sections were stained in
alcian blue/haematoxylin/eosin and scored for differentiated derivatives.
RESULTS
Isolation of somites with ectoderm and endoderm
Grafts were made of groups of four stage 9-12 posterior somites together with
the adjacent ectoderm and endoderm. These somites have been previously shown
(O'Hare, 1972) to consistently fail to differentiate cartilage when grafted in
isolation.
Effect of heterologous tissues on chondrogenesis
231
In one set of experiments grafts were prepared by manual dissection with
ectoderm and endoderm remaining undisturbed in association with the somites.
A second set of grafts was made in which somites plus ectoderm/endoderm
were treated with 3 % trypsin for 1 min before grafting. In the latter grafts the
ectoderm and endoderm were still loosely adherent to the surface of the somites,
but the close connexion between the tissues seen in vivo was largely disrupted.
Results of these experiments are presented in Table 1.
In the presence of undisturbed ectoderm and endoderm, stage 9-12 posterior
somites will differentiate cartilage in 21 % of grafts. Isolated somites consistently
fail to differentiate cartilage in spite of graft viability being demonstrated by
occasional nephric tubules found in the grafts.
Treatment of the somites plus ectoderm and endoderm with trypsin before
grafting results in a fall in the incidence of both cartilage and distinguishable
ectodermal derivatives to 10%. There is no correlation, however, between the
presence in the final 9-day graft of ectodermal (or endodermal) derivatives and
of cartilage.
Striated muscle and bone were not observed in any of these grafts. Nephric
tubules were found in about 10% of grafts of isolated somites and of somites
plus ectoderm and endoderm, but were unrelated to the presence of cartilage.
Recombination of somites with ectoderm
In these grafts, isolated stage 9-12 posterior somites in groups of four were
recombined with a piece of isolated ectoderm prepared from another part of the
embryo. The following sources of ectoderm were tested: 2-day trunk ectoderm,
3-day trunk ectoderm, 4-day trunk ectoderm, and 4-day limb-bud ectoderm.
Results are presented in Table 2.
The recombination of isolated somites with 2-day trunk ectoderm failed to
result in any cartilage differentiating from the somites. Recombination of somites
with 3- and 4-day trunk ectoderm did, however, result in a considerable incidence
of cartilage. The incidence of detectable differentiated ectodermal derivatives
Table 1. Differentiation of somites isolated with
ectoderm and endoderm
Isolated somites
Somites plus ectoderm and
endoderm
Somites plus ectoderm and
endoderm after trypsinizatioii
Cartilage
Ectoderm
Endoderm
0/89*
0%
17/81
21 V
—
—
15/81
19 °/
19/81
24%
14/48
29 °/
- 61 /o
5/48
10%
1
~ /o
5/48
10%
* Number of grafts positive/total grafts recovered.
z
-~ / o
232
M. J. O'HARE
rose from 42% with 3-day ectoderm to 60% with 4-day ectoderm plus
somites, while the incidence of cartilage fell from 30% to 23%. There was
no association of cartilage with differentiated ectodermal derivatives in the
final graft, cartilage being found in grafts both with and without ectodermal
derivatives.
The activity of 4-day limb-bud ectoderm, including apical ectoderm ridge,
was almost exactly the same as that of dorsal trunk ectoderm of the same age.
A total of 70 grafts of 3- and 4-day isolated ectoderm was made without
encountering any instances of cartilage differentiating due to accidental inclusion of mesenchyme. The incidence of distinguishable ectodermal derivatives
in these isolated ectoderm grafts was 30-40 %, being higher with older material.
Ectoderm usually differentiated as keratinized epithelial vesicles. In a few
instances, combined ectodermal/mesodermal structures such as feathers were
found in grafts of 2-day somites with 3- or 4-day ectoderm, but even in these
grafts ectoderm normally differentiated as keratinized vesicles.
The incidence of ectodermal differentiation reported here is probably an
underestimate, as only large ectodermal formations could be distinguished from
the keratinized epithelial 'pearls' that arise as non-specific manifestations of
chorioallantoic epithelial metaplasia (Moscona, 1959).
Somites plus heterologous tissues
A total of 164 grafts of stage 9-12 posterior somites in groups of four were
made in association with a variety of heterologous tissues. The tissues tested
included 3-day optic vesicle, 3-day otic vesicle, 4-day myocardium, 4-day mesonephros, 9-day heart ventricle, 9-day liver, 9-day intestine, 9-day choroid plexus,
9-day forebrain, 9-day spinal cord and polyoma transformed fibroblasts.
In all cases considerable growth and differentiation of these tissues took place
in the grafts, but in no case did cartilage differentiate from the somites. The
viability of the grafted 2-day tissues was demonstrated by the presence of nephric
tubules in 10% of the grafts.
Table 2. Differentiation of somites recombined with ectoderm
Somites plus 2-day
ectoderm
Somites plus 3-day
ectoderm
Somites plus 4-day
ectoderm
Somites plus 4-day
limb-bud ectoderm
Cartilage
Ectoderm
0/21
0%
10/33
30%
7/30
23%
7/29
24%
0/21
0%
14/33
42%
18/30
60%
13/29
45%
Effect of heterologous tissues on chondrogenesis
233
DISCUSSION
The results presented here show that the adjacent ectoderm and endoderm is
capable of promoting somite chondrogenesis. This is in agreement with the
results of Seno & Biiyiikozer (1958), and contrasts with the failure of somites
to undergo chondrogenesis when cultured in vitro with ectoderm (Stockdale et ah
1961; Lash, 1963). The possibility that the cartilage arising in grafts of somites
plus ectoderm and endoderm is of extra-somitic origin, as suggested by Stockdale et ah (1961), would seem most unlikely in the present study. There is no
association whatever between the incidence of nephric tubules and of cartilage
in the same grafts. Such an association would be expected if the cartilage arose
from accidentally included lateral mesoderm adjacent to the nephrotome.
Cartilage-promoting activity is even more clearly revealed in the grafts of
isolated somites recombined with 3- and 4-day ectoderm. A stage specificity in
cartilage-promoting activity by ectoderm is suggested by the fact that 2-day
ectoderm does not possess this activity, whereas 3- and 4-day are both active,
with 4-day ectoderm less active than 3-day ectoderm in spite of a higher
incidence of differentiated ectodermal derivatives.
Although the cartilage-promoting activity of ectoderm would appear to be
stage specific it is not apparently spatially specific. Thus limb-bud ectoderm
results in almost exactly the same incidence of cartilage differentiating in somite/
ectoderm grafts as dorsal trunk ectoderm of the same age. The specific role of
the limb-bud apical ectodermal ridge in the development of the limb mesenchyme (see Amprino, 1965) does not appear to influence its activity with respect
to somite chondrogenesis (apical ectodermal ridge was deliberately included in
all limb-bud ectoderm-containing grafts).
The effect of disrupting the ectoderm/endoderm-somite association by brief
trypsinization, together with the effect of recombination of somites with older
ectoderm, strongly suggests that the presence or absence of basement membrane
material (BMM) in association with the somite influences its subsequent differentiation. The origin and characteristics of BMM found in association with
somites and composed largely of sulphated glycosaminoglycans and collagen
will be discussed in detail in a subsequent communication, but it has been found
that the deposition of basement membrane material between somites and ectoderm is most marked in the 3-day embryo. This suggests that the activity of 3-day
ectoderm in promoting somite chondrogenesis is related to its ability to reform
BMM destroyed during isolation of the somites.
The importance of interface materials in other morphogenetic interactions
has been well documented (see Grobstein, 1967), and they appear to play an
important part in regulating the morphogenesis of epithelio-mesenchymally
derived organ systems. The effect of ectoderm described in the present paper, like
indeed all experimental interference in morphogenetic systems, may be artifactual
in the sense that the somite never suffers in vivo deprivation or disruption of
234
M. J. O ' H A R E
associated interface materials. Nevertheless, it is possible that the gradual
deposition of such materials around the somite may constitute an important
factor in temporal coordination of somite differentiation.
The absence of cartilage-promoting activity in any of the other tissues tested,
including 9-day spinal cord, demonstrates that the mere presence of actively
growing and metabolizing tissues (a situation not always attained in organ
cultures) is not of itself an adequate stimulus for somite chondrogenesis. This
may be contrasted with recent results concerning the specificity of the classic
interaction between the spinal cord and metanephrogenic mesenchyme in the
mouse which leads to the formation of kidney tubules. It would now appear that
a variety of non-specific neural tissues (Lombard & Grobstein, 1969) and unrelated mesenchymes (Unsworth & Grobstein, 1970) can act to 'induce' tubules.
The induction or promotion of somite chondrogenesis does, however, seem to
be restricted to tissues that have some relationship to the somites in vivo.
This, in turn, implies that these interactions do have genuine morphogenetic
significance in vivo.
I am grateful to Professor E. J. Ambrose for encouragement, and Drs G. C. Easty and
M. L. Ellison for helpful discussion. This investigation has been supported by grants to the
Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital)
from the Medical Research Council and the Cancer Campaign for Research.
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{Manuscript received 2 July 1971)