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/. Embryol exp. Morph. Vol. 27, 1, pp. 235-243, 1972
Printed in Great Britain
235
Aspects of spinal cord induction of chondrogenesis
in chick embryo somites
By M. J. O'HARE 1
From the Chester Beatty Research Institute, Institute of Cancer Research:
Royal Cancer Hospital
SUMMARY
The specificity of the chondrogenic inductive activity of chick embryonic spinal cord has
been examined in chorioallantoic culture. It has been found that the 2-day spinal cord is
capable of inducing somites to chondrify even after lethal X-irradiation (5000 rads,
i.e. 50 J/l kg); this dose of X-radiation resulting in the inhibition of mitosis in the spinal cord
and its complete necrosis within 48 h of irradiation.
The unirradiated spinal cord is capable of promoting the chondrogenesis of stage 9-12
posterior lateral mesoderm, but to a lesser extent than when it is combined with stage 9-12
posterior somite mesoderm. Irradiated spinal cord, however, possesses no chondrogenic
activity with respect to lateral mesoderm. Thus it would appear that the 2-day spinal cord
possesses a general cartilage-promoting activity dependent on its continued viability and
proliferation, in addition to a somite-specific chondrogenic activity that survives a lethal
dose of X-radiation.
The inclusion of enzymes in the Millipore filter used as a graft vehicle has been used to
demonstrate that collagenase and hyaluronidase when combined, but not individually, are
capable of interfering in the induction of somite chondrogenesis by irradiated spinal cord.
Grafts of somites with unirradiated spinal cord show that these enzymes do not directly
reduce the chondrogenic potential of the somites or interfere in the subsequent deposition of
cartilage matrix. The activity of the spinal cord in inducing somite chondrogenesis appears
to be associated with the synthesis of basement membrane materials by either or both of the
interacting tissues during the first 24 h of the interaction.
No influence of the 2-day spinal cord on the morphogenesis of non-somitic cartilage could
be detected.
INTRODUCTION
The embryonic spinal cord, together with the notochord, has been shown to
be involved in the morphogenesis of somite-derived vertebral cartilage in the
chick embryo in vivo (Watterson, Fowler & Fowler, 1954) and in vitro (Avery,
Chow & Holtzer, 1956), although the mechanisms whereby the interaction is
effected remains unknown (Holtzer, 1968).
Recent work on the response of isolated somites to in vitro organ culture
(Ellison, Ambrose & Easty, 1969) has shown that a variety of non-specific factors
in the organ culture environment are capable of influencing somite chondrogenesis, and that by the use of improved methods spontaneous chondrification
1
Author's address: Chester Beatty Research Institute, Institute of Cancer Research: Royal
Cancer Hospital, Fulham Road, London SW3 6JB, U.K.
236
M. J. O'HARE
of very young somites may be obtained. As the concept of the spinal cord as a
specific inducer of somite chondrogenesis is based on previous in vitro studies
(Avery et al. 1956; Lash, Holtzer & Holtzer, 1957), it is not clear whether the
in vitro activity of the spinal cord is analogous to these non-specific promoting
factors, or whether it does possess unique tissue-specific activity relevant to the
in vivo control of the onset of somite chondrogenesis.
Demonstration of specificity (or lack of specificity) in the activity of the spinal
cord is relevant to an assessment of the morphogenetic significance of in vitro
interactions. It has been demonstrated that spinal cord is unable to induce
cartilage in explants of differentiated myotomal striated-muscle cells (Stockdale,
Holtzer & Lash, 1961) but no observations have been reported on the activity
of the spinal cord with respect to non-somitic chondrogenic tissues.
It has been demonstrated previously that young posterior somites which fail
to undergo spontaneous chondrification in chorioallantoic (CAM) culture
(O'Hare, 1972a) will chondrify when grafted in association with unrelated
embryonic ectoderm (O'Hare, 1912b). This activity of the ectoderm has been
related to the synthesis of basement membrane material in association with
somite mesoderm, as the integrity of this extracellular matrix material appears
to be an important factor governing the chondrogenic potential of the somites
in culture.
Possible involvement of basement membrane material in the activity of spinal
cord with respect to somite chondrogenesis has been examined by interfering in
the interaction with specific enzymes. In addition, the proliferative activity of
the spinal cord has been dissociated from its metabolic activity by X-radiation.
The complete inhibition of mitosis achieved has enabled a distinction to be
made between the short- and long-term effects of the spinal cord on mesodermal
chondrogenesis.
As in previous experiments, a modified CAM grafting technique employing
Millipore filter as a graft vehicle has been used.
METHODS
CAM grafts of trypsin-dissociated tissues were prepared as detailed in a
previous paper (O'Hare, 1972a). Grafts were assembled on HA-grade Millipore
filter and transferred to the CAM with grafted tissues in direct contact with the
chorioallantoic epithelium, under the Millipore filter. After 9 days' growth, graft
sites were excised from the CAM, serially sectioned and stained for histological
examination as before.
Grafts to be treated with enzymes were assembled on Millipore filter that had
been impregnated with an agar solution of the appropriate enzyme. 0-1 ml of
a x 10 concentrated enzyme solution was added to 0-9 ml of a warm solution
of 1 % agar (Difco) in Simms BSS (balanced salt solution), while the latter was
still liquid. After thorough mixing, a piece of HA-grade Millipore filter was
Spinal cord induction of chondrogenesis
237
immersed in the liquid agar, which soaked up into the interstices of the filter.
The agar was allowed to set and the Millipore, now impregnated with an agar
solution of the enzyme, was removed from the gel.
The enzymes used were: collagenase (Cl. histolyticum), 125-200 units/mg
(Nutritional Biochemical Corp.); ovine testicular hyaluronidase, 350-500 i.u./
mg (B.D.H.); and bovine pancreatic RNase (protease-free), 40-50 Kunitz
units/mg (B.D.H.).
Irradiation of tissues was carried out in Simms BSS, a dose of 5000 rads
(1 rad = 001 J/l kg) of unfiltered X-radiation being given at 665 rads/min by a
Westinghouse therapy machine.
RESULTS
Somites plus spinal cord
The effect of X-irradiation of the spinal cord on its ability to induce cartilage
from young somites was examined as follows. Grafts were made of stage 9-12
posterior somites in groups of four in contact with a short segment of spinal
cord from an embryo of the same stage. Stage 9-12 posterior somites consistently fail to undergo spontaneous chondrification in CAM culture (O'Hare,
1972a). Embryos donating spinal cord were subjected to 5000 rads of X-radiation
prior to isolation of spinal cord segments. Control grafts were made of unirradiated spinal cord with unirradiated somites, and of unirradiated spinal cord
with irradiated somites. Results are presented in Table 1.
In the presence of unirradiated spinal cord the somites, as expected, gave rise
to cartilage in over 90 % of the grafts and to healthy striated muscle in 80 % of
the grafts. In these grafts the spinal cord formed large vesicular formations of
neural tissue, in the vicinity of which were found the cartilage nodules and
striated muscle bundles derived from the somite mesoderm.
Spinal cord irradiated with 5000 rads of X-radiation, on the other hand,
failed to give rise to any distinguishable neural derivatives in the final graft.
Examination of grafts at 1 and 2 days revealed that mitosis had been completely
inhibited in the irradiated spinal cord, and by 48 h the spinal cord was completely necrotic. In spite of this rapid degeneration of the irradiated spinal cord,
cartilage was found in over 50 % of these grafts. No striated muscle was found.
Table 1. Effect of X-irradiation on spinal cord induction of stage 9-12
posterior somites
Cartilage
Isolated somites
Somites + unirradiated spinal cord
Somites + irradiated spinal cord
Irradiated somites + spinal cord
0/89*, 0%
19/20,95%
15/29,52%
0/20, 0 %
Striated muscle
0/89,0%
16/20,80%
0/29,0%
0/20, 0 %
* Number of grafts positive/total grafts recovered.
Spinal cord
—
20/20,100%
0/29,0%
20/20,100 %
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M. J. O'HARE
The incidence of nephric tubules is 10-15 % in both types of grafts, and unrelated
to the presence or absence of cartilage. Irradiation of the somites instead of the
spinal cord results in their complete failure to form any differentiated derivatives.
Lateral mesoderm plus spinal cord
The effect of the embryonic spinal cord on the chondrogenic capabilities of
non-somite mesoderm was tested by grafts of irradiated and unirradiated spinal
cord with strips of isolated lateral mesoderm. This lateral mesoderm was prepared from the region adjacent to the posterior four somites of stage 9-12
embryos. The width of the strips was approximately equal to that of the somites
and part of the intermediate mesoderm was excluded from the grafts, ensuring
that they were not contaminated with somite mesoderm. Results are presented
in Table 2.
It is apparent that although isolated posterior lateral mesoderm from these
stages seldom differentiated cartilage when grafted alone, the inclusion of either
adjacent ectoderm and endoderm or unirradiated spinal cord results in differentiation of cartilage in about 40 % of the grafts. Grafts of spinal cord alone
reveal no instance of cartilage differentiating from accidentally included somite
mesoderm. The incidence of striated muscle in grafts of lateral mesoderm plus
spinal cord is 22 %, compared with 0 % in grafts of isolated lateral mesoderm.
When these strips of lateral mesoderm are grafted with irradiated spinal cord,
however, the incidence of cartilage (2 %) is similar to that observed in grafts of
lateral mesoderm alone. The irradiated spinal cord would thus appear to have
no influence on lateral mesodermal chondrogenesis.
The incidence of nephric tubules is 15-25% in all types of lateral mesodermal
grafts and is unrelated to the presence or absence of cartilage.
Effects of enzymes on spinal cord/somite grafts
In order to interfere with the transient inducing activity of irradiated spinal
cord, grafts were made in which various enzymes were included as agar solutions
within the Millipore filter. A considerable part of the volume of the filter is air
Table 2. Effect of spinal cord on stage 9-12 lateral mesoderm
Lateral mesoderm
Lateral mesoderm +
unirradiated ectoderm and
endoderm
Lateral mesoderm +
unirradiated spinal cord
Lateral mesoderm +
irradiated spinal cord
Spinal cord
Cartilage
Striated
muscle
1/23, 4 %
12/33, 37 %
0/23,0 %
0/33,0 %
19/45, 42 %
10/45, 22 %
42/45, 93%
11/45, 24%
1/56,2%
0/56,0%
0/56, 0%
12/56, 21%
0/20, 0 %
0/20,0%
20/20, 100%
Spinal cord
Nephric
tubules
6/23, 26%
5/33, 15%
Spinal cord induction of chondrogenesis
239
space (England, 1969), and in these grafts is occupied by the agar-enzyme
solution. It is evident that the activity of material included in the filter will be
transient, as the substance will diffuse out of the agar and will be dissipated by
the developing chorioallantoic circulation.
Enzymes included in the filter in this manner were collagenase, hyaluronidase
and RNase which degrade collagen, chondroitin sulphates A/C (chondroitin-4sulphate and chondroitin-6-sulphate) and ribonucleic acid respectively. It
was found by trial and error that 0-5% was the highest concentration of
enzyme that could be applied without affecting the overall viability of the
grafted somites. Grafts were prepared of irradiated spinal cord plus stage 9-12
posterior somites on Millipore impregnated with (1) 0-5 % collagenase, (2) 0-5%
hyaluronidase, (3) 0-5 % collagenase plus 0-5 % hyaluronidase, and (4) 0-5 %
RNase. Results are presented in Table 3.
In the grafts treated with collagenase, hyaluronidase and RNase independently, the incidence of cartilage is around 35 %, compared with 52 % in control
grafts. This depression of chondrogenesis may, however, be due to the fact that
the agar-impregnated Millipore is not adhesive towards the tissues, rendering it
more difficult to assemble grafts with contact between interacting tissues. In
grafts treated with collagenase plus hyaluronidase, however, the incidence of
cartilage fell to 14 %. This synergistic effect suggests that the effect may well be
specific. It is possible, however, that the enzymes might have a deleterious effect
on the viability of the grafted tissue in general, or that they might affect the
subsequent deposition of cartilage matrix. To test this, grafts were made of
unirradiated spinal cord plus somites on collagenase/hyaluronidase-impregnated
filters. Results are presented in Table 4.
Table 3. Effect of enzymes on irradiated spinal cordjsomite interaction
Cartilage
Irradiated spinal cord + somites
Plus 0-5 % hyaluronidase
Plus 0-5 % collagenase
Plus 0-5 % RNase
Plus 0-5 % collagenase + 0-5 %
hyaluronidase
15/29,52%
8/22, 36 %
10/30, 33 %
7/19, 37 %
5/35,14%
Nephric tubules
5/29, 17 %
1/22, 5 %
5/30, 17 %
2/19,10%
4/23,17%
Table 4. Effect of collagenase / hyaluronidase {CjH) on unirradiated
spinal cord/somite interaction
Irradiated spinal cord +
somites+ C/H
Unirradiated spinal cord +
somites+ C/H
Cartilage
Striated
muscle
Nephric
tubules
Spinal cord
5/35,14%
0/35,0%
4/35,11%
0/35,0 %
17/23,74%
9/23,39%
4/23,17%
19/23,82%
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M. J. O ' H A R E
Although the presence of the two enzymes marginally impairs the viability of
the grafts, as evidenced by the differentiation of spinal cord in only 82 % of
grafts, cartilage was present in 89 % of the grafts in which spinal cord was
present. It would thus appear that the enzymes have little or no effect on either
the induction of cartilage by unirradiated spinal cord, or on the deposition of
cartilage matrix. This finding is in accord with the expected transient nature of
the interference by the enzymes.
The incidence of striated muscle, however, fell from 80 % to 40 %, and it was
present only in small amounts. The collagenase/hyaluronidase did, therefore,
impair the myogenic ability of the somites. This was probably due to a dispersion
of the somite cells by the enzymes, resulting in the inhibition of myotube
formation by myoblast fusion.
Effect of spinal cord on limb-bud mesoderm
To test the effect of the spinal cord on the morphogenesis of non-somitic
cartilage, grafts were made of segments of stage 9-12 spinal cord with limb-bud
mesoderm cells from stage 22-25 limb-buds. These limb-buds were dissociated
into constituent cells with 0-25 % trypsin solution and pellets of dissociated cells
prepared by centrifugation. Fragments of these limb-bud cell pellets were
assembled on Millipore filter with a spinal cord and grafted on to the CAM.
Twenty grafts with spinal cord and 15 grafts of limb-bud cells alone were made.
Cartilage differentiated in all the control grafts of limb-bud cells alone but
no instances of striated muscle differentiation were observed. In contrast to
grafts of entire limb-buds, the cartilage formed did not resemble limb skeletal
elements. Large nodules of cartilage were formed around the Millipore filter in
the centre of the graft by the sorting out of chondrogenic cells in the dissociated
cell pellet.
The presence of spinal cord in the graft resulted in the differentiation of
striated muscle in 85 % of such grafts, but no influence of spinal cord on the
cartilage differentiating in the grafts could be observed. The differentiation and
morphogenesis of this limb-derived cartilage was apparently unrelated to the
presence of neural structures derived from spinal cord.
DISCUSSION
It is evident that the unirradiated spinal cord is capable of promoting chondrogenesis of stage 9-12 posterior lateral mesoderm. To this extent, the activity of
the spinal cord towards lateral mesoderm mimics its activity towards the somites.
There are, however, two important differences. In the first place, the incidence
of cartilage in spinal cord/somite grafts is over twice the incidence in spinal
cord/lateral mesoderm grafts. The activity of the spinal cord with respect to
lateral mesoderm is thus no greater than that of the adjacent ectoderm with
endoderm. Secondly, the irradiated spinal cord has no activity with respect to
Spinal cord induction of chondrogenesis
241
lateral mesoderm although it does result in the chondrification of over 50 %
of somite grafts of the same stage.
The activity of the irradiated spinal cord is in accord with the observation of
Lash et al. (1957) that only a limited period of association between spinal cord
and somites is necessary for induction of cartilage. In the present case less than
24 h elapse before the irradiated spinal cord is largely necrotic, and yet it is still
able to induce cartilage. The inductive activity of irradiated spinal cord demonstrates further that no proliferative activity on the part of the spinal cord is
necessary for induction to occur.
The young spinal cord would thus appear to possess two cartilage-promoting
activities. The first is a general cartilage-promoting activity in which it is necessary for the mitotically active spinal cord to be associated with the tissues for a
considerable period of time. The second activity is somite-specific and unrelated
to mitotic activity or continued viability of the spinal cord. The possession of
a general and a specific cartilage-promoting activity by the spinal cord is to
some extent reminiscent of the general and specific epithelium-promoting
activity of various embryonic mesenchymes, in which complete morphogenesis
of most epithelio-mesenchymally derived organs is achieved only with the
specific mesodermal component (Spooner & Wessells, 1970). In this case, however, it is the epithelially derived tissue (neurectoderm) that acts on mesoderm
(somites and lateral mesoderm).
The work of Pinot (1969) indicates that both the vertebrae and the ribs
together with abdominal and costal musculature are derived from the somites.
This leaves the sternal cartilage and muscles and also the appendicular cartilage
and muscle to be derived from lateral mesoderm, and the cartilage and muscle
observed in lateral mesoderm grafts must be related to these structures. In view
of the lateral and posterior location of prospective limb-bud mesenchyme at the
stages in question (Rosenquist, 1971), the posterior-medial lateral mesoderm
employed in this study probably gives rise to pectoral skeletal elements.
The present study did not provide any evidence that the young spinal cord is
capable of influencing the morphogenesis (as opposed to the initiation of differentiation) of unrelated cartilage. Thus, in spite of the complete disorganization
of the pellets of older limb-bud cells at the time of grafting, the chondrogenic
cells sorted out and formed substantial nodules of cartilage without reference to
the presence of proliferating neural structures derived from the spinal cord.
The morphogenetic activity of the spinal cord thus appears to be confined to
the somite-derived vertebrae, and even here it is clear that the spinal cord is only
one of a number of factors that influence the morphogenesis of the vertebrae
(Strudel, 1967).
The results obtained with agar-impregnated filters are more difficult to interpret. The enzyme contained in the filters will act on extracellular matrix material
or basement membrane material produced by either or both of the interacting
tissues. The sulphated glycosaminoglycans (mucopolysaccharides) associated
l6
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242
M. J. O ' H A R E
with the spinal cord and somites will be analysed in detail in a subsequent communication, but both somites and spinal cord are apparently capable of extracellular matrix material synthesis at the time in question, although activity is
more marked with respect to the spinal cord. Nevertheless, the fact that cartilage
arises in grafts of somites with unirradiated spinal cord plus collagenase/hyaluronidase does indicate that these enzymes are not acting either directly on the
chondrogenic potential of the somite cells themselves, or on the subsequent
deposition of cartilage matrix. The disruption of extracellular matrix material
synthesized in association with the spinal cord and somites appears to be the
most probable reason why collagenase and hyaluronidase act synergistically to
depress the incidence of somite chondrogenesis in grafts of somites plus
irradiated spinal cord.
Extracellular matrix material composed largely of sulphated glycosaminoglycans and collagen has been shown to be involved in a number of epitheliomesenchymal interactions (Grobstein, 1968), and there is autoradiographic and
ultrastructural evidence that the spinal cord is capable of synthesizing collagen
or collagen-like protein (Grobstein, 1959; Cohen & Hay, 1971), which is
deposited in the form of neural basement membrane. In the embryonic toothgerm epithelio-mesenchymal interaction RNA has been found in the basement
membrane material separating the interacting tissues, and it has been suggested
that this RNA may play some role in the interaction (Slavkin, Bringas & Bavetta,
1969; Slavkin, Bringas, Cameron, LeBaron & Bavetta, 1969). In the present
study RNase did not interfere in the spinal cord/somite interaction under conditions that revealed a significant interference in the interaction by collagenase
plus hyaluronidase. As the total number of grafts in each category of enzymetreated graft was relatively small (20-35) it is not possible to completely exclude
an effect of RNase on the interaction. It is clear, however, that under the present
conditions the effect of collagenase plus hyaluronidase is predominant.
A specific role of extracellular matrix material, even when dissociated from
its tissue of origin, has been demonstrated by Sigot & Marin (1970) with respect
to the activity of mesodermal extracellular matrix material towards gastric
epithelium.
The present results with specific enzymes are in accord with the concept that
the inductive activity of the spinal cord is related to the synthesis of the basement membrane material deposited between the neural tissue and the somites in
vivo, although they do not allow a distinction to be drawn between the effects of
the enzymes on material synthesized by somite cells and by spinal cord cells.
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.
Spinal cord induction of chondrogenesis
243
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