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 resultingin 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.

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 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 expiants 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, 1972b). 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.

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 × 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 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 = 0 · 01 J/l kg) of unfiltered X-radiation being given at 665 rads/min by a Westinghouse therapy machine.

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.

Table 1.

Effect of X-irradiation on spinal cord induction of stage 9 –12 posterior somites

Effect of X-irradiation on spinal cord induction of stage 9 –12 posterior somites
Effect of X-irradiation on spinal cord induction of stage 9 –12 posterior somites

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. 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.

Table 2.

Effect of spinal cord on stage 9 –12 lateral mesoderm

Effect of spinal cord on stage 9 –12 lateral mesoderm
Effect of spinal cord on stage 9 –12 lateral mesoderm

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 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-4-sulphate 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.

Table 3.

Effect of enzymes on irradiated spinal cord/somite interaction

Effect of enzymes on irradiated spinal cord/somite interaction
Effect of enzymes on irradiated spinal cord/somite interaction

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 4.

Effect of collagenase/hyaluronidase (C/H) on unirradiated spinal cord/somite interaction

Effect of collagenase/hyaluronidase (C/H) on unirradiated spinal cord/somite interaction
Effect of collagenase/hyaluronidase (C/H) on unirradiated spinal cord/somite interaction

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.

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 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 différentiation) 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 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 epithelio-mesenchymal 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 enzyme-treated 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.

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