An analysis was made of the differentiative capabilities of somites isolated from all regions of stage-8 (4-somite) to stage-18 (36-somite) chick embryos, with particular reference to the incidence of spontaneous chondrogenesis. A chorioallantoic grafting technique with Millipore filter as a graft vehicle was employed.

The youngest somites which spontaneously differentiated cartilage were the anterior somites of stage 11. Cartilage obtained from grafts of stage 8–10 anterior somites was probably derived from lateral rather than somite mesoderm. Younger, posterior somites consistently failed to differentiate cartilage in spite of the demonstrable viability of the grafts, as evidenced by the appearance of other differentiated derivatives.

Increasing the number of non-chondrifying somites grafted, from 4 to 16, did not result in their spontaneous chondrification when all such somites were derived from the same 4-somite region of embryos of the same stage.

All embryos from stage 12 to stage 18 showed a gradient of chondrogenic potential along the somite axis, with anterior somites always showing a higher incidence of cartilage than posterior somites of the same stage.

Bone and striated muscle derived from somite mesoderm, ganglion cells derived from trunk neural crest, and nephric tubules derived from nephrotome cells were also found in the grafts. Each differentiated type showed a distinct pattern of incidence along the somite axis. The incidences of nephric tubules and ganglion cells were unrelated to those of the differentiated derivatives of somite mesoderm, suggesting that the results reflect true differences of intrinsic differentiative potential.

The dependence of the in vivo morphogenesis of the vertebral column on the presence of the spinal cord and notochord was demonstrated in the chick embryo by Watterson, Fowler & Fowler (1954), following the demonstration of a similar relationship in the urodele by Holtzer (1951). Subsequent studies, employing mainly in vitro organ culture techniques, showed that isolated chick somites failed to chondrify unless spinal cord and/or notochord was included in the graft (Avery, Chow & Holtzer, 1956; Lash, Holtzer & Holtzer, 1957). Extracts of spinal cord/notochord were shown to possess cartilage ‘inducing’ activity (Lash, Hommes & Zilliken, 1962; Strudel, 1962), but attempts to identify the factor(s) responsible failed as fractionation of such extracts eventu-ally resulted in loss of activity (Zilliken, 1967). Furthermore, it was shown by Strudel (1963) that the differentiation of isolated somites could also be promoted by modification of the culture conditions.

The response of isolated somites to variations in the conditions of organ culturehas been extensively investigated by Ellison, Ambrose & Easty (1969a, b). These studies demonstrated that isolated somites previously thought ‘uninduced’ were found to be capable of chondrification under certain conditions. As a consequence the idea that the spinal cord or notochord acts as a specific inducer of somite chondrogenesis has given way to the concept of ‘stabilization’ of the phenotypic differentiation of predetermined somites. In this role the activity of the notochord can be mimicked by non-specific manipulations of the culture conditions (Ellison & Lash, not yet published).

It is clear from these improved culture methods that the in vitro experiments of Avery et al. (1956) and Lash et al. (1957) demonstrated a relative rather than an absolute dependence of somite chondrogenesis on the spinal cord and notochord. The present studies constitute an examination of the apparent specificity of this interaction, with a view to establishing whether the spinal cord does possess a unique tissue-specific activity in promoting or inducing somite chondrogenesis.

This paper reports an analysis of the intrinsic chondrogenic potential of isolated somites, undertaken in order to establish base-lines for subsequent interactive studies. Most previous workers have employed somites derived from a randomized pool of anterior and posterior somites of the same stage. An exception is Lash (1967), who, using large groups of stage 15–18 somites, reported that the posterior somites exhibited a higher chondrogenic potential in organ culture than the anterior somites. This finding is at variance with the state of differentiation of the somites at the time of explantation, as the anterior somites of all stages show a higher degree of morphological differentiation than posterior somites (see Fig. 3 and Williams, 1910). One of the objects of the present study was to ascertain if this anomalous gradient was exhibited with other culture conditions.

A modified chorioallantoic grafting technique was chosen for the present studies. Previous attempts at chorioallantoic grafts of chick somites (Seno & Büyükôzer, 1958) indicated that although a higher degree of morphogenetic differentiation could be obtained as compared with organ culture, graft ‘takes’ were erratic, with many grafts undetectable after the culture period. These drawbacks have been overcome by the use of cellulose ester (Millipore) filter material as a graft vehicle.

White Leghorn embryos were removed from the egg to Simms balanced salt solution (BSS), and staged according to Hamburger & Hamilton (1951). Trunk segments consisting of two groups of four adjacent somites, together with spinal cord, notochord, ectoderm and endoderm were excised and partially dissociated by treatment in 3% trypsin (Difco) in calcium/magnesium free salt solution for 1 min at room temperature. Trunk segments were then rinsed in 20% calf serum/Simms BSS to inactivate remaining trypsin and final dissociation into constituent tissues was carried out manually in Simms BSS.

Groups of isolated somites, free of ectoderm and endoderm, were assembled on pieces of pre-sterilized cellulose ester filter (Millipore Filter Corp. HA-grade, 150 μm thick, 0 ·45 μm pore size). Grafts were then transferred to the exposed chorioallantoic membrane (CAM) of 9- or 10-day incubated chick embryos and placed with grafted tissues between the filter and the CAM, i.e. in direct contact with chorioallantoic epithelium.

Grafts were grown for 9 days, at the end of which time the graft site was identified by the Millipore filter, which remained attached to or embedded in the CAM. Graft sites, together with the Millipore filter, were excised, fixed in Newcomer’s fluid, decalcified in 2% HCl/70% ethanol, embedded and serially sectioned. Sections were stained in 0 ·5% alcian blue 8GX (G. T. Gurr Ltd.) in 0 ·4 molar magnesium chloride solution, followed by Harris’s haematoxylin and eosin. All serial sections were,examined and each graft scored for differentiated derivatives identified therein.

Whole somite grafts

An analysis was made of the differentiation of isolated somites from stage 8 (4-somite) to stage 18 (36-somite) embryos, with 9-day chorioallantoic grafts being made of groups of four adjacent somites from a known position in the somite axis. All somites, with the exception of the anterior four somites of stage 14–18, were tested in this manner. Cartilage, bone and striated muscle derived from somitic mesoderm were identified in these grafts. In addition, ganglion cells derived from neural crest and nephric tubules derivedfrom nephrotome could be distinguished in some grafts.

A total of 893 grafts of isolated somites were recovered. No ectodermal or endodermal derivatives were detected in these grafts.

Cartilage

The incidence of cartilage in these grafts is presented in Table 1.

Table 1.

Incidence of cartilage in 4-somite grafts

Incidence of cartilage in 4-somite grafts
Incidence of cartilage in 4-somite grafts

In all embryos older than stage 12 there is a clear anterior-posterior gradient of chondrogenic potential, with the anterior somites always differentiating cartilage more often than posterior somites. By stage 15 nearly all grafts of anterior somites will differentiate cartilage and by stage 18 all somites with the exception of the posterior ten give rise to cartilage in all grafts.

The posterior four somites of stages 9–12 consistently failed to differentiate cartilage, whereas anterior somites of these stages did differentiate cartilage. The earliest stage at which cartilage was obtained from somite grafts was stage 8 (4-somite stage). Isolated stage-8 somites gave rise to cartilage in 30% of grafts. In contrast to later stages, however, the incidence of cartilage in grafts of young somites 1–4 falls as they get older, the anterior four somites of stage 11 giving only a 3 % incidence of cartilage. Grafts of bisected somites, to be reported later in this paper, indicate a possible explanation for this anomalous effect. With the exception of these very young anterior somites, all somites show a steady increase in chondrogenic potential as they mature.

The cartilage formed by these grafts usually took the form of discrete spherical or ovoid nodules (Fig. 2A). Such nodules were usually composed either entirely of immature proliferative cartilage, mature non-hypertrophied cartilage or of hypertrophied cartilage. Grafts were very seldom found with all stages of cartilage maturation present in a single nodule. The later stages of cartilage maturation were confined to grafts of older and/or more anterior somites.

In some of these 4-somite grafts, up to four discrete nodules of cartilage were found, and in grafts of stage 17–18 anterior somites fused masses of cartilage evidently derived from multiple foci of chondrification were found. When the incidence of cartilage was expressed as the mean number of nodules per graft, somites from stage 12 and older embryos showed the same anterior-posterior gradient demonstrated by the overall incidence of cartilage. The mean number of nodules per graft ranged from 0 ·03 for anterior stage 11 somites to 3 ·2 for anterior stage-18 somites. Thus older and/or anterior somite grafts showed a greater tendency to form multiple nodules.

Bone

Bone was found in association with cartilage in grafts of anterior somites of stage 15 and older, with an incidence rising to a maximum of 50% in grafts of anterior stage 18 somites.

In 42/45 grafts in which bone was observed it was present as perichondral bone associated with hypertrophied cartilage. In three grafts nodules of bone were found without the presence of cartilage.

Striated muscle

Striated muscle fibres (Fig. 2B) were detected in 32/893 isolated somite 9-day grafts and were confined to grafts of somites older than stage 15. The highest incidence of muscle was 50% in anterior stage 18 somite grafts.

A notable feature of the differentiation of striated muscle in these grafts was the degeneration associated with muscle derived from anterior somite grafts. Distinct degeneration could be seen in 8/16 cases of striated muscle differentiating from grafts of somites 5–16, pycnotic nuclei seen in bundles of necrotic fibrils, surrounding tissues being healthy. No degeneration was observed in the 16 cases of striated muscle differentiating from somites 17–36.

Ganglion cells

Groups of large cells with dense cytoplasm and large pale nuclei, associated in many cases with long cytoplasmic processes, were found in many somite grafts (Fig. 2C). In appearance these cells closely resembled ganglionic neuroblasts derived from neural crest cells. This identification as neural crest-derived cells is borne out by their distribution, which is given in Table 2.

Table 2.

Incidence of ganglion cells in 4-somite grafts

Incidence of ganglion cells in 4-somite grafts
Incidence of ganglion cells in 4-somite grafts

The incidence of ganglion cells (and nephric tubules) has been scored in some combination grafts in addition to isolated somite grafts, accounting for the greater numbers of early stage grafts recorded in Tables 2 and 3.

Table 3.

Incidence of nephric tubules in 4-somite grafts

Incidence of nephric tubules in 4-somite grafts
Incidence of nephric tubules in 4-somite grafts

The ganglion cells show a maximum incidence in grafts of anterior mid-trunk somites at all stages, falling off rapidly in the most anterior somite grafts and more gradually in posterior somite grafts. Ganglion cells are first seen in grafts of somites 5–8 of stage 12 (16-somite) embryos. This distribution of ganglion cells is quite distinct from that of cartilage in the same grafts (see Fig. 1). Similar patterns of cartilage, ganglion cell and nephric tubule differentiation to those illustrated in Fig. 1 are found at all stages from stage 12 to stage 18.

Fig. 1.

Differentiation of stage-15 somites in CAM culture. Somites grafted in groups of four. •—•, Ganglion cells; ▵—▵, cartilage; ▫—▫, nephric tubules.

Fig. 1.

Differentiation of stage-15 somites in CAM culture. Somites grafted in groups of four. •—•, Ganglion cells; ▵—▵, cartilage; ▫—▫, nephric tubules.

Figure 2.

Nine-day chorioallantoic grafts stained alcian blue, pH 0 ·5, and haematoxylin and eosin.

(A) Cartilage nodule (c) and ganglion cells (arrow).

(B) Striated muscle (sm) above Millipore filter (mf).

(C) Ganglion cells (arrow).

(D) Nephric tubule.

Figure 2.

Nine-day chorioallantoic grafts stained alcian blue, pH 0 ·5, and haematoxylin and eosin.

(A) Cartilage nodule (c) and ganglion cells (arrow).

(B) Striated muscle (sm) above Millipore filter (mf).

(C) Ganglion cells (arrow).

(D) Nephric tubule.

Figure 3.

Stages of somite differentiation: 5μm transverse sections stained with iron haematoxylin. Percentages indicate cartilage differentiating from 4-somite grafts of these regions. (Gaps between mesoderm and ectoderm/endoderm are fixation artifacts.)

(A) Somite 8, stage 10 (0 %).

(B) Somite 22, stage 14 (12 %).

(C) Somite 15, stage 14 (20 %).

(D) Somite 10, stage 14 (62 %).

(E) Somite 6, stage 14 (71 %).

(F) Somite 16, stage 18 (100 %).

Figure 3.

Stages of somite differentiation: 5μm transverse sections stained with iron haematoxylin. Percentages indicate cartilage differentiating from 4-somite grafts of these regions. (Gaps between mesoderm and ectoderm/endoderm are fixation artifacts.)

(A) Somite 8, stage 10 (0 %).

(B) Somite 22, stage 14 (12 %).

(C) Somite 15, stage 14 (20 %).

(D) Somite 10, stage 14 (62 %).

(E) Somite 6, stage 14 (71 %).

(F) Somite 16, stage 18 (100 %).

Nephric tubules

The incidence of nephric tubules (Fig. 2D) in these graftsis of some importance as the possibility exists that the inadvertent inclusion of nephrotome cells in the somite graft may also result in the inclusion of potentially chondrogenic lateral mesodermal cells from the prospective limb-bud regions (Lash, 1963).

It can be seen from Table 3 that nephric tubules appear most often in grafts of posterior mid-trunk somites at all stages. Nephric tubules very seldom differentiated in anterior somite grafts. If the incidence of nephric tubules can be taken to reflect inclusion of nephrotome cells in somite grafts, then it is clear that this bears no relationship to the incidence of cartilage in the same grafts.

Bisected somite grafts

It has been shown that the anterior somites of stages 8–10 show an unexpectedly high incidence of cartilage, unrelated to the otherwise uniform trend of increasing chondrogenic potential as somites mature. The possibility that this cartilage was derived from anterior lateral mesoderm rather than somite mesoderm was examined by means of bisected somite grafts.

Grafts were made of the anterior four somites of stages 8–10 after they had been bisected longitudinally into medial and lateral halves. Results are presented in Table 4.

Table 4.

Differentiation of longitudinally bisected somites 1–4, stages 8 and 9

Differentiation of longitudinally bisected somites 1–4, stages 8 and 9
Differentiation of longitudinally bisected somites 1–4, stages 8 and 9

The incidence of cartilage in the grafts of the lateral halves of these somites is approximately three-quarters of that with entire somites. None of the medial halves, however, ever gave rise to cartilage. Longitudinal bisection of older (stage 16) mid-trunk somites did not impair the chondrogenic potential of either lateral or medial halves, when this was compared with the incidence of cartilage in intact grafts. It is probable therefore that the cartilage that appears in grafts of stage 8–10 anterior somites is of lateral mesodermal origin, and that the earliest appearance of somite-derived cartilage is in stages 11–12. Anterior lateral mesoderm adjacent to the somites (in stages 8–10) may well be destined to form cartilage of the otic capsule.

The absence of cartilage in medial somite halves would appear to rule out the possible involvement of cranial neural crest, which is also capable of chondrogenesis, in the formation of cartilage in these young somite grafts.

Effect of graft mass

Grafts were made of 16 posterior stage 9–11 somites, each graft consisting of four equivalent groups of four somites (from two embryos), to ascertain if increasing the explant mass would result in spontaneous chondrification. In groups of four grafted in isolation, these somites never chondrify. In organ cultures of somites taken at random from the somite axis, Ellison et al. (1969a) have shown that increasing the explant mass markedly favours chondrogenesis. Twenty such 16-somite grafts were made, but in no case did cartilage differentiate, although the incidence of nephric tubules (35%) was four times that observed in grafts of four somites (7 %).

It is clear from these results that the modified chorioallantoic (CAM) grafting technique using Millipore filter as a graft vehicle represents a considerable improvement on the free grafting technique. Differentiated derivatives could be identified in grafts of the youngest material tested (stage 8) and in contrast to the results of Seno & Büyükôzer (1958), the differentiation of isolated somites in chorioallantoic culture proved possible by this method, with unique and distinctive patterns of anterior-posterior incidence exhibited by the different differentiated derivatives (see Fig. 1).

The incidence of cartilage in these grafts shows that, contrary to the results obtained by Lash (1967, 1968) in in vitro organ culture, anterior somites show a greater chondrogenic potential than posterior somites of the same stage. This is in accord with their morphological differentiation at the time of explantation (Fig. 3 A–F). The decline in the rate of DNA synthesis (Gordon, 1970) and in phosphoadenosinephosphosulphate synthesis (Gordon & Lash, 1970), observed when anterior somites are organ-cultured, indicates that these somites do not respond favourably to the organ culture environment. The reason for this differential response of anterior and posterior somites is not clear, although it has been suggested by Lash (1967) that anterior somites may fail to chondrify because they have not been exposed to the influence of the spinal cord and notochord for as long as the posterior somites at the time of explantation. In view of the marked effect that alteration of the organ culture environment has on somite chondrogenesis (Ellison et al. 1969 a), it seems more likely that some structural feature of the posterior somites predisposes them to survive the organ culture environment and subsequently to chondrify. It may be that the loss of the closepacked epithelioid ‘cortex’ of the young somite during the differentiation of the somite into sclerotome and dermomyotome (see Fig. 3) renders it susceptible to adverse conditions that may be encountered in organ culture. Anterior somites evidently do not respond adversely to chorioallantoic culture and therefore the failure of anterior somites to chondrify in organ culture cannot be a fundamental feature of somite differentiation.

Although cartilage was obtained from chorioallantoic grafts of stage 8 somites, experiments have been described which suggest that this cartilage is not of somitic origin. Excluding this cartilage, that formed by anterior somite of stage 11 embryos appears to represent the earliest example of spontaneous somite chondrogenesis. Thus the chorioallantoic graft with Millipore filter as graft vehicle permits spontaneous chondrogenesis by somites as young as those that will chondrify under the best organ culture conditions and younger than those shown by Seno & Büyüközer (1958) to undergo spontaneous chondrification in coelomic grafts. Although Ellison et al. (1969 d) did obtain cartilage from organ-cultured explants of stage 9 somites under the best organ culture conditions, the position of these somites in the somite axis was not recorded as they were taken at random. As it is possible that these explants contained anterior somites, and the present experiments suggest that cartilage of an extrasomitic origin may be obtained from such anterior somite grafts, it is possible that the cartilage obtained in these stage 9 explants was not derived from somite mesoderm. The accidental inclusion of anterior lateral mesoderm in stage 8–10 anterior somite grafts may be readily accounted for by the lack of a clear demarcation between the lateral edge of the somite and the adjacent lateral mesoderm at these stages in the anterior region.

The differentiation of up to four discrete nodules of cartilage from these 4-somite grafts suggests that at the time of explantation, each somite is capable of forming only one chondrogenic focus. This relationship holds for all the somites tested except the anterior somites of stages 17 and 18, in which fused masses of cartilage derived from multiple chondrogenic foci are sometimes seen. As each somite will eventually give rise to four separate chondrogenic foci (arcualia) during the development of the vertebral column (Goodrich, 1930), it seems that the separate arcualial elements are not determined until relatively late in the differentiation of the somite.

The consistent failure of the posterior four stage 9–12 somites to chondrify spontaneously when grafted in isolation appears to reflect a genuine immaturity of the chondrogenic tissues at the time of grafting. The possibility that this failure to chondrify is due to damage inflicted during isolation or culture seems improbable for the following reasons: (1) nephric tubules are found in a regular percentage of such grafts, demonstrating their overall viability; (2) cartilage can be obtained from grafts of younger material, albeit that such cartilage is probably not derived from somite mesoderm; (3) the use of collagenase and hyaluronidase (instead of trypsin) to effect isolation of the somites does not result in spontaneous chondrification; and (4) neither does increasing the mass of somites grafted from 4 to 16 result in chondrification. These young posterior somites are the ones that have been employed in all interactive experiments to be reported in subsequent papers.

The incidence of other differentiated derivatives does not bear directly on the problem of somite chondrogenesis, but some points are of interest.

The usual appearance of bone with hypertrophied cartilage indicated that the latter is normally the precursor or initiator of ossification, although the three instances of isolated bone nodules in grafts demonstrate that this is not an invariable relationship. It is of interest that hypertrophied cartilage and bone can appear in these CAM grafts within 5 days of culture (i.e. 8 days total somite age). In the intact embryo, ossification does not commence in the vertebrae until about the 16 th day of incubation (stage 40). Thus well-developed bone can appear in somite grafts not only long before the somites would have given rise to bone in vivo, but even before bone has begun to appear in the host embryo. This suggests that in the intact embryo factors that are absent in somite grafts operate to slow down the process of somite chondrification and ossification.

The suitability of the CAM grafts for the analysis of somite myogenesis is limited by the spontaneous degeneration of striated muscle that occurs between the fifth and ninth days of culture. Striated muscle degeneration of a ‘fatty’ type has been observed in CAM grafts of limb-buds (Hunt, 1932), where it is associated with the absence of innervation. Striated muscle degeneration in CAM grafts of somites did not present the ‘fatty’ appearance described by Hunt, but as grafts with neural tissue make clear, it is also associated with the absence to muscle innervation. Degeneration of striated muscle has not been reported in organ cultures of somites (Ellison et al. 1969 b), possibly because the maturation of striated muscle is delayed in such cultures as compared with CAM grafts.

The distribution of ganglion cells in the somite grafts conforms both in time and spatial location to the observations of Weston & Butler (1966) on the migration of trunk neural crest cells. The earliest appearance of ganglion cells in the grafts (stage 12) coincides precisely with the first observable migration of neural crest cells away from the neural axis (Weston, 1970).

Attention has been drawn by Lash (1963) to the possibility that the inclusion of nephrotome cells in somite grafts may also result in the inclusion of chondrogenic lateral mesoderm cells. In the present grafts, trends in the incidence of cartilage were not influenced by the presence or absence of nephric tubules (see Fig. 1). Thus it is unlikely that lateral mesoderm cells of limb-bud origin have been included in the grafts with the nephrotome cells. The culture of nephrotome cells without concomitant differentiation of limb-bud cartilage has been demonstrated by Strudel & Pinot (1965).

The chorioallantoic grafting technique with Millipore filter as a graft vehicle is evidently both a reliable and a sensitive method of revealing the differentiative potential of small groups of cells, and is capable for instance of permitting the differentiation of cells derived from the earliest stages of neural crest migration. It does not afford the defined culture environment of in vitro organ culture methods but neither is it subject to the great sensitivity of tissues cultured by the latter method to small and often undefined alterations in medium composition. It is particularly suited to interactive experiments in which the highest degree of morphogenetic differentiation is required from both tissues and in which the spatial relationships of interacting tissues can be readily observed.

I am grateful to Professor R. 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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