Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2·5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9·5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.
In the course of the normal development of multicellular organisms, individual cells have to be localized appropriately in an embryo according to their type of cell differentiation. A number of studies have shown that cell differentiation is affected by various factors such as other cells, tissues or extracellular matrices; and the genetic mechanisms regulating cell differentiation are being studied (Moscona & Monroy, 1983; Okada & Kondoh, 1986). On the other hand, numerous studies on morphogenesis have been restricted to a part of an embryo, for example limb development in birds and amphibia (Tickle et al. 1975; Summerbell, 1981), and there have been very few attempts to learn how the shape of a whole organism is determined, except for studies using invertebrates (in hydra, Gierer, 1977; Schaller et al. 1979). However, with the aid of recent developments in gene manipulation, genes controlling the morphogenesis of insects have been investigated (Gehring, 1987). These genes share a nearly identical base-pair sequence of DNA called a homeobox, which has been shown to be present in the genomes of some vertebrates (in Xenopus laevis, Carrasco et al. 1984; in mouse, McGinnis et al. 1984; in human, Levine et al. 1984). However, in vertebrates, the function of the genes containing a homeobox has not yet been clarified because there are no vertebrate mutants resembling the homeotic mutants in Drosophila.
Somites are the mesodermal structures that appear transiently along both sides of the neural tube in the course of development in vertebrates, and are the repetitive structures along the rostrocaudal axis that give rise to cartilage, muscle and dermis (Bellairs et al. 1986). The developmental fate of a somite depends on its location along the rostrocaudal axis. For example, only the somites at the thoracic level produce ribs; the others do not (Chevallier, 1975). On the other hand, the course of cell differentiation depends on where the cells are located in a somite (Lash & Ostrovsky, 1986; Hall, 1977). The cells situated ventromedially in a somite differentiate into the sclerotome which gives rise to cartilage. From the other part of a somite, the dermomyotome arises and differentiates into muscle and dermis. The rostral and caudal halves of a somite have different characteristics in terms of the cell density of the sclerotome (Stern et al. 1986), neural crest cell colonization (Rickmann et al. 1985) and motor nerve innervation (Keynes & Stern, 1984; Rickmann et al. 1985). Thus, somites are important units forming the fundamental structure of the animal’s body and a key to understanding how the animal body is formed.
Our aim in this study was to learn when the morphology of the cartilagenous skeleton is determined in somites or their derivatives and whether it is controlled by the intrinsic factors in a somite or by the extrinsic factors surrounding a somite or the migrating cells derived from a somite. We transplanted the somites of quail embryos at 19- to 30-somite stages into chick embryos at about the same stage, with the reversal of one or two axes, and traced the cells of the grafted somites with the quail cell nucleolar markers (Le Douarin, 1973). Our results showed that the morphological fate of the sclerotomal cells was determined before or at metamerization of the somite, and before and independently of the determination of cell differentiation. Abstracts of this report have been published elsewhere (Aoyama & Asamoto, 1987, 1988) .
Materials and methods
Fertilized eggs of the White Leghorn chick and the Japanese quail were purchased from Hokuriku Jikkenn Doubutsu (Kanazawa, Ishikawa, Japan) and Nihon Uzura (Toyohashi, Aichi, Japan), respectively. The eggs were incubated at 37 °C for about 2 ·5 days to reach the 18- to 31-somite stages (stages 14 –18, Hamburger & Hamilton, 1951). The long axis of the hen’s egg was always kept in a horizontal position during incubation. For the operation on the host chick embryo in ovo, the surface of the egg shell was sterilized with 70% ethanol. 2 ml of albumen was sucked up by a syringe with a 21-gauge needle through a hole made at the pointed end of the egg to make an artificial air chamber over the embryo which was covered by the vitelline membrane. An opening with a diameter of about 3 cm was made on the upper side of the egg by removing pieces of the shell and underlying shell membrane with a pair of forceps. Through this window, the following operation was carried out under a dissecting microscope (SMZ-10, Nikon, Tokyo) illuminated by a double-light guide (LGW; Olympus Optical Co. Ltd, Tokyo). In order to improve the contrast between the embryo and the yolk, a small amount of India ink (Fount India; Pelikan AG, D-3000 Hannover 1, Germany) was injected under the embryo. A few drops of Ca2 +-free and Mg2 +-free Puck’s solution (CMF) (Puck et al. 1958) were added to the vitelline membrane over the embryo and then the membrane was cut with a tungsten needle to expose the embryo. The addition of saline solution facilitates the removal of the vitelline membrane from the embryo and prevents the embryo from drying. A slit in the ectoderm just above the portion where the neural tube and the most caudal three somites were apposed was made with a microscalpel made from a sewing needle, and then the somites were separated from the neural tube and the overlying ectoderm with the microscalpel. The somites were removed from the embryo by sucking with a micropipette having a diameter of about 0 ·03 –0 ·05 mm made by drawing out the tip of a Pasteur pipette. Throughout this study, we transplanted the three most caudal somites, or the three somites situated more rostral by one somite level into the most caudal three somite levels or the three somite levels more rostral than that by one somite level. These three somites are referred to as the caudal somites, unless otherwise stated.
For the preparation of transplants of quail somites, the fertilized eggs of quail were incubated at 37 °C for about 2 ·5 days to reach the 19- to 30-somite stages. After sterilizing the surface of the egg shells with 70 % ethanol, the egg was broken into CMF in a Petri dish, where the embryo was removed from the yolk. The part of the embryo containing the three caudal somite pairs was cut out of the embryo in CMF using a scalpel under the dissecting microscope. In this piece of the embryo, segmental plates were always included as a marker for rostrocaudal orientation. After incubation in 500i.u. ml −1 Dispase (Godo shusei Co. Ltd, Tokyo) in Dulbecco’s modified Eagle’s MEM (Nissui, Tokyo) supplemented with 10 % fetal calf serum (Bocknek, Rexdale, Ontario, Canada) (DMEM) at room temperature for 20 –60min, the three caudal somites were isolated from the surrounding tissues using tungsten needles sharpened with molten sodium nitride. The three caudal somites were isolated in contact with each other by retaining only a small amount of intermediate mesoderm. The contacting sites between isolated somites indicate the orientation of their mediolateral axes. The orientation of the rostrocaudal axis of the somites was indicated by the difference in the size of the three somites. The dorsoventral axis of the somites was determined by the orientation of the other two axes, because it was evident they were on the left or right side in the donor embryo. The isolated somites were transferred into a drop of DMEM with a micropipette having a diameter of about 0 ·5 mm and were kept at room temperature for a maximum of 3 h. The isolated somites were then transferred into the host egg using the 0 ·5 mm diameter micropipette and were positioned in the required orientation with a tungsten needle in the hole made by the removal of the somites of the host embryo. After the operation, the window of the host egg shell was sealed with Scotch tape (Sekisui, Tokyo) and further incubated at 37 °C for a maximum of 7 days in an incubator (with cooling unit; CR-14; Hitachi Ltd, Tokyo) with water in a vessel to humidify the atmosphere.
The chimeric embryos were fixed 1, 2 or 7 days after the operation and were prepared for the following histological procedures. The cells derived from the transplanted quail somites were detected in the chimeric embryos after fixation with 3 ·5% formalin in 0 ·1 m-phosphate buffer (pH 7 ·4) followed by Feulgen staining of the paraffin sections. The quail cells were distinguishable from the host chick cells because of their prominent nucleoli (Le Douarin, 1973) after Feulgen staining. To visualize the rudiments of dorsal root ganglia (DRG) and nerves, the chimeric embryos incubated for 2 days after the operation (4·5-day-old embryos) were stained by the modified zinc iodide/osmium tetroxide method (Keynes & Stern, 1984). After staining, the chimeric embryos were dehydrated, embedded in paraffin and sectioned. The sections were cleared in xylene and were mounted with Entellan (E. Merck, Postfach 41 49 D-6100 Darmstadt 1) for observation. To investigate the morphology of the cartilaginous skeleton, some of the formalin-fixed 9 ·5-day-old chimeric embryos (7 days after the operation) were stained in toto by alcian blue (Chroma, Stuttgart, Unterturkheim, Germany) after removing the skin and the internal organs (Simons & Van Horn, 1971).
Reconstruction of three-dimensional images from sections
Sequential 7 μm paraffin sections were made from 9 ·5-day-old chimeric embryos (7 days after the operation). After Feulgen staining, the outline of the embryo, cartilage, muscle and ganglia were traced, and the distribution of the quail cells derived from the grafted somites was marked in every 10th section with the aid of a camera lucida. Three dimensional images of the chimeric embryos were reconstructed from tracings of serial sections, using Cosmozone 2S software (Nikon, Tokyo), in a personal computer, PC9801E (NEC, Tokyo) with an 8-inch floppy disc drive unit, PC9881K (NEC, Tokyo). The traces were input with a digitizer, MITABLET-II, KD-4030 (GRAPHTEC CORP. Tokyo).
In the present study, we transplanted the most caudal three somites, or the second to the fourth most caudal three somites at 19- to 30-somite stages of quail embryos into chick embryos at 18- to 31-somite stages. In some experiments, the transplantation was carried out so that the directions of one or two axes of the graft were reversed in the host embryo. The viability at 7 days after the operation was examined to determine the effects of the operation, including the axial reversals. In the control experiment, when the somites were transplanted in normal orientation, 22/65 = 34 % of chimeric embryos were alive 7 days after the operation. The viability of the chimeric embryos with reversed dorsoventral axis, with reversed rostrocaudal axis and with reversal of both dorsoventral and rostrocaudal axes was 2/5 = 40%, 35/78 = 45% and 1/4 = 25%, respectively. There was virtually no difference between the viability of the control chimeric embryos and that of the chimeric embryos with the rotated somites. These results suggested that the reversal of the somite axes did not affect embryonic development sufficiently to cause the death of chimeric embryos.
When is the type of cell differentiation of somite cells determined?
When somites are transplanted with reversed dorsoventral axis, the dorsal parts of the transplanted somites are composed of cells that should become sclerotome and the ventral parts of cells that should become dermomyotome. If transplantation is carried out after the determination of cell type, the sclerotome and the dermomyotome will be inverted dorsoventrally. Conversely, if the cell differentiation of the transplanted somites has not yet been determined, both sclerotomes and dermomyotomes should appear at their normal locations.
The most caudal three somites on the right sides of 2 ·5-day-old chick embryos (26- to 29-somite stages) were replaced by the most caudal three somites from the left side of quail embryos at about the same stages (26- to 30-somite stages), so as to invert only the dorsoventral axis of somites (4 cases) (see Fig. 1C). One day after the operation, the 3 ·5-day-old chimeric embryos were fixed. Transverse sections stained by the Feulgen method showed that the two more caudal transplanted somites gave rise to more or less normal sclerotomes and myotomes (right side in Fig. 2A,B), while the most rostral somite gave rise to a dermomyotome which was abnormally located (right side in Fig. 2E,F), with mesenchymal cells between it and the ectoderm. Since these mesenchymal cells were shown to be derived from the transplanted somites on the basis of the morphology of their nucleoli (Fig. 2H), they must be sclerotomal cells. On the other hand, the localization of the myotome in the dermomyotome was normal, that is the myotome was situated medially in the dermomyotome. In the control experiment, the most caudal three somites from the right sides of quail embryos (22- to 24-somite stages) were transplanted in place of the most caudal three somites on the right sides of chick embryos (17- to 26-somite stages), so as to maintain the original orientation. In this experiment (five cases), all three transplanted somites gave rise to normal sclerotomes and dermomyotomes. Fig. 3 shows the ventral part of the dermomyotome and the sclerotome derived from the most rostral transplanted quail somite. These results suggested that the determination of somite cell differentiation had not occurred immediately after the formation of the somites; and that sclerotomal cell differentiation or determination started while forming two new somites caudally (it takes 3h 6 min (±20 min) to form two somites according to our observation. This value was deduced from the time required to form 4 pairs of somites in each of 29 embryos at from 18- to 22-somite stages to 22- to 26-somite stages; 2 ·5 h according to Elias & Sandor, 1965 ; 4h according to Chernoff & Lash, 1981; 3h 50 min according to Menkes et al. 1969).
When is the rostrocaudal axis of somites determined?
In order to investigate whether the rostrocaudal axis of somites is determined before the determination of cell differentiation of the somite cells, the most caudal three somites on the right side of 2 ·5-day-old chick embryos (21- to 27-somite stages) were replaced with those from the left side of quail embryos at about the same stages (20- to 24-somite stages), with inversion of the rostrocaudal axis (Fig. 1A). Two days after the operation, the 4 ·5-day-old chimeric embryos were fixed with 3 ·5 % formalin and then stained with zinc iodide/osmium tetroxide (2 cases). Frontal sections of the chimeric embryos showed the localization of the rudiments of the DRG relative to the sclerotomes (Fig. 4). The DRG were formed between the neural tube and the rostral halves of the sclerotomes in a normal embryo (see Fig. 4A, left half) or in control chimeric embryos that received a graft with normal orientation (2 cases) (data not shown). In the chimeric embryos with the quail somites inverted rostrocaudally, the rudiments of the DRG faced the caudal halves, or the original rostral halves, of the sclerotomes derived from the transplanted and rostrocaudally inverted quail somites, while they faced the rostral halves of the host sclerotomes (Fig. 4A). The conclusion is that the rostrocaudal axis of the somites has already been determined at their segmentation from the segmental plate. Together with the results described in the previous section, this result suggests that the determination of the rostrocaudal axis precedes the determination of cell differentiation.
Is the rostrocaudal axis of somites determined only in the region of prospective sclerotomes?
Among the tissues derived from somites, only sclerotomes are shown to differentiate into two parts, the rostral and caudal halves, along the rostrocaudal axis. As stated above, when the rostrocaudal axis of somites is determined, the type of cell differentiation has not yet been determined. To investigate whether the rostrocaudal axis of somites is determined only in a prospective sclerotome or extends over a whole somite, the most caudal three somites on the right sides in 2 ·5-day-old chick embryos (at 19- to 29-somite stages) were replaced with the most caudal three somites from the right sides of the quail embryos at about the same stages (25- to 30-somite stages), with inversion of the somites both rostrocaudally and dorsoventrally. In these chimeric embryos (five cases), the myotome derived from the somite transplanted most caudally, which was originally the most rostral in the donor embryo and had been determined for cell differentiation at the time of transplantation, was localized more medially than the normal one (Fig. 5). This result indicated that the dorsoventral axis of the transplant was actually inverted. The dermomyotome and the sclerotome derived from the transplanted somite situated most rostrally were normally localized between the ectoderm and the neural tube. That is, this sclerotome was derived from the prospective dermomyotome. while the rudiment of the DRG faced the caudal half, or the original rostral half, of the sclerotome. Consequently, the sclerotome derived from the prospective dermomyotomal region had its rostrocaudal axis oriented as in the grafted somites. This suggested that the determination of the rostrocaudal axis in a somite was not restricted to the region of the prospective sclerotome but extended throughout the entire somite.
The effect on morphogenesis of reversing the rostrocaudal axis of somites
The differentiation of a sclerotome along the rostrocaudal axis directs the position of the DRG and the site of motor nerve innervation. To investigate the role of the rostrocaudal axis on morphogenesis at a later stage, we observed the morphology of the tissues derived from the transplants, especially the cartilaginous skeleton of 9 ·5-day-old chimeric embryos (7 days after the operation).
The chimeric embryos were made by replacing the three caudal somites on the right sides of chick embryos at 22- to 23-somite stages with those from quail embryos at about the same stage as chick embryos (19- to 26-somite stages), with a reversed rostrocaudal axis (Fig. 1A) in the experimental group (13 cases) or with normal orientation in controls (9 cases) (Fig. 1B,D). Since in this study we observed the morphology of vertebrae and ribs, the somites isolated from the thoracic region were transplanted into the thoracic region of the hosts.
The cells derived from the transplanted quail somites in chimeric embryos, both of the control group and of the experimental group, differentiated into three types of cells: cartilage, muscle and dermis (data not shown). There were no differences between the control group (4 cases) and the experimental group (2 cases) in the distribution of the quail cells derived from the transplanted somites. The quail cells were detected in the right halves of four vertebrae in all the chimeric embryos. Fig. 6 shows a sagittal section from a chimeric embryo in the control group. Among the vertebrae containing quail cells, these cells were localized in the caudal half of the most rostral one and in the rostral half of the most caudal one. Moreover, these regions of the vertebrae were exclusively formed from quail cells. This result confirmed that one hemivertebra was composed of two adjacent hemisclerotomes, the caudal half of the rostral one and the rostral half of the caudal one (Gray, 1985). The three-dimensional images reconstructed from serial sections (Fig. 7) showed that quail cells derived from transplanted somites were at most in three ribs (Fig. 7B, Table 1). Although Table 1 shows that some chimeric embryos had less than three ribs containing quail cells, this number did not appear to be affected by the rotation of the rostrocaudal axis. Quail cells were also detected in a part of the scapula (data not shown) and uncinate process (Fig. 7B, arrowhead). The quail cells in muscles were distributed in the region that covers the cartilage derived from transplanted somites (Fig. 7C). The quail cells in dermis were also distributed in the region covering the cartilage derived from the transplant (data not shown).
To observe the morphology of cartilage in detail, some chimeric embryos were stained with alcian blue in toto. Fig. 8 shows typical examples of the experimental and control groups. Since in this experiment we were not able to detect the quail cells by the Feulgen method, we devised the following transplantation method to establish whether the tissues were derived from the grafts. The somites at the upper thoracic level were transplanted into the lower thoracic level or the lumbar level in the chimeric embryo shown in Fig. 8. The chimeric embryo shown in Fig. 8A-C had the uncinate process on the sixth rib, which usually had no uncinate process (see Fig. 8A), on the experimental side (Fig. 8C, double arrowheads). This means that the sixth rib was derived from the transplanted quail somite. Fig. 8D-F shows the chimeric embryo with the eighth rib, that is an extra rib on the experimental side (Fig. 8F). In this embryo at least the first lumbar vertebra must contain the transplanted quail thoracic somite cells, since Chevallier (1975) showed that the somites in the prospective thoracic region gave rise to ribs when they were transplanted outside the thoracic region. The position of the transplanted somites that are considered to have given rise to half vertebrae and ribs is indicated between the two large arrows in Fig. 8B and E.
The morphology of the vertebrae was almost normal in three of the five chimeric embryos in the control group, while it was abnormal in all eleven cases in the experimental group. There were some common abnormalities in these experimental chimerae as follows. The transverse process was on the rostral half of the vertebra in normal embryos (see left, unoperated, side of chimeric embryos in Fig. 8B,E) or in chimeric embryos in the control group (Fig. 8B; right half of vertebrae between two arrows), while it was on the caudal half of the vertebra in the chimeric embryos with rostrocaudally inverted somites (Fig. 8E; arrowheads). The most rostral transverse process derived from the grafted somites appeared to fuse with the transverse process derived from the somite of the host (Fig. 8E; double arrowheads). Actually, in some 4 ·5-day-old chimeric embryos with somites inverted rostrocaudally, the rostral half of the sclerotome, i.e. the derivative of the original caudal half of the grafted somite, fused with the caudal half of the rostrally adjacent host sclerotome (Fig. 5; *). As to the articulation between the vertebrae, the superior articular process was situated lateral to the inferior articular process in normal embryos (see left side of chimeric embryos shown in Fig. 8B,E) or in the control chimeric embryos (Fig. 8B; right side between two large arrows), while the superior articular process was situated medial to the inferior articular process in the chimeric embryos in the experimental group (Fig. 8E; small arrows).
In Fig. 9, a part of the spine derived from three somites is shadowed in A, a normal embryo, and is inverted rostrocaudally in B. The shapes of the vertebrae in B resemble those in Fig. 8E. This suggests that the abnormal morphology of vertebrae in the chimeric embryo with rostrocaudally inverted somites may be caused by the rostrocaudal inversion of a part of the cartilage derived from the transplanted somites.
Finally, the morphology of the rib was shown, by the direction of the uncinate process, to be also inverted rostrocaudally in the chimeric embryos in the experimental group. The uncinate processes projected caudalomedially from ribs in the normal embryos (see Fig. 8A,D) or in the chimeric embryos of the control group (Fig. 8C), while they projected rostromedially from ribs in the experimental chimeric embryos with rostrocaudally inverted somites (Fig. 8F, arrowheads).
All of the above results suggested that the morphology of the cartilaginous skeleton was determined intrinsically in the somites when their rostrocaudal axis was determined, i.e. when they were formed from segmental plate, at the latest.
Gallera (1966) showed that the dermatome was formed from the original ventral side of the primitive streak or of segmental plates, when they were transplanted with inversion of the dorsoventral axis and that the ventral sides of somites gave rise to additional dermatomes when the endoderm was replaced by the ectoderm. These results lead to the conclusion that the differentiation of the dermatome is induced by the ectoderm. Following experiments involving the transplantation of the somites or the segmental plate in a dorsoventrally reversed manner, Jacob et al. (1974) claimed that the determination of somite cell differentiation occurs at segmentation. However, our more precise experiment showed that the fate of the prospective sclerotome was determined about 3h after the formation of the somite from the segmental plate; i.e. the more caudal two somites gave rise to sclerotomes and dermomyotomes in normal positions (Fig. 2A) in spite of their dorsoventral inversion, while the third somite from the caudal end of the row of segmented somites formed an ectopic sclerotome just beneath the ectoderm (Fig. 2B) after its dorsoventral inversion. In the latter case, however, the myotome was normally situated medial to the dermatome and moreover another sclerotome was formed in a normal position between the dermomyotome and the neural tube from the prospective dermomyotome. Consequently, in one somite, determination to form the sclerotomal cell occurred first, followed by the determination to form the dermomyotomal cell.
However, it should be pointed out that these experiments could not rule out the possibility of the migration of somite cells back to an appropriate position after their mislocation by the experiment. To resolve this problem the migration pathway of each somite cell would have to be precisely traced after its transplantation.
Although the somite cells are uniform just after segmentation from the segmental plate, one of their derivatives, the sclerotome, differentiates into two parts, the rostral half and the caudal half. These two parts can be distinguished from each other by differences in cell density (Stern et al. 1986), by their capacity for peanut agglutinin (PNA) binding (Stern et al. 1986) and, more clearly, by their relationship to neural cells (Keynes & Stern, 1984; Rickmann et al. 1985). Although PNA bound specifically to the caudal half of the sclerotome in chick embryos, it did not bind to any part of tissues derived from somites in quail embryos (Asamoto et al. 1988). On the other hand, in both species, neural crest cells settled and formed the rudiments of DRG between the rostral half of the sclerotomes and the neural tube, and motor nerves grew into the rostral half of sclerotomes (Keynes & Stern, 1984; Rickmann et al. 1985; Asamoto et al. 1988). Keynes & Stern (1984) showed that, if the segmental plate was inverted rostrocaudally in chicken embryos, axons grow through the caudal, originally rostral, half of each sclerotome, indicating that the rostrocaudal axis of the somites was determined at or before their segmentation. This agrees with our finding, using chick-quail chimerae. When the most caudal three somites were transplanted with an inversion of the rostrocaudal axis, the rudiments of the DRG were formed between the neural tube and the caudal halves of the sclerotomes; i.e. the original rostral halves of the transplanted somites (Fig. 4).
All of the above results suggest that the determination of somite cell differentiation was preceded by the determination of sclerotomal cell differentiation along the rostrocaudal axis. By inversion of somite axes dorsoventrally and rostrocaudally, we showed that sclerotome derived from prospective dermomyotome, which showed no apparent differentiation along rostrocaudal axis, kept the normal rostrocaudal axis. These results suggested that the establishment of the rostrocaudal axis of somites was independent of cell differentiation.
We observed the morphology of the cartilaginous skeleton in 9 ·5-day-old chimeric embryos, in which the caudal somites of the host embryo had been replaced with rostrocaudally inverted caudal somites of the donor. It was astonishing that the morphology of the cartilage was inverted along the rostrocaudal axis (Fig. 8E,F). The morphology of the articulation between vertebrae, the position of the transverse process on a vertebra and the direction of the projection of the uncinate process on a rib appeared to be inverted along the rostrocaudal axis (Fig. 8E,F and Fig. 9). These results strongly suggested that the morphology of the cartilage was controlled by factors intrinsic to each somite and was not much influenced by their circumstances. Chevallier (1975) showed that the segmental plate of the prospective thoracic region was able to give rise to ribs even if it was transplanted into the prospective cervical region or into the prospective lumbar region, and he concluded that ‘the somitic mesoderm is already regionalized at a stage slightly preceding its metamerization’. Our results show that the fine structures in a cartilage are also governed by intrinsic factors, which are realized as the rostrocaudal axis before, or at, metamerization of the somite. It is well known that cartilage cell differentiation from sclerotomal cells depends on neural tube and notochord (Hall, 1977). If morphogenesis of cartilage also depends on neural tube and notochord, the influence of these tissues on somitic mesoderm must be exerted before segmentation of the somite. Alternatively, morphogenesis may not depend at all on these tissues, unlike cell differentiation.
We have confirmed that one vertebra is composed of four parts derived from the four adjacent hemisclerotomes, the caudal halves of the right and left rostral sclerotomes, and the rostral halves of the right and left caudal sclerotomes (Gray, 1985). This was deduced from the fact that three consecutive somites transplanted in the chimeric embryo participated in forming four vertebrae (Figs 6, 9A). The most rostral vertebra containing transplanted quail cells had these in its caudal half on the experimental side. The most caudal vertebra containing transplanted quail cells had them in the rostral half on the experimental side. The experimental halves of the two vertebrae between the two just mentioned were totally composed of transplanted quail cells. Consequently, each somite must give rise to the caudal half of one half vertebra and the rostral half of the caudally adjacent half vertebra. As to ribs, the cells derived from three transplanted quail somites were detected in three ribs at most. This means that one rib is derived from one somite.
In the present study, we have shown that the differentiation of sclerotomal cells along the rostrocaudal axis is determined before, and independently of, the determination of somite cell differentiation. The rostrocaudal axis, which has been established in somites when they are formed by metamerization, guides the morphogenesis of cartilage derived from the somites.
Part of this study was financed by Grants-in-Aid for Scientific Research from The Ministry of Education, Science and Culture and by the grant from The Ichiro Kanehara Foundation to H.A.