= = = = = = = = = ANNALS
or ANATOMY = = = = = = = = =
From somites to vertebral column* Bodo Christ** and Jorg Wilting Institute of Anatomy, Albert-Ludwigs-University, AlbertstraBe 17, D-W-7800 Freiburg i. Br., Germany
Abstract. We report on the development and differentiation of the somites with respect to vertebral column formation in avian and human embryos. The somites, which are made up of different compartments, establish a segmental pattern which becomes transferred to adjacent structures such as the peripheral nervous system and the vascular system. Each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of vertebrae, whereas the vertebral body and the intervening disc develop from the axially-located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs. The neural arches form a frame for the neural tube and spinal ganglia. The boundary between head and vertebral column is located between the 5th and 6th somites. In the human embryo, proatlas, body of the atlas segment, and body of the axis fuse to form the axis. Key words: segmentation, somite, vertebral column, pattern formation, neural development
Introduction It is well known that the somites contribute to the formation of the vertebrae and the intervening discs. However, the precise relationship between the two generations of segmental structures has long been controversial. This also applies to the relationship between the somite boundaries and the
* This paper is dedicated to Professor Dr. K. **
V. Hinrichsen on occassion of his 65th birthday Main lecture at the 86th meeting of the Anatomische Gesellschaft in Szeged, Hungary, April 2 to April 5, 1991
)~I UMPElt2
Ann. Anat. (1992) 174: 23-32 Gustav Fischer Verlag Jena
vertebral bodies and the discs, as well as to the problem of how the somites take part in the development of the lateral elements (vertebral arches and ribs) of the vertebral column. Remak (1855) was the first to introduce the theory of resegmentation, which means that a vertebra is formed by the combination of the caudal half of one bilateral pair of somites with the cranial half of the immediately caudal pair of somites. This concept was later reinforced by von Ebner (1888), who used the term "Neugliederung" and received support from several other studies (reviewed by Verbout 1976). On the other hand, the concept of resegmentation has often be challenged (Blechschmidt 1957; Baur 1969; Christ et al. 1979, 1982; Verbout 1985). These authors described a development in which the vertebral bodies, the arches with their processes and the discs, arise from the beginning at their definitive sites. Recently, Bagnall et al. (1988, 1989a, b) provided experimental evidence that a single somite contributes to one half of each of the two adjacent vertebrae, as well as to the intervenning disc. However, the projection of the somite boundaries on to the vertebral column could not exactly be drawn because the results differed with respect to the marking method that was used. As was stated by Verbout (1985), the distinction between lateral and axial differentiation has not been taken into account in most of the studies on the development of the vertebral column. This is surprising, because distinct temporal and spatial differences can be found between the development of the arches and the formation of the vertebral bodies and intervening discs. Studying the development of the thoracic vertebrae in the mouse, Dalgleish (1985) emphasized that the axial parts of the vertebral column arise from a continous central tissue mass surrounding the notochord, whereas the neural arches develop from the caudal half of the sclerotome. This is in line with the conclusions drawn by Verbout (1985), who investigated the formation of the vertebral column in the sheep. In this paper, we review in detail the contribution of the
Results
sclerotome to the different parts of the vertebral column in the chick. We also describe the relationship between the sclerotomes and the neural elements. Eventually, we report on the formation of vertebral bodies and intervertebral discs in the avian and human embryo.
Material and
Somite Formation As a consequence of gastrulation two rods of mesoderm, the segmental plates arise. They are situated on each side of the neural tube and notochord. In the chick embryo, somites form from the cranial end of each segmental plate at an approximate rate of one pair every 100 min. The formation of somites is preceded by epithelialization of the segmental plate mesoderm (Christ et al. 1972). The still-unsegmented segmental plate, including its most caudal part, has been found to be destined to form somites in a cranio-caudal sequence even after inversion (Christ et al. 1972, 1974, 1975). This means that the segmental plate mesoderm has the ability to form somites from its birth in Hensen's node. The morphological manifestation of this predetermined state might be the so-called somitomeres seen by Meier (1979) with scanning electron microscopy.
Method~
Eggs of the White Leghorn strain of the common fowl (Gallus domesticus) were incubated at 3rC for periods between 2 and 8 days. After fIxation in 3-6% glutaraldehyd, in 0.12 M cacodylate buffer (pH 7.4) for periods of 4-24 h, the embryos were washed in Hanks' solution, dehydrated in graded acetone, and embedded in Durcupan ACM (Auka). Semithin sections were stained with methylene blue.
Each somite first appears as an epithelial sphere, whose radially-arranged cells line a small central lumen (somitocoel) which is reminiscent of the original mesenchyme (Fig. 1), and consists of about 2500 cells (personal communication, Ch. Ordahl, San Francisco). Somite-formation is accompanied by an increase in the expression of the calcium-dependent adhesion molecule N-cadherin (Duband et al. 1987). As was shown by Lash et al. (1984) and Jacob et al. (1991), fibronectin is involved in the epithelialization of the segmental plate mesoderm. The somite cells are polarized, with the Golgi zone located in the apical compartment and tight junctions at the luminal border (Revel et al. 1973; Lipton and Jacobson 1974). The newly-formed somite is surrounded by a dense basal lamina (Jacob et al. 1991).
Immunhistochemistry Specimens were fixed in 98 % ethanol and 2 % glacial acetic acid and then washed twice in ethanol and in xylene each for 15 min at room temperature. After embedding in paraplast, the embryos were serially sectioned. The sections were deparaffinated and rehydrated in xylene and graded ethanol, and rinsed in PBS buffer to which 0.05 % Tween-20 has been added. Section were incubated in a poly-clonal anti-fibronectin antibody (Euro-Diagnosticsll: 10,000) or an HNK-1 antibody (Becton Dickinson/I: 20) in PBS containing 0.05 % Tween-20, 1 % ovoalbumin and 0.2 % glycine. Overnight incubation was followed by washing in PBS containing Tween-20. In a second incubation step, the antigen-antibody complex was labelled with a second antibody to which horse radish peroxidase (HRP) was attached. These second antibodies were goat anti-rabbit and a goat anti-mouse antibodies (Dako Diagnostika) diluted 1: 300 in PBS. After incubation for 2 h, the sections were washed three times in buffer, and the staining reaction performed with diamino-benzidin (DAB)-HzO z for 10 min. The sections were washed again in buffer and subsequently stained in hematoxylin for 10 s. For indirect immunofluorescence, embryos were fIxed in 4 % paraformaldehyde in 0.1 M-potassium phosphate buffer (pH 7.6) overnight at 4 % C. Specimens were then rinsed in several changes of buffer and infIltrated with 5 % and 15 % sucrose in buffer, each overnight at 4 % C. Embryos were then embedded in OCT compound (Cambridge Instruments, Nussloch) and frozen. The sections were cut at 20 11m in a Reichert-Jung Model 2800 cryostat, and then collected on chrome alum-gelatin-coated slides, air dried and stained with a rabbit polyclonal anti-tenascin antibody (a gift from Drs. R. Chiquet-Ehrishmann and E. Mackie, Basel). After rinsing in buffer, the sections were incubated in FITC-conjugated goat antirabbit IgG (Dranova, Hamburg) for 1 h, and then rinsed and mounted in Hanks' saline: glycol (1: 1). For scanning electron microscopy (SEM), specimens were fixed in 4 % glutaraldehyde, dehydrated in graded acetone and criticalpoint-dried. After the specimens had been mounted on SEM stubs, the ectoderm was removed with tungsten needles. They were then sputtered with gold and examined with a Jeol JSM 35 R microscope. The human embryos were kindly provided by Dr. M. Grim (Praha). They were processed for semithin sections as described above for chick embryos.
The cells of the early epithelial somite are obviously assigned to at least six different compartments. Christ et al. (1978) have shown that the dorsal half of the somite gives rise to the dermornyotorne, while the ventral half is the source of the sclerotome. The studies of Keynes and Stem (1984), Rickmann et al. (1985), as well as those of Stem and Keynes (1987), habe provided strong evidence that each somite is subdivided into cranial and caudal halves. Motor axons and neural crest cells migrate through the cranial half of the sclerotome (Fig. 6 and 7). Selleck and Stem (1991) eventually demonstrates by DiI labelling of parts of Hensen's node and the primitive streak, that the somite can also be subdivided in a medial and a lateral half. The somitic precursors in Hensen's node only contribute to the medial halves of the somites, whereas the lateral halves are derived from a separate region of the primitive streak, caudal to the node itself. This finding received support from experiments concerning the origin of myoblasts (Ordahl and Le Douarin 1992) in which the lateral and medial halves were combined in different ways. Another important point is that the somites are predetermined with respect to the morphological features characteristic of the axial level. Thoracic somites heterotopic ally grafted give rise to ribs (Kieny et al. 1972; Jacob et al. 1975a). This means that the regionalization along the embry-
24
Fig. 1. Transverse section of a 2-day chick embryo. S somite; N neural tube; A aorta; asterisk: cell-free space between notochord and somite. x 450. Fig . 2. Lateral view of the neural tube of a 2-day chick embryo. Asterisks: unsegmented neural crest. X 550. Fig. 3. Sagittal section of a 2-day chick embryo. A aorta; arrows : intersomitic clefts containing blood vessels .
x
230.
Fig. 4. Frontal section of a 4-day chick embryo at the level of the ventral roots (arrows). M myotome; asterisk: mesenchymal condensation of the caudal sclerotome. x 300. Fig. 5. Frontal section of a 4-day chick embryo. Arrow: spinal nerve; asterisk: condensation of the caudal sclerotome; M myotome. x 300.
25
onic axis is a very early event during development. This regionalization is controlled by the expression of Hox genes at different axial levels (Kessel et al. 1990). Homeotic transformation in the mouse can be caused by an ectopic expression of Hox genes after the administration of retinoic acid (Kessel and Gruss 1991). According to these authors, the specificity of the paraxial mesoderm with respect to characteristic segmental identities depends on the formation of regional-specific Hox gene-expression domaines, the Hox codes. About 4 or 5 hours after the somite has formed, the migration of neural crest cells begins to take place (Fig. 2). At first, the neural crest cells form a continous and unsegmented strip (Fig. 2). The segmental arrangement of vessels (Fig. 3), nerves (Figs. 5 and 16), and spinal ganglia (Fig. 7) is caused by interaction between these structures, and the somites which had previously established the segmental pattern within the body wall of the early embryo (Christ 1975; Christ et al. 1979; Keynes and Stem 1988).
level of the spinal ganglia, and becomes surrounded by a highly vascularized perineural connective tissue. Figs. 4, 5 and 6 clearly show that the caudal boundary of the arch rudiment is marked by an intersegmental vessel. This observation points to the fact that the condensation originates only from the caudal part of each segment. A transverse section through the caudal half of the sclerotome reveals that the laterally-situated and denselypacked mesenchyme is triangular in shape. The edges represent the arcual process, the costal process, and the pedicle of the vertebra.
Formation of vertebral bodies and discs During the first steps of neural arch formation, the axial area is still cell-free. The notochord is surrounded by extracellular matrix material (Figs. 11 and 12). As has been shown by Jacob et al. (1975b), this material consists of radiallyoriented microfibrils and interstitial bodies. Fig. 12 shows that the fibrils are coated with fibronectin. It is very likely that these extracellular materials function as a substratum for the immigrating sclerotome cells which colonize the perinotochordal space. In this way the axially situated loosely-meshed mesenchyme originates, and does not show any signs of segmentation. As mentioned above, in this part of the sclerotome-derived mesenchyme (the perichordal tube) intra- and intersegmental fissures can never be found (Fig. 13). Fig. 16 shows several layers of mesenchymal cells which are concentrically oriented, surrounding the notochord. The segmentation of the axial mesenchyme begins to take place with parachordally located condensations, which represent the intervertebrate discs (Figs. 14 and 15). The course of this process, as well as its control mechanisms, are not yet well understood. However, with the appearance of the disc rudiments, the boundaries of the vertebral bodies become established. In order to form a unified vertebral column, the paired arch rudiments must connect the unpaired vertebral bodies. This process is shown in Figs. 15 and 16. That part of the arch rudiment that later represents the pedicle of the vertebra fuses with the extreme cranial part of the vertebral body (Fig. 15).
Somite differentiation During further development, the somites become subdivided in the mesenchymal sclerotome and the early epithelial arrangement of the dermomytome (Fig. 3). The latter subdivides further afterwards into the dermatome and the myotome (Figs. 6 and 13). While the myotome gives rise to postmitotic myoblasts, the dermatome is the source of still replicating myoblasts and the fibroblasts of the dermis of the back (Christ et al. 1983; Kaehn et al. 1988). The sclerotome is derived from the ventral half of the somite, which has lost its epithelial arrangement (Fig. 3). Though the later differentiation fate of the luminal cells of the somite is not yet quite clear, there is no doubt that they contribute to the formation of the sclerotome. The disaggregation of the somite wall is preceded by a loss of N-cadherin immunoreactivity in that region of the epithelium from which the sclerotome arises (Dub and et al. 1987). Following the sclerotome development, one has to distinguish between lateral and axial areas (Figs. 11, 13, 16,23).
Formation of neural arches and ribs Later stages of vertebral development
The formation of the vertebral column begins to take place laterally, with a morphologically conspicuous subdivision of the sclerotome into a densely packed caudal half and a more loosely structured cranial half (Fig. 13). Between these differently developing parts of the sclerotome, the so-called intrasegmental fissure (v. Ebner 1888) can be seen (Fig. 8). It separates the area of the developing arch and rib from the peripheral neural tissue. It is important to note that this "fissure" does not extend to the axial area, where at a later stage the vertebral bodies and discs arise. The alternation of arch elements and neural tissue is shown and Figs. 4-10 for the levels of spinal ganglia, ventral roots and spinal nerves. The peripheral neural tissue expands particularly at the
The vertebral body after fusion with the neural arches can be seen in Fig. 22. The neural arches of both sides then gradually surround the neural tube and fuse dorsally. Figs. 17, 18 and 21 show the development of vertebral bodies and discs in the human embryo. In the 12 mm embryo (Fig. 17) the perichordal mesenchyme has differentiated into discs and vertebral bodies. Within the disc rudiments the cells are densely packed, especially at the periphery. The anulus fibrosus develops, whereas in this area the central zone surrounding the notochord gives rise to the nucleus pulposus. Figs. 18 and 21 show the vertebral column of 30 mm embryo. The separation of the disc into
26
Fig. 6. Frontal section of a 3-day chick embryo at the level of the ventral roots (arrows). Staining with HNK-I antibody. M myotome. Asterisk: caudal part of the sclerotome. x 250. Fig. 7. Frontal section of a 3-day chick embryo at the level of the spinal ganglia (arrows). M myotome. Asterisk : caudal part of the sclerotome. x 250. Fig. 8. Sagittal section of a 3-day chick embryo. Big arrows : cranial half of the sclerotome with nervous elements; asterisks : caudal half of the sclerotome; small arrows: intersomitic vessels . X 200. Fig. 9. Sagittal cryosection of a 3-day chick embryo. Staining with anti-tenascin . Arrows: tenascin-positive cranial halves of the sclerotomes. x 110. Fig. 10. Higher magnification of Fig. 9. Arrows: tenascin-positive cranial halves of the sclerotomes. asterisks: condensed mesenchyme of the caudal halves. x 240.
27
The first somite disappears very early and the following 4 somites fuse to form the basioccipital bone. The cranial half of the 6th somite gives rise to the proatlas, which will later be incorporated into the apical part of the dens. The caudal half of the 6th somite fuses with the cranial half of the 7th somite to form a vertebral body, the dens axis, which later fuses with the body of the second vertebra, the axis. Fig 20 shows that dens and axis body are temporarily separated by an intervertebral disc, which later on disappears to form an unified bone. The main steps leading from the somites to the vertebral column are shown in the drawing in Fig. 23. According to our results, the neural arches, pedicles of vertebrae and ribs originate from the caudal half of one somite. Two adjacent somites seems to contribute to the formation of the vertebral body. The disc originates from the caudal half of the somite.
Discussion The results mentioned above can be summarized as follows. 1. The segmental plate mesoderm is very early determined with respect to the segmentation and cranio-caudal sequence in which the somites are formed. 2. The epithelial somite is subdivided into at least six different compartments. Each somite is regionalized with respect to the skeletal features characteristic of the axial level. 3. Each somite gives rise to the epithelial dermomyotome, and the mesenchymal sclerotome which provides the material for the vertebral column. 4. The vertebra arises from three sclerotomic areas. In the paired lateral areas the neural arch, the pedicle and the rib develop. The unpaired axial mesenchyme gives rise to the vertebral bodies and to the discs. These parts come together and form the vertebral column. 5. Projecting the somite boundaries onto the vertebral column we hold that the formation of the lateral vertebral elements takes place without resegmentation. On the other hand, formation of the body does not take place within the boundaries of one somite. 6. When the chondrification of the vertebral bodies begins, the discs start to differentiate into anulus fibrosus and nucleus pulposus. 7. The five cranial somites are included in the development of the head. The dens axis is homologous with the body of the atlas.
2 Fig. 11. Transverse section of a 2-day chick embryo. N neural tube; A aorta; asterisk: notochord; arrows: single cells migrating into the parachordal space. x 450. Fig. 12. Transverse section of a 2-day chick embryo. Staining with anti-fibronectin antibody. N neural tube; A aorta; asterisk: notochord; arrows: radially oriented extracellular matrix. x 450.
anulus fibrosus and nucleus pulposus is now clearly recognizable. The vertebral bodies are chondrified. It is worth mentioning that the notochord, which now becomes thicker at the disc levels and thinner at the body levels, is more ventral than central. Special features reveal the transitional zone between head and neck. As was shown by Christ et al. (1988), the boundary between head and neck corresponds to the boundary between the 5th and the 6th somite. This is true for the chick as well as for the human embryo.
In the embryo of higher vertebrates the somites have two important functions. They constitute the segmental pattern within the body wall, which is later transferred to other structures such as blood vessels and nerves (Christ et al. 1975; Stern und Keynes 1987). The other function is to specialize and to distribute cells. So, for instance, the muscle precursor cells become committed to two different lineages (Christ et al. 1990; Ordahl and Le Douarin 1992). As segmentation is a fundamental process of body organization, 28
Fig. 13. Sagittal section of a 3-day chick embryo. M myotome; asterisks: lateral condensation of the caudal sclerotome half. X 150. Fig. 14. Frontal section of a 4,5-day chick embryo. M myotome; asterisks: pedicle rudiments; arrows: anlagen of the discs. x 180. Fig. 15. Frontal section of a 5,5-day chick embryo. M myotome; asterisks: pedicles; arrows: intervertebral discs. Note the well vascularized perineural connective tissue in the foramen intervertebrale. x 130. Fig. 16. Transverse section of a 4-day chick embryo at the level of the caudal sclerotome half. M myotome; 1 neural arch; 2 pedicle; 3 rib. x 130. Fig. 17. Median section of a 12 mm human embryo. C notochord; arrows: intervertebral discs. x 100. Fig. 18. Median section of a 30 mm human embryo. C notochord; arrows: Anulus fibrosus of the intervertebral discs. x 70.
29
Fig. 19. Median section of a 24 mm human embryo. Head-neck transitional zone. Asterisk: oral cavity; Bb basioccipital bone; C notochord; arrow: proatlas; D Dens axis; Ax body of the axis. X 50. Fig. 20. Higher magnification of Fig. 19. C notochord; D dens axis; Ax body of the axis; Arrow: transient disc. x 130. Fig. 21. Transverse section of a 30 mm human embryo through a thoracic intervertebral disc: AF anulus fibosus; asterisk: notochord. x 90. Fig. 22. Transverse section of a 5-day chick embryo stained with anti-tenascin. N neural tube; asterisk: notochord; arrows: neural arches . x 120. Fig. 23 . Diagram showing the development of the vertebral column. A. Stage of epithelial sornites. The cranial half of the somite is loosely punctured and contains neural crest cells. B Differentiation of sclerotomes. Note the different development of the lateral and axial mesenchyme. Laterally, cranial and caudal halves give rise to different structures. Axially, the disc rudiments line the vertebral bodies. C Projection of the somite boundaries on to the differentiated vertebral column.
30
development of the dens, which arises by fusion of the axial body, the body of the atlas and the proatlas. This is in line with the findings of Reiter (1944), but does not support the hypothesis of Ludwig (1953, 1957) that the dens represents a structure sui generis developing from cranial processes of the axial body.
which is already realized in the earthwonn, it is not surprising that this pattern becomes detennined at a very early stage of development. As was shown by Christ et al. (1972, 1975) the newly developed parts of the segmental plate mesodenn are already destined to fonn somites. However, the shape of the developing somites depends upon the condition of the adjacent tissues. If, for instance, the neural tube is removed at the level of the still-unsegmented paraxial mesodenn, the plates fuse and fonn unpaired somites. It is of great interest that the somite is subdivided into different compartments. Selleck and Stern (1991) found that the progenitors of the medial half of the somite are derived from the lateral portion of Hensen's node, while the progenitors of the lateral half are derived from cells located in the primitive streak. The result points to the fact that the somite consists of already committed cells. Whether different compartments of the epithelial somite constitute different parts of the vertebra is still an open question. Verbout (1985) was right to emphasize that, in most of the studies dealing with the fonnation of the vertebral column, the differences between lateral and axial developing parts have not been taken into account. The controversial discussions on resegmentation may be founded on this deficiency. Experimental investigations on vertebral column fonnation must remain unsuccessful if the nonnal developmental steps are not fully taken into consideration. Interspecific replacements of single somites are not suitable for clarifying the problem of resegmentation, because the results could be falsified by injuries and regulation processes. Our observations support the view of Dalgleish (1985) and Verbout (1985) that the neural arch of the vertebra is derived from the caudo-Iateral part of one somite. We agree with Bagnall (1988, 1989a + b) that the vertebral body is made up of cells from two adjacent somites. However, the exact boundaries of somite participation are not yet clear. As shown by Christ (1975), Christ et al. (1979) and Rickmann et al. (1985), the segmental pattern is transferred by the somites to the blood vessels, the spinal ganglia, and the nerves. On the other hand, the segmental pattern of the neural arches develops in interaction with the spinal ganglia and nerves. Thus, a hierarchy of segmental structures can be suggested, in which the somites represent the primary, the spinal ganglia and nerves the secondary, and the neural arches the tertiary elements. The ribs, however, develop autonomously (Kieny et al. 1972; Jacob et al. 1975). The definite relationship between somites OIl the one hand and the vertebral bodies and discs on the other remains to be established. When the chondrification of the vertebral bodies begins to take place, the discs differentiate into anulus fibrosus and nucleus pulposus. The nonnal development of the axial parts of the vertebral column depends on interactions with the notochord (for review Christ, 1975). If the notochord has been experimentally removed the axial mesenchyme fonns ventral arches. The nonnal development of neural arches depends on the existence of the neural tube (Christ, 1975). The five cranial somites do not contribute to the vertebral column of human embryos. 2Yz somites participate in the
Concluding remarks Although important steps in the development of the vertebral column now seem to have been clarified, there are a lot of remaining problems still to be solved. A reliable method of experimentally labelling single somites or groups of somitic cells without destroying them would be highly desirable. Further genetic and molecular biological research is needed to deepen and extend our knowledge in this field.
References Bagnall KM, Higgins SJ, Sanders EJ (1988) The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimera model. Development 103: 69-85 Bagnall KM, Sanders EJ (1989a) The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo. Anat Embryol 180: 505-513 Bagnall KM, Higgins SJ, Sanders EJ (1989b) The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments. Development 107: 931-943 Baur R (1969) Zum Problem der Neugliederung der Wirbelsliule. Acta Anat 72: 321-356 Blechschmidt E (1957) Die Entwicklungsbewegungen der Somiten und ihre Bedeutung fUr die Gliederung der Wirbelsaule. Z Anat Entwickl-Gesch 120: 150-172 Christ B (1975) Die Entwicklung der Korperwandmetamerie . an Hiihnerembryonen, . Experimentelle Untersuchungen Habilitationsschrift, Bochum Christ B, Jacob HJ, Jacob M (1972) Experimentelle Untersuchungen zur Somitenentstehung beim Hiihnerembryo. Z Anat Entwickl-Gesch 138: 82-97 Christ B, Jacob HJ, Jacob M (1974) Die Somitogenese beim Hiihnerembryo. Zur Determination der Segmentierungsrichtung. Verh Anat Ges 68: 573-579 Christ B, Jacob HJ, Jacob M (1975) Autoradiographische Untersuchungen zur Somitenentstehung beim Hiihnerembryo. Verh Anat Ges 69: 259-261 Christ B, Jacob HJ, Jacob M (1979) Ober Gestaltungsfunktionen der Somiten bei der Entwicklung der Korperwand von Hiihnerembryonen. Verh Anat Ges 73: 509-518 Christ B, Jacob HJ, Jacob M (1978) On the formation of the myotomes in avian embryos . An experimental and scanning electron microscopic study. Experientia 34: 514-516 Christ B, Jacob M, Jacob HJ (1983) On the origin and development of the ventrolateral abdominal muscles in the avian embryo. Anat Embryoll66: 87-101 Christ B, Jacob HJ, Seifert R (1988) Ober die Entwicklung der zervikookzipitalen Obergangsregion. In: Hohmann D, Kiigelgen
31
B, Liebig K (eds) Springer, Berlin, Heidelberg, Neuroorthopadie 4 pp 13-22 Christ B, Brand-Saberi B, Jacob HJ, Jacob M, Seifert R (1990) Principles of early muscle development. In: Le Douarin N, Dieterlen-Lievre T, Simith J (eds) The avian model in developmental biology: From organism to genes. Edition du CNRS Paris Dalgleish AE (1985) A study of the development of thoracic vertebrae in the mouse assisted by autoradiography. Acta Anat 122: 91-98 Duband JL, Durfour S, Hatta K, Takeichi M, Edelmann E, Thiery JP (1987) Adhesion molecules during somitogenesis in the avian embryo. J Cell BioI 104: 1361-1374 Ebner von E (1888) Urwirbel und Neugliederung der Wirbelsaule. Sitzungsber Akad Wiss Wien I1I/97 194-206 Jacob M, Christ B, Jacob HJ (1975a) Uber die regionale Determination des paraxialen Mesoderms junger Htihnerembryonen. Verh Anat Ges 69: 263-269 Jacob M, Jacob HJ, Christ B (1975b) The early differentiation of the perinotochordal connective tissue. A scanning and transmission electron microscopic study on chick embryos. Experientia 31: 1083-1086 Jacob M, Christ B, Jacob HJ, Poelmann R (1991) The role of fibronectin and laminin in development and migration of the avian Wolffian duct with reference to somitogenesis. Anat Embryol 183: 385-395 Kaehn, K, Jacob HJ, Christ B, Hinrichsen K Poelmann R (1988) The onset of myotome faromation in the chick. Anat Embryol 177: 191-201 Kessel M, Balling R, Gruss P (1990) Variations of cervical vertebrae after expression of a Hox -1.1 transgene in mice. Cell 61: 301-308 Kessel M, Gruss P (1991) Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67 1-20 Keynes RJ, Stern CD (1984): Segmentation in the vertebrate nervous system. Nature, London 310: 786-785 Keynes RJ, Stern CD (1988) Mechanism of vertebrate segmentation. Development 103: 413-429
Kieny M, Mauger A, Sengel P (1972) Early regionalization of the somitic mesoderm as studied by the development of the axial skeleton of the chick embryo. Develop Bioi 142-161 Lash JW, Seitz AW, Cheney CM, Ostrowsky D (1984) On the role of fibronectin during the compaction stage of somitogenesis in the chick embryo. J exp Zool 232: 197-206 Lipton BH, Jacobsen AG (1974) Analysis of normal somite development. Develop Bioi 38: 73-90 Ludwig KS (1953) Die Friihentwicklung des Dens epistrophei und seiner Bander beim Menschen. Morphol Jb 93: 98-112 Ludwig KS (1957) Die Friihentwicklung des Atlas und der Occipitalwirbel beim Menschen. Acta Anat 30: 444-461 Meier S (1979) Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern. Develop Bioi 73: 25 -45 Ordahl PA, Le Douarin N (1992) Two myogenic lineages within the developing somite. Development (in press) Remak R (1855) Untersuchungen tiber die Entwicklung der Wirbeltiere. Reimer, Berlin Reiter A (1944) Die Friihentwicklung der menschlichen Wirbelsaule. II. Mitteilung: Die Entwicklung der Occipitalsegmente und der Halswirbelsaule. Z. Anat Entwickl-Gesch 113: 66-104 Revel JP, Yip P Chang LL (1973) Cell junctions in the early chick embryo - a freeze etch study. Develop Bioi 35: 302-317 Rickmann M, Fawcett J, Keynes RJ (1985) The migration of neural crest cells and the growth of motor axons through the ventral half of the chick somite. J Embryo1 exp Morph 90: 437-453 Selleck MA, Stern CD (1991) Fate mapping and cell lineage analysis of Hensen's node in the chick embryo. Development 112: 615-626 Stern CD, Keynes RJ (1987) Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo. J Embryol exp Morph 91: 209-226 Verbout AJ (1976) A critical review of the ,Neugliederung' concept in relation to the development of the vertebral colunm. Acta Biotheoret 25: 219-258 Verbout AJ (1985) The development of the vertebral colunm. Adv Anat Embryol Cell BioI 90: 1-122
32
or ANATOMY = = = = = = = = =
From somites to vertebral column* Bodo Christ** and Jorg Wilting Institute of Anatomy, Albert-Ludwigs-University, AlbertstraBe 17, D-W-7800 Freiburg i. Br., Germany
Abstract. We report on the development and differentiation of the somites with respect to vertebral column formation in avian and human embryos. The somites, which are made up of different compartments, establish a segmental pattern which becomes transferred to adjacent structures such as the peripheral nervous system and the vascular system. Each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of vertebrae, whereas the vertebral body and the intervening disc develop from the axially-located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs. The neural arches form a frame for the neural tube and spinal ganglia. The boundary between head and vertebral column is located between the 5th and 6th somites. In the human embryo, proatlas, body of the atlas segment, and body of the axis fuse to form the axis. Key words: segmentation, somite, vertebral column, pattern formation, neural development
Introduction It is well known that the somites contribute to the formation of the vertebrae and the intervening discs. However, the precise relationship between the two generations of segmental structures has long been controversial. This also applies to the relationship between the somite boundaries and the
* This paper is dedicated to Professor Dr. K. **
V. Hinrichsen on occassion of his 65th birthday Main lecture at the 86th meeting of the Anatomische Gesellschaft in Szeged, Hungary, April 2 to April 5, 1991
)~I UMPElt2
Ann. Anat. (1992) 174: 23-32 Gustav Fischer Verlag Jena
vertebral bodies and the discs, as well as to the problem of how the somites take part in the development of the lateral elements (vertebral arches and ribs) of the vertebral column. Remak (1855) was the first to introduce the theory of resegmentation, which means that a vertebra is formed by the combination of the caudal half of one bilateral pair of somites with the cranial half of the immediately caudal pair of somites. This concept was later reinforced by von Ebner (1888), who used the term "Neugliederung" and received support from several other studies (reviewed by Verbout 1976). On the other hand, the concept of resegmentation has often be challenged (Blechschmidt 1957; Baur 1969; Christ et al. 1979, 1982; Verbout 1985). These authors described a development in which the vertebral bodies, the arches with their processes and the discs, arise from the beginning at their definitive sites. Recently, Bagnall et al. (1988, 1989a, b) provided experimental evidence that a single somite contributes to one half of each of the two adjacent vertebrae, as well as to the intervenning disc. However, the projection of the somite boundaries on to the vertebral column could not exactly be drawn because the results differed with respect to the marking method that was used. As was stated by Verbout (1985), the distinction between lateral and axial differentiation has not been taken into account in most of the studies on the development of the vertebral column. This is surprising, because distinct temporal and spatial differences can be found between the development of the arches and the formation of the vertebral bodies and intervening discs. Studying the development of the thoracic vertebrae in the mouse, Dalgleish (1985) emphasized that the axial parts of the vertebral column arise from a continous central tissue mass surrounding the notochord, whereas the neural arches develop from the caudal half of the sclerotome. This is in line with the conclusions drawn by Verbout (1985), who investigated the formation of the vertebral column in the sheep. In this paper, we review in detail the contribution of the
Results
sclerotome to the different parts of the vertebral column in the chick. We also describe the relationship between the sclerotomes and the neural elements. Eventually, we report on the formation of vertebral bodies and intervertebral discs in the avian and human embryo.
Material and
Somite Formation As a consequence of gastrulation two rods of mesoderm, the segmental plates arise. They are situated on each side of the neural tube and notochord. In the chick embryo, somites form from the cranial end of each segmental plate at an approximate rate of one pair every 100 min. The formation of somites is preceded by epithelialization of the segmental plate mesoderm (Christ et al. 1972). The still-unsegmented segmental plate, including its most caudal part, has been found to be destined to form somites in a cranio-caudal sequence even after inversion (Christ et al. 1972, 1974, 1975). This means that the segmental plate mesoderm has the ability to form somites from its birth in Hensen's node. The morphological manifestation of this predetermined state might be the so-called somitomeres seen by Meier (1979) with scanning electron microscopy.
Method~
Eggs of the White Leghorn strain of the common fowl (Gallus domesticus) were incubated at 3rC for periods between 2 and 8 days. After fIxation in 3-6% glutaraldehyd, in 0.12 M cacodylate buffer (pH 7.4) for periods of 4-24 h, the embryos were washed in Hanks' solution, dehydrated in graded acetone, and embedded in Durcupan ACM (Auka). Semithin sections were stained with methylene blue.
Each somite first appears as an epithelial sphere, whose radially-arranged cells line a small central lumen (somitocoel) which is reminiscent of the original mesenchyme (Fig. 1), and consists of about 2500 cells (personal communication, Ch. Ordahl, San Francisco). Somite-formation is accompanied by an increase in the expression of the calcium-dependent adhesion molecule N-cadherin (Duband et al. 1987). As was shown by Lash et al. (1984) and Jacob et al. (1991), fibronectin is involved in the epithelialization of the segmental plate mesoderm. The somite cells are polarized, with the Golgi zone located in the apical compartment and tight junctions at the luminal border (Revel et al. 1973; Lipton and Jacobson 1974). The newly-formed somite is surrounded by a dense basal lamina (Jacob et al. 1991).
Immunhistochemistry Specimens were fixed in 98 % ethanol and 2 % glacial acetic acid and then washed twice in ethanol and in xylene each for 15 min at room temperature. After embedding in paraplast, the embryos were serially sectioned. The sections were deparaffinated and rehydrated in xylene and graded ethanol, and rinsed in PBS buffer to which 0.05 % Tween-20 has been added. Section were incubated in a poly-clonal anti-fibronectin antibody (Euro-Diagnosticsll: 10,000) or an HNK-1 antibody (Becton Dickinson/I: 20) in PBS containing 0.05 % Tween-20, 1 % ovoalbumin and 0.2 % glycine. Overnight incubation was followed by washing in PBS containing Tween-20. In a second incubation step, the antigen-antibody complex was labelled with a second antibody to which horse radish peroxidase (HRP) was attached. These second antibodies were goat anti-rabbit and a goat anti-mouse antibodies (Dako Diagnostika) diluted 1: 300 in PBS. After incubation for 2 h, the sections were washed three times in buffer, and the staining reaction performed with diamino-benzidin (DAB)-HzO z for 10 min. The sections were washed again in buffer and subsequently stained in hematoxylin for 10 s. For indirect immunofluorescence, embryos were fIxed in 4 % paraformaldehyde in 0.1 M-potassium phosphate buffer (pH 7.6) overnight at 4 % C. Specimens were then rinsed in several changes of buffer and infIltrated with 5 % and 15 % sucrose in buffer, each overnight at 4 % C. Embryos were then embedded in OCT compound (Cambridge Instruments, Nussloch) and frozen. The sections were cut at 20 11m in a Reichert-Jung Model 2800 cryostat, and then collected on chrome alum-gelatin-coated slides, air dried and stained with a rabbit polyclonal anti-tenascin antibody (a gift from Drs. R. Chiquet-Ehrishmann and E. Mackie, Basel). After rinsing in buffer, the sections were incubated in FITC-conjugated goat antirabbit IgG (Dranova, Hamburg) for 1 h, and then rinsed and mounted in Hanks' saline: glycol (1: 1). For scanning electron microscopy (SEM), specimens were fixed in 4 % glutaraldehyde, dehydrated in graded acetone and criticalpoint-dried. After the specimens had been mounted on SEM stubs, the ectoderm was removed with tungsten needles. They were then sputtered with gold and examined with a Jeol JSM 35 R microscope. The human embryos were kindly provided by Dr. M. Grim (Praha). They were processed for semithin sections as described above for chick embryos.
The cells of the early epithelial somite are obviously assigned to at least six different compartments. Christ et al. (1978) have shown that the dorsal half of the somite gives rise to the dermornyotorne, while the ventral half is the source of the sclerotome. The studies of Keynes and Stem (1984), Rickmann et al. (1985), as well as those of Stem and Keynes (1987), habe provided strong evidence that each somite is subdivided into cranial and caudal halves. Motor axons and neural crest cells migrate through the cranial half of the sclerotome (Fig. 6 and 7). Selleck and Stem (1991) eventually demonstrates by DiI labelling of parts of Hensen's node and the primitive streak, that the somite can also be subdivided in a medial and a lateral half. The somitic precursors in Hensen's node only contribute to the medial halves of the somites, whereas the lateral halves are derived from a separate region of the primitive streak, caudal to the node itself. This finding received support from experiments concerning the origin of myoblasts (Ordahl and Le Douarin 1992) in which the lateral and medial halves were combined in different ways. Another important point is that the somites are predetermined with respect to the morphological features characteristic of the axial level. Thoracic somites heterotopic ally grafted give rise to ribs (Kieny et al. 1972; Jacob et al. 1975a). This means that the regionalization along the embry-
24
Fig. 1. Transverse section of a 2-day chick embryo. S somite; N neural tube; A aorta; asterisk: cell-free space between notochord and somite. x 450. Fig . 2. Lateral view of the neural tube of a 2-day chick embryo. Asterisks: unsegmented neural crest. X 550. Fig. 3. Sagittal section of a 2-day chick embryo. A aorta; arrows : intersomitic clefts containing blood vessels .
x
230.
Fig. 4. Frontal section of a 4-day chick embryo at the level of the ventral roots (arrows). M myotome; asterisk: mesenchymal condensation of the caudal sclerotome. x 300. Fig. 5. Frontal section of a 4-day chick embryo. Arrow: spinal nerve; asterisk: condensation of the caudal sclerotome; M myotome. x 300.
25
onic axis is a very early event during development. This regionalization is controlled by the expression of Hox genes at different axial levels (Kessel et al. 1990). Homeotic transformation in the mouse can be caused by an ectopic expression of Hox genes after the administration of retinoic acid (Kessel and Gruss 1991). According to these authors, the specificity of the paraxial mesoderm with respect to characteristic segmental identities depends on the formation of regional-specific Hox gene-expression domaines, the Hox codes. About 4 or 5 hours after the somite has formed, the migration of neural crest cells begins to take place (Fig. 2). At first, the neural crest cells form a continous and unsegmented strip (Fig. 2). The segmental arrangement of vessels (Fig. 3), nerves (Figs. 5 and 16), and spinal ganglia (Fig. 7) is caused by interaction between these structures, and the somites which had previously established the segmental pattern within the body wall of the early embryo (Christ 1975; Christ et al. 1979; Keynes and Stem 1988).
level of the spinal ganglia, and becomes surrounded by a highly vascularized perineural connective tissue. Figs. 4, 5 and 6 clearly show that the caudal boundary of the arch rudiment is marked by an intersegmental vessel. This observation points to the fact that the condensation originates only from the caudal part of each segment. A transverse section through the caudal half of the sclerotome reveals that the laterally-situated and denselypacked mesenchyme is triangular in shape. The edges represent the arcual process, the costal process, and the pedicle of the vertebra.
Formation of vertebral bodies and discs During the first steps of neural arch formation, the axial area is still cell-free. The notochord is surrounded by extracellular matrix material (Figs. 11 and 12). As has been shown by Jacob et al. (1975b), this material consists of radiallyoriented microfibrils and interstitial bodies. Fig. 12 shows that the fibrils are coated with fibronectin. It is very likely that these extracellular materials function as a substratum for the immigrating sclerotome cells which colonize the perinotochordal space. In this way the axially situated loosely-meshed mesenchyme originates, and does not show any signs of segmentation. As mentioned above, in this part of the sclerotome-derived mesenchyme (the perichordal tube) intra- and intersegmental fissures can never be found (Fig. 13). Fig. 16 shows several layers of mesenchymal cells which are concentrically oriented, surrounding the notochord. The segmentation of the axial mesenchyme begins to take place with parachordally located condensations, which represent the intervertebrate discs (Figs. 14 and 15). The course of this process, as well as its control mechanisms, are not yet well understood. However, with the appearance of the disc rudiments, the boundaries of the vertebral bodies become established. In order to form a unified vertebral column, the paired arch rudiments must connect the unpaired vertebral bodies. This process is shown in Figs. 15 and 16. That part of the arch rudiment that later represents the pedicle of the vertebra fuses with the extreme cranial part of the vertebral body (Fig. 15).
Somite differentiation During further development, the somites become subdivided in the mesenchymal sclerotome and the early epithelial arrangement of the dermomytome (Fig. 3). The latter subdivides further afterwards into the dermatome and the myotome (Figs. 6 and 13). While the myotome gives rise to postmitotic myoblasts, the dermatome is the source of still replicating myoblasts and the fibroblasts of the dermis of the back (Christ et al. 1983; Kaehn et al. 1988). The sclerotome is derived from the ventral half of the somite, which has lost its epithelial arrangement (Fig. 3). Though the later differentiation fate of the luminal cells of the somite is not yet quite clear, there is no doubt that they contribute to the formation of the sclerotome. The disaggregation of the somite wall is preceded by a loss of N-cadherin immunoreactivity in that region of the epithelium from which the sclerotome arises (Dub and et al. 1987). Following the sclerotome development, one has to distinguish between lateral and axial areas (Figs. 11, 13, 16,23).
Formation of neural arches and ribs Later stages of vertebral development
The formation of the vertebral column begins to take place laterally, with a morphologically conspicuous subdivision of the sclerotome into a densely packed caudal half and a more loosely structured cranial half (Fig. 13). Between these differently developing parts of the sclerotome, the so-called intrasegmental fissure (v. Ebner 1888) can be seen (Fig. 8). It separates the area of the developing arch and rib from the peripheral neural tissue. It is important to note that this "fissure" does not extend to the axial area, where at a later stage the vertebral bodies and discs arise. The alternation of arch elements and neural tissue is shown and Figs. 4-10 for the levels of spinal ganglia, ventral roots and spinal nerves. The peripheral neural tissue expands particularly at the
The vertebral body after fusion with the neural arches can be seen in Fig. 22. The neural arches of both sides then gradually surround the neural tube and fuse dorsally. Figs. 17, 18 and 21 show the development of vertebral bodies and discs in the human embryo. In the 12 mm embryo (Fig. 17) the perichordal mesenchyme has differentiated into discs and vertebral bodies. Within the disc rudiments the cells are densely packed, especially at the periphery. The anulus fibrosus develops, whereas in this area the central zone surrounding the notochord gives rise to the nucleus pulposus. Figs. 18 and 21 show the vertebral column of 30 mm embryo. The separation of the disc into
26
Fig. 6. Frontal section of a 3-day chick embryo at the level of the ventral roots (arrows). Staining with HNK-I antibody. M myotome. Asterisk: caudal part of the sclerotome. x 250. Fig. 7. Frontal section of a 3-day chick embryo at the level of the spinal ganglia (arrows). M myotome. Asterisk : caudal part of the sclerotome. x 250. Fig. 8. Sagittal section of a 3-day chick embryo. Big arrows : cranial half of the sclerotome with nervous elements; asterisks : caudal half of the sclerotome; small arrows: intersomitic vessels . X 200. Fig. 9. Sagittal cryosection of a 3-day chick embryo. Staining with anti-tenascin . Arrows: tenascin-positive cranial halves of the sclerotomes. x 110. Fig. 10. Higher magnification of Fig. 9. Arrows: tenascin-positive cranial halves of the sclerotomes. asterisks: condensed mesenchyme of the caudal halves. x 240.
27
The first somite disappears very early and the following 4 somites fuse to form the basioccipital bone. The cranial half of the 6th somite gives rise to the proatlas, which will later be incorporated into the apical part of the dens. The caudal half of the 6th somite fuses with the cranial half of the 7th somite to form a vertebral body, the dens axis, which later fuses with the body of the second vertebra, the axis. Fig 20 shows that dens and axis body are temporarily separated by an intervertebral disc, which later on disappears to form an unified bone. The main steps leading from the somites to the vertebral column are shown in the drawing in Fig. 23. According to our results, the neural arches, pedicles of vertebrae and ribs originate from the caudal half of one somite. Two adjacent somites seems to contribute to the formation of the vertebral body. The disc originates from the caudal half of the somite.
Discussion The results mentioned above can be summarized as follows. 1. The segmental plate mesoderm is very early determined with respect to the segmentation and cranio-caudal sequence in which the somites are formed. 2. The epithelial somite is subdivided into at least six different compartments. Each somite is regionalized with respect to the skeletal features characteristic of the axial level. 3. Each somite gives rise to the epithelial dermomyotome, and the mesenchymal sclerotome which provides the material for the vertebral column. 4. The vertebra arises from three sclerotomic areas. In the paired lateral areas the neural arch, the pedicle and the rib develop. The unpaired axial mesenchyme gives rise to the vertebral bodies and to the discs. These parts come together and form the vertebral column. 5. Projecting the somite boundaries onto the vertebral column we hold that the formation of the lateral vertebral elements takes place without resegmentation. On the other hand, formation of the body does not take place within the boundaries of one somite. 6. When the chondrification of the vertebral bodies begins, the discs start to differentiate into anulus fibrosus and nucleus pulposus. 7. The five cranial somites are included in the development of the head. The dens axis is homologous with the body of the atlas.
2 Fig. 11. Transverse section of a 2-day chick embryo. N neural tube; A aorta; asterisk: notochord; arrows: single cells migrating into the parachordal space. x 450. Fig. 12. Transverse section of a 2-day chick embryo. Staining with anti-fibronectin antibody. N neural tube; A aorta; asterisk: notochord; arrows: radially oriented extracellular matrix. x 450.
anulus fibrosus and nucleus pulposus is now clearly recognizable. The vertebral bodies are chondrified. It is worth mentioning that the notochord, which now becomes thicker at the disc levels and thinner at the body levels, is more ventral than central. Special features reveal the transitional zone between head and neck. As was shown by Christ et al. (1988), the boundary between head and neck corresponds to the boundary between the 5th and the 6th somite. This is true for the chick as well as for the human embryo.
In the embryo of higher vertebrates the somites have two important functions. They constitute the segmental pattern within the body wall, which is later transferred to other structures such as blood vessels and nerves (Christ et al. 1975; Stern und Keynes 1987). The other function is to specialize and to distribute cells. So, for instance, the muscle precursor cells become committed to two different lineages (Christ et al. 1990; Ordahl and Le Douarin 1992). As segmentation is a fundamental process of body organization, 28
Fig. 13. Sagittal section of a 3-day chick embryo. M myotome; asterisks: lateral condensation of the caudal sclerotome half. X 150. Fig. 14. Frontal section of a 4,5-day chick embryo. M myotome; asterisks: pedicle rudiments; arrows: anlagen of the discs. x 180. Fig. 15. Frontal section of a 5,5-day chick embryo. M myotome; asterisks: pedicles; arrows: intervertebral discs. Note the well vascularized perineural connective tissue in the foramen intervertebrale. x 130. Fig. 16. Transverse section of a 4-day chick embryo at the level of the caudal sclerotome half. M myotome; 1 neural arch; 2 pedicle; 3 rib. x 130. Fig. 17. Median section of a 12 mm human embryo. C notochord; arrows: intervertebral discs. x 100. Fig. 18. Median section of a 30 mm human embryo. C notochord; arrows: Anulus fibrosus of the intervertebral discs. x 70.
29
Fig. 19. Median section of a 24 mm human embryo. Head-neck transitional zone. Asterisk: oral cavity; Bb basioccipital bone; C notochord; arrow: proatlas; D Dens axis; Ax body of the axis. X 50. Fig. 20. Higher magnification of Fig. 19. C notochord; D dens axis; Ax body of the axis; Arrow: transient disc. x 130. Fig. 21. Transverse section of a 30 mm human embryo through a thoracic intervertebral disc: AF anulus fibosus; asterisk: notochord. x 90. Fig. 22. Transverse section of a 5-day chick embryo stained with anti-tenascin. N neural tube; asterisk: notochord; arrows: neural arches . x 120. Fig. 23 . Diagram showing the development of the vertebral column. A. Stage of epithelial sornites. The cranial half of the somite is loosely punctured and contains neural crest cells. B Differentiation of sclerotomes. Note the different development of the lateral and axial mesenchyme. Laterally, cranial and caudal halves give rise to different structures. Axially, the disc rudiments line the vertebral bodies. C Projection of the somite boundaries on to the differentiated vertebral column.
30
development of the dens, which arises by fusion of the axial body, the body of the atlas and the proatlas. This is in line with the findings of Reiter (1944), but does not support the hypothesis of Ludwig (1953, 1957) that the dens represents a structure sui generis developing from cranial processes of the axial body.
which is already realized in the earthwonn, it is not surprising that this pattern becomes detennined at a very early stage of development. As was shown by Christ et al. (1972, 1975) the newly developed parts of the segmental plate mesodenn are already destined to fonn somites. However, the shape of the developing somites depends upon the condition of the adjacent tissues. If, for instance, the neural tube is removed at the level of the still-unsegmented paraxial mesodenn, the plates fuse and fonn unpaired somites. It is of great interest that the somite is subdivided into different compartments. Selleck and Stern (1991) found that the progenitors of the medial half of the somite are derived from the lateral portion of Hensen's node, while the progenitors of the lateral half are derived from cells located in the primitive streak. The result points to the fact that the somite consists of already committed cells. Whether different compartments of the epithelial somite constitute different parts of the vertebra is still an open question. Verbout (1985) was right to emphasize that, in most of the studies dealing with the fonnation of the vertebral column, the differences between lateral and axial developing parts have not been taken into account. The controversial discussions on resegmentation may be founded on this deficiency. Experimental investigations on vertebral column fonnation must remain unsuccessful if the nonnal developmental steps are not fully taken into consideration. Interspecific replacements of single somites are not suitable for clarifying the problem of resegmentation, because the results could be falsified by injuries and regulation processes. Our observations support the view of Dalgleish (1985) and Verbout (1985) that the neural arch of the vertebra is derived from the caudo-Iateral part of one somite. We agree with Bagnall (1988, 1989a + b) that the vertebral body is made up of cells from two adjacent somites. However, the exact boundaries of somite participation are not yet clear. As shown by Christ (1975), Christ et al. (1979) and Rickmann et al. (1985), the segmental pattern is transferred by the somites to the blood vessels, the spinal ganglia, and the nerves. On the other hand, the segmental pattern of the neural arches develops in interaction with the spinal ganglia and nerves. Thus, a hierarchy of segmental structures can be suggested, in which the somites represent the primary, the spinal ganglia and nerves the secondary, and the neural arches the tertiary elements. The ribs, however, develop autonomously (Kieny et al. 1972; Jacob et al. 1975). The definite relationship between somites OIl the one hand and the vertebral bodies and discs on the other remains to be established. When the chondrification of the vertebral bodies begins to take place, the discs differentiate into anulus fibrosus and nucleus pulposus. The nonnal development of the axial parts of the vertebral column depends on interactions with the notochord (for review Christ, 1975). If the notochord has been experimentally removed the axial mesenchyme fonns ventral arches. The nonnal development of neural arches depends on the existence of the neural tube (Christ, 1975). The five cranial somites do not contribute to the vertebral column of human embryos. 2Yz somites participate in the
Concluding remarks Although important steps in the development of the vertebral column now seem to have been clarified, there are a lot of remaining problems still to be solved. A reliable method of experimentally labelling single somites or groups of somitic cells without destroying them would be highly desirable. Further genetic and molecular biological research is needed to deepen and extend our knowledge in this field.
References Bagnall KM, Higgins SJ, Sanders EJ (1988) The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimera model. Development 103: 69-85 Bagnall KM, Sanders EJ (1989a) The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo. Anat Embryol 180: 505-513 Bagnall KM, Higgins SJ, Sanders EJ (1989b) The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments. Development 107: 931-943 Baur R (1969) Zum Problem der Neugliederung der Wirbelsliule. Acta Anat 72: 321-356 Blechschmidt E (1957) Die Entwicklungsbewegungen der Somiten und ihre Bedeutung fUr die Gliederung der Wirbelsaule. Z Anat Entwickl-Gesch 120: 150-172 Christ B (1975) Die Entwicklung der Korperwandmetamerie . an Hiihnerembryonen, . Experimentelle Untersuchungen Habilitationsschrift, Bochum Christ B, Jacob HJ, Jacob M (1972) Experimentelle Untersuchungen zur Somitenentstehung beim Hiihnerembryo. Z Anat Entwickl-Gesch 138: 82-97 Christ B, Jacob HJ, Jacob M (1974) Die Somitogenese beim Hiihnerembryo. Zur Determination der Segmentierungsrichtung. Verh Anat Ges 68: 573-579 Christ B, Jacob HJ, Jacob M (1975) Autoradiographische Untersuchungen zur Somitenentstehung beim Hiihnerembryo. Verh Anat Ges 69: 259-261 Christ B, Jacob HJ, Jacob M (1979) Ober Gestaltungsfunktionen der Somiten bei der Entwicklung der Korperwand von Hiihnerembryonen. Verh Anat Ges 73: 509-518 Christ B, Jacob HJ, Jacob M (1978) On the formation of the myotomes in avian embryos . An experimental and scanning electron microscopic study. Experientia 34: 514-516 Christ B, Jacob M, Jacob HJ (1983) On the origin and development of the ventrolateral abdominal muscles in the avian embryo. Anat Embryoll66: 87-101 Christ B, Jacob HJ, Seifert R (1988) Ober die Entwicklung der zervikookzipitalen Obergangsregion. In: Hohmann D, Kiigelgen
31
B, Liebig K (eds) Springer, Berlin, Heidelberg, Neuroorthopadie 4 pp 13-22 Christ B, Brand-Saberi B, Jacob HJ, Jacob M, Seifert R (1990) Principles of early muscle development. In: Le Douarin N, Dieterlen-Lievre T, Simith J (eds) The avian model in developmental biology: From organism to genes. Edition du CNRS Paris Dalgleish AE (1985) A study of the development of thoracic vertebrae in the mouse assisted by autoradiography. Acta Anat 122: 91-98 Duband JL, Durfour S, Hatta K, Takeichi M, Edelmann E, Thiery JP (1987) Adhesion molecules during somitogenesis in the avian embryo. J Cell BioI 104: 1361-1374 Ebner von E (1888) Urwirbel und Neugliederung der Wirbelsaule. Sitzungsber Akad Wiss Wien I1I/97 194-206 Jacob M, Christ B, Jacob HJ (1975a) Uber die regionale Determination des paraxialen Mesoderms junger Htihnerembryonen. Verh Anat Ges 69: 263-269 Jacob M, Jacob HJ, Christ B (1975b) The early differentiation of the perinotochordal connective tissue. A scanning and transmission electron microscopic study on chick embryos. Experientia 31: 1083-1086 Jacob M, Christ B, Jacob HJ, Poelmann R (1991) The role of fibronectin and laminin in development and migration of the avian Wolffian duct with reference to somitogenesis. Anat Embryol 183: 385-395 Kaehn, K, Jacob HJ, Christ B, Hinrichsen K Poelmann R (1988) The onset of myotome faromation in the chick. Anat Embryol 177: 191-201 Kessel M, Balling R, Gruss P (1990) Variations of cervical vertebrae after expression of a Hox -1.1 transgene in mice. Cell 61: 301-308 Kessel M, Gruss P (1991) Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67 1-20 Keynes RJ, Stern CD (1984): Segmentation in the vertebrate nervous system. Nature, London 310: 786-785 Keynes RJ, Stern CD (1988) Mechanism of vertebrate segmentation. Development 103: 413-429
Kieny M, Mauger A, Sengel P (1972) Early regionalization of the somitic mesoderm as studied by the development of the axial skeleton of the chick embryo. Develop Bioi 142-161 Lash JW, Seitz AW, Cheney CM, Ostrowsky D (1984) On the role of fibronectin during the compaction stage of somitogenesis in the chick embryo. J exp Zool 232: 197-206 Lipton BH, Jacobsen AG (1974) Analysis of normal somite development. Develop Bioi 38: 73-90 Ludwig KS (1953) Die Friihentwicklung des Dens epistrophei und seiner Bander beim Menschen. Morphol Jb 93: 98-112 Ludwig KS (1957) Die Friihentwicklung des Atlas und der Occipitalwirbel beim Menschen. Acta Anat 30: 444-461 Meier S (1979) Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern. Develop Bioi 73: 25 -45 Ordahl PA, Le Douarin N (1992) Two myogenic lineages within the developing somite. Development (in press) Remak R (1855) Untersuchungen tiber die Entwicklung der Wirbeltiere. Reimer, Berlin Reiter A (1944) Die Friihentwicklung der menschlichen Wirbelsaule. II. Mitteilung: Die Entwicklung der Occipitalsegmente und der Halswirbelsaule. Z. Anat Entwickl-Gesch 113: 66-104 Revel JP, Yip P Chang LL (1973) Cell junctions in the early chick embryo - a freeze etch study. Develop Bioi 35: 302-317 Rickmann M, Fawcett J, Keynes RJ (1985) The migration of neural crest cells and the growth of motor axons through the ventral half of the chick somite. J Embryo1 exp Morph 90: 437-453 Selleck MA, Stern CD (1991) Fate mapping and cell lineage analysis of Hensen's node in the chick embryo. Development 112: 615-626 Stern CD, Keynes RJ (1987) Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo. J Embryol exp Morph 91: 209-226 Verbout AJ (1976) A critical review of the ,Neugliederung' concept in relation to the development of the vertebral colunm. Acta Biotheoret 25: 219-258 Verbout AJ (1985) The development of the vertebral colunm. Adv Anat Embryol Cell BioI 90: 1-122
32
