Determination of epithelial half-somites in skeletal morphogenesis

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1 Development 116, (1992) Printed in Great Britain The Company of Biologists Limited Determination of epithelial half-somites in skeletal morphogenesis RONALD S. GOLDSTEIN * and CHAYA KALCHEIM Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School, P.O.B Jerusalem 91010, Israel * Present address: Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel To whom correspondence should be addressed Summary The segmental body plan of vertebrates arises from the metameric organization of the paraxial mesoderm into somites. Each mesodermal somite is subdivided into at least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of either somitic domain. In the present work, we have extended these studies by investigating the contribution of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each somitic half differ. The pedicle of the vertebral arch is almost continuous in caudal half-somite grafts and is virtually absent in rostral half-somite implants. Similarly, the intervertebral disk is present in rostral halfsomite chimeras, and much reduced or virtually absent in caudal somite chimeras. Thus, only the caudal half cells are committed to give rise to the vertebral pedicle, and only the rostral half cells are committed to give rise to the fibrocartilage of the intervertebral disk. Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal halfsomite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These data directly support the hypothesis of resegmentation, in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites. Key words: avian embryos, peripheral nervous system, quailchick chimeras, vertebral development. Introduction The somite is a segmentally repeating mesodermal structure common to vertebrates. It dissociates into the sclerotome, which gives rise to the axial skeleton, and the dermomyotome, which gives rise to the axial musculature and dermis. Various aspects of sclerotomal differentiation are determined while the somite is still an epithelial structure. For example, the division of the epithelial somite into rostral and caudal domains has been shown to be responsible for the segmental morphogenesis of the peripheral nervous system (PNS) of the trunk, including the nerves (Keynes and Stern, 1984), dorsal root ganglia (DRG; Rickmann et al., 1985; Bronner-Fraser, 1986; Kalcheim and Teillet, 1989; Lallier and Bronner-Fraser, 1988; Teillet et al., 1987) and sympathetic ganglia (SG; Goldstein and Kalcheim, 1991). The rostral somite is permissive to axonal outgrowth and neural crest cell migration, while the caudal portion of the somite inhibits axonal outgrowth by collapsing growth cones (Davies et al., 1990), and also prevents the entrance of neural crest cells. This division into rostral and caudal domains is already determined while the somite is epithelial, as demonstrated by replacing somites of chick hosts with multiple rostral (RS ) or caudal (CS) epithelial somitic halves from quail donors. Multiple half-somites retain their rostral or caudal character since RS grafts resulted in continuous, non-segmented PNS structures, including larger than normal DRG, and CS grafts in distorted segmentation of PNS structures and smaller than normal DRG (Goldstein et al., 1990; Kalcheim and Teillet, 1989). Certain aspects of vertebral morphology related to axial level, are also determined while the somite is still epithelial. For example, the dorsoventral axis of the sclerotome is determined three hours after somite segmentation from the segmental plate (Aoyama and Asamoto, 1988). In addition, replacement of cervical somites with those from thoracic levels, leads to the appearance of rib-like structures in the neck (Kieny et al., 1972). As to the possible molecular mechanisms controlling somite determination, members of the murine Hox gene family were shown to be expressed in the somites and their derivatives, along defined extents of the rostrocaudal axis (Kessel and Gruss, 1990; Mahon et al., 1988). Moreover, ectopic expression of the Hox 1.1 transgene caused specific variations of the cervical vertebra, suggesting that this homeobox gene plays a regulatory role in vertebral development (Kessel et al., 1990). Several groups have investigated whether the early deter-

2 442 R. S. Goldstein and C. Kalcheim mination of rostral and caudal half-somites is expressed in vertebral morphogenesis. Studies in which the fate of halfsomites was followed by either the quail marker (Stern and Keynes, 1987), or peanut lectin binding (Bagnall and Sanders, 1989), led to conflicting conclusions. Replacement of single chick half-somites by their quail counterparts showed that each somitic half can give rise to all vertebral components. In contrast, peanut lectin, which at early stages preferentially stains the caudal half of the sclerotome (Stern et al., 1986), at later stages stains specific vertebral components, such as the intervertebral (iv) disk. As part of our continuing studies on the mutual interactions between the paraxial mesoderm and the nervous system, we have reevaluated the issue of the contribution of half somites to vertebral morphogenesis, using grafts of multiple quail rostral or caudal somitic halves into chick hosts. We find that both rostral and caudal somitic halves can give rise to most parts of the axial skeleton, including the rib. However, the ability to give rise to two components of the spinal column is confined to distinct half-somites. The pedicle is derived from only the caudal somitic-half, and the iv disk from only the rostral. These results shed light on fundamental processes involved in the segmentation of axial structures in the verterbral trunk. Histology Embryos were fixed in Bouin s fluid on E9-E10, and embedded in paraplast. Serial 10 µm sections were mounted on gelatinized slides, and the tissue stained by one of two procedures. Cervicallevel chimeras were stained with Feulgen and counterstained with Fast green. Chimeras with brachial level half-somite grafts were stained with the monoclonal antibody HNK-1, which at this stage of development strongly labels both the central and peripheral nervous systems (Abo and Balch, 1981). The HNK-1 binding was visualized both with a goat anti-mouse secondary antibody coupled to horseradish peroxidase (Sigma) followed by diaminobenzidine treatment. The tissue was counterstained with Meyer s hematoxylin, which permitted distinction of the donor quail cells from the host cells almost as well as the more conventionally used Feulgen staining. The hematoxylin staining, however, had the advantage of being compatible with peroxidase cytochemistry, unlike the Feulgen technique. After initial analysis, some preparations were restained in 1% aqueous toluidine blue to facilitate distinction of hyaline and fibrous cartilage. Data analysis Three dimensional reconstructions of the neural and vertebral structures were made from alternate serial sections using the HVEM 3D program developed by the University of Colorado at Boulder Laboratory for High Voltage Electron Microscopy, and the images photographed from the computer monitor on Kodak Ektar 25 film. Materials and methods Embryos of chick (Gallus gallus) and Japanese quail (Coturnix coturnix Japonica) were used for this study. Eggs from commercial sources were kept in a humidified incubator at 38 ± 1 C. Embryonic microsurgery Surgery in this study was essentially the same as has been described (Kalcheim and Teillet, 1989). Briefly, rostral or caudal halves of the three or four most recently formed somites were excised from somite stage quail embryos. The sclerotome from somites at this level of the rostrocaudal axis generates cartilage that forms ribs (Kieny et al., 1972; Murillo-Ferrol, 1963; Seno, 1961). A drop of 50% pancreatin (Gibco) in phosphatebuffered saline (PBS) ph 7.4, was applied locally to facilitate dissection and ensure complete removal of the somitic mesoderm. Like somitic-halves from several donor quails were pooled in PBS until implanting. The three or four most recently formed somites on the right side of chick recipients were removed, and the space produced was filled with 2-5 quail rostral half-somites. In some animals, unsegmented paraxial mesoderm equivalent in length to 2 somites was removed, along with the last detached somite(s). Two types of chimeras were produced. In the first group, recipient embryos having somites at the time of implantation were used, resulting in modification of the mesoderm at the cervical level of the neuraxis. The second group of recipients had somite pairs at the time of the surgery, resulting in modification of the vertebra at the brachial level of the axis. Only embryos in which replacement of host mesoderm was complete in the vertebral column along the entire rostrocaudal extent of the graft were used for analysis. Serial section analysis of 14 and 8 embryos receiving rostral and caudal half-somite grafts respectively, was performed. Three-dimensional computer reconstructions were made from six chimeras, three with RS grafts, and three with CS grafts. Results Common properties of vertebrae in the chimeras Dissociation of the epithelial somites into dermomyotome and sclerotome occurs normally in multiple half-somite chimeras, but vertebral morphogenesis is modified (like that of PNS structures, Goldstein et al., 1990; Goldstein and Kalcheim, 1991; Kalcheim and Teillet, 1989). Vertebrae in both types of chimeras contained most of the appropriate components such as body, neural arches and costal processes, suggesting that both half-somite moieties can contribute to these structures in vivo. Both rostral and caudal chimeras prepared at cervical levels, were obtained by implanting half somites from brachial levels of the quail neuraxis. In both cases, long and thin rib-like cartilaginous structures developed from the implanted quail mesoderm (Fig. 1A, C) as observed by others implanting whole brachial somites in the neck (Kieny et al., 1972). These structures were caudal-pointing extensions of the small costal processes present in normal cervical vertebrae. This structure is homologous to the rib at brachial levels, which, however projects caudal instead of the anterior direction taken by normal ribs. Our observation of ectopic ribs in both types of grafts provides evidence that both rostral and caudal mesoderm can contribute to their formation. The caudal, but not the rostral half-somite, gives rise to the pedicle On the unoperated side of the chimeras, the DRG protrude between adjacent pedicles of the vertebra (Fig. 1A, C), as is the case in normal embryos. In contrast, the morpho-

3 Half somite grafts and vertebral development 443 genesis of the vertebrae in embryos containing grafts of multiple half-somites is altered. In the case of vertebrae formed from sclerotome derived from rostral half-somites, the morphogenesis of the lateral vertebral arch is incomplete due to the total absence of a pedicle connecting the neural arch with the vertebral body (n=13 out of 14 succesful replacements). Consequently, a continuous opening remains in the vertebra throughout the entire length of the graft (Figs 1A, 2A), creating a passage for the axons to extend from the spinal cord to the periphery (data not shown). In addition, the non-segmented DRG protrude through this lateral defect (Figs 1A, 2A). In contrast, in chimeras with CS grafts, an almost continuous pedicle-like structure develops from the grafted mesoderm throughout most of its length, enclosing the DRG within the vertebra (n=8; Fig. 1B and see also Kalcheim and Teillet, 1989). In each of the chimeras with CS grafts, a discrete opening in the lateral aspect of the vertebra could always be seen, through which peripheral nerves exited and the DRG sometimes partially protruded (Fig. 1B, C). The rostral half somite, but not the caudal, gives rise to the intervertebral disk The iv disk is composed of fibrocartilage containing more densely packed cells than the hyaline cartilage of the vertebrae. In toluidine blue-stained material, the iv disk stains blue, as opposed to the violet metachromatic stain of the vertebra (Fig. 2). In chimeras with multiple rostral half-somite grafts, the disk formed on both the normal, unoperated side (not shown) and on the operated side from cells of the grafted quail mesoderm (n=6, Fig. 2). The disks on the grafted side were at least as large (in terms of number of sections present) as the normal disks, and completely separated adjacent vertebral bodies. In each graft, the position of the disk sometimes matched that on the normal side, and sometimes did not. This result shows that individual vertebrae can form from a mesoderm composed exclusively of rostral halfsomites. In chimeras with multiple caudal somite-grafts, the iv disks were either much reduced, or entirely absent (n=5, Fig. 3). For example, in one brachial chimera with a graft replacing four chick somites, four disks were present on the control side, occupying a total of 63, 10 µm thick sections, whereas on the operated side, all cartilage in the area of the vertebral body was of normal hyaline morphology (Fig. 3A). In most caudal grafts, short pieces of fibrocartilage were present. These never completely separated the vertebral bodies, and were mostly confined to a small medial zone between the notochord and the spinal cord. In several grafts, small incursions of chick cells were seen in this mediodorsal area of the vertebral body into the operated side. Discussion We report here that experimental construction of a paraxial mesoderm composed exclusively of multiple rostral or caudal epithelial somite-halves, leads to the formation of Fig. 3. The caudal half of the somite does not give rise to the intervertebral (iv) disk. Serial section analysis of two embryos receiving caudal half-somite grafts in place of host somites Sections containing fibrocartilage (iv disk) are depicted by a thick line, and those with hyaline cartilage (vertebral body) with a thin line. In (A), no sections containing iv disk-like structures in the operated side were observed, in contrast to 63 sections in 4 iv disks on the control side. In (B), 3 short stretches of sections (a total of 23) containing fibrocartilage were present on the operated side of the chimera; however these cells were mostly confined to a medial zone between the notochord and spinal cord and never completely separated the vertebrae. On the control side of this embryo there were 4 iv disks spanning a total of 75 sections. vertebrae in which specific elements are missing. Multiple rostral grafts do not contain pedicle, while multiple caudal grafts lack iv disks. The differences in the morphogenesis of the vertebrae composed of only caudal or rostral moieties is likely to reflect intrinsic differences in the commitment of epithelial somite-halves: epithelial CS halves contain cells with the potential to give rise to the pedicle of the vertebrae, whereas the RS halves contain instead cells with the potential to form the iv disk. Consistent with this early somitic cell commitment to specific cartilaginous structures, we find that both rostral and caudal chimeras, prepared by implanting half somites from the rib-forming level of the axis into the neck, contain rib-like structures derived from the grafted mesoderm (see Fig. 1). This is in agreement with previous observations by Kieny et al. (1972) who performed implants of either unsegmented or segmented somitic mesoderm from thoracic into cervical regions and vice-versa. The formation of the iv disk from the rostral half of the somite is in apparent contradiction with another recent study (Bagnall and Sanders, 1989), in which the iv disk was heavily stained with peanut lectin, suggesting that it arises from the caudal somitic half. Expression of carbohydrate epitopes dynamically changes during ontogeny. Therefore, the observed staining with peanut lectin in the iv disk may be developmentally regulated rather than serving as a lineage marker to demonstrate the derivation of the iv disk from the caudal half of the somites. Although early determination of somite cells may be the mechanism that accounts for the present observations, it is still possible that the migration of sclerotomal cells and con-

4 444 R. S. Goldstein and C. Kalcheim Fig. 4. Early determination of half somites in vertebral morphogenesis. Immediately upon individualization, the epithelial somites are divided into at least two domains, rostral (R), and caudal (C). According to the model of resegmentation, each vertebra forms from the fusion of the caudal half of a given somite and the rostral half of the subsequent one, separated by iv disks. The vertebral body can be formed from both RS and CS halves (Fig. 1), (the horizontal line we have drawn separating the RS and CS contributions to the vertebral body, is an imaginary one, since our transplants of multiple half somites do not adress the exact position of this boundary, see Bagnall et al., 1988). In contrast, we show that the pedicle (Ped) derives exclusively from the CS (hatched) and the iv disk from the RS mesoderm (open). We therefore propose the following model for vertebral formation: each vertebra has a rostral portion containing a pedicle which, as shown here, derives from the CS, and a caudal portion deriving from the RS which lacks the pedicle and has, instead, an intervertebral foramen (F) into which neural structures protrude. According to this model, the iv disk is the caudalmost part of the vertebra, deriving from the RS half. The horizontal stripes define the spinous process of the vertebra whose somitic origin is unknown. NT, neural tube; SC, spinal cord. sequent skeletal morphogenesis also partly depend upon specific interactions taking place between these progenitors and neural elements in the rostral half of each somite. For example, the lack of pedicle in the RS chimeras might be due to competition for space between the expanding unsegmented DRG (Goldstein et al., 1990) and the sclerotomal cells. However, in RS grafts there are a few sections in which the pedicle is missing, although there is no DRG in that section. Conversely, DRG develop within the closed, almost continuous pedicle in CS chimeras, suggesting that competition is not the mechanism responsible for the lack of pedicle in RS chimeras. The DRG normally lies within the intervertebral foramen, resting on the pedicle of the vertebral arch of the corresponding segment. Based on the present results, we suggest that during normal development, the caudal somitic domain forms the vertebral pedicle, whereas the rostral half of a somite gives rise to the region corresponding to the intervertebral foramen, due to its inability to develop a pedicle (Fig. 4). This is consistent with the finding that both DRG and peripheral nerves exclusively form facing the rostral somitic domains in the early embryo, and lie in the intervertebral foramina in the mature animal. Since each normal vertebra contains a region with a pedicle located cranial to the corresponding pedicle-free, DRGcontaining zone, it may be inferred that each vertebra derives from the caudal half of a given somite and the rostral half of the subsequent one, in agreement with the resegmentation hypothesis (Fig. 4). Although some studies have questioned the validity of this hypothesis (Stern and Keynes, 1987, and discussed in Bagnall and Sanders, 1989; Keynes and Stern, 1988), others have supported it using different techniques (Aoyama and Asamoto, 1988; Bagnall, 1992; Bagnall and Sanders, 1989; Bagnall et al., 1988; Ewan and Everett, 1992). For example, in very recent studies, injection of a fluorescent dye or recombinant retrovirus into single somites, resulted in the labeling of components of two contiguous vertebrae (Bagnall, 1992; Ewan and Everett, 1992). The presence of a well developed iv disk in RS chimeras raises an important basic question about the source of segmentation cues in the development of axial structures of the trunk. It is believed that axial segmentation is caused by the alternation of RS and CS domains of the somites. The iv disk that separates two contiguous vertebrae, is an expression of this segmentation. The formation of the iv disks between the vertebrae derived exclusively from RS mesoderm in our chimeras, may be accounted for by several reasons. First, it is possible that the cue for segmentation of the grafted RS comes from the mesenchyme on the unoperated side of the embryo. This seems unlikely because in most chimeras there is a lack of bilateral correspondence in the position of the iv disks and also, iv disks are present on both sides of RS chimeras receiving bilateral grafts. Interestingly, the rostrocaudal position of the iv disks in the bilateral chimeras does not match (unpublished data). A second possibility is that the other major axial structures, the neural tube and/or notochord, provide specific segmentation cues to the paraxial mesoderm. This is also unlikely because of the bilateral asymmetry in iv disk formation in the RS chimeras. Finally, it is possible that the RS is subdivided into smaller domains committed to give rise to distinct vertebral components, such as the iv disk. This last notion extends the currently accepted view that vertebral segmentation results from the alternation of early committed somitic halves. We wish to express our gratitude to Professor A. Ornoy for valuable instruction in the details of the histology of developing cartilage and for helpful suggestions. We also thank Ms. Chana Carmeli for excellent technical assistance, Michal Pollak for help with the computer reconstructions and Gilat Brill for critical reading of the manuscript. This work was supported by grants from the National Council for Research and Development and the Commission for European Communities, the Familial Dysautonomia Foundation, and the Israel Academy of Sciences and Humanities to Chaya Kalcheim, and a grant from the Israel Institute for Psychobiology- Charles Smith foundation to Ron Goldstein. References Abo, T. and Balch, C. M. (1981). A differentiation antigen of human NK and K cells identified by a monoclonal antibody. J. Immunol. 127, Aoyama, H. and Asamoto, K. (1988). Determination of somite cells: independence of cell differentiation and morphogenesis. Development 104,

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6 Fig. 2. The rostral half of the somite gives rise to the intervertebral (iv) disk. A transverse section of an E9 chick embryo receiving a graft of 6 quail rostral half-somites in place of somites (A) On the operated, left side of the chimera, note the absence of the pedicle and the presence of a DRG (drg), in contrast to the closed vertebra and lack of DRG on the control (right) side. (B) The grafted mesoderm developed into fibrocartilage of an iv disk (blue) in place of the left half of the vertebral body. The right, control side is metachromatically stained purple for hyaline cartilage. (C) High magnification of the box in B showing that the iv disk is composed of quail (arrows), and the hyaline cartilage of chick cells. ch, chick; drg, dorsal root ganglion; N, notochord; SC, spinal cord; P, pedicle; q, quail. Bars: A, 100 µm; B, 40 µm; C, 15 µm. dev1923 colour tip-in