ABSTRACT
In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Further-more, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxy-lin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae.
After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipi-tal sclerotomes lack factors essential for normal sen-sory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.
Introduction
Segmentation in the peripheral nervous system of higher vertebrates arises as a result of interactions between developing nerve cells and the adjacent segmental mesoderm (Lehmann, 1927; Detwiler, 1934). In particular, because of intrinsic differences between the cranial and caudal halves of the somite-derived sclerotomes, axons and neural crest cells are constrained to grow through the cranial half of each sclerotome, and the dorsal root ganglia also develop exclusively in this position (Keynes & Stern, 1984, 1987).
The mechanisms underlying these phenomena have yet to be identified. A related issue, which we address here, is whether the craniocaudal differences within each sclerotome are identical along the cranio-caudal axis of the embryo as a whole. Presumptive sclerotome is regionally specified with respect to its skeletal derivatives; thoracic segmental plate, for example, gives rise to ribs when transplanted to the cervical region in chick embryos (Kieny, Mauger & Sengel, 1972). Perhaps, then, the sclerotome is also regionally specified in relation to neural develop-ment. In fact, it has been known for many years that in the occipital sclerotomes, caudal to the first somite, dorsal root ganglia are never fully formed. One or two ganglia do develop transiently, in the region of the caudal hypoglossal rootlets and the first cervical spinal nerve. These are known as ‘Froriep’s’ ganglia, so named by Wilhelm His (1888) after their dis-coverer (Froriep, 1882). The two to four sclerotomes still further cranial are usually devoid of dorsal root ganglion cells, with variation depending on the species (Chiarugi, 1889; R. M. Hunter, 1935).
In chick embryos, the ventral migration pathway of neural crest cells is thought not to be available at the level of the five most cranial somites. Instead, crest cells have been said to migrate over these somites, to colonize the developing gut and dif-ferentiate into postganglionic parasympathetic cells (Thiery, Duband & Delouvée, 1982). This being so, it is worth noting that, despite the apparent reluctance of crest cells to enter these cranial segments, motor axons still do, traversing each cranial half-sclerotome and then fasciculating to form the hypoglossal nerve. One interpretation of this is that these occipital sclerotomes differ from those of the trunk: they permit motor axon ingrowth, but not neural crest migration and sensory ganglion development, while trunk sclerotomes allow all three processes to occur. With the interesting implications that this would have for underlying mechanisms, it seemed worth-while distinguishing between this possibility and the alternative, namely that it is the neural tube/crest cells rather than the sclerotomes which vary along the craniocaudal axis. There is already evidence that occipital neural crest will differentiate into sensory ganglion cells when transplanted to the trunk region (Le Lievre, Schweizer, Ziller & Le Douarin, 1980). With the availability of the monoclonal antibody HNK-1 as a marker for neural crest cells (Tucker, Aoyama, Lipinski, Tursz & Thiery, 1984; Rickmann, Fawcett & Keynes, 1985), it was also of interest to examine in more detail the migration pathway of the crest and the development of sensory ganglia at occipital levels.
Materials and methods
Haematoxylin/eosin staining
Chick embryos were incubated at 38°C to stages 19-28 (Hamburger & Hamilton, 1951). They were then fixed in buffered formalin, paraffin-embedded, sectioned and stained by a standard procedure (McManus & Mowry, 1960).
Immunohistochemistry
Chick embryos were incubated to stage 15. After dissection from the membranes, they were fixed for 1 h in a solution of 1·25% glutaraldehyde, 1% paraformaldehyde and 3·5% sucrose in 0·1 M-phosphate buffer, pH 7·3, at 4°C. They were then immersed in 5 % sucrose in phosphate-buffered saline (PBS), pH 7·3, for 2 h at 4°C, and transferred to 15 % sucrose in PBS overnight at 4 °C. They were placed in 7-5 % gelatin (300 Bloom, Sigma) containing 15% sucrose/PBS at 37°C for 4-6 h, after which the sucrose/gelatin was allowed to set by lowering the temperature to 22°C. Blocks were frozen for cryostat sectioning in isopentane, cooled with liquid nitrogen and sectioned at 20-40μm.
Sections were mounted on gelatin-subbed slides and stained with HNK-1 monoclonal antibody by indirect immunoperoxidase as follows: sections were incubated for 1 h in 1% bovine serum albumin, for 3-4 h in HNK-1 antibody (Becton Dickinson, anti Leu-7,1:100), and for 6h with horseradish-peroxidase-conjugated anti-mouse IgG (Sigma, dilution 1:1000) at 37°C. They were then reacted for HRP by a modification of the method of Straus (1982). They were incubated for 15 min in 0·1 M-phosphate buffer, pH 7·4, containing 0 05% 3,3’-diaminobenzidine (Sigma) and 0·01 M-imidazole, at room temperature. 0· 015 % hydro-gen peroxide was then added to the incubation mixture and the sections reacted for a further 10 min at room tempera-ture. Finally, the slides were washed in distilled water and mounted in Hydramount (Gurr).
Zinc iodide/osmium tetroxide staining
Thirty normal chick embryos, stages 15-23, were stained according to the following procedure, a modification of the method of Akert & Sandri (1968). The embryos were pinned out on Sylgard dishes in Tyrode’s solution and bisected along the craniocaudal axis into right and left halves. They were then immersed in a freshly prepared mixture of 6 ml zinc iodide and 1·75 ml of 2% osmium tetroxide, to which had been added a drop of concentrated KI solution. The zinc iodide was made by combining 5g iodine with 15 g powdered zinc in 200 ml distilled water. They were then incubated at 55°C for Ih 40min, washed with distilled water, dehydrated in alcohols, cleared in xylene and whole mounted in Permount (Fisher) between two coverslips. Some embryos were immersed for a few seconds in a saturated potassium periodate solution before dehydration, to reduce background staining.
Chick/quail chimaeras
The host hens’ eggs were incubated at 38°C to stages 12-13 and were then prepared as follows: a window was cut with a scalpel blade and the embryo floated up to the level of the shell by adding calcium- and magnesium-free Tyrode’s solution (CMF) containing 1000i.u.ml-1 penicillin and 100gg ml-1 streptomycin. 0·1 ml of Indian ink suspension (Pelikan Fount India, diluted 1:10 in CMF) was injected into the sub-blastodermic space so that the embryo could be seen against a dark background. A rim of silicone grease was then placed around the edges of the window and a drop of CMF made to cover the embryo. Visibility was signifi-cantly enhanced with tangential fibre-optic illumination (Hara, 1970). The vitelline membrane was peeled back over the most caudal somites in the trunk region, and the neural tube and notochord were excised using a Week microsurgi-cal knife. The length of tissue removed was equivalent to five somites. 0·1 % trypsin (Difco, in CMF) was sometimes used to help separate the notochord from the endoderm.
Fertile quails’ eggs were incubated at 38°C to the 10-somite stage for operation, just before the onset of neural crest migration at the occipital level (Thiery et al. 1982). Each donor embryo was pinned out in a Sylgard dish with its ventral side uppermost. 0·1% trypsin was applied and the endoderm peeled off. The neural tube and notochord opposite somites 2-6 (counting somites in a caudal direc-tion from immediately caudal to the otic vesicle) were then removed, freed of adherent cells and transferred to the host with a siliconized glass pipette. After placing the graft in the gap left by the excised host neural tube/notochord, 1·5 ml of albumen were withdrawn to bring the embryo down into the egg once again. The egg was then sealed with PVC tape and incubated at 38°C to stages 24-30.
Chimaeric embryos were fixed in Zenker’s solution, dehydrated through alcohols and paraffin embedded. The blocks were sectioned transversely at 8μm, stained by Feulgen’s method (Le Douarin, 1973) and mounted in Permount.
Results
Normal embryos
(1) Haematoxylin/eosin staining
Transverse and sagittal sections of normal chick embryos between stages 19 and 28 were examined, four embryos at each stage. Somites were counted from immediately caudal to the otic vesicle. By this stage the most cranial somite has dissociated, without visibly dividing into cranial and caudal halves. It contains the combined proximal ganglia of the 9th and 10th cranial nerves (see Hinsch & Hamilton, 1956). Caudal to this segment, the most cranial ganglion-like aggregation of neural crest cells lies at the level of the 6th segment, being visible in the cranial half-sclerotome by stage 19. No presumptive dorsal root ganglia are visible in the segments still more cranial, while they are obvious in all the more caudal segments (Fig. 1A). In occipital segments, the region of sclerotome adjacent to the dorsolateral part of the neural tube is occupied instead by the emerging axons of the spinal accessory nerve and associated sheath cells (Fig. 1A). By stage 23 the aggregation in the 6th segment has disappeared, consistent with its designation as a ‘Froriep’s’ ganglion, and the most cranial spinal ganglion is now in the 7th segment. During stages 27 and 28, or 36-48 h later, the occipital sclerotomes dissociate to form the base of the skull (the caudal part of the parachordal plate) and the atlas and axis vertebrae. The most cranial dorsal root ganglion is now associated with the 3rd cervical spinal nerve, immediately caudal to the axis (Fig. IB).
(A) Sagittal section through a stage-19 chick embryo, stained with haematoxylin/eosin. Cranial is to the left, dorsal uppermost. The boundaries of the 5th sclerotome are marked with asterisks. Froriep’s ganglion (FG, arrowed) lies in the cranial half of the 6th sclerotome. The 1st permanent dorsal root ganglion (DRG 1) lies in the cranial half of the 7th sclerotome, while the spinal accessory nerve (XI) occupies the dorsal parts of the sclerotomes of segments 1-5, running adjacent to the neural tube towards the hind brain (HB). Bar, 100μm. (B) Sagittal section through the head region of a stage-28 chick embryo, stained with haematoxylin/eosin. The 1st (permanent) dorsal root ganglion (DRG 1) is arrowed. The parachordal plate is marked with an asterisk. Bar, 200μm.
(A) Sagittal section through a stage-19 chick embryo, stained with haematoxylin/eosin. Cranial is to the left, dorsal uppermost. The boundaries of the 5th sclerotome are marked with asterisks. Froriep’s ganglion (FG, arrowed) lies in the cranial half of the 6th sclerotome. The 1st permanent dorsal root ganglion (DRG 1) lies in the cranial half of the 7th sclerotome, while the spinal accessory nerve (XI) occupies the dorsal parts of the sclerotomes of segments 1-5, running adjacent to the neural tube towards the hind brain (HB). Bar, 100μm. (B) Sagittal section through the head region of a stage-28 chick embryo, stained with haematoxylin/eosin. The 1st (permanent) dorsal root ganglion (DRG 1) is arrowed. The parachordal plate is marked with an asterisk. Bar, 200μm.
(2) HNK-1 immunohistochemistry
Although ganglionic aggregations could not be seen in segments 2-6 with haematoxylin/eosin staining, the possibility remained that some neural crest cells nevertheless migrate into these segments, as they do in trunk regions of the embryo (Rickmann et al. 1985). This was assessed by immunoperoxidase stain-ing with HNK-1. Transverse and sagittal sections of ten stage-15 embryos showed that at occipital levels the distribution of HNK-1 positive cells in the sclero-tomes is very similar to that in the trunk region. The cranial half-sclerotomes of segments 2-6 are popu-lated by HNK-l-positive cells, while the caudal halves are not (Figs 2, 3A,B). Some cells, probably pre-sumptive postganglionic sympathetic cells, could also be seen around the dorsal aorta (Fig. 3A,B). We did not assess their fate, nor did we examine in any detail the dorsal pathway of crest migration (cf. Thiery et al. 1982). HNK-l-positive cells were also seen within the 1st segment, immediately caudal to the otic vesicle. Some of these cells were presumably destined to form the proximal sensory ganglia of the 9th and 10th cranial nerves, but again we did not assess this further (see Narayanan & Narayanan, 1980; D’Amico-Mar-tel & Noden, 1983).
Sagittal section through a stage-15 chick embryo, stained with HNK-1. Cranial is to the left, dorsal uppermost. HNK-1-positive cells are present in the cranial half of somite 2 (arrow) and similarly in the more caudal segments (right in the figure). The otic vesicle is marked with an asterisk.
Sagittal section through a stage-15 chick embryo, stained with HNK-1. Cranial is to the left, dorsal uppermost. HNK-1-positive cells are present in the cranial half of somite 2 (arrow) and similarly in the more caudal segments (right in the figure). The otic vesicle is marked with an asterisk.
(A) Transverse section through the cranial half of the 3rd segment in a stage-15 chick embryo, stained with HNK-1. Immunoreactive cells populate the sclerotome, extending ventrally to the region of the aorta. (B) Section through the caudal half of the same segment. HNK-l-positive cells do not penetrate the sclerotome at this level, being confined to the region between the dorsal neural tube and dermomyotome. Note the peri-aortal cells, which have migrated here from adjacent segmental levels.
(A) Transverse section through the cranial half of the 3rd segment in a stage-15 chick embryo, stained with HNK-1. Immunoreactive cells populate the sclerotome, extending ventrally to the region of the aorta. (B) Section through the caudal half of the same segment. HNK-l-positive cells do not penetrate the sclerotome at this level, being confined to the region between the dorsal neural tube and dermomyotome. Note the peri-aortal cells, which have migrated here from adjacent segmental levels.
(3) Zinc iodide/osmium tetroxide staining
Zinc iodide/osmium tetroxide staining of twelve whole-mounted stage-16 embryos showed that, by this stage, cells of presumed crest origin are sprouting axons within the cranial half-sclerotomes of segments 2 to 6 (Fig. 4). They could be distinguished from the motor axons, both by their presence in a different plane of focus and because their cell bodies were in the sclerotome rather than in the neural tube. At stage 19, in five embryos, small accumulations of cells and their axons were visible in segments 4 and 5, but not in segments 2 and 3. By stage 21, in eight embryos, no axons were visible within segments 2 to 6, while the ‘Froriep’s’ ganglion was still present in segment 6 (Fig. 5). Finally, by stage 23, in five embryos, the Froriep’s ganglion had also disap-peared, the most cranial dorsal root ganglion now appearing in segment 7.
Growth cone of a sprouting crest-derived cell in the cranial half-sclerotome of the 4th segment, stage-17 chick embryo, stained with zinc iodide/osmium tetroxide. The cell bodies of such sprouting cells could be seen within the sclerotome, in contrast to those of motor axons. Motor axon outgrowths could also be distinguished by their more ventral position. Bar, 10μm.
Growth cone of a sprouting crest-derived cell in the cranial half-sclerotome of the 4th segment, stage-17 chick embryo, stained with zinc iodide/osmium tetroxide. The cell bodies of such sprouting cells could be seen within the sclerotome, in contrast to those of motor axons. Motor axon outgrowths could also be distinguished by their more ventral position. Bar, 10μm.
Whole mount of a stage-21 chick embryo, occipital region, stained with zinc iodide/osmium tetroxide. Cranial is to the left, dorsal uppermost. The caudal halves of sclerotomes 5, 6 and 7 are indicated with the respective numbers. Motor axons can be seen ventrally, in the cranial halves of sclerotomes 5-8 (arrows). Sensory axons and dorsal root ganglia are visible in the cranial halves of sclerotomes 7 and 8, and Froriep’s ganglion is still present in sclerotome 6 (asterisks). However, no sensory axons are seen cranial to sclerotome 7. Bar, 50μm.
Whole mount of a stage-21 chick embryo, occipital region, stained with zinc iodide/osmium tetroxide. Cranial is to the left, dorsal uppermost. The caudal halves of sclerotomes 5, 6 and 7 are indicated with the respective numbers. Motor axons can be seen ventrally, in the cranial halves of sclerotomes 5-8 (arrows). Sensory axons and dorsal root ganglia are visible in the cranial halves of sclerotomes 7 and 8, and Froriep’s ganglion is still present in sclerotome 6 (asterisks). However, no sensory axons are seen cranial to sclerotome 7. Bar, 50μm.
Chick/quail chimaeras
Nine chick/quail chimaeras were constructed, in which quail occipital neural tube (opposite segments 2 to 6) was grafted into the trunk region of chick hosts, at wing level. The chimaeras were examined in Feulgen-stained transverse sections, at stages 24—30. In each case, dorsal root ganglia composed of quail cells, with or without additional chick cells, could be seen adjacent to the quail neural tube (Fig. 6). Comparison with the material stained with haema-toxylin/eosin and zinc iodide/osmium tetroxide showed that these chimaeric ganglia had survived beyond the stages at which the Froriep’s ganglion and the more cranial crest-derived cells disappear, that is beyond stage 23. It was noticeable, however, that they were not usually as large as the ganglia in the trunk region of normal embryos.
Transverse section through a chimaeric embryo wing region, stained by Feulgen’s method. The grafted quail neural tube is visible in the left of the picture, with a quail-derived dorsal root ganglion to its right, within the chick sclerotome. The prominent nucleolar marker of the quail cells distinguishes them from the host chick cells.
Transverse section through a chimaeric embryo wing region, stained by Feulgen’s method. The grafted quail neural tube is visible in the left of the picture, with a quail-derived dorsal root ganglion to its right, within the chick sclerotome. The prominent nucleolar marker of the quail cells distinguishes them from the host chick cells.
Discussion
As outlined already, the transient nature of dorsal root ganglion (DRG) development in the occipital segments of higher vertebrate embryos has been recognized for many years. Froriep (1882) first noticed this phenomenon in sheep embryos, finding a small ganglion associated with the most caudal hypo-glossal motor root, which later disappeared. Wilhelm His (1888) extended the observation to human em-bryos (but see O’Rahilly & Müller, 1984), and Chiar-ugi (1889) and Froriep & Beck (1895) went on to describe it in many other species. With some vari-ation between species, the usual pattern is for one or two adjacent Froriep’s ganglia to develop in the caudal hypoglossal and/or cranial cervical segments. The few segments still further cranial, to a level inclusive of the 2nd segment, are devoid of DRG cells, although they do allow the development of the motor roots of the hypoglossal and spinal accessory nerves. The proximal ganglia of the 9th and 10th cranial nerves are able to develop in the 1st segment.
In the chick embryo, using the conventional histo-logical techniques of the earlier investigators, no aggregates of DRG cells are visible in segments 2 to 5, and we see a Froriep’s ganglion in the 6th segment, which disappears by stage 23. In agreement with the observations of R. P. Hunter (1935), the most cranial (permanent) spinal DRG develops in the 7th seg-ment, associated with the 3rd cervical spinal nerve. Rogers (1965) also describes a Froriep’s ganglion in the 6th segment (the ‘fifth permanent somite’) and places the most cranial permanent dorsal root in the 7th segment, but differs in naming this the 2nd cervical spinal nerve.
It is striking that despite the failure of DRG formation in occipital segments, HNK-l-positive cells can be seen in their cranial half-sclerotomes, as they can in more caudal segments (Rickmann et al. 1985; Bronner-Fraser, 1986). In this respect our results differ from those of Thiery et al. (1982), who de-scribed the pathway of crest migration in this region using both the quail marker and fibronectin immuno-histochemistry, and who did not find evidence for ventral migration of neural crest cells at levels more cranial than the 6th segment. Nevertheless, in a recent immunohistochemical study Tucker, Ciment & Thiery (1986) do describe a pathway between the dermomyotome and sclerotome at the level of the 2nd somite.
Staining for axons with zinc iodide/osmium tetrox-ide also reveals that some crest cells are able to sprout axons within the cranial halves of the occipital sclero-tomes. What, then, is the cause of the failure of DRGs to form permanently at occipital segmental levels? Four major possibilities seem plausible: some deficiency in the neural crest or neural tube in this region, a failure of occipital sensory axons to find a suitable target for innervation, or an inability of the occipital sclerotomes, in particular their cranial halves, to support DRG survival.
An intrinsic crest deficiency is unlikely a priori given the results of Le Lievre et al. (1980). These authors grafted pieces of quail rhombencephalic crest, taken from levels cranial to the 7th somite, to a position between the neural tube and trunk somites in chick embryo hosts. The quail crest contributed neurones to the DRGs in the majority of cases.
A primary neural tube deficiency at occipital levels can also be ruled out. In agreement with Le Douarin & Teillet (1974) for autonomic derivatives, we find that when quail occipital neural tube is transplanted to the trunk level in chick hosts, the quail crest can differentiate according to its new position: quail-derived dorsal root ganglia form alongside the donor neural tube in the host trunk sclerotomes. It is important to note, however, that the DRGs that did develop in our grafting experiments were usually smaller than those found in the trunk region in normal embryos. While this may have resulted simply from adverse effects of explantation and grafting on the population of grafted quail cells, it could also reflect some additional inability on the part of occipi-tal neural tube to maintain normal DRG survival. There is evidence that the trunk neural tube does have a stimulatory influence on DRG development (Le Douarin, 1986; Kalcheim & Le Douarin, 1986).
Since DRG cell survival also depends upon the availability of a suitable target for innervation (Ham-burger & Levi-Montalcini, 1949), a further possibility is that such a target is lacking at occipital segmental levels. Two observations argue against this. First, in the zinc iodide/osmium tetroxide-stained material, sensory axons were not seen extending beyond the confines of the occipital sclerotomes, and so would not have reached their targets anyway. Second, cell death in the remaining (cervical) DRGs ends at about stage 30, well after the stage (23) by which cells have disappeared in the occipital segments (Hamburger & Levi-Montalcini, 1949). This implies that the cause of sensory cell loss in the occipital segments is different from that in trunk segments. ‘
The chief reason for the transitory nature of the existence of DRG cells in occipital segments must lie, therefore, with a property of the segments them-selves. Since the occipital sclerotomes do not dis-sociate until well after the disappearance of occipital sensory cells, the loss of these cells cannot result simply from an early dissolution of the sclerotomes at this level. Instead, we suggest that the cranial halves of the occipital sclerotomes lack factors essential for DRG cell survival and that these factors are corre-spondingly present in trunk sclerotomes. A role for somitic mesoderm in promoting DRG development was in fact originally proposed by Lehmann (1927) and Detwiler (1932), who studied the effects of somite removal on gangliogenesis in the trunk region of amphibian embryos. This suggestion does not exclude the additional importance of the neural tube in stimulating DRG development in trunk regions (Kalcheim & Le Douarin, 1986), and neural-tube-derived factors may also be diminished at occipital levels. Indeed, it seems likely that reciprocal interac-tions between all three tissues (DRG, sclerotome and neural tube) may be found to be necessary for the optimal development of each.
ACKNOWLEDGEMENTS
We thank Marie Watkins for expert technical assistance.