THE ANATOMICAL RECORD 22623-90 (1990)

Distribution of Cell Surface Glycoconjugates During Secondary Neurulation in the Chick Embryo C.M. GRIFFITH AND M.J. WILEY Department of Anatomy, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

ABSTRACT Lectin histochemistry was used to examine the expression of cell surface glycoconjugates during secondary neurulation in chick embryos. Fourteen lectins were applied to serial sections of the caudal region of embryos at the various stages of tail bud development. The lectins Bandeiraea simplicifolia, Dolichos biflorus agglutinin, Phaseolus vulgaris leukoagglutinin, soybean agglutinin, Sophora japonica agglutinin, Ulex europaeus agglutinin and succinylated wheat germ agglutinin (sWGA) showed very light or no binding to the developing medullary cord of the tail bud. With the other lectins, staining occurred throughout the early tail bud and solid medullary cord. During cavitation, however, differential expression of cell surface glycoconjugates by different cell populations was observed. The lectins concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin and WGA showed basic similarities in the distribution of lectin binding. Of these, the binding pattern of WGA was the most striking. As the medullary cord cells were separating into central mesenchymal and peripheral epithelial populations, WGA bound preferentially to the epithelial cells and the notochord. The lectin PNA, however, became preferentially bound to the mesenchymal cells. Heavy staining by WGA (specific for N-acetylglucosamine and sialic acid) where sWGA staining (specific for N-acetylglucosamine only) was faint suggested that WGA binding was due to the presence of sialic acid containing glycoconjugates. The neural tube in mammalian and avian embryos is formed by two separate and distinct processes. During primary neurulation, the portion of the neural tube that forms the brain and most of the spinal cord develops through folding of the neural plate. The remainder of the spinal cord forms during secondary neurulation by cell rearrangements and canalization in the tail bud (Criley, 1969; Jelinek et al., 1969; Schoenwolf, 1977, 1978, 1979; Schoenwolf and DeLongo, 1980). The tail bud is derived from the remains of Hensen’s node and the primitive streak following gastrulation. In avians, the cells in the dorsal midline of the tail bud aggregate to form the medullary cord, while laterally placed cells form the paraxial mesoderm. The cells of the medullary cord become further divided into a peripheral population and a centrally located population. Cavities subsequently appear at the boundary between the 2 cell populations. These enlarge while the central cells apparently merge with the peripheral cells. The cavities then coalesce and merge with the lumen of the primary neural tube (Schoenwolf, 1979; Schoenwolf and DeLongo, 1980). In chicken embryos, there is a well-defined zone of transition between primary and secondary mechanisms of neurulation at the level of the future lumbosacral region of the spinal cord (Criley, 1969; Jelinek e t al., 1969; Klika and Jelinek, 1969; Schoenwolf, 1977, 1978, 1979; Schoenwolf and DeLongo, 1980). Dryden (1980) described the relationship between the two 0 1990 ALAN R. LISS, INC

modes of neurulation in this zone as a reciprocal wedge in which the neural folds taper caudally, dorsal to the medullary cord. The medullary cord in t u r n tapers cranially, ventral to the neural folds. Consequently, within this zone, the dorsal aspect of the neural tube is formed from the neural folds of the posterior neuropore by primary neurulation, while the ventral aspect is formed from the medullary cord by secondary neurulation. Primary neurulation has been extensively investigated in many species, and i t has been established that changes in cellular morphology and adhesion are important factors (Moran and Rice, 1975; Lee, 1976; Lee et al., 1976a,b; McLone and Knepper, 1986; Sadler, 1978; Smits-van Prooije et al., 1986; Kapron-Bras and Trasler, 1988; Thorpe et al., 1988). Most recently, interest has been focussed on the role(s) of the carbohydrate moieties of cell surface glycoproteins andlor glycolipids in complex cellular interactions such a s apposition and fusion of the neural folds. The approach has been to study the binding and distribution of lectins by the ectoderm and neuroectoderm of the neural folds. Lectins are naturally occurring polypeptides that

Received January 4, 1989; accepted March 30, 1989.

82

C.M. GRIFFITH AND M.J. WILEY

TABLE 1. The lectins used in the studs and their specificities Lectin

Bandeiraea simplicifolia lectin I Concanavalin A

Dolichos biflorus agglutinin Lens culinaris agglutinin Peanut agglutinin

Pisum sativum agglutinin Phaseolus vulgaris erythroagglutinin Phaseolus vulgaris leukoagglutinin Ricinus communis agglutinin 120

Abbreviation BSL I Con A DBA LCA PNA PSA PVE PVL RCA 120

Wheat germ agglutinin

SBA SJA UEA I WGA

Succinylated WGA

sWGA

Soybean agglutinin

Sophora japonica agglutinin Ulex europaeus agglutinin I

bind specifically to carbohydrate (sugar) residues of glycoproteins and glycolipids. They have been used extensively as probes in studies of cell surface interactions in several developing systems (Damjanov, 1987), including the neural tube. For example, Currie et al. (1984) used various lectins to map the ectoderm surrounding the posterior neuropore of rat embryos at the time of closure. They reported that ectodermal cells, prior to their differentiation into neural tube, neural crest, and surface ectoderm, revealed their commitment to diverging cell lineages by expressing different lectin affinities. McLone and Knepper (1986) reported differences between normal mice and Splotch mutants in lectin binding patterns of neuroectoderm during closure of the posterior neuropore. The Splotch mice, which have a high spontaneous incidence of neurulation defects, showed a reduced affinity for the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A), in comparison to outbred animals. The reduced affinity may reflect characteristics of the cell surface in the mutant mice that lead to failure of the neural folds to fuse. The importance of the dynamic patterns of cell surface glycoconjugates during neurulation was further suggested by Lee (1976) and Lee e t al. (1976a,b), who were able to disrupt development of the primary neural tube by decreasing the amount of free cell surface glycoproteins and glycolipids using Con A to bind to available sugar residues. While the majority of investigations of glycoconjugates during neural tube development have dealt with primary neurulation, little is known of their role in secondary neurulation. The purpose of the present study was to investigate the characteristics and distribution of cell surface glycoconjugates during the development of the secondary neuraxis in chick embryos by means of lectin histochemistry.

Sugar specificity N-acetylgalactosamine D-glucose; D-mannose N-acety lgalactosamine D-glucose; D-mannose D-galactose; D-galactose-(~l-3)-N-acetylgalactosamine D-glucose; D-mannose N-acetylgalactosamine N-acetylgalactosamine D-galactose; N-acety lgalactosamine N-acetylgalactosamine N-acety lgalactosamine L-fucose N-acetylglucosamine; sialic acid (N-acetylneuraminic acid) N-acetylglucosamine

Lectin Staining

Embryos a t Hamburger-Hamilton (HH) stages 1315 or 17-19 were used in this study. At stages 13-15, cavitation of the medullary cord to form the secondary neural tube is just under way, while at stages 17-19, the secondary neural tube is well developed. These stages were therefore chosen in order to compare the lectin binding affinities of cell surface glycoconjugates in the immature and mature secondary neural tubes. Embryos were fixed in 4% paraformaldehyde in a 0.1 M phosphate buffer, pH 7.2-7.4 overnight at 4°C. They were then routinely processed for wax embedding. Four percent polyvinylpyrrolidone (PVP) was added to the fixative, buffer rinse, and graded ethanol series used in dehydration. PVP was used to protect against changes in osmolarity during tissue processing and has been shown to provide improved immunoreactivity of antigens in postembedment localization of antigens (Kuhlmann and Krishan, 1981). The affinities of the following lectins were studied: Bandeiraea simplicifolia lectin I (BSL I), concanavalin A (Con A), wheat germ agglutinin (WGA), succinylated WGA, Ricinus communis agglutinin 120 (RCA 120), Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA),peanut agglutinin (PNA),Lens culinaris agglutinin (LCA), Sophora japonica agglutinin (SJA), P i s u m sativum Agglutinin (PSA), Phaseolus vulgaris erythroagglutinin (PVE) and leukoagglutinin (PVL), and Ulex europaeus agglutinin I (UEA I). The sugar binding specificity of each lectin is shown in Table 1. Each lectin was tested in 5-12 embryos. Six-micron serial sections of the caudal portions of the embryos were deparaffinized in xylene, rehydrated, and placed in 1%hydrogen peroxide in methanol for 15 minutes to quench endogenous peroxidase activity. The sections were then washed in 0.1 M phosphate buffered saline (PBS) a t pH 7.2-7.4 for 20 minutes and then preincubated in 0.1 M PBS containing 4% fetal calf MATERIALS AND METHODS serum (FCS) (Grand Island Biological Co.) for 20 minFertile white leghorn eggs (Craig Hunter Farms, utes, to minimize nonspecific binding. A 0.02 mg/ml Stroud, Ontario) were incubated a t 38°C in a humidi- solution of each biotinylated lectin (Vector Labs) in fied forced-draft incubator for approximately 48 or 72 PBSiFCS was applied to sections mounted on a slide. hours. They were then windowed and staged according The lectins WGA, LCA, RCA, and PNA were also used to the descriptions of Hamburger and Hamilton (1951). a t a concentration of 0.01 mg/ml. The sections were

GLYCOCONJUGATES I N SECONDARY NEURULATION

incubated a t room temperature for 30 minutes in a humid atmosphere. They were then washed in PBS/ FCS for 30 minutes to remove unbound lectin, and reincubated in horseradish peroxidase-avidin D (HRPavidin D) (Vector Labs) at a concentration of 0.01 mg/ ml in PBS/FCS for 30 minutes. Unbound HRP-avidin D was rinsed off in PBS/FCS and the slides washed in 0.1 M Tris-HC1 buffer, pH 7.6, for 30 minutes. The HRPcomplexed lectins were visualized by reaction with a freshly prepared solution of 0.05% diaminobenzidine (DAB) and 0.03% hydrogen peroxide in the Tris-HC1 buffer for 5-10 minutes. Finally, the sections were washed in Tris-HC1 buffer for 15-20 minutes, dehydrated, cleared, and mounted in DPX mountant. To confirm the binding specificity of a lectin for a particular sugar, 0.2 M of a n appropriate competing sugar (Roth, 1978) was added to the solution of each lectin and allowed to react for 2 hours a t room temperature prior to use. Decreased lectin staining or inhibition of lectin staining was considered evidence of binding to the specific carbohydrate moiety. Nonspecific background staining in the sections was controlled by incubating sections in PBS/FCS only. The distribution and intensity of lectin staining in the different tissues of the developing medullary cord were examined. Comparisons were made among the various lectins, and also across the different types of tissues and stages in development. RESULTS

Secondary neurulation proceeds in a craniocaudal fashion with time. Consequently, by examining sections through the medullary cord in a caudal to cranial sequence, i t is possible to observe changes in the tail bud which reflect progressive differentiation and maturation (Schoenwolf and DeLongo, 1980). With the lectins BSL I, DBA, PVL, SBA, SJA, UEA I, and sWGA, little or no staining was observed in the solid or cavitating medullary cords, or neural tube. BSL, however, stained the extracellular matrix between the surface ectoderm and the paraxial mesoderm (Fig. 1).The faint staining by sWGA will be discussed below. Staining with the remaining lectins is summarized in Tables 2 and 3. In general, the intensity of lectin staining decreased gradually during tail bud development. The lectin RCA showed uniform staining throughout the developing medullary cord during stages 13-15 (Fig. 2A) and also in the late tail bud (stages 17-19) (Fig. 2B). The most intense staining with this lectin was in extracellular material between the surface ectoderm and the underlying mesoderm, and between the endoderm and overlying mesoderm. The lectins Con A, LCA, PSA, PVE, and WGA showed a similar basic pattern through the earlier stages of tail bud development (stages 13-15) (Table 2). The pattern was best exemplified by the staining by WGA. Before the appearance of the medullary cord and throughout the solid phase of medullary cord development, the cells were heavily and relatively uniformly labelled (Fig. 3A). The ventralmost part of the medullary cord, however, was more intensely labelled and was continuous, in more cranial sections, with a heavily labelled notochord. In addition, small, isolated, and heavily labelled areas could be identified (Fig. 3B)

83

Fig. 1. Section through the cavitating medullary cord (MC), stained by BSL I. The extracellular matrix between the paraxial mesoderm (PM) and its overlying surface ectoderm (SE) is intensely stained. x 300.

which marked the boundary between peripheral and central populations of cells of the medullary cord. These areas also marked the sites a t which lumina develop during cavitation. With the onset of cavitation, the cells at the periphery of the medullary cord assumed a pseudostratified appearance, and became radially oriented with respect to mesenchymal-shaped cells a t the centre of the medullary cord (Fig. 3C). The labelling in the peripheral domain of medullary cord was less intense than the staining of the medullary cord a t earlier stages. In addition, the central cells were more lightly stained than the peripheral cells or not stained at all (Fig. 3D,E). The linings of the lumina formed by cavitation a t the boundary between the central and peripheral cell groups were heavily labelled, and retained the labelling a s they enlarged and coalesced to form the single lumen of the secondary neural tube. (Fig. 3). The apical lining of the primary neural tube was also heavily stained by these lectins, and in the transitional or overlap zone, where primary and secondary neurulation occur concurrently, staining was observed in the primary lumen as well as in the lumina of the cavitating medullary cord, and secondary neural tube (Fig. 3F). Staining by WGA was found to decrease during the course of caudal axial development, but it did not disappear entirely. By stages 17-19, WGA staining was confined mainly to the extracellular matrix around the notochord and its basement membrane (Fig. 4). Where labelling by WGA was intense, sWGA labelling was faint. This was particularly striking in the ventral medullary cord and more cranially, in the notochord (Fig. 5). Labelling by sWGA was confined mainly to the linings of lumina during earlier stages of tail bud development (stages 13-15), and subsequently disappeared at later stages (stages 17-19). WGA also stained the lumina1 linings at stages 13-15, but the binding was more intense. While Con A, LCA, and PSA are all specific for sugar

C.M. GRIFFITH AND M.J. WILEY

84

TABLE 2. Binding of lectins in the chick tail bud during HH stages 13-15' Lectin

Solid medullary cord

(inhibitory sugar)

Dorsal

Con A LCA PSA (D-Glc, D-Man) PVE (GalNAc) RCA (D-gal, galNAc) WGA (GlcNAc, Sialic acid) sWGA (GlcNAc) PNA (D-gal)

Middle

Secondary portion of neural tube

Cavitating medullary cord

Ventral

Luminal

Basal

border

border

PC

CC

Ventral

Luminal

Basal

margin

border

border

Notochord Basal

Cells

border

Cells

+

+ + + + + ++ + + ++ ++++ ++++ ++++ +++++ +++++ ++++ +++ ++++ +++++ +++++ ++++ +++++ ++++ ++ ++ ++ +++ +++ ++ + +++ +++ ++ +++ ++

+

++

++

++

+++

+++

++

+

++

+++

-

++

+++

+++

+++

++++

++++

++

++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++

+

++++

+++

t + f

+t

+

+

+

+

+

-

+

+++

+++

-

+

+

+++

+++

+++

++++

++++

++++

++

++++

++++

+

+++

++

~

+

'Staining intensity is based on a subjectively estimated scale from 0 to PC = peripheral cells; CC = central cells.

-

+ + i t

+++

+ i t + ++t

+ + + + + , with - being negative and + + + + + being most positive.

TABLE 3. Binding of lectins in the chick tail bud during HH stages 17-19.'

Lectin Con A LCA PSA PVE RCA WGA sWGA PNA

Luminal border -

++ ++ + ++ ++ + ++

Cavitating tail bud (caudalmost region of tail) Basal border PC CC

+ ++++ +++ + +++ +++ + +++

+ +++ +-+ ++ ++ +

+ +++ + ++ + ++

Ventral margin

+ +++ + -+ + ++ + -+ + ++

Mature secondary neural tube Luminal Basal border border Cells

+++ +++ + ++ ++ + ++

+ ++++ +++ +++ +++ ++ + +++

-

+++ +-+ ++ + -

-

Notochord Basal border Cells

+ ++++ +++ ++ +++ +++ + ++

+ +++ + +- + ++ +-

'Staining intensity is based on a subjectively estimated scale from 0 to PC = peripheral cells; CC = central cells.

+ + + + + , with - being negative and + + + + + being most positive;

Fig. 2,Transverse sections through the tail bud a t two different stages in secondary neurulation, stained with RCA. x 300. A Section through the cavitating medullary cord at stages 13-15 of development. CC, central cells; NC, notochord; PC, peripheral cells; AR-

ROWS, lumina formed by cavitation; P, primary portion of developing neural tube. B: Section through the tail bud at stages 17-19, showing the mature secondary neural tube (NT). NC, notochord; TG, developing tail gut.

GLYCOCONJUGATES I N SECONDARY NEURULATION

residues of D-mannose and D-glucose, their binding affinities in comparable tissues were different. Binding by LCA tended to be heavy and fairly uniformly distributed throughout the developing tail bud at both earlier and later stages (Fig. 6). Binding by PSA was slightly more discriminating (Fig. 7) but was still relatively uniformly distributed throughout tail bud development. Con A bound mainly to the peripheral cells of the cavitating medullary cord and cells of the more anteriorly located neural tube, but not to the lumina1 or basal borders (Fig. 8). Staining by Con A was slightly more intense a t the subapical regions of the developing neuroepithelial cells. By stages 17-19, only trace amounts of Con A staining were observed in tail bud tissues. Of the lectins that stained the differentiating medullary cord, the staining pattern of Con A was the least typical in that it was not heavily bound to the borders of the primary lumen or the lumina of cavitation. However, binding of Con A was increased a t the subapices of cells that were arranged around these lumina, in comparison with the binding patterns of the other lectins. PNA staining was intense throughout the tail bud and early medullary cord (Fig. 9A). During cavitation, PNA staining of the elongated peripheral cells decreased in intensity, while the central cells retained the heavy staining (Fig. 9B,C). The staining intensity of the ventral region of the medullary cord decreased during development, and the notochord was very lightly stained or not stained a t all (Fig. 9D). In older (stage 17-19) embryos, the neuroepithelium of secondary neurulation was comparatively unstained but the mesenchymal components of the tail bud still retained an affinity for PNA (Fig. 10). DISCUSSION

It has been suggested that many of the events leading to morphogenesis and differentiation in the embryo are triggered by the interaction of the carbohydrate moieties of cell surface glycoproteins andlor glycolipids (Moscona, 1974; Raedler e t al., 1981; Currie e t al., 1984; Raedler and Raedler, 1985). The present investigation undertook to study the binding of a variety of lectins during the development of the secondary neural tube. This was done to determine whether or not the changes in cell shape and orientation which occur during secondary neurulation are accompanied by changes in the distribution of cell surface glycoconjugates. During secondary neurulation in avians, the remains of the primitive streak coalesce into a solid rod of mesenchymal cells, known as the medullary cord. Subsequently, the cells of the medullary cord become arranged into two domains. In the outer, peripheral domain, the cells become elongated and resemble the pseudostratified columnar arrangement of the neuroectoderm in the primary neural tube. The more centrally placed cells, on the other hand, retain a more stellate mesenchymal appearance. In the present study, the boundary between the central and peripheral cell populations was found to be marked by small areas heavily labelled with Con A, LCA, PSA, PVE, RCA 120, and WGA. These areas were continuous cranially with the cavities that develop during secondary neurulation. As the lumen of the secondary neural tube develops during cavitation, the central cells are lost,

85

most probably by intercalation with the cells of the periphery. We have shown that these morphological changes and rearrangements are accompanied by changes in the affinities of the tail bud tissues for certain lectins. Lee (1976) and Lee et al. (1976a,b) treated chick embryos in culture undergoing primary neurulation with the lectin Con A, thereby effectively eliminating the availability of free cell surface glycoconjugates containing mannose (the sugar residue to which Con A binds specifically). This resulted in the failure of the primary neural tube to close and suggested that mannose-bearing cell surface glycoconjugates are probably involved in primary neurulation. Mannose residues are also recognized by LCA and PSA. In our study, LCA binding was uniform and heavy, while PSA and Con A showed less affinity overall, but tended to stain tail bud tissues differentially. The reasons for the differences between these mannose-specific lectins are most likely due to stereochemical features of the glycoconjugates (Damjanov, 19871, but the results do demonstrate that large concentrations of mannosylated surface molecules are a feature common to both primary and secondary neurulation. A reciprocal affinity of tail bud tissues was observed between PNA and several other lectins. While the mesenchymal-like central cells of the cavitating medullary cord were relatively unstained by Con A, PVE, PSA, RCA, and WGA, these cells were heavily stained by PNA. In contrast, the epithelial-like peripheral cells, which were preferentially stained by the other lectins, they were lightly stained by PNA. A preferential staining of mesenchymal cells by PNA and neuroepithelial cells by WGA was also observed during primary neural tube development in Bantam chick embryos by Takahashi (1988). It has been suggested that the presence of receptors for PNA on a cell type may be a n indication of the undifferentiated state of the cell andior functional immaturity of the cell (Takahashi, 1988). WGA proved to be one of the most effective lectins for labelling cell surfaces in the tail bud and particularly in the medullary cord. While the staining intensity of the lectins RCA, Con A, LCA, or PSA tended to be fairly uniform throughout secondary neurulation, staining by the WGA became more selective during the course of secondary neuraxis development, becoming confined to the extracellular matrix andlor basement membranes of the neural tube and notochord. Since WGA showed the most striking changes in distribution through secondary neuraxis development, this suggests a special role for the glycoconjugates binding the lectin during secondary neurulation. WGA binds to both sialic acid and N-acetylglucosamine moieities. Succinylated WGA (sWGA), however, binds only to Nacetylglucosamine residues. Since sWGA labelled only the lumina of the cavitating medullary cord and the lumen of the primary neural tube, it appears that the binding of WGA by the caudal neural tube and notochord was most likely due to sialic acid. The neural cell adhesion molecule (NCAM) is a membrane associated glycoprotein which has been implicated in mesenchymal condensation and adhesion leading to organogenesis (Klein e t al., 1988).While various forms of NCAM are known, the highly sialylated form, contain-

GLYCOCONJUGATES IN SECONDARY NEURULATION

Fig. 4.Sections through the tail bud at stages 17-19, stained with WGA. x 280. A Oblique section through the caudal-most portion of the tail bud, where cavitation is still underway. CC, central cells; PC, peripheral cells; ARROW, cells which were continuous with the an-

87

teriorly located notochord. B: Section through a more cranial level, showing the mature secondary neural tube (NT), notochord (NC), and developing tailgut (TG).

ing up t o 30%sialic acid by weight, is the predominant form in immature tissues (Rutishauser et al., 1985, 1988; Rutishauser and Goridis, 1986; Klein et al., 1988). In chickens, it has been reported to be present in embryos from stages 10 to 45, with high levels maintained between stages 15 and 45 (Sunshine et al., 1987). During the histogenesis of the chick central nervous system, NCAM with a low sialic acid content is thought to maintain tissue integrity under conditions of mechanical stress, e.g., the primary neuroepithelium during primary neural tube formation, development of flexures and evaginations, while highly sialylated NCAM provides plasticity in subsequent cellular interactions (Sunshine et al., 1987). The results of our study suggest that NCAM may be involved in the process of cell aggregation and rearrangement in secondary neurulation. ACKNOWLEDGMENTS Fig. 5.Cavitating medullary cord (MC) stained with sWGA. Faint staining is confined to the lumina1 linings of the cavities formed during secondary neurulation (arrows), and to a lesser extent, the notochord. This is in contrast to the comparatively heavy labelling seen with WGA (Fig. 3). x 250.

We thank Mr. Battista Calvieri for his technical assistance.This work was supported by Grant Number MA6419 from the Research Of Canada. Ms. Griffith is supported by an MRC Studentship.

Fig. 3.Transverse sections through the tail bud and overlap zone of a n embryo during early secondary neurulation (stages 13-15). They are arranged in a caudal to cranial order, showing the sequential changes in lectin binding during the development of the secondary neuraxis. Stained with WGA. ~ 3 3 0 A: . Section through the solid medullary cord (MC). WGA staining is fairly even throughout but is slightly more intense at the dorsal and ventral margins. PM, paraxial mesoderm. B Oblique section through the medullary cord a t a stage just prior to or at the very beginning of cavitation. The small spots of intense staining (arrows) mark sites at which lumina appear in more cranial sections. The compact group of intensely stained cells at the ventral margin of the medullary is continuous with the notochord

cranially. C: The medullary cord during early cavitation. The cells have differentiated into a pseudostratified peripheral and a mesenchymal central population. The dorsalmost lumen (PL) is the caudal end of the primary neural tube lumen. PC, peripheral cells; CC, central cells; arrows, lumina of cavitation; NC, notochord. D, E: Sections through the medullary cord a t different stages of cavitation, showing heavily stained peripheral cells and relatively unstained central cells. The borders of the primary lumen and lumina from cavitation are also very intensely stained, as is the notochord. F: Late cavitation in the neurulation overlap zone, showing almost complete fusion between the primary (dorsal half) (P) and secondary (ventral half) (S)neural tubes.

Figs. 6-8. Sections through the tail bud at stages 13-15 and stages 17-19, stained with lectins specific for D-mannose and D-glucose. MC, medullary cord; NC, notochord; NT, mature secondary neural tube; TG, tailgut. x 280. Fig. 6. A Section through stage 13-15 tail bud stained with LCA, showing uniform heavy staining of the medullary cord and developing notochord. B: Section through an equivalent level of the tail bud a t stages 17-19 showing decreased staining in the mature secondary neural tube and notochord.

Fig. 7. A Stage 13-15 tail bud section stained with PSA. Staining is fairly homogenous, with intense labelling at the lumina1 borders and basal border of the medullary cord. B: Section through stage 17-19 tail bud, stained with PSA. Fig. 8. A Section through stage 13-15 tail bud stained with Con A, showing concentration of staining a t the subapices of cells (arrows) around both the dorsally placed primary neural tube lumen and lumina formed by cavitation. B Stage 17-19 tail bud, unstained by Con A.

Fig. 9.Transverse sections through the medullary cord of stage 1315 embryos, stained with PNA, arranged in a caudal to cranial sequence, representing different stages in the development of the secondary neuraxis. x 330. A Section through the medullary cord (MC) prior to cavitation, showing intense but fairly uniform binding, except for the ventralmost portion which appears more heavily stained. B, C: Sections through the cavitating medullary cord, showing the progressive loss of PNA affinity in the elongated peripheral cells (PC).The mesenchymal central cells (CC);however, retain the intense staining. Staining in the notochord (NC) can be seen to decrease progressively (from B to C). PL, lumen of primary neutral tube. D Fusing primary

(P)and secondary (S) portions of the neural tube. The dorsal primary neural tube region is more darkly stained than the ventral secondary neural tube. The notochordal cells are unstained. Fig. 10Sections through the tail bud of a stage 17-19 embryo, stained with PNA. x 430. A Section through the caudalmost region of the tail bud, showing cavitation. CC, central cells; PC, peripheral cells; arrow, cells which are continuous with the anteriorly located notochord. B: Section through the mature secondary neural tube (NT),taken from a more cranial level of the tail bud. TG, tailgut; NC, notochord.

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LITERATURE CITED