J. Anat. (1982), 134, 3, pp. 491-506 491 With 16figures Printed in Great Britain Culture of rat embryos with p-D-xyloside: evidence of a role for proteoglycans in

GILLIAN M. MORRISS-KAY AND BETH CRUTCH Department ofHuman Anatomy, University of Oxford (Accepted 1 May 1981)

INTRODUCTION The synthesis of sulphated glycosaminoglycans by early post-implantation rat embryos occurs at very low levels until the onset of neurulation, at which time there is an increase in the level ofsynthesis (as indicated by [3H]glucosamine incorporation) of chondroitin/chondroitin sulphate and heparan sulphate (Solursh & Morriss, 1977). In embryos undergoing neurulation, histochemical staining techniques have indicated that these sulphated glycosaminoglycans are localised in the ectodermal basement membrane and in the extracellular matrix and cell surface-associated material of the mesenchyme. The staining intensity is higher in the region than elsewhere in the embryo. They are probably present in the form of proteoglycan in association with hyaluronate (Morriss & Solursh, 1978a). In order to investigate further these stage and position-related histochemical differences, we have cultured rat embryos in vitro during neurulation and early somitogenesis in the presence of the xylose-derivative, /8-D-xyloside. This substance has been shown to bring about a reduction in the level of synthesis of protein-bound chondroitin sulphate and an increase in free chondroitin sulphate chains in both chondrogenic and non-chondrogenic systems (Schwartz, Ho & Dorfman, 1976; Galligani, Hopwood, Schwartz & Dorfman, 1975). Its activity is due to interference with the sequence of normal chondroitin sulphate-proteoglycan synthesis, as fol- lows. Prior to chondroitin sulphate chain synthesis, three sugar molecules, xylose and two galactose molecules, are added sequentially to certain serines of the core protein (for review, see Roden & Schwartz, 1975). /5-D-xyloside competes with protein-bound xylose for the enzyme galactosyl transferase, and thereby acts as an initiation site for galactose-linked chondroitin sulphate chain synthesis (Fukanaga et al. 1975; Schwartz, 1977). Schwartz (1979) found that in chondroitin sulphate- proteoglycan formed by cultured chondrocytes in the presence of 1 mM ,-D-xyloside, the degree of substitution of core protein serine residues with chondroitin sulphate chains was reduced by 60 %, but there were only minor reductions in chain length and degree of sulphation. Furthermore, there was an increase of serine-bound chondroitin sulphate within the cells, suggesting that transport of the low-carbo- hydrate proteoglycan molecules to the extracellular matrix was impaired. /?-D-xylo- side itself stimulates chondroitin sulphate synthesis (Galligani et al. 1975). These free chondroitin sulphate chains differ from the protein-bound chains in being short and under-sulphated (Gibson, Segen & Audhya, 1977). ,f-D-xyloside-induced reduction of proteoglycan synthesis has also been demon- strated in whole embryo systems: chick (Gibson, Segen & Doller, 1979) and sea urchin (Kinoshita & Saiga, 1979), where it was associated with abnormal morpho- genesis. In the present study, we have analysed its effect on development, particularly 492 GILLIAN M. MORRISS-KAY AND BETH CRUTCH of the cranial neural folds, in rat embryos. Our purpose was to elucidate the morpho- genetic significance of the sulphated glycosaminoglycans present at this stage, and to discover whether they are present in the form of proteoglycan. These results were presented briefly to the Anatomical Society in December 1980 (Morriss-Kay & Crutch, 1981).

MATERIALS AND METHODS Embryo culture Wistar strain rat embryos were explanted in Tyrode's saline on the afternoon of day 9 ofpregnancy (day ofpositive vaginal smear = day 0) and Reichert's membrane was opened. They were cultured at 38 °C in 60 ml bottles, each containing 5 ml medium. The bottles were rotated at 30 rev/min. The culture medium consisted of 2-5 ml Tyrode's saline (with or without 8-D-xyloside) and 2 5 ml immediately centrifuged, heat-inactivated rat serum (Steele & New, 1974) with 50 pg/ml strepto- mycin. 8l-D-xyloside (p-nitrophenyl-,8-D-xylopyranoside, Koch-Light) was added from a stock solution (1I35 mg in 100 ,ul Tyrode's saline) to give a final concentra- tion of 1 mm. The explanted embryos were at the late presomite to very early somite stage (flat to slightly convex neural folds; Morriss & Solursh, 1978b), and were carefully matched for allocation to control and experimental groups, with a maximum of six embryos per bottle. 95 control and 101 experimental embryos were cultured for periods ranging from 15 to 30 hours. The bottles were first gassed with 5 %20/5 % C02/90 % N2 (New, Coppola & Cockroft, 1976a, b). Cultures continued after 24 hours were regassed with 5 % CO2 in air. When the cultures were terminated the embryos were washed in Tyrode saline and the membranes dissected off. They were examined and photographed whole, either unfixed (prior to prepara- tion for paraffin embedding) or after glutaraldehyde fixation (prior to preparation for electron microscopy). In order to confirm the specificity of /-D-xyloside in this system, two related p-nitrophenol derivatives (p-nitrophenyl-fi-D-glucopyranoside and p-nitrophenyl- ,8-D-galactopyranoside) and free xylose were tested at 1 mm in 24 hour cultures (other details as above). Each of these cultures contained four day 9 embryos. After 24 hours the membranes were dissected off and the embryos were examined with the dissecting microscope in order to assess development (somite numbers, development and form of the neural folds, etc.) in comparison with co-cultured control embryos. They were not processed further. For histology, embryos were fixed in one quarter-strength Bouin's fluid, paraffin- embedded, cut at 5 /zm, and stained with haematoxylin and eosin. For histochemistry, five control and five /J-D-xyloside-cultured embryos were fixed after a 3 hour culture period in Bouin's fluid (one embryo), Carnoy's fluid (two embryos), and Carnoy's fluid containing 100 mg/ml cetylpyridinium chloride (CPC) (two embryos). They were paraffin-embedded, cut at 8 #zm and mounted four sections per slide. Adjacent slides were either stained immediately with alcian blue at pH 1, or stained with alcian blue at pH 1 following 3 hours incubation with 0-1 M phosphate buffer (pH 5 6) with or without 400 units/ml purified, protease-free testicular hyaluronidase (Worthington; degrades hyaluronate, chondroitin, and chondroitin sulphatesA & C). Six control and five experimental embryos, cultured for 15 hours, and five control and five experimental embryos, cultured for 24 hours, were used for electron microscopy. They were fixed with 2-5 % cacodylate-buffered glutaraldehyde (0-1 M, pH 7-2, with fl-D-xyloside: effects on neurulation 493

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Fig. 1. Embryos cultured for 17 hours. Control (upper) has 9 pairs of somites; the cranial neural epithelium has a V-shaped profile and its junction with the surface (arrow) is clear. Anteriorly, the two sides of the anterior forebrain are approaching each other. The f6-D-xyloside- cultured embryo (lower), with 8 pairs of somites, has a much broader neural epithelium whose lateral edge forms the border of the head from this viewpoint. In the anterior forebrain region, the neural epithelium is everted instead of concave. Fig. 2. The same embryos as in Fig. 1, to show clearly outlined somites in the control (upper) compared with less distinct somitic mesoderm in the 8l-D-xyloside-cultured embryo (lower). Both embryos show close apposition, probably fusion, of the neural folds to form in the somitic region. 494 GILLIAN M. MORRISS-KAY AND BETH CRUTCH 2 mm CaCl2), washed in buffer, post-fixed in 1 % cacodylate-buffered osmium tetroxide, dehydrated, and embedded individually in Spurr resin. They were orienta- ted at this stage, and during cutting, so that all sections were cut perpendicular to the long axis of the embryo. 1 ,um sections were mounted on glass slides and stained with 05 % methylene blue/0 5 % azure II in 0 5 % borax for reference; thin sections were stained with lead citrate and uranyl acetate.

RESULTS Embryos cultured for 24 hours in the presence ofp-nitrophenol derivatives other than .,-D'-xyloside (p-nitrophenyl-/6-D-glucopyranoside and p-nitrophenyl-f6-D- galactopyranoside) and free xylose were examined with the dissecting microscope after removal of their membranes. They showed no morphological abnormalities when compared with co-cultured control embryos. The morphological abnormalities reported: below are therefore regarded as a specific effect of fl-D-xyloside on em- bryonic. development. Morphogepesis ofcultured embryos During formation of the first four pairs of somites, the cranial neural folds of control embryos developed as increasingly convex structures. Subsequently, up to the 9 somite stage, the cranial neural plate/ boundary formed an angle wich became progressively sharper as the neural folds increased in height and became V-shaped in profile (Fig. 1). Cranial neural tube closure was completed at the 14 somnite stage, after approximately 24 hours ofculture. (This sequence of events is described and illustrated more fully in Morriss & New, 1979.) The earliest (convex) stages of cranial neural fold formation in /5-D-xyloside- cultured embry6o were apparently normal, although somite formation proceeded at a slower rate than in controls, and the somites themselves were less distinct (Fig. 2). Subseq'uently, the cranial neural folds continued to enlarge but retained their convex shape, so that in contrast to control embryos, a clear boundary did not develop between neural and surface ectoderms in embryos cultured for 15-17 hours; these embryos had 5-8 pairs of somites, cf. 6-10 in controls (Fig. 1). By 24 hours a neural/surface ectodermal angle had developed, but the neural ectoderm was itself still convex in profile.; embryos cultured for longer periods (up to 30 hours) did not progress further'towkrds closure of the cranial neural tube. The anterior forebrain region developed as. two increasingly concave hemispheres from the 6 somite stage until their apposition at the 10 somite stage. In ,8-D-xyloside-cultured embryos, the forebrain neural''ectoderm remained broadly convex and became everted at later stages; the optic solci appeared very late. Histological sections (Figs. 3-7) of the cranial neural folds revealed that in control embryos, the conversion from biconvex shape to V-shaped profile was accompanied by an increase in thhk.ness of the neural epithelium, i.e. the cells increased in height and the epitheliui'm chu9ged from a columnar to a pseudostratified organization. As the cranial neural Plte'increased in thickness, its breadth concomitantly decreased (Figs. 3, 5). Thickening of the neural epithelium did not occur in embryos cultured in fl-D-xyloside for 15 hours, and the cranial neural folds remained broad (Fig. 4). By 24 hours there:was-som thickening (Fig. 6) but not as much as in control embryos (Fig. 5). Also, thbe cranial mesenchymal cells of /-D-xyloside-cultured embryos were more widely separated by extracellular matrix than were those of the controls. fI-D-xyloside: effects on neurulation 495

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Fig. 3. Control embryo, 15 hour culture, 6 pairs of somites: transverse section of cranial region at the level of the heart. The neural epithelium is thick except close to the mid-line, and the neural/surface ectodermal junction is distinct. a, first aortic arch; f, foregut; h, heart in peri- cardial cavity; n, . Fig. 4. 15 hour ,-D-xyloside-cultured embryo, 5 pairs of somites, cut at an equivalent level to the control. The neural epithelium is abnormally thin, and thejunction with the surface ectoderm is indistinct. The neural fold shape is more convex than that of the control. Fig. 5. Control embryo, 24 hour culture, 10 pairs ofsomites. The broad cranial neural fold region has a V-shaped profile, and the epithelium has thickened and narrowed. At the lateral edges, the epithelium has begun to curve towards the mid-line, and cells (nc) have begun to migrate away. The anterior forebrain region shows deep optic sulci (arrows) and the two sides are approaching each other in the mid-line. Fig. 6. 24 hour , D-xyloside-cultured embryo, 10 pairs of somites. The neural epithelium is relatively thin in the broad cranial neural fold region, and the neural fold shape is broad and convex. The anterior forebrain region is everted, and there are no optic sulci. Foregut shape differs from that of the control, and the notochord appears to be less well organized. The mesenchymal cells are more widely separated by extracellular matrix. 496 GILLIAN M. MORRISS-KAY AND BETH CRUTCH 7

J''r} 0 mm Fig. 7. 30 hour ,f-D-xyloside-cultured embryo, 9 pairs of somites (cf. 18 in co-cultured controls): transverse section of broad cranial neural fold region, heart level. The neural epithelium is now thicker, but still somewhat convex in profile. The lateral edges have curved towards the mid- line and neural crest cells (nc) are migrating. a, first aortic arch; f, foregut; n, notochord. During the later stages of cranial neural tube formation in control embryos (10-13 somite stages), neural crest cells could be seen emigrating from the lateral neural ectoderm region while the lateral edges themselves curved mediad and moved towards each other in the mid-line (Fig. 5). In ,8-D-xyloside-cultured embryos, neural crest migration and mediad curvature ofthe lateral neural epitheliurn occurred (some hours later than in controls) even though the major part of the cranial neural epithelium was still slightly convex in profile, and the lateral edges remained widely separated from each other (Fig. 7). Closure of the spinal neural tube began at the 7 somite stage in control embryos and progressed in a caudal direction throughout the period of culture, with the concomitant addition of new somites. In fl-D-xyloside-cultured embryos, the spinal neuroepithelial surface was abnormally convex during neurulation; the time of onset of neural tube closure was very variable between embryos, and in some the whole neural tube remained open. The process of somitogenesis was represented by the formation of paired segmental clumps of mesenchymal cells, which failed to undergo the normal conversion into epithelial structures. The notochord was less well organized than in control embryos. The cardiovascular system was also affected: the heart and major vessels were larger than those of controls, and the heart rate was slower. The yolk sac circulation developed later than in control embryos and was later in coalescing with the intra-embryonic circulation, which therefore lacked erythrocytes until relatively late stages. ,f-D-xyloside: effects on neurulation 497

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Fig. 10. Apical neuroepithelial surface region of a 6 somite control embryo. Desmosome-like junctions (j) are orientated in a plane perpendicular to the epithelial surface. Fig. 11. Similar region from a 6 somite fl-D-xyloside-cultured embryo. The cells are broader than in controls. fl-D-xyloside: effects on neurulation 499 Alcian blue histochemistry (Figs. 8, 9) The pattern of alcian blue staining at pH 1 was consistent in all control embryos regardless of the fixative used. The following areas were stained: neuroepithelial basement membrane, notochordal sheath (strongest staining), mesenchymal cell surfaces and extracellular matrix (especially in the region adjacent to the notochord and neural tube), foregut basement membrane, heart and blood vessels. The surface ectoderm basement membrane stained faintly except for the otic pit region which was strongly stained. Following testicular hyaluronidase incubation, very little staining could be detected except, at a greatly reduced intensity, in the neuroectodermal basement membrane and notochordal sheath; this faint staining is likely to be due to the presence of small amounts of heparan sulphate (Morriss & Solursh, 1978a). In f-D-xyloside-cultured embryos there was, overall, only a very feeble staining reaction. Only two embryos (one Bouin-, one Carnoy-fixed) showed slightly stronger staining in the neuroectodermal basement membrane than elsewhere. Perinoto- chordal staining was not detectable. No staining could be detected in sections pre- incubated with testicular hyaluronidase, i.e. heparan sulphate was not detectable histochemically. Ultrastructure of the neuroepithelium Ultrastructural abnormalities in fl-D-xyloside-cultured embryos were investigated only in the neuroepithelium. Embryos of 6 somite stage (15 hours) and 10 somite stage (21 hours) were selected for comparison. Apical region. In control 6 somite stage embryos, most of the apical desmosome- like junctions were orientated in a plane perpendicular to the epithelial surface (Fig. 10). The overall contour of the surface was fairly flat at this stage, but with many projecting microvilli. The neuroepithelial cells of 6 somite stage f6-D-xyloside- cultured embryos lacked the narrow neck region of the controls, so that each cell had a greater apical surface area (Fig. 11). In 10 somite stage control embryos, the neuroepithelial cells were more elongated than at earlier stages; their necks were narrower, and there was considerable over- lapping of the apical surfaces so that many of the desmosome-like junctions were orientated parallel to the epithelial surface. Microfilament bands associated with these junctions were prominent, and the most apical portions of all neuroepithelial cells (except those of the lateral edge region, from which neural crest cells were migrating) bulged above the line formed by the microfilaments and junctions (Fig. 12). In contrast, the apical neuroepithelial surface region of 10 somite stage /6-D- xyloside-cultured embryos remained broad and flat (Figs. 13, 14). There was, however, some overlapping of cells, with the consequent realignment of junctions parallel to the surface. Junction-associated microfilament bands were present, but they followed the contours of either the junctions themselves (Fig. 14) or the plasma membrane (Fig. 13), presenting an appearance of slackness. Basal region. The basal neuroepithelial surface of 6 somite and 10 somite stage control embryos was smooth and regular except close to the notochord and the lateral (neural crest) regions. ln f6-D-xyloside-cultured embryos, the basal neuro- epithelial surface was irregular, with many discontinuities ofthe basement membrane. There were herniations of parts of the adjacent cells through these discontinuities (Fig. 15), and some separation of cells (Fig. 16). 500 GILLIAN M. MORRISS-KAY AND BETH CRUTCH

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DISCUSSION The purpose of this study was to elucidate the roles of proteoglycans in morpho- genetic processes occurring during neurulation. The method used was to culture rat embryos during this stage of development in medium containing 1 mmp-nitrophenyl- fl-D-xyloside, a substance which primes the synthesis ofchondroitin sulphate, thereby inhibiting the synthesis of protein-linked chondroitin sulphate chains. Embryos cultured in this medium showed growth retardation, oedema, and abnormal morpho- genesis. Of these effects, abnormal morphogenesis will be discussed last and in the greatest detail. ,/-D-xyloside has previously been used to study the role of chondroitin sulphate- proteoglycan in the development of chick embryos (Gibson, Doller & Hoar, 1978). It was injected into the amniotic sac of 9 days old eggs (Hamburger & Hamilton stage 35, i.e. after most organogenesis is complete). The main effects were overall growth retardation and a general oedema; at this relatively late stage of develop- ment, the only indication of abnormal morphogenesis was the reduction in number and abnormal shape of the feathers. Kinoshita & Saiga (1979) used fl-D-xyloside to observe early stages in the development of sea urchin embryos. They observed abnormal morphogenesis in the early larva, and found that development was blocked by the reagent when it was present at a concentration sufficient to inhibit proteoglycan synthesis by about 50 %. We have not investigated the biochemistry of ,8-D-xyloside-treated rat embryos, but our alcian blue histochemical study indicates that the material which stains at pH 1 is drastically reduced. This material was shown in a previous study to consist largely of chondroitin sulphate, with some heparan sulphate (Morriss & Solursh, 1978 a). Heparan sulphate is not susceptible to the testicular hyaluronidase preparation used here, yet all alcian blue pH 1-stainable material was removed from sections of fi-D-xyloside-cultured embryos pre-incubated with testicular hyaluronidase; this observation suggests that f-D-xyloside reduces the level of heparan sulphate below that which is detectable histochemically. The mechanism of this effect is likely to be the same as for the reduction in chondroitin sulphate levels, since heparan sulphate is linked to its protein core by the same linkage sequence (Krecht, Cifonelli & Dorfman, 1967). The overall reduction in stainable material is unlikely to be due to a decrease in the rate of movement of low- carbohydrate proteoglycan out of the cells (as reported in f-D-xyloside-cultured chondroblasts by Schwartz, 1979), since there was no intracellular staining. The feebleness of the staining reaction may be due to a rapid loss of free (,/-D-xyloside- linked) glycosaminoglycans from their site of synthesis, or it may indicate that the free chains are undersulphated and short (Scott & Stockwell, 1967). Our observations of growth retardation, oedema, and abnormal morphogenesis may be due in part to some unknown direct effect of fl-D-xyloside. However, in the light of the known effects of fi-D-xyloside on proteoglycan synthesis, the above observations enable some deductions to be made on the roles ofchondroitin sulphate and heparan sulphate in normal morphogenesis. First, as a general point, the effects of fl-D-xyloside indicate that the normal morphogenetic activity of these glycos- aminoglycans in rat embryos during neurulation depends on their being present in the form of proteoglycan. This may be explained either as the whole, intact, macro- molecule being essential for the morphogenetic activity of part of it, or to the protein core (perhaps linked to hyaluronate) providing an anchorage site in the basement membrane and elsewhere for the morphogenetically active glycosaminoglycans, ,I-D-xyloside: effects on neurulation 503 which would otherwise be degraded or leached out of their normal sites. This finding is consistent with the results of Cohn, Banerjee & Bernfield (1977), which indicated that in the developing salivary gland the morphogenetically active glycosaminoglycan is organised as proteoglycan/hyaluronate aggregates within the basement membrane. The observed growth retardation in f8-D-xyloside-cultured embryos is likely to be related to a function of chondroitin sulphate-proteoglycan in the control of cell proliferation. Takeuchi (1968) found that chondroitin sulphate stimulates the growth of solid ascites tumours in mice when injected at the same time as the ascites cells; Ninomiya & Nagai (1979) found that during cell proliferation in cultures of em- bryonic chick tendon cells, chondroitin sulphate increased rapidly, whereas dermatan sulphate and heparan sulphate increased only gradually. In rat embryos undergoing neurulation, an effect on cell proliferation rates could contribute to abnormal morphogenesis as well as to growth retardation. This is under investigation. The alcian blue-stainable extracellular material forms a continuous meshwork within the basement membrane, mesenchymal extracellular matrix, and mesen- chymal cell surfaces (Morriss & Solursh, 1978 b). It is probably in the form of proteo- glycan in association with hyaluronate (Morriss & Solursh, 1978 a). The apparent oedema of the mesenchymal matrix observed in ,-D-xyloside-cultured embryos may be the result of an osmotic effect related to the abnormal biochemistry of this mesh- work of material. Hyaluronate levels are high in this region of the embryo (Solursh & Morriss, 1977), and a decrease in the availability of proteoglycan may lead to an increase in water binding by this molecule. However, it is also possible that greater than normal separation of the cranial mesenchymal cells was concomitant with failure of decrease in breadth/thickening of the adjacent cranial neural epithelium. Abnormal morphogenesis in f6-D-xyloside-cultured embryos was observed in several systems; only that of the neural folds will be considered in detail. As in normal embryos, the early cranial neural folds developed a biconvex shape; how- ever, conversion to a V-shaped profile with concomitant thickening of the neural epithelium was delayed and incomplete. The final stage of neural tube formation in this region normally involves the loss of neural crest cells from the most lateral regions of the neural epithelium, as the lateral edges of the two sides curve towards each other and meet in the mid-line (Morriss & New, 1979). In ,f-D-xyloside-cultured embryos, neural crest cells emigrated from the lateral regions even though the two sides were widely separated and the major part of the neural epithelium at that time was still slightly convex. These observations indicate that thickening of the neural epithelium (conversion from columnar to pseudostratified organization) and its conversion from convex to flat form is dependent on the presence of proteoglycan in the subjacent basement membrane, but that the lateral edge specializations occur independently of both proteoglycan and of the morphogenetic changes occurring in the rest of the epithelium. Independence of the lateral edge changes from artificial alterations in proteoglycan synthesis is not surprising in view of the earlier observa- tion that alcian blue-stainable material in the neuroepithelial basement membrane does not extend into the lateral region (Morriss & Solursh, 1978 a), and that apical microfilament bundles are absent from the cells (Morriss & New, 1979). Delayed and deficient conversion of the convex, columnar neural epithelium to a flat, pseudo- stratified form in /-D-xyloside-cultured embryos indicates that the presence of normal levels of proteoglycan is essential for these aspects of neural fold morpho- genesis. Since f-D-xyloside affected the histochemical detectability of all the sulphated 504 GILLIAN M. MORRISS-KAY AND BETH CRUTCH glycosaminoglycans which stain with alcian blue at pH 1, it is not possible to separate the roles of these components when interpreting the results. However, evidence from a murine cell system suggests that cell surface heparan sulphate may mediate cell adhesion (Rollins & Culp, 1979). Heifetz, Lennarz, Libbus & Hsu (1980) observed a considerable increase in heparan sulphate synthesis in cultured stage mouse embryos. These observations may be relevant to those presented here, in that con- version of the neural epithelium from columnar to pseudostratified form involves an increase in the cell surface contact area. Deficiency and retardation of this conversion in fi-D-xyloside-cultured embryos may be correlated with the observed absence of heparan sulphate-specific staining in these embryos. The ultrastructural observations showed that fl-D-xyloside-induced low levels of proteoglycan were associated with breaks in the neuroepithelial basement membrane. There were also effects in the apical region: microfilament bundles, which are a characteristic feature during neuroepithelial thickening and conversion from convex to flat/concave form (Morriss & New, 1979), were present in /?-D-xyloside-cultured embryos but were poorly organized, and positioned immediately below the plasma membrane instead of more deeply. The breadth of the apical region of the cells suggested that the microfilament bundles were not under tension. When comparing control with f8-D-xyloside-cultured embryos, and the major part of the neuroepithelium with the lateral (neural crest) region, there is a correlation between the presence of well organized sub-apical microfilament bundles and proteoglycan in the subjacent basement membrane. A similar correlation has been observed in the developing salivary gland (Bernfield, Banerjee & Cohn, 1972; Banerjee, Cohn & Bernfield, 1977). In that system, newly synthesised proteoglycans (mainly chondroitin sulphate) were found to accumulate in the basement membrane at incipient points of epithelial curvature, where basal microfilament bundles con- comitantly appeared in the overlying cells. The results therefore suggest that proteoglycans have a morphogenetic function in rat embryos during the period of cranial neurulation. Chondroitin sulphate- proteoglycan appears to have a role in relation to epithelial cell shape and cell proliferation, while heparan sulphate may be involved in cell adhesion.

SUMMARY Day 9 rat embryos were cultured during the period of cranial neurulation in medium containing 1 mM f-D-xyloside, a substance which inhibits proteoglycan synthesis while stimulating the synthesis of free chains of chondroitin sulphate. The purpose of this investigation was to elucidate the morphogenetic role of chondroitin sulphate, a component of the neuroepithelial basement membrane and other extra- cellular regions, and to discover whether it was present in the form of proteoglycan. The histochemical results indicated a great reduction in chondroitin/chondroitin sulphate and in heparan sulphate in the neuroepithelial basement membrane and elsewhere, in /1-D-xyloside-cultured embryos. Ultrastructural studies showed an effect on the structural integrity of the neuroepithelium, with breaks in the basement membrane and abnormal form of apical microfilament bundles. These observations were correlated morphogenetically with failure of the convex neural folds to be converted to flat/concave structures, and to change their epithelial organization from columnar to pseudostratified. Neural crest cell migration was slightly retarded but apparently normal. ,/-D-xyloside: effects on neurulation 505 The results are interpreted as indicating that the sulphated glycosaminoglycans, chondroitin/chondroitin sulphate and heparan sulphate, are present in the form of proteoglycan in the neuroepithelial basement membrane and elsewhere in the cranial region of day 9/day 10 rat embryos, and that they have a morphogenetic function during cranial neurulation. We wish to thank M. Barker for technical assistance, B. Archer and J. Lloyd for photographic assistance, and the Medical Research Council and the Royal Society for financial support.

REFERENCES BANERJEE, S. D., CoHN, R. H. & BERNFIELD, M. R. (1977). Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology. Journal of Cell Biology 73, 445-463. BERNFIELD, M. R., BANERJEE, S. D. & COHN, R. H. (1972). Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. Journal of Cell Biology 52, 674-689. COHN, R. H., BANERJEE, S. D. & BERNFIELD, M. R. (1977). Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials. Journal of Cell Biology 73, 464A-478. FUKUNAGA, Y., SOBUE, M., SUZUKI, N., KUSHIDA, H., SUZUKI, S. & SUZUKI, S. (1975). Synthesis of a fluorogenic mucopolysaccharide by chondrocytes in cell culture with 4 methyl-umbelliferyl-,8-D- xyloside. Biochimica et biophysica acta 381, 443-447. GALLIGANI, L., HOPWOOD, J., SCHWARTZ, N. B. & DORFMAN, A. (1975). Stimulation of synthesis of free chondroitin sulfate chains by f8-D-xylosides in cultured cells. Journal of Biological Chemistry 250, 5400-5406. GIBSON, K. D., DOLLER, H. J. & HOAR, R. M. (1978). fi-D-xylosides cause abnormalities of growth and development in chick embryos. Nature 273, 151-154. GIBSON, K. D., SEGEN, B. J. & AUDHYA, T. K. (1977). The effect of ,8-D-xylosides on chondroitin sulfate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors. Biochemical Journal 162, 217-233. GIBSON, K. D., SEGEN, B. J. & DOLLER, H. J. (1979). Changes in chemical composition of chick embryos treated with a ,8-xyloside and a lathyrogen. Teratology 19, 345-356. HEIFETZ, A., LENNARZ, W. J., LIBBUS, B. & Hsu, Y-C. (1980). Synthesis of glycoconjugates during the development of mouse embryos in vitro. 80, 398-408. KJNOSHITA, S. & SAIGA, H. (1979). The role of proteoglycan in the development of sea urchins. I. Abnor- mal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis. Experi- mental Cell Research 123, 229-236. KRECHT, J., CIFONELLI, J. A. & DORFMAN, A. (1967). Structural studies on heparitin sulfate of normal and Hurler tissues. Journal ofBiological Chemistry 242, 4652-4661. MORRIss-KAY, G. M. & CRUTCH, B. (1981). Culture of rat embryos with ,/-D-xyloside: evidence of a role for proteoglycans in cranial neurulation. Journal of Anatomy 132, 450. MORRISS, G. M. & NEW, D. A. T. (1979). Effect of oxygen concentration on morphogenesis of cranial neural folds and neural crest in cultured rat embryos. Journal of and Experimental Morphology 54, 17-35. MORRISS, G. M. & SOLURSH, M. (1978a). Regional differences in mesenchymal cell morphology and glycosaminoglycans in early neural-fold stage rat embryos. Journal of Embryology and Experimental Morphology 46, 37-52. MORRISS, G. M. & SOLURSH, M. (1978b). The role of primary mesenchyme in normal and abnormal morphogenesis of mammalian neural folds. Zoon 6, 33-38. NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (1976a). Improved development of head-fold rat embryos in culture resulting from low oxygen and modifications of the culture serum. Journal of Reproduction and Fertility 48, 219-222. NEW, D. A. T., COPPOLA, P. T. & CocKROFr, D. L. (1976b). Comparison of growth in vitro and in vivo of post-implantation rat embryos. Journal of Embryology and Experimental Morphology 36, 133-144. NINOMIYA, Y. & NAGAI, Y. (1979). Modulation of glycosaminoglycan synthesis during cell growth as observed in an embryonic chick tendon cell culture. Journal of Biochemistry 86, 111-119. RODtN, L. & SCHWARTZ, N. B. (1975). Biosynthesis of connective tissue proteoglycans. In Biochemistry of Carbohydrates (ed. W. J. Whelan), vol. 5, pp. 96-152. M.T.P. International Review of Science, Biochemistry Series One. London: Butterworth & Co. ROLLINS, B. J. & CULP, L. A. (1979). Glycosaminoglycans in the substrate adhesion sites of normal and virus-transformed murine cells. Bioclhemistry 18, 141-148. 506 GILLIAN M. MORRISS-KAY AND BETH CRUTCH SCHWARTZ, N. B. (1977). Regulation of chondroitin sulfate synthesis. Effect of 8-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains and core protein. Journal ofBiological Chemistry 252, 6316-6321. SCHWARTZ, N. B. (1979). Synthesis and secretion of an altered chondroitin sulfate proteoglycan. Journal ofBiological Chemistry 254, 2271-2277. SCHWARTZ, N. B., Ho, P-L. & DORFMAN, A. (1976). Effect of 8-xylosides on synthesis of cartilage- specific proteoglycan in chondrocyte cultures. Biochemical and Biophysical Research Communications 71, 851-856. Scorr, J. E. & STOCKWELL, R. A. (1967). On the use and abuse of the critical electrolyte concentration approach to the localization of tissue polyanions. Journal of Histochemistry and Cytochemistry 15, 111-113. SOLURSH, M. & MORRIss, G. M. (1977). Glycosaminoglycan synthesis in rat embryos during the forma- tion of the primary mesenchyme and neural folds. Developmental Biology 57, 75-86. STEELE, C. E. & NEW, D. A. T. (1974). Serum variants causing the formation of double hearts and other abnormalities in explanted rat embryos. Journal of Embryology and Experimental Morphology 31, 707-719. TAKEUCHI, J. (1968). Effect of chondroitin sulfate on the growth of solid Ehrlich ascites tumor under the influences of other intestinal components. Cancer Research 28, 1520-1523.