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 neurulation 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 neural fold 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 neural plate 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 -5mm~~~~~~~~ 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 ectoderm (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 neural tube 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/surface ectoderm 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.