/. Embryol. exp. Morph. Vol. 28, 2, pp. 437-448, 1972 437 Printed in Great Britain Surface modifications of neural epithelial cells during formation of the neural tube in the rat embryo By BRUCE G. FREEMAN1 From the Department of Anatomy, The University of Tennessee Medical Units, Memphis, Tennessee SUMMARY The apical (juxtaluminal) ends of the neural epithelial cells of rat embryos were examined using light and electron microscopy during varying stages of neural tube formation. At the neural-plate stage the apical surfaces exhibit numerous microvilli. At the presomite neurula stage the microvilli are longer and more irregular. Filaments of approximately 40-60 A diameter appear in the apical cytoplasm. By the neural-groove stage, cytoplasmic protrusions containing various organelles have begun to appear. Apical filaments are present. At the beginning of closure the apical surfaces are characterized by large, irregular protrusions that are still associated with apical filaments. Finally, at the time of neural closure, the apical protrusions as well as the apical filaments have disappeared and the apical surfaces of the neural epithelial cells are relatively smooth. These observations bear out the proposal that contraction of the apical filaments is responsible for the folding of the neural plate and the production of apical protrusions. INTRODUCTION It is generally agreed that certain congenital abnormalities of the central nervous system (exencephaly, anencephaly, myeloschisis) are due to a failure of normal formation of the neural tube. In spite of the fact that a large number of chemicals and drugs have been used to cause abnormal neural development (Kalter, 1968), there is little or no consensus on the factors responsible for normal neurulation in many species. Since the process of neurulation is funda- mental to the development of the central nervous system, it would be highly desirable to have as much information about it as possible. The following investigation was undertaken to study, at the fine structural level, the normal morphology of the embryonic rat neural epithelial cells during neurulation in order to gain some insight as to the mechanism of neural tube formation under normal conditions. Structural modifications of the apical ends of cells undergoing neurulation or 1 Author's address: Department of Anatomy, Case Western Reserve University School of Medicine, 2119 Abington Road, Cleveland, Ohio 44016, U.S.A. 438 B. G. FREEMAN neurulation-like movements have been described by a number of authors. Balinsky (1961) was among the first to report protrusions from the apical ends of neural epithelial cells during neurulation in frog embryos. Since then, Baker & Schroeder (1967) and Schroeder (1970) noted' apical protrusions' in neurulat- ing amphibian cells, Wrenn & Wessells (1969) noted 'finger-like projections' in invaginating mouse lens, and Pearce & Zwaan (1970) noted 'apical protusions' in invaginating chick lens. However, to this date, the changes seen in the apical (juxtaluminal) surfaces of the neural epithelial cells of the rat during neurulation have not been described. This study will deal with the observed changes in the apical ends of the neural epithelial cells of the rat during formation of the neural tube. The possible significance of these changes in the mechanisms of closure will be discussed. MATERIALS AND METHODS Sprague-Dawley rats were obtained from Zivic-Miller Laboratories, Allison Park, Pa., at varying days of pregnancy. Both uterine horns were removed under ether anesthesia and transferred to Tyrode's solution. Embryos were removed under Tyrode's and staged according to Witschi (1956). The embryos were then fixed in toto in 4 % glutaraldehyde or in 2 % OsO4, both buffered to pH 7-5 with 0-2 M cacodylate. After 2-4 h of fixation the embryos fixed in glutaraldehyde were washed for an equivalent amount of time in buffer and postosmicated in 2 % osmium tetroxide buffered to pH 7-5 with 0-2 M cacodylate. The embryos were then dehydrated in an ascending series of concentrations of methanol, passed through propylene oxide, and embedded in Epon 812. Thick FIGURE 1 Fig. 1. Light micrographs of transverse sections of embryos at Witschi stages 12,13, 14, 15, and 16. All embryos were embedded in Epon and sectioned in the transverse plane at levels approximating one-half of the length of the embryo in stages 12 and 13 and approximately mid- to high-thoracic in stages 14,15, and 16. Sections for electron microscopy were taken from the same levels. (A) Stage 12 (primitive streak), day 9 of gestation. PRO = proamniotic cavity; PNE = primitive neural epithelium; END = endoderm; glutaraldehyde-osmium fixation, x 120. (B) Stage 13 (presomite neurula), day 9-5 of gestation. NG = neural groove; NEP = neural epithelium; arrows = mitotic figures; osmium fixation, x 225. (C) Stage 14 (1-4 somites), day 10 of gestation. NG = neural groove; NEP = neural epithelium; AP = apical protrusions; arrows = mitotic figures; glutaralde- hyde-osmium fixation, x 225. (D) Stage 15 (5-12 somites), day 10-5 of gestation. NG = neural groove; NEP = neural epithelium; AP = apical protrusions; arrows = mitotic figures, glutaralde- hyde-osmium fixation, x 225. (E) Stage 16 (13-20 somites), day 11 of gestation. NEP = neural epithelium; osmium fixation, x 200. Mammalian neurulation 439 440 B. G. FREEMAN Fig. 2. Higher magnification view of a transverse section through the apical ends of primitive neural ectoderm cells at stage 12. L = lumen; MV = microvilli; P = plasma membrane vesicles; M = mitochondria; JC = junctional complex; glutar- aldehyde-osmium fixation, x 20000. and thin transverse sections from approximately half-way through the neurula, neural plate, neural groove and high thoracic levels in older embryos were cut on a Sorval MT1 ultramicrotome fitted with a diamond knife. Thick (0-5-1 /an) sections of whole embryos were made and stained with Mallory azure II- methylene blue for purposes of orientation. Thin sections were floated on distilled water, picked up on 150-mesh carbon- coated grids and contrasted with uranyl acetate and lead citrate. Specimens were examined in an RCA EMU 3F electron microscope equipped with a heated objective aperture or an Hitachi HU 11A electron microscope, both operated at 50 kV. Micrographs were made on prepumped Cronar, Ortholitho, Type A sheet film at original magnifications of 5000-20000 and photographically enlarged up to 4 times. Mammalian neurulation 441 Fig. 3. (A) View of apical ends of neural epithelial cells in transverse section at stage 13. L = lumen; MV — microvilli; P = plasma membrane vesicles; M = mito- chondria; JC = junctional complex; W = mitochondrial whorl; glutaraldehyde- osmium fixation, x 200000. (B) Higher magnification view of apical ends of neural epithelial cells in transverse section at stage 13. L = lumen; JC = junctional com- plex; F = apical filaments; osmium fixation, x 40000. 442 B. G. FREEMAN Fig. 4. Transverse section through apical ends of neural epithelial cells at stage 14. L = lumen; MV = microvilli; M = mitochondria; JC = junctional complex; AP = apical protrusion; F = apical filaments; glutaraldehyde-osmium fixation, x 20000. In all, 17 dams were used to provide a minimum of three dams for each stage of development. A minimum of three embryos were examined from each dam for this investigation. RESULTS The changes in the appearance of the neural epithehum during formation of the neural tube are evident in light micrographs taken from midneural-plate sections in younger embryos to approximately midthoracic levels in older embryos (Fig. 1A-E). At stage 12 the cells are arranged in a low pseudostratifled columnar epithelium (Fig. 1 A). They contain numerous free ribosomes as well as ribosomal aggre- Mammalian neurulation 443 Fig. 5. Transverse section through apical ends of neural epithelial cells at stage 15. AP = apical protrusion; L = lumen; M = mitochondria; JC = junctional com- plex; F = apical filaments; glutaraldehyde-osmium fixation, x 20000. gates. Mitochondria are numerous. The apical surface is seen to be quite irregular and to exhibit numerous microvilli. Some profiles of shed plasma membranes can be seen in the lumen (Fig. 2). By stage 13 the neural groove has already formed and the neural epithelial cells have become somewhat taller (Fig. 1B). The apical surfaces of the neural epithelial cells exhibit numerous microvilli, some of which contain filaments that continue into the cytoplasm (Fig. 3 A). Junctional complexes are present and well developed. The cytoplasm contains large numbers of polysomes but relatively few profiles of granular endoplasmic reticulum. Mitochondria are numerous and a few contain 'membranous whorls' resembling those described by Jurand & Yamada (1967) in degenerating mitochondria. 20 EMB 28 444 B. G. FREEMAN Fig. 6. Transverse section through apical ends of neural epithelial cells at stage 16. L = lumen; JC = junctional complex; EX = small extrusion; C = centriole; CI = developing cilium; glutaraldehyde-osmium fixation, x 20000. Beginning at stage 13, a system of filaments, approximately 40-60 A in diameter, appears in the apical cytoplasm (Fig. 3A, B). These filaments are usually seen to be associated with the junctional complexes of the neural epi- thelial cells. At stage 14 the neural groove has deepened and the neural folds have begun to approximate each other (Fig. 1C). The apical ends of the neural epithelial cells have undergone observable changes. These include a decrease in the number of microvilli and the appearance of protrusions of the apical cytoplasm into the presumptive lumen (Figs. 1C, 4). These protrusions appear as small buds containing cytoplasmic matrix and ribosomes or as large 'blebs' containing cytoplasmic matrix, ribosomes and mitochondria (Fig. 4). The cytoplasmic Mammalian neurulation 445 protrusions are usually associated with rather complex arrangements of junc- tional complexes and apical filaments (Fig. 4). By stage 15 the neural tube has usually closed in low cervical and high thoracic levels, although Fig. ID shows a neural tube that is slightly open. At this stage the apical surfaces of the neural epithelial cells are almost completely devoid of microvilli.