SEM observations of surface alterations associated with neural tube closure in the mouse and hamster.код для вставкиСкачать
BRIEF COMMUNICATION SEM Observations of Surface Alterations Associated with Neural Tube Closure in the Mouse and Hamster ROBERT EARLE WATERMAN Department of A n a t o m y , T h e University of N e w Mexico School of Medicine, Albuquerque, N e w Mexico 871 31 Vertebrate neural tube formation and differentiation have been extensively examined. Particular interest has been paid to : ( 1) the induction of a thickened neural plate by subjacent chorda-mesoderm, (2) factors responsible for the appearance of the neural folds and neural grove, ( 3 ) the migration of neural crest cells from the neural folds and, ( 4 ) patterns of mitotic activity and cellular differentiation within the walls of the neural tube leading to the complex functional morphology of the central nervous system. Relatively little attention has been given to the details of fusion between the neural folds resulting in formation of a closed neural tube (MarinPadilla, ’ 7 0 ) . Small membrane “ruffles” associated with cells of the neural folds during neural tube closure in the chick were recently observed with the scanning electron microscope (SEM) (Revel, ’74). It was suggested that these structures may play a role in forming new adhesions between the surfaces of apposed neural folds. The present study reports the presence of similar surface alterations during fusion of the neural folds in embryonic mice and golden hamsters. MATERIALS AND METHODS Timed pregnant CD-1 mice and golden hamsters were sacrificed by cervical dislocation during day 8 of gestation. The uterine horns were removed and placed in physiological saline. Embryos were rapidly dissected free of uterine and extraembryonic tissue and immersed in a 0.2 M phosphate-buffered 2% paraformaldehyde, 2% glutaraldehyde solution containing 0.01% trinitro phenol (pH 7.3-7.4) (It0 ANAT. REC., 183: 95-98. and Karnovsky, ’68) for periods from three hours to three days. Specimens were then dehydrated in ethanol, dried by the critical point method using liquid CO,, coated with gold-palladium in a vacuum evaporator and viewed with an ETEC autoscan scanning electron microscope operated at 1020 kV. Thirteen mouse and 10 hamster embryos were observed. RESULTS All embryos exhibited partially fused neural tubes (fig. 1). A narrow band of altered surface appearance was observed for a variable distance along the neural folds in both species. This band is located between the larger flattened cells of the body surface and the smaller apical profiles of the cells of the presumptive neural tube. It occurs both anterior and posterior to the fused portion of the neural tube, but is most prominent immediately anterior to the point of fusion. In the mouse (fig. 2 ) , the altered band anterior to the area of fusion is characterized by overlapping attenuated cells and numerous lamellopodial membrane extensions or “ruffles.” The same relative area in the hamster, however, exhibits more long cytoplasmic extensions and filipodia in addition to flattened cell surfaces (fig. 3 ) . Presumably lysing cells and rounded cell “blebs” associated with the altered zone are also more numerous in the hamster than in the mouse, Rounded cellular profiles are likewise more characteristic of the hamster (fig. 4), and membrane “ruffles” more prevalant in the mouse (fig. 5 ) , posterior to the area of fusion. Received May 6, ’75. Accepted May 13, ’75. 95 96 ROBERT EARLE WATERMAN Figures 1-3 SEM OF NEURAL TUBE CLOSURE 97 Fig. 4 Neural folds of hamster embryo posterior to the fused neural tube. Small blebs, possible cell lysis and some flattened cells ( * > are seen between the surface cells and cells of the presumptive neural tube. SEM. X 630. Fig. 5 Small membrane “ruffles” associated with the posterior point of fusion between the neural folds in a mouse embryo. SEM. X 2,650. DISCUSSION The results of this study reveal the presence of a localized zone of altered surface appearance along the presumptive area of fusion between opposed neural folds prior Fig. 1 Dorsal view of an 8-day old hamster embryo. The neural folds have fused along the mid portion of the body and are open anteriorly and posteriorly. This stage represents the typical appearance of both hamster and mouse embryos examined. SEM. x 31. Fig. 2 Portion of the neural folds anterior to the area of fusion in an 8.25 day old mouse embryo similar to that illustrated in figure 1. A narrow band of surface alteration can be seen between cells of the body surface and presumptive neural tube. SEM. x 510. Inset: Area of right neural fold enclosed by rectangle in figure 2 is seen at higher magnification. Zone of surface alteration is present between the neural plate and presumptive surface cells. SEM. x 1,270. Fig. 3 Stero pair showing slender filipodiumlike cellular extensions and some “blebs” along the neural folds immediately anterior to the point of fusion in a hamster embryo. (To be viewed with stero glasses). SEM. x 2,710. to their contact and adhesion. Examination of the surface appearance of the neural folds from neural plate to closed neural tube stages is currently in progress. Preliminary results indicate there is a progressive increase i n the degree of alteration along the neural fold which is correlated with the fusion process (Waterman, ’75). One feature of this alteration appears to be changes in the membrane properties of the cells which are expressed as increased numbers of membranous extensions. I n this respect, the “ruffles” observed in the mouse are more similar to those observed during neurulation in the chick (Revel, ’74) than the filipodium-like processes observed in the hamster. The demonstration of similar localized membrane activity in the chick, mouse and hamster suggests this may represent a common feature of neural tube closure. The fact that the zone of alteration is restricted to the area of the neural fold involved in fusion strongly suggests that 98 ROBERT EARLE WATERMAN observed surface changes may be func- is gratefully acknowledged. Supported by tionally involved in the adhesion process. N.I.D.R. Grant DE 03897-01. Similar localized epithelial alterations have LITERATURE CITED also been observed along the medial edge of palatal shelves prior to fusion of the Ito, S., and M. .T. Karnovsky 1968 Formaldehyde-glutaraldehyde fixatives containing trinisecondary palate in mouse (Waterman et tro compounds. J. Cell Biol., 39: 168a-169a. al., '73) and human embryos (Waterman Marin-Padilla, M. 1970 The closure of the and Meller, '74). Because the membrane neural tube in the golden hamster. Teratology, ruffles and extensions seen along the neu3: 39-46. ral fold appear similar to those commonly Nelsen, 0. E. 1953 Comparative Embryology of exhibited by many migratory cells in vitro, the Vertebrates. McGraw-Hill, New York. it has been proposed that they may aid in Revel, J. P. 1974 Scanning electron microscope establishing initial contacts between the studies of cell surface morphology and labelling, in situ and in vitro. IITRI, SEM, 1974, contralateral folds (Revel, '74). It is also pp. 542-548. noteworthy that the zone of alteration corresponds in large part to the area between Waterman, R. E. 1975 Scanning electron microscopic observations of neural tube closure in presumptive surface cells and neural plate the embryonic mouse and hamster. Anat. Rec., from which the neural crest cells arise 181: 506 (abstract). (Marin-Padilla, '70; Nelsen, '53). Whether Waterman, R. E., L. M. Ross and S. M. Meller the observed surface features may be pre1973 Alterations in the epithelial surface of requisite for adhesion between the neural A/Jax mouse palatal shelves prior to and during palatal fusion: A scanning electron microfolds, and the possibility that they are scopic study. Anat. Rec., 176: 361-376. directly related to neural crest formation Waterman, R. E., and S. M. Meller 1974 Alterneed to be more fully explored. ACKNOWLEDGMENTS The technical expertise of Susan Palmer ations in the epithelial surface of human palatal shelves prior to and during fusion: A scanning electron microscopic study. Anat. Rec., 180: 111-136.