fine structure of the lumbosacral neural folds in the ... · lumbosacral region reveal a...

/ . Embryo!, exp. Morph. Vol. 55, pp. 279-290, 1980 2 7 9Printed in Great Britain © Company of Biologists Limited 1980

Fine structure of the lumbosacral neural foldsin the mouse embryo

By DORIS B.WILSON1 AND LAUREL A. FINTA1

From the Division of Anatomy, Department of Surgery,University of California

SUMMARY

The neural folds in the lumbosacral region of the normal 8-day and 9-day mouse embryowere studied by means of transmission electron microscopy with and without lanthanumtreatment. The cells showed an abundance of ribosomes, microtubules arranged parallelto the long axes of the cells, and microfilaments extending across the apices. At the luminalborder junctional complexes were common, and an occasional midbody was seen stretchingbetween adjacent cells nearing the end of telophase. In the 8-day embryos, gap junctionalvesicles (annular nexuses) bounded by layered membranes and containing cytoplasm withribosome-like material were commonly observed; at 9 days the vesicles were relativelyrare. The lanthanum-treated material demonstrated that the tracer was able to pass throughthe subluminal junctional complexes and throughout the intercellular spaces. However, thespace between the membranes of the gap junctional vesicles lacked lanthanum and thusapparently did not communicate with the intercellular space.

INTRODUCTION

The formation of the neural tube has been the subject of numerous trans-mission electron microscopic (TEM) studies on normal embryos of the amphibian(Schroeder, 1970; Burnside, 1971, 1973; Karfunkel, 1974; Decker & Friend,1974; Mak, 1978) and chick (Bellairs, 1959; Fujita & Fujita, 1963; Handel &Roth, 1971; Karfunkel, 1972; Revel, 1974; Bancroft & Bellairs, 1975; Revel& Brown, 1976; Camatini & Ranzi, 1976). In contrast, comparable TEMstudies on early rodent embryos are relatively sparse (Hinds & Ruffett, 1971;Freeman, 1972; Sadler, 1978). In the mammal, the early stages of neural tubeclosure, particularly the formation and elevation of the neural folds, are ofspecial importance in the lumbosacral region, since this is a site frequentlyaffected by non-closure malformations (dysraphism) (Auerbach, 1954; Wilson,1974; Lemire, Loeser, Leech & Ellsworth, 1975). In view of the paucity ofTEM information on the lumbosacral neural folds in normal mammalianembryos, the current study was undertaken on normal 8-day and 9-day miceso as to establish a basis for future fine structural analyses of this region inabnormal mouse embryos at comparable stages of development.

1 Authors'1 address: Division of Anatomy, M-004, Department of Surgery, School ofMedicine, University of California, San Diego, La Jolla, California 92093, U.SA.

280 D. B. WILSON AND L. A. FINTA

MATERIALS AND METHODS

C57BL/6J mice were bred, females were checked daily for vaginal plugsand killed by cervical dislocation early on the eighth or ninth day post-plug(day of plug = day 0), and the embryos were removed in saline. In addition,8- and 9-day embryos were obtained from matings of C57BL/6J and normal( + / + ) individuals of the splotch (Sp) mutant mouse maintained on a C57BL/6Jbackground. Six embryos corresponding to developmental stage 12 (Theiler,1972) were selected from three litters, and four embryos at developmentalstage 14 were selected from three litters. The lumbosacral region of eachembryo was fixed for 1 h in cold (4 °C) half-strength Karnovsky's solution(Karnovsky, 1965), rinsed in 0-1 M cacodylate buffer (pH 7-2) and postfixedin cold 1 % osmium tetroxide-Ol M cacodylate buffer for 1 h. The specimenswere then dehydrated in graded ethanols and propylene oxide and flat-embeddedin Epon-Araldite. Thick sections for orientation with light microscopy werestained with methylene blue-azure II. Thin sections were placed on naked200 mesh copper grids and stained with uranyl acetate (Watson, 1958) for20 min and lead citrate (Reynolds, 1963) for 10 min.

For the lanthanum studies, the technique of Revel & Karnovsky (1967) wasused. A 4 % solution of lanthanum nitrate was brought to pH 7-8 by meansof vigorous stirring with 0-02 N-NaOH and was added to the above formalde-hyde-glutaraldehyde mixture to give a final concentration of 1 % lanthanum.The colloidal lanthanum was not used in the buffered rinses, in the osmiumtetroxide, or during dehydration.

Observations were made with a Zeiss 9S-2 electron microscope at directmagnifications up to x 28000.

FIGURES 1-6

Fig. 1. Cross section of lumbosacral region of neural groove at 8 days' gestation.L, presumptive lumen, x 180.Fig. 2. Higher magnification of neuroepithelium at 8 days' gestation. Arrow indicatesinternal cellular process, x 600.Fig. 3. Cilium in apical portion of neuroepithelial cell in lateral region of lumbo-sacral neural groove at 8 days' gestation, x 28 500.Fig. 4. Microvillous projection (small arrow) at margin of neuroepithelial cellat 8 days' gestation. Large arrow, junctional complex, x 28 500.Fig. 5. Lanthanum in intercellular space (small arrows) between two neuro-epithelial cells at 8 days' gestation. Large arrow, possible gap junction, x 84000.Fig. 6. Midbody at the end of a mitotic division. Small arrows, microtubules. L,presumptive lumen. Large arrow, dense band, x 28 500.

Neural folds in mouse embryos 281


RESULTS

No differences were noted at the light microscopic or electron microscopiclevel between offspring of C57BL x C57BL and those of C57BL x normal (4- / +)individuals of Sp/ + parentage. Hence the following description applies toall embryos analyzed in the current study.

Light microscopy

Epon-Araldite thick sections of both the upper and lower lumbosacralregions of 8-day and 9-day embryos were studied. At 8 days the sectionsdemonstrate a neural groove that is relatively wide and V-shaped (Fig. 1). Theneurectodermal cells of the groove are tall and columnar. At the lateral edgesof the groove the cells change to cuboidal. Mitotic figures are scattered alongthe presumptive luminal border, and deeper lying nonmitotic cells maintaincontact with the lumen by means of a slender internal cellular process (Fig. 2).In sections taken from the upper lumbosacral region the cell surfaces tend tobulge slightly upwards into the lumen, whereas sections removed from thelower lumbosacral region show more flattened luminal cell surfaces. In bothregions of the groove the cells appear to be separated from one another sub-luminally by extracellular spaces of varying size, except for points of contactby means of small lateral projections. These contact points and extracellularspaces may be exaggerated by shrinkage due to tissue preparation.

In the upper lumbar region of 9-day embryos the neural folds have fused toform a neural tube. Lower lumbar and sacral regions show varying degrees ofclosure. In open regions the neural groove is horseshoe-shaped and the medialaspects of the folds are concave.

Electron microscopy

8 days. At the electron microscopic level low magnifications of the 8-daylumbosacral region reveal a neuroepithelium with nuclei at varying levels andmitotic nuclei interspersed between the columnar cells at the presumptiveluminal border. Short microvilli are scattered over the luminal surface, andcentrally located, single apical cilia are occasionally present (Fig. 3). Junctionalcomplexes occur between adjacent cells at the luminal border, and microfilamentscan sometimes be seen extending outward from the junctions across the apicesof the cells. In most cases a long microvillous projection also extends into thelumen at the cell margin adjacent to the junctional complex (Fig. 4).

The intercellular junctional complexes are permeable to lanthanum, asevidenced by the presence of the tracer throughout the intercellular spaces. Insome instances the lanthanum-filled space is narrowed and suggestive of a gapjunction (Fig. 5).

Midbodies are common at the luminal surface and consist of a narrowcytoplasmic bridge between two cells in the process of completing a mitotic


Fig. 7. Internal cellular process of neuroepithelial cell at 8 days' gestation. Notenumerous microtubules (small arrows). Large arrow, gap junctional vesicle,x 28 500.Fig. 8. Gap junctional vesicle (arrow) in mitotic cell at 8 days' gestation, x 28500.Fig. 9. Gap junctional vesicle in lanthanum-treated embryo at 8 days' gestation.Note presence of lanthanum in adjacent intercellular spaces but not betweenmembranes of the vesicle, x 38000.

division. Within the cytoplasmic bridge are parallel stacks of microtubuleswith a csntrally located dense band (Fig. 6). A few cells also show largeirregular blebs on the luminal surface.

The neuroepithelial cells of the upper and lower lumbosacral neural groovecontain a variety of cytoplasmic organelles including free ribosomes, poly-ribosomes, rough endoplasmic reticulum (RER), and small, dense mitochondria.In some cells the rough endoplasmic reticulum exhibits circular or whorledzones. Pinocytotic invaginations are frequently found at the luminal surfaceof the internal processes of the nonmitotic cells. These processes also shownumerous microtubules running parallel to the long axes of the cells (Fig. 7).

A notable feature of both upper and lower lumbosacral neural groove is thepresence of gap junctional vesicles (annular gap junctions or annular nexuses)(Figs. 7-9). The vesicles are bounded by a dense, layered membrane and


contain ribosome-like material. Most vesicles have an electron-lucent zonelocated within the vesicle subjacent to its membrane.

The gap junctional vesicles are most frequently found near the luminalsurface in the internal cellular processes of nonmitotic cells, but they have alsobeen observed at deeper levels in the cellular processes, near the basal laminaof the neuroepithelium, and in mitotic cells. The occurrence of the vesicles issimilar in both upper and lower lumbosacral regions, and each section ofneural groove examined has at least one gap junctional vesicle. Some sectionshave as many as seven individual vesicles, with an occasional cell having twovesicles. Examination of adjacent sections confirms that the vesicles are distinctand separate from one another and not sections of the same vesicle. Thelanthanum preparations show that the space between the membranes of thevesicles lacks lanthanum and that the vesicles are not in communication withthe intercellular space (Fig. 9).

9 days. At 9 days of gestation the mitotic nuclei are situated at the luminalborder and are interspersed among the internal cellular processes extendingfrom the deeper nonmitotic cells (Fig. 10). The luminal borders of many ofthe ventricular cells and of the internal cellular processes bulge prominently.At higher magnifications, thick bundles of microfilaments are commonly seenspanning the apices of these cells (Fig. 11). Midbodies are also common. Cilia,cytoplasmic blebs and microvilli project into the lumen and long microvillousprojections at the cell margins adjacent to junctional complexes are similar insize and location to those noted in the 8-day embryos. The appearance anddistribution of intracellular organelles are also similar to those observed at8 days.

In regions where the neural folds have begun to fuse dorsally, the cells atthe apices of the folds approach one another and become apposed. Longundulating cytoplasmic processes extend between the dorsal aspects of theapposing cells, forming an interdigitating tangled web (Fig. 12).

In the 9-day lanthanum preparations, the tracer penetrated the junctionalcomplexes at the luminal borders of the neuroepithelial cells and was presentin the intercellular spaces. However, in rare instances the lanthanum failed topenetrate into the intercellular space subjacent to an apical junction.

FIGURES 10-12

Fig. 10. Luminal aspect of lumbosacral groove at 9 days' gestation. The surfacesshow prominent bulges and projections into the lumen. M, mitotic cell. Arrowindicates a rare gap junctional vesicle, x 3900.Fig. 11. Higher magnification of apex of a ventricular cell at 9 days' gestationshowing apical bulging into the lumen (L). Arrows indicate bundles of micro-filaments, x 24000.Fig. 12. Low magnification of dorsal neural folds in the process of fusing witheach other at 9 days' gestation. L, Lumen, x 5400.


EMB 55


With respect to gap junctional vesicles, these structures are relatively rareat 9 days (Fig. 10). In a total of 20 sections, only four vesicles could be found.The structure of these did not show any apparent differences from that in the8-day embryos, and their membranes did not contain lanthanum. The locationsof the vesicles were also similar to those seen at 8 days, i.e. near the lumen,within an internal cellular process, and in a mitotic cell.

DISCUSSION

The C57BL mouse was chosen for this study primarily because it has beencommonly used as a normal base line in neurological investigations (Sidman,Angevine & Pierce, 1971). Moreover, it also represents a genetic backgroundupon which several neurological mutants of the mouse occur (Sidman, Green& Appel, 1965).

The neuroepithelial cells in the lumbosacral folds in the normal 8-day and9-day mouse embryos used in the current study show features similar to thosedescribed in various other regions of the chick and rodent neural folds duringearly development (Freeman, 1972; Karfunkel, 1972; Bancroft & Bellairs,1975; Camatini & Ranzi, 1976; Revel & Brown, 1976). These features includemicrotubules arranged parallel to the long axes of the cells, especially in theinternal cellular processes, and an abundance of free ribosomes and poly-ribosomes. Apical cilia are also present but are not as frequent or well developedas during later stages of neural development (Sotelo & Trujillo-Cenoz, 1958;Bancroft & Bellairs, 1975; Wilson, 1978).

The bundles of microfilaments extending across the apical regions of thecells are not as prominent or densely arranged in the 8-day mouse as at 9 dayswhen the medial aspects of the neural folds become concave. The arrangementof these microfilaments is similar to that seen in the amphibian and chick, andtheir role has been postulated as producing the apical constriction necessaryfor changes in the shape of the cells during neurulation (Karfunkel, 1972, 1974;Burnside, 1973).

The observation that the luminal surfaces of the 8-day neuroepithelial cellsbulge somewhat in the upper lumbosacral region, whereas those in the lowerregion tend to be more flat, most likely reflects the fact that the upper regionis slightly more advanced than the lower region at any given stage of develop-ment. This bulging, as well as the formation of apical blebs, becomes moreprominent as the folds elevate and begin to approach one another at 9 days;similar protrusions were observed during elevation of rat neural folds (Freeman,1972).

The luminal surfaces of the neuroepithelial cells at 8 and 9 days alsoexhibit an occasional cytoplasmic bridge between two cells nearing the end oftelophase. Once the cells separate completely the cytoplasmic bridge is pinchedoff and portions remain as debris at the lumen. These bridges have been

Neural folds in mouse embryos 287termed midbodies (Allenspach & Roth, 1967), although the term has also beenused more restrictively to designate remnants of the mitotic spindle or thedense band in the center of the bundles of spindle microtubules (Buck, 1963;Krystal, Rattner & Hamkalo, 1978). The presence of midbodies along theluminal aspects of the 8- and 9-day neuroepithelium reflects the mitotic activityof these cells, and these structures are particularly common after closure ofthe neural tube (Allenspach & Roth, 1967; Bancroft & Bellairs, 1975;Wilson, 1978).

An impressive array of long finger-like interdigitations was observed at 9 daysin those regions where the neural folds had approximated one another andwere beginning to fuse. These cytoplasmic processes appear to be similar tothose observed by means of scanning electron microscopy in the mouse andhamster (Waterman, 1976) and in the chick (Revel & Brown, 1976). Trans-mission electron microscopy in the chick (Bancroft & Bellairs, 1975) and inthe amphibian (Moran & Rice, 1975) has also demonstrated these structures,and it is possible that they provide a means of initial contact and/or maintenanceof fusion of the folds.

In the 8- and 9-day unfused neural folds, the apical regions of the neuro-epithelial cells are bound to one another by means of junctional complexes.However, there is a relatively large amount of extracellular space subapicallyin the 8-day mouse neural tube, and this is similar to that observed in thechick at a comparable stage of development (Bancroft & Bellairs, 1975). Whilethis may be an artifact produced by the aldehyde fixative, there is some evidencethat the extracellular space may well be extensive in immature neural tissue(Sumi, 1969; Hinds & Ruffett, 1971), and this may allow for the relativelyrapid changes in cell shape and movement which occur during the earlystages.

Although the exact nature of the junctional complexes could not be confirmedin the current study without special techniques such as freeze-fracture, thejunctions at the luminal surface appear to be gap junctional in nature, sincelanthanum passed freely through the intercellular spaces to deeper levels. Inthe amphibian, Decker & Friend (1974) noted that gap junctions becomewidely distributed in the neural folds during closure. Likewise in the chick,Revel & Brown (1976) describe small gap junctions or gap junction-likestructures in the gutter stage of the neural groove. An occasional juxtaluminalzonula occludens was observed in more mature regions of the developingneural tube in the chick (Revel & Brown, 1976); this agrees with our observationof an occasional junction which was not permeable to lanthanum in our 9-daymouse material. Although gap junctions have been cited as a means of cell tocell communication and are particularly common during embryonic differenti-ation (Decker & Friend, 1974; Fisher & Linberg, 1975; Hayes, 1977), tightjunctions and gap junctions are often closely associated with one another indeveloping tissue, and the complex changing patterns of these junctions in the

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neural folds and neural tube preclude a clear definition of their nature andfunction (Revel & Brown, 1976).

Of special interest in the current study is the presence of gap junctionalvesicles (annular nexuses). Although the role and significance of the vesiclesare unknown, these unusual structures have been shown by means of freeze-fracture and tracer techniques to bud off from finger-like projections of gapjunctions into the cytoplasm, and it has been postulated that the vesicles mayrepresent a means of interiorizing and disposing of gap junctions (Albertini,Fawcett & Olds, 1975). Gap junctional vesicles have been noted in a varietyof cells including ovarian granulosa cells (Espey & Stutts, 1972; Merk, Albright& Botticelli, 1973; Albertini et al. 1975; Coons & Espey, 1977; Tung & Larsen,1979) and various adenocarcinoma cells, particularly during dissociation experi-ments (Leibovitz et al. 1973; Letourneau, Li, Rosen & Ville, 1975; Murray,Larsen & O'Donnell, 1978; Murray, 1979). In the current study at least oneof these vesicles was found per section of lumbosacral neural folds in thenormal 8-day mouse embryo; in contrast, gap junctional vesicles were rarelyfound in the normal 9-day lumbosacral folds, suggesting that they may playa role in mediating a normal loss of cell to cell contact and/or communicationat this critical stage of neural tube closure.

In the loop-tail (Lp) and splotch (Sp) mutant mouse, homozygous individualsshow closure defects of the neural tube. Although fine structural characteristicsof microtubules, microfilaments, midbodies, and junctional complexes in the9-day abnormal embryos are similar to those seen in their normal litter-matesand in the normal 9-day C57BL individuals of the current study, one strikingdifference is the increased number of gap junctional vesicles in the abnormalneural tubes (Wilson & Finta, 1979; Wilson, 1979). Whether this representsa cause or an effect of the abnormality remains to be explored in these mutants,particularly during the eighth day of development. The relationship of gapjunctional vesicles to gap junctions and tight junctions during normal as wellas abnormal neural development would also seem to warrant further attention.

This research was supported by National Institutes of Health grant no. HD09562 fromthe National Institute of Child Health and Human Development.

REFERENCES

ALBERTINI, D. F., FAWCETT, D. W. & OLDS, P. J. (1975). Morphological variations in gapjunctions of ovarian granulosa cells. Tissue & Cell 7, 389-405.

ALLENSPACH, A. L. & ROTH, L. E. (1967). Structural variations during mitosis in the chickembryo. / . Cell Biol. 33, 179-176.

AUERBACH, R. (1954). Analysis of the developmental effects of a lethal mutation in thehouse mouse. / . exp. Zool. 127, 305-329.

BANCROFT, M. & BELLAIRS, R. (1975). Differentiation of the neural plate and neural tubein the young chick embryo. A study by scanning and transmission electron microscopy.Anat. Embryol. 147, 309-355.

BELLAIRS, R. (1959). The development of the nervous system in chick embryos, studied byelectron microscopy. / . Embryol. exp. Morph. 7, 94-116.

Neural folds in mouse embryos 289BUCK, R. C. (1963). The central spindle and the cleavage furrow. In The Cell in Mitosis

(ed. L. Levine), pp. 55-65. New York: Academic Press.BURNSIDE, B. (1971). Microtubules and microfilaments in newt neurulation. Devi Biol. 26,

416-441.BURNSIDE, B. (1973). Microtubules and microfilaments in amphibian neurulation. Amer.

Zool. 13, 989-1006.CAMATINI, M. & RANZI, S. (1976). Ultrastructural analysis of the morphogenesis of the

neural tube, optic vesicle and optic cup in chick embryo. Acta Embryol. Exp. 1, 81-113.COONS, L. W. & ESPEY, L. L. (1977). Quantitation of nexus junctions in the granulosa cell

layer of rabbit ovarian follicles during ovulation. J. Cell Biol. 74, 321-325.DECKER, R. S. & FRIEND, D. S. (1974). Assembly of gap junctions during amphibian neuru-

lation. / . Cell Biol. 62, 32-47.ESPEY, L. L. & STUTTS, R. H. (1972). Exchange of cytoplasm between cells of the membrana

granulosa in rabbit ovarian follicles. Biol. Reprod. 6, 168-175.FISHER, S. K. & LINBERG, K. A. (1975). Intercellular junctions in the early human embryonic

retina. /. Ultrastruct. Res. 51, 69-78.FREEMAN, B. G. (1972). Surface modifications of neural epithelial cells during formation

of the neural tube in the rat embryo. / . Embryol. exp. Morph. 28, 437-448.FUJITA, H. & FUJITA, S. (1963). Electron microscopic studies on neuroblast differentiation

in the central nervous system of domestic fowl. Z. Zellforsch. mikrosk. Anat. 60, 463-478.

HANDEL, M. A. & ROTH, L. E. (1971). Cell shape and morphology of the neural tube:Implications for microtubule function. Devi Biol. 25, 78-95.

HAYES, B. P. (1977). Intercellular gap junctions in the developing retina and pigmentepithelium of the chick. Anat. Embryol. 151, 325-333.

HINDS, J. W. & RUFFETT, T. L. (1971). Cell proliferation in the neural tube: an electronmicroscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. mikrosk.Anat. 115, 226-264.

KARFUNKEL, P. (1972). The activity of microtubules and microfilaments in neurulation inthe chick. / . exp. Zool. 181, 289-302.

KARFUNKEL, P. (1974). The mechanisms of neural tube formation. Int. Rev. Cytol. 38,245-271.

KARNOVSKY, J. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity foruse in electron microscopy. / . Cell Biol. 27, 137A.

KRYSTAL, G., RATTNER, J. B. & HAMKALO, B. A. (1978). Partial purification and characteri-zation of the intercellular bridge from cultured mouse cells. Proc. natn. Acad. Sci., U.S.A.75,4977-4981.

LEIBOVITZ, R., MCCOMBS, W. B., JOHNSTON, D., MCCOY, C. & STINSON, J. C. (1973). Newhuman cancer cell culture lines. I. SW-13 small cell carcinoma of the adrenal cortex.J. natn. Cancer Inst. 51, 691-697.

LEMIRE, R. J., LOESER, J. D., LEECH, R. W. & ELLSWORTH, C. A., JR. (1975). Normal andAbnormal Development of the Human Nervous System. Hagerstown: Harper & Row.

LETOURNEAU, R. J., Lr, J. J., ROSEN, S. & VILLE, C. (1975). Junctional specialization inestrogen-induced renal adenocarcinoma of the golden hamster. Cancer Res. 35, 6-10.

MAK, L. L. (1978). Ultrastructural studies of amphibian neural fold fusion. Devi Biol.65, 435-446.

MERK, F. B., ALBRIGHT, J. T. & BOTTICELLI, C. R. (1973). The fine structure of granulosacell nexuses in rat ovarian follicles. Anat. Rec. 175, 107-126.

MORAN, D. & RICE, R. W. (1975). An ultrastructural examination of the role of cellmembrane. Surface coat material during neurulation. / . Cell Biol. 64, 172-180.

MURRAY, S. A., LARSEN, W. J. & O'DONNELL, S. (1978). Gap junctional endocytosis andloss of cell cohesion in cultured human adenocarcinoma of adrenal cortex. Anat. Rec.190, 489.

MURRAY, S. A. (1979). The relationship of gap junctions to cell behavior in cultured humanadenocarcinoma of the adrenal cortex. Anat. Rec. 193, 632.

REVEL, J. P. (1974). The Cell Surface in Development (ed. A. A. Moscona), pp. 51-66.New York: John Wiley & Sons, Inc.


REVEL, J. P. & BROWN, S. S. (1976). Cell junctions in development, with particular referenceto the neural tube. Cold Springs Harbor Symp. Quant. Biol. 40, 433-455.

REVEL, J. P. & KARNOVSKY, M. J. (1967). Hexagonal array of subunits in intercellular junctionsof the mouse heart and liver. / . Cell Biol. 33, C7-C12.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. / . Cell Biol. 7, 208-212.

SADLER, T. W. (1978). Distribution of surface coat material on fusing neural folds of mouseembryos during neurulation. Anat. Rec. 191, 345-350.

SCHROEDER, T. E. (1970). Neurulation in Xenopus laevis: an analysis and model based uponlight and electron microscopy. J. Embryol. exp. Morph. 23, 427-462.

SIDMAN, R. L., ANGEVINE, J. B. & PIERCE, E. T. (1971). Atlas of the Mouse Brain and SpinalCord. Cambridge: Harvard University Press.

SIDMAN, R. L., GREEN, M. C. & APPEL, S. H. (1965). Catalog of the Neurological Mutants ofthe Mouse. Cambridge: Harvard University Press.

SOTELO, J. R. & TRUJILLO-CENOZ, O. (1958). Electron microscope study on the developmentof ciliary components of the neural epithelium of the chick embryo. Z. Zellforsch. mikrosk.Anat. 49, 1-12.

SUMI, S. M. (1969). The extracellular space in the developing rat brain: its variation withchanges in osmolarity of the fixative, method of fixation and maturation. / . Ultrastruct.Res. 29, 398-425.

THEILER, K. (1972). The House Mouse (Development and Normal Stages from Fertilizationto Four Weeks of Age). Berlin: Springer-Verlag.

TUNG, H. N. & LARSEN, W. J. (1979). Cytoplasmic filaments, microtubules, and the endo-cytosis of gap junctions in rabbit granulosa cells. Anat. Rec. 193, 706.

WATERMAN, R. E. (1976). Topographical changes along the neural fold associated withneurulation in the hamster and mouse. Am. J. Anat. 146, 151-172.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavymetals. / . biophys. biochem. Cytol. 4, 475-478.

WILSON, D. B. (1974). Proliferation in the neural tube of the splotch (Sp) mutant mouse./ . comp. Neurol. 154, 249-256.

WILSON, D. B. (1978). The fine structure of ventricular cells in the brains of mouse embryoshomozygous for the loop-tail gene. Teratology 17, 115-136.

WILSON, D. B. (1979). Ventricular cell defects in the hindbrain of the loop-tail (Lp) mutantmouse at nine days of gestation. Teratology 19, 54A.

WILSON, D. B. & FINTA, L. A. (1979). Gap junctional vesicles in the neural tube of thesplotch (Sp) mutant mouse. Teratology 19, 337-340.

{Received 18 May 1979, revised 30 July 1979)

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