The neural folds in the lumbosacral region of the normal 8-day and 9-day mouse embryo were studied by means of transmission electron microscopy with and without lanthanum treatment. The cells showed an abundance of ribosomes, microtubules arranged parallel to the long axes of the cells, and microfilaments extending across the apices. At the luminal border junctional complexes were common, and an occasional midbody was seen stretching between adjacent cells nearing the end of telophase. In the 8-day embryos, gap junctional vesicles (annular nexuses) bounded by layered membranes and containing cytoplasm with ribosome-like material were commonly observed; at 9 days the vesicles were relatively rare. The lanthanum-treated material demonstrated that the tracer was able to pass through the subluminal junctional complexes and throughout the intercellular spaces. However, the space between the membranes of the gap junctional vesicles lacked lanthanum and thus apparently did not communicate with the intercellular space.

The formation of the neural tube has been the subject of numerous transmission 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 TEM studies on early rodent embryos are relatively sparse (Hinds & Ruffett, 1971 ; Freeman, 1972; Sadler, 1978). In the mammal, the early stages of neural tube closure, particularly the formation and elevation of the neural folds, are of special importance in the lumbosacral region, since this is a site frequently affected by non-closure malformations (dysraphism) (Auerbach, 1954; Wilson, 1974; Lemire, Loeser, Leech & Ellsworth, 1975). In view of the paucity of TEM information on the lumbosacral neural folds in normal mammalian embryos, the current study was undertaken on normal 8-day and 9-day mice so as to establish a basis for future fine structural analyses of this region in abnormal mouse embryos at comparable stages of development.

C57BL/6J mice were bred, females were checked daily for vaginal plugs and 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/6J background. Six embryos corresponding to developmental stage 12 (Theiler, 1972) were selected from three litters, and four embryos at developmental stage 14 were selected from three litters. The lumbosacral region of each embryo 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 postfixed in cold 1 % osmium tetroxide-0·1 M cacodylate buffer for 1 h. The specimens were then dehydrated in graded ethanols and propylene oxide and flat-embedded in Epon-Araldite. Thick sections for orientation with light microscopy were stained with methylene blue-azure II. Thin sections were placed on naked 200 mesh copper grids and stained with uranyl acetate (Watson, 1958) for 20 min and lead citrate (Reynolds, 1963) for 10 min.

For the lanthanum studies, the technique of Revel & Karnovsky (1967) was used. A 4 % solution of lanthanum nitrate was brought to pH 7·8 by means of 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 osmium tetroxide, or during dehydration.

Observations were made with a Zeiss 9S-2 electron microscope at direct magnifications up to × 28000.

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

Light microscopy

Epon-Araldite thick sections of both the upper and lower lumbosacral regions of 8-day and 9-day embryos were studied. At 8 days the sections demonstrate a neural groove that is relatively wide and V-shaped (Fig. 1). The neurectodermal cells of the groove are tall and columnar. At the lateral edges of the groove the cells change to cuboidal. Mitotic figures are scattered along the presumptive luminal border, and deeper lying nonmitotic cells maintain contact 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 to bulge slightly upwards into the lumen, whereas sections removed from the lower lumbosacral region show more flattened luminal cell surfaces. In both regions of the groove the cells appear to be separated from one another sub-luminally by extracellular spaces of varying size, except for points of contact by means of small lateral projections. These contact points and extracellular spaces may be exaggerated by shrinkage due to tissue preparation.

FIGURE 1–6

Fig. 1. Cross section of lumbosacral region of neural groove at 8 days’ gestation. L, presumptive lumen, × 180.

Fig. 2. Higher magnification of neuroepithelium at 8 days’ gestation. Arrow indicates internal cellular process, × 600.

Fig. 3. Cilium in apical portion of neuroepithelial cell in lateral region of lumbosacral neural groove at 8 days’ gestation, × 28 500.

Fig. 4. Microvillous projection (small arrow) at margin of neuroepithelial cell at 8 days’ gestation. Large arrow, junctional complex, × 28 500.

Fig. 5. Lanthanum in intercellular space (small arrows) between two neuroepithelial cells at 8 days’ gestation. Large arrow, possible gap junction, × 84000.

Fig. 6. Midbody at the end of a mitotic division. Small arrows, microtubules. L, presumptive lumen. Large arrow, dense band, × 28 500.

FIGURE 1–6

Fig. 1. Cross section of lumbosacral region of neural groove at 8 days’ gestation. L, presumptive lumen, × 180.

Fig. 2. Higher magnification of neuroepithelium at 8 days’ gestation. Arrow indicates internal cellular process, × 600.

Fig. 3. Cilium in apical portion of neuroepithelial cell in lateral region of lumbosacral neural groove at 8 days’ gestation, × 28 500.

Fig. 4. Microvillous projection (small arrow) at margin of neuroepithelial cell at 8 days’ gestation. Large arrow, junctional complex, × 28 500.

Fig. 5. Lanthanum in intercellular space (small arrows) between two neuroepithelial cells at 8 days’ gestation. Large arrow, possible gap junction, × 84000.

Fig. 6. Midbody at the end of a mitotic division. Small arrows, microtubules. L, presumptive lumen. Large arrow, dense band, × 28 500.

In the upper lumbar region of 9-day embryos the neural folds have fused to form a neural tube. Lower lumbar and sacral regions show varying degrees of closure. In open regions the neural groove is horseshoe-shaped and the medial aspects of the folds are concave.

Electron microscopy

8 days

At the electron microscopic level low magnifications of the 8-day lumbosacral region reveal a neuroepithelium with nuclei at varying levels and mitotic nuclei interspersed between the columnar cells at the presumptive luminal border. Short microvilli are scattered over the luminal surface, and centrally located, single apical cilia are occasionally present (Fig. 3). Junctional complexes occur between adjacent cells at the luminal border, and microfilaments can sometimes be seen extending outward from the junctions across the apices of the cells. In most cases a long microvillous projection also extends into the lumen at the cell margin adjacent to the junctional complex (Fig. 4).

The intercellular junctional complexes are permeable to lanthanum, as evidenced by the presence of the tracer throughout the intercellular spaces. In some instances the lanthanum-filled space is narrowed and suggestive of a gap junction (Fig. 5).

Midbodies are common at the luminal surface and consist of a narrow cytoplasmic bridge between two cells in the process of completing a mitotic division. Within the cytoplasmic bridge are parallel stacks of microtubules with a centrally located dense band (Fig. 6). A few cells also show large irregular blebs on the luminal surface.

The neuroepithelial cells of the upper and lower lumbosacral neural groove contain a variety of cytoplasmic organelles including free ribosomes, polyribosomes, rough endoplasmic reticulum (RER), and small, dense mitochondria. In some cells the rough endoplasmic reticulum exhibits circular or whorled zones. Pinocytotic invaginations are frequently found at the luminal surface of the internal processes of the nonmitotic cells. These processes also show numerous microtubules running parallel to the long axes of the cells (Fig. 7).

Fig. 7.

Internal cellular process of neuroepithelial cell at 8 days’ gestation. Note numerous microtubules (small arrows). Large arrow, gap junctional vesicle. × 28 500.

Fig. 7.

Internal cellular process of neuroepithelial cell at 8 days’ gestation. Note numerous microtubules (small arrows). Large arrow, gap junctional vesicle. × 28 500.

A notable feature of both upper and lower lumbosacral neural groove is the presence of gap junctional vesicles (annular gap junctions or annular nexuses) (Figs. 79). The vesicles are bounded by a dense, layered membrane and contain ribosome-like material. Most vesicles have an electron-lucent zone located within the vesicle subjacent to its membrane.

Fig. 8.

Gap junctional vesicle (arrow) in mitotic cell at 8 days’ gestation, × 28500.

Fig. 8.

Gap junctional vesicle (arrow) in mitotic cell at 8 days’ gestation, × 28500.

Fig. 9.

Gap junctional vesicle in lanthanum-treated embryo at 8 days’ gestation. Note presence of lanthanum in adjacent intercellular spaces but not between membranes of the vesicle, × 38000.

Fig. 9.

Gap junctional vesicle in lanthanum-treated embryo at 8 days’ gestation. Note presence of lanthanum in adjacent intercellular spaces but not between membranes of the vesicle, × 38000.

The gap junctional vesicles are most frequently found near the luminal surface in the internal cellular processes of nonmitotic cells, but they have also been observed at deeper levels in the cellular processes, near the basal lamina of the neuroepithelium, and in mitotic cells. The occurrence of the vesicles is similar in both upper and lower lumbosacral regions, and each section of neural groove examined has at least one gap junctional vesicle. Some sections have as many as seven individual vesicles, with an occasional cell having two vesicles. Examination of adjacent sections confirms that the vesicles are distinct and separate from one another and not sections of the same vesicle. The lanthanum preparations show that the space between the membranes of the vesicles lacks lanthanum and that the vesicles are not in communication with the intercellular space (Fig. 9).

9 days

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

FIGURE 10–12

Fig. 10. Luminal aspect of lumbosacral groove at 9 days’ gestation. The surfaces show prominent bulges and projections into the lumen. M, mitotic cell. Arrow indicates a rare gap junctional vesicle, × 3900.

Fig. 11. Higher magnification of apex of a ventricular cell at 9 days’ gestation showing apical bulging into the lumen (L). Arrows indicate bundles of microfilaments. × 24000.

Fig. 12. Low magnification of dorsal neural folds in the process of fusing with each other at 9 days’ gestation. L, Lumen, × 5400.

FIGURE 10–12

Fig. 10. Luminal aspect of lumbosacral groove at 9 days’ gestation. The surfaces show prominent bulges and projections into the lumen. M, mitotic cell. Arrow indicates a rare gap junctional vesicle, × 3900.

Fig. 11. Higher magnification of apex of a ventricular cell at 9 days’ gestation showing apical bulging into the lumen (L). Arrows indicate bundles of microfilaments. × 24000.

Fig. 12. Low magnification of dorsal neural folds in the process of fusing with each other at 9 days’ gestation. L, Lumen, × 5400.

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

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

With respect to gap junctional vesicles, these structures are relatively rare at 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 the 8-day embryos, and their membranes did not contain lanthanum. The locations of 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.

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

The neuroepithelial cells in the lumbosacral folds in the normal 8-day and 9-day mouse embryos used in the current study show features similar to those described in various other regions of the chick and rodent neural folds during early development (Freeman, 1972; Karfunkel, 1972; Bancroft & Bellairs, 1975; Camatini & Ranzi, 1976; Revel & Brown, 1976). These features include microtubules arranged parallel to the long axes of the cells, especially in the internal cellular processes, and an abundance of free ribosomes and polyribosomes. Apical cilia are also present but are not as frequent or well developed as 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 the cells are not as prominent or densely arranged in the 8-day mouse as at 9 days when the medial aspects of the neural folds become concave. The arrangement of these microfilaments is similar to that seen in the amphibian and chick, and their role has been postulated as producing the apical constriction necessary for 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 cells bulge somewhat in the upper lumbosacral region, whereas those in the lower region tend to be more flat, most likely reflects the fact that the upper region is slightly more advanced than the lower region at any given stage of development. This bulging, as well as the formation of apical blebs, becomes more prominent 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 also exhibit an occasional cytoplasmic bridge between two cells nearing the end of telophase. Once the cells separate completely the cytoplasmic bridge is pinched off and portions remain as debris at the lumen. These bridges have been termed midbodies (Allenspach & Roth, 1967), although the term has also been used more restrictively to designate remnants of the mitotic spindle or the dense band in the center of the bundles of spindle microtubules (Buck, 1963; Krystal, Rattner & Hamkalo, 1978). The presence of midbodies along the luminal aspects of the 8- and 9-day neuroepithelium reflects the mitotic activity of these cells, and these structures are particularly common after closure of the neural tube (Allenspach & Roth, 1967; Bancroft & Bellairs, 1975; Wilson, 1978).

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

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

Although the exact nature of the junctional complexes could not be confirmed in the current study without special techniques such as freeze-fracture, the junctions at the luminal surface appear to be gap junctional in nature, since lanthanum passed freely through the intercellular spaces to deeper levels. In the amphibian, Decker & Friend (1974) noted that gap junctions become widely distributed in the neural folds during closure. Likewise in the chick, Revel & Brown (1976) describe small gap junctions or gap junction-like structures in the gutter stage of the neural groove. An occasional juxtaluminal zonula occludens was observed in more mature regions of the developing neural tube in the chick (Revel & Brown, 1976); this agrees with our observation of an occasional junction which was not permeable to lanthanum in our 9-day mouse material. Although gap junctions have been cited as a means of cell to cell communication and are particularly common during embryonic differentiation (Decker & Friend, 1974; Fisher & Linberg, 1975; Hayes, 1977), tight junctions and gap junctions are often closely associated with one another in developing tissue, and the complex changing patterns of these junctions in the neural folds and neural tube preclude a clear definition of their nature and function (Revel & Brown, 1976).

Of special interest in the current study is the presence of gap junctional vesicles (annular nexuses). Although the role and significance of the vesicles are unknown, these unusual structures have been shown by means of freezefracture and tracer techniques to bud off from finger-like projections of gap junctions into the cytoplasm, and it has been postulated that the vesicles may represent a means of interiorizing and disposing of gap junctions (Albertini, Fawcett & Olds, 1975). Gap junctional vesicles have been noted in a variety of 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 experiments (Leibovitz et al. 1973; Letourneau, Li, Rosen & Ville, 1975; Murray, Larsen & O’Donnell, 1978; Murray, 1979). In the current study at least one of these vesicles was found per section of lumbosacral neural folds in the normal 8-day mouse embryo; in contrast, gap junctional vesicles were rarely found in the normal 9-day lumbosacral folds, suggesting that they may play a role in mediating a normal loss of cell to cell contact and/or communication at this critical stage of neural tube closure.

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

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

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