Distribution of Surface Coat Material on Fusing Neural Folds of Mouse Embryos during Neurulation

T. W. SADLER ' Department of Anatomy, School of Medicine, University of Virginia, Charlottesuille, Virginia 22902

ABSTRACT Fusing and non-fusing regions of neural folds from mouse em- bryos were examined during neurulation for the distribution of extracellular macromolecules (surface coats) prior to and a t the time of closure. Ruthenium red staining of 10th day ICR/DUB mouse embryos was used to detect the dis- tribution of surface coat material. Light microscopic examination of fusing and non-fusing regions in the midbrain, hindbrain, and spinal cord showed a con- sistent increase in ruthenium red positive material immediately prior to closure. Heavy deposits of positive staining material were present along apical neural fold borders and overlying ectoderm cells. This staining pattern was con- sistent in the three regions examined, but the pattern of initial contact be- tween opposing neural folds differed. In mid- and hindbrain areas contact was initiated by overlying ectoderm, whereas in spinal cord regions contact was first established by neuroepithelial cells. Once contact between opposing neural folds was initiated a decrease in stainable material was observed.

The distribution of carbohydrate-rich sur- face coat material along prospective zones of fusion in the palate and nasal processes of rat and mouse embryos has led to the hypothesis that these macromolecules are essential for normal epithelial contact and adherence. Dur- ing palatogenesis, surface coat material has been shown to increase dramatically over me- dial-edge epithelial cells prior to fusion and is concentrated in prospective contact regions (Pratt e t al., '73; Greene and Kochhar, '74; Prat t and Hassell, '75; Souchon, '75; Greene and Pratt, '76). Surface coats have also been observed during fusion of medial and lateral nasal processes in mouse embryos (Gaare and Langman, '77; Smuts, '77) and in fusing neu- roepithelium of chick (Lee e t al., '76b) and am- phibian (Moran and Rice, '75) embryos. In both the nasal and neuroepithelial areas, in- creased amounts of surface coat material over prospective fusion zones was observed imme- diately prior to contact. These findings sug- gest that extracellular materials may be important for normal fusion and adhesion be- tween epithelial surfaces during organogen- esis (Greene and Pratt, '76). This view is sub- stantiated by results indicating that inhib- itors of surface coat synthesis such as DON (6-diazo-5-oxonorleucine) prevent adhesion of

ANAT. REC. (1978) 191: 345-350.

epithelial surfaces during palatogenesis in rats (Greene and Pratt , '76, '77).

To further investigate the role of surface coat material during epithelial adhesion, fus- ing neural folds in mouse embryos were exam- ined for the presence, and distribution of sac- charide-rich surface macromolecules. Neural tube closure in mouse embryos was selected because the fusion process is a continuum, beginning in the cervical region and proceed- ing rostrally and caudally, thereby permitting observations of fusing and non-fusing regions in the same embryo. Ruthenium red, a stain used to demonstrate surface coat carbohy- drates, was (Luft, '71a,b), used to demonstrate the presence of surface coat material.

MATERIALS AND METHODS

Random-bred ICR/DUB mice (Flow Labora- tories, Dublin, Virginia) were used for all in- vestigations and animals were sacrificed a t 10 and 10.5 days of gestation (plug day=day 1). Following removal from the uterus, embryos were fixed for one hour in cacodylate-buffered modified Karnovsky's fixative (2% glutaralde-

Received Aug. 9, '77. Accepted Feb. 16, '78. ' This work was supported by NIH Grant 5132 DE07037-02 for

craniofacial development and by Biomedical Research Support Grant 5S07RR5431-15.

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346 T. W. SADLER

hyde, 2'11 paraformaldehyde) containing 4,500 ppm ruthenium red (Sigma). After fixation, embryos were rinsed briefly in 0.1 M cacody- late buffer containing 4,500 ppm ruthenium red and post-fixed for three hours in cacody- late buffered OsO,, also containing 4,500 ppm ruthenium red (Luft, '71a,b). The tissue was then dehydrated in alcohol and embedded in araldite (502). Thick (1 pm) sections were made on an LKB-Huxley ultramicrotome, stained with toluidine blue, and observed with the light microscope.

RESULTS

Neural tube closure in ICR mouse embryos was initiated on the ninth day of gestation and continued to completion over the next 24 hours. Fusion of hind- and midbrain areas was similar morphologically and in regard to dis- tribution of ruthenium red positive material. Morphologically, neural folds from these re- gions elevated from the neural plate to a verti- cal position in which opposing folds were par- allel (fig. 1). At this point a bend occurred near the middle and at the apex of each fold which tended to approximate the apices from either side. Concomitantly, the apices, which were rounded in the parallel position, began to taper a s they approached each other (figs. 2, 3). However, tapering was more pronounced in hindbrain (figs. 2, 3) than midbrain (fig. 5) a reas which re ta ined a rounded shape throughout closure. Overlying the tips of each neural fold was a n ectodermal cell layer which was continuous with ectoderm surrounding the entire embryo (figs. 1-41, Ectoderm cells appeared cuboidal except over the uppermost area of approximating neural folds where these cells were elongated. A t closure, ecto- derm cells made initial contact between opposing neural folds and formed a cellular plug situated between adjacent neuroepi- thelial cells from each fold (fig. 4). Finally, neuroepithelial cells made contact beneath the ectodermal plug, thereby forming a con- tinuous layer of neuroepithelium around the lumen. Following neuroepithelial fusion, over- lying ectoderm cells resumed their cuboidal shape.

Distribution of ruthenium red positive ma- terial was similar in hind- and midbrain re- gions. Approximately 100-120 p m anterior to a fusion zone, in either of these regions, no ru- thenium red stained material was observed with light microscopy. However, as a n area of

fusion was approached a n accumulation of stain was observed. Initially, stained material was present uniformly over the entire luminal surface of neural folds and overlying ecto- derm. This staining pattern was maintained until approximately 40 p m anterior to fusion at which point a concentration of positively staining material accumulated along luminal surfaces of the upper third of opposing neural folds (figs. 2, 3, 5). The deposition of stain was heaviest a t the apices of neural folds and occa- sionally extended over ectoderm cells immedi- ately overlying apical neuroepithelial cells. Following contact between neuroepithelium from adjacent folds and a return of ectoderm to a cuboidal shape, staining decreased until only a small, evenly distributed deposit of ru- thenium red positive material was observed over the luminal surface and overlying ecto- derm.

Neural tube closure in spinal cord regions differed morphologically from hind- and mid- brain fusion, but the pattern of ruthenium red staining remained the same. For example, during spinal cord closure, neural fold apices were rounded, not tapered as those from hind- brain areas (fig. 6). Furthermore, overlying ectoderm cells did not completely cover apical areas and were not involved in initial contact. However, staining patterns in tail regions were similar to hind- and midbrain areas and positively staining material was initially ob- served 100-120 p m prior to contact in a n even distribution along luminal surfaces. The amount of staining then increased along upper luminal borders and overlying ectoderm as opposing folds drew closer together (fig. 6).

DISCUSSION

Neural tube closure in ICR mouse embryos encompasses a 24-hour period beginning on the ninth and extending into the tenth day of gestation. Closure begins in the cervical re- gion and proceeds cranially and caudally. In cranial regions, increased folding and taper- ing of neural folds occurs, especially in hind- brain regions, and initial contact appears to be between overlying ectoderm. Tail regions show very little folding and no tapering, with initial contact being accomplished by neu- roepithelial cells from opposing neural folds. This pattern of fusion in cranial areas is in contrast to scanning electron microscopic ob- servations of CD-1 mouse and hamster neu- rulation in which initial contact was observed

SURFACE COAT FORMATION DURING MOUSE NEURULATION 347

between neuroepithelial cells (as we observed in tail sections) followed by ectodermal fusion (Waterman, '76).

Although differences occur between cranial and caudal segments regarding alterations in neural fold contours, the distribution of sur- face coat material is similar in all regions. Both cranial and caudal areas show increased deposition of surface carbohydrates, which a re concentrated over prospective zones of fusion immediately prior to contact. After contact is initiated, surface coat material diminishes in the fusion zones and returns to levels found in non-fusing areas of neuroepithelium. These results a re similar to those obtained from lanthanum stained sections of neural folds during amphibian (Ambystoma maculatum) (Moran and Rice, '75) and chick (Lee e t al., '76) neurulation which also demonstrated the presence of increased surface coat material along prospective zones of neural fold fu- sion prior to contact. Lanthanum positive "bridges" of cell surface material were also ob- served in amphibians and were thought to par- ticipate in the closure process by pulling the folds together (Moran and Rice, '75). However, no surface coat "bridges" were observed in mouse embryos and no evidence was obtained which would suggest t ha t surface coat mate- rial plays a n active role in drawing the neural folds toget her.

The distribution and location of surface coat material in the mouse neural tube is consist- en t with the hypothesis t ha t surface coats play a role in epithelial fusion and adhesion during organogenesis. Similar accumulations of extracellular materials have been observed in other systems during epithelial fusion in- cluding rat and mouse palatogenesis (Pratt e t al., '73; Greene and Kochhar, '74; Pratt and Hassell, '75; Souchon, '75; Greene and Pratt, '76) and fusion of nasal processes in mice (Gaare and Langman, '77; Smuts, '77). In each of these systems increased deposition of sur- face macromolecules was observed in prospec- tive fusion areas prior to contact, followed by a decrease in stainable material after fusion. The mechanism whereby surface coats medi- a t e epithelial fusion in these systems is unknown although surface macromolecules may provide initial adherence until more per- m a n e n t cell contac ts can be established (Greene and Pra t t , '76). I t is known, however, t h a t interference with existing cell surface

macromolecules during chick neurulation (by exposure to concanavalin A) (Lee e t al., '76a,b), or inhibition of synthetic processes re- sponsible for surface coat production (treat- ment with DON) (Greene and Pratt, '77) pre- vents fusion in the chick neural tube and ra t palate respectively. .

ACKNOWLEDGMENTS

Thanks to Robert Cushing for his skillful technical assistance.

LITERATURE CITED

Gaare, J., and Jan Langman 1977 Fusion of nasal swellings in the mouse embryo. I. Surface coat and initial contact. Am. J. Anat., 150: 461-476.

Greene, R. M., and D. M. Kochhar 1974 Surface coat on the epithelium of developing palatine shelves in the mouse as revealed by electron microscopy. J. Embryol. Exp. Morph., 31: 683-692.

Greene, R. M., and R. M. Prat t 1976 Developmental aspects of secondary palate formation. J. Embryol. Exp. Morph., 36: 225-245.

1977 Inhibition by diazo-0x0-norleucine (DON) of ra t palatal glycoprotein synthesis and epithelial cell adhesion in uitro. Exp. Cell Res., 105: 27-37.

Lee, H., R. G. Nagele, Jr. and G. W. Kalmus 1976a Further studies on neural tube defects caused by concanavalin A in early chick embryos. Experientia, 32: 1050-1052.

Lee, H., J. B. Sheffield, R. G. Nagele and G. W. Kalmus 1976b The role of extracellular material in chick neu- rulation. I. Effects of concanavalin A, J. Exp. Morphol., 198: 261-266.

Luft, J. H. 1971a Ruthenium red and violet. I. Chemis- try, purification, methods of use for electron microscopy, and mechanism of action. Anat. Rec., 171: 347-368.

Ruthenium red and violet. 11. Fine struc- tural localization in animal tissues. Anat. Rec., 171: 369-416.

Moran, D., and R. W. Rice 1975 An ultrastructural exami- nation of the role of cell membrane surface coat material during neurulation. J. Cell Biol., 64: 172-181.

Pratt, R. M., W. A. Gibson and J. R. Hassell 1973 Con- canavalin A binding to the secondary palate of the embry- onic rat. J. Dent. Res., 52: 111A.

Pratt, R. M., and R. M. Greene 1975 The effects of diazo- 0x0-norleucine (DON) on development of the palatal epi- theliumin uitro. In: New Approaches to the Evaluation of Abnormal Development. D. Neubert and H. J. Merker, eds. Georg Thieme Publishers, Stuttgart, pp. 648-658.

Pratt, R. M., and J. R. Hassell 1975 Appearance and dis- tribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf. Dev. Biol., 45: 192-198.

Smuts, M. S. 1977 Concanavalin A binding to the epithe- lial surface of the developing mouse olfactory placode. Anat. Rec., 188: 29-38.

Souchon, R. 1975 Surface coat of the palatal shelf epi- thelium during palatogenesis in mouse embryos. Anat. Embryol., 147: 133-142.

Waterman, R. E. 1976 Topographical changes along the neural fold associated with neurulation in the hamster and mouse. Am. J. Anat., 146: 151-172.

1971b

PLATE 1

EXPLANATION OF FIGURES

1-4 Cross section through the hindbrain region of tenth day embryos showing the par- allel position (fig. l), the tapered configuration (figs. 2, 31, and initial contact (fig. 4) between opposing neural folds. The increased distribution of ruthenium red pos- itive material is also demonstrated along the upper luminal borders of neural folds. Ectoderm (El, mitotic figures (M); neural crest, (NO; neural folds, (NF); pyknotic cell, (PC); pyknotic debris, (PD). One micron ruthenium red-toluidine blue. Figure 1 X 400; figures 2-4 X 1,100.

5 Midbrain region of a tenth day embryo prior to fusion, showing slight tapering of neural folds (NF) and a heavy deposit of ruthenium red positive material over neural fold apices and overlying ectoderm (E). One micron ruthenium red-tolu- idine blue. X 1,600.

6 Cross section from the spinal cord of a tenth day embryo near the point of fusion showing increased amounts of ruthenium red positive material over upper neural fold regions (NF) and overlying ectoderm (E). Mitotic figures (MI; pyknotic cell (PC). One micron ruthenium red-toluidine blue. x 1,100.

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SURFACE COAT FORMATION DURING MOUSE NEURULATION T. W. Sadler

PLATE 1

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