J. Embryol. exp. Morph. 78, 211-228 (1983) 2 1 1Printed in Great Britain © The Company of Biologists Limited 1983

Occlusion of the neural lumen in early mouseembryos analysed by light and electron microscopy

By M. H. KAUFMANFrom the Department of Anatomy, University of Cambridge

SUMMARYA histological account of neural tube occlusion during early mouse embryogenesis is presen-

ted here from an analysis of sections taken from plastic-embedded material. Because the overallpattern of neuroepithelial apposition and the duration of luminal occlusion appears to varyslightly from one embryo to another at otherwise similar stages of development, only a generalguide to the events occurring in the mouse embryo between about midday on the 9th to lateon the 10th day of gestation can be given. The earliest evidence of complete luminal occlusionwas seen when the cephalic and caudal extremities of the neural tube were still widely open.

An ultrastructural analysis of the morphological appearance of the closely apposed luminalcells in zones of partial and complete occlusion has demonstrated that occlusion is broughtabout initially by the interdigitation of processes from closely apposed neuroepithelial cells.This initial event is followed by direct contact over a much more extensive area between thesurfaces of apposed cells with a characteristically flattened luminal border. Apposition andluminal occlusion is probably facilitated by the presence of viscous extracellular material.Finally, complete occlusion involving an extensive region of the lumen occurs. The latterphenomenon is a transient event lasting 1 or at most 2 days in the mouse. At no stage werejunctional complexes observed between closely apposed neuroepithelial cells in regions inwhich the neural lumen appeared to be completely occluded, though they were apparentbetween adjacent neuroepithelial cells.

Observations on the underlying mechanism(s) of cellular fusion are considered in the lightof the ultrastructural findings. These results are compared with findings from analyses ofvarious other sites of cellular fusion during embryogenesis. Attention is also drawn to thesimilarities and differences observed in the timing and overall pattern of events occurringduring the early development of the neural tube in mouse and human embryos.

INTRODUCTION

A recent study has clearly demonstrated that occlusion of the lumen of theneural tube - eventually involving up to about 60 % of its length - occurs as anormal event during early embryogenesis in man (Desmond, 1982). Further-more, it was proposed that complete occlusion of the neural canal, which wasfirst apparent at stage 11 (when embryos possessed 13-20 pairs of somites, seeStreeter, 1942; O'Rahilly & Gardner, 1979), plays an important part in facilita-ting enlargement of the brain (Desmond & Jacobson, 1977). Indeed, theseauthors suggested that the latter would only occur once the neural tube hadbecome a closed compartment filled with cerebrospinal fluid. Evidence from

1 Author's address: Department of Anatomy, University of Cambridge, Downing Street,Cambridge CB2 3DY, U.K.

212 M. H. KAUFMAN

chick embryo studies in which the cerebrospinal fluid pressure within the neuralsystem was markedly reduced following intubation of the neural tube (Desmond& Jacobson, 1977), eye (Coulombre, 1956) or myelencephalon (Coulombre &Coulombre, 1958) has clearly demonstrated that these experimental proceduresinvariably lead to the abnormal morphogenesis of both the brain and eye.

Apart from the detailed histological analysis of the human embryonic materialpresented by Desmond (1982), observations on neural luminal occlusion in thechick embryo (Desmond & Jacobson, 1977), and the isolated example of luminalocclusion in the thoracic region of an early rat embryo illustrated by Freeman(1972), very little appears to be known about the occurrence and possible sig-nificance of this phenomenon.

In the present study, a histological account of neural tube occlusion during earlymouse embryogenesis is given, detailing the period of development during whichthis phenomenon may be observed. Unlike the situation in the human embryowhere occlusion occurs 'subsequent to the formation of a closed tube' (Desmond,1982), in the mouse, at least, occlusion may be observed in embryos with only10-12 pairs of somites present, when the cephalic and caudal regions of the neuraltube are still widely open. Similarly, while the situation described in the humanembryo suggests that in man the onset and sequential events associated withneural tube occlusion are remarkably uniform from embryo to embryo, thepresent findings tend to indicate that this does not appear to be the case in themouse. For the latter reason, the present account can only serve as a very generalguide to the events occurring in the early mouse embryo as development proceedsbetween about midday on the 9th to late on the 10th day of gestation.

Only sections from plastic-embedded material are presented here, asappropriately fixed material embedded in this way shows little in the way ofshrinkage artefacts as are commonly observed in conventional paraffin-embedded material. An ultrastructural account of the morphological ap-pearance of the closely apposed luminal cells in zones of partial or completeocclusion is also presented.

The present findings of occlusion of an extensive, but variable, segment of theneural lumen in the mouse embryo would seem to confirm the contention thatthis phenomenon is probably a normal occurrence in many vertebrate species(Desmond, 1982). However, the detailed timing of this event, the overall patternof neuroepithelial apposition and the duration of luminal occlusion appear tovary from one species to another, and in the mouse at least, from one embryoto another at otherwise similar stages of development.

MATERIALS AND METHODS

Female CFLP mice (Hacking and Churchill Ltd) were mated with males of thesame strain and isolated on the morning of finding a vaginal plug (designated thefirst day of pregnancy). Between the early afternoon of the 9th day to late on the

Neural luminal occlusion in mouse embryos 21310th day of gestation individual females were killed by cervical dislocation andthe embryos isolated in phosphate-buffered saline (pH7-3). A total of 30 em-bryos were selected for this study, being divided in groups according to theapproximate number of somites present, their degree of 'turning', and thepresence of an obvious forelimb bud (the most advanced group). Between fiveand ten embryos were assigned to each group, though a small degree of overlapwas inevitable using these somewhat arbitrary morphological criteria. Anadditional group of three embryos was isolated early in the morning on the 11thday of gestation. All of the embryos were dissected free of their membranes andfixed in 2 % glutaraldehyde in 0-1 M-sodium cacodylate buffer containing 10 gmsucrose/100 ml. After about 2h the embryos were transferred to 0-1 M-sodiumcacodylate buffer containing 3gm sucrose/100 ml. After l h the material waspostfixed in 1 % osmium tetroxide containing 5gm sucrose/100 ml for 25min.The material was then dehydrated through a graded ethanol series, and eventu-ally embedded in epoxy resin (Spurr, 1969) and semithin transverse sections(thickness about 0-5-0-75 jum) taken and stained with methylene blue. Thinsections at appropriate levels were cut with a Huxley ultramicrotome, doublestained with uranyl acetate and lead citrate and viewed in a Philips EM 300transmission electron microscope.

An additional group of five embryos was used to determine whether extra-cellular material was detectable on the neuroepithelial cell surface at sites ofclose cell-cell apposition. The cephalic region of embryos in this group wasremoved prior to their fixation (for 2 h) in 2 % glutaraldehyde in 0-1 M-sodiumcacodylate buffer containing 3mM-Ca2+ (pH7-2). Decapitation was carried outin order to allow the ruthenium red access to the neuroepithelial cells lining theneural lumen in all embryos in this group, whether the rostral neuropore hadclosed or not. The embryos were then post-fixed in 1 % osmium tetroxide either(i) with or (ii) without ruthenium red (2mg/ml) in 0-1 M-sodium cacodylatebuffer. This material was then dehydrated through a graded ethanol series,embedded, and semithin and thin sections taken as described above. The thinsections were viewed unstained in the Philips EM 300 transmission electronmicroscope orientated at either 20 or 40 kV.

The author's collection of Bouin- and Susa-fixed paraffin-embedded materialcovering embryonic development between the 10th-12th days of gestation wasalso examined, and the incidence of luminal occlusion in this material is alsobriefly reported. The latter information is included here as it confirms theauthor's contention that this phenomenon is most clearly seen in appropriatelyfixed plastic-embedded material.

RESULTS

1. Histological appearance of neural tube

Because of the considerable degree of variability observed in neural tube

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morphology in mouse embryos that appear, at least externally, to be almostidentical, it will only be possible to provide an approximate guide to the sequenceof events occurring with respect to side-side apposition and occlusion of theneural lumen. For this reason, an overview of this phenomenon in the mouse willbe presented here, using representative histological sections through the neuraltube at different levels along the neuraxis in a selection of embryos isolatedbetween the early afternoon of the 9th day to late on the 10th day of gestation.

The earliest embryo in which a moderate degree of occlusion was observed,had about 10-12 pairs of somites present, and had been isolated in the afternoonon the 9th day of gestation. This embryo was still 'unturned', but from its overallappearance was likely to have 'turned' to adopt the characteristic foetal positionwithin a matter of hours. Only a small region of complete luminal occlusion ispresent, though extensive areas of side-side apposition and partial luminalocclusion are evident at various levels along the spinal axis. Representativesections through the neural tube at different levels are illustrated in Fig. 1.

The appearance of the neural lumen in an embryo at a slightly later stage ofdevelopment, isolated in the evening on the 9th day of gestation, will now beconsidered. The cephalic neural folds in this embryo had yet to become apposedand fused in the regions overlying the presumptive fore-, mid- and hindbrain.The embryo was partially 'turned' and had approximately 12-14 pairs of somitespresent. As in the previous embryo, complete luminal occlusion is only evidentover a relatively short segment, though side-side apposition and partial luminalocclusion involving extensive regions of the neural tube are clearly seen.Representative sections through the neural tube at different levels along thespinal axis in this embryo are illustrated in Fig. 2.

The next embryo in this series was almost completely 'turned' and had alsobeen isolated in the evening of the 9th day of gestation. The embryo had about

Fig. 1. Representative transverse sections through the neural tube of an 'unturned'embryo with 10-12 pairs of somites present, isolated in the afternoon on the 9th dayof gestation. Sections stained with methylene blue. (A) Section through the neuraltube at the midcardiac level. Note that the neural lumen is widely patent, and thenotochord (arrowed) clearly seen. (B) Low-magnification view through the 'thor-acic' region of this embryo. The section is taken through the caudal one third of theheart, and through the midtail region. Key: a, atrium; v, ventricle; y, yolk sac; m,amnion. (C) Higher magnification view through the neural tube in the 'thoracic'region at a level identical to that illustrated in B. A slight indication of side-sideapposition is apparent at this level. (D) Section through the neural tube at the levelof the sinus venosus, at the caudal extremity of the heart. A considerable degree ofside-side apposition is seen, particularly in the central region of the neural tube,though the neural lumen is still patent at this level. (E) Section through the neuraltube about half way between the sections illustrated in D above and F below, somedistance proximal to the U-shaped lordotic segment in this 'unturned' embryo. Themiddle third of the neural lumen (region between arrows) appears to be completelyoccluded. (F) Section through the neural tube just proximal to the lordotic segment.The dorsal half of the lumen appers to be completely occluded whereas the ventralsegment is still patent. Bar = lOOjton.

Neural luminal occlusion in mouse embryos 21520 pairs of somites present, and the cephalic neural folds were completely fused.The region of apposition and occlusion in this embryo was also quite extensive,involving the neural tube from the hindbrain region just rostral to the otic pits,at about the level of the middle of the first branchial arch, caudally almost as faras the caudal neuropore. The appearance of the neural tube in this embryo isillustrated in Fig. 3.

The next embryo was isolated at about midday on the 10th day, had obviousforelimb buds and about 25 pairs of somites present. This embryo also had anextensive region of close side-side apposition with intermittent regions of partial

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luminal occlusion. At no level, however, did the neural lumen appear to becompletely occluded. The appearance of the neural tube in this embryo isillustrated in Fig. 4. The transmission electron micrographs which illustrate thesites of apposition and occlusion in more detail were all taken from this embryo.

The next embryo in this series was also isolated at about midday on the 10thday of gestation, had about 25 pairs of somites and easily recognisable forelimbbuds present. A moderate degree of luminal apposition was present in thisembryo involving principally the ventral third of the neural canal. The apposed

Fig. 2

Neural luminal occlusion in mouse embryos 217Fig. 2. Representative transverse sections through the neural tube of a partially'turned' embryo with 12-14 pairs of somites present, isolated in the evening on the9th day of gestation. Sections stained with methylene blue. (A) low-magnificationview at the level of the rostral one-third of the heart. Some degree of side-sideapposition of the walls of the neural tube are apparent. Key: f, foregut; v, ventricle;w, 'thoracic' wall. (B) Low-magnification view of section though the midcardiaclevel. Note the considerable diminution in the volume of the foregut compared to thesituation illustrated in A above, and the almost complete obliteration of the neurallumen. Key: a, atrium, v, ventricle; f, foregut. (C) Low-magnification view throughthe mid-U region of the neural tube. In the proximal (lower) segment the lumen isvirtually occluded, whereas in the distal (upper) segment the ventral half of thelumen is widely patent. Large blocks of somites (arrowed) are apparent on either sideof the neural tube. (D) Higher magnification view of section through the neural tubeat the level of the rostral one-third of the heart, just distal to the section illustratedin A above. A degree of side to side apposition is apparent. E. Section through theneural tube at a similar level to that illustrated in B above. Apart from a short dorsalsegment (above), the majority of the neural lumen at this level appears to be com-pletely occluded. (F) Section through the neural tube just rostral to the lordoticregion. The neural lumen appears to be completely occluded. Bar = 100 (xm.

Fig. 3. Representative transverse sections through the neural tube of an almostcompletely 'turned' embryo with about 20 pairs of somites present, isolated in theevening on the 9th day of gestation. Sections stained with methylene blue. (A)Section through the cephalic region just rostral to the otic pits and slightly distal tothe origin of the 1st branchial arches (arrowed). Some degree of dorsal and par-ticularly ventral side to side apposition is apparent in the hindbrain region (h). Key:f, forebrain; g, foregut. (B) Section through the hindbrain at the level of the otic pits(arrowed). Note that the neural lumen is completely occluded. Key: f, foregut; 1,first branchial arch; 2, origin of the second branchial arch. Bar = 300 jjm. (C) Sectionthrough the 'thoracic' region at the level of the distal one-third of the heart. Theventral two-thirds of the neural lumen is occluded at this level. Key: a, atrium, priorto division into right and left sides; v, ventricle. (D) Section through embryo at thelevel of the two horns of the sinus venosus (s). The neural tube is virtually completelyoccluded at this level. (E) Slightly oblique section through embryo just above theforelimb bud (arrow). The lumen in the ventral half of the proximal region of theneural tube (upper) is almost completely occluded, while in the distal region (lower)the lumen, though narrow, is completely patent. (F) Section through embryo justbelow the forelimb bud. The middle two-thirds of the neural tube shown hererepresents the occluded proximal ventral segment of the lumen illustrated in Eabove. The paraxial blocks of somites are clearly seen at this level.

Fig. 4. Representative transverse sections through the neural tube of an embryowith approximately 25 pairs of somites present, isolated at about midday on the 10thday of gestation. Sections stained with methylene blue. (A) Section through fore-and hindbrain regions of embryo at the level of the first branchial arch. Key: f,forebrain; h, hindbrain; 1, first branchial arch; t, tail region. (B) Section through'thoracic' region at the outflow of the heart. This section also passes through thehindbrain at the level of the otocysts. Key: o, otocyst, b, bulbus cordis; v, ventricle.(C) Slightly oblique section through embryo at the level of the forelimb bud (arrow).(D) Higher magnification view of neural tube in hindbrain region at the levelillustrated in A above. A considerable degree of side to side apposition is apparentin the ventral third of the neural tube. Note that the notochord is adherent to theendodermal lining of the oropharynx at this level (arrow). (E) Neural tube in hind-brain region at level illustrated in B above. The middle segment of the neural lumenis completely occluded. (F) Neural tube proximal to the forelimb bud at levelillustrated in C above, bar = 200 jum. The majority of the ventral half of the neurallumen is completely occluded. (G) Neural tube distal to the forelimb bud at levelillustrated in C above. (H) Neural tube in lower trunk region at level illustrated inA above. (I) Neural tube in lower trunk region just proximal to the caudalneuropore.

218 M. H. KAUFMAN

Fig. 3. For legend see p. 217.

Neural luminal occlusion in mouse embryos

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219

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Fig. 4. For legend see p. 217.

220 M. H. KAUFMAN

segment extended for a short distance both rostrally and caudally from the levelof the forelimb bud. This, and the previous embryo, appeared externally to beat an almost identical stage of development, but the extent and overall patternof apposition and luminal occlusion in these two embryos were obviously quitedissimilar. The pattern seen in this, the last embryo in this series, however,represents the more typical situation observed at this period of embryogenesis.The appearance of the neural tube in this embryo is illustrated in Fig. 5.

Of three plastic-embedded embryos examined that had been isolated early onthe 11th day of gestation, no evidence of occlusion, or even of close side-sideapposition was observed.

Reference to the author's collection of paraffin-embedded material coveringthe period studied, however, would appear to suggest that the incidence ofluminal occlusion observed in embryos fixed on the 9th and 10th days is extreme-ly low: only 3 out of 58 embryos had evidence of partial luminal occlusion. In allthree cases, the embryos had about 25 pairs of somites present. Only a shortpartially occluded segment was evident, being located at the level of the forelimbbuds. No evidence of partial occlusion was observed in the material isolated atearlier stages of embryogenesis.

Out of 34 paraffin-embedded embryos isolated throughout the 11th day, 3showed a limited degree of partial luminal occlusion. In all of these embryos,only a short segment of the neural tube appeared to be involved, being locatedeither proximal to the level of the hindlimb bud, at the level of the hindlimb bud,or between the fore- and hindlimb buds.

Somewhat surprisingly, out of five 12th day embryos examined, two hadevidence of partial luminal occlusion with the zone of apposition being locatedin one embryo at the level of the hindlimb buds, and in the second embryo,between the fore- and hindlimb buds. In all but one of the eight paraffin-embedded embryos in which a zone of side-side 'fusion' was present, the lumenwas patent dorsal and ventral to the site of apposition. In only one embryo wasthe dorsal half of the canal completely occluded. Fixation of embryos for 4h inSusa produced considerably less evidence of shrinkage artefacts, particularly inthe more advanced groups studied than 12-24 h fixation in full-strength or half-strength Bouin solution.

It is of interest that a section through the neural tube of a rat embryo with13-20 pairs of somites present, at approximately the mid- to high-thoracic level,appears to demonstrate that complete neural tube occlusion also occurs in the ratembryo at this site (see Figure 1, E, Freeman, 1972). No additional informationon the extent or overall pattern of neural tube occlusion observed in this speciesis, however, available.

2. Ultrastructural appearance of neuroepithelial cell surfaces at sites of closecell-cell apposition

The observations presented in this section are largely derived from an analysis

Neural luminal occlusion in mouse embryos 221

Fig. 5. Representative transverse sections through the neural tube of a second em-bryo with approximately 25 pairs of somites present, isolated at about midday on the10th day of gestation. Sections stained with methylene blue. (A) Section through the'thoracic' region at the level of the distal one-third of the heart. Key: h, distal regionof hindbrain; f, foregut; a, atrium; prior to division into right and left sides; v,ventricle; t, mid-tail region. (B) Section through embryo at the level of the forelimbbud. Canalization of the midgut is just visible (between arrows). Bar = 500/mi. (C)Section through embryo distal to the forelimb bud. (D) Higher magnification viewof neural tube at similar level to that illustrated in A above. (E) Neural tube proximalto the forelimb bud at level illustrated in B above. (F) Neural tube in the lower trunkregion just proximal to the segment illustrated in C above.

EMB78

222 M. H. KAUFMAN

of the electron micrographs taken at various locations along the neural axis fromthe embryo illustrated in Fig. 4. Other embryos at slightly different stages ofdevelopment (both more and less advanced) were also examined, but therepresentative sections from this embryo serve to illustrate the principalultrastructural features observed at the neuroepithelial cell surface in embryosin which occlusion of the neural lumen is taking place.

In locations where cellular contact had been made across the midline, a narrowluminal slit was still evident. Initial contacts were usually established betweenrelatively small or occasionally quite large cellular protruberances. Characteris-tically, the majority of the apposing cell surfaces were either completely flat-tened or had a few small undulations (see Fig. 6A, B). Even the occasional smallcellular protrusion observed appeared to have a relatively wide cross section (seeFig. 6A). At locations of very close apposition (e.g. see Fig. 6C), only a few areaswhere minimal 'point' contact was established were generally seen. At the cellsurface, the occasional presence of coated pits was noted, while in the subcorticalzone, small numbers of coated vesicles were also apparent.

In areas where extensive cell-cell contact had been established, only smallpockets of luminal fluid remained between these sites of neuroepithelial cell-cell'fusion'. Despite a detailed search along lengthy stretches where the lumen wascompletely occluded (see Fig. 6D-E), no convincing evidence of junctionalcomplexes linking the two sides was seen, even though the apical complexesbetween adjacent neuroepithelial cells were clearly apparent.

Cellular contact was generally established between two non-dividing cells, butwas also not uncommonly observed between.a non-dividing and a dividing cell(see Fig. 6C-D).

In the ruthenium-red-treated group, a thin layer of positively staining materialwas usually apparent on the neuroepithelial cell surface in regions of closecell-cell apposition, and extended distally until sites of complete neural tubeocclusion were encountered. In those embryos in which there was complete

Fig. 6. Transmission electron micrographs illustrating appearance ofneuroepithelial cell surfaces at sites of cell-cell apposition and luminal occlusion.These are representative thin sections from various sites along the neural axis of theembryo illustrated in Fig. 4.(A) and (B) Apposing neuroepithelial surfaces bridged by relatively large cellularprotrusions. Between these sites of initial contact, the cell surfaces are either com-pletely flattened or only slightly undulating. Both micrographs X2600. (C) Extensiveregion of close apposition in which a narrow luminal channel is still visible. Note theoccasional presence of coated pits (arrowheads) and coated vesicles (arrows). X3700.(D) Extensive regions in which the neural lumen is completely obliterated, inter-spersed with small lacunae containing 'spinal' fluid. x3100. (E) Higher magnificationview of region in which the neural lumen is completely obliterated. Note the presenceof coated pits and vesicles with granular contents (arrowed) in close proximity to theneuroepithelial cell surfaces, and the absence of junctional complexes between theapposed neuroepithelial cell surfaces. In this, as in A-D above, apical junctionalcomplexes are observed between adjacent neuroepithelial cells, x 12 500.

Neural luminal occlusion in mouse embryos 223

r* > •

224 M. H. KAUFMAN

7A

Neural luminal occlusion in mouse embryos 225occlusion of the neural lumen, however, the neuroepithelial cell surfaces in non-occluded regions just distal to the initial sites of occlusion generally remainedunstained, presumably because the ruthenium red failed to penetrate beyond theoccluded segment. Examples of electron micrographs of the neuroepithelial cellsurface in ruthenium-red-stained and unstained control material are presentedin Fig. 7.

DISCUSSION

It seems evident from this and previous experimental and descriptive studieson neural tube occlusion (chick: Desmond & Jacobson, 1977; man: Desmond,1982) that there are considerable species differences in the overall pattern ofneuroepithelial cell apposition and luminal occlusion along the spinal axis. Theunderlying morphogenetic processes which eventually bring about thisphenomenon are obviously rather complex, and presumably involve an interplaybetween a series of morphological changes which must occur within the cellularcomponents of the neural tube, and extrinsic cellular and extracellular 'forces'which act on the neural tube from without (see, for example, Schroeder, 1971;Karfunkel, 1974). Once medial migration of the walls of the neural tube has beeninitiated, close apposition and eventual, albeit transient, 'fusion' may be theinevitable consequence. It is possible that the first contact may be establishedacross the midline by the interdigitation of relatively small diameter projectionswhich protrude from the surfaces of the many neuroepithelial cells whose apicesabut on the spinal lumen. The initial contact and adhesion may in addition befacilitated by the presence of viscous ruthenium-red-positive extracellularmaterial at the luminal surface of these cells.

An increase in ruthenium-red-positive material has been demonstrated alongapical neural fold borders and on the overlying ectoderm cells in regions im-mediately prior to and at the time of neurulation (Moran & Rice 1975; Sadler,1978), and its removal at this time can interfere with neural tube closure (Lee,Sheffield, Nagele & Kalmus, 1976; O'Shea & Kaufman, 1980). The presence ofcarbohydrate-rich surface coat material is also observed along prospective zonesof fusion in the palate (Greene & Kochhar, 1974; Pratt & Hassell, 1975) andduring fusion of the medial and lateral nasal processes (Gaare & Langman, 1977;

Fig. 7. Unstained transmission electronmicrographs from two embryos with about15-20 pairs of somites present to illustrate the presence of extracellular material atthe surface of neuroepithelial cells in sites of close cell-cell apposition. Both em-bryos were isolated at about 6 p.m. in the evening on the 9th day of gestation. (A)Ruthenium-red-stained section showing the presence of a thin coating of positivelystained material at the neuroepithelial cell surface which apparently fails topenetrate beyond the apical junctional complexes between adjacent cells. x6100.(B) Higher magnification view of ruthenium-red-positively staining material at theneuroepithelial cell surface, x 14 250. (C) Appearance of neuroepithelial cell surfacein unstained control embryo, x 14 500.

226 M. H. KAUFMAN

Smuts, 1977). In all of these locations, a decrease in the distribution of surfacemacromolecules is observed after fusion.

Possibly slightly later, after initial side-side contact has taken place, the cellswith mound-like and flattened surfaces become more closely apposed, and allowcontact to be established over much more extensive, though still localized, areas.Concomitant with the progressive increase which occurs in the surface area ofcontact, the luminal volume necessarily decreases.

The underlying mechanism(s) which eventually lead to the complete occlusionof the spinal lumen is still far from clear, though the absence of large numbersof pinocytic vesicles in the subcortical region of the neuroepithelial cells tendsto suggest that absorption of the luminal fluid probably plays only a minor rolein its removal from this site. The presence of moderate numbers of coatedvesicles with granular contents in the subcortical zone and coated pits, which maywell be their precursors (see Pratten, Duncan & Lloyd, 1980), at the cell surface,may be indicative of their role — possibly facilitating the removal of excess extra-cellular matrix material at the fusion site - during the final stages of appositionand cell-cell adhesion.

Curiously, despite the presence of obvious junctional complexes between theapical zones of adjacent neuroepithelial cells, no convincing complexes of anytype could be discerned at the fusion interface. While this does not unequivocallyexclude the possibility that some type(s) of specialized complexes are in factformed, clearly other techniques e.g. freeze fracture analysis, would be requiredto demonstrate them.

In the mouse, unlike the situation in the human embryo (see Desmond, 1982),since apposition and fusion is first apparent in embryos with about 10 pairs ofsomites when both the rostral and caudal neuropores are still widely open, thiswould seem to be evidence in favour of the hypothesis that at least at thisrelatively early stage of embryogenesis most of the luminal fluid is probablydisplaced caudally or cranially (into the amniotic cavity) rather than resorbedlocally. However, once the rostral neuropore, in particular, has closed (fortiming, see Geelen & Langman, 1977; Kaufman, 1979), and complete luminalocclusion occurred some distance caudally, it seems likely that the productionand only limited resorption of cerebrospinal fluid would facilitate the dilation ofthe brain and optic vesicles. While a situation similar to this appears to occur inman and in the chick embryo, this is obviously a considerable oversimplification,as in the majority of mouse embryos, for example, the neural lumen becomespatent along its entire length just before the caudal neuropore eventually closes(for timing, see Copp, Seller & Polani, 1982). Presumably, once 'cerebral' dila-tion has been initiated, the equalization between the CSF pressure and theamniotic fluid pressure - which occurs after the lumen becomes patent along itsentire length, and remains thus at least until the caudal neuropore closes - doesnot appear to be detrimental in any way, at least in the mouse.

While the above account is undeniably somewhat speculative, at least as far

Neural luminal occlusion in mouse embryos 227as providing a detailed picture of the underlying mechanism of cell-cell fusionin this location, it does appear to confirm the contention that neural luminalocclusion probably plays an important morphogenetic role in the developmentof the vertebrate nervous system.

Apart from answering certain questions regarding the timing and overall pat-tern of events in the mouse, this study raises other important questions. Forexample, it would be of considerable interest to know whether chemical messen-gers play a role at any stage in guiding the two morphologically indistinguishableneuroepithelial cell surfaces together. Equally, an analysis of the events occur-ring when the two sides separate once more late on the 10th or early on the 11thday (in the mouse) would be instructive. Similarly, descriptive and experimentalobservations on neuroepithelial cell-cell apposition and luminal occlusion inother vertebrate species might enable both the full significance of thisphenomenon and its evolutionary history to be established.

This work was supported by a grant from the National Fund for Research into CripplingDiseases. I thank Mr. J. Skepper and Mr. M. Wombwell for their expert technical assistance,and Mr. J. Skepper additionally for his invaluable advice on the interpretation of the electron-micrographs.

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{Accepted 24 August 1983)