Cong. Anom., 30: 5-16,1990 original

Morphological analysis of defects: Chlorambucil- induced exencephaly in mice

Osamu TANAKA, Natsumi YOSHIOKA, Takafumi YOSHIOKA, Hiroyuki NAORA and Toshihisa HATTA Department of Anatomy, Shimane Medical University, Enya-cho 89-1, Izumo 693, Japan

ABSTRACT Exencephaly has been induced in mouse embryos by chlorambucil (CA), a cytotoxic agent. To understand the course of development of this mal- formation, open neural tube defects in CA-treated mice were examined using light and scanning electron microscopes (SEM). CA was given to the pregnant mice on day 7.4 of gestation. Embryos were removed and futed on gestational days 9.3- 9.4, 9.7-9.8 and 10.4, and compared to control embryos from untreated mice. By gestational day 9.4, all control embryos had closed neural tubes, except for the posterior neuropores, and well developed brain vesicles. By contrast, in the ex- perimental embryos the frequencies of open neural tube were 26/33 (78.8%), 32/ 40 (80.0%) and 23/66 (34.8%) on each day examined, respectively. Open neural tube defects were classified into six patterns according to the location and magni- tude of the open area. The patterns of open neural tube on day 9.7-9.8 were diverse; however, in almost all cases on day 10.4 the open neural tube appeared in a region from the caudal forebrain to the rostral hindbrain. It was evident that an unusual pattern of closure of the neural tube was involved in forming the cranial neural tube. The present study shows that failure of closure of the cranial neural tube in the CA-treated mouse embryos can be defined as a primary neural tube defect (NTD), which can be, in part, repaired by unusual closure of the neural tube. At high magnifications of SEM, the neuroectodermal surfaces of the 9.0-day affected embryos often had a number of slender processes projecting from the neuroepithelial cells. “Ruffles” and “blebs” at the lateral edges of the neural folds were observed in both control and affected embryos. Key words: Exencephaly--Mouseembryo-Chlorambucil-

Neural tube closure defect (NTD) is one of the most common congenital malformations in hu- mans. NTD can be induced experimentally by a variety of exogenous teratogens (Morrissey and Mottet, 1980). Although it has been described that the teratogens affect primarily the neuroepi- thelium or the mesenchyme lining the neural tissue (Marin-Padilla, 1966; Morris, 1973; Theodosis 6 0. Tanaka et a1 and Fraser, 1978; Geelen et al., 1980; Wiley, 1980; Frantel et al., 1981; Nagele et al., 1981; Willhite, 1981; Horton and Sadler, 1983; Iijima et al., 1983; Yoshioka et al., 1984; Shinohara et al., 1985), the morphogenetic mechanism of the genesis of the NTD remains unclear (Tanaka, 1988). In addi- tion, it is difficult to determine whether the abnormalities observed during early neurulation are in fact early stages in the development of exencephaly because the occurrence of exencephaly in most experimental situations is never 100% (Putz and Morriss-Key, 1981). On the other hand, it is in- adequate to analyse the genesis of the exencephaly based on observations made during the late stages of development when the malformations have been fully expressed (Morris, 1972; Jurand, 1973; Randall and Taylor, 1979; Putz et al., 1980; Smith et al., 1982; Morrissey and Mottet, 1983). Exencephaly in mice, which was either induced by chemical teratogens such as oxygen (Morris and New, 1979), ochratoxin or concanavalin A (Hayasaka et al., 1986) and cadmium (Webster and Messerle, 1980) or observed in mutants with systemic chromosomal disorder, (Putz and Morris-Key, 1981), resulted from failure of the midbrain neural folds to appose or fuse. In general, experimental- ly induced exencephaly carried the defect from the caudal forebrain to the rostral hindbrain. We have previously reported that exencephaly can be induced in mice by chlorambucil, a type of nitrogen mustard, which causes rapid cell death and subsequent increase of extracellular space in the neuroepithelium (Yoshioka et al., 1984; Shinohara et al., 1985). As with other experimental- ly induced exencephaly, there is a neural overgrowth in the region from the caudal forebrain to the rostral hindbrain. Why does the exencephaly in mice often appear in regular region of the brain? To elucidate the course of closure of the cranial neural tube, embryos with chlorambucil-induced NTD were examined using a scanning electron microscope.

MATERIALS AND METHODS

Jc1:ICR virgin mice, 8-12 weeks old, were placed overnight with males of the same strain. The beginning of pregnancy was timed to 0:OO of the day when the vaginal plug was found and 12:OO of the same day was defined as 0.5 day. Chlorambucil (CA) (SIGMA Chemical Co.) suspended in cotton seed oil was given per 0s to the pregnant mice on approximately gestational day 7.4. On days 9.3-9.4, 9.7-9.8 and 10.4, embryos were removed in Tyrode solution and were rapidly trans- ferred to a half-strength Karnovsky’s fixative. The number of freely collected embryos was 33 (open neural tube: 26, closed neural tube: 7) from 5 litters at 9.3-9.4 days, 40 (open neural tube: 32, closed neural tube: 8) from 4 litters at 9.7-9.8 days and 66 (open neural tube: 22, closed neural tube: 42) from 5 litters at 10.4 days. Embryos with an open neural tube were examined using a scanning electron microscope (SEM) and embryos with a closed neural tube were observed by light microscopy. Embryos for SEM were fixed initially for periods from three hours to three days, then washed in phosphate buffer (O.lM, pH 7.2). Materials were postfixed with a 0.1M phosphate-buffered 1% osmium tetroxide for two hours, rinsed with buffer solution, stained overnight with 1% tannic acid, rinsed again and stained with O.1M phosphate-buffered 1% osmium tetroxide for one hour (Murakami, 1976). After dehydration in a graded series of ethanol they were treated with isoamyl acetate and dried using the critical point method with liquid COz. The dried specimens were coated with platinum-palladium in a vacuum evaporator and viewed under a Hitachi S-450 SEM operated at Morphological analysis of neural tube defects in mice I

10-20 kV. For light microscopy, the fixed materials were rinsed with phosphate-buffered solution, post- futed with the phosphate-buffered 1% osmium tetroxide for two or three hours, dehydrated in a graded series of ethanol and finally embedded in Epoxy resin. Sections 0.5-1.0 /.irn thick were ob- tained using a ultramicrotome (OmU4, Reichert-Jung) and then stained with 1% phosphate-buffered toluidine blue. Intact embryos at 8.4-8.8 and 9.4 days of gestation were obtained from untreated pregnant mice and examined by SEM and light microscope.

RESULTS

A. Normal neurulation in the cephalic region Normal neural folds during late neurulation were observed by SEM. In all embryos at 8.7 days of gestation, the fusion which had initiated from the caudal hindbrain (Sakai, 1989) proceeded to the level of the otic pit in the hindbrain (Figs. 1C and D). On the other hand, independent of the hindbrain closure, neural folds of the caudal forebrain were fused or nearly apposed, but the lower part of the was still open (Figs. 1A and B). Neural folds of the midbrain and the rostral hindbrain had a concave shape, apposed in each dorsal midline. When the lateral edges of the closing neural folds were examined at a higher magnification, the lamellopoidal membrane extensions “ruffles” were frequently present. The rounded cellular profiles “blebs” were also often visible on the lateral edges and the lateral surfaces of neural folds. By gestational day 9.4 all intact embryos had a completely closed neural tube, both in the cranial and spinal regions, except for the posterior neuropore. The otic pits were well developed as deep holes, and the pharyngeal arches were distinct.

B. Topography of the of the embryo with open neural tube 1. Low magnification SEM examinations of embryos with open neural tubes at three different stages, i.e., 9.3-9.4, 9.7-9.8 and 10.4 days of gestation, revealed either a delay or failure of cranial neural folds develop- ment and of the morphogenetic movement. Open neural tubes were classified into six patterns according to location and magnitude of the open area (Fig. 2). Table 1 shows the distribution of embryos with open neural tubes according to the stages of examination and patterns of the open neural tube. At 9.3-9.4 days of gestation, embryos displayed all six patterns of open neural tube with fair frequencies (Table 1). Regardless of the patterns, the neural folds of the midbrain in almost all embryos were opened and the dorsal edges of the folds were seen to be curved outward, whereas the forebrain neural folds showed an apparent concave shape resulting from the formation of well-shaped optic vesicles (Fig. 3A). As an exception, the optic vesicles in two embryos were not formed and their neural folds were convex in shape (Fig. 3B). Otic pits and pharyngeal arches were as developed as in the controls at same stage. At 9.7-9.8 days, the frequency of patterns 1 and 4 decreased, while that of pattern 2 increased. That is, at this stage a greater number of embryos with open neural tubes had a closed forebrain rostral to the optic vesicle and a developed telencephalic hemisphere (Fig. 4A). The frequency 8 0. Tanaka et al.

Fig. 1 Normal neural folds during late neurulation in an 8.7-day intact mouse embryo. A) Anterior view. XlOO B) High-magnification of A). X1300 C) Dorsal view. X120 D) High-magnification of C). X900

of embryos with open rostral forebrain decreased from 38% at 9.3-9.4 days to 6% at 9.7-9.8 days. The neural folds of patterns 1, 2 and 3 had slightly biconvex shapes (Fig. 4A). The open areas of pattern 6 in 9.7-9.8-day embryos were smaller than those in 9.3-9.4-day embryos (Fig. 4B). Otic pits observed at the preceding stage had developed into otic vesicles. Morphological analysis of neural tube defects in mice 9

Table 1 The number of embryos with neural tube closure defect

No. of No. of No. of Patterns of ONT* Stage litters embryos embryos examined with ONT* 123456

9.3 - 9.4 5 33 26 752345

9.7 - 9.8 4 40 32 2 17 3 37 10.4 5 66 22 1 8 12 1

ONT*: open neural tube

@ A N anterior-neuropore FM : The boundary of forebrain and midbrain 0 P : otic pit

,...... :Open area --- ' - - :Variation Fig. 2 Illustration of patterns of open neural tube. Pattern 1: All the anterior region from the rostral part of the hindbrain is open. Pattern 2: The area from the caudal forebrain or the boundary between forebrain and midbrain (FM) to the caudal end of the midbrain or rostra1 hindbrain is open. Pattern 3: The area from the rostral midbrain to the caudal end of the midbrain or rostral hindbrain is open and the region of midbrain near FM is closed. Pattern 4: All the anterior region from the apex of midbrain is open. Pattern 5: The rostral midbrain with/without the caudal forebrain is open. Pattern 6:Small area in the midbrain is open.

At 10.4 days, the number of embryos with open neural tubes.was less than the two preceding stages and almost all affected embryos had open neural tubes of patterns 2 and 3 (Table 1). The opening of the neural tube was seen in the domain (area) from the caudal forebrain (the 3rd ventricle) or the rostral midbrain to the caudal end of the midbrain or the rostral hindbrain. This area coin- cides with that of exencephaly in later periods of gestation. At this stage, typical pattern 2 neural tube openings were observed. In the forebrain region, the open 3rd ventricles were recognized showing on one or both sides the convex-shaped thalamic region, and the telencephalic hemispheres 10 0. Tanaka et al.

Fig. 3 A) Anterior view of a 9.3-9.4-day embryo with pattern 1 open neural tube. X90 B) Anterior view of a 9.3-9.4- day embryo with pattern 1 open neural tube. X130 Note the convex shape of neural fold in the forebrain.

Fig. 4 A) Anterior view of a 9.7-9.8-day embryo with pattern 2 open neural tube. X90 B) Anterior view of the 9.7-9.8-day embryo with pattern 6 open neural tube. X70

were developed. The unfused neural folds in the midbrain were remarkably everted and the mid- brain floor, which lies on top of the head, was exposed. Half of the embryos had an open rostra1 hindbrain (Figs. 5A and SB). At this stage, almost all embryos had completely closed spinal neural tubes including the posterior neuropore. There were two embryos with patterns 1 and 5. In the Morphological analysis of neural tube defects in mice 11

Fig. 5 A) Lateral view of a 10.4-day embryo with pattern 2 open neural tube. X40 B) Lateral view of a 10.4-day embryo with pattern 3 open neural tube. X35

Fig. 6 A) The at the inferior part of the optic vesicle in a 9.8-day embryo. X800 B) The roof and lateral walls of the rostra1 hindbrain in a 9.7-9.8-day embryo. X1800 embryo with pattern 1, the telencephalic floor was biconvex in shape and exposed anterioly. For- mation of the optic vesicles in this embryo was nil. 2. High magnification Detailed observations with higher magnifications revealed that the closure form of the neural 12 0. Tanaka et al.

Fig. 7 Light micrographs of transverse sections of embryos with closed neural tube. Toluidine blue. A) The level of the forebrain in a 9.4-day intact embryo. X80 B) The level of the forebrain in a 9.7-9.8-day treated em- bryo. XI30 C) The level of the midbrain in a 9.7-9.8-day treated embryo. XlOO D) High magnification of B. X215

tube and the surface topography of neuroectodermal cells in the affected embryos differed from those of the control embryos. Fig. 6A shows the ectodermal surface of the rostral forebrain of a 9.8-day embryo. The occasionally formed cellular bridges over the lateral edge, which fused with the opposite neural folds at several points. The neuroectodermal surface of the 9.7-9.8-day affected embryos often had a number of slender processes projecting from the neuro- epithelial cells (Fig. 6B). These processes were in bundles and occasionally intertwined. These struc- tures were not observed in 10.4-day treated embryos or in normally neurulating ones. “Ruffles” and “blebs” observed during normal neurulation were also evident in the affected embryos.

C. Histology of the embryo with closed neural tube When the heads of treated embryos with closed neural tube were examined in semi-thin sections with light microscope, an abnormal morphogenesis of the ventricles was often observed. In contrast to the symmetrical development of the brain vesicles in intact embryos (Fig. 7A), the affected brain exhibited severe asymmetry (Figs. 7B and 7C). In almost all cases, some of the vessels forming peri- neural vascular plexus were extremely enlarged, and consequently, the neuroepithelial walls were Morphological analysis of neural tube defects in mice 13 seen to protrude into the ventricles (Fig. 7B). The neuroepithelial cells in the abnormal brain vesicles appeared to be in a pseudostratified arrangement, as in the intact brains. Densely stained intracellular inclusions, which might be degenerated cell materials phagocytosed by neighboring cells (Shinohara et al., 1985), were found in protruding regions of the neuroepithelial wall. In addition, the ventri- cular surface of affected brains was rugged and a small number of cells departed from the neuro- epithelial layer into the ventricle (Figs. 7B and 7D). As shown in Fig. 7D, those dispossessed cells seen at the region where the ventricular surfaces of both neuroepithe1,ial walls were closely apposed as a result of the abnormal bulging of the walls, appeared to form cellular bridges across the ventricle between the neuroepithelial walls.

DISCUSSION

Two possible gross morphogenetic mechanisms involved in the genesis of exencephaly have been proposed: reopening of a previously closed neural tube (Gardner, 1973; Webster and Messerle, 1980) and failure of the neural tube to close (Morrissey and Mottet, 1980; Putz and Morriss-Key, 1981). The present investigation revealed that exencephaly induced by treatment with chlorambucil is due to a primary nonclosure of the cranial neural tube. The behaviors of neural folds during normal cranial neurulation have been described in rats, hamsters and mice (Waterman, 1975 and 1976; Geelen and Langman, 1977; Putz and Morris-Key, 1981; Jacobson and Tam, 1982). More recently, the course of cranial neurulation in mice was described in details by Sakai (1989). The first closure occurs in the cervical region and proceeds both rostrally and caudally, the second starts at the rostral end of and proceeds rosto- dorsally, and the third occurs in the caudal diencephalon and proceeds both rostrally and caudally. Independently, MacDonald et al. (1989) described that, when the first closure of the neural folds proceeding rostrally from the cervical region reaches the level of the otic pit, the second closure begins at the region between the fore- and midbrain and proceeds caudally and rostrally until the fusions reach the rostral portion of the hindbrain and the anterior neuropore, respectively. Thus, the closure process of cranial neurulation continues in a regular manner (one or two directions) from the independent points along the neural axis. This moFphogenetic pattern was also observed in the present study, that is, the cranial neural folds in 8.7-day intact embryos were fused at the caudal hindbrain and forebrain and the neural folds of the open area were concave in shape. In the 9.3-9.4-day treated mouse embryos, various patterns of open cranial neural tubes were identified. Most of them were deviated from the sequence of morphogenetic movement of normal neurulation. Therefore, we can define the failure of closure of the cranial neural tube in 9.3-9.4- day embryo as the primary NTD. The various patterns of open neural tube we observed also suggest that the progression of fusion of the neural folds does not necessarily require a regular sequence of neurulation, as noted in the intact embryo (Sakai, 1989; MacDonald et al., 1989). Some embryos with pattern 2 showed closure of the rostral forebrain, without fusion of the caudal forebrain. In case of patterns 4 and 5, closure of the caudal midbrain preceded that of the rostral midbrain and/or forebrain. In some with pattern 6, the region between the fore- and midbrain which was expected to close earlier remained open. Excluding the anterior neuropore, the last part to close during normal cranial neurulation is the 14 0. Tanaka et al.

region between the caudal midbrain and rostral hindbrain. Morriss and New (1979) suggested that neurulation of this region might be associated with a longitudinal stretch force, that is, a longitudinal pull at the surface ectoderm level, which is generated by an increase in cranial . However, in the present study, the observed patterns of open neural tube in 9.3-9.4-day embryos have features which are incompatible with this suggestion. As shown in eleven embryos with patterns 4 and 5, the region rostral to the apex of midbrain is open, while the caudal midbrain and rostral hindbrain are closed. The following postulations are proposed for the unusual closure of the cranial neural folds: 1) if the third fusion in the caudal forebrain fails, the preceding first and second closures may progress over the promised area during normal neurulation, 2) the fourth fusion and subsequent closure may occur in the rostral hindbrain, as shown by MacDonald et al. (1989), and 3) there are other forms of fusion of the cranial neural folds different from the normal neurulation. If it is true, these events may be due to intrinsic forces such as contraction of apical surfaces of the neuro- epithelial cells using microfilaments and elongation of these cells by the microtubules (Karfunkel, 1974) and/or extrinsic forces such as the formation of the optic vesicles (Kaufman, 1979; Putz and Morriss-Key, 198 1). The developmental transition in the patterns of open area during the examined stages and the decrease in the rate of embryos with an open neural tube during the different stages, indicate that the affected neural tubes may be repaired by unusual closures of neural folds. Indeed, in embryos with patterns 4, 5 and 6, the open area became smaller and further smaller at 9.7-9.8 days and 10.4 days, respectively. By gestational day 10.4, only one embryo had these patterns. Therefore, it seems that the open areas in these patterns may be closed during the following development. It is, how- ever, evident that the embryos with a closed neural tube had an abnormal development of the brain vesicles. Other investigators proposed various conditions responsible for the fusion of cephalic neural folds, such as differences in the modes of closure between fore-, mid- and hindbrain (Geelen and Langman, 1979), the initial contact at the level of the presumptive neural-crest cells (Marin- Padilla, 1970; Waterman, 1976), and extensive surface alterations (ruffles or blebs) of neuroepithelial cells in lateral edges (Waterman, 1975; 1976). These conditions may or may not be needed for the new modified neural tube closure. In the present experiment, the open neural tube, examined at 10.4 days, was localized in the midbrain area, and resembled the morphology of the fully developed exencephaly in the late fetal periods. Jacobson and Tam (1982) described that the early appearance of cranial flexure imposed a mechanical impediment to neural tube closure in the midbrain region. The present study shows that the primary NTDs which are caused by direct or indirect effects of a teratogen can be repaired by unusual patterns of neural tube closure. The typical exencephaly, identified in later stages, may be generated by the combination of direct effects of teratogens on the neural tissue and the extrinsic forces described by Jacobson and Tam (1982). The overall pattern of cephalic neurulation and optic vesicle formation in mice is very different from that of humans. This is especially noted in the pattern of forebrain closure and the location of the anterior neuropore (Kaufman, 1979; O’Rahilly and Gardner, 1979). It is, therefore, suggested that the pathogenesis of exencephaly in mice is also different from that of humans, even when the exencephaly is induced by a chemical teratogen. Morphological analysis of neural tube defects in mice 15

ACKNOWLEDGMENTS

The authors are indebted to F. Satow, Shimane Medical University, for technical assistance in preparation of the specimens and to Dr. Antoine Abu Musa for critical comments. This study was supported by a grant (62A - 5) from National Center of Neurology and Psychiatry (NCN) of the Ministry of Health and Welfare, Japan.

REFERENCES

Frantel, A.G., Greenway, J.C., Shepard, T.H., Juchau, Am. J. Anat., 155: 425-444. M.R. and Selleck. S.B. (1981) The teratogenicity MacDonald. K.B., Juriloff, D.M. and Harris. M.J. of cytochalasin D and its inhibition by drug meta- (1989) Developmental study of neural tube bolism. Teratology, 23: 223-231. closure in a mouse stock with a high incidence of Gardner, W.J. (1973) The Dysraphic States from esencephaly. Teratology. 39: 195-213. Syringomyelia to Anencephaly. Excerpta Medica, Marin-Padilla, M. (1966) Mesodermal alterations Amsterdam, pp. 1-14. induced by methyl sulfoside. P. S. E. B. M.. 122: Geelen, J.A.G. and Langman, J. (1977) Closure of 717-720. the neural tube in the cephalic region of the mouse Marin-Padilla, M. (1970) The closure of the neural embryo. Anat. Rec., 189: 625-640. tube in the golden hamster. Teratolog).. 3: 39-46. Geelen, J.A.G. and Langman, J. (1979) Ultrastruc- Morriss, G.M. (1972) Morphogenesis of the mal- tural observations on closure of the neural tube in formations induced in rat embryos by maternal the mouse. Anat. Embryol., 156: 73-88. hypervitaminosis. Am. J. Anat., 113: 241-250. Geelen, J.A.G., Langman, J. and Lowdon, J.D. (1980) Morriss, G.M. (1973) The ultrastructural effects The influence of excess vitamin A on neural tube of excess maternal vitamin A on the primitive closure in the mouse embryo. Anat. Embryol., streak stage rat embryo. J. Embryol. esp. Morph., 159: 223-233. 30: 219-242. Hayasaka, I., Hoshino, K. and Kameyama, Y. (1986) Morriss, G.M. and New, D.A.T. (1979) Effect of Pathogenesis of ochratoxin A- and concanavalin oxygen concentration on morphogenesis of cranial A-induced exencephalies in mice. Cong. Anom., neural folds and in cultured rat em- 26: 11-24. bryos. J. Embryol. exp. Morph.. 54: 17-35. Horton, W.E. and Sadler, T.W. (1983) Effects of ma- Morrissey, R.E. and Mottet, N.K. (1980) Neural tube ternal diabetes on early embryogenesis. Diabetes, defects and brain anomalies: a review of selected 32: 610-616. teratogens and their possible modes of action. Iijima, S., Matsumoto, N. and Lu, C.C. (1983) Trans- Neurotoxicol., 2: 125-162. fer of chromic chloride to embryonic mice and Morrissey, R.E. and Mottet, N.K. (1983) Arsenic- changes in the embryonic mouse neuroepithelium. induced exencephaly in the mouse and associated Toxicology, 26: 257-265. lesions occurring during neurulation. Teratology, Jacobson, A.G. and Tam, P.P.L. (1982) Cephalic 28: 399-41 1. neurulation in the mouse embryo analyzed by Murakami, T. (1976) A revised tannin-osmium meth- SEM and morphology. Anat. Rec., 203: 375-396. od for non-coated scanning electron microscope Jurand, A. (1973) Teratogenic activity of metadone specimens. Arch. Histol. Jpn., 36: 189-193. hydrochloride in mouse and chick embryos. J. Nagele, R.G., Pietrolungo, J.F., Lee, H. and Roisen, Embryol. exp. Morph., 30: 449-458. F. (1981) Diazepan-induced neural tube closure Karfunkel, P. (1974) The mechanism of neural tube defects in explanted early chick embryos. Tera- formation. Int. Rev. Cytol., 38: 245-271. tology, 23: 343-349. Kaufman, M. (1979) Cephalic neurulation and optic O’Rahilly, R. and Gardner, E. (1979) The initial vesicle formation in the early mouse embryo. development of the human brain. Acta anat., 104: 16 0. Tanaka et al.

123-133. Theodosis, D.T. and Fraser, F.C. (1978) Early Putz, B., Kause, G., Garde, T. and Gropp, A. (1980) changes in the mouse neuroepithelium preceding A comparison between trisoiny 12 and vitamin A exencephaly induced by hypervitaminosis A. induced exencephaly and associated malformations Teratology, 18: 219-232. in the mouse embryo. Virchows Arch. Path. Anat. Waterman, R.E. (1975) SEM observations of surface Histol., 368: 65-80. alterations associated with neural tube closure in Putz, B., Morriss-Key, G. (1981) Abnormal neural the mouse and hamster. Anat. Rec., 183: 95- development in trisomy 12 and trisomy 14 mouse 98. embryos. J. Embryol. exp. Morph., 66: 141- Waterman, R.E. (1976) Topographical changes along 158. the neural fold associated with neurulation in Randall, C.L. and Taylor, W.J. (1979) Prenatal etha- the hamster and mouse. Am. J. Anat., 146: nol exposure in mice: teratogenic effects. Tera- 151-172. tology, 19: 305-312. Webster, W.S. and Messerle, K. (1980) Changes in Sakai, Y.' (1989) Neurulation in the mouse: Manner the mouse neuroepithelium associated with and timing of neural tube closure. Anat. Rec., cadminium-induced neural tube defects. Tera- 223: 194-203. tology, 21: 79-88. Shinohara, H., Yoshioka, T. and Tanaka, 0. (1985) Wiley, M.J. (1980) The effects of cytochalasins on Effects of chlorambucil on the ultrastructure of the ultrastructure of neurulating hamster em- neuroepithelial cells of neurulating mouse em- bryos in vitro. Teratology, 22: 59-69. bryos. Shimane J. Med. Sci., 9: 12-20. Willhite, C.C. (198 1) Arsenic-induced axial skeletal Smith, M.T., Wood, L.R. and Honig, S.R. (1982) (dysraphic) disorders. Exp. Molecul. Pathol., Scanning electron microscopy of experimental 34: 145-158. anencephaly development. Neurology, 32: 992- Yoshioka, N., Tanaka, O., Yoshioka, T., Satow, F. 999. and Tatewaki, R. (1984) Experimental study of Tanaka, 0. (1988) Abnormalities of brain differ- open neural tube defects in mouse embryos in- entiation induced by chemical agents. No To duced by chlorambucil. Teratology, 30: 8A Hattatsu. 20: 105-1 14. (Japanese) (Abst.).