Cong. Anom., 30: 5-16,1990 original Morphological analysis of neural tube 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-Neurulation-Mouseembryo-Chlorambucil-Neuroectoderm 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 optic vesicle 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 neural fold 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.