differentiation, neuroblast, notochord. Introduction. The generation of neuroblasts in the spinal cord has been the subject of many investigations, which have.
Development 107, 793-803 (1989) Printed in Great Britain © T h e Company of Biologists Limited 1989
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Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo
H. W. M. VAN STRAATEN, J. W. M. HEKKING, J. P. W. M. BEURSGENS, E. TERWINDTROUWENHORST and J. DRUKKER Department of Anatomy/Embryology,
Medical Faculty, National University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands
Summary After implantation of a notochord fragment lateral to the neural tube in a 2-day chick embryo, at 4 days the ipsilateral neural tube half was increased in size and axons left the neural tube in a broad dorsoventral area (van Straaten et al. 1985). This enlargement appears to coincide with an increased area of AChE-positive basal plate neuroblasts, as determined with scan-cytophotometry. The effect was ipsilateral and local: clear effects were seen only when the implant was localized less than 80 fan from the neural tube and over 120 fan from the ventral notochord. In order to investigate the expected enhancement of proliferation, the mitotic density and the number of cells at the site of the implant at 3 days was determined and the mitotic index calculated. All three parameters showed an increase. It was con-
cluded that the cell cycle was shorter in the implant area relative to the control area, at least during the third day. At 4 days the number of cells was still increased, predominantly in the basal plate. It appeared that the numerical increase was for the larger part due to neuroblasts. The synergism of two notochords thus resulted in enhancement of proliferation and differentiation in the neural tube. It is suggested that the notochord merely regulates and arranges the surrounding sclerenchymal cells, which are the effective cells in the regulation of neural tube development.
Introduction
Especially the notochord is involved in several developmental processes of the neural tube. After the induction of the neural plate, the notochord is involved in the differentiation of the floor plate of the neural tube (Weiss, 1955; Watterson et al. 1955; van Limborgh, 1956; van Straaten et al. 1988a), and plays a role in the closure of the neural tube in amphibians (Jacobson, 1984). We have demonstrated that the notochord is also involved in the subsequent morphogenesis of the neural tube. After implantation of an isochronous notochord between the neural tube and somite in a chick embryo of 2 days of age, the size of the ipsilateral neural tube appeared to be increased and axons left the neural tube in a broad dorsoventral area. It was suggested that proliferation and differentiation of basal plate neuroblasts was increased and that the notochord played an important role in the determination of axonal exits (van Straaten et al. 1985). In the present study, we have investigated whether the increase of size of the neural tube after the addition of a notochord is indeed due to an increased number of neuroblasts, and if this phenomenon is preceeded by an enhanced proliferation of the neural tube matrix layer. Acetylcholinesterase appears a useful marker of neuroblasts in the chick neural tube during our period of investigation (Mizo-
The generation of neuroblasts in the spinal cord has been the subject of many investigations, which have resulted in a detailed description of their development. In the thoracic region of the neural tube of the chick embryo, the first neuroblasts arise in the basal plate between 1.5 and 2 days (e.g Fujita, 1963; Lyser, 1964; Miki et al. 1981; Masuko and Shimada, 1983; Bennett and DiLullo, 1985). From 2 to 4 days, the area of highest mitotic density shifts gradually from the basal to the alar plate (Hamburger, 1948; Corliss and Robertson, 1963). In the former, the amount of postmitotic neuroblasts increases steadily, and at 4 days 95 % of the future ventral horn motoneurons are present (Hollyday and Hamburger, 1977). These are located in the ventrolateral region, and form a longitudinal, almost uniform column (Lunn et al. 1987; Layer et al. 1988). Their axons leave the neural tube in the same area. Several investigations demonstrate that development of the early neural tube is modulated by its surroundings. After dorsoventral rotation of the neural tube, this structure adapted completely (Steding, 1962), partially (Martin, 1977) or not (Jacob etal. 1976) to its surroundings, probably depending on the age of the embryo.
Key words: chick embryo, neural tube, proliferation, differentiation, neuroblast, notochord.
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guti and Miki, 1985; Layer et al. 1988), and allows photometrical determination of the size of the area of neuroblasts at 4 days. Additionally, the number of neuroblasts was counted. Proliferation was measured by determining the mitotic density and mitotic index at 3 days, after notochordal implantation at 2 days. The results confirm our hypothesis, and the mechanism by which the notochord may exert these effects is discussed.
Materials and methods
Implantation of a notochordal fragment Chick embryos (White leghorn) of 2 days of incubation were used (20 to 25 somites, stage 13/14 according to Hamburger and Hamilton, 1951). From the donor embryo, a notochord fragment of 150-300 /mi of length from the area between somite 14 and prospective somite 28 was dissected with tungsten needles. The host embryo was operated in ovo. With a tungsten needle a longitudinal slit was made between the neural tube and the somite at the right side in the region of the somites 21-24 and the notochord fragment was inserted into it. Sham operations (nothing to be inserted) were performed in the first set of experiments (used for demonstration of AChE). Since this resulted in essentially normal embryos, in the subsequent experiments unoperated instead of shamoperated embryos were used as controls. Embryos were killed at 3 or 4 days and used for one of the following techniques.
AChE histotechniqueIscan-cytophotometry At 4 days, 31 experimental embryos were fixed in Holt's solution, embedded in Technovit 71001 and cut at 10/an. The sections were incubated for the acetylcholinesterase (AChE) reaction according to the direct colouring method of Karnovsky and Roots (1964) at pH5.6 during 6h at 37°C. This technique was adapted for plastic sections (van Straaten et al. 1986). For morphometrical and scan-cytophotometrical purposes, the sections were screened for good histological and enzyme histochemical quality, resulting in the selection of 15 embryos with the implant present and 3 sham-operated embryos. Every second or fifth section was measured as follows. The sectional area of both neural tube halves as well as the distance between the borders of implant and the neural tube and of implant and notochord were determined with a digitizer. Subsequently, the sectional area of the AChEpositive cells was determined with a scan-cytophotometer. Using a wavelength of 480 nm and an objective magnification of 16 times, the left and right neural tube area of the section was scanned with steps of 10/mi. The diameter of the photodetector spot equaled 3/an of the section. The gained values (intensity per step) were plotted in an intensity histogram. High-intensity values from the neural tube matrix, its lumen and surrounding tissues were cut off by a threshold, which had a high fidelity since it matched a clear dip of the histogram. Values from other AChE-positive structures, like peripheral nerves, myotome and spinal ganglia, were removed on a digitalized picture. The remainder of the values belonged to the AChE-positive area to be determined. Data 'manufacturers address: Kulzer & Co. GmbH, Philipp-ReisStr. 8, D-6393 Wehrheim (Taunus) 1.
were graphically depicted for each embryo (see Fig. 2). The range of the total methodical error as determined by intraindividual duplicate measurements was less than 5 % for the sectional area values. The area in which the implant was present was designated one of three locations. An example of each location is shown in Fig. 1. In Fig. 1A the implant is at a lateral location (distance implant - neural tube 120/un), in Fig. IB at a ventrolateral location (distance implant - neural tube
