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 μm from the neural tube and over 120 μm 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 coneluded 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.

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. 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 profiferation of the neural tube matrix layer. Acetylcholinesterase appears a useful marker of neuroblasts in the chick neural tube during our period of investigation (Mizoguti 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.

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 μm 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 histotechnique/scan-cytophotometry

At 4 days, 31 experimental embryos were fixed in Holt’s solution, embedded in Technovit 71001 and cut at 10 μm. 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 μm. The diameter of the photodetector spot equaled 3 μm 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 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 <80 μm; implant - notochord >120 – m), in Fig. 1B at a ventrolateral location (distance implant – neural tube <80μm; distance implant – notochord <120 μm) and in Fig. 1C in a remote position (implant located beyond these limits). In the 15 embryos with the implant present, one or two areas per embryo were determined according to the locations specified above: 8 lateral, 6 ventrolateral and 6 remote areas. From each area, data from 3 consecutive sections were averaged, the ratio of the values from the right and left side (R/L) calculated, arithmetically averaged for each location and presented in Table 1. A ratio eliminates certain biological and histotechnical variations, and it was used since the contralateral areas appeared hardly affected by the implant. With respect to the 3 sham-operated embryos, neither the photographs nor the graphs revealed deviations. Therefore 3 areas (of 3 sections each) were randomly selected per embryo, the R/L ratio calculated and averaged for the three embryos. Significance of differences was calculated using the t-test.

Table 1.

Effect of the location of the notochordal implant on the sectional area of the neural tube and of the AChE-positive cells

Effect of the location of the notochordal implant on the sectional area of the neural tube and of the AChE-positive cells
Effect of the location of the notochordal implant on the sectional area of the neural tube and of the AChE-positive cells
Fig. 1.

Transverse sections of the thoracic region of the 4-day chick embryo, with enzyme histochemical demonstration of AChE (acetylcholinesterase) activity. An additional notochord fragment, implanted lateral to the neural tube at two days, appears present at various locations in A, B, and C.

(A) Implant located laterally, near the neural tube. An ipsilateral enlargement of the neural tube and of the AChE-positive area is present. The area of axonal exits extends dorsoventrally.

(B) Implant located ventrolatcrally, near the neural tube. A partial dorsal shift in the distribution of AChE-positive cells and axonal exits is seen. The AChE-positive area is not increased in size.

(C) Implant located in a remote position. A slight tilt of the neural tube is present. A slight increase in size of the ipsilateral neural tube and AChE-positive area is present.

(D) Section of a sham-operated embryo in the implant area. Neither disturbing histological aspects at the right side nor right– left differences can be seen. ap, alar plate; bp, basal plate; i, implanted notochord fragment; m, myotome; n, notochord; nt, neural tube; s, sclerenchyme; sg, spinal ganglion; arrowheads, axonal exits. Bar=100μm.

Fig. 1.

Transverse sections of the thoracic region of the 4-day chick embryo, with enzyme histochemical demonstration of AChE (acetylcholinesterase) activity. An additional notochord fragment, implanted lateral to the neural tube at two days, appears present at various locations in A, B, and C.

(A) Implant located laterally, near the neural tube. An ipsilateral enlargement of the neural tube and of the AChE-positive area is present. The area of axonal exits extends dorsoventrally.

(B) Implant located ventrolatcrally, near the neural tube. A partial dorsal shift in the distribution of AChE-positive cells and axonal exits is seen. The AChE-positive area is not increased in size.

(C) Implant located in a remote position. A slight tilt of the neural tube is present. A slight increase in size of the ipsilateral neural tube and AChE-positive area is present.

(D) Section of a sham-operated embryo in the implant area. Neither disturbing histological aspects at the right side nor right– left differences can be seen. ap, alar plate; bp, basal plate; i, implanted notochord fragment; m, myotome; n, notochord; nt, neural tube; s, sclerenchyme; sg, spinal ganglion; arrowheads, axonal exits. Bar=100μm.

Determination of proliferation

At 3 days, 37 experimental and 8 control embryos were fixed in Bodian’s fluid for 16h, dehydrated in alcohol and embedded in Technovit 7100. From a large trajectory, including the implant area, 5μm sections were cut perpendicular to the neural axis. Every section was collected, hydrolysed in 1 N-HC1 at 60°C and stained with a 0.1 % solution of toluidin blue in 1 % borax (van Straaten et al. 1988b).

In 9 out of the 37 embryos, the implant appeared to be in a lateral location. These embryos, as well as 8 controls, were used for determination of cell numbers, of mitotic density and of mitotic index. In alternating sections, the mitotic figures were determined using a digitizer: a pointlight, which was mounted in the center of digitizer cursor, was projected onto the microscopic slide by the interposition of a microscopic drawing device. By this, data from the microscopic section could be collected directly; they were subsequently processed by a Digital PDP 11/73 minicomputer. The mitotic figures were plotted onto the unfolded surface of the internal limiting membrane of the neural tube (see Fig. 3). On the plots, a rectangle with the implant in its center was indicated, with a dorsoventral extension (h) of 200 μm and a longitudinal extension (1) of the length of the implant including about 50μm at both ends (250– 400μm). In this area, the mitotic density was determined, expressed as the number of mitotic figures per 2000 μm2. The average number of mitotic figures per section was determined over the length (1) and over the whole fragment of neural tube (750– 1000μm). For the determination of the mitotic index, 5 (out of 9) embryos with the highest mitotic density in their implant area were selected. Over the length (1) the number of nuclei was counted in 3 randomly selected sections. A mitotic index was calculated, being expressed as the number of mitotic figures per section divided by the number of nuclei per section. They were not corrected for section thickness. The above determinations were also performed in matching contralateral areas, and in same-sized areas in 5 randomly selected control embryos. Since sham-operated areas could not be traced back histologically (see above), we used unoperated embryos as controls. Data were presented in two ways: a bar diagram showing the averages and S.D of data from homologuous areas (Fig. 4), and a table showing the averages of ratios from the right (R; ipsilateral) and left (L; contralateral) matching areas. Significance of differences was calculated using the t-test.

Fig. 2.

Longitudinal graphs (cranial=right) of three representative embryos showing sectional areas of the neural tube halves (upper pair of lines), of the AChE-positive areas (lower pair of lines) and of the area of the remainder of the neural tube (middle pair of lines). Dashed line, right (ipsilateral) neural tube half, solid line, left half; striped bar, presence of the implant; I, lateral; vl, ventrolateral; r, remote location.

(A) 2 implants present, at different locations. The left implant is lateral to the neural tube, as in Fig. 1A. An ipsilateral (dashed line) increased neural tube and AChE-positive area are present. The right implant (ventrolateral, as in Fig. 1B) shows no increased areas. Immediately cranially and caudally to both implants increased areas are seen. The remainder area (middle lines) shows minor changes. The contralateral areas (solid lines) appear hardly affected by the implant.

(B) An area with a large notochordal implant, obliquely positioned. The ventrolateral location has no effect on the area values. The shift to the lateral position is paralleled by an increase of neural tube and AChE-positive area. The values decrease when the implant becomes remote.

(C) Implant area of a sham-operated embryo (as in Fig. 1D). No clear right– left differences can be seen.

Fig. 2.

Longitudinal graphs (cranial=right) of three representative embryos showing sectional areas of the neural tube halves (upper pair of lines), of the AChE-positive areas (lower pair of lines) and of the area of the remainder of the neural tube (middle pair of lines). Dashed line, right (ipsilateral) neural tube half, solid line, left half; striped bar, presence of the implant; I, lateral; vl, ventrolateral; r, remote location.

(A) 2 implants present, at different locations. The left implant is lateral to the neural tube, as in Fig. 1A. An ipsilateral (dashed line) increased neural tube and AChE-positive area are present. The right implant (ventrolateral, as in Fig. 1B) shows no increased areas. Immediately cranially and caudally to both implants increased areas are seen. The remainder area (middle lines) shows minor changes. The contralateral areas (solid lines) appear hardly affected by the implant.

(B) An area with a large notochordal implant, obliquely positioned. The ventrolateral location has no effect on the area values. The shift to the lateral position is paralleled by an increase of neural tube and AChE-positive area. The values decrease when the implant becomes remote.

(C) Implant area of a sham-operated embryo (as in Fig. 1D). No clear right– left differences can be seen.

Fig. 3.

Projection drawing of mitotic figures (dots) onto the unfolded left (L) and right (R) internal limiting membrane of the neural tube of a 3-day embryo. The projection of the implant is indicated on the right side. An increased mitotic density is seen around the implant. The marked bulging of the right border line at ‘1’ indicates an increased surface of the internal limiting membrane, as can be seen in Fig. 1A. ‘1’ and ‘h’ mark an area in which mitotic density and index were determined.

Fig. 3.

Projection drawing of mitotic figures (dots) onto the unfolded left (L) and right (R) internal limiting membrane of the neural tube of a 3-day embryo. The projection of the implant is indicated on the right side. An increased mitotic density is seen around the implant. The marked bulging of the right border line at ‘1’ indicates an increased surface of the internal limiting membrane, as can be seen in Fig. 1A. ‘1’ and ‘h’ mark an area in which mitotic density and index were determined.

Fig. 4.

Effect of a notochordal implant on the proliferation of the neural tube at three days. Average and S.D. of homologous areas of the right and left side of the neural tube are plotted. E=experimental, C=control embryos. The numbers used are indicated in Table 2.

(A) Mitotic density for the areal × h (see Fig. 3), expressed as number of mitotic figures per 2000μm2.

(B) Number of mitotic figures per section for the length ‘1’ in experimental embryos (El) and for the whole fragment of neural tube in experimental (E2) and control embryos (C).

(C) Number of nuclei per section for the length ‘1’.

(D) Mitotic index for the length ‘1’, expressed as the number of mitotic figures per section divided by the number of nuclei per section, × 100. In all histograms, in the experimental embryos an increase of the right vs. the left side is seen. In control embryos also some preference for the right side is seen.

Fig. 4.

Effect of a notochordal implant on the proliferation of the neural tube at three days. Average and S.D. of homologous areas of the right and left side of the neural tube are plotted. E=experimental, C=control embryos. The numbers used are indicated in Table 2.

(A) Mitotic density for the areal × h (see Fig. 3), expressed as number of mitotic figures per 2000μm2.

(B) Number of mitotic figures per section for the length ‘1’ in experimental embryos (El) and for the whole fragment of neural tube in experimental (E2) and control embryos (C).

(C) Number of nuclei per section for the length ‘1’.

(D) Mitotic index for the length ‘1’, expressed as the number of mitotic figures per section divided by the number of nuclei per section, × 100. In all histograms, in the experimental embryos an increase of the right vs. the left side is seen. In control embryos also some preference for the right side is seen.

Table 2.

Average and S.D. of the R/L ratio for proliferation parameters of the neural tube at 3 days

Average and S.D. of the R/L ratio for proliferation parameters of the neural tube at 3 days
Average and S.D. of the R/L ratio for proliferation parameters of the neural tube at 3 days

Determination of nuclear parameters

In order to confirm that the increase of the AChE-positive area at 4 days was due to an increase in cell number, the number of AChE-positive and -negative cells had to be counted. However, discrimination of individual cells was not possible in AChE sections, and therefore toluidin-blue-stained plastic sections from 4 experimental and 4 control embryos of 4 days (collected during previous experiments, van Straaten et al. 1988a) were used. These embryos were killed at 4 days and histotechnically treated like the foregoing 3-day group. Per embryo one section was selected in which the implant was lateral to the neural tube and the ipsilateral enlargement of the neural tube was evident (average enlargement R/L=1.14, S.D.±0.03). In this section, no spinal artery and no disturbing histological artefacts were present (Fig. 5A). The perimeters of the internal and external limiting membrane of the neural tube were digitized. In the basal plate, all nuclear equatorial planes present (determined by a through-focus movement, using a Zeiss oil-immersion objective 63 ×, numeric aperture 1.4) were circumscribed with the point-light cursor. From these perimeter data, several nuclear parameters were calculated: nuclear sectional area, lehgth of the long and short axis of the nucleus and its ratio (nuclear shape), the orientation of the long axis, and the location of the nucleus within the neural tube. A correlation diagram of sectional area and shape of the nucleus was plotted, and the cluster of points was separated by two lines into 3 fractions: (1) A fraction of about 42% of large circular nuclei, which topographically corresponded with AChE-positive cells, and are indicated here as ‘neuroblasts’; (2) a fraction of about 33 % of small elliptic nuclei, located mainly in the matrix layer and floor plate, and indicated as ‘matrix’ and (3) an intermediate fraction (about 25%), which appeared intermediate in shape and size, and was scattered topographically, though the larger part of these nuclei were found in the zone in between both foregoing fractions. These threshold settings were used afterwards. The long axis of ‘neuroblast’ and ‘matrix’ nuclei were plotted (see Fig. 5C). This method was performed in the basal plate only, since the shape of the alar plate nuclei did not correlate with specific cell types. To separate the alar plate from the basal plate, a border was taken perpendicular to the dorsoventral axis of the alar plate, intersecting the left outer neural membrane slightly dorsally to the basal plate area (see Fig. 5C). Per neural tube half the number of nuclei in the neural tube half, in the alar and basal plate, and in the fractions of the basal plate were determined. Data were represented as the average number of ‘neuroblasts’ and of remainder (‘matrix’+ intermediate fraction) per neural tube side (Fig. 6), and as average ± S.D. of R/L ratios per fraction (Table 3). Significance of differences was calculated using a t- test. Procedural intake errors were determined by a 20-time intraindividual repetition of the measurement of a mediumsized elliptic nucleus. The range for sectional area was 4% and of the excentricity 8% of the average. Since identical fractions were compared, correction for section thickness (Holmes correction) was not applied.

Table 3.

Average R/L ratio of the number of nuclei in the neural tube at 4 days

Average R/L ratio of the number of nuclei in the neural tube at 4 days
Average R/L ratio of the number of nuclei in the neural tube at 4 days
Fig 5.

Determination of cell types in the basal plate of the neural tube of the 4-day chick embryo, based on nuclear shape.

(A) Transverse plastic section of 5 μm of the thoracic region. The implant is lateral to the neural tube, and the enlargement of the ipsilateral neural tube half is evident. Bar=100μm.

(B) Basal plate area. In general, matrix cells show small elliptic nuclei, while neuroblasts show large circular nuclei. Bar=20μm.

(C) Projection drawing from the section shown in A. The long axes of small elliptic nuclei are projected as bars. These nuclei are supposed to belong mostly to the proliferating matrix layer and the floor plate cells. The center of the large circular nuclei are plotted as dots; these nuclei mostly belong to postmitotic neuroblasts. A numerical increase on the right side as compared to the left side can be seen for both nuclear fractions. Nuclei from the intermediate fraction are not plotted. Bar=100μm.ap, alar plate; bp, basal plate; i, notochordal implant; m, matrix cells; n, notochord; nb, neuroblasts; nt, neural tube.

Fig 5.

Determination of cell types in the basal plate of the neural tube of the 4-day chick embryo, based on nuclear shape.

(A) Transverse plastic section of 5 μm of the thoracic region. The implant is lateral to the neural tube, and the enlargement of the ipsilateral neural tube half is evident. Bar=100μm.

(B) Basal plate area. In general, matrix cells show small elliptic nuclei, while neuroblasts show large circular nuclei. Bar=20μm.

(C) Projection drawing from the section shown in A. The long axes of small elliptic nuclei are projected as bars. These nuclei are supposed to belong mostly to the proliferating matrix layer and the floor plate cells. The center of the large circular nuclei are plotted as dots; these nuclei mostly belong to postmitotic neuroblasts. A numerical increase on the right side as compared to the left side can be seen for both nuclear fractions. Nuclei from the intermediate fraction are not plotted. Bar=100μm.ap, alar plate; bp, basal plate; i, notochordal implant; m, matrix cells; n, notochord; nb, neuroblasts; nt, neural tube.

Fig. 6.

Effect of the notochordal implant on the number of nuclei in the neural tube at 4 days. Average number and S.D. for the right and left side of 4 embryos are presented. The total number of nuclei in control (C) and experimental (E) embryos is presented as the left bars. The total number from the experimental embryos is split into numbers for the alar and the basal plate (middle bars), according to the separation indicated in Fig. 5B, and the number of nuclei of the basal plate is split into ‘neuroblasts’ and remainder (right bars). The numerical increase as seen at the right vs. left side of the experimental embryos appears mainly to be caused by the basal plate nuclei. In the latter, both fractions contribute to this increase.

Fig. 6.

Effect of the notochordal implant on the number of nuclei in the neural tube at 4 days. Average number and S.D. for the right and left side of 4 embryos are presented. The total number of nuclei in control (C) and experimental (E) embryos is presented as the left bars. The total number from the experimental embryos is split into numbers for the alar and the basal plate (middle bars), according to the separation indicated in Fig. 5B, and the number of nuclei of the basal plate is split into ‘neuroblasts’ and remainder (right bars). The numerical increase as seen at the right vs. left side of the experimental embryos appears mainly to be caused by the basal plate nuclei. In the latter, both fractions contribute to this increase.

In chick embryos of two days of age, a fragment of notochord was implanted lateral to the neural tube at the level of somites 21– 24. The implant was found in the area lateral to the neural tube and notochord up to the dorsolateral ectoderm. Histologically, the implant resembled the natural notochord with respect to the total size, the pattern and size of its vacuoles and the concentric orientation of mesenchymal cells around the notochord. In Fig. 1, examples of implanted notochords as well as their different locations can be seen. Also the pink staining of the sheath in toluidin-blue-stained sections, indicating the presence of glycosaminoglycans, is present in both implanted and natural notochord (not shown).

Area of neural tube and AChE-positive cells

One group of embryos was killed at 4 days and used for the enzyme-histochemical demonstration of AChE.

In Fig. 1A, a section of an embryo is shown in which the implant is located lateral (see Materials and methods for definition) to the neural tube. Invariably with this location, an increased size of the ipsilateral neural tube half is seen, as well as a markedly enlarged AChE-positive area in the basal plate. Moreover, axons leave the neural tube in a broad dorsoventral area. In Fig. 2, the sectional areas of both halves of the neural tube and of the AChE-positive cells are shown for three embryos. In Fig. 2A, the left implant is located laterally (1) to the neural tube. Increased sizes of the total neural tube area (upper dashed line) and of the AChE-positive area (lower dashed line) are seen, which extend beyond the cranial and caudal limits of the implant. The contralateral side (represented by the solid lines) appears unaffected. The remainder of the neural tube (neural tube area minus AChE-positive area, middle lines) shows neither pronounced nor consistent differences between right and left area. In Table 1, the average of the ratio from the right and left side (R/L) is presented. It appears that both neural tube area and AChE-positive area are significantly increased in the case of a lateral implant.

In Fig. 1B, the implant appears ventrolateral to the neural tube. The location of the AChE-positive cells is shifted dorsally. Neither the neural tube nor the AChEpositive area are changed in size (Fig. 2A, right side; Table 1, 2nd row). Cranial and caudal to the implant area both total neural tube area and AChE-positive area are increased (Fig. 2A); in the middle of the figure the enlargements due to both implants blend.

In Fig. 1C, the implant is in a remote position, and enlargements are present but small. The average values of areas show an increase of the AChE-positive area only (Table 1, 3rd row).

In Fig. 2B, an example is shown of an embryo in which a large notochordal fragment was implanted obliquely. In a ventrolateral (vl) location no effects are seen. In a lateral (1) location, enlargements of the neural tube area and the AChE-positive area are seen. In a remote location (r), the previous effects of enlargement are diminished.

In sham-operated embryos (Fig. 1D), the right/left variation of the AChE-positive area is small (Fig. 2C), as is the symmetrical deviation (Table 1).

Mitotic density

Another group of embryos was killed at 3 days and used for the determination of mitotic figures of the neuroepithelium. In Fig. 3, a representative projection drawing from one embryo is shown. An area with an increased mitotic density is present around the implant, when compared to other areas on the ipsi- and contralateral side. Mitotic density was determined on the right and left side of the neural tube in defined areas (indicated by ‘1’ x ‘h’ in Fig. 3). Fig. 4A shows that the average value of the mitotic density is increased in the implant area (right) when compared to both the contralateral (left) as to both control (C) areas. This effect appears significant when data are expressed as R/L ratios (Table 2, lst row). The average number of mitotic figures per section for the length ‘1’ (Fig. 4B, El) and for the whole neural tube fragment (E2) shows an almost identical pattern as is seen in Fig. 4A. The R/L ratios are significantly increased over control values (Table 2, 2,nd and 3rd row). Determination of the number of nuclei per section reveals that both the average number of nuclei per section (Fig. 4C) as the R/L ratio (Table 2, 4th row) have a slight increase in favor of the implant side. The mitotic index (calculated from the mitotic density and the number of nuclei) shows a significant increase for the right side, though it does not differ significantly from the control values (Fig. 4D). Also the averaged R/L values are significantly increased (Table 2, lower row).

Number of nuclei at 4 days

In another group of embryos, killed at 4 days, the number of nuclei were counted. In the experimental embryos, the total number of nuclei per neural tube half at the right side appears significantly increased as compared to the left side and as compared to the control embryos (Fig. 6, left bars; Table 3). The basal plate contributes for the larger part to this numerical increase (Fig. 6, middle bars; Table 3). Counts of distinctive cell types in the basal plate (Fig. 5C), based on their nuclear shape and size (Fig. 5B), reveal that the ‘neuroblasts’ and the remainder both contribute to this increase (Fig. 6, right bars). In Table 3 the averages of the R/L ratios of these data are expressed. In the basal plate, the percentage increase of the neuroblasts is about twice that of the remainder.

In 4 control embryos, the right/left differences in number of nuclei in the basal plate and its three fractions is less than 5 %, and for statistical purposes the R/L ratio is therefore regarded as 1.00 in Table 3.

The presence of an additional notochord lateral to the neural tube of the chick embryo from 2 days onwards results in an increase in the sectional area of the ipsilateral neural tube half, which is clear at 4 days. This increase is mainly due to an increased area of AChE positive cells, which is indicative of an increased area of neuroblasts. Parallel to this, an increase in cells with large circular nuclei, which are mainly located in the basal plate area, is seen; they are supposedly postmitotic neuroblasts. Axons leave the neural tube over a broad dorsoventral area. The mitotic density, number of cells and mitotic index are increased in the neural tube implant area at 3 days. The effects are restricted to the ipsilateral neural tube area, and less than 200 μm away from the implant.

Increased proliferation in the implant area

In the neural tube area close to the notochordal implant, the mitotic density, mitotic index and cell numbers are increased at 1 day after the implantation as compared to the contralateral side. Mitotic density and mitotic index are useful parameters of proliferation, but their value is restricted since they depend on the growth fraction (the percentage of proliferating cells), on the duration of the cell cycle and on the duration of its M-phase. The growth fraction is close to 100% since only a few neuroblasts are developed at this age (Hollyday and Hamburger, 1977; Miki et al. 1981). A prolonged M-phase, resulting in more mitotic figures than expected in histological sections (Wilson, 1974), is not likely, because this would not result in an increase of cell numbers. Therefore, we conclude that the cell cycle time is shorter on the implant side as compared to the contralateral side. The ipsilaterally increased number of cells at 3 days indicates that this cycle time has been shorter during the third day. The increased mitotic index at 3 days indicates that this difference in cycle time is still present. It confirms the hypothesis that implantation of a notochord fragment results in an enhanced proliferation of the neural tube matrix layer. During normal development of the neural tube, the cell cycle time appears to lengthen in the chick (Fujita, 1962; Wilson, 1973) and mouse embryo (Kaufman, 1968). It is therefore possible that the absolute cycle time at the implant side is not decreased, but has merely remained at the 2-day level. This is under current investigation.

Value of nuclear size parameters

The use of nuclear dimensions as parameters for specific cell types is based on the finding that the nuclei of developing neuroblasts enlarge and round up during development, while matrix cells have small elliptic nuclei. (Fig. 5B; Lyser, 1964; Holley et al. 1982). Indeed, most of the nuclei were located where expected (Fig. 5C). However, two methodical errors have to be considered. (1) In the matrix layer, early prophase cells have a large circular nucleus, and in the mantle layer some neuroblasts have spindle-shaped nuclei. Both types of nuclei can be seen ‘misplaced’ in Fig. 5C. Their contribution to the total amount of nuclei is small. (2) Nuclei from the intermediate fraction, about 25 %, are scattered mainly in the zone between matrix and neuroblasts, and they may belong to both. In Table 3 this fraction is added to the ‘matrix’ fraction, leaving a distinct ‘neuroblast’ fraction. If this intermediate fraction is added to the ‘neuroblast’ fraction, then the percentage increase of neuroblasts at the implant side still appears the largest. It is therefore concluded that the enlargement of the basal plate at 4 days as a result of a notochordal implant is for the larger part caused by a percentage increase of neuroblasts. This confirms the hypothesis that, after implantation of a notochord fragment, the increase of the total area of AChE positive neuroblasts is the result of a numerical increase.

Interaction of the notochord with the neural tube

The implanted notochord resembles the natural notochord with respect to several histological parameters (van Straaten et al. 1985, 1988a, present results). Moreover, BrdU-labeling has demonstrated a normal cell cycle (preliminary data). It is therefore likely that the implanted notochord develops and behaves like the natural notochord.

In our experiments, a synergism of two notochords results in enhancement of proliferation and differentiation in the neural tube. This leads to the supposition that stimulation of proliferation and differentiation belongs to the regular actions of the natural notochord on the neural tube. This supposition is confirmed by several experiments: development of the neural tube is reduced after removal of the notochord (van Straaten and Drukker, 1987), and amphibian chordamesodennal cells exert a beneficial influence on neuroblast differentiation in vitro (Duprat et al. 1985). Notochord also stimulates gut development; chick gut epithelium differentiates fully as coelomic graft only when cocultured with notochord (Wiertz-Hoessels et al. 1987). A proliferation-enhancing action of the notochord is contradicted by other investigations. After implanting a notochord alongside the neural groove, a local reduction of mitotic density and a floor plate-like structure in the neural tube was found (van Straaten et al. 1988a). In the floor plate, an increased cycle time and a reduced M-phase has been reported (Smith and Schoenwolf, 1987, 1988). Thus, during early development, the notochord appears involved in inhibition of proliferation of the neural plate and in development of the floor plate. This specific role of the notochord was already suggested by Weiss (1955), Watterson et al. (1955) and van Limborgh (1956). Later in development, the notochord is involved in stimulation of proliferation of the neural tube and differentiation of neuroblasts, as appears from our present experiments. Now direct contact, as seen in floor plate induction, appears not necessary, but, on the other hand, the effects of an implanted notochord on the neural tube are clear only up to 80 μm from the implant; they do not extend beyond 200 μm and hardly affect the contralateral side. These findings point to an indirect, but rather local effect. We suggest that the notochord merely regulates and/or arranges surrounding mesenchymal cells (sclerenchyme), which are then the effective cells in regulating the development of the neuroblasts. This is supported by our results that in the case of an lateral implant the clear effects on the neural tube are paralleled by a relative abundance of mesenchyme surrounding both notochords.

Mesenchyme

The role of mesenchyme in the proliferation of neural tissue was suggested by Weiss (1955) and Watterson et al. (1955). Takaya (1977) reported that mesenchyme does stimulate the development of amphibian neural tissue in vitro. Rothman etal. (1987) found an ipsilateral enlarged spinal cord after implanting gut wall (derived from visceral mesoderm) between somite and neural tube. The mesenchyme may exert its proliferationenhancing effect by hyaluronate (HA). This is synthesized by the sclerenchyme (Vasan et al. 1986b) and present from the time of dispersion of the sclerotome onwards (van Straaten et al. 1989). Extensive work on morphogenesis of mesenchymal tissues implicates HA in regulating cell proliferation and delaying differentiation (e.g. Underhill and Dorfman, 1978; Toole et al. 1984; Brecht et al. 1986). Our present results confirm the above suggestion: proliferation is enhanced in that area of the neural tube, where a relative abundance of sclerenchyme is found in between both notochords. (Fig. 1A, 1C).

Sulphated glycosaminoglycans (sGAGs), on the other hand, are correlated with differentiation and with a reduction in hyaluronate accumulation (e.g. Cohn et al. 1976; Underhill and Dorfman, 1978; Bemfield et al. 1984; Toole et al. 1984; Vasan et al. 1986a,b). In the mouse embryo, an increasing amount of sGAGs and a decreasing amount of HA are synthesized in craniocaudal direction (opp and Bemfield, 1988), which is indicative of a developmental shift in GAG ratio. Notochord and sclerenchyme synthesize sGAGs during their interaction (Kosher and Lash, 1975; Vasan, 1983), and in somite/notochord explants the initial HA synthesis is gradually replaced by sGAG synthesis (Vasan et al. 1986b).

In the implant area, both increased proliferation and differentiation can thus be explained by the increased amount of sclerenchyme. Changes in synthetic patterns in this area are under current investigation.

Axonal outgrowth

An early feature of the neuroblast is the development of its axon, which leaves the neural tube at the ventrolateral site (Fig. 1D). This point may be determined simply by the location of the matrix cell that differentiates into a neuroblast first, and axons of subsequent neuroblasts follow this first axon. From our experiments, where a dorsoventrally extended area of axonal exits is seen, we propose that several neuroblasts arise simultaneously in a broad dorsoventral area of the basal plate as a result of the extended area of sclerenchyme between both notochords.

Also in subsequent axonal outgrowth the sclerenchyme appears important as a modulating factor. Axons grow through the cranial part of the somite only (Keynes and Stem, 1984; Layer et al. 1988; Tosney, 1988b), and this segmental pattern is lost after somites are removed (Tosney, 1988a). The sclerenchyme surrounding the notochord acts as a barrier to axon guidance, (van Straaten et al. 1985; van Straaten and Drukker, 1987; Tosney, 1988a; Fig. 1A, B, C).

Although several factors in the regulation of the development of the early neural tube remain unclear, it is likely that the sclerenchyme is involved in processes like regulation of proliferation, differentiation of neuroblasts, determination of the site of axonal exits and the pattern of subsequent axonal outgrowth. The notochord probably regulates and arranges the sclerenchyme and is as such involved in developmental processes of the neural tube.

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