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. TERWINDT-
ROUWENHORST 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 tothe neural tube in a 2-day chick embryo, at 4 days theipsilateral neural tube half was increased in size andaxons left the neural tube in a broad dorsoventral area(van Straaten et al. 1985). This enlargement appears tocoincide with an increased area of AChE-positive basalplate neuroblasts, as determined with scan-cytopho-tometry. The effect was ipsilateral and local: cleareffects were seen only when the implant was localizedless than 80 fan from the neural tube and over 120 fanfrom the ventral notochord. In order to investigate theexpected enhancement of proliferation, the mitotic den-sity and the number of cells at the site of the implant at 3days 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 arearelative 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 thenumerical increase was for the larger part due toneuroblasts. The synergism of two notochords thusresulted in enhancement of proliferation and differen-tiation in the neural tube. It is suggested that thenotochord merely regulates and arranges the surround-ing sclerenchymal cells, which are the effective cells inthe regulation of neural tube development.
The generation of neuroblasts in the spinal cord hasbeen the subject of many investigations, which haveresulted in a detailed description of their development.In the thoracic region of the neural tube of the chickembryo, the first neuroblasts arise in the basal platebetween 1.5 and 2 days (e.g Fujita, 1963; Lyser, 1964;Miki et al. 1981; Masuko and Shimada, 1983; Bennettand DiLullo, 1985). From 2 to 4 days, the area ofhighest mitotic density shifts gradually from the basal tothe alar plate (Hamburger, 1948; Corliss and Robert-son, 1963). In the former, the amount of postmitoticneuroblasts increases steadily, and at 4 days 95 % of thefuture ventral horn motoneurons are present (Hollydayand Hamburger, 1977). These are located in the ventro-lateral region, and form a longitudinal, almost uniformcolumn (Lunn et al. 1987; Layer et al. 1988). Theiraxons leave the neural tube in the same area.
Several investigations demonstrate that developmentof the early neural tube is modulated by its surround-ings. After dorsoventral rotation of the neural tube, thisstructure adapted completely (Steding, 1962), partially(Martin, 1977) or not (Jacob etal. 1976) to its surround-ings, probably depending on the age of the embryo.
Especially the notochord is involved in several develop-mental processes of the neural tube. After the inductionof the neural plate, the notochord is involved in thedifferentiation 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 theclosure of the neural tube in amphibians (Jacobson,1984). We have demonstrated that the notochord is alsoinvolved in the subsequent morphogenesis of the neuraltube. After implantation of an isochronous notochordbetween the neural tube and somite in a chick embryoof 2 days of age, the size of the ipsilateral neural tubeappeared to be increased and axons left the neural tubein a broad dorsoventral area. It was suggested thatproliferation and differentiation of basal plate neuro-blasts was increased and that the notochord played animportant role in the determination of axonal exits (vanStraaten et al. 1985). In the present study, we haveinvestigated whether the increase of size of the neuraltube after the addition of a notochord is indeed due toan increased number of neuroblasts, and if thisphenomenon is preceeded by an enhanced proliferationof the neural tube matrix layer. Acetylcholinesteraseappears a useful marker of neuroblasts in the chickneural tube during our period of investigation (Mizo-
794 H. W. M. van Straaten and others
guti and Miki, 1985; Layer et al. 1988), and allowsphotometrical determination of the size of the area ofneuroblasts at 4 days. Additionally, the number ofneuroblasts was counted. Proliferation was measuredby determining the mitotic density and mitotic index at3 days, after notochordal implantation at 2 days. Theresults confirm our hypothesis, and the mechanism bywhich the notochord may exert these effects is dis-cussed.
Materials and methods
Implantation of a notochordal fragmentChick embryos (White leghorn) of 2 days of incubation wereused (20 to 25 somites, stage 13/14 according to Hamburgerand Hamilton, 1951). From the donor embryo, a notochordfragment of 150-300 /mi of length from the area betweensomite 14 and prospective somite 28 was dissected withtungsten needles. The host embryo was operated in ovo. Witha tungsten needle a longitudinal slit was made between theneural tube and the somite at the right side in the region of thesomites 21-24 and the notochord fragment was inserted intoit. Sham operations (nothing to be inserted) were performedin the first set of experiments (used for demonstration ofAChE). Since this resulted in essentially normal embryos, inthe subsequent experiments unoperated instead of sham-operated embryos were used as controls. Embryos were killedat 3 or 4 days and used for one of the following techniques.
AChE histotechniqueIscan-cytophotometryAt 4 days, 31 experimental embryos were fixed in Holt'ssolution, embedded in Technovit 71001 and cut at 10/an. Thesections were incubated for the acetylcholinesterase (AChE)reaction according to the direct colouring method of Kar-novsky and Roots (1964) at pH5.6 during 6h at 37°C. Thistechnique was adapted for plastic sections (van Straaten et al.1986). For morphometrical and scan-cytophotometrical pur-poses, the sections were screened for good histological andenzyme histochemical quality, resulting in the selection of 15embryos with the implant present and 3 sham-operatedembryos. Every second or fifth section was measured asfollows. The sectional area of both neural tube halves as wellas the distance between the borders of implant and the neuraltube and of implant and notochord were determined with adigitizer. Subsequently, the sectional area of the AChE-positive cells was determined with a scan-cytophotometer.Using a wavelength of 480 nm and an objective magnificationof 16 times, the left and right neural tube area of the sectionwas scanned with steps of 10/mi. The diameter of thephotodetector spot equaled 3/an of the section. The gainedvalues (intensity per step) were plotted in an intensityhistogram. 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 thehistogram. Values from other AChE-positive structures, likeperipheral nerves, myotome and spinal ganglia, wereremoved on a digitalized picture. The remainder of the valuesbelonged to the AChE-positive area to be determined. Data
were graphically depicted for each embryo (see Fig. 2). Therange of the total methodical error as determined by intraindi-vidual duplicate measurements was less than 5 % for thesectional area values.
The area in which the implant was present was designatedone of three locations. An example of each location is shownin Fig. 1. In Fig. 1A the implant is at a lateral location(distance implant - neural tube <80/mi; implant - notochord>120/un), in Fig. IB at a ventrolateral location (distanceimplant - neural tube <80/zm; distance implant - notochord< 120 /mi) and in Fig. 1C in a remote position (implant locatedbeyond these limits). In the 15 embryos with the implantpresent, one or two areas per embryo were determinedaccording to the locations specified above: 8 lateral, 6ventrolateral and 6 remote areas. From each area, data from 3consecutive sections were averaged, the ratio of the valuesfrom the right and left side (R/L) calculated, arithmeticallyaveraged for each location and presented in Table 1. A ratioeliminates certain biological and histotechnical variations,and it was used since the contralateral areas appeared hardlyaffected by the implant. With respect to the 3 sham-operatedembryos, neither the photographs nor the graphs revealeddeviations. Therefore 3 areas (of 3 sections each) wererandomly selected per embryo, the R/L ratio calculated andaveraged for the three embryos. Significance of differenceswas calculated using the r-test.
Determination of proliferationAt 3 days, 37 experimental and 8 control embryos were fixedin Bodian's fluid for 16h, dehydrated in alcohol and embed-ded in Technovit 7100. From a large trajectory, including theimplant area, 5/un sections were cut perpendicular to theneural axis. Every section was collected, hydrolysed in 1 N-HC1 at 60°C and stained with a 0.1 % solution of toluidin bluein 1 % borax (van Straaten et al. 1988ft).
In 9 out of the 37 embryos, the implant appeared to be in alateral location. These embryos, as well as 8 controls, wereused for determination of cell numbers, of mitotic density andof mitotic index. In alternating sections, the mitotic figureswere determined using a digitizer: a pointlight, which wasmounted in the center of digitizer cursor, was projected ontothe microscopic slide by the interposition of a microscopicdrawing device. By this, data from the microscopic sectioncould be collected directly; they were subsequently processedby a Digital PDP 11/73 minicomputer. The mitotic figureswere plotted onto the unfolded surface of the internal limitingmembrane of the neural tube (see Fig. 3). On the plots, arectangle with the implant in its center was indicated, with adorsoventral extension (h) of 200/<m and a longitudinalextension (1) of the length of the implant including about50 /tin at both ends (250-400/on). In this area, the mitoticdensity was determined, expressed as the number of mitoticfigures per 2000 /mi2. The average number of mitotic figuresper section was determined over the length (1) and over thewhole fragment of neural tube (750-1000/<m). For the deter-mination of the mitotic index, 5 (out of 9) embryos with thehighest mitotic density in their implant area were selected.Over the length (1) the number of nuclei was counted in 3randomly selected sections. A mitotic index was calculated,being expressed as the number of mitotic figures per sectiondivided by the number of nuclei per section. They were notcorrected for section thickness. The above determinationswere also performed in matching contralateral areas, and insame-sized areas in 5 randomly selected control embryos.Since sham-operated areas could not be traced back histologi-
Notochord and neural tube development 795
cally (see above), we used unoperated embryos as controls.Data were presented in two ways: a bar diagram showing theaverages 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. Signifi-cance of differences was calculated using the Mest.
s
B
sg
I >;•••-
n
Fig. 1. Transverse sections of the thoracic region of the 4-day chick embryo, with enzyme histochemical demonstration ofAChE (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-positivearea is present. The area of axonal exits extends dorsoventrally.(B) Implant located ventrolaterally, near the neural tube. A partial dorsal shift in the distribution of AChE-positive cells andaxonal 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 ipsilateralneural 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 norright-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=100jan.
796 H. W. M. van Straaten and others
Determination of nuclear parametersIn order to confirm that the increase of the AChE-positivearea at 4 days was due to an increase in cell number, thenumber of AChE-positive and -negative cells had to becounted. However, discrimination of individual cells was notpossible in AChE sections, and therefore toluidin-blue-stained plastic sections from 4 experimental and 4 controlembryos of 4 days (collected during previous experiments,van Straaten et al. 1988a) were used. These embryos werekilled at 4 days and histotechnically treated like the foregoing3-day group. Per embryo one section was selected in whichthe implant was lateral to the neural tube and the ipsilateralenlargement of the neural tube was evident (average enlarge-ment R/L=1.14, S.D.±0.03). In this section, no spinal arteryand no disturbing histological artefacts were present(Fig. 5A). The perimeters of the internal and external limitingmembrane of the neural tube were digitized. In the basalplate, all nuclear equatorial planes present (determined by athrough-focus movement, using a Zeiss oil-immersion objec-tive 63 x, numeric aperture 1.4) were circumscribed with thepoint-light cursor. From these perimeter data, several nuclearparameters were calculated: nuclear sectional area, length ofthe long and short axis of the nucleus and its ratio (nuclearshape), the orientation of the long axis, and the location of thenucleus within the neural tube. A correlation diagram ofsectional area and shape of the nucleus was plotted, and thecluster of points was separated by two lines into 3 fractions:(1) A fraction of about 42% of large circular nuclei, whichtopographically corresponded with AChE-positive cells, andare indicated here as 'neuroblasts'; (2) a fraction of about33 % of small elliptic nuclei, located mainly in the matrix layerand floor plate, and indicated as 'matrix' and (3) an intermedi-ate fraction (about 25%), which appeared intermediate inshape and size, and was scattered topographically, though thelarger part of these nuclei were found in the zone in betweenboth foregoing fractions. These threshold settings were usedafterwards. The long axis of 'neuroblast' and 'matrix' nucleiwere plotted (see Fig. 5C). This method was performed in thebasal plate only, since the shape of the alar plate nuclei did notcorrelate with specific cell types. To separate the alar platefrom the basal plate, a border was taken perpendicular to thedorsoventral axis of the alar plate, intersecting the left outerneural membrane slightly dorsally to the basal plate area (seeFig. 5C). Per neural tube half the number of nuclei in theneural tube half, in the alar and basal plate, and in thefractions of the basal plate were determined. Data wererepresented as the average number of 'neuroblasts' and ofremainder ('matrix' + intermediate fraction) per neural tubeside (Fig. 6), and as averagets.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-timeintraindividual repetition of the measurement of a medium-sized elliptic nucleus. The range for sectional area was 4%and of the excentricity 8% of the average. Since identicalfractions were compared, correction for section thickness(Holmes correction) was not applied.
Results
In chick embryos of two days of age, a fragment ofnotochord was implanted lateral to the neural tube atthe level of somites 21-24. The implant was found in thearea lateral to the neural tube and notochord up to thedorsolateral ectoderm. Histologically, the implant re-sembled the natural notochord with respect to the total
size, the pattern and size of its vacuoles and theconcentric orientation of mesenchymal cells around thenotochord. In Fig. 1, examples of implanted noto-chords 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 glycos-aminoglycans, is present in both implanted and naturalnotochord (not shown).
Area of neural tube and AChE-positive cellsOne group of embryos was killed at 4 days and used forthe enzyme-histochemical demonstration of AChE.
In Fig. 1A, a section of an embryo is shown in whichthe implant is located lateral (see Materials andmethods for definition) to the neural tube. Invariablywith this location, an increased size of the ipsilateralneural tube half is seen, as well as a markedly enlargedAChE-positive area in the basal plate. Moreover, axonsleave the neural tube in a broad dorsoventral area. InFig. 2, the sectional areas of both halves of the neuraltube and of the AChE-positive cells are shown for threeembryos. In Fig. 2A, the left implant is located laterally(1) to the neural tube. Increased sizes of the total neuraltube area (upper dashed line) and of the AChE-positivearea (lower dashed line) are seen, which extend beyondthe cranial and caudal limits of the implant. Thecontralateral side (represented by the solid lines) ap-pears unaffected. The remainder of the neural tube(neural tube area minus AChE-positive area, middleLines) shows neither pronounced nor consistent differ-ences between right and left area. In Table 1, theaverage of the ratio from the right and left side (R/L) ispresented. It appears that both neural tube area andAChE-positive area are significantly increased in thecase of a lateral implant.
In Fig. IB, the implant appears ventrolateral to theneural tube. The location of the AChE-positive cells isshifted dorsally. Neither the neural tube nor the AChE-positive area are changed in size (Fig. 2A, right side;Table 1, 2nd row). Cranial and caudal to the implantarea both total neural tube area and AChE-positivearea are increased (Fig. 2A); in the middle of the figurethe enlargements due to both implants blend.
In Fig. 1C, the implant is in a remote position, and
Table 1. Effect of the location of the notochordalimplant on the sectional area of the neural tube and of
the AChE-positive cells
Implantlocation
lateralventrolateralremotesham
n
8669
Sectional area
neural tubeR/L ±S.D.
1.149**0.9981.0131.021
0.0670.0180.0450.022
AChER/L
1.535"1.0081.107*0.981
±S.D.
0.2700.0720.1330.067
The effect is expressed as the average and S.D. of the ratiosbetween the ipsilateral and contralateral sides (R/L). Significanceis arithmetically calculated as compared to the data of the shamexperiment, using the f-test. *P<0.05; "P<0.01.
Notochord and neural tube development 797
400
200-
Oo
200 400 600 800 1000 m
4 0 0
200-
Vl
200 400 200 400 600
Fig. 2. Longitudinal graphs (cranial=right) of three representative embryos showing sectional areas of the neural tubehalves (upper pair of lines), of the AChE-positive areas (lower pair of lines) and of the area of the remainder of the neuraltube (middle pair of lines). Dashed line, right (ipsilateral) neural tube half, solid line, left half; striped bar, presence of theimplant; 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. IB)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 areavalues. The shift to the lateral position is paralleled by an increase of neural tube and AChE-positive area. The valuesdecrease when the implant becomes remote.(C) Implant area of a sham-operated embryo (as in Fig. ID). No clear right-left differences can be seen.
enlargements are present but small. The average valuesof areas show an increase of the AChE-positive areaonly (Table 1, 3rd row).
In Fig. 2B, an example is shown of an embryo inwhich a large notochordal fragment was implantedobliquely. In a ventrolateral (vl) location no effects areseen. In a lateral (1) location, enlargements of theneural tube area and the AChE-positive area are seen.In a remote location (r), the previous effects of enlarge-ment are diminished.
In sham-operated embryos (Fig. ID), the right/leftvariation of the AChE-positive area is small (Fig. 2C),as is the symmetrical deviation (Table 1).
Mitotic densityAnother group of embryos was killed at 3 days and usedfor the determination of mitotic figures of the neuroepi-thelium. In Fig. 3, a representative projection drawingfrom one embryo is shown. An area with an increasedmitotic density is present around the implant, whencompared to other areas on the ipsi- and contralateralside. Mitotic density was determined on the right andleft side of the neural tube in denned areas (indicated byT x 'h' in Fig. 3). Fig. 4A shows that the average valueof 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
798 H. W. M. van Straaten and others
400-
200-
0-
200-
400-
caudal
200
cranial
400urn
600 800
Fig. 3. Projection drawing of mitotic figures (dots) onto theunfolded left (L) and right (R) internal limiting membraneof the neural tube of a 3-day embryo. The projection of theimplant is indicated on the right side. An increased mitoticdensity is seen around the implant. The marked bulging ofthe right border line at '1' indicates an increased surface ofthe internal limiting membrane, as can be seen in Fig. 1A.T and 'h' mark an area in which mitotic density and indexwere determined.
significant when data are expressed as R/L ratios(Table 2, 1st row). The average number of mitoticfigures per section for the length T (Fig. 4B, El) andfor the whole neural tube fragment (E2) shows analmost identical pattern as is seen in Fig. 4A. The R/Lratios are significantly increased over control values(Table 2, 2nd and 3rd row). Determination of thenumber of nuclei per section reveals that both theaverage number of nuclei per section (Fig. 4C) as theR/L ratio (Table 2, 4th row) have a slight increase infavor of the implant side. The mitotic index (calculatedfrom the mitotic density and the number of nuclei)shows a significant increase for the right side, though itdoes not differ significantly from the control values
B
mitoticdensity
number ofmitoses /section
number ofnuclei /section
mitoticindex
16 _
1 2 .
8 .
4 .
[**
• right
12
8_
4_
Ei
left
E2
800
600 .
400 .
200. I114 _
3 .
2_
1 .
Fig. 4. Effect of a notochordal implant on the proliferation of the neural tube at three days. Average and S.D. ofhomologous areas of the right and left side of the neural tube are plotted. E=experimental, C=control embryos. Thenumbers used are indicated in Table 2.(A) Mitotic density for the area I x h (see Fig. 3), expressed as number of mitotic figures per 2000 ftm2.(B) Number of mitotic figures per section for the length T in experimental embryos (El) and for the whole fragment ofneural tube in experimental (E2) and control embryos (C).(C) Number of nuclei per section for the length 'I'.(D) Mitotic index for the length T, expressed as the number of mitotic figures per section divided by the number of nucleiper section,x 100.In all histograms, in the experimental embryos an increase of the right vs. the left side is seen. In control embryos also somepreference for the right side is seen.
Notochord and neural tube development 799
(Fig. 4D). Also the averaged R/L values are signifi-cantly increased (Table 2, lower row).
Number of nuclei at 4 daysIn another group of embryos, killed at 4 days, thenumber of nuclei were counted. In the experimentalembryos, the total number of nuclei per neural tube halfat the right side appears significantly increased ascompared to the left side and as compared to thecontrol embryos (Fig. 6, left bars; Table 3). The basalplate contributes for the larger part to this numericalincrease (Fig. 6, middle bars; Table 3). Counts of dis-tinctive cell types in the basal plate (Fig. 5C), based ontheir nuclear shape and size (Fig. 5B), reveal that the'neuroblasts' and the remainder both contribute to thisincrease (Fig. 6, right bars). In Table 3 the averages ofthe R/L ratios of these data are expressed. In the basal
plate, the percentage increase of the neuroblasts isabout twice that of the remainder.
In 4 control embryos, the right/left differences innumber of nuclei in the basal plate and its threefractions is less than 5 %, and for statistical purposes theR/L ratio is therefore regarded as 1.00 in Table 3.
Discussion
The presence of an additional notochord lateral to theneural tube of the chick embryo from 2 days onwardsresults in an increase in the sectional area of theipsilateral neural tube half, which is clear at 4 days. Thisincrease is mainly due to an increased area of AChE-positive cells, which is indicative of an increased area ofneuroblasts. Parallel to this, an increase in cells withlarge circular nuclei, which are mainly located in the
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/OTI of the thoracic region. The implant is lateral to the neural tube, and the enlargementof the ipsilateral neural tube half is evident. Bar=100/«n.(B) Basal plate area. In general, matrix cells show small elliptic nuclei, while neuroblasts show large circular nuclei.Bar=20^m.(C) Projection drawing from the section shown in A. The long axes of small elliptic nuclei are projected as bars. Thesenuclei are supposed to belong mostly to the proliferating matrix layer and the floor plate cells. The center of the largecircular nuclei are plotted as dots; these nuclei mostly belong to postmitotic neuroblasts. A numerical increase on the rightside as compared to the left side can be seen for both nuclear fractions. Nuclei from the intermediate fraction are notplotted. Bar=100/im.ap, alar plate; bp, basal plate; i, notochordal implant; m, matrix cells; n, notochord; nb, neuroblasts; nt, neural tube.
800 H. W. M. van Straaten and others
Table 2. A verage and s. D. of the R/L ratio for proliferation parameters of the neural tube at 3 days
Experimental Control
Proliferation parameter
mitotic density around implantmitotic figures/section/length Tmitotic figures/section/neural tubenuclei/section/length Tmitotic index/length '1'
Significance is calculated between experimental and control data, using the r-test. *P<0.05; •*/3<0.01.
n
9995
R/L
1.38**1.31**1.22**1.16**1.25*
±S.D.
0.160.180.100.070.11
n
88855
R/L
1.051.091.090.991.09
±S.D.
0.080.050.050.030.10
Table 3. Average R/L ratio of the number of nuclei inthe neural tube at 4 days
Nuclei were counted in total neural tube half, in its alar andbasal plate an in fractions of the basal plate ('neuroblasts' andremainder). In control embryos the numbers in the total halveswere counted. Significance: the upper two rows are compared toeach other, using the r-test. For the lower 4 rows the paired f-testwas used. *P<0.05; **P<0.025; ***P<0.01.
800-,
600-
oc
400-
200-
Total
D right El left
alar basal "rteuro- remainderplate plate blasts"
Fig. 6. Effect of the notochordal implant on the number ofnuclei in the neural tube at 4 days. Average number ands.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 numberfrom the experimental embryos is split into numbers for thealar and the basal plate (middle bars), according to theseparation indicated in Fig. 5B, and the number of nuclei ofthe basal plate is split into 'neuroblasts' and remainder(right bars).The numerical increase as seen at the right vs. left side ofthe experimental embryos appears mainly to be caused bythe basal plate nuclei. In the latter, both fractionscontribute to this increase.
basal plate area, is seen; they are supposedly postmi-totic neuroblasts. Axons leave the neural tube over abroad dorsoventral area. The mitotic density, numberof cells and mitotic index are increased in the neuraltube implant area at 3 days. The effects are restricted tothe ipsilateral neural tube area, and less than 200 [anaway from the implant.
Increased proliferation in the implant areaIn the neural tube area close to the notochordalimplant, the mitotic density, mitotic index and cellnumbers are increased at 1 day after the implantation ascompared to the contralateral side. Mitotic density andmitotic index are useful parameters of proliferation, buttheir value is restricted since they depend on the growthfraction (the percentage of proliferating cells), on theduration of the cell cycle and on the duration of itsM-phase. The growth fraction is close to 100% sinceonly a few neuroblasts are developed at this age(Hollyday and Hamburger, 1977; Miki et al. 1981). Aprolonged M-phase, resulting in more mitotic figuresthan expected in histological sections (Wilson, 1974), isnot likely, because this would not result in an increaseof cell numbers. Therefore, we conclude that the cellcycle time is shorter on the implant side as compared tothe contralateral side. The ipsilaterally increased num-ber of cells at 3 days indicates that this cycle time hasbeen shorter during the third day. The increased mitoticindex at 3 days indicates that this difference in cycletime is still present. It confirms the hypothesis thatimplantation of a notochord fragment results in anenhanced proliferation of the neural tube matrix layer.During normal development of the neural tube, the cellcycle time appears to lengthen in the chick (Fujita,1962; Wilson, 1973) and mouse embryo (Kaufman,1968). It is therefore possible that the absolute cycletime at the implant side is not decreased, but has merelyremained at the 2-day level. This is under currentinvestigation.
Value of nuclear size parametersThe use of nuclear dimensions as parameters forspecific cell types is based on the finding that the nucleiof developing neuroblasts enlarge and round up duringdevelopment, while matrix cells have small ellipticnuclei. (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
Notochord and neural tube development 801
considered. (1) In the matrix layer, early prophase cellshave a large circular nucleus, and in the mantle layersome neuroblasts have spindle-shaped nuclei. Bothtypes of nuclei can be seen 'misplaced' in Fig. 5C. Theircontribution to the total amount of nuclei is small.(2) Nuclei from the intermediate fraction, about 25 %,are scattered mainly in the zone between matrix andneuroblasts, and they may belong to both. In Table 3this fraction is added to the 'matrix' fraction, leaving adistinct 'neuroblast' fraction. If this intermediate frac-tion is added to the 'neuroblast' fraction, then thepercentage increase of neuroblasts at the implant sidestill appears the largest. It is therefore concluded thatthe enlargement of the basal plate at 4 days as a result ofa notochordal implant is for the larger part caused by apercentage increase of neuroblasts. This confirms thehypothesis that, after implantation of a notochordfragment, the increase of the total area of AChE-positive neuroblasts is the result of a numerical in-crease.
Interaction of the notochord with the neural tubeThe implanted notochord resembles the natural noto-chord with respect to several histological parameters(van Straaten et al. 1985, 1988a, present results).Moreover, BrdU-labeling has demonstrated a normalcell cycle (preliminary data). It is therefore likely thatthe implanted notochord develops and behaves like thenatural notochord.
In our experiments, a synergism of two notochordsresults in enhancement of proliferation and differen-tiation in the neural tube. This leads to the suppositionthat stimulation of proliferation and differentiationbelongs to the regular actions of the natural notochordon the neural tube. This supposition is confirmed byseveral experiments: development of the neural tube isreduced after removal of the notochord (van Straatenand Drukker, 1987), and amphibian chordamesodermalcells exert a beneficial influence on neuroblast differen-tiation in vitro (Duprat et al. 1985). Notochord alsostimulates gut development; chick gut epithelium dif-ferentiates fully as coelomic gTaft only when coculturedwith notochord (Wiertz-Hoessels et al. 1987). A pro-liferation-enhancing action of the notochord is contra-dicted by other investigations. After implanting a noto-chord alongside the neural groove, a local reduction ofmitotic density and a floor plate-like structure in theneural tube was found (van Straaten et al. 1988a). In thefloor plate, an increased cycle time and a reducedM-phase has been reported (Smith and Schoenwolf,1987, 1988). Thus, during early development, the noto-chord appears involved in inhibition of proliferation ofthe neural plate and in development of the floor plate.This specific role of the notochord was already sugges-ted by Weiss (1955), Watterson et al. (1955) and vanLimborgh (1956). Later in development, the notochordis involved in stimulation of proliferation of the neuraltube and differentiation of neuroblasts, as appears fromour present experiments. Now direct contact, as seen infloor plate induction, appears not necessary, but, on theother hand, the effects of an implanted notochord on
the neural tube are clear only up to 80 ̂ m from theimplant; they do not extend beyond 200 ;Um and hardlyaffect the contralateral side. These findings point to anindirect, but rather local effect. We suggest that thenotochord merely regulates and/or arranges surround-ing mesenchymal cells (sclerenchyme), which are thenthe effective cells in regulating the development of theneuroblasts. This is supported by our results that in thecase of an lateral implant the clear effects on the neuraltube are paralleled by a relative abundance of mesen-chyme surrounding both notochords.
MesenchymeThe role of mesenchyme in the proliferation of neuraltissue was suggested by Weiss (1955) and Watterson etal. (1955). Takaya (1977) reported that mesenchymedoes stimulate the development of amphibian neuraltissue in vitro. Rothman etal. (1987) found an ipsilateralenlarged spinal cord after implanting gut wall (derivedfrom visceral mesoderm) between somite and neuraltube. The mesenchyme may exert its proliferation-enhancing effect by hyaluronate (HA). This is syn-thesized by the sclerenchyme (Vasan et al. 19866) andpresent from the time of dispersion of the sclerotomeonwards (van Straaten et al. 1989). Extensive work onmorphogenesis of mesenchymal tissues implicates HAin regulating cell proliferation and delaying differen-tiation (e.g. Underhill and Dorfman, 1978; Toole et al.1984; Brecht et al. 1986). Our present results confirmthe above suggestion: proliferation is enhanced in thatarea of the neural tube, where a relative abundance ofsclerenchyme is found in between both notochords.(Fig. 1A, 1C).
Sulphated glycosaminoglycans (sGAGs), on theother hand, are correlated with differentiation and witha reduction in hyaluronate accumulation (e.g. Cohn etal. 1976; Underhill and Dorfman, 1978; Bernfield et al.1984; Toole et al. 1984; Vasan et al. 1986a,6). In themouse embryo, an increasing amount of sGAGs and adecreasing amount of HA are synthesized in craniocau-dal direction (Copp and Bernfield, 1988), which isindicative of a developmental shift in GAG ratio.Notochord and sclerenchyme synthesize sGAGs duringtheir interaction (Kosher and Lash, 1975; Vasan, 1983),and in somite/notochord explants the initial HA syn-thesis is gradually replaced by sGAG synthesis (Vasanetal. 19866).
In the implant area, both increased proliferation anddifferentiation can thus be explained by the increasedamount of sclerenchyme. Changes in synthetic patternsin this area are under current investigation.
Axonal outgrowthAn early feature of the neuroblast is the development ofits axon, which leaves the neural tube at the ventro-lateral site (Fig. ID). This point may be determinedsimply by the location of the matrix cell that differen-tiates into a neuroblast first, and axons of subsequentneuroblasts follow this first axon. From our exper-iments, where a dorsoventrally extended area of axonalexits is seen, we propose that several neuroblasts arise
802 H. W. M. van Straaten and others
simultaneously in a broad dorsoventral area of the basalplate as a result of the extended area of sclerenchymebetween both notochords.
Also in subsequent axonal outgrowth the scleren-chyme appears important as a modulating factor.Axons grow through the cranial part of the somite only(Keynes and Stern, 1984; Layer et al. 1988; Tosney,1988b), and this segmental pattern is lost after somitesare removed (Tosney, 1988a). The sclerenchyme sur-rounding the notochord acts as a barrier to axonguidance, (van Straaten et al. 1985; van Straaten andDrukker, 1987; Tosney, 1988a; Fig. 1A, B, C).
Although several factors in the regulation of thedevelopment of the early neural tube remain unclear, itis likely that the sclerenchyme is involved in processeslike regulation of proliferation, differentiation ofneuroblasts, determination of the site of axonal exitsand the pattern of subsequent axonal outgrowth. Thenotochord probably regulates and arranges the scleren-chyme and is as such involved in developmental pro-cesses of the neural tube.
References
BENNETT, G. S. AND DILULLO, C. (1985). Expression of aneurofilament protein by the precursors of a subpopulation ofventral spinal cord neurons. Devi Bwl. 107, 94-106.
BERNFIELD, M. R., BANERJEE, S. D., KODA, J. E. AND RAPRAEGER,
A. C. (1984). Remodeling of the basement membrane as amechanism of morphogenetic tissue interaction. In The Role ofExtracellular Matrix in Development. (L. Trelstad, ed.). Alan R.Liss Inc. New York. pp. 545-572.
BRECHT, M., MAYER, U., SCHLOSSER, E. AND PREHM, P. (1986).
Hyaluronate synthesis is required for fibroblast detachment andmitosis. Bioch. J. 239, 445-450.
COHN, R. H., CASSIMAN, J. J. AND BERNFIELD, M. R. (1976).Relationship of transformation, cell density and growth controlto the cellular distribution of newly synthesizedglycosaminoglycan. / . Cell Biol. 71, 280-294.
COPP, A. J. AND BERNFIELD, M. (1988). Glycoconjugateaccumulation varies along the neuraxis during spinal neurulationin the mouse embryo. Devi Biol. 130, 573-582.
CORLISS, C. E. AND ROBERTSON, G. G. (1963). The pattern ofmitotic density in the early chick neural epithelium. J. exp. Zoo!.153, 125-141.
DUPRAT, A. M., KAN, P., GUALANDRIS, L., FOULQUIER, F., MARTY,J. AND WEBER, M. (1985). Neural induction: embryonicdetermination elicits full expression of specific neuronal traits.J. Embryol. exp. Morph. 89 Supplement 167-183.
FUJITA, S. (1962). Kinetics of cellular proliferation. Expl Cell Res.28, 52-69.
FUJITA, S. (1963). The matrix cell and cytogenesis in the developingcentral nervous system. / . comp. Neurol. 120, 37-42.
HAMBURGER, V. (1948). The mitotic patterns in the spinal cord ofthe chick embryo and their relation to histogenetic processes.J. comp. Neurol. 88, 221-284.
HAMBURGER, V. AND HAMILTON, H. G. A. (1951). A series ofnormal stages in the development of the chick embryo. / .Morph. 88, 49-92.
HOLLEY, J. A., WIMER, C. C. AND VAUGHN, J. E. (1982).
Quantitative analyses of neuronal development in the lateralmotor column of the mouse spinal cord. II. Development ofmotor neuronal organelles. / . comp. Neurol. 207, 322-332.
HOLLYDAY, M. AND HAMBURGER, V. (1977). An autoradiographicstudy on the formation of the lateral motor column in the chickembryo. Brain Res. 170, 132-208.
JACOB, H. J., CHRIST, B., JACOB, M. AND AHLSTROM, P. (1976).Differenzierung des neuralrohres. Acta anat. 94, 204-220.
JACOBSON, A. G. (1984). Further evidence that formation of theneural tube requires elongation of the nervous system. J. exp.Zool. 230, 23-28.
KARNOVSKY, M. J. AND ROOTS, L. (1964). A "direct coloring"thiocholin method for cholinesterase. J. Histochem. Cytochem.12, 219.
KAUFFMAN, S. L. (1968). Lengthening of the generation cycleduring embryonic differentiation of the mouse neural tube. ExplCell Res. 49, 420-424.
KEYNES, R. J. AND STERN, C. D. (1984). Segmentation in thevertebrate nervous system. Nature, Lond. 310, 786-789.
KOSHER, R. A. AND LASH, J. W. (1975). Notochordal stimulation ofin-vitro somite chondrogenesis before and after enzymaticremoval of perinotochordal materials. Devi Biol. 42, 362-378.
LAYER, P. G., ALBER, R. AND RATHJEN, F. G. (1988). Sequential
activation of butyrylcholinesterase in rostral half somites andacetylcholinesterase in motoneurones and myotomes precedinggrowth of motor axons. Development 102, 387-396.
LUNN, E . R . , SCOURFIELD, J . , KEYNES, R. J. AND STERN, C. D .
(1987). The neural tube origin of ventral root sheath cells in thechick embryo. Development 101, 247-254.
LYSER, K. M. (1964). Early differentiation of motor neuroblasts inthe chick embryo as studied by electron microscopy. I. Generalaspects. Devi Biol. 10, 433-466.
MARTIN, A. H. (1977). Neural tube-somite interaction, effect ofneural tube reversal upon neural development. Acta Embryol.exp. 3, 305-313.
MASUKO, S. AND SHIMADA, Y. (1983). Neural cell-surface specificantigen(s) is expressed during the terminal mitosis of cellsdestined to become neuroblasts. Devi Biol. 96, 396-404.
MIKI, A., ATOJI, Y. AND MIZOGUTI, H. (1981). Time of origin ofthe neuroblasts in the neural tube of the chick embryodetermined by histochemical observation of acetylcholinesterase.Acta Histochem. Cytochem. 14, 641-653.
MIZOGUTI, H. AND MIKI, A. (1985). Interrelationship among theproliferating ability, morphological development andacetylcholinesterase activity of the neural tube cells in early chickembryos. Acta Histoch. Cytochem. 18, 85-95.
ROTHMAN, T. P., GERSHON, M. D., FONTAINE-PERUS, J. C ,
CHANCONIE, M. AND LE DOUARIN, N. M. (1987). The effect ofback-transplants of the embryonic gut wall on growth of theneural tube. Devi Biol. 124, 331-346.
SMITH, J. L. AND SCHOENWOLF, G. C. (1987). Cell cycle andneuroepithelial cell shape during bending of the chick neuralplate. Anat. Rec. 218, 196-206.
SMITH, J. L. AND SCHOENWOLF, G. C. (1988). Role of cell-cycle inregulating neuroepithelial cell shape during bending of the chickneural plate. Cell Tissue Res. 252, 491-500.
STEDING, G. (1962). Experimente zur morphogenese desruckenmarkes. Acta Anat. 49, 199-231.
TAKAYA, H. (1977). Types of neural tissues induced through thepresumptive notochord of newt embryo. Differentiation 7,187-192.
TOOLE, B. P., GOLDBERG, R. L., CHI-ROSSO, G., UNDERHILL, C. B.
AND ORKIN, R. W. (1984). Hyaluronate-cell interactions. In TheRole of Extracellular Matrix in Development, (ed. L. Trelstad).Alan Riss, Inc. New York.
TOSNEY, K. W. (1988a). Proximal tissues and patterned neuriteoutgrowth at the lumbosacral level of the chick embryo: partialand complete deletion of the somite. Devi Biol. 127, 266-286.
TOSNEY, K. W. (19886). Somites and axon guidance. ScanningMicrosc. 2, 427-442.
UNDERHILL, C. B. AND DORFMAN, A. (1978). The role of hyaluronicacid in intercellular adhesion of cultured mouse cells. Expl CellRes. 117, 155-164.
VAN LIMBORGH, J. (1956). The influence of adjacent mesodermalstructures upon the shape of the neural tube and neural plate inbird embryos. Acta Morphol. Neerl. Scand. 1, 155-166.
VAN STRAATEN, H. W. M. AND DRUKKER, J. (1987). Influence of thenotochord on the morphogenesis of the neural tube. InMesenchymal-epithelial Interactions m Neural Development, (ed.JR Wolff et al). NATO ASI series, pp. 153-162. Springer-VerlagBerlin Heidelberg.
VAN STRAATEN, H. W. M., HEKKING, J. W. M. AND DRUKKER, J.
Notochord and neural tube development 803
(1986). The demonstration of acetylcholinesterase in plasticsections. Its application as a marker of early neuronaldevelopment. Ada Histochem. [Suppl] Jena 32, 185-190.
VAN STRAATEN, H. W. M., HEKKING, J. W. M. AND DRUKKER, J.(19886). A HCl-toluidine blue staining procedure specific fornuclei, mitotic figures and axons in GMA embedded embryonicneural tissue. Stain Technol. 65, 360-362.
VAN STRAATEN, H. W. M., HEKKING, J. W. M., WIERTZ-HOESSELS,E. L., THORS, F. AND DRUKKER, J. (1988a). Effect of thenotochord on the differentiation of a floor plate area in theneural tube of the chick embryo. Anat. Embryol. Berl. Yll,317-324.
VAN STRAATEN, H. W. M., HOOPER, K. AND BERNFIELD, M. (1989).Hyaluronate disappears intercellularly and appears at thebasement membrane during formation of embryonic epithelia.Development (submitted).
VAN STRAATEN, H. W. M., THORS, F., WIERTZ-HOESSELS, L.,HEKKING, J. W. M. AND DRUKKER, J. (1985). Effect of anotochordal implant on the early morphogenesis of the neuraltube and neuroblasts: histometrical and histolological results.Devi Biol. 110, 247-254.
VASAN, N. S. (1983). Analysis of perinotochordal materials. 2.Studies on the influence of proteoglycans in somitechondrogenesis. J. Embryol. exp. Morph. 73, 263-274.
VASAN, N. S., LAMB, K. M. AND LA MANNA, O. (1986a). Somite
chondrogenesis in vitro: 1. Alterations in proteoglycan synthesis.Cell Differ. 18, 79-90.
VASAN, N. S., LAMB, K. M. AND LA MANNA, O. (1986t). Somite
chondrogenesis in vitro: 2. Changes in the hyaluronic acidsynthesis. Cell Differ. 18, 91-99.
WATTERSON, R. L., GOODHEART, C. R. AND LINDBERG, G. (1955).
The influence of adjacent structures upon the shape of the neuraltube and neural plate of chick embryos. Anat. Rec. 122, 539-559.
WEISS, P. (1955). Nervous system. In Analysis of Development.(ed. Willier, B.H., Weiss, P. and Hamburger, V.). Section VII,Chapter 1, pp. 346-401. Saunders Philadelphia.
WIERTZ-HOESSELS, E. L., HARA, K., HEKKING, J. W. M., VAN
STRAATEN, H. W. M., THORS, F. AND DRUKKER, J. (1987).
Differentiation of gut endoderm in dependence of the notochord.Anat. Embryol. Berl. 176, 337-343.
WILSON, D. B. (1973). Chronological changes in the cell cycle ofchick neuroepithelial cells. J. Embryol. exp. Morph. 29, 745-751.
WILSON, D. B. (1974). The cell cycle of ventricular cells in theovergrown optic tectum. Brain Res. 69, 41-48.
