J. Embryol. exp. Morph. Vol. 23, 3, pp. 751-759, 1970
Printed in Great Britain
751
Differentiation in small grafts of the median region
of the presumptive prosencephalon
By ELZE C. B O T E R E N B R O O D 1
From the Hubrecht Laboratory, Utrecht
SUMMARY
In early neurulae of Triturus alpestris the cranio-caudal distribution of differentiation
tendencies in the presumptive prosencephalon was tested.
Small grafts made from a median strip excised from the region of the neural plate and
transverse neural fold were transplanted to a pocket made under the cranio-ventral ectoderm
of early neurulae.
Grafts made from the middle portion of the strip showed almost exclusively eye differentiation, whereas more cranial as well as more caudal grafts showed relatively more telencephalic
differentiation, and only little diencephalic differentiation.
The results indicate that the process of pattern formation in the central region of the prospective prosencephalon is advanced with respect to the surrounding areas.
INTRODUCTION
The maps of presumptive brain areas in the open neural plate of Triturus
alpestris (von Woellwarth, 1960) show that material of the future telencephalon
is localized in the transverse anterior neural fold, that the eye primordia are
situated as two almost circular areas just behind the telencephalic region, and
that the future diencephalon arises from the remaining region lying anterior to
the slightly V-shaped junction with the future mesencephalon.
In order to obtain an insight into the formation of this pattern, Nieuwkoop,
Niermeijer & Jansen (1964) tested the actual state of the pattern at the open
neural plate stage (stage 14^, Harrison, 1969). The presumptive prosencephalic
area was subdivided into five transverse, slightly V-shaped segments, running
parallel to the boundary with the presumptive mesencephalic area. Portions
comprising an increasing number of segments were isolated and transplanted
into host embryos or cultured in vitro. Any portion turned out be be able to
form all three prosencephalic elements, which means that at the open neural
plate stage the pattern is still far removed from the ultimate 'mosaic'. A quantitative analysis of the volumes of the structures formed showed, however, a
relatively enhanced telencephalic differentiation in grafts including more anterior
parts, and a relatively enhanced diencephalic differentiation in grafts including
1
Author's address: Hubrecht Laboratory, Uppsalalaan 1, Universiteitscentrum
Uithof', Utrecht, The Netherlands.
'De
752
E. C. BOTERENBROOD
more posterior parts of the presumptive prosencephalic area. In a later, unpublished series of experiments by P. D. Nieuwkoop, M. H. M. de Bruine, G. J. E.
Heilbron & L. Duran (1965) three different regions of the presumptive prosencephalic area were excised at Harrison stages 13^, 15|and 16^-, respectively, and
transplanted to host embryos (see Fig. 4). The localization of each of these regions
at the various stages had previously been determined by means of vital staining.
Notwithstanding the rather close correspondence between the transplanted
regions and the various brain and eye primordia the results are essentially
similar to those of Nieuwkoop et al. (1964). The quantitative data (see Table 2)
show that even prior to a clear demarcation of the neural plate (stage 13^)
regional differences in the relative volumes of each of the prosencephalic elements
formed are already evident. In more advanced stages of development the distribution of differentiation tendencies becomes more and more similar to the final
pattern.
The fact that at the early neurula stage any region of the presumptive prosencephalon is able to form telencephalic, eye and diencephalic structures indicates that at an early phase of pattern formation the cells have the potentiality to
differentiate in various directions. In certain regions, however, the tendency for
one of these differentiations seems to be stronger than that for the others. The
distribution of differentiation tendencies in a given region is thought to find its
expression in the relative volumes of the various structures formed by this
region after grafting to a neutral environment. This quantitative composition,
however, represents the average for the entire grafted region. Therefore it was
thought desirable to repeat the experiments of Nieuwkoop et al. with much
smaller grafts, so as to arrive at a more selective test for the regional distribution
of differentiation tendencies in the presumptive prosencephalic area.
In the present report the results of preliminary experiments are presented,
in which small squares, situated along the median line of the anterior neural
plate and transverse neural wall, are examined as to their differentiation
tendencies.
MATERIAL AND METHODS
The experiments were carried out with eggs of Triturus alpestris at stage 14-^
of Harrison, when the neural walls are just clearly elevated and the caudal part
of the plate has not yet begun to narrow. With the help of an ocular grid consisting of regular squares, parts of equal size could be accurately marked out on
the surface of the egg according to the scheme given in Fig. 1. The border
between the neural plate and the anterior transverse neural fold was taken as a
landmark, forming the borderline between segments 2 and 3. Superficial
transverse and longitudinal scratches, made with a sharp tungsten needle and
affecting only the 'surface coat' of the egg, marked the positions of the definitive cuts by which six segments of 0-12x0-24 mm each were to be excised.
The scratches had to be made quickly in order to avoid disturbing the excision
Grafts of presumptive prosencephalon
753
scheme by contractions of the tissue resulting from damage of the surface coat.
The whole strip of segments was then excised without the underlying prechordal
plate material, and divided into individual segments along the transverse
scratches.
Grafts belonging to some series were directly implanted into a pocket made
under the ectoderm overlying the cranio-ventral mosoderm-free region of host
embryos of stages 13^-14^. Each graft was transplanted into a separate host,
except for a few cases in which one host was used for two grafts. Grafts belonging to other series were allowed to fuse with pieces of ectoderm before
implantation. Ventro-cranial ectoderm of either stage 14^- or stage 12 was used.
0 7 0-6 0-5 0-4 0-3 0-2 0-1 0 0 0-1 0-2 0-3 0-4 0-5 0-6 0-7
T
Fig. 1. Diagram of the presumptive prosencephalic area of a neurula stage 14^ of
average size of Triturus alpestris, indicating the segments 1-6, which were used as
grafts. The localization of the presumptive brain structures and eye Anlagen is based
upon unpublished data of P. D. Nieuwkoop et al. (1965).
Host embryos were cultured in Niu and Twitty's solution for 10 days at ± 23 °C,
then fixed in Smith's fixative, block-stained with borax-carmine, sectioned at
10 /A, and counter-stained with Aniline Blue-Orange G.
For each graft the volume of each of the various differentiated structures was
determined with the help of a grid consisting of perpendicular rows of dots
placed 3 mm apart. This was placed over drawings made at x 100 enlargement.
The total number of dots falling within the boundaries of a given structure in the
drawings of all sections of the graft series provides a relative measure of the
volume of the structure in question, expressed in grid units.
754
E. C. B O T E R E N B R O O D
RESULTS
A. Qualitative analysis
Grafts are usually found to lie between the host's pericardium and the ectoderm; they are sometimes lying inside the pericardium, in the lower jaw or in
rare cases in a closed ectodermal vesicle attached to the belly of the host.
Eyes, consisting of both retina and tapetum are often located with the tapeturn bordering the host's ectoderm, and the invaginated retina and lens situated
more internally. Eyes lying in the pericardium never contain tapetum. Lenses
are often formed from the adjacent ectoderm, but in a number of cases their
location suggests a formation from the retina itself, since they are completely
surrounded by retina tissue.
Telencephalic structures are recognizable by one or more of the following
features :
(i) Adjacent induced olfactory placode.
(ii) Sickle-shaped ventricle located eccentrically in the mass of neural cells.
(iii) Presence of a paraphysis.
(iv) Tendency to become separated from eye and diencephalic structures by
constriction.
(v) Rather irregular transition from nuclear to fibrous parts.
(vi) Contact with eye structures in the tapetum region only.
Diencephalic structures are recognizable by the following features:
(i) The fibrous part is larger, more regularly shaped, and more clearly
peripherally located, as compared with telencephalic structures.
(ii) Contact with the retina region of the eye structures.
B. Quantitative analysis
A total of 82 grafts was transplanted, originating from 14 donors. [In two
donors segment 1 was not transplanted. In one of the series segments 1 and 2,
implanted into the same host, had fused; this graft has been included in the
calculation of average percentage composition of the combinations of segments 1
and 2 (Table 2), but not in that of average volume and percentage composition
of separate segments (Table 1 ; Figs. 2, 3). In one case segment 2 was divided
into an anterior and a posterior half before implantation; these grafts have been
considered separately.] Of the 82 grafts, seven were not retrieved in the hosts.
These grafts, all consisting of segment 1, probably formed no neural tissue. The
remaining 75 grafts were analysed quantitatively. Thirty-three grafts contain
eye structures only, while five grafts contain telencephalic structures only.
There are no cases consisting of diencephalic structures only. Eye together with
telencephalon is found in 22 grafts, eye together with diencephalon in six
grafts, eye together with telencephalon and diencephalon in seven grafts, and
telencephalon together with diencephalon in two grafts.
Grafts of segment 1, in which differentiation has taken place (only four cases),
Grafts of presumptive prosencephalon
755
Table 1. Average volume and average percentage composition of
neural masses of grafts of various segments, ± s.E.
Segment
no.
No. of
differentiated
grafts
Average
volume
(g.u.)*
Average
% diene.
Average
%eye
1
2
3
4
5
6
4
14
14
14
14
14
323 ±165
1023 ±208
1166 ±134
1186± 97
1275 ±143
730 ±106
—
5-6±3-5
01 ± 0 1
—
8-6 ±2-9
4-4 ±2-9
—
58-3 ±10-6
99-6 ± 0-3
95-3 ± 2-2
83-7 ± 5-2
5 9 0 ± 8-7
Average
% telene.
100
361 ± 9 0
0-3 ±0-3
4-7 ±2-2
7-7 ±4-2
36-6 ±6-7
* (3rid units (see text)
100 r
100
90
90
80
80 -
70
70 60
60
^
50
^
50
40
40
30
30 h
20
20
10
Segment:
n:
^
1
4
2
14
3
14
4
14
5
14
1
6
14
10h
Segment: 1
n:4
2
14
3
14
4
14
5
14
6
14
Ë53
telene.
eye
diene.
Fig. 2
telene.
eye
diene.
Fig. 3
Fig. 2. Occurrence of telencephalic, eye, and diencephalic structures in grafts of different segments, expressed as percentages of the total number (n) of differentiated
grafts of the segment concerned.
Fig. 3. Average composition of neural masses of different segments, expressed in
volume percentages.
consist exclusively of telencephalon. Segment 2, on the contrary, shows much
variation. Four grafts consist of eye structures only, eight grafts contain a
varying amount of eye structures, either together with telencephalon (six cases)
or together with telencephalon and diencephalon (two cases). There are two
grafts containing telencephalon and diencephalon only. None formed telencephalic structures only. The differentiation of segment 3 is very uniform. Usually
only eye structures are formed, and there are only three cases with very small
brain structures (1-3 vol. %). Segment 4 gives a rather similar picture, but the
756
E. C. B O T E R E N B R O O D
brain structures formed in four cases are larger: 9-24 vol. % (only telencephalon). Likewise, all grafts of segment 5 always contain eye structures, but
their average volume is lower than in segment 4. The number of grafts containing eye as well as brain structures has increased to eight cases, with volumes
of brain structures varying between 12 % and 66 %. Finally, segment 6 again
shows a considerable variation, the results being very similar to those of segment 2.
The average volume of eye structures and of diencephalic structures is lower than
in segment 5, whereas that of telencephalic structures is considerably higher.
The average percentage occurrence of telencephalic, eye and diencephalic
structures in grafts of different segments is given in Fig. 2. The average quantitative composition of the grafts arranged according to their origin is given in
Table 1 and in Fig. 3. The quantitative results of these experiments show on
application of the sign test significant differences in volume percentages of
telencephalic and eye structures in segments 2 and 6 as compared with those in
segments 3-5 (P ^ 004).
DISCUSSION
With the exception of segment 1, which forms telencephalic structures only,
each segment is able to form at least two, and usually all three prosencephalic
elements. There is, however, a considerable regional variation among the various
segments (see Table 1 and Figs. 2,3). Assuming, as mentioned in the Introduction,
that the average structural composition of a grafted region is an expression of the
distribution of differentiation tendencies in that region, the results give the impression that the various segments fall into three different categories, namely:
(i) The three differentiation tendencies are well represented, so that all
prosencephalic elements are formed. This situation is seen in segments 2 and 6.
(ii) A situation intermediate between (i) and (iii). This is found in segment 5.
(iii) The differentiation tendencies are completely or almost completely
restricted to only one of the elements. This situation is seen in segment 1, where
only telencephalon is formed, and in segments 3 and 4, where eye structures are
formed almost exclusively.
Very probably these three categories represent subsequent stages in prosencephalic pattern formation: at an early stage the cells possess only a 'nonspecific' prosencephalic differentiation tendency, which implies that the cellular
material may still differentiate in various directions, resulting in the formation of
telencephalic as well as eye and diencephalic structures. In a later stage the
cellular material acquires 'specific', mutually exclusive differentiation tendencies for the development of either telencephalic, eye, or diencephalic structures
(see Boterenbrood, 1962). From the present results it may be concluded that at
the time of operation there are regional differences in the progress of prosencephalic pattern formation.
The transition from regions with 'specific' to regions with 'non-specific'
differentiation tendencies is apparently gradual when going from segments 3
Grafts of presumptive prosencephalon
757
Table 2. Average composition of neural masses of grafts of median regions
(present investigations, see Fig. 1) and of grafts of corresponding medio-lateral
regions (P. D. Nieuwkoop et al., unpublished observations 1965, see Fig. 4) of the
anterior neural plate and transverse neural fold
]Data
of the
nt investigation
Stage 141
Combination
of segments
1 +2
3+4
Average
composition
(vol. %)
Diene. 6
Eye
45
Telene. 49
(n = 19)
Diene. —
Eye
97
Telene. 3
(n = 28)
Data of P. D. Nieuwkoop et al.
r-
Stage 13*
Segment
A
B
Average
composition
(vol. %)
Diene. 5
Eye
40
Telene. 55
(n = 27)
Diene. 16
Eye
66
Telene. 17
(/» = 27)
C
5+6
Diene. 7
Eye
71
Telene. 22
(n = 28)
Stage 151
Segment
A
B
C
Diene. 66
Eye
21
Telene. 13
(n = 27)
Average
composition
(vol. %)
Diene. 1
Eye
19
Telene. 80
(« = 29)
Diene. 4
Eye
84
Telene. 12
(n = 29)
Mesenc. 1
Diene. 59
Eye
28
Telene. 12
(n = 29)
Stage 13j
Stage 15i
Stage 16£
Stage 16£
Stage I6i-
Segment
A
B
C
Average
composition
(vol. %)
Diene. 1
Eye
5
Telene. 94
(n = 27)
Diene. 23
Eye
65
Telene. 12
(/» = 27)
Mesenc. 6
Diene. 85
Eye
7
Telene. 2
{n = 27)
Fig. 4. Diagram illustrating the regions of the anterior neural plate and transverse
neural fold excised by P. D. Nieuwkoop et al. (unpublished observations, 1965) at
stages 13*, .151 and 161 ' n Triturus alpestris. Region A contains the presumptive
telencephalon, region B corresponds to the presumptive eye areas and the surrounding
presumptive anterior diencephalon, and region C to the remaining posterior part of
the presumptive diencephalon.
758
E. C. BOTERENBROOD
and 4 posteriorly, but sudden when going anteriorly. In the latter case the transition coincides with the borderline between the anterior neural plate and the
transverse neural fold. It seems that there is also a sudden transition between the
regions of segment 2 and segment 1, but the number of grafts of segment 1
showing differentiation is too low to allow a definite conclusion.
Assuming that the realization of the morphological pattern is due to a change
of an original situation of equilibrium between the various differentiation
tendencies to a situation where one of the tendencies predominates over the
others, it may be concluded that the realization of the normal prosencephalic
pattern begins in the centre of the presumptive area with the predominance of the
tendency for eye differentiation. Both more anteriorly and more posteriorly the
process of pattern formation is apparently less far advanced. This probably
holds also for more lateral regions (see below). Using the same criterion it may
be suggested that the anterior periphery of the presumptive prosencephalon is
another area where the process is more advanced, the tendency for the telencephalic differentiation being predominant in this case. However, at present the
data are too limited for a definite statement.
When comparing the results of the present investigation with those of P. D.
Nieuwkoop et al. (unpublished observations, 1965), two differences in the present
experimental set-up have to be considered, namely the median origin, and the
small size of the grafts. For a meaningful comparison (see Table 2) the results of
segments 1+2, 3 + 4 and 5 + 6 have to be combined, since these combinations
are approximately equal to the median areas of the segments A, B, and C of
Nieuwkoop's study (compare Fig. 1 and Fig. 4). Of that study the data pertaining to stages 13^ and 15^ are most relevant. The composition of segments
[1 +2] does not differ markedly from that of segment A. The segments [3 + 4]
show a considerably higher percentage of eye structures than segment B. This
may be due to the presence of lateral regions in grafts of segment B, since
Adelmann (1930) found that lateral parts of the anterior neural plate, grafted to
the ventral side of a neurula, formed eye structures in only 11 % of the cases,
whereas median parts did so in 71 % of the cases. Apparently in the lateral
regions the cellular material possesses only 'non-specific' differentiation tendencies, so that on an average eye differentiation in segment B is reduced as
compared with segments [3 + 4] .This is another indication that in the presumptive prosencephalon regions can be distinguished which are not equally far
advanced as far as pattern formation is concerned. A most remarkable aspect of
the present investigation is the differentiation of segments [5 + 6], which shows a
low volume percentage of diencephalic structures and a high percentage of
telencephalic and eye structures in comparison with segment C of Nieuwkoop et
al. This result may be related to the fact that the segment C of Nieuwkoop et al.
included more lateral material, but very probably the small size of the grafts
used in the present investigation has also played a role. In previous investigations on reaggregates of presumptive prosencephalic material (Boterenbrood,
Grafts of presumptive prosencephalon
759
1962) it was found that in smaller neural masses relatively more telencephalic
structures were formed. Apparently a special, not yet analysed condition prevailing in the peripheral zone of a neural cell mass favours telencephalic differentiation. This 'peripheral condition' may have played a role in the small
grafts of the present investigation. Such a shift of differentiation is only conceivable as long as the cells still have 'non-specific' prosencephalic differentiation
tendencies. The fact that in the segments 3 and 4 a similar change towards a
'peripheral condition' has not led to a relative increase of telencephalic differentiation is another indication that at stage 14+ the cells of these segments
have already acquired differentiation tendencies for the development of eye
structures.
RÉSUMÉ
Différenciation
de greffons provenant de la région médiane
prosencéphale
présomptif
du
Dans la jeune neurula de Triturus alpestris, la distribution cranio-caudale des tendances à
la différenciation dans le prosencéphale présomptif, a été recherchée.
Les greffons sont obtenus par excision de bandes médianes prélevées dans la région de la
plaque neurale et du repli neural transverse. Ils sont transplantés dans une poche pratiquée
sous l'ectoderme cranioventral de la jeune neurula.
Les greffons provenant de la partie centrale de la bande excisée montrent presque exclusivement une différenciation de type 'œil', tandis que ceux provenant de la partie craniale ou
caudale donnent lieu à relativement plus de différenciation télencéphalique et peu de différenciation diencéphalique.
Les résultats indiquent que le processus de la régionalisation des ébauches dans la zone
centrale du prosencéphale présomptif est en avance par rapport à celui des aires qui entourent
cette zone.
The author wants to thank Dr C. von Woellwarth, Heiligenberg, for the supply of
Triturus alpestris, Miss A. de Wit for the very accurate execution of the volume determinations,
and Dr J. Faber for the correction of the English text.
REFERENCES
ADELMANN, H. B. (1930). III. The effect of the substrate ('Unterlagerung') on the heterotopic development of median and lateral strips of the anterior end of the neural plate of
Amblystoma. J. exp. Zool. 57, 223-81.
BOTERENBROOD, E. C. (1962). On Pattern Formation in the Prosencephalon. An Investigation on
Disaggregated and Reaggregated Presumptive Prosencephalic Material of Neurulae of
Triturus alpestris. Thesis, Utrecht.
HARRISON, R. G. (1969). Harrison stages and description of the spotted salamander,
Amblystoma punctatum (L.). In Organization and Development of the Embryo (ed. S. Wilens),
pp. 44-66. New Haven and London: Yale University Press.
NIEUWKOOP, P. D., NIERMEIJER, E. K. & JANSEN, W. F. (1964). When and how pattern for-
mation begins in the presumptive prosencephalon. / . Anim. Morph. Physiol. 11, 1, 21-44.
WOELLWARTH, C. VON (1960). Über das Anlagenmuster und die Kinematik des Ektoderms,
insbesondere der präsumptiven Epidermis, im Neurula und Schwanzknospenstadium
von Triturus alpestris. Wilhelm Roux Arch. EntwMech. Org. 152, 602-31.
(Manuscript received 28 July 1969)