/ . EmbryoL exp. Morph. Vol. 24, 3, pp. 467-478, 1970
467
Printed in Great Britain
Is the presumptive notochord responsible for
somite genesis in the chick?
By G. NICOLET 1
From the Institut d'Histologie et cTEmbryologie,
Laboratoire d'Embryologie experimentale, Ecole de Medecine
de VUniversite de Geneve, Switzerland
SUMMARY
Fragments of Hensen's node or head process were implanted into the primitive streak
0-8 mm behind the node. The posterior region was isolated from all influences emanating
from the anterior part of the blastoderm, either by cutting off a large rectangular piece just in
front of the implant or by explanting the region combined with the notochord implant into
the area opaca. As controls, corresponding regions of the primitive streak were isolated or
explanted.
In controls, somite formation never took place, while the implantation of presumptive
notochord into this posterior region was followed by the formation of somites in most cases.
In these experiments, the action of the presumptive notochord in its new situation was
similar to that of the chorda-factor in amphibians.
INTRODUCTION
Several authors have suggested that a role may be ascribed to Hensen's node
in somite formation (Peebles, 1898; Wetzel, 1929; Fraser, 1954). In the present
work, we have attempted to analyse how far the presumptive notochord, which
is one of the major components of the node, may be implicated in this process
by implanting fragments of presumptive notochord into the posterior parts of
the primitive streak, as performed in slightly different conditions by Bellairs
(1963). Adequate controls were carried out to show that the grafts of presumptive notochord were not contaminated by presumptive somitic cells and that
the posterior parts used were unable to form somites spontaneously.
MATERIALS AND METHODS
Chick blastoderms cultured in vitro (Gallera & Nicolet, 1961) were employed.
The normal stages of development were defined according to the tables of
Hamburger & Hamilton (1951). As mentioned in Table 1, our research is
divided into two experimental systems. In the experimental series, we sometimes
used labelled grafts to recognize accurately the structures provided by the
1
Author's address: Institut d'Histologie, Ecole de Medecine, 1211, Geneve 4, Switzerland.
468
G. NICOLET
implants and to be sure that they did not contain any presumptive somitic cells.
In experimental series as well as in controls, the operation was performed at
stage 5, when the length of the head process was about 0-5 mm.
Table 1. Design of experiments
Experimental system 1
Experimental system 2
Control 1: 8 cases
Experiment 1: 8 (5)* cases
Experiment 2:18 (6) ""cases
Control 2: 8 cases
Control 3: 16 cases
Experiment 3: 5 (4)* cases
Experiment 4: 9 (4)* cases
* The numbers in parentheses represent the cases in which labelled grafts were implanted.
In each series, the surgical operation required several steps as indicated by
Fig. 1 for the first experimental system. The control series 1 consisted of extirpating a rectangular area containing the anterior part of the primitive streak.
After its antero-posterior axis had been rotated through 180°, it was transplanted into the area opaca. The germ-wall at the site of explantation was carefully eliminated before we applied the ventral side of the explant to the ventral
surface of the peripheral ectoblast. The injury made in the blastoderm was
covered by a piece of a thick millipore filter (150 fi) to prevent its enlargement.
As two regions were useful for our purposes, they are designated as control
series \{d) and (b). In Exps 1 and 2, a small square of primitive streak was cut
off 0-8 mm behind the node and was exchanged for a graft of similar size coming
either from the young head process (Exp. 1) or from Hensen's node (Exp. 2).
After 45 min, the implant was sufficiently attached to the neighbouring tissues
to make it possible to extirpate the anterior part of the primitive streak and
cover the hole by a piece of millipore filter.
In the second experimental system, various regions of the primitive streak
were explanted on the area opaca (Fig. 2). They were mainly composed of
invaginating mesoblastic cells but also contained the few endoblastic cells which
cover the ventral side of the primitive streak. On the other hand, some part of
the neural tissue surrounding the node was involuntarily cut off with the graft
in a few cases. Later, this neural tissue was found on sections as a small mass
tightly attached to the notochord. As in control series l(a), the ventral side of
explants was applied to the ectoblast and their cephalocaudal axis rotated
through 180°. A piece of millipore filter was put on the hole, so that it was
possible to use only one host embryo for all the experimental process. In
Exps 3 and 4, the explantation was preceded by the implantation of a notochord
piece of two different origins (Fig. 2). The implant was inserted 0-8 mm behind
the node. The host Hensen's node was also excised to slow down the regression
movement during the time required for the healing. A lapse of time of 2 h was
Notochord and somite genesis
469
necessary to assure a complete integration of the implant to its new environment. After this delay, the primitive streak region combined with the notochord
graft was explanted into the area opaca in the same way as in control series 2
and 3.
Control 1
H,
H,
Experiment 1
Experiment 2
Fig. 1. Diagram showing the operations performed in the first experimental system. See
text for detailed comments. D = donor; H ^ h o s t (Exp. 1); H2 = host (Exp. 2).
470
G. NICOLET
Several donors were labelled with [3H]thymidine. These embryos were used as
donors and were explanted some time before others, since 8 h were necessary
for good labelling of all nuclei. The medium contained 5 /*Ci of [3H]thymidine
Control 2
u
s
2(h)
H4
Experiment 3
Experiment 4
Fig. 2. Diagram showing the four kinds of explantation performed in the second
experimental system. See text for further comments. D = donor; H3 = host (Exp. 3);
H4 = host (Exp. 4).
Notochord and somite genesis
471
(Radiochemical Centre, Amersham, England). For the auto radiography, the
technique described by Ficq (1959) was used and Ilford emulsion K2. A correct
exposure of the autoradiographic plates required about 30 days.
All the host embryos were incubated for about 36 h (including the incubation
time in ovo). They were fixed with Carnoy, drawn in toto in the camera lucida and
often photographed. The histological study has been made on serial sections of
8 /( stained with kernechtrot.
RESULTS
In control series I (a), isolates gave rise to well-differentiated trunks as
formerly observed in particular by Spratt (1955) (Fig. 3 A). On the contrary,
the development was somewhat different in control series 1 (b). Since the rim
of the hole adhered to the millipore filter, the posterior part of the blastoderm
did not become V-shaped, as noticed by many authors (Waddington, 1935;
Jacobson, 1938; Butros, 1962; Bellairs, 1963). Nevertheless, a typical regression
took place. It led to the formation of incomplete trunks in which the presence
of a reduced spinal cord was observed. These trunks contained neither notochord nor somite, and the mesoblast lying on each side of the reduced spinal
cord must have been lateral mesoblast since it was sometimes segregated into
lateral plate (Figs. 3 C, D; 4). Posteriorly, the reduced spinal cord and the two
lateral plates fused together and were continued by the shortened primitive
streak. Such topographical relationships suggest that normogenesis has occurred
in this system, since a more or less normal sinus rhomboiidalis has been formed
in spite of the heavy loss of material from the spinal cord and the complete
absence of notochord and somites. In Exps 1 and 2, the implanted notochord
elicited the formation of one or two rows of somites (Figs. 3B, E; 4). Examination of hosts in which labelled graft was implanted, shows that all nuclei of the
notochord and a few of the endoblastic nuclei were labelled. Somite material
was never labelled. The spinal cord of these embryos remained as reduced as in
control series 1 (6). Somite segmentation began just behind the posterior edge of
the hole and progressed backwards. The anterior tip of the notochord was
generally lying behind the first pairs of somites.
Let us now examine the results of the second experimental system (Fig. 5).
Isolates of the anterior part of the primitive streak gave rise mainly to axial and
paraxial mesoblast (Fig. 3F). Sometimes, a small mass of neural tissue was
observed tightly attached to the notochord. Posterior isolates of the primitive
streak differentiated into extra-embryonic mesoblast (Fig. 3G). In Exps 3 and 4,
an intermediate response was obtained after implantation of presumptive notochord: a short embryonic axis included in a small area vasculosa (Fig. 3H, I).
The mesoblast adjacent to the somites was segregated into lateral plate. After
implantation of a labelled graft, the labelling was again exclusively found in the
notochord (Fig. 3 J), and in a few endoblastic cells. As in control series 2, a small
neural mass was present in a few cases.
30
E M B 24
472
G. NICOLET
Notochord and somite genesis
473
The results are expressed in a semi-quantitative way in two tables (Tables 2, 3).
Table 2 shows that no somite formation took place in the posterior region (see
control series 1 (b) and 3) unless it was combined with a notochord fragment,
but somite formation was observed whenever Hensen's node was implanted.
On the other hand, the young head process did not succeed in provoking somite
segmentation in all cases.
Examination of Table 3 shows how far the somitic response was linked either
to the presence of notochord or to that of neural tissue. The notochord was
Table 2. Frequency of somite formation as observed in experimental
series and their respective controls
Series
Number
of
cases
Control 1 (b)
Experiment I
Experiment 2
Control 3
Experiment 3
Experiment 4
8
8
18
16
5
9
Presence
of
Frequency
(%)
somites
0
6
18
0
4
9
0
75
100
0
80
100
Fig. 3 (A). This isolate explanted in the control series l(a) differentiated into a
typical trunk. A well-differentiated, widely open neural tube is lying over the
mesoblast. x 25.
(B). This case concerns Exp. 2. Segmented somites are formed along the implanted
notochord. x 10.
(C). In control series 1(6), the posterior region gives rise only to an incomplete
trunk devoid of notochord and somites, x 10.
(D). Section across the incomplete trunk seen in (C). The floor of the reduced spinal
cord lies directly on the endoblast. On each side, the mesoblast is not well organized
into lateral plate, x 220.
(E). Section through the embryo shown in (B). The topographical relationships
between the various Anlage are almost normal, except that the notochord is not
attached to the neural tube, x 200.
(F). This explant coming from the anterior part of the primitive streak differentiates
mainly into somites and notochord. x 25.
(G). In control series 3, the explant has a posterior origin and yields only extraembryonic mesoblast. x 25.
(H). In Exp. 4, the cells, which are in close contact with the notochord implant,
differentiate into somites. The mesoblast contiguous with the somites is converted
into lateral plate, whereas the peripheral mesoblast gives rise to a small area
vasculosa. x25.
(I). Section through a differentiated explant in Exp. 3. The segmented somite is
closely associated with the fragment of the implanted head process, x 165.
(J). In Exp. 4, almost all the labelling is condensed in the notochord, after implantation of a labelled graft. In the present case, typical lateral plates are adjacent to the
somites, x 165.
30-2
474
G. NICOLET
Table 3. The formation of somites, notochord and neural tissue in
control and experimental series
Structures
(
Tslntn
IN UlU
K
\
Total
Series
chord
Somites
Control 1 (a)
Control 1 (b)
Experiment 1
Experiment 2
Control 2
Control 3
Experiment 3
Experiment 4
8
0
8
18
8
0
5
9
8
0
6
18
8
0
4
9
tissue*
Number of segmented somites
of
rNvUid.1
cases
1-5
6-10
8
8
8
18
8
16
5
9
0
4
0
0
_
3
2
0
2
7
1
1
3
8+ +
8+
8+
18 +
8+
0
2±
3±
11-15 16-20
0
0
6
5
0
4
8
0
5
2
0
0
* + + , Well-developed spinal cord; + , reduced spinal cord; ± , smal 1 mass of neural
tissue.
1 mm
Control 1 b
Experiment 1
Experiment 2
Fig. 4. Diagram showing the structures observed in the isolated posterior part
combined or not combined with an implant of notochord. The same magnifications
have been used in these three drawings. Structures are designated by numbers,
namely: 1 = reduced spinal cord; 2 = notochord; 3 = segmented somites;
4 = unsegmented somitic mesoblast; 5 = lateral plate; 7 = shortened primitive
streak. See text for further comments.
Notochord and somite genesis
475
always present when somite formation occurred, whereas the presence or
absence of neural tissue had apparently no effect upon it. In the right part of
Table 3, the number of segmented somites counted in each case have been distributed into four categories. The reliability of this method is limited, since the
size of somites is not constant and their number depends on the stage attained
by the embryo at the time of fixation. In spite of these restrictions, this quantitative expression of the somitic response was in agreement with the morphological
observations. The impression was gained that, in both experimental systems,
Control 2
Control 3
Experiment 3
Experiment 4
Fig. 5. Diagram showing the structures differentiated from the four kinds of explants
used in the second experimental system. All have been drawn at the same magnification
and represent the typical response obtained in each series. Structures are designated
by the following numbers: 1 = neural tissue; 2 = notochord; 3 = segmented
somite; 4 = unsegmented somitic mesoblast; 5 = lateral plate; 6 = extraembryonic mesoblast.
Hensen's node produced a stronger somitic response than the young head
process. This rule seems clearly verified by the quantitative analysis. However,
further investigations are needed to explain this quantitative difference, for
several factors may be involved. First of all, we must be sure that the young head
process is incorporated into the primitive streak as well as Hensen's node before
we assume that the node indeed stimulates somite formation more actively.
Finally, the somitic response was higher in control series 2 than in Exp. 4,
indicating, as expected, that somite differentiation is less activated in the
heterogenous combination than in the normal association.
476
G. NICOLET
DISCUSSION
Whenever labelled grafts were used it was observed that they were not
contaminated by presumptive somitic cells, so that it is very likely that this
condition was also fulfilled when implants of similar size came from unlabelled
donors. The differentiated notochords found in posterior isolates were entirely
composed of labelled nuclei, but not all labelled nuclei were contained in them
since a few endoblastic cells and sometimes presumptive neural cells were cut
off with the grafts.
In the first experimental system, a typical pattern of regression was observed
in controls as well as in experimental series. Therefore, as already assumed by
Waddington (1935, 1952) and Butros (1962, 1967), an autonomous movement
of regression occurs in posterior isolates. In fact, this result was expected, since
several authors have shown that backward movements were observed at all
levels of the primitive streak (Spratt, 1947; Vakaet, 1960; Nicolet, 1970).
The posterior isolates used could not differentiate into somites by themselves.
In the first experimental system, posterior remnants of the primitive streak gave
rise to lateral plate according to their normal prospective significance (Wolff,
1936; Nicolet, 1970). On the other hand, after explantation into the area opaca
(see control 3), they were no longer able to differentiate into lateral plate, but
only into extra-embryonic mesoblast. The implantation of presumptive notochord
in these posterior isolates elicited somite formation in most cases, in agreement
with the results obtained by Bellairs (1963). Hence, it is concluded that the
presumptive notochord has modified the prospective significance of some of
these cells. The chorda bulb as well as the head process acted in a similar way,
though the latter was less efficient as activator.
In our opinion, the major interest of the present experiments is in showing
clearly that the action of the presumptive notochord of the chick in this new
situation is similar to that of the chorda-factor discovered in amphibians
(Yamada, 1940; Niazi, 1969). At first sight, it seems unlikely that the presumptive notochord has the same action in the normal development of birds because
it has been demonstrated that its presence is not required for somite differentiation after stage 5 (Waddington, 1932; Spratt, 1955). However, the possibility
cannot be excluded that it may play a role in the earlier steps of somitic determination, since the presumptive notochordal cells start to congregate in
Hensen's node as soon as stage 3 (Gallera & Nicolet, 1969; Nicolet, 1970).
Hence, this accumulation of the presumptive notochord in the node begins
earlier than the invagination of the presumptive somitic cells through the
primitive streak.
Notochord and somite genesis
All
RESUME
La chorde presomptive intervient-elle directement dans la differentiation
des somites chez le poulet ?
Les experiences consistent a implanter un fragment de noeud de Hensen ou de prolongement cephalique au sein de la ligne primitive a 0-8 mm derriere Je noeud de Hensen. Pour
isoler la region posterieure de toutes influences emanant de la region anterieure du blastoderme, nous procedons soit a l'excision d'un vaste territoire quadrangulaire situe juste devant
1'implantat de chorde presomptive, soit a l'explantation de la region combinee avec
Pimplantat dans 1'aire opaque. Comme controles, des regions correspondantes de la ligne
primitive ont ete isolees ou explantees.
Chez les controles, la formation des somites n'a jamais lieu, alors que l'implantation de
materiel chordal dans le territoire posterieur est presque toujours suivie par l'apparition de
somites.
Dans ces experiences, la chorde presomptive agit sur son nouvel environnement comme le
ferait le facteur chordal qui a ete decouvert chez les Amphibiens.
This work was generously supported by the Fonds national suisse de la Recherche
scientifique, Berne, Switzerland.
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{Manuscript received 20 January 1970)