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.

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.

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 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

Design of experiments
Design of experiments

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 μ) to prevent its enlargement. As two regions were useful for our purposes, they are designated as control series 1 (a) 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.

Fig. 1.

Diagram showing the operations performed in the first experimental system. See text for detailed comments. D = donor; H1 = host (Exp. 1); H2 = host (Exp. 2).

Fig. 1.

Diagram showing the operations performed in the first experimental system. See text for detailed comments. D = donor; H1 = host (Exp. 1); H2 = host (Exp. 2).

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 1 (a), the ventral side of expiants 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 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.

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).

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).

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 μCi of [3H]thymidine (Radiochemical Centre, Amersham, England). For the autoradiography, 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 μ stained with kernechtrot.

In control series 1 (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. 3C, 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 rhomboïdalis 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 (b). 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.

Fig. 3.

(A). This isolate explanted in the control series 1(a) differentiated into a typical trunk. A well-differentiated, widely open neural tube is lying over the mesoblast, ×25.

(B). This case concerns Exp. 2. Segmented somites are formed along the implanted notochord, × 10.

(C). In control series 1(6), the posterior region gives rise only to an incomplete trunk devoid of notochord and somites, × 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, × 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, × 200.

(F). This explant coming from the anterior part of the primitive streak differentiates mainly into somites and notochord, × 25.

(G). In control series 3, the explant has a posterior origin and yields only extraembryonic mesoblast, × 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, × 25.

(I). Section through a differentiated explant in Exp. 3. The segmented somite is closely associated with the fragment of the implanted head process, ×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. ×165.

Fig. 3.

(A). This isolate explanted in the control series 1(a) differentiated into a typical trunk. A well-differentiated, widely open neural tube is lying over the mesoblast, ×25.

(B). This case concerns Exp. 2. Segmented somites are formed along the implanted notochord, × 10.

(C). In control series 1(6), the posterior region gives rise only to an incomplete trunk devoid of notochord and somites, × 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, × 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, × 200.

(F). This explant coming from the anterior part of the primitive streak differentiates mainly into somites and notochord, × 25.

(G). In control series 3, the explant has a posterior origin and yields only extraembryonic mesoblast, × 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, × 25.

(I). Section through a differentiated explant in Exp. 3. The segmented somite is closely associated with the fragment of the implanted head process, ×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. ×165.

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.

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.

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.

Fig. 5.

Diagram showing the structures differentiated from the four kinds of expiants 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.

Fig. 5.

Diagram showing the structures differentiated from the four kinds of expiants 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.

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.

Table 2.

Frequency of somite formation as observed in experimental series and their respective controls

Frequency of somite formation as observed in experimental series and their respective controls
Frequency of somite formation as observed in experimental series and their respective controls
Table 3.

The formation of somites, notochord and neural tissue in control and experimental series

The formation of somites, notochord and neural tissue in control and experimental series
The formation of somites, notochord and neural tissue in control and experimental series

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 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,

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.

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.

La chorde présomptive intervient-elle directement dans la différenciation des somites chez le poulet ?

Les expériences consistent à implanter un fragment de noeud de Hensen ou de prolongement céphalique au sein de la ligne primitive à 0·8 mm derrière le noeud de Hensen. Pour isoler la région postérieure de toutes influences émanant de la région antérieure du blastoderme, nous procédons soit à l’excision d’un vaste territoire quadrangulaire situé juste devant l’implantat de chorde présomptive, soit à l’explantation de la région combinée avec l’implantat dans l’aire opaque. Comme contrôles, des régions correspondantes de la ligne primitive ont été isolées ou explantées.

Chez les contrôles, la formation des somites n’a jamais lieu, alors que l’implantation de matériel chordal dans le territoire postérieur est presque toujours suivie par l’apparition de somites.

Dans ces expériences, la chorde présomptive agit sur son nouvel environnement comme le ferait le facteur chordal qui a été découvert chez les Amphibiens.

This work was generously supported by the Fonds national suisse de la Recherche scientifique, Berne, Switzerland.

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