/. Embryol. exp. Morph. Vol. 28, 3, pp. 547-558, 1972
Printed in Great Britain
547
Studies on self-differentiating
and induction capacities of Hensen's node using
intracoelomic grafting technique
By J. R. VISWANATH 1 AND LEELA MULHERKAR 1
From the Department of Zoology, University of Poona
SUMMARY
Living Hensen's node of the definitive primitive streak of chick embryo was prepared into
'sandwiches' with the competent ectoderm and the sandwich grafts were transplated into the
2-5 day chick embryo using the intracoelomic grafting technique of Hamburger.
One hundred and twenty-four grafts were prepared and transplanted intracoelomically,
28 grafts were lost due to the death of the host embryos, 63 grafts did not differentiate at all,
but 33 well-defined grafts were recovered, after cultivating the transplanted hosts for 12-14
days.
All kinds of tissues from feather germs to neural tissue were found to have differentiated
in the grafts. The more frequently occurring tissues were feather germs, epidermal vesicle,
neural tissue, kidney and muscle. Other differentiations were the cartilage notochord and gut.
No definite combination pattern has emerged from the tissues. But when the tissues were
traced to their germ-layer derivation, 22 of them belonged to the mesodermal complex, 11 to
the ectodermal complex and 8 to the endodermal complex.
In the light of the above results, the probable existence of a mesodermal factor and an
ectodermal factor independently responsible for the respective differentiations, as also the
competence of the ectoderm, is discussed.
INTRODUCTION
The capacity of Hensen's node for self-differentiation and induction has been
investigated using different techniques by various investigators. Wetzel (1929),
Hunt (1931), Willier & Rawles (1931) and Rudnick (1948) claimed that Hensen's
node alone has a capacity to form axial structures and is therefore an organizer.
It was not until 1932 that Waddington, using intrablastodermal grafting technique, showed that the anterior third of the primitive streak possesses organizing
capacity in chick embryo cultivated in vitro. Spratt (1942, 1948, 1952), using in
vitro techniques, has also reported the differentiating capacity of the parts of the
early chick blastoderm, including Hensen's node. However, no work has so far
been reported on the potentialities of Hensen's node using intracoelomic
grafting technique; although the studies on killed node using the same technique
have been reported (Viswanath, Leikola & Rostedt, 1968). The present investi1
Authors' address: Department of Zoology, University of Poona, Poona 7, India.
35"=
548
J. R. VISWANATH AND L. MULHERKAR
gation has been undertaken to study self-differentiation and induction capacities
of living Hensen's node using anintracoelomic grafting technique and to compare
them with those of the killed node where the same technique has been employed
(Viswanath et aJ. 1968).
MATERIAL AND METHODS
Freshly laid White Leghorn eggs supplied by a local hatchery were incubated
at 37-5 °C. Eggs used as donors were incubated to definitive primitive streak
stage (Hamburger & Hamilton (1951), stage 4) and those that were used as hosts
were incubated to 60-70 h embryos (Hamburger & Hamilton (1951), stage
15-16). The blastoderms from the donor eggs were separated from the underlying yolk mass and spread in Locke solution in the Petri dish with agar base.
Competent ectodermal pieces antero-lateral to the Hensen's node were excised
by carefully stripping off the endo- and mesodermal layers by means of sharp
tungsten needles. Simultaneously the region of Hensen's node was excised,
isolated and pipetted into a separate dish with Locke solution, Yamamoto (1949).
Inner side of the already prepared competent ectoderm piece was brought in
intimate contact with the inner surface of the Hensen's node to make a ' sandwich' which was kept for 30 min at room temperature to ensure proper
attachment and then incubated at 37-5 °C for 3-6 h in order to facilitate contact
between the reactive ectoderm and the mesoderm of the node. This was absolutely
essential, since the contact time required is at least 6 h to produce a neuroid
reaction in the ectoderm of area opaca (Gallera, 1965). In the area pellucida
ectoderm typical neural inductions were obtained after 3-4 hours of contact
(Gallera, 1970; Hara, 1961; Leikola & McCallion, 1967; Viswanath et al. 1968).
Preparation of hosts
Eggs incubated to 2-5 days, by which time the amniotic fold has reached the
omphalomesenteric blood vessels, were used as hosts. That would be stage 16
of the Hamburger & Hamilton series. The intracoelomic grafting technique of
Hamburger (1960) has been used throughout and guidelines envisaged by Hara
(1961) and Rao (1968) have been mainly followed. The air chamber was
punctured, a window was made on the shell and Locke solution was pipetted
in. Part of the vitelline membrane covering the lateral somatopleure on either side
of the embryo in the vicinity of the omphalomesenteric blood vessels was removed with the aid of tungsten needles. The cultured sandwich was gently
pipetted close to the slit made in the embryo and inserted into the coelom.
The hosts were cultivated generally for 12-14 days, but sometimes for 8-9 days
in the incubator depending upon their condition, then they were sacrificed
and opened. The grafts found inside the coelomic cavity were excised, fixed
with Bouin, sectioned, stained with haematoxylin/eosin and examined histologically.
1. Neural tissue
2. Notochord
3. Muscle
4. Cartilage
5. Kidney
6. Blood vessels
7. Gut
8. Respiratory
tubules
9. Epidermal
vesicles
10. Feather germs
1. Neural tissue
2. Notochord
3. Muscle
4. Cartilage
5. Kidney
6. Blood vessels
7. Gut
8. Respiratory
tubules
9. Epidermal
vesicles
10. Feather germs
Tissues induced
or differentiated
18
19
20
21
4
23
6
24
7
25
+
26
+
9
27
10
+ = Tissues induced or differentiated.
22
5
28
11
29
12
+
+
31
+
+
+
+
+
14
30
+
+
+
+
+
+
13
-
32
+
+
+
15
25
10
+
12
3
19
6
10
8
5
7
Total
+
+
+
+
+
+
+
+
+
33
+
+
+
+
+
+
+
17
+
16
Table 1. Showing the number of instances of the induced or differentiated tissues and their combination
pattern in grafts 1-33
a
§
citie
550
J. R. VISWANATH AND L. MULHERKAR
Capacities of Hensen s node
551
In control operations only the competent ectoderm without the inductor kept
for 3-6 h in a minimal amount of Locke solution at 37-5 °C was inserted into
the host coelom.
RESULTS
Of a total of 124 grafts prepared and transplanted intracoelomically 28 grafts
were lost due to the death of the host embryos, 63 grafts did not differentiate at
all, but 33 well-defined grafts were recovered mostly on the posterior aspect of
the coelomic cavity. Usually they were found attached to the body wall of the
host embryo or to the mesentery. The grafts could be distinguished clearly from
the host tissue by its spherical or bulbous structure with distinct feather germs
noticed on the surface. All the identified tissues observed in the 33 grafts are
detailed in Table 1. Out of 33 hosts showing grafts, 22 were alive till the 14th
day, 5 up to the 10th day, while the rest lived less than 10 days.
All kinds of tissues from neural to feather germs were found to have differentiated in the grafts as categorized in Table 1. The more frequently occurring
tissues were epidermal vesicle (Figs. 6, 9), muscle (Figs. 1, 2, 6, 8), neural tissue
(Figs. 1, 4), kidney (Fig. 9) and feather germs (Fig. 1). As can be made out from
Table 1, no definite combination pattern has emerged from the tissues. Virtually
all kinds of tissues occurred as such, but when these tissue patterns were traced
to their germ-layer derivation, 22 of them belonged to the mesodermal complex,
11 to the ectodermal complex and 8 to the endodermal complex (Table 1).
Of the 21 control experiments only 3 cases showed small epidermal vesicles
and the remaining 18 were probably absorbed in the host tissue, or another
possible explanation would be the failure of the control grafts to adhere to the
body wall of the hosts.
DISCUSSION
It can be seen from the results that a number of structures from neural tissue
to feather germs have been differentiated in the sandwiches (of Hensen's node
and competent ectoderm) grown intracoelomically. The difficulty of distinguishing between the node (inductor) and the reacting ectoderm makes it
impossible to state with certainty which tissues have differentiated as a result of
the self-differentiating capacity of the node and those which are formed as a
ABBREVIATIONS ON FIGURES
Ch = Notochord, Ct = cartilage, Ev = epidermal vesicle, FG = feather germs,
G = gut, K— Kidney tubules, M= muscle, TV = neural tissue.
Fig. 1. Almost the entire graft in section, showing neural tissue, cartilage, muscle,
gut and feather germs, x 48.
Fig. 2. Graft showing cartilage, muscle and gut. x 48.
Fig. 3. Part of the cartilage from Fig. 2 magnified, x 135.
552
J. R. VISWANATH AND L. MULHERKAR
Fig. 4. Part of the graft showing neural tube, x 165.
Fig. 5. Part of the neural tube from Fig. 4 magnified, x 750.
Capacities of Henserfs node
M
Fig. 6. Graft showing notochord, muscle and epidermal vesicle, x 54.
Fig. 7. Part of the notochord from Fig. 6 magnified, x 155.
Fig. 8. Part of the graft showing notochord and muscle, x 54.
553
554
J. R. VISWANATH AND L. MULHERKAR
Fig. 9. Graft showing kidney tubules with blood vessels, and also an epidermal
vesicle on the side, x 135.
Fig. 10. Part of the graft showing the gut. x 135.
Capacities of HenserCs node
555
result of induction by the node. It is probable that the reactive ectoderm in
contact with Hensen's node is induced into neural tissue (Figs. 1, 4, 5). Existence
of mesodermal competence in the epiblast as shown by Waddington & Taylor
(1937) might have resulted in the formation and differentiation of muscles and
cartilage (Figs. 3, 6, 8). It is equally possible that the presence of notochord and
somite centres in the node (Spratt, 1955, 1957a, b, 1958) have attributed the selfdifferentiation of these structures - somites differentiating in course of time into
dermatome giving rise to dermis, sclerotome to cartilage andmyotome to muscles.
According to Waddington (1952, p. 87), feather germs can originate from ectoderm independent of inductive stimulus from the axial mesoderm. However,
some mesodermal cells might migrate out of the dermatome to form feather
germs (Fig. 1) as the dermis has been shown to be the inductor of feather
differentiation (Sengel, 1958). Endodermal derivatives such as gut (Fig. 10) or
respiratory tubules in the present case may either be due to the inductive interaction between the competent ectoderm and the axial mesoderm of the node
(Waddington, 1952, p. 87; Rudnick & Rawles, 1937) or to the presence and
differentiation of endoblast in the node at stage 4 (Nicolet, 1970, 1971, p. 246).
Bellairs (1953) and Nicolet (1970), using short-term culturing technique, have
found the differentiation of foregut from the node. However, Rudnick &
Rawles (1937) got the differentiation of small or large intestine from the fragments of node cultivated for 8-10 days on chorio allantois. In the present work
also gut resembling small intestine (Fig. 10) was formed probably by the
differentiation of the grafted node. The difference in the differentiation of the
level of gut (pharynx or intestine) may be attributed to the use of different
techniques as suggested by Bellairs (1953).
As the grafted sandwich is grown in the coelomic environment of the host, the
question arises whether the environment has influenced the differentiation or
induction of these structures. Waddington (1935) as well as Holtfreter (1934)
suggested the possibility of blood or chick embryonic extract possessing the
factor for neuralization in amphibians. No occurrence, however, of neuralizing
factor in the intracoelomic environment was reported by Hara (1961), although
he does not completely rule out such a possibility. Moreover, the reacting
ectodermal pieces without the inductor in the control series grown in the
same environment do not undergo any differentiation, indicating the neutrality
of the environment.
It will be interesting to compare at this stage the potentialities of the living
node with those of the killed node. The grafts of the killed node were unable to
induce neuralization in the reacting ectoderm (Pasternak & McCallion, 1962)
and therefore they concluded that the inductor is probably a component derived
from the mesoderm of the living nodes. Gallera, Nicolet & Baumann (1968) and
Gallera(l971) also failed to get neural reaction in the ectoderm of area opaca with
killed node. Waddington (1933, 1934) got only two cases of neuralization with
heat-coagulated chick organizer while Leikola & McCallion (1968) reported
556
J. R. VISWANATH AND L. MULHERKAR
neuralization in 17 out of 24 cases with alcohol-treated nodes. The property of
induction by killed nodes has been shown to be similar to that shown by heatcoagulated dorsal lip of blastopore in Amphibia (Bautzman, Holtfreter, Spemann
& Mangold, 1932). The self-differentiated capacity of the killed node has not
been reported by any of these investigators. In fact, according to Waddington
(1933, 1934), the dead implant is soon covered by mesenchyme cells of the host.
All the investigators used short-term culture techniques, and therefore differentiation, if any, into cartilage or muscles which arise late in development could
not be studied. A variety of structures exactly as shown by the living node in the
present work has been shown to be differentiated or induced by the ethanol-killed
node using intracoelomic grafting technique (Viswanath et ah 1968). If it is so it
means there is no qualitative difference in the differentiation and induction by
the living and killed node, or Hensen's node, which was immersed in 70%
ethanol only for 3-5 min, was not completely killed and that the cells which
were still alive evoked induction in competent ectoderm or self-differentiated
into various structures as seen in the living node. A similar case has been reported
by Gallera (1971, p. 172), in which the Hensen's node was incompletely killed
by freezing. The problem will have to be reconsidered before the comparison
is made. It will also be interesting to study the potentialities of the regressing
node and the last third of the primitive streak using an intracoelomic grafting
technique.
The authors wish to thank Professor Sulo Toivonen, Head, Laboratory of Experimental
Embryology, University of Helsinki, Helsinki, Finland, for reading the manuscript and constructive criticism. One of the authors (J.R. V.) also wishes to thank the U.G.C., New Delhi,
for the award of a Junior Research Fellowship and the Government of Mysore for granting
study leave. Thanks are also due to Mr N. K. Naik for his photographic assistance.
REFERENCES
H., HOLTFRETER, J., SPEMANN, H. & MANGOLD, O. (1932). Versuche zur Analyse
der Induktionmittel in der Embryonalentwicklung. Naturwissenschaften 20, 972-974.
BELLAIRS, R. (1953). Studies on the development of the foregut in the chick blastoderm. 1.
The presumptive foregut area. / . Embryol. exp. Morph. 1, 115-124.
GALLERA, J. (1965). Quelle est la duree necessaire pour declencher des inductions neurales
chez le Poulet? Experientia 21, 218-219.
GALLERA, J. (1970). Difference de reactivite a l'inducteur neurogene entre l'ectoblaste de
l'aire opaque et celui ce l'aire pellucide chez le Poulet. Experientia 26, 1353-1354.
GALLERA, J. (1971). Primary embryonic induction in birds. Adv. Morphog. 9, 172.
GALLERA, J., NICOLET, G. & BAUMANN, M. (1968). Induction neurale chez les Oiseaux a
travers un filtre millipore: etude au microscope optique et electronique. /. Embryol. exp.
Morph. 19, 439-450.
HAMBURGER, V. (1960). A Manual of Experimental Embryology. Chicago: University of
Chicago Press.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HARA, K. (1961). Regional Neural Differentiation Induced by Prechordal and Presumptive
Chordal Mesoderm in the Chick Embryo. Ph.D. Thesis, Utrecht.
BAUTZMANN,
Capacities of Hensen's node
557
J. (1934). Ober die Verbreitung induzierender Substanzen und ihre Leistungen
im Tritonlceim. Wilhelm Roux Arch. EntwMech Org. 132, 307-383.
HUNT, T. E. (1931). An experimental study of the independent differentiation of the isolated
Hensen's node and its relation to the formation of axial and non-axial parts in the chick
embryos. /. exp. Zool. 59, 395-427.
LEIKOLA, A. & MCCALLION, D. J. (1967). Time required for heterogeneous induction in
chick embryo ectoderm. Experientia 23, 869-870.
LEIKOLA, A. & MCCALLION, D. J. (1968). Inductive capacity of killed Hensen's node and head
process in the chick embryo. Can. J. Zool. 46, 205-206.
NICOLET, G. (1970). Analyse autoradiographique de la localisation des differentes ebauches
presomptives dans la ligne primitive de l'embryon de Poulet. /. Embryol. exp. Morph. 23,
79-108.
NICOLET, G. (1971). Avian gastrulation. Adv. Morphog. 9, 246.
PASTERNAK, L. & MCCALLION, D. J. (1962). Heterogeneous inductions in the chick embryo.
Can. J. Zool. 40, 585-591.
RAO, B. R. (1968). The appearance and extension of neural differentiation tendencies in the
neuroectoderm of the early chick embryo. Wilhelm Roux Arch. EntwMech. Org. 160,
187-236.
RUDNICK, D. (1948). Prospective areas and differentiation potencies in the chick blastoderm.
Ann. N.Y. Acad. Sci. 49, 761-772.
RUDNICK, D. & RAWLES, M. E. (1937). Differentiation of the gut in chorio-allantoic grafts
from chick blastoderms. Physiol. Zool. 10, 381.
SENGEL, P. (1958). Recherches experimentales sur la differentiation des germes plumaires et
du pigment de la peau de l'embryon de Poulet en culture in vitro. Annls Sci. nat. {Zool.)
20, 431-514. Cited in 'The organogenesis and arrangement of cutaneous appendages in
birds', p. 182. Adv. Morphog. 9 (1971).
SPRATT, N. T. (1942). Location of organ-specific regions and their relationship to the development of the primitive streak in the early chick blastoderm. /. exp. Zool. 89, 69.
SPRATT, N. T. (1948). Development of the early chick blastoderm on synthetic media. /. exp.
Zool. 107, 39.
SPRATT, N. T. (1952). Localization of the prospective neural plate in the early chick blastoderm. /. exp. Zool. 120, 109-130.
SPRATT, N. T. (1955). Analysts of the organizer center in the early chick embryo. I. Localization of prospective notochord and somite cells. /. exp. Zool. 128,121-164.
SPRATT, N. T. (1957 a). Analysis of the organizer center in the early chick embryo. II. Studies
of the mechanism of notochord elongation and somite formation. /. exp. Zool. 134,
577-612.
SPRATT, N. T. (19576). Analysis of the organizer center in the early chick embryo. III.
Regulative properties of the chorda and somite centers. /. exp. Zool. 135, 319-353.
SPRATT, N. T. (1958). Analysis of the organizer center in the early chick embryo. IV. Some
differential enzyme activities of node center cells. /. exp. Zool. 138, 51-80.
VISWANATH, J. R., LEIKOLA, A. & ROSTEDT, I. (1968). Induction by killed Hensen's node in
chick embryo ectoderm. Ann. Zool. Fenn. 5, 384-388.
WADDINGTON, C. H. (1932). Experiments on the development of chick and duck embryos,
cultivated in vitro. Phil. Trans. R. Soc. B 221, 179-230.
WADDINGTON, C. H. (1933). Induction by coagulated organisers in the chick embryo. Nature,
Lond. 131, 275-276.
WADDINGTON, C. H. (1934). Experiments on embryonic induction. II. Experiments on coagulated organisers in the chick. /. exp. Biol. 11, 218-223.
WADDINGTON, C. H. (1935). The development of isolated parts of the chick blastoderm. /.
exp. Zool. 71, 273-291.
WADDINGTON, C. H. (1952). The Epigenetics of Birds. Cambridge University Press.
WADDINGTON, C. H. & TAYLOR, J. (1937). Conversion of presumptive ectoderm to mesoderm in the chick. /. exp. Biol. 14, 335.
WETZEL, R. (1929). Untersuchungen am Hunchen. Die Entwicklung des Keims wahrend der
ersten beiden Bruttage. Wilhelm Roux Arch. EntwMech. Org. 119, 188-321.
HOLTFRETER,
558
J. R. VISWANATH AND L. MULHERKAR
B. H. & RAWLES, M. E. (1931). The relation of Hensen's node to the differentiating
capacity of whole chick blastoderms as studied in choric allantoic grafts. /. exp. Zool.
59, 429-465.
YAMAMOTO, T. (1949). A Manual of Experimental Physiology (in Japanese), Tokyo, p. 212
(cited by Hara, 1961, p. 44).
WILLTER,
{Manuscript received 1 November 1971, revised 7 July 1972)