/. Embryo/, exp. Morph. Vol 36, 2, pp. 315-320, 1976
Printed in Great Britain
315
Fish swimbladder: an excellent meso dermal
inductor in primary embryonic induction
IZUMI KAWAKAMI1
From the Department of Biology, Faculty of Science,
Kyushu University
SUMMARY
Swimbladder of the crucian carp, Carassius auratus, was found to be better as a vegetalizing tissue than other tissues, such as guinea-pig bone marrow, when presumptive ectoderm of
Triturus gastrulae was used as reacting tissue.
Swimbladder usually induced assemblies of highly organized mesodermal tissues, such as
notochord, somites and pronephric tubules, some of which were covered by mesodermal
epithelium without an epidermal covering.
A special character of the effect of swimbladder was the rather frequent induction of solid
balls of undifferentiated cells, which were identified as mesodermal or mesodermal and
probably endodermal. Thesefindingsshow that swimbladder has a strong and fast spreading
vegetalizing effect on the responding presumptive ectoderm.
INTRODUCTION
There have been many papers on the inductive capacities of heterologous
tissues in embryonic primary induction, but there have been few on the inductive capacities of extracellular substances. Recently we reported (Kawakami,
Sato & Osako, 1976)"a difference in the inductive capacities of liver with and
without the perisinusoidal basement membrane, and we suggested a mesodermal inductive capacity for the membrane.
Thinking that the major or essential agent in heterologous tissues with the
capacity for primary induction must be an extracellular, amorphous substance,
we examined the inductive capacities of various tissues in the hope of rinding
tissues suitable for experiments on the mechanisms of primary induction.
Among the tissues examined fish swimbladder was found to be the best for
mesodermal induction, having advantages over guinea-pig bone marrow,
used previously (Toivonen, 1953).
This paper reports studies showing that fish swimbladder is a good material to
use in analysis of induction mechanisms.
1
Author's address: Department of Biology, Faculty of Science, Kyushu University,
Fukuolea, Japan.
316
I. KAWAKAMI
Table 1. Inductions observed in four experiments with fish
swimbladder as inductor
Experiment
i
Neural tissue
Notochord
Somites muscle
Myoblasts
Pronephros
Mesenchyme
Mesoblasts
Yolky cells
Positive/total
1
2
3
4
0
10
10
2
19
5
9
8
6
35
37
2
4
8
2
0
1
11
15
0
3
5
8
4
1
12
7
7
14
3
6
2
34/34
43/43
23/23
26/26
MATERIALS AND METHODS
The inductive capacity of swimbladder of the crucian carp (Carassius auratus)
was examined by the usual sandwich method with presumptive ectoderm from
gastrulae of Triturus pyrrhogaster as reacting tissue. The procedures used were
as described in the previous paper (Kawakami et al. 1976). Pieces of the swimbladder were grafted after being fixed in 90 % ethanol.
The wall of swimbladder consists of outer serosa, middle fibrous layers and
inner thin endodermal epithelium. The fibrous layers are composed of collagen,
elastic fibers and amorphous matrix, among which a few slender cells are
scattered.
The middle fibrous part of the tissue was used as inductor after removing the
serosa and inner epithelium, because preliminary experiments showed that all
layers of the fibrous part had similar inductive effects.
RESULTS
Fresh and ethanol-fixed swimbladders showed similar rates and types of induction, so for simplicity only the results of experiments with ethanol-fixed tissue
are described here (Table 1). The inductions observed were usually purely mesodermal, with a very few cases of fragmentary neural inductions, which were
probably due to secondary inductions by the induced mesodermal tissues.
The histological features of the induced mesodermal tissues were in general
similar to those of tissues induced by guinea-pig bone marrow (Toivonen, 1953),
though with a few characteristic features.
Formation of the pronephros occurred frequently, but the induction frequency varied in different series of experiments, even when the same components
of the swimbladder were used (Table 1). In some cases the pronephros differentiated alone with the inductor piece at the centre of the explants (Fig. 1 A). The
Fish swimbladder as mesodermal inductor
317
m.ep.
op
pron
Fig. 1. Inductions by fish swimbladder {bid).
(A, B) Explants consist mostly of propephric tubules {pron), except for lateral
mesoderm {lm) in (B). The covering epithelium is identified as mesodermal {m.ep)
by an opening {op) of a pronephric tubule in the pithelium. A, x 80; B, x 60.
(C) Notochord {not) and myoblast {myb) induced in an explant without covering
epidermis. The nuclei of myoblasts are arranged concentrically, x 100.
(D) A mass of undifferentiated mesodermal cells or mesoblasts {mbl). x 100.
(E, F) Explants with an epidermal covering have a normally arranged notochord
{not), somites {som) and pronephric tubules {pron). x 60.
21
EMB 36
318
I. KAWAKAMI
induced pronephros was usually well differentiated, though it was only partially
differentiated in some specimens and in others it was observed as a compact
mass of undifferentiated cells, in which twisted double rows of nuclei suggested
poorly developed tubules.
Masses of undifferentiated mesodermal and yolky cells were observed in some
explants. In some of these the nuclei were arranged concentrically while in others
they were randomly distributed (Fig. 1C, D). They may be indentified as myoblasts and mesoblasts respectively. Some explants, in which pronephric tubules
were observed, had undifferentiated cells which were identified as the lateral
plate of mesoderm (Fig. IB).
Most explants developed assemblies of mesodermal tissues, such as the notochord, somites and pronephros. Some of these explants were partially or completely devoid of an ectodermal covering, but some were covered with what
seemed to be mesodermal epithelium because of the presence of the openings of
nephric tubules in it. Perhaps because they lacked covering epidermis many of
the explants did not develop any trunk-tail structure, but remained spheroidal
although they contained caudal tissues.
In general explants with an epidermal covering had a normally arranged
notochord, somites and pronephros. In these explants the notochord was far
from the inductor graft and the pronephros was usually ventrally positioned
near the graft (Fig. IE, F).
DISCUSSION
The value of using swimbladder in experiments on primary induction will be
demonstrated in subsequent papers. Here only the special characters of the induction by this tissue will be considered.
The present results show clearly that swimbladder has the capacity to induce
mesodermal tissue. Fragmentary neural tissues were occasionally observed in
explants. However, these were probably due to secondary induction by the
induced mesodermal tissues and particularly the notochord, because in a preliminary experiment only mesodermal induction was observed when presumptive ectoderm from late gastrulae with a small yolk-plug was used as reacting
tissue. In the latter the neural competence of the older presumptive ectoderm
had presumably disappeared before the primarily mesodermalized ectoderm
became inductive. (Sasaki, Iwamoto, Noda & Kawakami, 1976).
Frequent and often bulk induction of the pronephros was noted, although the
frequency of its induction varied in different series of experiments (Table 1).
Attempts to find conditions favouring induction of only the pronephros or more
frequent induction of the pronephros were unsuccesful. Further investigations
on this might provide an understanding of the mechanisms involved in differentiation of a special tissue.
Another interesting finding was the induction of massive balls of undifferentiated cells. Judging from the presence of myoblastic cells and pronephros-like
Fish swimbladder as mesodermal inductor
319
tubular structures, these cells were taken to be mesodermal. The high frequencies of inductions of these masses of undifferentiated and of poorly differentiated cells without an epidermal cover suggests that the swimbladder has
a strong and rapidly spreading mesodermalizing effect.
The explants were not cultured for more than 10 days, so no clearly differentiated endodermal tissues could be identified, but the masses of yolky cells found
separated from mesoblastic cell masses were probably endodermal. This conclusion is supported by results, described in a following paper where explants
were examined after 30 days of cultivation. Collateral induction of endodermal
tissues with mesodermal ones by mesoderm inductive materials has been reported (Takata & Yamada, 1960; Tiedemann, 1966). Thus, simultaneous
inductions of both mesodermal and endodermal tissues may be common effects
of mesoderm inductive agents irrespective of their origin. This suggests that
single substances obtained from different materials, even though they differ in
chemical structure, may be competent to induce both mesodermal and endodermal structure. Thus, as advocated by Tiedemann (1966), these inductive
materials may be termed vegetalizing factors.
Explants were usually ellipsoidal, even when they showed differentiation of a
notochord and somites, and formation of a long spinocaudal structure was
rare. Sections of these explants usually showed the bilateral arrangement of the
notochord and somites and a pronephros located ventrally near the grafted
inductor tissue. A similar kind of induction was observed with tooth matrix.
(Tanaka et al. unpublished). This type of induction is probably due to the very
strong mesoderm-inducing capacity of these tissues, the fact that the grafted
inductive tissues are flat and probably also to some additional factor, which
operates in the induction itself in the early phase of differentiation.
The swimbladder is very useful for experiments on primary induction because
it has a very strong vegetalizing effect. It is also better than other known vegetalizing tissues such as guinea-pig bone marrow because only a short contact
period with presumptive ectoderm is required to cause definite induction, because
it can exert its vegetalizing effect across a filter (Sasaki et al. unpublished), and
because it is relatively easy to isolate a large amount of a fraction having vegetalizing capacity. (Yamada, 1958; Tiedemann & Tiedemann, 1959; Kawakami
& Iyeiri, 1964). Further studies on the vegetalizing effects of the swim-bladder
will be reported in following papers.
REFERENCES
I. & IYEIRI, S. (1964). Isolation of mesoderm-inducing protein from chick
embryos. Expl Cell Res. 33, 516-522.
KAWAKAMI, I., SATO, A. & OSAKA, N. (1976). Differencein inductive effect of liver tissues with
and without perisinusoidal bssement membrane. Devi. Growth and Diff. (in press).
SASAKI, N., IWAMOTO, K., NODA, S. & KAWAKAMI, I. (1976). The staging of the newt blastula
embryos and the first appearance of the primary mesodermal competence. Devi. Growth
and Diff (in press).
KAWAKANH,
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I. KAWAKAMI
C. & YAMADA, T. (1960). Endodermal tissues developed from the isolated newt
ectoderm under the influence of guinea-pig bone marrow. Embryologia 5, 8-20.
TIEDEMANN, H. & TIEDEMANN, H. (1959). Versuche zur Gewinnung eines mesodermalen
Induktionsstoffe aus Hiihnerembryonen. Hoppe-Seyler''s Z'. physiol. Chem. 314, 160-176.
TIEDEMANN, H. (1966). The molecular basis of differentiation in early development of amphibian embryos. In Current Topics in Developmental Biology vol 1 (ed. A. Monroy &
A. A. Moscona), pp. 85-112. New York and London: Academic Press.
TOIVONEN, S. (1953). Bone-marrow of the guinea pig as a mesodermal inductor in implantation experiments with embryos of Triturus. J. Embryol. exp. Morph. 1, 97-104.
YAMADA, T. (1958). Embryonic induction. In A Symposium on the Chemical Basis oj Development . (ed. W. McElroy & B. Glass) pp. 217-238. Baltimore: John Hopkins Press.
TAKATA,
(Received 20 March, revised 18 May 1976)