J. Embryol. exp. Morph., Vol. 14, Part 3, pp. 281-287, December 1965
Printed in Great Britain
Action of rat submaxillary gland extracts on neural
tube growth in organ culture
by RUBEN ADLER and ROBERTO NARBAITZ1
From the Instituto de Anatomia General y Embryiologia, Facultad de Medicina, Buenos Aires.
WITH ONE PLATE
INTRODUCTION
FOR a long time embryologists have been interested in mechanisms which control
growth of the neural tube. The results of experiments made on amphibian and
chick embryos suggested that the differential growth of different portions of the
neural tube is dependent both on genetic factors and on the degree of development of peripheral tissues (see Weiss, 1955). The mechanism of this peripheral
action has not yet been elucidated. It has been attributed to diffusible substances
from peripheral tissues acting on the growing neural tube. However, according
to Hamburger (1958), the junction between neurons and peripheral tissue is
necessary for this growth effect to occur.
Studies on a neural growth factor found in mouse sarcoma 180 tissue, snake
venom and mouse and rat submaxillary glands (Levi Montalcini, 1959) has
renewed interest in this problem. However, this factor acts only on sympathetic
and spinal ganglia and not on neural tube derivatives.
Recently organ culture techniques have been used with increasing frequency
by embryologists, and their use has broadened our understanding of many
aspects of morphogenesis (see Borghese, 1958). However, to the best of our
knowledge, these techniques have not so far been used with success in the analysis
of neural tube growth.
In the present work, organ cultures of portions of chick embryo neural tube
together with surrounding tissues, were made to test the action of other tissues in
nearby culture, or of organ extracts added to the medium.
MATERIALS AND METHODS
White Leghorn embryos were used in all our experiments. The explants were
removed at stages 11 to 13 (Hamburger & Hamilton, 1951). Seven series of experiments were made, as described in Table 1. As shown in the plate, Fig. A, the
1
Authors' address: Instituto de Anatomia General y Embriologia, Facultad de Medicina,
Paraguay 2155, Buenos Aires, Argentina.
R. ADLER and R. NARBAITZ
282
' simple explants' were four somites long and the ectoderm and endoderm were
cut laterally at the external limit of the somites. In the' combined explants', wing
or leg primordia were dissected from embryos in stages 18 to 19 (Hamburger &
Hamilton, 1951), sectioned into small pieces and cultured in apposition to neural
tube explants. Isolated limb bud pieces were cultured as controls.
TABLE 1
The different series of experiments
2
3
Type of
explant
Simple
Combined
Simple
4
Simple
36
5
Simple
444*
6
Simple
72
7
Limb bud
36
Series
1
No
Media
860
A,B
108
360
A
A plus submaxillary gland
extract (S.G.E.)
A plus S.G.E. treated with
iodine
A plus extracts of rat brain,]
lung, heart, liver and kid- [•
ney, and chicken brainj
A plus purified ' nerve growth
factor'
A
Duration of
Culture
6-24-72-144 hr
72-144 hr.
6-24-72 hr.
6-24-72 hr.
72 hr.
72 hr.
72 hr.
* 74 with each kind of organ extract.
Cultures were made following the technique of Wolff & Haffen (1952), using
embryological watch glasses. The medium was made solid by the addition of 1 per
cent agar. The following two media were used:
A. 1 per cent agar in Hanks' solution, 10 ml.; E.E. 50, 5 ml.; Eagle's basal
medium, with double amount of dextrose, 5 ml.; Sodium penicillin,
20,000 U.
B. 1 per cent in Hanks' solution, 10 m l ; Horse serum, 1 ml.; Eagle's basal
medium with double amount of dextrose, 9 ml.; Sodium penicillin,
20,000 U.
In both cases these amounts were enough for ten cultures.
Extracts from chicken brain, and from rat brain, liver, submaxillary gland,
kidney, heart and lung were all prepared in the same way. The tissues were homogenized with Hanks' solution in a cool Omni-mixer; homogenates were then centrifuged at 4000 r.p.m. for 15 min. and supernatants were added in a proportion of
0-025 ml./ml. to the media. Both chicken and rat brain extracts were also tested
at a higher concentration (0-14 ml./ml.). In one of the series iodine was added to
submaxillary gland extract up to a final concentration of 0-0025 to 0-005 M as
suggested by Sreenby, Meyer & Bachem (1955) in order to inhibit proteases
J. Embryol. exp. Morph.
Vol. 14, Part 3
100 >x
FIG. A. Drawing of a twelve somites embryo. The pieces which were used as' simple explants'
are indicated.
FIG. B. 'Simple explant' cultured during three days. H.&.E. 180x .
FIG. C. 'Combined explant'cultured during three days. H.&E. 100x.
FIG. D. 'Combined explant' cultured during six days. H.&.E. 100 x
FIG. E. 'Simpleexplant' cultured during six hours. Medium with submaxillary gland extract.
H.&E. 180x.
FIG. F. 'Simple explant' cultured during three days. Medium with submaxillary gland
extract. H.&E. 230 x.
RUBEN ADLER and ROBERTO NARBAITZ
(Facing page 283)
Neural tube growth in organ culture
283
contained in the extract. This iodine treated extract was left half an hour at room
temperature before medium preparation. Purified 'nerve growth factor' (kindly
supplied to Dr De Robertis by Dr Levi Montalcini) was added to the media in
amounts varying from 0-015 to 16-6y/ml.
Cultures were maintained at 37° C. being observed under a dissecting microscope at various intervals. The explants were fixed in Bouin's fluid and prepared
with routine histological procedures. An approximate measure of the volume of
neural tissue cultivated was obtained by projection and drawing of the 10/A serial
sections on thick paper and weighing the resulting paper images (paper weight).
In order to establish the extent to which peripheral tissues were innervated several
explants were stained with a silver technique (Cajal & De Castro, 1933).
RESULTS
' Simple explants'
After 3 days of culture, the ectoderm and endoderm fuse forming a continuous
layer of cubical cells around the explant. Neural tubes differentiate normally and
the three layers (germinal, mantle and marginal) characteristic of normal neural
tube development are present (Plate, Fig. B). After 6 days of culture, neural
tubes continue to grow keeping the same type of organization. In some of the
explants the mantle layer looses its uniform width showing thickenings which
simulate the ventral horns of the spinal cord. In very few explants fibre bundles
were seen to arise from the neural tube. After 3 days culture somites lose their
organization forming a mesenchymal type of tissue which fills the whole explant.
Myoblasts are seen arranged in tight groups at both sides of the neural tube; some
of them fuse forming multinucleated masses. After 6 days culture, myoblasts have
continued differentiating and typical spontaneous, irregularly spaced contractions are detected.
Medium l A' gave the most consistent results. Explants cultured in medium' B',
in which embryo extract was replaced by horse serum failed on several occasions
to develop. However, in other cases the culture succeeded in a similar way as with
medium 'A'.
' Combined explants'
In experiments in which pieces of limb bud and' simple explants' were cultured
together, both explants fused completely and became surrounded by a single
layer of cuboidal cells. The mesoderm of the limb bud after three days culture
became transformed into a mass of closely packed pre-cartilaginous cells (Plate,
Fig. C). After 6 days, large masses of cartilage were observed (Plate, Fig. D).
Nerve fibres connecting the neural tubes with the added limb bud tissues were
never found. The volume of neural tissue developed in the 'combined explants',
as judged by the paper weight, is expressed in Table 2. The difference from that
developed in the 'simple explants' was not statistically significant.
284
R. ADLER and R. NARBAITZ
Addition of organ extracts to culture media
The addition of submaxillary gland extract produced the dorsal opening of
neural tubes during the first 6 hours of culture (Plate, Fig. E). After 3 days,
neural tubes showed an exuberant and irregular growth (Plate, Fig. F). As
showed in Table 2 neural tissues volume as judged by its paper weight is greater
in these explants than in the simple ones, this difference being statistically significant. The mesodermal tissue showed a comparatively poor development, the
bulk of the cultured explants being formed by neural tissue.
TABLE 2
Mean paper weight of neural tissue*
Type of
explants
Combined
Medium
A
No
20
Paper
weight
l-05g.-^
>
013g.f
Simple
A
29
0-92 g . <
A plus S.G.E.
28
>
2-37 g . ^
l-45g.J
Simple
Difference
* Weight of paper used:20-4 mg./sq.c.; magnification: 125 x .
t Statistically non significant.
j Statistically significant (p < 0-0001).
The addition of chicken and rat-brain extracts, even at high doses did not
produce morphological changes in the development of the explants, and the same
negative results were obtained with other organ extracts such as kidney, heart,
lung and liver.
Explants cultured in media containing purified nerve growth factor showed no
morphological difference from the controls.
DISCUSSION
Our results show that the neural tube of the chick embryo is capable of growth
and differentiation while kept in organ culture. However, both processes progress
at slower pace than in ovo. The fact that for several days the tissue keeps its typical
shape and organization is an important advantage over histotypical cultures for
the study of morphogenesis of the neural tube.
Hamburger & Keefe (1944) showed that the addition of an extra limb bud to
chick embryos produces a rise in the mitotic index of neural tube cells. Nevertheless, later results from the same laboratory (Hamburger, 1958) cast some doubt on
that conclusion by demonstrating that the action of the limb bud on the size of the
neural tube depends more on prevention of late cell degeneration than on early
growth stimulation. This author believes that the junction between neurones and
peripheral tissues is necessary for this action to take place. This would explain
the absence of peripheral action on neural tubes in our combined explants in
Neural tube growth in organ culture
285
which innervation does not exist, at least in the period in which our experiments
were performed.
Our most interesting observation is undoubtedly the powerful action that the
submaxillary gland extracts exert on the neural tube. Two main effects of these
extracts were observed: an early dorsal opening of neural tubes and a marked
increase in neural tissue volume. The assumption that this greater volume in the
explants treated with submaxillary extracts is indicative of an increased number
of cells is acceptable only if it is known that cellular density is similar in both types
of explants. Although no cell count was made, an examination of all the explants
indicated that if there is a difference in cellular density those cultured with submaxillary extracts are denser, in which cells appear to be more closely packed
than those in control explants. Thus, it appears to us that increase in paper weight
observed in submaxillary extract treated explants indicates a real increase in cell
population and not merely cellular dispersal. Nevertheless, it remains to be
demonstrated that cell proliferation is an independent phenomenon and not a
consequence of early neural tube opening and disorganization.
Cohen (1960) found in submaxillary extracts a factor which stimulates growth
of sympathetic and spinal ganglia, and could isolate a protein fraction in which
the activity was highly concentrated. Levi Montalcini (1964) postulated that this
nerve growth factor is specific for the neurons of the ganglia, and has no action on
neural tubes derivatives. This is in agreement with our experiments in which
'nerve growth factor' showed no action on neural tube development. The action
over neural tubes found by us, would then be dependent on some component of the
submaxillary extract different from'Nerve growth factor' and also from submaxillary protease activity, as suggested by the fact that iodine, that was showed
(Sreenby, Meyer & Bachem, 1955) to be an inhibitor of those proteases did not
inhibit the action of submaxillary extracts on neural tubes.
Cohen (1962) has isolated from mouse submaxillary extract another factor
with an action on incisor eruption and eyelid opening in the newborn mouse.
The existence of such a variety of substances with morphogenetic action in rat
and mouse submaxillary glands is difficult to understand. It might be that these
substances are unrelated to normal development, but some of them may prove to
be important tools in the investigation of the mechanisms of neural tube growth
and differentiation. In this respect the action studied by us may be of interest in
relation to the understanding of mechanisms involved in neural tube closure and
its abnormalities.
SUMMARY
1. Organ cultures of portions of chick embryo neural tube together with surrounding tissues, were made in order to test the action of other tissues in nearby
culture, and of organ extracts added to the medium.
2. Explants four somites long, dissected from embryos at Stages 11 to 13
(Hamburger & Hamilton), developed and differentiated normally in vitro. Pieces
286
R. ADLER and
R. N A R B A I T Z
of limb bud from older embryos, added to the explants, fused with them and
differentiated as usual. The addition of the limb bud pieces did not influence the
size of the cultured neural tube.
3. The addition of submaxillary gland extract to the medium produced during
the first 6 hours of culture a dorsal opening of the neural tubes and, thereafter, an
exuberant and irregular growth of them. Other organ extracts, purified 'nerve
growth factor' and iodine treated submaxillary gland extract failed to produce the
same action.
4. The presence in submaxillary extracts of an active factor independent of
'nerve growth factor' and protease activity is deduced from our results.
RESUME
Action d'extraits de giande sous-maxillaire de rat sur la croissance du tube nerveux
en culture d'organes.
1. Pour eprouver l'action d'autres tissus en culture avoisinante, et celle
d'extraits d'organes ajoutes au milieu, on a realise des cultures de tube nerveux
d'embryon de poulet et de tissus environnants.
2. Des explants correspondant a la longueur de quatre somites, preleves sur
des embryons aux stades 11 a 13 (Hamburger et Hamilton), se sont developpes et
se sont differencies normalement in vitro. Des fragments de bourgeon de membre
d'embryons plus ages, ajoutes aux explants, se sont fusionnes avec eux et se sont
differencies selon le mode habituel. L'adjonction de fragments de bourgeon de
membre n'a pas influence la taille du tube nerveux cultive.
3. L'adjonction d'extrait de glande sous-maxillaire au milieu de culture a
provoque pendant les six premieres heures de culture un percement dorsal et par
la suite une croissance exuberante et irreguliere des tubes nerveux. D'autres
extraits d'organes, du 'facteur de croissance nerveuse' purifie, et de l'extrait de
glande sous-maxillaire traite a l'iode n'ont pas eu la meme action.
4. Nous deduisons de nos resultats la presence dans les extraits sous-maxillaires d'un facteur actif independent du ' facteur de croissance nerveuse' et de
l'activite proteasique.
ACKNOWLEDGEMENTS
The authors thank Dr E. De Robertis, who read the manuscript and made important
suggestions.
This work was supported by a grant of the Consejo Nacional de Investigaciones Cientificas
y Tecnicas de Argentina. Dr Adler holds a Fellowship and Dr Narbaitz a permanent position
in the same Institution.
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{Manuscript received 26th March 1965; revised 17th June 1965)