/. Embryo/, exp. Morph. Vol. 23, J, pp. 237-243, 1970
Printed in Great Britain
237
Experimental studies
on morphogenetic factors localized in the first and
the second polar lobe of Dentalium eggs
By W. L. M. G E I L E N K I R C H E N , 1 N. H. V E R D O N K 1
AND L. P. M. T I M M E R M A N S 1
From the Zoological Laboratory, University of Utrecht
and the Stazione Zoologica at Naples
During the development of several annelids and molluscs, a lobe of protoplasm
is observed to protrude from the vegetal pole of the egg during the first cleavages.
Removal of this polar lobe causes characteristic defects in the developing larvae
as has been observed in the scaphopod, Dentalium (Wilson, 1904; Verdonk,
1968), the annelid Sabellaria (Hatt, 1932; Novikoff, 1938), the gastropod
Jlyanassa obsoleta (Crampton, 1896; Clement, 1952) and the lamellibranch,
Mytilus edulis (Rattenbury & Berg, 1954).
In Dentalium a polar lobe is formed at the first, the second and the third
cleavage. The lobes contain part of the cytoplasm, which later is confined to the
D blastomere. After removal of the first polar lobe, embryos develop in which
the post-trochal region and the apical tuft are absent. After removal of the
second polar lobe the post-trochal region of the embryos is greatly reduced, but
they develop an apical tuft (Wilson, 1904).
Apparently, a factor essential for development of an apical tuft is present in
the first polar lobe but not in the second. Yerdonk (1968) centrifuged the eggs
shortly before first cleavage and removed the first polar lobe. Independent of
the stratification and the resulting altered cytoplasmic composition of the polar
lobe, the embryos never developed an apical tuft after removal of the lobe.
Repeating the classic experiments of Wilson (1904), in which the first or the
second polar lobes were removed, we discovered that the first and the second
lobe differ considerably in volume. It appeared that the volume of the second
polar lobe measures about 60 % of the volume of the first lobe. Thus it is possible
that the factor essential for apical tuft formation, present in the first polar lobe,
is not extruded with the second polar lobe but remains in the D cell at second
cleavage.
The present experiments were designed to answer the question as to where the
factor essential for apical tuft formation is localized during the first cleavages.
1
Authors' address: Zoological Laboratory, Janskerkhof 3, Utrecht, The Netherlands.
238
W . L. M. G E I L E N K I R C H E N A N D
OTHERS
MATERIAL AND METHODS
The experiments were carried out at the Zoological Station in Naples in May
and June 1968 with the eggs of Dentalium dentale L. The animals were kept in
running sea water on a layer of sand. Gametes were obtained by allowing the
animals to spawn spontaneously in separate dishes of sea water. Oocytes could
also be obtained in an artificial way by breaking the shell and opening the ovaries.
Polar lobes were removed with pointed hairs. These were also used to remove
parts of cells or parts of polar lobes.
The eggs were cultured in filtered and boiled sea water in Boveri dishes at a
temperature of 20-21 °C.
RESULTS
1. After removal of the first lobe free-swimming trochophores develop. In all
50 lobeless embryos the post-trochal region and the apical tuft proved to be
missing (Fig. 1 A). After removal of the second polar lobe, all 15 lobeless embryos
developed into trochophores with a distinct apical tuft and a greatly reduced
post-trochal region (Fig. 1B).
Table 1.
Average vol.
xlOV
Vol. smallest egg
xlOV
Vol.. largest egg
xlOV
Uncleaved egg
1
2
3
3474
4017
3955
3369
3764
3500
4084
4189
4100
1st polar lobe
1
2
3
4
1
2
3
4
939
875
778
893
577
521
614
603
796
769
716
790
1046
987
867
1029
739
612
624
659
2nd polar lobe
493
421
600
555
Average volumes of uncleaved eggs, first and second polar lobes per batch of eggs used.
The arithmetic means are taken of at least ten eggs or polar lobes.
2. Volume measurements were made of uncleaved eggs, first polar lobes and
second polar lobes (Table 1). The volumes were calculated from the measured
diameters of the spherical cells and of removed and rounded-off polar lobes or
parts of polar lobes.
3. In a first set of experiments, fragments of the first polar lobe were removed.
After removal, the fragments became rounded off and the diameters could be
measured. The resulting embryos were grouped in two classes: (a) embryos in
which up to 60 % of the first polar lobe was removed and (b) embryos in which
Morphogenetic
factors
239
60 % to 80 % of the first polar lobe was removed (see Fig. 1 C). It is obvious
from these results that up to 60 % of the vegetal side of the first polar lobe can be
removed without affecting apical tuft formation. These results show that the
factor essential for apical tuft formation is either localized in the animal part
of the first polar lobe, or if it is a substance present in the whole lobe, only a
fraction of it is needed for apical tuft formation.
Total
Trochophores
w i t h an
apical tuft
Trochophores
w i t h o u t an
apical tuft
Post-trochal
region absent
or reduced ( + )
50
+
• p.i.1
50
— Pl- 2
15
15
+
28
19
+
a Part removed
< 6 0 % of p.I. 1
b c<
b Part removed
between 60 and
8 0 % of p.l. 1
Part removed
~ 1 - 5 x vol.
p.1.1
+
20
20
f a Part removed
£J
[b
<p.l.1
12
Part removed
>p.l.1
12
10
Fig. I. Schema of the experiments performed and the results; p.l. = polar lobe.
4. In a second set of experiments the eggs were allowed to develop until the
first polar lobe had regressed and partially fused with the CD blastomere
(Fig. ID). In these experiments a part of the vegetal side of the CD blastomere
was removed which was larger than the first polar lobe. The results show that if
the removed part is larger than the first polar lobe, a tuft does not develop.
Apparently, the factor essential for apical tuft development resides at this
developmental stage in the vegetal half of the CD cell.
5. In a third set of experiments the embryos were allowed to develop until
after second cleavage, when the second polar lobe had partially fused with the
240
W. L. M. G E I L E N K I R C H E N AND OTHERS
D blastomere. The vegetal half of the D blastomere was removed (Fig. IE). The
developing embryos were grouped in two classes: (a) the embryos developing
from egg cells in which the part removed from the D blastomere was equal to or
smaller than the first polar lobe, and (b) the embryos developing from egg cells
in which the part removed was considerably larger than the first polar lobe. The
results show that in 75 % of all cases trochophores develop with an apical tuft.
These results indicate that the factor essential for apical tuft formation no
longer resides in the vegetal half of the D blastomere.
DISCUSSION
The experiments show that the factor essential for apical tuft formation moves
from the first polar lobe, which forms at the vegetal side, towards regions in the
D blastomere situated at the animal side. These results are consistent with the
results obtained by Wilson (1904) in experiments in which the developmental
capacities of isolated micro- and macromeres of the eight-cell stage were studied.
All these cells may develop into actively swimming larvae; only the Id micromere derivatives will develop an apical tuft. The data obtained in the present
study and those obtained by Wilson together prove that between the second and
third cleavage the factor essential for polar lobe formation moves from the
vegetal side upwards to the animal side where the Id micromere will be
split off.
After centrifugation it is possible to obtain in Dentalium polar lobes filled
with yolk granules or fat droplets (Verdonk, 1968), whereas normally the cytoplasm in the polar lobe is almost devoid of yolk granules. However, in centrifuged eggs, after removal of the first polar lobe, an apical tuft is not developed.
This result shows that the essential factor has not been displaced by centrifugation. Comparably in Ilyanassa, vegetal halves with lobes filled with clear
cytoplasm instead of the normal filling with yolk granules may develop lobedependent structures (Clement, 1966,1968). This means that it is not a substance
contained in the bulk of the cytoplasm that is important for apical tuft development. Our experiments show, moreover, that only a small portion of the lobe is
essential for apical tuft formation. These data are suggestive of a cortical factor.
It remains undecided, however, whether this factor is part of the surface structures as such or consists of cytoplasmic units bound in some way or other to
the surface membrane.
Davidson et al. (1965) showed that removal of the polar lobe formed at first
cleavage in eggs of Ilyanassa depresses gene activation in a later stage of development. They suggested that gene activation in early embryogenesis may be
mediated by material localized in the cytoplasm, viz. in the polar lobe, thus
accounting for initial nuclear differentiation.
A suggestion of similar purport was made by Brächet (1965 a, b) for the dorsal
cortex of the fertilized amphibian egg: 'The dorsal cortex of the fertilized egg
Morphogenetic factors
241
must, ultimately, control the activation (or de-repression) of certain genes in the
dorsal lip of the blastoporus (organizer). It is conceivable that this de-repression
might be mediated, as suggested by Frenster (1965), by a DNA-like RNA, which
should be, in the present case, synthesized on this cytoplasmic DNA template'.
The cytoplasmic DNA template in Brachet's statement consists of extranuclear DNA which, after being produced during maturation of the oocyte, is
localized in the dorsal cortex.
The citations on Ilyanassa and amphibians are given because in Dentalium
we clearly observe, as in Ilyanassa, that elimination of the first polar lobe leads
to embryos which lack specific organs or structures. Davidson's conclusion for
Ilyanassa is in all probability also valid for Dentalium.
Furthermore, we observed (Timmermans, Geilenkirchen & Verdonk, 1970)
that during maturation in Dentalium oocytes Feulgen-positive granules—
indicative of DNA—are localized in the cortical region at the vegetal pole,
where at first cleavage the polar lobe will protrude. These observations are
clearly comparable to the observations made by Brächet in amphibian oocytes.
At first cleavage the Feulgen-positive granules observed in Dentalium oocytes
are still present in the same cortical region, which is now part of the vegetal
side of the first polar lobe (Timmermans et al. 1970). As shown above this part
of the lobe can be removed without consequences for apical tuft formation. If
the Feulgen-positive granules have a template function, this function must
have been exerted before first cleavage and derived determinants for apical tuft
formation must then be localized in the animal half of the polar lobe cortex.
From all these data the following hypothesis may be presented for the development of Dentalium:
1. During maturation of the oocytes DNA-containing granules are deposited
in a selected area of the egg, the vegetal pole region.
2. These granules are morphogenetic determinants (or their precursors) in
the egg.
3. The determinants are essential for apical tuft formation and differentiation
of a post-trochal region.
4. The determinants for the apical tuft are located in the vegetal one-third
of the unfertilized egg. Factors derived from these determinants afterwards
become localized in the animal half of the first polar lobe. After second cleavage
they move towards the animal pole. Ultimately they are distributed among the
derivatives of the 1 d micromere.
5. The determinants account for nuclear differentiation at later stages of
development.
SUMMARY
1. The effect of removing the first polar lobe, the second polar lobe, parts of
the first polar lobe, parts of the CD or D blastomere in eggs of Dentalium dentale
L. has been studied.
242
W. L. M. G E I L E N K I R C H E N AND OTHERS
2. Removal of the first polar lobe produces larvae which develop without the
apical tuft and the post-trochal region.
3. After removal of 60 % of the vegetal side of the first polar lobe, larvae
develop with an apical tuft and a reduced post-trochal region.
4. Removal of 80 % of the first polar lobe gives rise to larvae without apical
tuft and post-trochal region.
5. The factor essential for apical tuft formation is localized in the animal
half of the first polar lobe.
6. After first cleavage the factor is still localized in the vegetal half of the CD
blastomere.
7. After second cleavage the factor moves towards the animal side of the D
blastomere.
8. The hypothesis is presented that the factor essential for the apical tuft
formation is related to extranuclear DNA appearing at the vegetal pole during
maturation of the oocytes.
RESUME
Etudes expérimentales sur des facteurs morphogénétiques localisés dans le
premier et le second lobe polaire des oeufs de Dentalium.
1. Les effets de l'ablation du premier lobe polaire, du second lobe polaire, de
parties du premier lobe, de parties de blastomères CD ou D ont été étudiés sur
des œufs de Dentalium dentale L.
2. L'ablation du premier lobe polaire produit des larves se développent sans
touffe apicale ni région post-trochale.
3. Après ablation de 60 pour cent du lobe polaire, prélevé à sa partie végétative, les larves se développent avec une touffe apicale et avec une région posttrochale réduite.
4. L'ablation de 80 pour cent du premier lobe polaire produit des larves sans
touffe apicale ni région post-trochale.
5. Le facteur essentiel pour la formation de la touffe apicale se localise dans
la moitié animale du premier lobe polaire.
6. Après la première division, le facteur est toujours localisé dans la moitié
végétative du blastomere CD.
7. Après la seconde division, le facteur se déplace vers le côté animal du
blastomere D.
8. On présente comme hypothèse que le facteur essential pour la formation
de la touffe apicale est en relation avec l'ADN extranucléaire qui apparaît au
pôle végétatif au cours de la maturation des oocytes.
This work has been supported by a travel grant from the Netherlands Ministry of
Education and Sciences.
Morphogenetic factors
243
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(Manuscript received 8 April 1969)
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