/ . Embryol. exp. Morph. Vol. 25, 1, pp. 57-63, 1971
Printed in Great Britain
57
The localization of morphogenetic factors
in uncleaved eggs of Dentalium
N. H. VERDONK, 1 W. L. M. GEILENKIRCHEN 1
AND L. P. M. TIMMERMANSi
From the Zoological Laboratory, University of Utrecht
and the Stazione Zoologica at Naples
SUMMARY
The effect of removing parts of unfertilized and fertilized eggs of Dentalium has been
studied.
Up to 70 % of the volume of a 1st polar lobe can be removed from the vegetal side of both
unfertilized and fertilized eggs, without influencing apical tuft formation. The post-trochal
region is reduced. These eggs form a reduced polar lobe at first cleavage.
After removal of more than 70 % of the volume of a 1st polar lobe from the vegetal side
of an uncleaved egg, before or after fertilization, no polar lobe is formed and larvae develop
without apical tuft or post-trochal region.
The polar lobe area is quantitatively determined in the uncleaved egg, but after fertilization of isolated vegetal fragments regulation occurs and the polar lobe becomes proportional in size to the fragment.
Feulgen-positive granules, present at the vegetal side of the uncleaved egg, can be removed
without influencing apical tuft formation. They may be important as determinants for the
formation of the post-trochal region or adult structures.
INTRODUCTION
The morphogenetic role of the polar lobe in the development of several
annelids and molluscs is well established by experiments in which removal of
the polar lobe produces larvae with characteristic defects. In Dentalium, after
removal of the first lobe, a larva develops without apical tuft and post-trochal
region; after removal of the polar lobe at second cleavage an apical tuft is present,,
but the post-trochal region is greatly reduced (Wilson, 1904; Verdonk, 1968).
We have recently shown (Geilenkirchen, Verdonk & Timmermans, 1970)
that about 60 % of the vegetal side of the first polar lobe in Dentalium can be
removed without influencing the formation of an apical tuft, whereas removal
of 80 % or more of the first lobe causes the apical tuft to be absent in the larva.
After first cleavage the factor for apical tuft formation is still localized in the
vegetal half of the CD blastomere, but after second cleavage it moves towards,
the animal side of the D blastomere.
1
Authors' address: Zoological Laboratory, Janskerkhof 3, Utrecht, The Netherlands.
58
N. H. VERDONK AND OTHERS
In order to study the localization of this factor in the uncleaved egg, welldefined parts of the egg were removed in experiments described in this paper.
MATERIAL AND METHODS
The present experiments were carried out with eggs of Dentalium dentale L. at
the Zoological Station in Naples in June and July 1969. The animals were kept
in running sea water in a layer of sand. Oocytes and sperm were obtained by
allowing the animals to spawn spontaneously in separate dishes of sea water.
Pointed hairs were used for making lesions or removing parts of eggs or polar
lobes. All operations were carried out in solid watch glasses. After the operation
the egg and the part removed soon rounded off again. The operated eggs were
transferred separately to small Boveri dishes in which they were cultured in
filtered and boiled sea water at a temperature of 25 °C.
Within 12 h a free-swimming larva is formed from normally developing eggs.
As mentioned already by Wilson (1904) a certain percentage of the eggs develop abnormally; the post-trochal region is abnormal in most of these, and
the pre-trochal region may also be affected. The apical tuft is absent in most
of the abnormal embryos.
The parts removed were measured after having rounded off. From each batch
of eggs also some polar lobes were removed at first cleavage and measured. In
this way it was possible to compare the volume of the parts removed with the
average volume of the polar lobes of the same batch of eggs. Details of the
absolute volumes of uncleaved eggs, first and second polar lobes, are given in
a preceding paper (Geilenkirchen et al. 1970).
RESULTS
1. Localized lesions of the cell surface. In order to investigate the influence
of a wound on development, lesions of the surface were made without removing
part of the egg. In each experiment the effect of damaging the surface at the
vegetal pole and in the equatorial region of eggs was compared before and after
fertilization. With a pointed hair the surface was cut open over a distance of
about one-eighth of the circumference. Protoplasm bulged from the wound, but
soon the egg rounded off again. Fig. 1 shows that the egg surface is damaged and
that the cytoplasm is exposed. In several experiments subsequent development
was completely normal. This proved that lesion of the cell surface as such before
or after fertilization has no influence on development.
2. Removal of part of the vegetal half before fertilization. When part of the egg
is removed at the vegetal pole before fertilization the egg may form a polar lobe
at first cleavage; this depends on the volume of the part removed (Table 1).
A part as large as 70 % of a first polar lobe can be removed and in most cases a
polar lobe is still formed at first cleavage (Fig. 2B). This lobe, however, is reduced in size; the greater the part removed, the smaller the polar lobe. From
Localization of morphogenetic factors
59
eighteen eggs which formed a polar lobe at first cleavage, twelve swimming
trochophores originated, eleven of which showed an apical tuft. The development of six eggs was very abnormal.
Fig. 1. Electron micrograph showing a lesion of the cell surface of an uncleaved
Dentalium egg. To the left of arrow undamaged surface, to the right damaged
surface, x 8750. (Courtesy of Dr P. F. Elbers.)
Table 1. The effect of removal of part of the vegetal half
of the egg before fertilization
Part removed
< 70 %ofa 1st polar lobe
> 70% of a 1st polar lobe
Total
no.
eggs
21
34
Polar lobe Trochophores Post-trochal
present at
with an region absent
1st cleavage apical tuft
or reduced
18
2
11
1
20
34
Table 2. The effect of removal of part of the vegetal half
of the egg after fertilization
Part removed
< 70% of a 1st polar lobe
> 70% of a 1st polar lobe
Total
no.
eggs
14
13
Polar lobe Trochophores Post-trochal
present at
with an region absent
1st cleavage apical tuft
or reduced
12
2
14
13
60
N. H. VERDONK AND OTHERS
Fig. 2. Diagrammatic representation of the results.
(A) Normal first cleavage and normal trochophore with apical tuft and posttrochal region.
(B) After removal of less than 70 % of the volume of a polar lobe in unfertilized
and fertilized eggs a reduced polar lobe is formed. The trochophore has an apical
tuft: the post-trochal region is reduced.
(C) Removal of more than 70 % of the volume of a polar lobe in unfertilized and
fertilized eggs. No polar lobe is formed. Apical tuft and post-trochal region are
absent in the larva.
(D) Removal of a large amount of the vegetal part of an unfertilized egg. After
fertilization the animal fragment cleaves without a polar lobe; the vegetal fragment
forms a polar lobe proportional to the blastomeres. From the animal fragment a
typical lobeless larva originates, whereas the vegetal fragment forms a larva of
reduced size.
(E) After removal of a large amount of the vegetal part from a fertilized egg the
animal fragment cleaves, without forming a polar lobe. The vegetal fragment
forms a polar lobe of normal size but does not cleave.
Localization of morphogenetic factors
61
The post-trochal region was always markedly reduced in size except in one
case, in which the part removed was not larger than 20 % of a polar lobe.
3. Removal of part of the vegetal half after fertilization. Dependent on the
volume of the part removed, eggs operated on after fertilization may or may not
form a polar lobe at first cleavage (cf. Table 2). If present, it was in all cases
reduced in size proportional to the volume of the part removed. The trochophores originating from eggs with a polar lobe at first cleavage mostly showed
an apical tuft, but the post-trochal region was smaller than normal (Fig. 2B).
When, however, the volume of the removed fragment was larger than 70 % of
a normal polar lobe of the same batch of eggs, a polar lobe was hardly ever
formed at first cleavage and the trochophores did not have an apical tuft except
in one case (Fig. 2C).
4. Removal of part of the animal side before fertilization. In thirteen cases a part
of the animal side was removed varying from 5 to 52 % of the total egg volume.
After fertilization the vegetal parts of the egg formed a polar lobe and a larva
with an apical tuft and a post-trochal region. Generally the animal part did not
cleave, but in two eggs which were cut in nearly equal animal and vegetal parts
both halves cleaved after fertilization. The animal parts did not form a polar
lobe and in the larvae the apical tuft and post-trochal region were absent. The
vegetal parts formed a lobe proportional to the size of the fragment and in the
larvae the apical tuft and a post-trochal region appeared to be present
(Fig. 2D).
5. The behaviour of isolated vegetal parts. When a vegetal part including the
polar area is removed before fertilization, it rounds off; if not fertilized it does
not show any further activity. However, when a part of the vegetal hemisphere
is isolated immediately after fertilization, i.e. within 5 min after administering
sperm to the eggs, this fragment shows a distinct constriction at the moment
when the corresponding animal part starts cleaving (Fig. 2E). Subsequently, the
isolated part rounds off again and the phenomenon recurs at second cleavage.
DISCUSSION
From his classical experiments Wilson (1904) concluded that the vegetal part
of the egg of Dentalium is essential for the formation of an apical tuft and a
complete post-trochal region. The present experiments corroborate this conclusion as far as the post-trochal region is concerned. However, for the apical tuft
the situation is more complex as about 70 % of the volume of a normal polar
lobe may be cut off from the vegetal pole of an unfertilized or fertilized egg without influencing apical tuft formation. This is in agreement with our previous
experiments, which demonstrated that larvae with an apical tuft can be obtained after removal of about 60 % of a first polar lobe (Geilenkirchen et ah
1970).
From the observation that the size of the polar lobe at first cleavage of an
62
N. H. VERDONK AND OTHERS
isolated and subsequently fertilized vegetal egg fragment, which included the
polar area, was proportional to the size of the fragment, Wilson (1904) concluded 'that the predetermination of this area is qualitative but not quantitative
or only quantitative in so far as it is subject to regulative control of other
factors'. From our experiments it appears that (1) after removal of the total
polar lobe area in the uncleaved egg, no polar lobe is formed; (2) after removal
of only a part of this area a reduced lobe is formed, the reduction being more or
less proportional in size to that of the part removed. This suggests a quantitative determination. However, the polar area can also be subject to regulation,
as isolated and subsequently fertilized vegetal parts form a polar lobe proportional to the size of the part isolated. At fertilization a sperm nucleus comes into
the vegetal part and during cleavage a spindle is formed which lies very near or
even in the polar area and may influence the process of cleavage and polar lobe
formation. When a nucleus is not present, as in isolated vegetal fragments of
fertilized eggs, no regulation takes place and a polar lobe of normal size is
formed.
The formation of a polar lobe in vegetal fragments without a nucleus, isolated
immediately after fertilization, indicates that the constriction of the polar lobe
area at first and second cleavage is an autonomous process. This is in agreement
with earlier observations of Wilson (1904) for Dentalium and Morgan (1933)
for Ilycmassa. From the present experiments, however, it becomes clear that this
process must be activated at fertilization, since pieces isolated before fertilization do not show any activity.
In Dentalium Feulgen-positive granules are localized in the cortical region at
the vegetal pole of the uncleaved egg; these granules are found in the trefoil stage
at the vegetal side of thefirstpolar lobe (Timmermans, Geilenkirchen &Verdonk,
1970). From data presented in a preceding paper (Geilenkirchen et al. 1970) we
concluded that 'if these granules are to be related with the morphogenesis of the
apical tuft, their primary influence must have been exerted before first cleavage'.
From the present experiments it becomes clear that already, immediately after
oviposition, the part of the egg which contains these Feulgen-positive granules
can be removed without influencing the formation of an apical tuft. This does
not imply that these granules are of no importance for normal development.
When the polar area or even part of it is removed the larvae are defective. The
same was observed in Ilyanassa (Crampton, 1896; Clement, 1952). It is conceivable that the Feulgen-positive granules—indicative of DNA—have a relation
to the morphogenesis of these structures.
Localization of morphogenetic factors
63
RESUME
La localisation de facteurs morphogenetiques dans des ceufs
non-segmentes de Dentalium
Les effects de l'ablation de parties d'ceufs fecondes et non fecondes ont ete etudies.
Jusqu'a 70 % du volume d'un premier lobe polaire peut etre preleve a la partie vegetative
d'un oeuf feconde ou non feconde sans consequence pour la formation d'une touffe apicale.
La region post-trochale est reduite. Pendant la premiere segmentation ces oeufs forment un
lobe polaire reduit.
Apres l'ablation de plus que 70 % du volume d'un premier lobe polaire a la partie vegetative d'un ceuf non-segmente, avant ou apres la fecondation, un lobe polaire ne se forme plus
et les Iarves se developpent depourvues de touffe apicale et de region post-trochale.
Le lobe polaire est determine quantitativement dans l'ceuf non-segmente, mais apres fertilisation de fragments vegetatives isoles une regulation se produit et le lobe polaire se proportionne au volume de chaque fragment.
Des granules Feulgen-positives, presentes a la partie vegetative d'ceufs non-segmentes,
peuvent etre preleves sans influencer la formation d'un touffe apicale. Elles peuvent avoir
une certaine importance dans la formation d'une region post-trochale ou des structures
adultes.
This work has been supported by a travel grant from the Netherlands Ministry of
Education and Sciences.
REFERENCES
A. C. (1952). Experimental studies on germinal localization in Ilyanassa. I. The
role of the polar lobe in determination of the cleavage pattern and its influence in later
development. /. exp. Zool. 121, 593-626.
CRAMPTON, H. E. (1896). Experimental studies on gastropod development. Arch. EntwMech.
Org. 3, 1-19.
GEILENKIRCHEN, W. L. M., VERDONK, N. H. & TIMMERMANS, L. P. M. (1970). Experimental
studies on morphogenetic factors localized in the first and the second polar lobe of Dentalium eggs. /. Embryo/, exp. Morph. 23, 237-243.
MORGAN, T. H. (1933). The formation of the antipolar lobe in Ilyanassa. J. exp. Zool. 64,
433-467.
TIMMERMANS, L. P. M., GEILENKIRCHEN, W. L. M. & VERDONK, N. H. (1970). Local accumulation of Feulgen-positive granules in the egg cortex of Dentalium dentate L. /. Embryol.
exp. Morph. 23, 245-252.
VERDONK, N. H. (1968). The effect of removing the polar lobe in centrifuged eggs of Dentalium. J. Embryol. exp. Morph. 19, 33-42.
WILSON, E. B. (1904). I. The germ-regions in the egg of Dentalium. J. exp. Zool. 1, 1-74.
CLEMENT,
{Manuscript received 19 June 1970)