The localization of morphogenetic factors in uncleaved eggs of Dentalium

0 Authors' address: Zoological Laboratory , Janskerkhof 3, Utrecht , The Netherlands 1 From the Zoological Laboratory, University of Utrecht 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. - 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. 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). 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 ofpart 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 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.) < 70 %ofa 1st polar lobe > 70% of a 1st polar lobe < 70% of a 1st polar lobe > 70% of a 1st polar lobe Polar lobe Trochophores Post-trochal present at with an region absent 1st cleavage apical tuft or reduced Polar lobe Trochophores Post-trochal present at with an region absent 1st cleavage apical tuft or reduced 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. 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 vegeta (...truncated)