Experimental studies on morphogenetic factors localized in the first and the second polar lobe of Dentalium eggs

0 Authors' address: Zoological Laboratory , Janskerkhof 3, Utrecht , The Netherlands 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. 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. 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). 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. 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 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. 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 cyto plasm 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 centrifuga tion. Comparably in Ilyanassa, vegetal halves with lobes filled with clear cytoplasm instead of the normal filling with yolk granules may develop lo (...truncated)