ALBERT

Notes: Summary Three different types of vesicles present before fertilization in the sea urchin egg were examined. The “a-type” corresponds to rough endoplasmic vesicles; the vesicles of “b-type” are rather smooth but may have vestigial granules within their membranes; the “c-type” belongs to the multivesicular bodies. Upon fertilization, vesicles appear in the outer cortical zone (vesicles of “d-type”). The majority of them arises by budding from the vesicles ofb-type. The budding occurs mainly at the basis of or within ridges of the cell surface; they may also be present in broader microvilli. The distance between the ridges was varied by early transfer of the eggs to calcium-free sea water. An inducing effect of the ridges on location of theb-vesicles and on the formation ofd-vesicles by budding could thus be demonstrated. Thed-vesicles appearing upon fertilization resemble in size and structure Golgi vesicles formed by budding from Golgi bodies present in the interior cortex or below it. Also theb-vesicles have their counterpart in the Golgi bodies. Theb andd-vesicles are therefore regarded as a Golgi system in which theb-vesicles represent dispersed Golgi bodies and thed- vesicles Golgi vesicles. Thed-vesicles may be designated as cortical Golgi vesicles, (c.G.v.). In thec-vesicles i.e. the multivesicular bodies (m.v.b.), a nucleid was observed which may be subdivided. A compartmentation of m.v.b. was observed which may lead to a detachment of vesicles of about the same size as thed-vesicles (c.G.v.) but probably of a different character. The differentiation of the fertilization membrane of the sea urchin egg occurs in two stages: the assembly and the solidification stage. The tentative conclusion is drawn that a secretion from the c.G.v. functions as an agent in the solidification process. The c.G.v. may also act upon the hyaline layer, both in its early formation and its maintenance during the course of embryonic development.

Notes: Summary The question about the nature of the effect of lithium on early sea urchin development is reexamined. Essential features of the morphogenetic changes of lithium treated embryos are followed with continuous comparisons with control embryos. The key to the lithium effect is the greater cytoplasmic susceptibility in the animal polar region as compared with the vegetal one. This is diagrammed in Fig. 23a-c on the basis of the double gradient concept. There is a decline of the animal gradient with increasing lithium concentration. As a consequence the level of differentiation of the terminal region becomes more and more vegetal, seean/veg-values in Fig. 23. The region suppressed by a prolonged exposure to lithium, e.g. 9–16 hours, cannot be restored. Nevertheless, there are data from previous research supporting the view that the primary effects of lithium are reversible within certain limits. However, when the normal balance is disturbed by decline of the animal gradient and particularly by suppression of its higher levels, there is a compensatory enhancement of the vegetal gradient system which stabilizes the suppression. As a consequence of the suppression of the higher animal levels, a certain accumulation of cells in an anterior direction has taken place in the blastula stage. The degree of accumulation reflects the degree of vegetalization. Later there is to varying extent a backflow of cells in the vegetal direction. It was shown how a great part of the blastula wall may have the aspect of an attachment zone (Fig. 7). The primary mesenchyme cells attach themselves only to a certain level of the ectoderm in which the relativean/veg-value is around 0.7 according to the conventions behind Fig. 23. Sections of Carnoy fixed embryos were exposed to trypsin. It proved that the external cytoplasm of blastodermic cells in lithium treated embryos was more strongly attacked than the internal one. The latter showed a strong resistance to tryptic action. On the other hand, in the control embryos the inner part of blastodermic cells was completely digested with exception of the vegetal region including the attachment zone. The trypsin resistant structure may be preformed and may be responsible for the higher rigidity of the cytoplasm in lithium treated embryos (section IIIf). It is proposed that in the period of lithium susceptibility, the colloidal state is most affected in the animal region, thereby creating a block to the diffusion of the animalizing substances which results in the shifts diagrammed in Fig. 23.

Notes: Summary The demonstration by Hörstadiuset al. (1967) that animalizing and vegetalizing agents may be extracted from unfertilized eggs of the sea urchinParacentrotus lividus gave support to the view that a double gradient system of endogenous animalizing and vegetalizing agents regulate the early sea urchin development. The way of action of the endogenous morphogenetic agents may now be approached in a more direct way. A fraction of the egg extract (L. Josefsson) proved to have a pronounced animalizing effect. In sixteen cell stage isolated vegetal half embryos served as reaction systems. Trypsin which acts as an exogenous animalizing agent was tested in parallel way. Actinomycin D which is known as a specific inhibitor of transcription processes removed or almost removed the effect of the animalizing fraction and of trypsin on vegetal half embryos. From this is tentatively inferred that animalizing agents control the transcription of genes specifying the animal differentiation of the embryo.

Notes: Summary Sixteen-cell stages of the sea urchin Paracentrotus lividus were separated into animal and vegetal halves. The former were reared in four different media: pure sea water, sea water with 10 μg/ml actinomycin D, sea water with 0.043 mol lithium, sea water containing the same concentration of lithium together with 10 μg/ml actinomycin D. The main result of the study was that the development had a more animal character after combined exposure to actinomycin D and lithium than after exposure to lithium alone. The inference from this result is that transcription processes are involved in the vegetalization of lithium treated sea urchin embryos. The differentiation of the animal halves occurring in normal sea water is widely repressed in the presence of actinomycin D. Particularly obvious is the fact that the transformation of the acron with ensuing substitution of the long stereocilia by short motile cilia fails to occur in the presence of actinomycin D. The transformation of the acron and its stereocilia seem thus to be dependent on transcription processes. In animal halves reared in normal medium droplets, dark in phase contrast, may appear near the tip of the stereocilia. This is followed very soon by the shedding of the stereocilia. In animal halves treated with actinomycin D, the droplets appear in a somewhat later stage than in the halves reared in normal sea water. After their appearance, the stereo cilia acquire a certain motility. The droplet formation may correspond to the elimination of a component which stiffens the stereocilia. The elimination of this component does not seem to be directly dependent of transcription processes. On the other hand, the stability of the stereocilia decreases with the distance from the animal pole. The data obtained in this investigation may be integrated into the double gradient concept, also in its recent elaboration (Runnström, 1967). The present investigation was carried out at Stazione Zoologica, Naples. We are deeply indebted to Professor M. Pantaleo, head of this institution, for his interest and generous support. We express also our gratitude to Professors R. Martin and R. Rocca for their help and friendly interest. One of us expresses his gratitude to Professor M. Pantaleo for the award of a research grant from the Stazione. He recognizes also his indebtedness to Professor M. De Vincentiis, director of the Department of Histology and Embryology at the University of Naples. The second of us expresses his thanks for financial support from the “Swedish Natural Sciences Research Council”, from the “Swedish Cancer Society” and from the “Research group of embryology for the study of cellular differentiation ” of the Department of Zoology, University of Rome. He expresses his sincere thanks to the Director of this Department, Professor P. Pasquini, for his stimulating interest.