Berndt E. Hagstrom ¹ and Sunniva Lonning ²

¹ Wenner-Gren Institute, University of Stockholm, Norrtullsgaten 16, S-11345 Stockholm, Sweden, and
² Institute of Physiology, University of Bergen, Arstadveien 19, N-5000, Bergen, Norway

The cleavage pattern of the young sea urchin embryo was studied by means of light and electron microscopy.

The micromeres, which are known to have a strong organizing effect on the embryo, were found to form a syncytium with their neighboring micromeres and with the macromeres. The cell walls between these cells were observed to be incomplete while there were interphase nuclei with intact nuclear membranes in the micro- and the macromeres.

Similar phenomena with a break down of of the cell membranes were not observed between macro- and mesomeres while there were intact interphase nuclei in these cells.

Micromeres implanted on macromeres or mesomeres were found to coalesce with these latter cells in the course of a few minutes. During interphase, when the nuclei of both micro- and mesomere (macromere) had intact nuclear membranes, there was also a breakdown of the cell walls and a syncytium was formed by the "host cell" and the implanted micromere (see Fig. 6).

The primary mesenchyme cells, which are regarded as the descendants of the micromeres, were also studied and were likewise found to form true syncytia.

The importance to embryogenesis of this unique formation of syncytia is discussed.

The micromeres of the sea urchin larva, which are formed at the fourth cleavage of the egg, are known to have a strong influence on the development of the embryo. It was demonstrated that the micromeres have a strong morphogenetic effect and play a role of particular significance in the differentiation of the larva (Horstadius 1935, 1939). However, in spite of intense experimentation, the true cause of the peculiar effects ascribed to the micromeres has remained hidden.

In 1963, we started an investigation of cleavage and development in the individual blastomeres of the young sea urchin (Hagstrom and Lonning, 1964). We had already observed that so-called "animalizing and vegetalizing treatments" of the embryo brought about a change in the relative rate of cleavage within the larva, which in some instances led to the production of larva with aberrant numbers of blastomeres (Hagstrom 1963, cf. also Hagstrom and Lonning 1966, 1967).

In our first paper in this series we "observed, that micromeres cleave in a very divergent manner, which may give an explanation of their morphogenetic effect" (Hagstrom and Lonning 1964, p. 22). In a succeeding investigation (Hagstrom and Lonning 1965) we established that the micromeres are dependent upon their neighboring cells and that they do not seem to have the capacity to form blastulae if they are separated by an operation at the 16-cell stage and if the micromeres are cultured independently. We also found that the micromeres are characterized by an evident tendency towards formation of pseudopodia.   However, of still more far-reaching consequences was the observation that the cell membranes between micromeres and macromeres became temporarily broken up during interphase and that there was too a fusion between the small and big micromeres (Hagstrom and Lonning 1965).

In the present investigation we have made a detailed study of the cleavage and development of the micromeres and their descendants, the primary mesenchyme cells.

2. Materials and Methods:
...
3. Results:
...
4. Discussion:

As is already indicated by their name, the micromeres are smaller than the other blastomeres of the young embryo, but they also differ in several other respects. Theel (1892), Kuhl and Kuhl (1949), and Zeuthen (1951)  demonstrated that these cells cleave considerably more slowly that the other blasteromeres. This has the consequence that the multiplication of cells in the so-called vegetal half of the normal larva is slower than in the animal half.

The micromeres have a pronged interphase and they likewise show an intense synthesis of RNA (Agrell 1958; Cowden and Lehman 1963). The problem of the significance of the micromeres and their production of RNA was recently subjected to an elaborate study by Czihak (1965, 1966, Czihak et al, 1967) who, by using i. a. autoradiographic methods, was able to demonstrate that the label found in the RNA synthesized by the micromeres is soon also found in the macro- and mesomeres. The importance of Czihak's results is obvious and for a conclusive discussion the reader is referred to Czihak's recent papers (1965, 1966, Czihak et al, 1967).

The morphogenetic effect of the micromeres still needs an explanation, and of particular significance id the mechanism of transport which is instrumental in the transfer of active substances, for instance, RNA from the micromeres to the other blastomeres (cf. Czihak, loc. cit.). Our previous and present results on the cleavage of the micromeres and their neighboring cells  (cf. Hagstrom and Lonning 1964, 1965) have an obvious bearing on this problem and seem to afford an explanation.

The open cytoplasmic connection which we have discovered between micromeres and macromeres during interphase represents a rapidly working mechanism for transport between cells. The cytoplasmic streamings, the formation of filaments and pseudopodia and also the pulsating movements of the nuclei are probably morphological indications of an exchange taking place between the cells. Through time-lapse experiments we have been able to verify that there is such an exchange and that particles , possibly mitochondria and yolk granules , are transferred from cell to cell. It is interesting to note that there is no incorporation of labelled uridine in macro- and mesomeres during mitosis; first when these cells have reached interphase is it possible to detect an incorporation of marked substance (Czihak 1965). This coincides very well with our findings that the cell membranes were dissolved during interphase.

Our observation that the cell walls between the primary mesenchyme cells regularly dissolve is in congruence with the similar observations of the ancestors of the primary mesenchyme, viz. the micromeres. Characteristic are the filaments and pseudopodia formed also by the mesenchyme cells. It was already emphasized by Theel (1892) that some of the mesenchyme cells participate in the formation of the skeleton and that the pseudopodia of the mesenchyme cells may fuse. The formation of the skeleton was investigated by von Urisch (1937) and more recently reinvestigated by Okazaki (1960, 1965), Gustafson and Wolpert (1961), and Wolpert and Gustafson, 1961). The fact that the primary mesenchyme cells form true syncytia seems not to have been reported earlier. It is obvious that the tendency of the primary mesenchyme to form syncytia may be advantageous for one of their main objectives, the formation of the skeleton.

In our search for reasonable explanations of the cleavage pattern and general behaviour of micromeres and primary mesenchyme cells we have reinvestigated some older statements which indicated that these cells are distinguishable from the other blastomeres of the young embryo, but we have not been able to substantiate any of these older opinions. As we have so far not found any other differences in the cytoplasm or nuclei of the micromeres distinguishing these cells from the other blastomeres, we think that the distinctive differences are associated with the cleavage, the unique dissolution of cell membranes and the mobility of the cytoplasm and that these phenomena may essentially account for several aspects of the observed physiological and organizing effects exerted by the micromeres.

Acknowledgements:

Our sincere gratitude is due to "Biologisk stasjon", University of Bergen, Norway, and "Stazione Zoologica", Naples, Italy, for working facilities and supply of materials, and to the Anatomical Institute, University of Bergen, for use of the electron microscope. This investigation was supported by grants from the Swedish Natural Science Research Council (B.E.H.) and the Norwegian Research Council for Science and the Humanities (S.L.) which are gratefully acknowledged. Thanks are due also to Mrs. Aud Heilund for skillful technical assistance.

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