Gerhard Czihak and Sven Horstadius

Max Planck Institut fur Zellbiologie, Tubingen, Germany, and Zoologiska Institutionen, Uppsala, Sweden.

Micromeres of 16-cell stages of sea urchin embryos which had incorporated uridine-¹⁴C were transplanted into unlabeled animal halves.

Labeled substance then moved from the micromeres into other cells. This was not influenced by the presence of large amounts of cold uridine in the medium. The labeled substance is located in the cytoplasm of the cells in the animal halves, and is not soluble in ethanol-acetic acid, dilute acetic acid, or ditilled water. Since the micromeres are known to synthesize RNA, it is probable that RNA is the substance that moves from the micromeres to the neighboring cells. This may be responsible for the inductive action of micromeres on animal-half cells.

Isolated animal halves of sea urchin eggs, separated from their vegetal counterparts in early cleavage stages, do not gastrulate. They will not have an archenteron and they will remain ectodermal. In most cases they do not even form a stomodeum and ciliated band, and they develop only to ciliated blastulae. A group of micromeres implanted into an animal cell in the 32- or 64-cell stage will be incorporated in the blastula wall. The descendents of the micrmeres later migrate into the blastocoele, forming the primary mesenchyme, which will produce the skeletal spicules. During contact with the wall, the implanted cells exert such an influence upon the cells in the neighborhood that these differentiate into endoderm, invaginate as archenteron, and eventually form a typical digestive tract. Besides this process of induction the micromeres also cause the formation of  stomodeum and ciliated band. The ectoderm is now also capable of forming arms in cooperation with the outgrowing skeleton. The formation of the archenteron, etc., is a striking case of induction (Horstadius, 1935), a process in which differentiation of a group of cells is initiated by other cells which themselves never perform this differentiation.

The four micromeres of the 16-cell stage have a conspicuous amount of cytochemically easily detectable RNA (Agrell, 1958). At the end of the 16-cell stage, only the micromeres are synthesizing RNA, or they do so to a much greater extent than other blastomeres (Fig.1) (Czihak, 1965a, b). Embryos kept in 8-azaguanine or 6-azauridine for 20-30 minutes in this period of RNA-synthesis do not invaginate an archenteron or have only a reduced one (Fig.2) (Czihak, 1965a, b). Azaguanine is exclusively incorporated into RNA, not only in sea urchin embryos (Bamberger, et al, 1963), but also in bacteria (Creaser, 1956) and in mammalian liver (Lasnitzki, et al, 1954). The disturbance of RNA synthesis in the micromeres in the presence RNA antimetabolites , however, does not influence the differentiation and activity of the micromeres themselves, as they are still able to form spicules (Fig. 2). Their RNA may be produced for differentiation of the adjacent cells, and perhaps this represents the starting point for the induction process. The first step toward elucidating whether this RNA might be the inductive substance, is the transplantation into unlabeled animal halves of micromeres that have incorporated labeled uridine and synthesized radioactive RNA, with subsequent observation of the destiny of the radioactive substance.

Summary:

Micromeres of 16-cell stages of sea urchin embryos which had incorporated uridine-¹⁴C were transplanted into unlabeled animal halves.

Labeled substance then moved from the micromeres into other cells. This was not influenced by the presence of large amounts of cold uridine in the medium. The labeled substance is located in the cytoplasm of the cells in the animal halves, and is not soluble in ethanol-acetic acid, dilute acetic acid, or ditilled water. Since the micromeres are known to synthesize RNA, it is probable that RNA is the substance that moves from the micromeres to the neighboring cells. This may be responsible for the inductive action of micromeres on animal-half cells.

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Czihak G, (1965a), "Evidence for Inductive Properties of the Micromere-RNA in Sea-urchin Embryos", Naturwissenschaften, vol. 52, no. 6, pp. 141-142.

Czihak G, (1965b), "Entwicklungsphysiologische Untersuchen an Echiniden. Ribonucleinsaure-Synthese in dem Mikromeren und Entodermdifferenzierrung. Ein Beitrag zum Problem der Induktion", Wilhelm Roux' Arch. Entwicklungsmechan.Organismen vol. 156, pp. 504-524.

Czihak G, Wittmann HG, and Hindennach I, (1967), "Uridineinbau in die nucleinsauren von Furchungsstadien  der Eier des Seeigels Paracentrotus lividis", Z. Naturforsch. vol. 22b, 1176-1182.

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Horstadius S, (1935), "Uber die Determination im Verlaufe der Eiachse bei Seeigeln.", Publ. Staz. Xool. Napoli, vol. 14, pp. 251-479.

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Piatigorsky J, and Whiteley AW, (1965), "A change in permeability and uptake of [¹⁴C] uridine in response to fertilization in Strongylocentrotus purpuratus eggs", Biochim. Biophys. Acta vol. 108, 404-418.

1. Czihak G, "Evidence for Inductive Properties of the Micromere-RNA in Sea-urchin Embryos", Naturwissenschaften, vol. 52, no. 6, pp. 141-142 (1965).

2. Czihak G, "Entwicklungsphysiologische Untersuchen an Echiniden. Ribonnucleinsaure-Synthese in dem
Mikromeren und Entodermdifferenzierrung. Ein Beitrag zum Problem der Induktion", Wilhelm Roux' Arch.
Entwicklungsmechan.Organismen vol. 156, pp. 504-524 (1965).

3. Hagstrom BE, and Lonning S, "Time-lapse and Electron Microscopic Studies of Sea Urchin Micromeres", Protoplasma vol. 68, no. 3, pp. 271-288 (1969).

4. Kronenberg LH, and Humphreys T, "Double-Stranded Ribonucleic Acid in Sea Urchin Embryos", Biochemistry, vol. 11, no. 11, pp. 2020-2026 (1972).

Additional Chromatin References:

1. Frenster JH, Allfrey VG, and Mirsky, AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci., USA, vol. 50, no. 6, pp. 1026-1032 (Dec. 1963):

2. Frenster JH, "Ultrastructural Continuity between Active and Repressed Chromatin", Nature, vol. 205, no. 4978, pp. 1341-1342 (March 27, 1965).

3. Frenster JH, "Nuclear Polyanions as De-repressors of Synthesis of Ribonucleic Acid", Nature, vol. 206, no. 4985, pp. 680-683 (May 15, 1965).

4. Frenster JH, "A Model of Specific De-repression within Interphase Chromatin", Nature, vol. 206, no. 4990, pp. 1269-1270 (June 19, 1965 ).

5. Frenster JH, "Localized Strand Separations within Deoxyribonucleic Acid during Selective Transcription", Nature, vol. 208: no. 5013, pp. 894-896 (November 27, 1965).

6. Frenster JH, "Correlation of the Binding to DNA Loops or to DNA Helices with the Effect on RNA Synthesis", Nature, vol. 208, no. 5015, p. 1093 (December 11, 1965).

7. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin", In-Vitro, vol. 1, pp. 78-101 (1965).

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