DEVELOPMENTAL MODIFICATIONS IN ARBACIA PUNCTULATA BY VARIOUS METABOLIC SUBSTANCES

¹ Department of Biology, New York University, New York New York 10003, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543

1. For clarity, brevity, and accuracy in comparisons, a series of stages in the early development of the sea urchin, Arbacia punctulata, is presented. Each major morphological event is identified by a symbol.

2. Concentrations of ethanol and methanol of 1% or less have no visible effects on embryogenesis except for a slight growth retardation in ethanol. At 5% development was arrested at the morula stage, while at levels greater than 5% fixation of the cells occurred at the 2-cell stage.

3. The effects of papain (1 mg./ml.) on fertilized eggs were similar to but harsher than those of trypsin in arresting development and effecting dispersal of blastomeres. The fertilization membrane, however, was more resistant to dissolution than in trypsin. Upon lysis of the fertilization membrane, naked larvae often fell apart to blastomeres.

4. Sperm remained motile for several hours in pronase solutions (0.25 mg./ml.) whereas egg cells showed striking cortical changes followed by cytolysis. The fertilized egg, however, was more resistant and underwent cleavage in solutions 10 times more concentrated. Pronase often lysed the fertilization membrane within two hours post-fertilization; subsequently, separation of blastomeres occurred. When the membrane remained intact, normal plutei developed. It is suggested that pronase is a more suitable cell-dispersing enzyme than trypsin.

5. Sodium taurocholate was ineffective at 0.2 mg./ml. but at 2 mg./ml., by virtue of surface and internal changes (increasing lipolysis), brought about a rapid death to zygotes.

6. Exogenous DNA, rather than enhancing cleavage, causes a growth retardation at 0.1 mg./ml. with some lethal effects. Differentiation of mesodermal structures, however, continues although the pluteus shape is inhibited. The consensus is that nuclear aberrations are produced.

7. In general, the addition of DN-ase elicits a retardation of activity and cleavage in Arbacia. A concentration of 0.2 mg./ml. brings about death in 48 hours without any morphogenetic alterations. These effects may be attributed to a depletion of DNA by the enzyme.

8. At 0.1 mg./ml. and greater of RNA, an inhibition of cleavage is characteristic, but no specific malformations develop. Small levels of RNA (10 µg./ml.) accelerate slightly the rate of early cleavage, probably by a contribution to the cytoplasmic pool of RNA from which it is metabolized.

9. When physiological doses of RN-ase (0.1-0.12 mg./ml.) of RN-ase are added to sea water, Arbacia embryos exhibit only a slight retardation in rate of cleavage and a sluggishness in movement, and pass successfully through gastrulation. Larger amounts are cytotoxic, probably due to a massive degradation of cellular RNA.

10. Thymine, in concentrations of 0.04 mg./ml. to 0.4 mg./ml., inhibits development, the effects increasing with increasing doses. The block at gastrulation is striking at 0.4 mg./ml. Recovery by washing is rapid.

11. Cobalt chloride and cobalt glycine are lethal to Arbacia embryos at the blastula (0.2 mg./ml.) and at the morula (1 mg./ml.). In below-lethal levels (0.01 to 0.02 mg./ml.), the vegetalization-radialization is striking. Cleavage planes are disrupted, resulting in rough-surfaced embryos. The drastic aberration, however, becomes evident at gastrulation when bloated embryos develop as exogastruleae. The skeleton is miniaturized. RNA and RN-ase very slightly ameliorate the effect, but RNA and DNA and DN-ase do not. Washing within five hours of immersion prevents exogastrulation.

12. The low solubility of actinomycin D in sea water and its slight precipitation upon standing must be considered in assaying for its dose-dependent retardation of cleavage and differentiation in sea urchins.

13. Accordingly, 4 µg./ml. of actinomycin D effectively proves to inhibit full development beyond the blastula. The sequelae, blastular arrest, inhibition of spicule and arm formation, are well defined at 8 µg./ml. but this constitutes a toxic overdose. The first morphological evidence, aside from retardation of cleavage, was a disruption in the alignment of blastomeres, even before the morula stage. Reversal of the effect by washing, DN-ase, RNA or RN-ase was not obtained, but some amelioration was provided by DNA or thymine. Because of these events the binding of actinomycin probably occurs at specific sites.

14. Similar to actinomycin D, at 5 µg./ml. 4-nitroquinoline N-oxide, acted as a powerful inhibitor of gastrulation. Sperm cells, however, were viable for several hours at 0.0125 mg./ml. It is suggested that the metabolic block is inoperable in the sperm. The mechanism, inhibition of -SH groups or lack of RNA polymerase in sperm cells, needs to be investigated.

15. Coumarin (ca. 0.5 mg./ml.) can effectively act as a block to gastrulation. Although development is highly abnormal, the overall activity, swimming in eccentric orbits, is not curtailed. Which of the multiple physiological effects of coumarin is operable cannot be stated.

16. Mescaline at 1 mg./ml. is lethal to embryos at the gastrula stage. The most striking effect of mescaline (0.1 mg./ml.), aside from its growth-retarding one, is the production of a miniaturized skeleton in a normal-sized pluteus larva.

17. In non-lethal concentrations (0.02 to 1 mg./ml.) thalidomide permits normal although slower morphogenesis through the pluteus. The retardation, however, is not reversible by washing or the addition of RNA, RN-ase, DNA, DN-ase, etc. In many instances the fertilization membrane persisted about the larvae.

18. In evaluating the overall developmental effect of compounds on embryogenesis, substances may be identified, with some degree of overlapping, as: normal metabolites, animalizing, vegetalizing, or radializing agents, as mitotic arrestors or inhibitors, genotropic or genostatic or toxic.