Classical merotomy and grafting experiments in Acetabularia (Dasycladales, Dasycladaceae)

Brian Wysor

Southampton College
Spring 1995

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Acetabularia is a unicellular green alga of the order Dasycladales found in warm waters of sheltered lagoons (Lee, 1980). It is characterized by radial symmetry of a nonseptate axis with whorls of laterals (Bold and Wynn, 1985). The conspicuous nucleus, referred to as the giant or primary nucleus, is situated in the basal rhizoid of the 1-6 cm thallus (Puiseux-Dao, 1970). This distinctive uninucleate condition has provided biologists with an excellent system for the study of nucleocytoplasmic interactions (Hammerling, 1953; berger et al., 1987; Bonotto, 1994).
 
Historically the formation of the cellular materials was thought to be under the control of the nucleus, either directly or indirectly; however, the cytoplasm has been identified as a source of environmental stimulus for the nucleus such that parts of the genetic code can be turned on or off (Lobban et al.l, 1985). Enucleation of plant and algal cells has been widely used to assess the developmental influences of the cytoplasm and the nucleus on each other. Acetabularia lends itself particularly well to studies of this nature because the nucleus is large (approximately 100-150 um in diameter [Schweiger, 1969; Bonotto, 1994]) and conspicuously located. Hammerling pioneered the exploitation of Acetabularia in nucleo-cytoplasmic interaction studies in the 1930s when he discovered that the alga had enormous regenerative potential and was capable of surviving for many weeks after enucleation of the cell. He also showed that inter- and intraspecific grafting was possible (Puiseux-Dao, 1970).
 


Acetabularia acetabulum and Acetabularia crenulata

 

Because species of Acetabularia are are identified by the number, shape and degree of ray adhesion in the cap, as well as the morphological features of the corona (Richter, 1962), characteristics of progeny of merotomy and grafting experiments are easily identified in a grafted specimen.

 


Successful interspecific grafts.

 
With a bit of patience, high grafting success is achievable. Grafts are made as if putting two straws together; the smaller end of either a stalk or a rhizoid is slipped inside the cell wall of it's "mate". Cells hold up very well despite manipulation and, as is evident in the second image, even when cells are disturbed, they may still fuse successfully.
 
When Hammerling grafted a nucleate portion of A. wettsteinii to an enucleate, decapitated stalk of A. mediterranea, he disovered that the resulting cell could regenerate a cap and the cap initially retained the morphological characteristics associated with A. mediterranea (stalk portion). However, if the resultant cap was removed or the cell was allowed to age he discovered that the new cap acquired morphological characteristics resembling A. wettsteinii (nucleated portion). In another experiment Hammerling placed enucleate apical and basal portions of the stalk in culture and observed the regenrative potential of each portion. He discovered that new caps could be regenerated, however the potential to do so seemed to be greater in the apical portions than that for the basal portions (Lee, 1980). Similarly, he observed that morphogenetic capacity of long pieces of the stalk was greater than that of short pieces taken from the same region o f the thallus. Furthermore, Hammerling concluded that nucleated portions of Acetabularia retained full morphogenetic capacity and could be made to regenerate repeatedly (Hammerling, 1953).
 
The results of the extensive series of grafting experiments performed by Hammerling, led him to the conclusion that morphogenetic substances are "more than nucleus controlled." Because caps were regenerated, and posessed characteristics of the nucleus-donor species in both intra- and interspecific grafts and, that intermediate caps were first developed in interspecific grafts suggested that morphogenetic substances didn't simply induce development along a fixed pathway, but determined the direction of development itself. Hammerling thus compared these morphogenetic substances with the effects of products of gene action regulating cap morphology and concluded that morphogenetic substances had to be considered products of gene action (Hammerling, 1953). Future studies which considered the biochemistry of Acetabularia have elaborated these ideas.
 
In summary, evidence suggesting that genetic information for species specific mophogenesis originates in the cell was first derived from surgery experiments using various species of the genus Acetabularia. Further experimentation led to the conjecture that morphogenetic information originated in the nucleus and then transferred to the cytoplasm and a site where it would be expressed. The implication of these experiments is that regulation occurs at the level of translation rather than at the transcriptional level as previously proposed (Schweiger, 1969; Berger et al., 1987; Bonotto, 1994).
 


First regenerative cap of an interpecific graft of an A. crenulata stalk and an A. acetablum rhizoid.

 
 
Literature Cited
 
Berger, S., E. de Groot, G. Heuhaus and M. Schweiger. 1987. Acetabularia: a giant single cell organism with valuable advantages for cell biology. Eur. J. Cell Biol. 44: 349-370.
 
Bold, H. and M. Wynn. 1985. An Introduction to the Algae. Prentice Hall, Inc., New Jersey, 720 p.
 
Bonotto, S. 1994. Developmental biology of Acetabularia. J. mar. biol. Ass. U.K. 74: 93-106.
 
Hammerling, J. 1953. Nucleo-cytoplasmic relationships in the development of Acetabularia. J. Intern. Rev. Cytol. 2: 475-498.
 
Lee, R. 1980. Phycology. Cambridge University Press, Cambridge, 478 p.
 
Lobban, C., P. Harrison and M. Duncan. 1985. The Physiological Ecology of Seaweeds. Cambridge University Press, New York, 242 p.
 
Puiseux-Dao, S. 1970. Acetabularia and Cell Biology. Springer-Verlag new York, Inc. Logos Press Limited, Great Britain, 162 p.
 
Schweiger, H. 1969. Cell biology of Acetabularia. Current Topics in Microbiology and Immunology, 50: 1-36

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