Membrane excitability in cleavage-arrested, differentiated cells

cytoplasm coincided with the assignment of the fate of cells in those regions. Although these results constitute a fate map, showing the positional origins of different cell types in normal development, they do not show whether cell fate is assigned in a mosaic fashion. However, .in subsequent experiments at the 4- and 8-cell stages, cell ablation produced incomplete embryos, revealing the absence of regulation, while isolated individual cells were able to develop autonomously into structures appropriate to their position. These direct tests confirmed the predictions of mosaicism and were further supported by the striking finding by Whittaker and coworkers3, that blastomeres of cleavage-arrested embryos could produce terminal differentiation products if left for the equivalent time to hatching as control embryos. The presence of acetylcholinesterase in blastomeres destined to become muscle, for example, implied the segregation of cytoplasmic factors or determinants capable of influencing gene activity, even prior to the 64-cell stage, when cell fate is uniquely determined.

Membrane excitability in cleavagearrested, differentiated cells A rare glimpse of patterns of cell determination in the early embryo is provided by a recent article in the Journal of Physiology, in which T. Hirano and colleague? report that in, cleavage-arrested ascidian embryos, lefi the equivalent time to hatching, identified blastomeres differentiate to produce daughter cells with one of four welldefined types of membrane excitability; neural, epidermal, muscular or non-excitable. This interesting system contributes to our understanding of mosaicism and regulation in early development, and may enable detailed study of the development of membrane excitability.

The ascidian is a sessile marine animal, whose tadpole resembles that of vertebrate species and is therefore classified in the phylum Chordata (see Fig. 1). Embryological interest has centred on the ascidian embryo partly because it is designated as a classical ‘mosaic’. This term refers to an embryo in which, at an early stage, different regions become irreversibly committed to develop into different cell types. The mosaic embryo is envisaged as discrete blocks of cells, behaving independently and without interaction. This lack of plasticity means

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that it cannot replace missing parts, in contrast with a ‘regulative’ embryo, e.g. the mouse, in which cells are less rigidly committed to their developmental fates and can accommodate change. Cytoplasmic factors, partitioned early on and localized in various parts of the embryo, are thought to be responsible for eliciting these mosaic patterns of differentiation. The ascidian’s mosaic status was first suggested by experiments of Conklin’ who showed that, during development, the partitioning of regions of coloured

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The differentiation of properties in the cleavage-arrested embryo thus affords the opportunity to explore early events in development, a potential which has been exploited by T. Hirano and colleagues. They have used voltage clamp and constant current analysis to examine membrane excitability in identified blastomeres of l-32 cell ascidian embryos, cleavage-arrested with cytochalasin B and left until control embryos of similar age hatched. They describe four excitability types, neural, epidermal, muscular and non-excitable. Only one of these was found to be expressed per blastomere. Each of these excitabilities was characterized on the basis of the shape and ionic dependence of its action potential (see Fig. 2), muscular type blastomeres also being defined by acetylcholinesterase staining. Quantitative analysis of the distribution of types showed that, in general, neural blastomeres were situated in regions destined to become brain, muscular in presumptive muscle, epidermal in endoderm, and non-excitable in vegetal, regions, which form non-excitable tissue. The muscular excitability response had an action potential closely resembling that of adult muscle cells, but all the other types were defined by their position in the fate map, giving them uncertain developmental significance. Furthermore, the distribution map varied between embryos at the same stage; there was only a rough correspondence between the excitability map determined in the cleavage-arrested embryo and the fate map determined in the control. In 41% of 16-cell embryos, for example, no neural-type blastomeres were present, while in some embryos muscular responses were evoked in blastomeres from vegetal regions. A sequence of decision was indicated both by blastomere distribution and electrical coupling patterns. Up to and including the 4-cell stage, all blastomeres gave epidermaltype responses and the embryo behaved as an electrical continuum. From the &cell stage onwards, a number of different excitability types emerged, while the spread of current became compartmentalized between blastomeres of similar type, regardless of their position. This elegant study provides some interesting indications of cell determination, but conclusions drawn must take into account the limitations of the method. The authors attach importance, for example, to the observation that one blastomere showed one type of excitability exclusively, despite its several

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possible developmental fates. The constraints of the fluid membrane make it unlikely, however, that a single blastomere could express multiple types of action potential in a spatial sense; mosaic excitability which might arise through the co-existence of various different channels within one membrane (e.g. a mixed population of egg and adult channels) cannot be resolved by these experiments. What the results do indicate. is that the ascidian is not a mosaic in terms of the assignment of final differentiation in a p.ositionally invariant way. The membrane excitability types described here probably do not represent terminal differentiation; their current channels are different from those in the egg, but their action potentials are not identical with those of the adult, except in the case of muscle. There is also evidence of differences between the action potentials of similar type blastomeres, arrested at different times in development. There appears to be a decisional hierarchy of ,potential, with the initially uniform epidermal response diversifying as different excitabilities are expressed and ‘coinciding with restriction of electrical communication. The correlation of the sequence of restriction of current with restriction of developmental fate may suggest control at the junctional level and might be resolved by electrophysiological analysis. All the results in this paper indicate more regulation and cell interaction than occurs in the determinate cell divisions of the nematode, but far less than in vertebrates. Cytoplasmic localization per se does not rule out regulation; indeed it has been described in most other animals, up to a moderate grade of chordate evolution. Thus the ascidian cannot be considered purely in mosaic

terms; like other species its development consists of a regulative, followed by a mosaic phase, differences between species residing in the relative durations of these periods. These experiments open up many possibilities for future work. The virtue of the ascidian system is that it allows the study of determination in identified cells, long before their differentiation becomes visually apparent. Tracing neural differentiation at the single cell level, for example, is impossible in vertebrate species, where reliable identification of a neuron is based on the expression of differentiated properties. Obvious extensions of this work on single blastomeres may enable elucidation of the sequence of development of excitability, and the characteristics and insertion of single channels into membranes. The construction of a map of cell commitment by Hirano et al. also raises the possibility of investigating the roles of different cell types in early cell interactions. Thus, despite the apparent atypicality of the ascidian, research derived from it may prove relevant to a wide range of vertebrate and invertebrate species.

Reading list Conklin, E. G. (1905) .I. Acad. Nat. Sci. Phila. 13, l-119 2 Hirano, T., Takahashi, K. and Yamashita, N. (1984) J. Physiol. (London) 347, 301-325 3 Whittaker, J. R. (1973) Proc. Nat1 Acad. Sci. 1

USA 70, 2096-2100

4 Slack, J. M. W. (1983) From Egg to Embryo p. 116, Cambridge University Press, Cambridge

SARAH C. GUTHRIE

Dept College,

of Anatomy and Embryology, Cower St, London WCIE

6BT,

University UK,