JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 7:85-89 (1987)

Effect of Antibodies Against Gap Junction Protein on Gap Junction Formation in Early Amphibian Embryogenesis

MIBAI ZENG (MI-PAI TSENG) Shanghai Institute of Cell Bwlogy, Chinese Academy of Sciences, Shanghai, People’s Republic of China

KEY WORDS Gap junctions, Amphibia

ABSTRACT By electron microscopic observations of freeze-etching repli- cas, the gap junctions of ectoderm cells of early gastrulae of Xenopus leauis, injected with antibodies against gap-junction protein at an earlier stage, were compared with those of normal gastrulae. Most of the gap junctions found in the antibody-injected series were of very small size and consisted of fewer than 40 connexons, while those of the normal gastrulae usually have gap junctions with over 100 connexons. It was concluded that the normal assembly of intra- membranous particles to form gap junctions was attenuated by microinjection - _ _ of the antibodies.

INTRODUCTION

Since the early work of Potter, Furshpan, and Lennox (1966) on squid embryos to dem- onstrate the parallel restrictions of cell cou- pling and range of cell interaction in development, important evidence has accum- ulated on both the early occurrence of gap junctions (Dasgupta and Udai, 1982; Dorres- teijn et al., 1981, 1982; Ducibella et al., 1975; Hanna et al., 1980) and the correlation be- tween gap-junctional changes and some em- bryonic events (Gilula et al., 1978; Guthrie, 1984; Lo and Gilula, 1979a,b; Tseng and Wang, 1984). However, unequivocal data are still lacking to show that gap junctions pro- vide a pathway for cell-to-cell transmission of signals in embryonic differentiation and development.

We have carried out a series of experiments with embryos of Cynops orientalis and of Xenopus leauis. The system of embryonic in- duction and differentiation of induced struc- tures has been chosen.

Gap junctions in embryonic cells before and after induction were studied and compared by using the freeze-etching technique and morphometry (Tseng et al., 1986). In both cases, gap junctions were present, but they differ in their topographical locations, junc- tion sizes, packing densities of the connexons as well as their configuration. In the induced neuroepithelium as well as the induced me- sodermal cells, the connexons are arranged loosely and irregularly, the condition repre- sentative of a functional state.

On the other hand, when microinjections of antibodies against gap-junction protein to early blastomeres of Xenopus leauis were fol- lowed by treatment with a mesodermal in- ductor, it was found that induction was not affected by the injection of the antibodies, but differentiation of notochord and muscle were impeded to different degrees (Zeng et al., in press [a]). It appears that in the differ- entiation of induced cells, cell interactions via gap-junction communication are in- volved, and the microinjection of antibodies disturbs the differentiation process.

By electron microscopic observations of freeze-etching replicas of gap junctions of cells from an embryo injected at an earlier stage and those from normal embryos, evi- dent morphological changes have been re- corded.

MATERIALS AND METHODS Embryological manipulations

Embryos of Xenopus leavis were used and staged according to Nieuwkoop and Faber (1956). The antibodies used were affinity-pu- rified polyclonal rabbit antibodies against mouse liver 26KD gap junction protein (Der- mietzel et al., 1984).l

‘Provided by Prof. Klaus Willecke of Essen University.

Received June 23,1987; accepted July 11, 1987. Address reprint requests to Mibai Zeng, Shanghai Institute of

Cell Biology, Chinese Academy of Sciences, Shanghai, People’s Republic of China.

0 1987 ALAN R. LISS, INC.

86 M. ZENG

Microinjections were made into the two dorsal animal blastomeres of the eight-cell stage which are destined to form ectoderm. For all other details of the manipulation re- fer to Zeng et al. (in press [b]).

When the injected embryo reached early gastrula, a piece of dorsal ectoderm was extirpated and fixed for freeze-etching pro- cessing. For the control, a piece of dorsal ectoderm was taken from normal gastrula without any injection.

Freezeetching preparations The extirpated ectoderm pieces were fixed

in phosphate buffered (pH 7.4) 2.5% glutar- aldehyde at 4"C, washed with buffer, and soaked in 30% glycerol for at least 2 hours. The freeze-etching preparations were carried out in a Balzer apparatus (BAF-400D) and replicas were observed with a Zeiss EM109.

RESULTS

In the freeze-etching replicas of ectoderm cells of Xenopus leauis, both from the normal and from the antibody-injected gastrulae, gap junctions were observed just like those found in the ectoderm cells of other amphibia, e.g., Cynops orientalis (Tseng et al., 1986). They were situated in the lateral membrane or beneath the tight junctions but always at a distance from them (Figs. 1,8).

If the number of the connexons is tenta- tively divided into > 100,100-40,40-20, and < 20 subdivisions, most of the gap junctions of the normal gastrula ectoderm had connex- ons of more than 40 with some over 100 (Table 1; Figs. 1-7). In the ectoderm cells of antibody-injected gastrula, most of the gap junctions found were of very small size (Table 1). Of the 20 gap junctions recorded, 4 of them had more than 40 connexons (Figs. 9-10); the other 16 had less than 40 connex- ons, and 14 of them had less than 20 (Figs. 11-16).

DISCUSSION

Since the use of antibody probes in the study of gap-junctional communication, be-

sides the correlation data of the morphologi- cal changes of gap junctions and the occurrence of some developmental events, di- rect demonstrations have been provided that gap junctions are indeed involved in cell-to- cell communication. Warner et al. (1984) re- ported that antibodies to the rat liver gap junction protein inhibited dye transfer, elec- trical coupling, and normal morphogenesis. By using cultured cells, Hertzberg et al. (1985) found that microinjections of antibod- ies inhibited both dye and electrical cou- plings. Our previous works have shown that antibodies to mouse liver gap junction pro- tein, when injected into different vegetal blastomeres of Xenopus embryos, brought about asymmetric malformation of sense or- gans on the injected side (Zeng et al., in press [a]). In the induction system, it was found that differentiation of the induced cells was affected by the antibody microinjection (Zeng et al., in press [b]).

With all these effects brought about by the antibody microinjection, no records on mor- phological changes have previously been reported.

The results of the present work have dem- onstrated that gap junctions of ectoderm cells of the antibody-microinjected gastrula dif- fered from those found in the normal gas- trula ectoderm cells in the number of gap junction connexons. In most of the cells, only a few connexons came together to from a cluster. These clusters remind one of the gap- junction formation during normal embryonic development. In the preimplantation mam- malian embryo (Magnuson et al., 1977) and before amphibian gastrulation (Sanders and Dicaprio, 19761, the earliest stage in the gap- junction formation was represented by the appearance of small groups of intramem- branous particles. As development pro- ceeded, gap junction plaques of different sizes became frequent. Similar events have been reported during cardiac development (Gros et al., 1978; Shibata et al., 1980) and in re- generating liver (Yee and Revel, 1978). By using reaggregated Novikoff hepatoma cells,

TABLE 1. Number of connexons ofgap junctions recorded

No. of No. of connexons gap junctions

Series recorded > 100 100-40 40-20 <20

Normal 29 6 13 7 3

Gastrula ectoderm gastrula ectoderm

after 20 4 2 14 antibody injection

ANTI-GAP JUNCTION PROTEIN AND GAP JUNCTION FORMATION

Figs. 1-6. Bar represents 0.1 pm. All micrographs are at the same magnification.

Figs. 1-7. Freeze-etching replicas of ectoderm cells of normal gastrulae.

Fig. 1. A gap junction of 152 connexons is situated beneath the tight junction.

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88 M. ZENG

Figs. 8-16. Freeze-etching replicas of ectoderm cells of Figs. 9-10, Gap junctions each with more than 40 gastrulae injected with antibodies. connexons.

Fig. 8. A gap junction of 21 connexons is situated near Figs. 11-16. Gap junctions each with less than 40 the tight junction. connexons.

ANTI-GAP JUNCTION PROTEIN AND GAP JUNCTION FORMATION 89

Johnson et al. (1974) provided evidence that the first indication of the genesis of new junc- tions was the appearance of small aggrega- tions of particles on the so-called formation plaque. It seems that after the injection of antibodies, the normal assembly of intra- membranous particles to form the gap junc- tions is impeded and the degree of impediment differs. In the case of small clus- ters, further recruitment and increase in junction size is more severely affected, while in the minority of the cases, gap junctions were found to consist of more than 40 connexons.

Microinjection was carried out at the eight- cell stage, and the ectoderm explant exam- ined was taken from early gastrula. At the culture temperature (22°C 1 "C), it took about 6 hours for the eight-cell embryo to reach early gastrula. How long the effect of antibody lasts depends upon the biosynthesis of new junction protein as well as the half- life of the antibody protein. It was reported that mouse liver gap junction protein has a half-life of 5 hours (Fallon and Goodenough, 1981), but none of these parameters is known at present for Xenopus embryos.

The polyclonal antibodies used were shown on immunoblots to react with a 26-KD pro- tein from membrane of Xenopus oocytes and of 32-cell embryos, but they also react with a few other denatured proteins (Zeng et al., in press [a]). Although high conservation of gap junction proteins was demonstrated in a wide variety of species (Hertzberg and Skibbens, 1984), the use of antibodies to Xenopus gap junction protein would no doubt provide more relevant evidence.

ACKNOWLEDGMENTS

This work was supported by the Science Fund of the Chinese Academy of Sciences.

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