Anat Embryol (1981) 163:321 330 Anatomy and Embryology �9 Springer-Verlag 1981

Effects of Colchicine on Gap Junction Formation During Retinal Neurogenesis

Karl Meller Institut ftir Anatomie, Arbeitsgruppe ffir Experimentelle Cytologie, Ruhr-Universit~it Bochum, Bundesrepublik Deutschland

Summary. Colchicine, injected into the amniotic cavity of 3 to 10 day-old chick embryos, has the following effects on the developing retina:

1. Cells in arrested metaphase accumulate in the ventricular portion of the matrix region. If colchicine is applied at the end of the first week of incubation, the kinetic migration of the cell nuclei is inhibited.

2. The cells lose their typical slender, bipolar shape to become ovoid to spherical. These morphological changes are more obvious when colchicine acts during the first week of incubation.

3. The formation of temporary gap junctions between the matrix cells of the retina occurs during the first week of development. When colchicine is administered around the 3rd day of incubation, the assembly of intramem- branous particles (IMPs) during gap junction formation is incomplete.

These results suggest that an intact microtubular system within the sub- membranous cytoskeleton is essential for the assembly of these intercellular contacts.

Key words: Gap junction formation - Retinal neurogenesis - Colchicine

Introduction

Previous studies have shown that developing neural retinal cells are connected by intercellular junctions (Dixon and Cronly-Dillon 1972; Fusijawa et al. 1976; Hayes 1976). Gap junctions are formed during the proliferation phase in the early stages of development and disappear at the end of proliferation. They

Offprint requests to : Prof. Dr. Karl Meller, Institut ftir Anatomie, Arbeitsgruppe ffir Experimentelle Cytologie, Ruhr-Universitfit Bochum, Universitfitsstr. 150, 4630 Bochum 1, Federal Republic of Germany

This work was supported by grants from the Ministerium ffir Wissenschaft und Forschung des Landes Nordrhein-Westfalen (IIB 5-FA 7470) and from the Stiftung Volkswagenwerk (Az 11 2977). The author thanks R. Maeder and E. Descher for the excellent technical assistance and K. Donberg for preparing the photographs. I also thank K. Rascher for her help with the English translation and C. Bloch for typing the manuscript

0340-2061/81/0163/0321/$02.00

322 K. Meller

are morphological ly different f rom contacts present in the adul t animals (Raviola and Gilula 1973). These structures probably represent the intercytoplasmic path- ways for the transfer of ions and small molecules between the developing cells (Bennett 1973 ; Powers and Tupper 1977 ; Bennet t and G o o d e n o u g h 1978 ; Perac- chia 1980). The fact that their appearance is temporal ly coincidenta l with crucial neurogenet ic events suggests their impor tance for the "genesis of posi t ional i n f o r m a t i o n " between the retinal cells (Jacobson 1976).

The fo rmat ion of cellular contacts dur ing development depends on the mobi- lity and assembly of IMPs in the cell m e m b r a n e (Revel et al. 1973; Decker and Fr iend 1974; Yee and Revel 1978; Meller 1979). There is a growing body of evidence that suggests an in terdependence between cell surface receptors, the mobi l i ty of IMPs in the cell membrane , and the s u b m e m b r a n o u s cytoplasmic network (Aubin et al. 1975; Edelman et al. 1973; Poste et al. 1975; Yahara and Ede lman 1975; Carraway et al. 1979; McCla in and Ede lman 1979; Pick and Wilner 1979). The growth of gap junc t ions by accret ion of particles or by some other mechanism could theoretically be control led by cytoplasmic fi laments or other cytoskeletal structures (Yancey and Revel 1978).

The present study examines the effects of the an t imic ro tubu la r drug colchi- cine on the development of the retina. These results suggest an interact ion between micro tubula r structures and IMPs dur ing the assembly phase of gap junct ions . Fur thermore , the inhibi t ion of gap j unc t i on fo rmat ion might be in- volved in the subsequent death of colchicine-arrested metaphase cells.

Material and Methods

0.5 ml of colchicine (Sigma, Miinchen) at a concentration of 1 x 10 6 g/ml was injected into the amniotic cavity of 3 to 10 day-old chick embryos.

Electron Microscopy (TEM). The eyes of chick embryos (3 to 10 days of incubation) were enucleated and opened by an equatorial incision. The vitreous body was removed from the posterior hemisphere and the eyecups fixed in 2.5% glutaraldehyde in phosphate buffer at pH 7.4. After dissection, the retina was postfixed in OsO4, dehydrated in ethanol and embedded in Epon (Serva, Heidelberg). Semithin sections were stained with methylene blue and ultrathin ones were contrasted with lead citrate (Venable and Coggeshall 1964) and examined with a Siemens Elmiskop 1A at 80 kV.

Scanning Electron Microscopy (SEM). The eyecups were fixed in buffered glutaraldehyde for 12 h, rinsed in buffer and postfixed in OsO4 for several hours. After fixation, the neuroretina was dissected out and fractured with the aid of small forceps. Dehydration was carried out in methanol. The specimens were transferred to Freon 11 and finally to Freon 13 in a critical point apparatus. They were coated with gold and examined in a Jeol JSM-35 at 25 kV.

Freeze-etching. After fixation, the retina was washed in phosphate buffer and immersed in 10 to 30% glycerol buffer. The specimens were mounted on Balzer's gold discs, rapidly frozen in liquid nitrogen-cooled Freon 22 and stored in liquid nitrogen. Freeze fracturing and etching was performed in a Balzers' apparatus (BAF 300). Replicas were cleaned in sodium hypochlorite for 24 h, washed in distilled water and mounted on Formvar-coated copper grids.

Organ Culture. The control retinas and those of colchicine-injected embryos were removed five hours after treatment under sterile conditions in Hank's solution. Small pieces of tissue were placed in a culture medium (Minimum essential medium (Flow Laboratories, Bonn), 10% horse serum, 1% embryonic extract, 6 mg/ml glucose and antibiotics) and cultivated in rotating Erlenmeyer flasks in a CO2-enriched incubator at 36.5 ~ C for 1 to 3 days.

Colchicine Effects on Retinal Neurogenesis 323

Results

The effects of colchicine on the retina vary, depending on the embryonic stage of development at drug application. When colchicine is administered to 3 to 5 day-old chick embryos it affects the retinal cells during the proliferative phase. Six to eight hours after colchicine injection, cells arrested in metaphase accumu- late in the external region of the matrix zone beneath the pigment layer. This concentration of arrested metaphase cells is comparable to that described in another region of the CNS (e.g. neural tube) after colchicine administration (Watterson 1965) (Fig. 2a). When colchicine is administered at later stages (7 to 10 days of incubation) there is almost no accumulation of metaphase cells. Some nuclei divide without having undergone kinetic migration to the ventricular zone; they remain in the proximity of the vitreal surface (Fig. 4d).

SEM examination of the 3 to 6 day-old treated embryos revealed alterations in the shapes of the retinal elements. The generally bipolar matrix cells become more spherical (Fig. 1A, B). The apical and basal cell ends that collectively limit the ventricular and vitreal retinal surfaces protrude balloon-like at these free surfaces. This is shown in Fig. 2b where the basement membrane is detached from the cell protrusions oriented toward the vitreal surface. These morphologi- cal changes are more obvious when colchicine is administered in early stages of development. During the second week of incubation modifications of this kind, produced by colchicine, were not observed (Fig. 1 C).

During the first week of incubation (up to 6 days) gap junctions develop between the matrix cells. They are localized in the apical regions of the cells beneath the ventricular surface, but can also be found on the cell body and between the slender vitreal processes. As shown in Fig. 3a, b, these contacts are round to oval patches of variable diameter, consisting of particles arranged in parallel rows, which confer to the macula a mosaic-like appearance. After the 7th day these gap junctions progressively disappear and become rare on the 9th day of incubation. Colchicine affects gap junctions only when it is administered during gap junction formation around the 3rd day of incubation, but not at later periods of development. The effect is brought about by the inhibition of the assembly of IMPs into the patches described above. Figures 3c-e show examples of this incomplete assembly of gap junctions in the 6 day-old retina, three days after colchicine application. Small groups or rows of particles are present only in the contact zones between the matrix cells. In contrast, the controls (Fig. 3 a, b) possess fully-developed contacts.

A typical consequence of colchicine administration is the degeneration of some cells located in the apical zone of the matrix. This localization suggests that the degeneration of these cells is a consequence of mitotic arrest. Their cytoplasm appears vacuolated and their nuclei are in different stages of pyknosis (Fig. 4a). However, the subsequent organization of the retina does not seem to be affected by this cell degeneration and the typical three cell layers can be recognized in the 9th to 10th day of incubation (Fig. 4c).

Retinas of colchicine-injected 3 day-old embryos and those cultivated in vitro show that the metaphase arrested cells become pyknotic during the first day of cultivation (Fig. 4b). Surviving ceils later become arranged in the rosettes typical of retinal cell cultures (compare Hild and Callas 1967).

Fig. 1A-C. Chick embryo retina. A 5 days old, 8 h after colchicine administration. B 5 days old, 6 h after colchicine administration. C 7 days old, 8 h after colchicine administration. Observe in A and B the changes in the shapes of the bipolar matrix cells. These are less pronounced in C .A • x840;C •

Fig. 2a, b. Chick embryo retina, a 5 days old, 6 h after colchicine administration. Accumulat ion of cells arrested in metaphase beneath the outer surface of the retina, x4,000, b 6 days old, 3 h after colchicine administration. Removal of the basement membrane from the vitreal surface reveals balloon-like protrusions of the cell basal prolongations, x 2,800

Fig. 3a-e. Chick embryo retina 6 days old. a Apical portion of a matrix cell beneath the pigment layer. Arrow: gap junction, x50,000, b Temporary gap junct ion situated in the apical region of a matrix cell. Note characteristic mosaic-like arrangement of IMPs. x 75,000. c, d, e 3 days after colchicine administration. Assembly of gap junct ions is incomplete. Compare with a and b. • 75,000

Fig. 4. a Chick embryo retina 10 days old: 3 days after colchicine administration. Arrows: Degenera- ted matrix cells with pyknotic nuclei and vacuolated cytoplasm, x 5,000. b Retina organ culture. Portion of a retinal rosette after administrat ion of colchicine. Arrows: Degenerated cells, x 840. e Chick embryo retina 8 days old, 6 h after colchicine administration. Observe the normal develop- ment of the retinal cell layers, x 400. d Chick embryo retina 7 days old, 6 h after colchicine administration. Arrow: Cell in mitotic arrest beneath the vitreal surface. • 2,750

328 K. Meller

Discussion

Temporary gap junctions present during specific periods of embryonic develop- ment are probably a common finding. They have been described not only in the developing retina (Dixon and Cronly-Dillon 1972; Fujisawa et al. 1976; Hayes 1976) but also in the neural tube (Schoenwolf and Kelley 1980), in the spinal ganglia (Pannese et al. 1977), in the cortical plate of mice (Meller, unpublished), and even in Schwann cells during the regeneration of sciatic nerves (Tetzlaff 1981). This ubiquity and correspondence with a definite time of development suggests that these junctions coordinate both 1) the proliferative cycle, probably regulating the relative number of neurons of each type (Cowan 1979) and 2) positional information between the developing cells (Jacobson 1976).

The formation of specific junctions is, among other factors, dependent upon the mobility of the intramembranous particles (IMPs).

The assembly of IMPs during the formation of cell junctions can be disturbed by drugs that bind to receptor sites on the cell membrane, such as concanavalin A (Meller 1979) or that act on microtubules as reported by Sang et al. (1980) who demonstrated the inhibition of tight junction formation by colcemide,

The present results show that colchicine disrupts the formation of gap junc- tions precisely at the stage when their assembly through accretion of IMPs takes place. Colchicine 'administered after this stage (around the 3rd day of development) does not appear to affect the structure of gap junctions.

Colchicine caused not only antimitotic but also other biological effects such as alterations in cell form and movement, i.e., axonal transport, neurite growth etc. (Daniels 1972; Jones and Partridge 1974; Hanson and Edstr6m 1978). The major effects are due to the disruption of microtubules through the binding of colchicine to the protein tubulin (Wilson and Friedkin 1964; Borisy and Taylor 1974 ; Wilson et al. 1974). Nicolson (1976, 1979) postulated that cytoskele- tal elements such as microtubules (MT) and microfilaments (MF) linked to the plasma membrane proteins and glycoproteins mediate cytoplasmic control of cell surface phenomena. Carraway et al. (1979) assume two possibilities: microtubules and microfilaments are both linked to the membrane or the micro- tubules are linked to microfilaments which in turn are linked to the membrane; a perturbation of the former alters the structure of the latter. Edelman and coworkers (Edelman et al. 1973; Yahara and Edelman 1975; Edelman 1976; McClain and Edelman 1979) proposed that surface movements are regulated by microtubules or related structures and surface events affect the state of colchicine-sensitive structures. Poste et al. (1975) claimed that colchicine treat- ment increases the mobility of Con A receptors and this supports the concept of an interplay between the two systems: surface receptors and cytoskeletal components. This possible control of surface receptors via cytoskeletal structures is corroborated by other authors (Albertini and Anderson 1977 ; Pick and Wilner 1979).

The relationship between colchicine effect and inhibition of gap junction formation suggests that cytoskeletal structures play a role in the assembly of junctional contacts, but the factors governing the association of MF and MT with the cell membrane are as yet only poorly understood.

Colchicine Effects on Retinal Neurogenesis 329

With the freeze etching technique used in the present study it has so far

no t been possible to demonst ra te that cytoskeletal structures are anchored to IMPs.

Gap junc t ions may mediate the control of prol i ferat ion and regulate intercel- lular commun ica t i on between coupled cells. It is probable that the act ion of colchicine on gap junc t ions leads to the degenerat ion and subsequent pyknosis of cells in mitot ic arrest. The f inding that also cul tured retinal cells, arrested in metaphase, degenerate may indicate that there is a causal relat ionship between dis turbed gap junc t ion assembly and cellular degenerat ion.

Two points must finally be stressed. First, gap j unc t i on fo rmat ion seems to depend on an intact mic ro tubu la r system, a l though its role dur ing gap j unc t i on assembly remains to be elucidated. More precise techniques are necessary to answer the quest ion of whether the mobi l i ty of IMPs in the cell m e m b r a n e depends on possible anchorage points. Secondly, a no t d is turbed intercellular commun ica t i on at a critical period of neurogenesis seems to be necessary for cell survival. In this connec t ion it could be speculated that " n o r m a l " cell death (see review by Cowan 1979) dur ing neurogenesis , which reaches a m a x i m u m in the ret ina dur ing the second week of incubat ion , is regulated by a similar process of regression in the n u m b e r of gap junct ions .

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Accepted October 10, 1981