Cell Tiss. Res. 154, 103--108 (1974) �9 by Springer-Verlag 1974

A New Membrane Organelle in Developing Amphibian Neurones

Alan Roberts and Br ian P. Hayes *

Department of Zoology, University of Bristol, England

Received August 17, 1974

Summary. Membrane specialisations have been found on neurones in embryos of the clawed toad Xenopus laevis. The specialisations have been called dense membrane knobs and consist of an outpushing of the plasma membrane with a slight increase in its density. The out- pushing forms a spherical knob with an amorphous dense core and a total diameter of 500 to 600 A. The knobs are found on axons and dendrites both in the spinal cord and peripherally.

Key words: Plasma membrane specialisations - - Developing neurons - - Xenopus laevis - - Electron microscopy.

Introduction

We have found what may prove to be a new type of membrane organelle which is common in the developing spinal cord of the amphib ian Xenopus laevis (South African Clawed Toad.). I n the course of a s tudy of nervous development, the u l t ras t ruc ture of the spinal cord and some other tissues was examined in embryos of Xenopus from the t ime of neurula t ion unt i l short ly after the tadpole has hatched. Many of the differentiat ing nerve cell processes were found to have dense pro- jections from their plasma membranes. Similar dense membrane knobs occurred outside the spinal cord on nerve axons and some skin cell processes. This paper presents a brief account of their characteristics and dis t r ibut ion in Xenopus embryos.

Materials and Methods Embryos were obtained by injecting pairs of adult Xenopus laevis. They were fixed at pH

7.3 in 4 % glutaraldehyde and 1% formaldehyde and then postfixed in 2 % osmium tetroxide. Several stage 27 embryos were stained "in the block" with a 2% solution of uranyl acetate buffered with sodium hydrogen maleate (Brightman and Reese, 1969). Sections were stained with alkaline lead citrate (Reynolds, 1963) and those not stained "in the block" were also stained with 10% uranyl acetate in 50% ethanol. Further details of the specimen treatment can be found in Hayes and Roberts (1973).

Results and Discussion

The appearance of the dense membrane knobs can be seen in Figs. 1 to 8. The basic s tructure is a convex outpnshing of dense plasma membrane into the extra- cellular space with an under ly ing region of dense cytoplasm. In transverse sections of the spinal cord these outpushings were seen as par t ly or completely circular bodies from 300 to 1000 A in diameter bu t for the most par t (64% of the total)

Send o//print requests to: Dr. Alan Roberts, Department of Zoology, University, Bristol BS 8 lUG, England. * Brian P. Hayes, Institute of Ophthalmology, University of London, Judd Street, London, WC 1 H 9 QS, England.

Figs. 1--5. Dense membrane knobs in the late embryo spinal cord

Fig. 1. Dense membrane knob on an axon protrudes between two neighbouring axons. The knob is 550 A in diameter

Fig. 2. Dense membrane knob on an axon separated from an adjacent axon by a narrow (50 A) extracellular space. The knob has a short constricted neck and is 580/~ in diameter

Fig. 3. Dense knob on a fine process in the tract neuropil of the ventrolateral cord. The process may be an axon filopodium. The knob is 500 A in diameter and clearly shows a double plasma

membrane

Fig. 4. Dense knob on an axon with microfilaments. The knob is 500 • in diameter and is 60 A from the adjacent process

Fig. 5. Dense membrane knob on a presynaptic axon. The synaptic junction is on the left. The axon on the r ight shows a slight indentat ion of its plasma membrane opposite the dense membrane knob which is 540 A in diameter. Magnification is 128000. Scale lines are 0,1 [z

A New Membrane Organelle in Neurons 105

500 to 600 A in diameter. When serial sections were available the knobs occupied one or two sections, suggesting a near spherical shape. In some 10% a dense core was separated from the cell plasma membrane by an electron lucent zone (see Figs. 2 and 3). This was particularly noticeable where the knob was sectioned in a plane parallel to that of its parent plasma membrane and therefore not in continuity with a process in the section. Most dense membrane knobs were on slight out- pushings of the normal plasma membrane (Figs. 1, 4) or had a short constricted neck joining them to their parent process (Figs. 2, 3, 5, 7, 8). However, in the tail region at stage 27 of Nieuwkoop and Faber (1956), a third were on the ends of fine processes or filopodia (Figs. 2, 3). Sections from material stained "in the block" with uranyl acetate showed that the knobs were bounded by a double leaflet of normal plasma membrane (Figs. 1 to 4) rather than by a five layered structure which has been reported in cases where vesicle extrusion may be occurring (e.g. Heuser, Katz and Miledi, 1971).

Examination of the distribution of dense membrane knobs showed that most were on processes which could be identified as axons by their microtubules, micro- filaments, elongated mitochondria and 500 A vesicles. One third were on pre- synaptic axons (Fig. 5) or those with "membrane-vesicle clusters" (see Hayes and Roberts, 1973, 1974). Only 9% were on processes identified as dendrites by their postsynaptie position, numerous dense granules (probably ribosomes), large mitochondria and infrequent microtubules and microfilaments. No dense membrane knobs were found on muscle cells or on undifferentiated ventricular cells or their processes. The knobs first appear in the cervical spinal cord in the late neurula (stage 20 of Nieuwkoop and Faber, 1956) which is also the stage when differentiat- ing neuronal processes, "membrane-vesicle clusters" and synapses first appear (see Hayes and Roberts, 1973). They were then found at all stages up to and including stage 35, the free swimming tadpole, which was the last stage examined. Another way to relate the dense membrane knobs to neural differentiation is to look at the gradient of development along the spinal cord. In a late embryo (stage 27) knobs were found on axons throughout the length of the spinal cord. They first occurred on dendrites 1300 ~z from the tip of the tail and were in- creasingly common on dendrites more rostrally so that in the anterior trunk one third were on the mainly radial dendrites and their branches (see Hayes and Roberts, 1974). In the tail region at stage 27 dense knobs were also found on neuronal processes outside the cord lying between the skin and the myotomes. These processes arose from the dorsolateral tract neuropil of the spinal cord and were probably therefore sensory. Membrane knobs have also been found on pre- synaptie profiles at neuromuscular junctions (Fig. 6). Very rarely dense membrane knobs were found on the interdigitating processes of cells in the deep layer of the skin.

There was no clear relationship between dense membrane knobs and other intracellular organelles or other cell plasma membranes. In general the knobs faced into wide extracellular spaces, quite often into the space between two nearby processes. Sometimes the space between the knob and neighbouring pro- cesses was quite narrow as in Figs. 2 and 4. Rarely there was some sign of speciali- sation in the nearby plasma membrane of another cell as in the examples of Fig. 5 and 6 where the knob is indented into the plasma membrane.

106 A. Roberts and B. P. Hayes

:Figs. 6--8. Dense membrane knobs in the late embryo

A New Membrane Organelle in Neurons 107

Fig. 6. Dense membrane knob (arrowed) on a motor end plate forming a synaptic junction with a mid-trunk myotome at stage 27. The knob is 500 A in diameter. An indentation can be

seen in the muscle plasma membrane opposite the knob. Magnification is 40000

Fig. 7. Part of the tract neuropil in the lateral, mid-trunk spinal cord at stage 27. Most of the processes are axons running along the spinal cord but some are the external processes of ventricular cells (v). Two dense knobs occur on a large process, probably an axon varicosity or growth cone, immediately beneath the basal lamina (asterisk) at the outer surface of the cord. A dense knob on an axon varicosity can be seen at the bottom left and three dense knobs with their parent processes out of the section at the bottom right. Dense knobs are arrowed.

Magnification is 25000

Fig. 8. Two dense knobs on an axon varicosity in the tract neuropil of the domolateral spinal cord. The knobs are 550 to 600 A in diameter. Magnification is 102000. Scale lines are 0.1

Dense membrane knobs appear to be a feature of cell processes which are growing and establishing s t ructural relationships with other cells. They consist of an outpushing of the plasma membrane of the cell with a slight increase in the densi ty of the membrane in this region. Wi th in the outpushing the cytoplasm is also more electron dense bu t no structures can be resolved .The knobs are usual ly 500 to 600/~ in diameter, near ly spherical and can be on a variable length of stalk. They are found on axons and dendrites of nerve cells and rarely on the processes of skin cells. I t seems unl ikely t ha t these dense knobs are a product of our pre- parat ive techniques. The presence of other fine filopodia which lack dense knobs argues against their being a result of shrinkage of fine processes. However i t is possible tha t a relationship of the knobs with other cell membranes could have been upset dur ing fixation. We can only guess about their function. I t is possible tha t the knobs could serve as points of adhesion for growing processes. Alterna- t ively, there may be no art ifact in their separat ion from other profiles and they could be sites for membrane proliferation or reabsorption. There is no evidence for a funct ion a t present so we must wait for more information. We can find no pictures of similar s tructures in developing neural tissue in chick or mammals (e.g. Bunge, 1973; Skoff and Hamburger , 1974; Tennyson, 1970) bu t one of Lentz ' (1967) figures of the regenerating newt l imb shows an axon with what may be dense membrane knobs. There is a certain resemblance between the dense knobs and some virus particles bu t the la t ter t end to be larger, to have a denser and more s t ructured core, and to be concentra ted at the cell body rather t han be confined to cell processes.

Acknowledgements. This work was supported by a grant from the Medical Research Council. We are grateful to Drs. A. R. Lieberman and K. E. Webster for their comments on drafts of the manuscript.

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108 A. Roberts and B. P. Hayes

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