The formation of the sensory neurite plexus on the basal lamina of trunk skin in Xenopus embryos has been examined using the scanning electron microscope. It is formed by Rohon-Beard and extramedullary neurons which provide the first sensory innervation of the skin. By observing the distribution of growth cones on the inside surface of the skin of embryos at different ages, the development of the plexus has been followed and related to the development of sensitivity to sensory stimulation. The general features of the plexus are illustrated using a photomontage taken at x 1100. Measurements on neurites from this, and of growth cone orientations demonstrate a general ventral growth pattern with some small regional variations. Interactions of neurites within the plexus are examined. Neurites meeting at shallow angles tend to fasciculate, while those meeting at close to 90° tend to cross each other. Angles of incidence and separation of neurites show few angles less than 30°, which suggests that active adjustments occur after a growth cone meets or leaves another neurite. The observations allow comparison of behaviour of growing neurites in vivo and in vitro. Our evidence suggests that adhesion between growth cones and neurites is stronger than that between growth cones and the basal lamina of the skin.

Factors affecting the growth of peripheral neurites in vivo have been studied by Harrison (1910) and Spiedel (1932, 1935). Constant reference to these two classical papers on amphibians confirms their stature and the general lack of information on the initial stages of growth of nerve pathways in whole embryos. This contrasts with a wealth of information on cultured neurons (reviewed recently by Johnston & Wessells (1980)). We have used the scanning electron microscope to study the first establishment of innervation fields in Xenopus laevis embryos (Davies, Kitson & Roberts, in preparation ; Taylor & Roberts, in preparation). In this paper we examine the growth of the network, or plexus of neurites formed on the inside surface of trunk skin by Rohon-Beard and extramedullary cells (Harrison, 1910; Hughes, 1957; Roberts & Hayes, 1977). While most of Spiedel’s observations were on regenerating neurites in the tails of older tadpoles, our results concern the first establishment of sensory innervation of the trunk skin by unmyelinated neurites. We have been concerned throughout to evaluate factors which might influence the direction of growth of these neurites and to obtain observations which would allow comparison of our in vivo preparation with in vitro results. A preliminary note has been published in which the form of Xenopus sensory neurite growth cones is described (Roberts, 1976).

Embryos from stages 22 to 32 (Nieuwkoop & Faber, 1956) were removed from their egg membranes and fixed in 4 % glutaraldehyde in 0·5 M cacodylate buffer at pH 7·3 for 1-2 h. After fixation they were dissected in buffer using fine tungsten needles. The skin was removed in a single sheet from each side of the body. After dehydration in ethanol, specimens in dry acetone were critical-point dried using CO2, mounted on stubs and sputter coated with gold. The skinned trunk and inside surface of the skin were then examined in a Cambridge S4 stereoscan.

Figs. 1 and 2 show examples of the trunk and skin pieces at low magnification. Both surfaces were examined at x 1100 or more to look for neurites and growth cones. The preservation was generally good for the neuronal tissue. Some tearing of neurites was noticed and was clear, since neurites were seen detached from their substrate. Similar detachment of growth cone processes was also seen occasionally (e.g. Fig. 7). Pitting of the basal lamina of the skin is probably a preservation artifact.

Figs. 1 and 2

general view of trunk and peeled skin. Fig. 1. Lateral view of the rostral trunk of a stage-27 embryo after removal of the skin. Dorsal up, rostral to the right, x 100.

Figs. 1 and 2

general view of trunk and peeled skin. Fig. 1. Lateral view of the rostral trunk of a stage-27 embryo after removal of the skin. Dorsal up, rostral to the right, x 100.

Fig. 2.

Inner surface of a skin piece removed from the rostral trunk of a stage-27 embryo. Dorsal up, rostral to the left, x 100.

Fig. 2.

Inner surface of a skin piece removed from the rostral trunk of a stage-27 embryo. Dorsal up, rostral to the left, x 100.

Measurements were made from photographic prints taken with the long axis of the embryo horizontal on the microscope screen. Angles were measured to ±5° with a protractor. For one animal a photomontage of 173 pictures at x 1100 was made of the trunk skin (Fig. 6). The results are based on examination of over 25 embryos.

General pattern of development

Examination of the inside surface of the trunk skin showed that neurites and growth cones first appear over the myotome region at stage 24 or 25. These neurites belong to Rohon-Beard or extramedullary cells and arise from the dorsal spinal cord (Roberts & Clarke, 1982; Taylor & Roberts, in preparation). By mapping the distribution of the growth cones of these neurites at sequential embryonic stages, the development of the neurite network under the skin was followed (Fig. 3). After their first appearance over rostral myotomes, growth cones were found more caudally, and ventrally as development proceeded. They had reached the tail and ventral belly by stage 29 to 30. Outgrowth of neurites to the skin is not synchronous at any one location. As the pioneers grow away from the spinal cord later growth cones appear dorsally over the myotomes. The number of growth cones is increased by these additions to reach a maximum at about stage 26 (inset in Fig. 3). By stage 29 there are no growth cones over the myotomes and the total number has dropped. Outgrowth of neurites from the cord must stop just before this stage.

Fig. 3.

Distribution of growth cones at different stages (left-hand diagrams) in relation to sensitivity to stimulation (right-hand diagrams). At each stage large dots indicate the distribution of growth cones. Diagrams of stages 24, 26 and 28 include data from more than one skin specimen. The graph plots the number of growth cones on skin specimens at different stages. Stippled areas in right-hand diagrams show areas of skin where gentle mechanical stimulation evokes movements (based on Fig. 5 in Roberts & Smyth (1974)). Note that data were not available for the skin of the tail region.

Fig. 3.

Distribution of growth cones at different stages (left-hand diagrams) in relation to sensitivity to stimulation (right-hand diagrams). At each stage large dots indicate the distribution of growth cones. Diagrams of stages 24, 26 and 28 include data from more than one skin specimen. The graph plots the number of growth cones on skin specimens at different stages. Stippled areas in right-hand diagrams show areas of skin where gentle mechanical stimulation evokes movements (based on Fig. 5 in Roberts & Smyth (1974)). Note that data were not available for the skin of the tail region.

Between stages 24 and 28 the ventral border of the growth cones in the mid trunk moves about 500 μm. These stages are 6 h apart, indicating a growth rate of about 80 μm per h at 22-24 °C. A comparison of the distribution of growth cones and the areas of the body which when stimulated mechanically will initiate movements (Fig. 3) shows that there is a marked delay of 3-5 h between the arrival of growth cones under the skin and the establishment of sensory function.

The details of the neurite network formed on the basal lamina of the skin can be seen at magnifications from 1000 to 2500 (Figs 4 and 5). As Harrison (1910) noted, neurites branch, cross each other, fasciculate and separate again to form a dense plexus. A general view of one side of one embryo at stage 26/27 was obtained by making a photomontage of pictures at × 1100 and tracing the neurites revealed (Fig. 6). Since the neurites arise dorsally in the spinal cord and most of the skin to be innervated lies ventral to the cord, it is clear that growth must be primarily ventral in orientation. However, while showing such a general trend the map (Fig. 6) also shows the complexity of the plexus. Neurites lie in many orientations and there may be regional differences. Measures of orientation will be considered in the next section.

Fig. 4 and 5.

The plexus on the basal lamina. Fig. 4. Examples of fasciculation and crossing of neurites at stage 29/30. There is no branching. A growth cone is arrowed, x 2500.

Fig. 4 and 5.

The plexus on the basal lamina. Fig. 4. Examples of fasciculation and crossing of neurites at stage 29/30. There is no branching. A growth cone is arrowed, x 2500.

Fig. 5.

Branching (arrowhead), fasciculation and crossing of neurites at stage 28. x2300.

Fig. 5.

Branching (arrowhead), fasciculation and crossing of neurites at stage 28. x2300.

Fig. 6.

Neurite plexus on the basal lamina of trunk skin of a stage 26-27 embryo. The location of the map of the plexus is shown on the diagram of the embryo. The map is a reduced tracing from a photomontage. The dorsal and ventral edges of the myotomes (dotted) were clear from indentations in the skin. The most rostral myotome is the second post-otic. Further details in the text. Scale bar 300μm.

Fig. 6.

Neurite plexus on the basal lamina of trunk skin of a stage 26-27 embryo. The location of the map of the plexus is shown on the diagram of the embryo. The map is a reduced tracing from a photomontage. The dorsal and ventral edges of the myotomes (dotted) were clear from indentations in the skin. The most rostral myotome is the second post-otic. Further details in the text. Scale bar 300μm.

On the basal lamina the main structural feature that neurites encounter is other neurites. Figure 6 shows that features such as the indentations formed in the skin by the myotomes do not noticeably affect the course of the neurites. However, changes in the direction of growth results after fasciculation, separation of fasciculated neurites or branching (Figs. 4, 5). Factors which influence branching remain unclear, but fasciculation appears to occur when neurites approach at shallow angles. When they approach at right angles they usually cross each other (Wessells et al. 1980). That these patterns seen in established parts of the plexus are primarily a result of the behaviour of growth cones rather than later adjustment can be seen by examining the growing ends of neurites. When growth cones approach a neurite at nearly 90° they usually cross (Figs. 7, 8) but if they make contact at shallower angles (Figs. 8, 9 and 10) fasciculation results. Many observations of this type suggest that the layout of the plexus is established by the way it grows. Later adjustment of the positions of neurites is unlikely to be extensive since they appear to be anchored to the basal lamina by many small attachments (see also Roberts, 1976). Measurements on the occurrence of fasciculation are reported below.

Fig. 7-10.

Growth cones interacting with neurites on the basal lamina. Fig. 7. Example of a growth cone which meets a neurite at nearly 90° and crosses, rather than fasciculating. Note mutual attachment as growth cone crosses neurite at a varicosity, and some damaged micropodia on growth cone (arrowhead), x 2220.

Fig. 7-10.

Growth cones interacting with neurites on the basal lamina. Fig. 7. Example of a growth cone which meets a neurite at nearly 90° and crosses, rather than fasciculating. Note mutual attachment as growth cone crosses neurite at a varicosity, and some damaged micropodia on growth cone (arrowhead), x 2220.

Fig. 8.

Example showing crossing and fasciculation. Two neurites (top middle) fasciculate and then separate. A neurite from top left shows a simple growth cone with one micropodium extending along the contacted neurite. This would probably lead to fasciculation. The right-hand growth cone meets at 90° and crosses, x 2220.

Fig. 8.

Example showing crossing and fasciculation. Two neurites (top middle) fasciculate and then separate. A neurite from top left shows a simple growth cone with one micropodium extending along the contacted neurite. This would probably lead to fasciculation. The right-hand growth cone meets at 90° and crosses, x 2220.

Fig. 9.

Two growth cones. The upper one meets at a shallow angle and will probably fasciculate. Note attachment of neurites to substrate. * indicates artifact, x 2220.

Fig. 9.

Two growth cones. The upper one meets at a shallow angle and will probably fasciculate. Note attachment of neurites to substrate. * indicates artifact, x 2220.

Fig. 10.

Growth cone fasciculating with a fine neurite (arrowhead) which it appears to pull towards itself, x 2220.

Fig. 10.

Growth cone fasciculating with a fine neurite (arrowhead) which it appears to pull towards itself, x 2220.

Orientation of growth

In analysing orientation, measures were first made on growth cones. Since the plexus map (Fig. 6) suggested that there could be regional variation in orientation, growth cone angles were related to position on the trunk (Fig. 11). These plots show an overall ventral orientation of the growing tips of the neurites (306 angles, mean 186°). The angles are significantly non-uniform in distribution (probability of uniformity less than 0·1 % by Rayleigh’s test). The V1 test (Durand & Greenwood, 1958) also gives a better than 0·1 % probability in testing for closeness to an a priori angle of 180°. All but one of the dorsally oriented growth cones in the dorsal regions were in embryos older than stage 26. These growth cones also have a characteristic morphology (Roberts & Taylor, in preparation). Over the ventral myotomes and belly skin growth is broadly ventral. No rostral or caudal tendencies are clear.

Fig. 11.

Orientation of growth cones at stages 24-33. Polar plots showing numbers of growth cones in each 10° segment. The trunk skin was divided into three equal longitudinal regions. Each of these was subdivided into dorsal (fin and dorsal myotome), mid (ventral myotome and dorsal belly), and ventral (belly) skin giving nine regions. The shortest segment length is one growth cone. 0° dorsal ; 90° caudal ; 180° ventral; 270° rostral.

Fig. 11.

Orientation of growth cones at stages 24-33. Polar plots showing numbers of growth cones in each 10° segment. The trunk skin was divided into three equal longitudinal regions. Each of these was subdivided into dorsal (fin and dorsal myotome), mid (ventral myotome and dorsal belly), and ventral (belly) skin giving nine regions. The shortest segment length is one growth cone. 0° dorsal ; 90° caudal ; 180° ventral; 270° rostral.

Orientation of neurites in the plexus was examined for the neurite map of Fig. 6. A regular 10 cm grid was drawn on the original map at x 1100 magnification. An 8 cm diameter sampling circle was centred in turn on each junction of the grid. The number of neurites crossing 1 cm spaced parallel lines in the circle was counted : with the lines longitudinal (XA) ; turned 45° clockwise (XB), and turned 45° anticlockwise (Xc) (see Fig. 6). The following ratios were then calculated :
It was assumed, on the basis of the growth cone orientation measures, that growth in the midmyotome and dorsal belly regions was in a broadly ventral direction. These ratios are related to longitudinal position in Fig. 12. The density of neurites falls caudally, making the orientation measure insignificant behind the 15th myotome. Rostral to the 6th myotome there is a significant rostral orientation of neurites (Binomial test 99 % confidence). Caudal to the 15th myotome neurites may have a longitudinal tendency, but rostral to this the ventral orientation is clear (Biomial test 99 % confidence).
Fig. 12.

Measures of orientation and density of neurites as a function of longitudinal position. Samples were taken at mid-myotome level (closed circles) and dorsal belly level (open circles). Caudal to the 19th post-otic myotome segmentation (S) is in progress. Further details in the text. Stages 26-27.

Fig. 12.

Measures of orientation and density of neurites as a function of longitudinal position. Samples were taken at mid-myotome level (closed circles) and dorsal belly level (open circles). Caudal to the 19th post-otic myotome segmentation (S) is in progress. Further details in the text. Stages 26-27.

Neurite interactions

The neurite map (Fig. 6) was used to evaluate the behaviour of neurites when they meet and separate. Random behaviour would suggest weak interactions, but our qualitative observations led us to expect mutual attraction between neurites and growth cones. The direction of growth must be known to allow distinction of incidence from separation, so we have only used the area of skin ventral to the dorsal edge of the myotomes where the neurites arise (see Fig. 6 and Taylor & Roberts, in preparation). Even in this area measurements were excluded if there was any uncertainty about direction of growth. Angles of neurite incidence and separation were measured (Fig. 13) and after incidence, the number of neurites crossing or fasciculating was recorded (Fig. 14).

Fig. 13.

Distribution of angles of incidence and separation of neurites. For incidence N = 68 and angles over 90° were excluded because of possible ambiguity about the direction of growth. For separation N = 138, S.D. = 21·6, Mean 68°.

Fig. 13.

Distribution of angles of incidence and separation of neurites. For incidence N = 68 and angles over 90° were excluded because of possible ambiguity about the direction of growth. For separation N = 138, S.D. = 21·6, Mean 68°.

Fig. 14.

Relationship between the angle of incidence of two neurites and whether the neurites fasciculate or cross. The numbers of measurements at each angle are indicated. The line through the points is based on a weighted linear regression analysis (slope 0·9128 ±0·182, intercept 0·6116 ±10·4, r = 77·5%,F(l,66) = 99.12, N = 68) and the dashed lines indicate the 95 % confidence limits.

Fig. 14.

Relationship between the angle of incidence of two neurites and whether the neurites fasciculate or cross. The numbers of measurements at each angle are indicated. The line through the points is based on a weighted linear regression analysis (slope 0·9128 ±0·182, intercept 0·6116 ±10·4, r = 77·5%,F(l,66) = 99.12, N = 68) and the dashed lines indicate the 95 % confidence limits.

If there was no active interaction between neurites one might expect angles of incidence to be randomly distributed, and Figure 13 shows that from 30° to 90° this seems a reasonable expectation (x2 test). However, there is a significant lack of shallow angles of incidence, which is particularly surprising as we have shown that the neurites show a general ventral orientation. This might have resulted in more, rather than less shallow angles of incidence. Active interaction of neurites is therefore implicated. After meeting, the data of Fig. 14 shows that shallow angles of incidence lead to fasciculation while steeper angles result in neurites crossing. The final measurements were on angles of separation of neurites. The majority of cases were defasciculations but branching was nqt excluded, since in both cases neurites have to separate. The mean angle of separation was 68° and the angles were normally distributed with few angles less than 30° or greater than 110° (Fig. 13). Again, the absence of shallow angles suggests active interaction between neurites.

The present SEM observations on fixed preparations of normal embryos show neurite structure and behaviour very similar to that seen in earlier studies on cultured frog neurites (Harrison, 1910) and on regenerating unmyelinated neurites in older tree frog larvae (Spiedel, 1933). Later studies on a wide range of cultured neurons show strikingly similar behaviour in the growing neurites. These similarities can give us confidence in all these methods of observation. The rate of neurite growth (80 μm h-1 at 22-24 °C) in the Xenopus embryos is faster than in the other amphibians (up to 56 μm h-1 (Harrison, 1910) ; 30-40 μm h-1 (Spiedel, 1932)). This could result partly from expansion of skin behind growth cones. The delay between arrival of growth cones under the skin and the establishment of sensory function (3-5 h) seems long. At present we have no satisfactory method to follow the growth between the skin cells. This problem also makes factors influencing branching hard to assess since many branches go through the basal lamina into the skin and would be hard to detect with our SEM technique. It is in fact difficult to distinguish between branching and the separation of two intimately joined neurites (Fig. 5).

Orientation

It seems likely that the early overall ventral orientation of growth results from the way the neurites emerge from the spinal cord and then grow over the myotomes whose curvature directs the neurites ventrally (Taylor & Roberts, in preparation). The later phase of dorsal growth (Fig. 11 and Roberts & Taylor in preparation) could depend on the changed morphology of the cord, myotomes and skin by stage 27, but more observations are required to resolve this. Small regional differences in orientation could result from towing of neurites by relative movements of the skin and myotomes. The neurites on the basal lamina of the skin have a general ventral orientation but this is far from strict, suggesting fairly weak determining factors rather than a single, powerful orienting force. It seems plausible at present to suggest that the innervation pattern (Roberts & Hayes, 1977) is determined by gross morphological features coupled with the fact that each sensory Rohon-Beard cell has some limit to the area of skin that it can grow to innervate.

Interaction, of neurites

The factors which determine whether neurites cross or fasciculate when they meet has been a major concern in this study. It has allowed close comparisons to be made between in vivo and in vitro observations. Before attempting to interpret our observations we need to consider the relevant properties of the neurites and growth cones (reviewed by Johnston & Wessels, 1980).

(1) Growth cones and neurites in some way adhere to the substrate and in the case of neurites on the basal lamina this may be by small branchlets (Figs. 7-10; Roberts, 1976).

(2) Behind growth cones the neurite can remain active, still capable of extending filopodia for some distance (Figs. 7-10; Roberts, 1976; Roberts & Taylor, in preparation).

(3) Growth cones grow over each other and other neurites to which they appear to adhere (Harrison, 1910; Spiedel, 1933; Nakai, 1960; Nakajima, 1965; Roberts, 1976; Wessells et al. 1980, Figs. 7-10). In Xenopus we suggest that other growth cones and neurites are more adhesive than the basal lamina.

(4) Growth cones, their processes and nearby neurites generate tension between points which are anchored by adhesion (Bray, 1979; Nakai, 1960).

On the basis of this list of properties or assumptions, we can now interpret our observations on neurite interactions.

Crossing occurs more as the angle of incidence approaches normality (Fig. 13) At normal incidence the neurite provides a narrow area of greater adhesion equally distributed on either side of the advancing growth cone. Tension will be equally distributed and there is, therefore, no tendency for the growth cone to change course and fasciculate. Fasciculation occurs with shallower angles of incidence as the adhesive surface of the neurite becomes more unequal in its effect on the growth cone. Filopodia will extend preferentially along the adhesive surface of the neurite (C. F. Letourneau, 1975). Fasciculation can occur either on initial contact of the growth cone and neurite, or the growth cone can cross and then be pulled back to fasciculate by filopodia contacting the neurite after crossing. Two processes could contribute to the absence of shallow angles of incidence of fasciculating neurites (Fig. 14). If the neurite is not firmly anchored at the point where it is contacted, the growth cone could pull on it. After contact the active region behind the growth cone could pull further, zipping-up the two neurites into closer contact (Fig. 15). Such processes may be occurring in Figs. 9 and 10 and have been illustrated by Nakai (1960) and Spiedel (1933) who also shows that crossings of neurites can be transformed into short fasciculations by the second of these two processes.

Fig. 15.

Interactions of neurites and growth cones, (a) Fasciculation: (1) contact, (2) growing neurite pulls static neurite, (3) area of attachment is increased to change apparent site of incidence and to widen the angle of incidence, (b) Separation of a growth cone which then (3) pulls the neurite to increase the apparent angle of separation. Points of attachment are shown as little branchlets.

Fig. 15.

Interactions of neurites and growth cones, (a) Fasciculation: (1) contact, (2) growing neurite pulls static neurite, (3) area of attachment is increased to change apparent site of incidence and to widen the angle of incidence, (b) Separation of a growth cone which then (3) pulls the neurite to increase the apparent angle of separation. Points of attachment are shown as little branchlets.

Separation of neurites implies that the adhesion offered by the basal lamina is not much less than that of the neurite surface. Clearly the available area of basal lamina is larger, so if a sufficient number of growth cone processes contact the basal lamina, the growth cone can escape provided the angle of separation is sufficient. As in the case of incidence, angles of separation can be increased by pulling (Fig. 15 b) and zipping-up of neurites.

It seems that differential adhesion could, therefore, account for most of Our observations. Rutishauser, Gall & Edelman (1978) have shown in vitro that chick ganglion cells fasciculate less when cultures are on a more adhesive substrate. It will be of interest to see whether, in more natural, whole embryo situations, the degree of fasciculation is dependent on (a) the adhesion of the substrate, (b) the shape of the substrate (a narrow tube would provide less chance Of a growth cone escaping fasciculation), (c) numbers or density of neurites (more neurites offer a larger adhesive surface). Perhaps these factors alone provide sufficient variables to account for the variety of behaviour seen in growing neurites, like those considered here which come from Rohon-Beard and extramedullary neurons. Centrally, these form a compact dorsal sensory tract (Roberts & Clarke, 1982). On emerging from the cord their neurites form bundles which when they reach the skin break up to form the plexus we have described (Taylor & Roberts, in preparation). Such apparently complex behaviour could be based on a few limited rules. For example, Katz & Lasek (1979) have described how retinal ganglion cell axons from eye primordia transplanted to the tail of Xenopus embryos grow axons to ascend in the spinal sensory tracts to the brain. This could be entirely dependent on the ganglion cell axons fasciculating with Rohon-Beard neurites which they then followed back to the spinal cord and up to the brain.

We thank: L. Balch for making the photomontage of the plexus; B. Porter, S. Martin and K. S. Williams for technical assistance; Drs J. Rayner and G. M. Jarman for statistical advice; Professor P. Haggett and N. French for discussions on measurements; the MRC for financial support.

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