/. Embryol. exp. Morph. 86, 53-70 (1985)
Printed in Great Britain. © Company of Biologists Limited 1985
53
In vitro analysis of interactions between sensory
neurons and skin: evidence for selective innervation of
dermis and epidermis
J.-M. VERNA
Groupe de Neurobiologie du Developpement, U.A-C.N.R.S n°682, Laboratoire
de Zoologie et Biologie Animate, Universite Scientifique et Medicate de Grenoble,
B.P. 68, 38402 St Martin D'Heres Cedex, France
SUMMARY
Axons from dorsal root ganglion cells cultured in a serum-free medium on poly-L-lysine or
collagen substrates interact differently with dermis and epidermis. The orientation of neurite
growth is not changed by encountering mesenchymal cells migrating from the outgrowth zone of
a dermal explant, and neurites form close membrane associations with some dermal cells; in
contrast, neurites strongly avoid epidermis and deviate around the edge of an epidermal explant.
When cultures are grown on polylysine this avoidance behaviour occurs at a distance from the
epidermis. It is suppressed in the presence of necrotic epidermal cells. We suggest that this
avoidance is due to epidermal diffusible factor(s) which bind preferentially to polylysine. The
possibility of an absence of specific recognition cues between neurites and epidermal cells is
discussed.
INTRODUCTION
The importance of interactions with the local environment for the extension and
guidance of nerve fibres during neurogenesis has been demonstrated in vivo as well
as in vitro (see Goodman, Raper, Ho & Chang, 1982; Johnston & Wessells, 1980;
Katz & Lasek, 1980, for reviews). In particular, many in vitro studies have employed neurons from the peripheral nervous system to try to understand the part
played by target tissues in nerve growth control (Bonhoeffer & Huf, 1980; Ebendal,
1981; Ebendal & Jacobson, 1977; Pollack, Muhlach & Liebig, 1981 . . . ) .
Few investigations, however, have been dedicated to the detailed analysis of
interactions between nerve fibres and skin cells in vitro (Andres & Van der Loos,
1983; Ebendal, 1977; Lumsden & Davies, 1983; Verna & Saxod, 1979a). Although
conclusions derived from the artificial environment of tissue culture necessarily
remain questionable, in vitro studies may provide suggestive glimpses of events
occurring during the patterning of cutaneous nerves and the development of sensory receptors. In birds, for instance, the nature of the morphogenetic interactions
between sensory nerve fibres and mesenchymal cells which lead to the formation
of sensory end organs (such as Herbst corpuscule; see Saxod, 1978 for a review) and
Keywords: Chick embryo, dorsal root ganglia, sensory innervation, skin.
54
J.-M. VERNA
the origin and significance of the scarce intraepidermal innervation are still unknown.
In order to extend previous studies (Saxod & Mauger, 1976; Verna & Saxod, 19796)
on the development and regulation of the pattern of bird cutaneous nerves we have
analysed the interactions between dorsal root sensory neurons and cutaneous cells
from chick embryos in serum-free cocultures. We used serum-free supplemented
medium in order to reduce the heavy outgrowth of non-neuronal cells of the dorsal
root ganglion which hinders quantitative estimations of nerve fibre growth, and to
prevent modifications in the interactions between the cultivated cells due to binding of
serum factors on the cell surface. Wefindthat neurons interact differently with dermal
than with epidermal cells. While nerve fibres readily extend over dermal cells, forming close membrane associations with some of them, they demonstrate a strong
avoidance reaction with epidermal cells by changing their direction of extension. A
brief preliminary note on these results was recently published (Verna & Saxod, 1983).
MATERIALS AND METHODS
Materials
L15 culture medium and soybean trypsin inhibitor were purchased from Boehringer Mannheim
France, Nerve Growth Factor from Laref (Switzerland); transferrin and insulin from
Collaborative Research, Inc. (U.S.A.); progesterone, putrescine and seleneous acid from Fluka
AG (Switzerland); and poly-L-lysine HBr (MT150000-300000) from Sigma.
Cocultures
Lumbosacral dorsal root sensory ganglia (DRG) and dorsal skin were dissected from 7-day
chick embryos.
Skin was suspended for 15 mins at 4°C in Ca2+-Mg2+-free PBS containing 0-5 % trypsin, then
separated into dermis and epidermis. These tissues were rinsed in Ca2+-Mg2+-free PBS and
allowed to stand for 5 mins in L15 medium containing 0-33 % trypsin inhibitor.
Using fine forceps, each tissue was dissociated into small pieces (lmm diameter). Seven to
eight tissue fragments of either dermis or epidermis were cultured with the same number of DRG
fragments in such a way that each explant of cutaneous tissue was adjacent to two DRG at a
distance of about 1 mm. In order to avoid disturbance in the positioning of the explants, a piece
of dialysis cellophane was laid over them for the first 24 h (Verna & Saxod, 1979a).
The serum-free supplemented culture medium was prepared according to Bottenstein,
Skaper, Varon & Sato (1980) and consisted of: L15 medium (x 1); glucose (6mg/ml); penicillin
(125i.u./ml); N.G.F. (10/ig/ml); transferrin (5/ig/ml); insulin (5/ig/ml); progesterone
(6-3ng/ml); putrescine (8-8jug/ml) and seleneous acid (4ng/ml).
35 mm tissue culture dishes (Falcon) were either coated with a thin layer of soluble bovine skin
collagen (gift from theC.E.R. A. D.,Lyon, France) or exposed to a poly-L-lysine solution(10jUg/ml
in distilled water). Collagen coating was achieved by spreading one drop of a 0-5 mg/ml collagen
solution in distilled water on to each dish and allowing it to dry for 2-3 h. Culture dishes were
exposed for 3 h to the poly-L-lysine solution and rinsed with L15 medium just before use.
The cultures were incubated at 37 °C in a humidified atmosphere and maintained for 1 to 10
days without change of medium.
Time-lapse analysis
Time-lapse cinemicrography was used to analyse neurite behaviour and growth. For this purpose, selected areas of the cultures were observed with a Leitz Diavert inverted microscope and
Interaction between chick sensory neurons and skin
55
filmed at one frame every 5-15 minutes with a 16 mm Bolex camera using Codex pan rapid film.
Recordings were made from the 1st until the 4th day of culture. Cinematographic records were
projected, frame by frame, on an x-y coordinate digitizing tablet (Hipad) connected to an Apple
III microcomputer. The coordinates of successive positions of the growth cone were recorded
with the aid of the tablet electronic pen, and stored in the computer. These data were then
processed (using a computer program written by Y. Usson) to calculate various parameters of the
neuritic growth.
Two velocity components were used to describe the rate of neurite extension. The instantaneous velocity (Vi) is defined as the average of velocities recorded between each two
consecutive positions of the growth cone
V i - C j , Cj =
Cj, velocity between two consecutive positions (Xj, Xj + 1) of the growth cone; d, distance
between these two positions; t, time interval between 2 successive measurements.
The mean velocity (Vm) represents the velocity between the first and last positions of the
growth cone during the film sequence
d, distance between the first (XI) and the last position (Xn) of the growth cone; T, total
duration of the observation.
To express the directional changes in growth cone extension, a mean change in angle (Am) was
calculated as
N
Am = N " 1 2
| «i |;
i= l
a; is the angle between the directions of two successive segments, two consecutive positions
of the growth cone defining a segment; N is the number of angles.
Student's t-test and Fisher's f-test were used to compare means and variances of data respectively.
Growth parameters were calculated for a total of 122 nerve fibres in cocultures (47 with dermis
and 75 with epidermis).
Transmission electron microscopy
Cultures were quickly rinsed in a 0-1 M-phosphate buffer (pH7-4), fixed for 30mins at room
temperature in a 0-1 M-phosphate buffer containing 6-25 % glutaraldehyde and postfixed for 1 h
in 1 % osmium tetroxide in phosphate buffer. Some cultures were then stained lOmins in 0-3 %
tannic acid (according to Rees, 1978).
Areas of particular interest were selected, cut and stained with uranyl acetate and lead citrate.
RESULTS
1) Development of cutaneous tissues in L15 serum-free supplemented medium
a) Dermis
This medium formulation, as reported for the non-neuronal cells of peripheral
ganglia (Bottenstein etal. 1980) strongly reduces the proliferation and migration of
dermal cells. Consequently, despite a good survival of these cells after 10 days of
culture, confluency is never reached and the singly migrating cells remain close to
the e^plant periphery (Fig. 1). The cells display a fibroblastic morphology, with
56
J . - M . VERNA
4r*
•
Figs 1-3
Interaction between chick sensory neurons and skin
57
a flat triangular or polygonal shape on both culture substrates. A well-developed
endoplasmic reticulum, microtubules and bundles of submembraneous microfilaments are common features of their cytoplasm. Cell migration is characterized by
short-range translocation periods separated by stationary phases during which the
dermal cells nevertheless exhibit an intense protusive activity.
b) Epidermis
During the first days of culture, the lack of serum factors in the medium does not
hinder the survival and development of the epidermis. By day 4, however, some
cells begin to die and a rapid increase in rate of cell death leads to almost complete
necrosis of the explant after 8 days of culture.
After plating, epidermal explants attach quickly and spread on the culture substrate. The cells are tightly associated and migrate in sheets (Fig. 2). The spreading
and the motile activity of epidermis is far greater on collagen than on poly-L-lysine.
Consequently, on collagen the epidermal sheet is large and rarely consists of more
than two cell layers. On the other hand, epidermis grown on poly-L-lysine is thicker
(three or more cell layers), in particular on the edge of the sheet, and often contracts
as the cultures age.
The uppermost layer of the epidermal sheet is made of typical peridermal cells
with numerous microvilli on the surface facing the culture medium (Fig. 3).
Keratinocytes are joined one to another by well-differentiated desmosomes. From
place to place, melanoblasts lie between keratinocytes.
2) Growth of sensory neurons in cocultures
The radially directed neurite outgrowth from spinal ganglion explants occurs
within 12 hours of culture. Extending nerve fibres may be isolated, or grouped in
bundles. Because of the serum-free culture medium, the early neuritic outgrowth
takes place without the accompanying non-neuronal cells (Bottenstein et al., 1980;
Yavin & Yavin, 1980) which, as cultures age, will migrate out from the explant.
However the population of migrating non-neuronal cells remains low throughout
Fig. 1. 7-day chick embryo culture of dermal mesenchyme. (A) 2-day culture; (B) 7-day
culture. Collagen substrate. Because of the absence of serum from the culture medium,
the outgrowth zone (star) is restricted to the close periphery of the explant (D). Phase
optics. Scale bar equals 50jum.
Fig. 2. 7-day chick embryo culture of epidermis. (A) collagen substrate. 4-day culture;
(B) poly-L-lysine substrate. 2-day culture. Epidermal cells migrate as a sheet of closely
associated cells. On collagen substrate (A), the cell sheet (star) appears as a monolayer
with flattened cells at the edge (arrow). In contrast, the epidermal sheet is thicker on
poly-L-lysine substrate (B), particularly at the edge (arrow). Phase optics. Scale bar
equals 50/zm.
Fig. 3. Transmission electron micrograph of the upper cell layers of epidermis grown
4 days on poly-L-lysine substrate, d, desmosome; mi, microvilli; p, peridermal cell.
Scale bar equals 0-
35
J . - M . VERNA
Table 1. Quantitative analysis of nerve fibre growth in the presence of dermis.
Instantaneous
velocity *
Vi±s.D.
Mean
velocity *
Vm + s.D.
Mean change
in angle *
Am±s.D.
Polylysine (n = 27)
61-4±17-0/zm/h
53-4 ± 18-4 ptm/h
34-7° ± 19-0°
Collagen (n = 20)
35-9 ± 14-1 tm/h
27-8 ± 14-7 (jm/h
51-3° ±24-2°
Significant
P<001
Significant
P<0-01
Significant
P<0-02
Culture
substrate
Difference between
the means.
Student t-test
n, number of nerve fibres.
S.D., standard deviation.
* see Materials & Methods.
Interval between successive measurements = 15min.
The duration of the period of observation varies from 150 min to 540 min depending upon how
long a given growth cone can be followed.
the period of culture and their displacement is restricted to the periphery of the
ganglion explant.
a) Cocultures with dermis
The initial orientation of neurite extension is not changed by encountering cells
migrating from the periphery of the dermal explant (Fig. 4). Transient contacts are
established between growth cones and dermal cells, and later on neurites end up in
the mass of the dermal explant. Neurites can elongate both over and under dermal
cells as well as over the culture substrate between them without significantly modifying their directions of extension.
The rate of neuritic growth was significantly greater on poly-L-lysine than on
collagen (Vi, 61-4jum/h versus 35-9jum/h; Vm, 53-4 ^m/h versus 27-8jum/h) and
the extension more directionally consistent (Am, 34-7° on polylysine versus 51-3°
on collagen) (Table 1).
We saw no significant modifications in these growth parameters before and after
nerve fibres encountered dermal cells, but as our sample of neurites was small this
observation deserves a more detailed study.
Fig. 4. Coculture of dorsal root ganglia and dermis. 2-day culture on collagen substrate.
Time interval between A and B is 3h. The initial orientation of neurite (n) extension
is not changed by encountering cells (d) migrating from the periphery of the dermal
explant. Phase optics. Scale bar equals lOO/rni.
Fig. 5. Transmission electron micrographs of a contact between a neurite growth cone
(c) and a dermal cell (d). No particular junctional specialization is formed. Coculture
of dorsal root ganglia and dermis. Collagen substrate; 3-day culture. A) scale bar equals
; B) enlargement of box in A, scale bar equals 0-
Interaction between chick sensory neurons and skin
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Figs 4-5
59
60
J.-M. VERNA
When the filopodia of a growth cone come into contact with a dermal cell, either
they withdraw within a few minutes and the neurite keeps elongating and ignores
the cell, or a closer apposition is realized between the membranes of the growth
cone and the cell. In this case, membranes run parallel for a length of 5 to 15 \xm
and are kept apart by an intercellular space of 20 nm (Fig. 5). No particular junctional specialization is observed. Slight changes of neurite trajectory can occur
when, after a contact, the neurite is dragged along by a dermal cell during its phase
of short-range translocation (Verna & Saxod, 1979a). Thus neurite-dermal cell
contact does not prevent the dermal cell migration and is strong enough to suggest
a possible role in nerve fibre growth. Moreover, it appears that these contacts are
not formed with every cell encountered and it will be of special interest to determine
whether growth cones are distinguishing a special class of dermal cells.
b) Cocultures with epidermis
When spinal ganglia and epidermal explants are cultured side by side on the
substrate, no neurites extend from the side of the ganglion touching the epidermis
(Fig. 6). Moreover, in epidermal cultures plated with a ganglion cell suspension, no
neurons attach and extend neurites on the epidermal sheet. These events occur only
on the culture substrate (Fig. 7).
In order to determine the fine growth behaviour of neurites during their encounters with epidermis, spinal ganglia explants were put 1 mm away from the epidermal
tissue. The initial radially directed outgrowth of neurites was similar to that observed in cocultures with dermis. However, when growth cones reach the close vicinity
of the epidermal sheet, nerve fibres modify their direction of growth and deviate
around the epidermis (Fig. 8). Neurites never extended over the epidermis, and the
great majority did not penetrate deeply into it. The expression of this avoidance
behaviour is particularly obvious when tissues are grown on poly-L-lysine. Comparison of data gathered from quantitative time-lapse analysis reveals a significant
decrease in the rate of neurite extension in the presence of epidermis. This is
associated with a more erratic ordering of the direction of neurite extension than
in the presence of dermis (Vi and Vm decrease from 61-4 and 53-4 jum/h respectively to 45-9 and 29-4 //m/h; Am rises from 34-7° with dermis to 58-1° with epidermis;
Table 1 and 2A). In order to determine whether these modifications were actually
due to the epidermis, growth parameters were calculated for neurites whose growth
Figs 6 to 8. Cocultures of dorsal root ganglia and epidermis. 2-day culture.
Fig. 6. No neurites (n) extend from the side of the ganglion explant (G) touching the
epidermis (£). Poly-L-lysine substrate. Darkfield.Scale bar equals 200./on.
Fig. 7. Epidermis cultured with a ganglion cell suspension. No ganglion cells (g) are
apparent on the epidermal cell sheet (£). Collagen substrate. Phase optics. Scale bar
equals 50 pm.
Fig. 8. Neurites (n) arriving in the vicinity of the epidermis (E) deviate around it. Some
neurites however contact the epidermis (arrows). Poly-L-lysine substrate. Phase optics.
Scale bar equals 100 (jm.
Interaction between chick sensory neurons and skin
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61
62
J.-M. VERNA
Table 1. Quantitative analysis of nerve fibre growth in the presence of epidermis (cocultures grown onpoly-L-lysine substrate).
A.
Neurite extension
toward epidermis
(n = 47)
B.
1. Neurite extension
(d>100ium)
(n = 20)
2. Neurite extension
Instantaneous
velocity *
Vi±s.D.
Mean
velocity *
Vm + s.D.
Mean change
in angle *
Am + s.D.
45-9 ± 20-0 |um/h
29-4±20-l]um/h
58-1° ±31-4°
56-4 ± 18-4/an/h
48-6 ± 20-2^/11
38-9° ±18-9°
41-6±22-5^m/h
Significant
(t test)
P>0-01
21-9±22-3^m/h
Significant
(t test)
P<0-01
70-4° ± 33-6°
Significant
(t test)
P<0-01
(d < lOOjum)
(n = 33)
Statistical tests:
means, Student t;
d, distance between growth cone and epidermis.
n, number of nerve fibres.
S.D., standard deviation.
* see Materials & Methods.
Interval between successive measurements = 15min.
The duration of the period of observation varies from 150 min to 540 min depending upon how
long a given growth cone can be followed.
cones were located 100/im or less from the epidermis and compared with those
calculated for neurites with growth cones further away (Table 2B). In the vicinity
of epidermis the growth of nerve fibres was slowed (Vi decreased from 56-4 to
41-6 jum/h and Vm from 48-6 to 21-9 /im/h), and the directionality of extension was
markedly altered (Am increased from 38-9° to 70-4°). These changes in neuritic
Fig. 9. Interactions between epidermal cells (E) and neurites in a 4-day coculture on
collagen substrate. 16 mm time-lapse recordings; time interval between two consecutive
frames: 30 minutes (except between E and F: 120 minutes). Neurite 1 after contacting
an epidermal cell becomes positioned on it by the movement of the cell (C to D); the
flattened morphology of the growth cone then disappears (arrow) andfinallythe neurite
withdraws (F). Neurite 2 quickly withdraws after contact with an epidermal cell (A to
C) and starts elongating again in a slightly different direction. Neurite 3 apparently turns
before contacting epidermal cells. Phase optics. Scale bar equals 25 jum.
Fig. 10. Growth of neurites (n) in the presence of necrotic epidermis (E). Epidermis
was cultivated 1 day and then killed by X-irradiation before plating with dorsal root
ganglia explants. Collagen substrate. A and B, same area: A, 3 days and B, 4 days after
plating of the ganglia. No avoidance reaction of neurites to epidermis occurs and the
necrotic epidermal explant is quickly invaded by the growing nervefibres.Phase optics.
Scale bar equal: A, 100 jum; B, 50jum.
Interaction between chick sensory neurons and skin
63
fir
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64
J . - M . VERNA
growth behaviour were seen at distances varying from 5 to 120 /im (average distance: 42 ± 4//m) from the epidermal border. A majority of neurites (65 %) stop
growing before reaching the epidermis, and either retract or exhibit successive
short-range movements of extension and retraction. In most instances, growth is
later reinitiated, but generally not towards the epidermis, so that neurites deviate
around it. Nerve fibres reaching the epidermis (35 %) immediately suspend their
further extension, and quickly withdraw to progress along the epidermal border.
Later-arriving nerve fibres fasciculate with first ('pioneering') nerve fibres giving
rise to a network of nerve fibre bundles around the sides of the epidermal explant
(Fig. 8).
Similar quantitative analysis of cocultures grown on collagen substrates has not
provided such clear evidence of significant differences in neuritic growth
parameters between cocultures with dermis and epidermis. Nor were these values
modified when neurites got closer to the epidermis. Such analyses were made
difficult by the intensive migratory activity of the epidermal sheet on collagen which
could, in part, account for the discrepancy with results obtained on poly-L-lysine.
Indeed, moving epidermal cells generally come rapidly into contact with extending
growth cones (the distance between the epidermal sheet and the neurite tip is
limited by the field of view of the microscope) and this could happen before the
neuritic growth rate could change. Nevertheless, nerve fibres coming into contact
with the epidermis grow no further. Growth cones then withdraw and no close or
long-lasting associations (such as those observed with dermal cells) are formed.
Later on, after several unfruitful attempts to maintain their forward extension and
to pass over the epidermal barrier, the growth cones are diverted along the edge of
the epidermis (Fig. 9). If the movement of the epidermal sheet results in a growth
cone being on its surface, filopodia are withdrawn, protrusive activity is abolished
and the growth cone loses its fan-shape morphology and becomes bulbous.
In order to determine whether neurite 'avoidance' behaviour requires the
presence of living epidermal cells, epidermis was precultivated 1 to-3 days, then
killed with a lethal dose of X-rays (lOOOOrads using a Secasi Tubic X-ray apparatus), and spinal ganglia were added to the culture. Epidermal cell death begins
within a few hours of X-irradiation with a progressive increase in rate. Development and survival of neurons do not appear to be affected by this necrosis as judged
by microscopic observation. However, neurite behaviour is significantly different,
and no change in the direction of extension occurs either in the vicinity of epidermis, or after contact with the remaining necrotic epidermal cells (Fig. 10).
DISCUSSION
In a previous study (Verna & Saxod, 1979), we described contacts occurring
between neurites and dermal cells cocultured in serum supplemented media and
observed that neurites can be dragged away by the mesenchymal cells. Similar
results were obtained in the present study with cells grown in serum-free medium.
Interaction between chick sensory neurons and skin
65
Furthermore, the encounter with dermal cells led neither to a change in the direction of axon growth nor to a withdrawal of the nerve fibres. Neurites commonly use
the dermal cell surface as a substrate on which to grow. The motility of growth cones
from dorsal root ganglia neurons is therefore not inhibited in vitro by contact with
dermal cells. Similar absence of contact inhibition of movement was reported by
Wessells et al. (1980) during in vitro contacts between neurites and non-neuronal
cells derived from the same ganglion, whereas Dunn (1971) and Ebendal (1976)
observed contact inhibition in their cultures. It thus appears, as recently demonstrated by Nuttall & Zinsmeister (1983) that the neuronal response varies significantly depending on the type of non-neuronal cell encountered. Although
growth cones appear to be able to adhere to any dermal cell surfaces, close association seems restricted to only a subpopulation of cells. Moreover, the resulting
cellular membrane adhesion is strong enough to resist to the tension created by the
displacement of the dermal cell. Taken together, these observations suggest a
certain degree of mutual membrane affinity between growth cone and particular
dermal cell. However, the absence of morphological criteria allowing us to distinguish these 'target' cells from their neighbours makes it difficult to comment on
the specificity of these contacts. In birds, cutaneous sensory corpuscules are exclusively located in dermis. It was shown (Saxod, 1978) that their development, and
especially that of Herbst corpuscules, requires interactions between the somatosensory nerve endings and the mesenchymal tissue. However, the exact nature of the
morphogenetic events involved in the corpuscule histogenesis still remains unknown. That at least some of the contacts observed represent some of these morphogenetic interactions has to be considered.
Ebendal (1977), in an in vivo study of spinal cord ventral root axon growth in the
chick embryo, gave evidence of morphologically similar contacts between growth
cones and surrounding mesenchymal cells. Based on these and other observations,
this author suggested that the axons might be directed by contacts with the surrounding cell surfaces. More recently, different in vivo studies (Al-Gaith & Lewis,
1982) have demonstrated, especially in the insect embryo, that some peculiar cells,
located along the neuronal pathways, might serve as 'guiding cues' to growing
neurites (Bate, 1976; Ho & Goodman, 1982; Goodman et al, 1982; Taghert,
Bastiani, Ho & Goodman, 1982; Bentley & Keshishian, 1982). It thus would be of
great interest to determine in our culture system whether such a 'guiding' role is
devoted to some dermal cells and then to study these cellular interactions more
precisely in vitro.
Many studies (Chamley, Goller & Burnstock, 1973; Chamley & Dowel, 1975;
Ebendal & Jacobson, 1977; Ebendal, Jordell-Kylberg & Soderstrom, 1978; Eranko
& Lathinen, 1978; Pollack et al., 1981; Muhlach & Pollack, 1982) on the influence
of target tissues on axonal growth, have demonstrated a preferential extension of
neurites towards target cells, so that the 'avoidance' behaviour of nerve fibres with
respect to epidermis was unexpected. Nevertheless, such behaviour has been
described in cocultures associating tissues of different parts of the nervous system
66
J.-M. VERNA
(Bray, Wood & Bunge, 1980; Ebendal, 1980; Crain & Peterson, 1982; Peterson &
Crain, 1982; Smalheiser, Peterson & Crain, 1981, 1982). In these cocultures, the
turning response of neurites generally occurs after the contact with the associated
tissues, leading to the assumption that the paucity or the absence of specific recognition cues within the inappropriate tissue explants might be responsible for the
change in the direction of axon extension. However, this hypothesis does not
explain our results, especially those from cocultures on poly-L-lysine substrate. On
this substrate, the deflection in neurite extension is, for the majority of nerve fibres,
triggered at a distance from epidermis (some of the nerve fibres contacting the
epidermal layer may extend from spinal ganglia neurons which normally provide
the intraepidermal innervation). This behaviour still occurs in cocultures of spinal
ganglia and whole skin in which nerve fibres elongate over migrating dermal cells
located at the periphery of the epidermal sheet but yet deviate around it at a
distance. The disappearance of this deviation reaction if epidermal cells were
necrotic indicates that normal metabolic activity of these cells is an important
prerequisite for the appearance of this phenomenon. A possible explanation of
these results is the production by epidermal cells of one or several substances which,
once released in the culture medium, affect neurite growth behaviour. Following
the discovery of the nerve growth factor (Levi-Montalcini & Hamburger, 1953) and
its action on nerve fibre extension, numerous studies have been dedicated to the
characterization of other substances displaying such trophic effects (Lumsden &
Davies, 1983; see Varon & Adler, 1980; Berg, 1984 for review). Although there is
still no clear demonstration, there is a growing body of evidence for the existence
of such chemotactic gradients within the developing embryo. However, these
putative substances always display a positive neurotrophic influence leading to an
oriented growth of neurites towards the source. It is thus important to investigate
further the peculiar behaviour reported in this study, and to define the mechanisms
involved in the 'avoidance' reaction.
On a collagen substrate, the absence of neurite deflection at a distance from the
epidermis might be due to the strong motility of epidermal cells, which could bring
them into contact with the growth cones well before a change in neurite behaviour
took place. However, considering the hypothesis that a substance is released from
epidermal cells, its concentration in the close vicinity of the edge of epidermis will
be lower (also, the epidermal sheet edge is much thinner on collagen than on polyL-lysine) and consequently have less effect on neurite behaviour than in cocultures
on polylysine. On the other hand, as observed for some neurite-promoting growth
factors found in conditioned media (Collins, 1978; Collins & Garrett, 1980; Adler
& Varon, 1980, 1981), such a substance may only act when bound to a peculiar
substrate and there may be a preferential binding to polylysine rather than to
collagen. In the embryo, where no polylysine is found, this substance may bind to
some non-collagenous component of the extracellular matrix.
Nerve fibres contacting the epidermis formed no close membrane adhesions with
epidermal cells, and most of the time neurites neither extended over it nor
Interaction between chick sensory neurons and skin
67
penetrated deeply within it, but progressed on the culture substrate along the edge
of the epidermal cell sheet. Similar results in the mouse system, on a collagen
substrate, have been recently obtained by E. Peterson (N. Smalheiser, personal
communication). To explain this, one can assume that:
i) in agreement with Letourneau's observations (1975, 1979), nerve fibres
preferentially remain and extend on the culture substrate because of its higher
adhesiveness (growth cone-epidermal cell adhesiveness might be decreased by
changes in the growth cone plasma membrane triggered by some epidermal
factor; see Schubert et al. 1978 who demonstrated the influence of N.G.F. on
P.C. 12 cell-substratum adhesiveness);
ii) that the lack of some adhesion molecules (such as CAM, see Edelman, 1983,
1984 for reviews) or the absence of recognition cues on the epidermal cell surface
impede the anchorage and thus the progression of growth cones.
The observations reported here give rise to many questions, in particular whether
such an 'avoidance' mechanism acts during the development of sensory cutaneous
innervation in vivo, or is due in vitro to a peculiar state of epidermal cells in
response to serum-free culture conditions. In this latter case, epidermal cells may
then release some chemical agents responsible for the 'avoidance' reaction observed. Nevertheless, the ultrastructural morphology of epidermal cells closely
resembled that described in the epidermis of 7-day chick embryos by Mottet &
Jensen, 1968. Moreover, the behaviour and the morphology of these cells do not
profoundly differ, at least during the first four days of culture, from those occurring
in serum medium (in which the nerve fibres still deviate around the epidermis;
Verna, unpublished data) and in various other culture conditions (for a review, see
Holbrook & Hennings, 1983).
In considering the possible role of this phenomenon in the normal development
of bird cutaneous innervation, it should be noted that birds are peculiar among
vertebrates with respect to cutaneous innervation in that: i) 'Merkel corpuscules'
are found exclusively in the dermis and ii) intraepidermal nerve fibres giving rise
to free nerve endings (most of them, if not all, being cold receptors) are not very
numerous (see Saxod, 1978 for a review). In this latter case, what impedes the
penetration of nerve fibres into the epidermis? This could be due to the presence
of a mechanical barrier (such as the basement membrane); to the absence of
putative target cells in the epidermis (such as Merkel cells, supposed to act as
targets for arriving nerves, Scott, Cooper & Diamond, 1981); or, finally, to some
'repulsion' effect triggered by contact with epidermal cells or by some diffusible
factors released by them. Recently, Feinberg, Repo & Saunders showed that the
ectoderm of early chick embryos plays a role in the establishment and maintenance
of the avascular zone (100 ± 20 /im thick) in the underlying mesodermis by controlling the position of the blood vessels. It is therefore conceivable that epidermis
also controls the cutaneous nerve pattern formation. Thus, the avoidance of epidermis by nerve fibres in vitro may well reflect events which occur normally in the
embryo, and, therefore give insights into the way the dermal and epidermal
68
J.-M. VERNA
innervation is realized. In order to correlate these in vitro observations with normal
development, an in vivo study of the location of nerve fibres in the dorsal skin of
the chick embryo is currently in progress. Preliminary histological results show that,
in 6- to 7-day embryos, main nerve fibre bundles are located beneath the epidermis
at an average distance of 24 ± 4 jum (n = 20) and it is important now to determine
the fine positions of nerve fibre terminals in the skin.
This work was supported by M.I.R. 83-V099 and 82E0680 grants.
REFERENCES
ADLER, R., MANTHORPE, M., SKAPER, S. & VARON, S. (1981). Polyornithine-attached neuritepromoting factors (PNPFs). Culture sources and responsive neurons. Brain Res. 206,129-144.
ADLER, R. & VARON, S. (1980). Cholinergic neurotrophic factors. V. Segregation of survival-and
neurite-promoting activities in heart-conditioned media. Brain Res. 188, 437-448.
ADLER, R. & VARON, S. (1981). Neuritic guidance by polyornithine-attached materials of ganglionic origin. Devi Biol. 81, 1-11.
AL-GAITH, L. K. & LEWIS, J. H. (1982). Pioneer growth cones in virgin mesenchyme: an electronmicroscope study in the developing chick wing. /. Embryol. exp. Morph. 68, 149-160.
ANDRES, F. L. & VAN DER LOOS, H. (1983). Cultured embryonic non-innervated mouse muzzle
is capable of generating a whisker pattern. Int. J. devl. Neuroscience 1, 319-338.
BATE, C. M. (1976). Pioneer neurons in an insect embryo. Nature 260, 54-56.
BENTLEY, D. & KESHISHIAN, H. (1982). Pioneer neurons and pathways in insect appendages.
T.I.N.S. 5, 364-367.
BERG, D. K. (1984). New neuronal growth factors. Ann. Rev. Neurosc. 7,149-170.
BONHOEFFER, F. & HUF, J. (1980). Recognition of cell types by axonal growth cones in vitro.
Nature 288, 162-164.
BOTTENSTEIN, J. E., SKAPER, S. D., VARON, S. S. & SATO, G. H. (1980). Selective survival of
neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented
medium. Expl Cell Res. 125, 183-190.
BRAY, D., WOOD, P. & BUNGE, R. P. (1980). Selective fasciculation of nerve fibres in culture.
Expl Cell Res. 130, 241-250.
CHAMLEY, J. H., GOLLER, I. & BURNSTOCK, G. (1973). Selective growth of sympathetic nerve
fibers to explants of normally densely innervated autonomic effector organs in tissue culture.
Devi Biol. 31, 362-379.
CHAMLEY, J. H. & DOWEL, J. J. (1975). Specificity of nerve fiber "attraction" to autonomic
effector organs in tissue culture. Expl Cell Res. 90, 1-7.
COLLINS, F. (1978). Axon initiation by ciliary neurons in culture. Devi Biol. 65, 50-57.
COLLINS , F. & GARRETT, J. E. (1980). Elongating nervefibersare guided by a pathway of material
released from embryonic nonneuronal cells. Proc. natn. Acad. Sci., U.S.A. 77, 6226-6228.
CRAIN, S. M. & PETERSON, E. M. (1982). Selective innervation of target regions within fetal
mouse spinal cord and medulla explants by isolated dorsal root ganglia in organotypic cocultures. Devi Brain Res. 2, 341-362.
DUNN, G. A. (1971). Mutual contact inhibition of extension of chick sensory nervefibersin vitro.
J. comp. Neurol. 143, 491-508.
EBENDAL, T. (1976). The relative role of contact inhibition and contact guidance in orientation
of axons extending on aligned collagen fibrils in vitro. Expl Cell Res. 98, 159-169.
EBENDAL, T. (1977). Extracellular matrix and cell contacts in the chick embryo. Possible role in
orientation of cell migration and axon extension. Cell Tiss. Res. 175, 439-458.
EBENDAL, T. (1980). Control of neurite extension by target tissues in culture. I.S.D.N., 1st Intern.
Meeting, Strasbourg 162.
EBENDAL, T. (1981). Control of neurite extension by embryonic heart explants. /. Embryol. exp.
Morph. 61, 289-301.
Interaction between chick sensory neurons and skin
69
T. & JACOBSON, C. O. (1977). Tissue explants affecting extension and orientation of
axons in cultured chick embryo ganglia. Expl Cell Res. 105, 379-387.
EBENDAL, T., JORDELL-KYLBERG, A. & SODERSTROM, S. (1978). Stimulation by tissue explants on
nerve fibre outgrowth in culture. Zoon 6, 235-243.
EDELMAN, G. M. (1983). Cell adhesion molecules. Science 219, 450-457.
EDELMAN, G. M. (1984). Cell surface modulation and marker multiplicity in neural patterning.
T.I.N.S. 7, 478-484.
ERANKO, O. & LATHINEN, T. (1978). Attraction of nerve fiber outgrowth from sympathetic
ganglia to heart auricles in tissue culture. Acta Physiol. Scand. 103, 394-403.
FEINBERG, R. N., REPO, M. A. & SAUNDERS, J. W., Jr. (1983). Ectodermal control of the
avascular zone of the peripheral mesoderm in the chick embryo. /. exp. Zool. 226, 391-398.
GOODMAN, C. S., RAPER, J. A., Ho, R. K. & CHANG, S. (1982). Pathfinding by neuronal growth
cones in grasshopper embryo. In Developmental Order: Its Origin and Regulation, pp.
275-316. New York: Alan R. Liss, Inc.
Ho, R. K. & GOODMAN, C. S. (1982). Peripheral pathways are pioneered by an array of central
and peripheral neurones in grasshopper embryos. Nature 297, 404-406.
HOLBROOK, K. A. & HENNINGS, H. (1983). Phenotypic expression of epidermal cells in vitro: a
review. J. invest. Dermatol. 81, lls-24s.
JOHNSTON, R. N. & WESSELLS, N. K. (1980). Regulation of the elongating nerve fiber. Curr.
Topics devl Biol. 16, 165-206.
KATZ, M. J. & LASER, R. J. (1980). Guidance cue patterns and cell migration in multicellular
organisms. Cell Motility 1, 141-157.
LETOURNEAU, P. C. (1975). Cell-to-substratum adhesion and guidance of axonal elongation. Devi
Biol. 44, 92-101.
LETOURNEAU, P. C. (1979). Cell-to-substratum adhesion of neurite growth cones, and its role in
neurite elongation. Expl Cell Res. 124, 127-138.
LEVI-MONTALCINI, R. & HAMBURGER, V. (1953). A diffusible agent of mouse sarcoma, producing
hyperplasia of sympathetic ganglia and hyperneurotization of viscera in the chick embryo.
/. exp. Zool. 123, 233-238.
LUMSDEN, A. G. S. & DAVIES, A. M. (1983). Earliest sensory nervefibersare guided to peripheral
targets by attractants other than nerve growth factor. Nature 306, 786-788.
MOTTET, N. K. & JENSEN, H. N. (1968). The differentiation of chick embryonic skin. An electron
microscopic study with a description of a peculiar epidermal cytoplasmic ultrastructure. Expl
Cell Res. 52, 261-283.
MUHLACH, W. L. & POLLACK, E. D. (1982). Target tissue control of nerve fiber growth rate and
periodicity in vitro. Devi Brain Res. 4, 361-364.
NUTTALL, R. P. & ZINSMEISTER, P. P. (1983). Differential response to contact during embryonic
nerve-nonnerve cell interactions. Cell Motility 3, 307-320.
PETERSON, E. R. & CRAIN, S. M. (1982). Preferential growth of neurites from isolated fetal mouse
dorsal root ganglia in relation to specific regions of co-cultured spinal cord explants. Devi Brain
Res. 2, 363-382.
POLLACK, E. G., MUHLACH, W. L. & LIEBIG, V. (1981). Neurotrophic influence of mesenchymal
limb target tissue on spinal cord neurite growth in vitro. J. comp. Neurol. 200, 393-405.
REES, R. P. (1978). Structure of cell coats during initial stages of synapse formation on isolated
cultured sympathetic neurons. J. Neurocytol. 7, 679-691.
SAXOD, R. (1978). Development of cutaneous sensory receptors in birds. In Handbook of Sensory
Physiology (ed. M. Jacobson), pp. 337-417, Berlin: Springer-Verlag.
SAXOD, R. & MAUGER, A. (1976). DeVeloppement de l'innervation cutanSe des Oiseaux. Etude
descriptive et exp6rimentale. Bull. Soc. Zool. 101, 155.
SCHUBERT, D., LACORBIERE, M., WHITLOCK, C. & STALLCUP, W. (1978). Alteration in properties
of cells responsive to nerve growth factor. Nature 273, 718-723.
SCOTT, S. A., COOPER, E. & DIAMOND, J. (1981). Merkel cells as targets of the mechanosensory
nerves in salamander skin. Proc. Soc. Lond., B 211, 455-470.
SMALHEISER, N., PETERSON, E. M. & CRAIN, S. M. (1981). Neurites from mouse retina and dorsal
root ganglion explants show specific behavior within co-cultured tectum or spinal cord. Brain
Res. 208, 499-505.
EBENDAL,
70
J . - M . VERNA
N., PETERSON, E. M. & CRAIN, S. M. (1982). Specific neuritic pathways and arborizations formed by fetal mouse dorsal root ganglion cells within organized spinal cord
explants in culture. Devi Brain Res. 2, 383-395.
SMALHEISER, N., PETERSON, E. M. & CRAIN, S. M. (1982). Specific neuritic pathways and arborizations formed by fetal mouse dorsal root ganglion cells within organized spinal cord
explants in culture. Devi Brain Res. 2, 383-395.
TAGHERT, P. H., BASTIANI, M. J., HO, R. K. & GOODMAN, C. S. (1982). Guidance of pioneer
growth cones: Filopodial contacts and coupling revealed with an antibody to lucifer yellow.
Devi Biol. 94, 391-399.
VARON, S. & ADLER, R. (1980). Nerve growth factors and control of nerve growth. Curr. Topics
devl. Biol. 16, 207-252.
VERNA, J.-M. & SAXOD, R. (1979a). Co-cultures de neurones ganglionnaires spinaux et de
me"senchyme dermique d'embryons d'oiseaux. Biol. Cellulaire 35, 233-242.
VERNA, J.-M. & SAXOD, R. (19796). De"veloppement de l'innervation cutan6e chez le poulet:
analyse ultrastructurale et quantitative. Archs Anat. micr. Morph. exp. 68, 1-16.
VERNA, J.-M. & SAXOD, R. (1983). Analyse microcin6matographique des interactions entre
cellules nerveuses sensorielles et tissus cutan6s in vitro: mise en evidence d'une difference de
comportement desfibresnerveuses vis a vis des cellules dermiques et 6pidermiques. C. r. hebd.
stanc. Acad. Sci., Paris 297, 241-246.
SMALHEISER,
WESSELLS, N. K.,
GEIDUSCHECK, J.
LETOURNEAU, P.
C,
NUTALL, R.
P.,
LUDUENA-ANDERSON, M.
&
M. (1980). Responses to cell contacts between growth cones, neurites and
ganglionic non-neuronal cells. /. Neurocytol. 9, 647-664.
YAVIN, Z. & YAVIN, E. (1980). Survival and maturation of cerebral neurons on poly(L-lysine)
surfaces in the absence of serum. Devi Biol. 75, 454-459.
{Accepted 7 November 1984)