Download Differentiation in vitro of sympathetic cells from chick

/ . Embryol. exp. Morph. Vol. 33, 1, pp. 43-56, 1975
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43
Differentiation in vitro of sympathetic cells
from chick embryo sensory ganglia
By D. F. NEWGREEN AND R. O. JONES 1
From the Department of Zoology
University of Melbourne
SUMMARY
This study was carried out in order to determine what factors control the differentiation
of certain neural crest cells in the chick embryo. Emphasis was placed on the morphologically
and biochemically divergent sensory and sympathetic pathways of differentiation. Embryos
were precisely staged according to Hamburger & Hamilton (1951) and it was observed that
sensory ganglia with somites, explanted at stages 21-24, gave rise to cells showing formaldehyde-induced fluorescence in more than 25% of explants. These cells were identical in
properties to thefluorescentcells of the sympathetic system of embryos of similar age, and
appeared by 12 days in vitro. Thesefluorescentcells did not appear when somites and sensory
ganglia explants were maintained separately.
The incidence of fluorescent cells in combined explants was considerably reduced or
absent when cultures were maintained for 7 days or less, or when the explants were obtained
from stage 25-26 embryos. Furthermore, when neural tube was also included in the cultures,
the appearance of fluorescent cells was markedly inhibited. The requirement for somitic
tissue to induce fluorescent cells in combined explants can be replaced by forelimb-bud
tissue.
The origin of these cells and the factors that control their differentiation in vitro are discussed
with reference to the neural crest origin of the sensory ganglion, and the possible conditions
pertaining in vivo in this region.
INTRODUCTION
After their initial condensation above the neural tube, cells of the vertebrate
neural crest undergo a period of extensive migration, becoming widely dispersed
from their original position. Derivatives of the neural crest are diverse, including
sensory ganglion neurons, sympathetic neurons and the chromafiin cells of
the suprarenal medullary tissue. The neural crest also gives rise to the intrinsic
neurons of the heart, lungs and gut, some Schwann cells and possibly some
supportive cells of the ganglia of the peripheral nervous system, non-retinal
pigment cells, as well as a variety of mesenchymal cell types (see Weston,
1970, for review). After localization, the development of diverse morphological
and histochemical properties enable neural crest cells to be easily identified
in many cases (Hamburger & Levi-Montalcini, 1949; Gunn, 1951; Strumia &
1
Author's address: Department of Zoology, University of Melbourne, Parkville, Vic.
3052, Australia.
44
D. F. NEWGREEN AND R. O. JONES
Baima-Bollone, 1964; Enemar, Falck & Hakanson, 1965; Pearse & Polak,
1971; Polak, Rost & Pearse, 1971; Fernholm, 1971, 1972).
Despite knowledge of the extent of neural crest developmental potential,
and detailed study of the differentiated state of many derivatives, the factors
controlling the differentiation of any specific cell type or the choice between
one developmental pathway rather than another are still largely unknown.
Heterochronic grafting of chick neural tube and crest suggest that, at any
one level trunk neural crest cells are labile with respect to their later differentiation, at least along the morphologically and biochemically divergent sensory
and sympathetic pathways (Weston & Butler, 1966). There is a preponderance
of pigment cell differentiation in vitro from amphibian (Model & Dalton,
1968), and chick (Dorris, 1938) neural crest. This can also be interpreted from
the view that at any particular axial level the environment in which neural
crest cells find themselves during and after migration, controls their subsequent
differentiation (see Weston, 1970). However, it has been shown that some
regionalization exists down the embryonic axis, e.g. cartilage can be formed
only from head neural crest in amphibians (Chibon, 1967), and possibly also
in the chick (Johnston, 1966). Similarly chick suprarenal medullary cells are
apparently provided only by trunk neural crest between somites 12 and 29
(Chevallier, 1972).
Initial direct experimental work on factors controlling neural crest cell
differentiation consisted of grafting onto the chorio-allantoic membrane (CAM)
(Cohen, 1972), or growing in tissue culture (Norr, 1973) various portions of
the Vr to 2-day-old chick embryo.
These indicated that cells, apparently identical to sympathetic neurons, still
appeared even when the normal region of localization of the primary sympathetic chain was not included in the graft. Furthermore a requirement for
somitic mesenchyme in this developmental pathway which could not be replaced
by either heart or limb-bud mesenchyme in these conditions was also demonstrated. Ventral neural tube also appeared to favour this line of differentiation.
However, neural tube and crest in an organ culture system (Bjerre, 1973)
produced sympathetic-like cells even when explanted alone, suggesting that
an inductive interaction with somitic tissue may not be absolutely necessary
for sympathetic cell differentiation.
The degree to which the lability of neural crest differentiation is retained
after localization has been examined by Cowell & Weston (1970). In an
analysis of cells of the 4-day-old chick embryo sensory ganglion in vitro, it
was observed that many cells differentiated as melanocytes, although normally
these cells are fated to develop as sensory neurons or their supporting cells.
This ability declined with donor age, no melanocytes being observed in explants
from donors older than six days' incubation. Older ganglion cells also spread
far less in culture than the younger ganglion cells. A reduced amount of
pigmentation was also observed when the younger ganglia were explanted on
Sympathetic cells from chick sensory ganglia
45
agar, which restricts cellular outgrowth. This suggests that cell dispersal may
be a necessary prerequisite for the melanocyte trait to appear. In addition,
some intrinsic restriction on the ability to form this unusual cell type intervenes.
This investigation is concerned with the ability of the chick sensory ganglion
to produce sympathetic-like cells in vitro, and the effect of other embryonic
structures on this unusual developmental pathway.
MATERIALS AND METHODS
Preparation
White Leghorn-Black Australorp cross chick embryos of 3^-5 days' incubation (stages 21-26, Hamburger & Hamilton, 1951) were transversely sectioned
into three somite-wide blocks between somite 11 and somite 22. Each piece
was then frontally sectioned immediately ventral to the neural tube. The
dorsal and ventral pieces so obtained were then divided along the mid-line.
Further dissection of the ventral piece (VP) was restricted to trimming away
lateral somitic mesenchyme, heart, gut, limb-buds and notochord where these
occurred. The dorsal piece, consisting of half neural tube (NT), three sensory
ganglia (SG) and dorsal somites with ectoderm (S), could then be divided into
these respective components as desired for various recombination experiments.
Occasionally a reduced amount of neural tube (nt) of only one somite length
was used. Forelimb-bud tissue (FLB) used in some experiments was obtained
from the middle section of the limb-bud, and was approximately the same
size as the somite pieces.
The dissections were performed with cataract knives under a binocular
dissecting microscope, with the tissues completely immersed in a dissecting
medium consisting of Eagle's Basal Medium with 10 % horse serum (Commonwealth Serum Laboratories, Melbourne, hereafter referred to as C.S.L.).
Tissue culture
Tissue fragments were explanted singly or recombined in Rose (1954)
chambers on glass coverslips (Lomb Co. Sydney) under 1 cm wide strips of
dialysis cellophane (Visking Co. Chicago, size 27/32, average pore radius
2-4 nm), as described by Rose, Pomerat, Shindler & Trunnell (1958). Cultures
were maintained for between 4 and 19 days at 37 °C in a medium of 10 %
horse serum and 4 % 9-day-old chick embryo extract in Eagle's Basal Medium
(C.S.L.). Some cultures were provided with medium 199 (Salk, Younger &
Ward, 1954) with 20 % foetal calf serum (C.S.L.); 5 mg/ml glucose; 0-05 units/ml
insulin; 100 units/ml penicillin; and 1 unit/ml Nerve Growth Factor (Burroughs
Wellcome, U.K.). The culture medium was changed every three days.
Explants were routinely examined with a Wild M 40 inverted phase contrast
microscope. Living cultures were photographed with a Zeiss Ikon camera
mounted on a Zeiss Standard RA microscope.
46
i
D. F. NEWGREEN AND R. O. JONES
Sympathetic cells from chick sensory ganglia
47
Histochemistry
Formaldehyde-induced fluorescence (FIF) of catecholamines was demonstrated after removing the strip of dialysis cellophane and washing the cultures
briefly in balanced salt solution. Cultures were dried on the coverslips in a
vacuum dessicator over phosphorus pentoxide for at least 1 h, and then
incubated over paraformaldehyde powder (Merck, Darmstadt) at 80 °C for
1 h. The coverslips were mounted on microscope slides with liquid paraffin
and examined with a Leitz Ortholux microscope with HBO 200 mercury lamp
and 3 mm BG 38, 3 mm BG 12 and K 530 filters, and a light field condenser.
A Leitz Orthomat automatic camera was used for photomicrography.
Non-specific autofluorescence was detected by incubating some cultures
without paraformaldehyde. In addition, all cultures with, fluorescent cells were
washed gently with running water for 1-2 min then re-examined. Structural
autofluorescence is not diminished by this treatment whereas the intensity of
catecholamine fluorescence declines markedly.
After FIF treatment, some cultures were stained with toluidine blue
(Humason, 1962) to reveal cartilage, or a von Kossa method (Mallory, 1961),
slightly modified, to detect calcium deposits.
RESULTS
(1) Explants of ventral piece (VP)
Cultures of VP from stages 21-26 showed rapid cellular outgrowth within
a few days, and myotubes and cartilage nodules were prominent. FIF cells
were present in large numbers in all cases after a period of between 4 and
12 days in vitro. Long and complex fluorescent axon networks were frequently
observed, especially in the older cultures (Fig. 1). The average nuclear diameter
of fluorescent cells, as measured from fluorescence photomicrographs (Fig. 2),
was 6-3 /«n (range 5-0-8-8/im; 53 cells; 12 days in vitro). The average nuclear
diameter of neurons and neuroblasts from phase contrast photomicrographs
was 6-0 (range 5-0-7-3 jtim; 35 cells; 12 days in vitro). Not all neurons in
Fig. 1. VP explant of stage 21 donor, 12 days in culture after FIF treatment. Note
fluorescent cells and long processes. Scale = 100 jim.
Fig. 2. VP explant of stage 23 donor, 12 days in culture after FIF treatment. A
small group of fluorescent cells. Scale = 10 /*m.
Fig. 3. Same area as Fig. 2, phase contrast optics. Fluorescence is more frequently
displayed by the larger cells of the group (cf Fig. 2). Scale = 10 /*m.
Fig. 4. SG explant of stage 21 donor, 5 days in culture, phase contrast optics.
Non-fluorescent sensory neurons and supporting cells are shown. Note the size
difference compared to the fluorescent cells of Fig. 3. Scale = 10/*m.
Fig. 5. S:SG explant of stage 23 donor, 12 days in culture, after FIF treatment.
Single fusiform fluorescent cell. Scale = 10 ftm.
48
D. F. NEWGREEN AND R. O. JONES
culture were fluorescent (Fig. 3). These cells were similar in appearance to
their fluorescent neighbours, although they tended to be slightly smaller when
examined with phase contrast optics. It is possible that these non-fluorescent
cells were not adrenergic neurons, or they may have been metabolically resting
(Yamauchi, Lever & Kemp, 1973; Benitez, Murray & Cote, 1973), or simply
have been immature. The fluorescent cells could not be divided into groups
on the basis of their fluorescence intensity or size: all appeared small and
brightly fluorescent (cf. Chamley, Mark, Campbell & Burnstock, 1972 a).
(2) Explants of somite and sensory ganglia
Sensory ganglia (SG) explanted alone gave considerable numbers of neurons
that normally occurred in smaller groups than those typically observed in
VP cultures, and in addition the cells had much larger nuclei of 8-9 jam average
diameter (range 7-0-11-3 jtim; 125 cells; 12 days in vitro; phase contrast optics).
The cells also appeared to have a smaller nucleo-cytoplasmic ratio. These
groups of neurons were interconnected by a very extensive axonal network,
which was usually accompanied by rather flattened supporting cells (Fig. 4),
although definitive Schwann cells closely applied to axons and glial cells were
also observed. A sheet of fibroblast-like cells separated neurons and axonal
networks from the glass of the culture chamber. No clearly identifiable melanocytes were observed beneath the strip of dialysis cellophane (cf. Cowell &
Weston, 1970), although occasionally distinct black or brown pigment cells
were observed at the edges of the cellophane. Cultures containing even one
cell with FIF after 12 days in culture were very rare (see Table 1).
Somite explants (S) showed large numbers of myotubes by 6 days in vitro,
but spontaneous contractions were rarely observed. Cartilage nodules were
infrequently observed in explants from donors of stage 23 or younger, even
after 19 days in vitro. Older donors produced cartilage far more frequently
and often in large amounts. In addition to these differentiation end states,
almost all cultures initially showed condensed flattened bars or strands of
contiguous cells, which by 7 days in vitro usually displayed a definite cell free
central core with a surrounding layer of flattened cells. After 12 days in
culture this central core was shown to have accumulated considerable amounts
of calcium salts, and probably therefore represents a premature differentiation
of sclerotomal cells along an osteogenic pathway. These explants were surrounded by flattened fibroblastic cells, and occasionally a few large, presumably
sensory neurons could be seen, but FIF cells were rare even after 12 days
in vitro (see Table 1).
Somite and sensory ganglia explants cultured as one piece (S:SG) showed
a combination of the differentiation end states already described, although
the neurons were confined largely to the outer fibroblastic layer. One significant
deviation from the above observations, however, was the appearance of cells
with FIF in over 25 % of cultures from stage 21-24 donors after 12 days
Sympathetic cells from chick sensory ganglia
49
Table 1. Occurrence of FIF cells in cultures of chick embryo tissue
Explant
VP
VP
VP
VP
SG
S
S:SG
S:SG
S:SG
S:SG
S/SG
S/SG
S:SG(M199)t
FLB
FLB/SG
FLB/SG
S:SG:NT
S:SG/NT
S:SG:NT
S:SG:nt
Donor age*
21-24
21-24
25-26
25-26
21-24
21-24
21-24
21-24
21-24
25-26
21-24
25-25
21-24
21-24
21-24
25-26
21-24
21-24
21-24
21-24
Period
in culture
(days)
4-7
12
7
12
12
12
7
12
19
12
12
12
12
12
12
12
12
12
19
12
Cultures with
FIF cells/total
cultures
10/10
27/27
4/4
4/4
2/37
1/46
1/29
22/77
6/16
1/22
14/31
0/12
5/12
0/25
7/22
0/8
0/11
0/12
0/25
3/55
* Hamburger & Hamilton (1951) stages.
t Culture medium containing foetal calf serum, NGF, etc. (see Methods).
in vitro (S: SG versus S, P < 0-1 % per cent; S: SG versus SG, P = 1 %, using x2
test with Yates' correction; see Table 1). Furthermore, virtually no fluorescent
cells were observed in explants from stages 21-24 in the first 7 days in vitro
(12 days S:SG versus 7 day S:SG, P = 1 %), or when the explants were
taken from embryonic stages 25-26 (stages 21-24 S:SG versus stages 25-26
S: SG, P < 5 %). In addition a slightly increased frequency of FIF cellcontaining cultures was observed on prolonged cultivation of explants (19
days), or by the utilization of enriched medium, or by dividing and then
recombining somite and sensory ganglia explants prior to cultivation (S/SG).
These increases however were not significant in the numbers performed.
These fluorescent cells when present, occurred in the same axonal network
as the large non-fluorescent ganglion cells, and were in small numbers (average
8-3 per culture, range 1-33), in one or two loose groups confined to a small
proportion of the total neural area. These fluorescent cells were of small size
(average nuclear diameter 6-7 /an, range 5-5-8-5/tin, 27 cells measured after
FIF treatment), and were often fusiform in outline (Fig. 5) although cells
with larger numbers of short fluorescent processes were also common (Fig. 6).
Cells with fluorescent processes exceeding 50 /an in length were very rare, but it
4
EMB 33
50
D. F. NEWGREEN AND R. O. JONES
Fig. 6. S:SG explant of stage 23 donor, 19 days in culture, after FIF treatment.
Group of cells with short fluorescent processes. Scale = 10/*m.
Fig. 7. FLB/SG explant of stage 23 donor, 12 days in culture, after FIF treatment.
Single cell with long fluorescent process. Scale = 10/*m.
could not be established whether this was due to the absence of such long
processes or merely to the lack of catecholamine. No increase in the frequency
of long fluorescent cell processes was observed even in cultures of 19 days
duration.
(3) Explants offorelimb-bud {FLB) and foreUmb-bud
combined with sensory ganglia (FLB/SG)
Fragments of FLB from embryos of stages 21-24 showed marked similarities
in cell types represented, to explants of somite, although cartilage was present
in great amounts even in explants grown from stage 21 embryos. Exposure to
formaldehyde gas after 12 days in vitro revealed no fluorescent cells. Similar fragments when combined with sensory ganglion (FLB/SG) produced fluorescent
cells identical in appearance and distribution to those observed in somite
recombined with sensory ganglia (S/SG) cultures (Fig. 7). Recombinants from
donors of stages 25-26 again did not produce fluorescent cells (Table 1).
(4) Explants involving neural tube
Explants of stages 21-24 S:SG with attached neural tube (S:SG:NT)
showed mesenchymal derivatives similar to S: SG cultures, although contractions
of myotubes were common after 6 days in vitro. As anticipated, the axonal
networks were much more extensive as compared to S:SG cultures, with a
large percentage of axons apparently without supporting cells (cf. SG and
S:SG cultures). Large neurons similar in appearance to the non-fluorescent
ganglion cells of SG explants, occurred in small groups near to the neural
tube, apparently in similar or slightly fewer numbers as compared to explants
Sympathetic cells from chick sensory ganglia
51
without NT. It is possible that some neurons were overlooked due to their
proximity to the neural tube. After 12 days in vitro no fluorescent cells could
be found in these cultures.
Explants of S: SG with NT dissected free and recombined at explantation in
random orientation to the other tissue fragment (S: SG/NT), also showed this
lack of fluorescent cells after 12 days in vitro. Extension of the culture period
to 19 days gave the same result, but reduction of the amount of NT tissue to
about one third (S:SG:nt) showed fluorescent cells in a few cultures. This
was far below the frequency of fluorescent cells observed in explants without
neural tube (S:SG versus S:SG:nt, P < 1 %), and suggests that the neural
tube has an inhibitory effect on the differentiation of F1F cells, at least in
these conditions.
DISCUSSION
Standard histological techniques have demonstrated a clear separation
between the cell aggregates of the sensory ganglion and the primary sympathetic
chain in the chick embryo, at least from about stage 19 onwards. Auto radiography has revealed the presence of a few neural crest or neural tube derived
cells lying along the ventral nerve roots. These cells are probably presumptive
Schwann cells, rather than neuronal precursors (Weston, 1963).
Cells containing catecholamine are confined to the primary sympathetic
chain and its ventral extensions into the adrenal medulla, between day 3 and
4 of incubation (Enemar et al. 1965). No cells with demonstrable FIF occur in
the sensory ganglion even after loading with DOPA, a catecholamine precursor
(Polak et al. 1971). Migration of cells from the primary sympathetic chain
to form the secondary sympathetic complex closer to the sensory ganglion
has commenced after 4 days' incubation (about stage 25), when fluorescent
cells can be seen extending dorsally from the primary position (Enemar et al.
1965). It is possible however that non-fluorescent cells have commenced this
migration at an earlier stage though these cannot be revealed by loading with
a-methyl noradrenaline or DOPA (Allen & Newgreen, unpublished observations). The formation of the secondary chain and increased recruitment of
FIF cells into it, continues from day 5 to day 7 of incubation (stage 26 onwards;
Enemar et al. 1965). It is therefore unlikely that sympathetic precursor cells
are included in the dorsal explants at this time of development (stages 21-26).
The scarcity of FIF cells in explants of somite (S) or sensory ganglia (SG)
tends to confirm this, since sympathetic ganglion cells survive well for many
weeks in vitro even when denied pre- and post-ganglionic connexions (Chamley
et al. 1972a; Benitez et al. 1973). Indeed the definitive sympathetic system of
embryos of the same age showed no significant decrease in fluorescent cell
numbers between 4 and 19 days in culture.
The fluorescent cells observed on culturing somite (S) combined with sensory
ganglia (SG) are therefore unlikely to be derived from cells normally fated as
4-2
52
D. F. NEWGREEN AND R. O. JONES
sympathetic neurons, although they bear a great resemblance to the latter.
Their exact status could not be precisely established since both these cells and
the normal sympathetic cells in culture resembled SIF cells in their small size
and bright fluorescence (cf. Chamley et al. 19726). The absence of long
fluorescent processes was also common to many true sympathetic cells.
The most likely source of those FIF cells is therefore the sensory ganglion.
This view is supported by the FLB/SG results, since the mid-FLB explants
used here could be expected to contain virtually no neural crest derived cells
except for the first of the melanoblasts or their precursors in the epidermis,
before stage 24 (Fox, 1949). This view parallels the observation that the
sensory ganglion at a similar stage in development contains a population of
cells capable of differentiating as melanocytes under certain conditions, rather
than as sensory neurons or their supporting cells as would be the case normally
(Cowell & Weston, 1970). Presumably these cells are numbered amongst the
medio-dorsal cells of Hamburger & Levi-Montalcini (1949). The length of
time necessary for the appearance of FIF cells, greater than 7 days in vitro, is
consistent with the view that these cells are expressing an entirely new cell
phenotype as a result of the conditions in culture. Again, like the production
of melanocytes from the sensory ganglion (Cowell & Weston, 1970), the
appearance of FIF cells is dependent upon the age of the donor, although
this ability declines with age even more rapidly than that of melanin synthesis.
In these explants of sensory ganglion, comparable in age to Cowell &
Weston's (1970) study, but in different culture conditions, overtly differentiated
pigment cells were lacking. This cannot be due to genetic factors (see Dorris
(1938) on pigment cell differentiation in vitro from this breed), nor by a
prevention of dispersal of their cellular precursors as occurs in explants on
agar (Cowell & Weston, 1970) or on the CAM (Dorris, 1941). On the contrary,
dispersal seems to be enhanced under the cellophane strip. It is therefore of
interest to note the occasional presence of pigment in some cells that had
escaped from under the cellophane. It is uncertain whether the absence of
pigmented melanocytes indicates that more cells could be available for other
lines of differentiation, but evidently none or few of these express themselves
as catecholamine synthesizing cells in SG explants. In plasma clot cultures the
decrease in pigmentation observed when somitic tissue is explanted with sensory
ganglion could indicate that fewer cells embark upon this line of differentiation,
and in fact mature ganglion cell numbers seem to be increased (Peterson &
Murray, 1955). A reciprocal relationship between pigmentation and neuronal
development does seem to exist in sensory ganglia in vitro, the degree of cell
dispersal being influential in the development of one phenotype rather than
the other (Weston, 1971).
Nevertheless, the small numbers of fluorescent cells in cultures involving
three sensory ganglia as well as somite, compared with the large numbers of
Sympathetic cells from chick sensory ganglia
53
pigment cells obtained by Cowell & Weston (1970), from similar aged donors,
indicate that only a small percentage of cells potentially available for transformation actually expresses the sympathetic line. This is unlikely to be due to
immaturity of the catecholamine synthesizing or storage mechanisms (see
Ignarro & Shideman, 1968), since 19 day cultures showed little increase in
fluorescent cell numbers over 12 day cultures. It is known however that not
all adrenergic cells in sympathetic ganglia exhibit FIF at any one time, both
in vivo (Yamauchi et al. 1973) and in vitro (Benitez et al. 1973). Rather than
the possibility that the sensory ganglion is inherently less capable of producing
fluorescent cells than melanocytes, it is more likely that this culture system
does not easily allow this line of differentiation, in much the same way as
melanocyte differentiation is inhibited at some level in the same cultures. It is
clear, however, from observations of VP cultures, that the method is capable
of cell maintenance if not the expression of final differentiated end states. The
experiments of Cohen (1972) and Norr (1973) indicate that early somitic
tissue and ventral neural tube induce normal sympathetic differentiation,
although Bjerre (1973) has reported the appearance of fluorescent cells in
cultures of hind brain neural crest alone. In the present cultures, slightly older
somitic mesenchyme could also elicit this type of differentiation, but the disorder produced by explantation and the distribution of fluorescent cells in
small isolated groups suggest that the mere presence of somitic tissue is
insufficient in itself. Perhaps more precise microenvironmental factors must
be present, which are met by only a relatively few cells potentially capable of
responding to them and subsequently expressing FIF differentiation.
Limb-bud or heart mesenchyme could not duplicate the effects of early
somitic mesenchyme in promoting sympathetic differentiation from neural
crest on the CAM (Cohen, 1972), although crest cell migration was extensive.
In the present work, forelimb-bud tissue proved just as effective as older somitic
mesenchyme in the development of sympathetic cells from the sensory ganglion.
It is uncertain whether this represents some response factor inherent to the
sensory ganglion. However, the similarities of somite and forelimb-bud explants
under these culture conditions have already been noted.
Although somite and neural tube are thought to be important for sympathetic
cell differentiation (Cohen, 1972) the sensory ganglion, lying adjacent to the
neural tube and embedded in the somite, shows a complete absence of these
characteristics. No fluorescent cells can be detected from the time when these
first appear in the primary sympathetic chain (Enemar et al. 1965), even after
loading with the catecholamine precursor DOPA (Polak et ah 1971) or with
a-methyl noradrenaline (Allen & Newgreen, unpublished observations). The
ability to take up catecholamines is lacking at later stages (Burdman, 1968;
England & Goldstein, 1969).
The different characteristics of sensory and sympathetically fated cells
appear to be enforced only after these cells have left the neural crest (Weston
54
D. F. NEWGREEN AND R. O. JONES
& Butler, 1966). Although derived from the same axial levels (Yntema &
Hammond, 1954, 1955), the cells ultimately destined as sympathetic cells
migrate before those fated to form the sensory ganglion (Weston, 1963).
Sympathetic cell precursors pass through the somite close to the neural tube
before localizing near the dorsal aorta, where the differentiated state in the
form of catecholamine accumulation is normally first manifested (Enemar et
al. 1965). Neural crest cells destined to form the sensory ganglion do not
migrate so extensively, and aggregate next to the neural tube with which they
are continuously associated. In tissue culture, the presence of the neural tube
drastically inhibited the appearance of FIF cells from explants of sensory
ganglion and somites (Table 1). It is possible that the neural tube has a similar
inhibitory effect in vivo. It should be noted that the migration of sympathetic
cells to the position of the secondary sympathetic chain, closer to the neural
tube, occurs only after most of the cells have to a large degree differentiated,
and are already capable of displaying fluorescence.
The action of the neural tube however may not be this specific. In these
cultures, neural tube did not suppress FIF cells by significantly promoting
sensory neuron differentiation from the limited pool of cells available. Peterson
& Murray (1955) found that central nervous tissue actually increased the
number of degenerating neurons in explants of embryonic sensory ganglia.
Thus the suppression of FIF cells by the neural tube may merely reflect an
inhibition of neuronal differentiation on a population of cells which, differentiating from a relatively labile state entirely in vitro, may be especially sensitive
to its action. The condensation of neural crest cells may be promoted adjacent
to the neural tube (Weston, 1970), but subsequent differentiation, although
restricted to neuronal lines (Weston, 1971), may be inhibited or retarded.
Indeed, sensory neuronal differentiation appears first in the ganglion cells
furthest from the neural tube (Hamburger & Levi-Montalcini, 1949), and
possibly lends further support to the concept of the selective inhibitory action
of the neural tube on the differentiation of various neuronal elements.
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{Received 2 April 1974, revised 5 June 1974)