/. Embryol. exp. Morph. Vol. 21, 2, pp. 331-40, April 1969
Printed in Great Britain
331
Chondrogenesis in chick embryo somites in vitro
By M. L. ELLISON, 1 E. J. AMBROSE 1 & G. C. EASTY 1
From the Chester Beatty Research Institute, London
During the embryonic development of vertebral cartilages, cells from the
somite mesoderm differentiate into chondrocytes around the spinal cord and
notochord. Grafting experiments in amphibian and chick embryos have
indicated that the spinal cord and notochord have some influence on this
differentiation of somite cells to cartilage (Holtzer & Detwiler, 1953; Watterson,
Fowler & Fowler, 1954). Further analysis in vitro has established that, under
specified culture conditions, cartilage formation from somites is, in fact,
dependent on the presence of either spinal cord and/or notochord or their
extracts (Grobstein & Parker, 1954; Grobstein & Holtzer, 1955; Strudel, 1962,
1963; Lash, 1963).
For example, Lash (1963) showed that in his system on a liquid medium
stage 16 (Hamburger & Hamilton, 1951, stages) somites alone formed no
cartilage, but that cartilage did develop when notochord or spinal cord was
cultured with the somites. This suggested that the spinal cord and notochord
were 'inducing' somite mesoderm cells to form chondrocytes.
However, more recent observations showed that in some instances cartilage
production by somites in culture is independent of 'inducing' tissues, and this
independence varies with the conditions.
Lash (1964) noted that a percentage of 56 h chick somite explants became
able to differentiate without inducer when the liquid medium of the culture
system was solidified with agar. Strudel (1963) was able to reduce the minimum
age for ' autodifferentiation' to an equivalent of stage 12 (17 somites stage) by
enclosing the explant in vitelline membrane. Holtzer's (1964) analysis of the
effect of environmental factors on the somites showed that, while the minimum
age for differentiation independent of inducers was stage 18 when plasma
clots were used, stage 16 gave positives on agar medium, and even stage 14
material could differentiate when the size of the explant was increased from
10 to 30 somites.
These findings raise questions concerning the state of the somites on explantation and the role of the interaction between spinal cord and notochord, and the
somites in combined cultures.
Are the environmental factors which seem to favour cartilage formation in
1
Authors' address: Chester Beatty Research Institute, Institute of Cancer Research, Royal
Cancer Hospital, London, S.W.3.
22
J EEM 21
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M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
vitro mimicking the action of the tissue inducers, or are the somites already
induced—that is, 'determined' (Waddington, 1966)—at the time that they are
explanted ? If they are already determined, what role has the environment in the
phenotypic expression of cartilage ?
In these experiments a study was made of the reaction of chick somites,
explanted without any spinal cord or notochord, to the following culture
variables: different nutrient media, different sizes of explant, the presence of an
oily layer covering the explant.
MATERIALS AND METHODS
Somites taken from chick embryos of stages 9-17 (Hamburger & Hamilton,
1951) were prepared for culture by a method modified from that of Avery, Chow
& Holtzer (1956).
The embryos were removed from the eggs into sterile Simms BSS, trimmed of
extra-embryonic membranes, endoderm and ventral mesoderm, and treated
with 3 % Trypsin (Moscona, 1952) for 3-4 min. After transfer to a Simms/calf
serum mixture, the somites could readily be dissected free from all notochord,
spinal cord, epidermis, mesonephros fragments and lateral mesoderm. Each
explant for culturing consisted of a group of somites taken from a pool of the
10 hindmost pairs from each of the chicks of the same stage, and clustered on
rafts of Type TA Millipore filter (25 [i thick, 0-8 [i pore size). For experiments in
which the effect of the medium was under study, each cluster consisted of eight
somites, but where the explant size effect was being tested, the medium was kept
constant, and the number of somites per cluster varied between 1 and 32.
Basically, the culture medium consisted of a 1 % solution of Agar (DIFCO)
in a 1:1 mixture of NCTC109 and Simms BSS (modified by addition of a tris
buffering system and a double quantity of glucose). Antibiotics were included
at final medium concentrations of 100 i.u./ml sodium benzylpenicillin, 37-5
i.u./ml streptomycin sulphate, and 2-5/*g/ml Amphotericin B deoxycholate.
One or more of the following protein constituents were also added: horse serum
(Burroughs Wellcome no. 5), foetal calf serum (Microbiological Associates),
and egg, which was prepared by beating together, thoroughly, the yolk and
albumen of an unincubated hen's egg. Both fertile and infertile eggs were tested,
but no difference was seen in the results. On the other hand, different batches
of foetal calf serum were found to give slight variations, so that it was necessary
to use a single batch for all of these experiments. The final protein concentrations
were: 20 % horse serum (HS medium), 20 % foetal calf serum (FCS medium),
16 % horse serum+ 20 % egg homogenate (HS + egg medium), or 16 % foetal
calf serum + 20 % egg homogenate (FCS + egg medium).
Somite explants on their Millipore rafts were transferred to the surface of the
solidified medium, about eight explants per Petri dish containing 5 ml of
medium. The dishes were placed inside humidified boxes with a gas phase of
Chondrogenesis in chick embryo
333
5 % C0 2 in air, for incubation at 37 °C. After 8-11 days culture the explants on
the filter rafts were fixed in phosphate-buffered glutaraldehyde, stained 2 min
in Bismarck Brown (1 % in 1 % Ethanol), 2-4 h in Toluidine Blue (0-1 % in 30 %
ethanol), dehydrated, cleared and whole-mounted for microscopy.
Each explant was scored for the presence of cells within a metachromatic
matrix, this being taken as the criterion for the differentiation of chondrocytes.
High-power examination of the stained and cleared whole mounts permitted
the identification of positives represented by very small numbers of cells, so that
the incidence of differentiation in this system cannot be compared directly with
that obtained by the scoring of living cultures for nodules.
RESULTS
It was clear that this culture system was able to support cartilage differentiation from somites in the absence of inducing tissues. The material produced
rarely appeared as morphotypic cartilages, but varied from very small islands
or sheets of matrix-producing cells, to distinct nodules, or to masses of a rather
soft matrix which tended to precipitate into strands on fixation. Histochemical
investigations (kindly undertaken by Mr M. J. O'Hare of the Chester Beatty
Research Institute) showed that the matrix, which stained with Alcian Blue at
pH 0-5 and was totally labile to testicular hyaluronidase, contained only
chondroitin sulphate A and/or C as the mucopolysaccharide component. No
keratosulphate was detected. The soft matrix had a weak diastase-resistant
PAS reactivity, indicating perhaps a lower collagen content than in vivo cartilage
of equivalent age.
When cultured somites were scored for presence or absence of metachromatic
matrix rather than for type, it was clear that the incidence of cartilage varied
with the conditions.
Medium constituents
Table 1 illustrates the influence of different media over a range of stages in
which each explant consisted of eight somites. Taking as an example the values
for stage 15: on HS medium cartilage was not able to develop, addition of egg
permitted a low incidence, but replacement of horse serum by foetal calf serum
enabled a majority of the explants to make cartilage, while addition of egg to the
FCS medium further increased the incidence so that all of the explants differentiated.
This general trend was repeated through all of the stages (11-17) in which
cartilage was able to differentiate in the absence of spinal cord and notochord.
It is significant that the favourable effect of egg which is seen on its addition to
horse serum is also seen when it is added to foetal calf serum, itself a strongly
favourable factor. This indicates that two factors, each favourable individually,
can supplement one another's effect.
334
M. L. ELLISON. E. J. AMBROSE & G. C. EASTY
Explant size
Another trend is also seen in Table 1—an increase in cartilage with increase
in age of the chicks from which the somites were explanted. This could be due
to the difference in size of the explants, since, for example, a group of eight
somites from stage 9 is considerably smaller than a similar group from stage 17.
Table 1. Proportion of somite explants differentiating cartilage on varying media
Medium
Chick stages
Horse serum
HS + egg
—
—
—
—
—
—
—
—
—
—
—
0/6
0/11
3/23
0/17
2/12
4/12
7/15
9
10
11
12
13
14
15
16
17
Foetal calf
serum
FCS + egg
0/6
0/9
0/5
0/7
1/29
3/16
2/10
11/16
22/24
24/24
25/28
1/22
4/13
8/15
12/14
10/10
13/13
13/13
Table 2. The proportion of somite explants differentiating cartilage
in explants of varying sizes
No. of somites per explant
Chick
stages
9
10
11
12
13
14
15
16
17
r
1
2
4
6
8
12
16
24
32
—
—
0/5
0/7
1/5
3/9
—
—
0/6
0/6
1/5
5/9
0/6
0/9
0/5
0/5
3/8
0/5
0/6
0/5
—
—
—
—
—
0/8
0/12
4/15
4/11
6/17
6/14
6/15
8/13
11/14
9/12
8/10
10/11
9/11
0/5
0/6
3/9
—
—
—
—
—
—
0/5
1/9
1/7
—
—
—
—
—
—
1/29
3/16
2/10
11/16
22/24
24/24
25/28
2/12
3/6
9/10
11/11
—
—
4/10
4/12
4/7
7/7
—
—
—
Therefore, this possible size effect was tested by growing explants of different
numbers of somites on a single medium (FCS medium). Comparison of explant
size and somite number per explant is valid within any one stage, though not
between different stages. Table 2 summarizes the results of this test.
Taking again stage 15 as the example, no cartilage developed in single somite
explants, but over 50 % of the cultures differentiated at 4 times that volume, and
when 12 somites were clustered together, there was a 100 % incidence of
differentiation. Again, this pattern—increasing ability to differentiate with
Chondrogenesis in chick embryo
335
increasing amount of tissue—tends to be repeated through all of the later stages.
The reason for the variable results in the younger stages is not clear, though it
may be associated with a greater sensitivity of young material to damage during
preparation and the relatively large surface area open to this damage.
The behaviour of stage 17 somites, showing a reduced incidence of cartilage
when compared with stage 16, may relate to an effect noted by Lash (1967) that
posterior somites were much more inclined to chondrogenesis in vitro than were
anterior somites. If this property is associated with the structure of the somites,
as Lash suggests, stage 17 is the only stage represented in these experiments which
contains 'anterior type', non-chondrogenic, somites in the 10hindmost pairs.
This could also account for the difference between the mass effect observed
here and that observed by Lash (1968) where no enhancement of chondrogenesis could be obtained with increasing cluster size. The cultures represented
in Table 2 correspond, not with the whole range of explants used by Lash, but
more nearly with the 'posterior type' for which Lash has 100 % incidence of
cartilage at the smallest grouping in his system.
Table 3. The proportion of somite explants differentiating cartilage
under the influence of a liquid paraffin layer
Explants
Medium
•\
Stage and somite no.
Stage 9, 8 somites/explant
Stage 10, 8 somites/explant
Stage 15, 1 somite/explant
Foetal calf serum
0/6
0/9
0/12
Foetal calf serum
with liquid paraffin
layer
3/8
4/20
13/15
Liquid paraffin layer
One further culture variable was tested following the suggestion by Dr J. W.
Lash (personal communication) that the general conditions could be improved
by covering the explant and the agar surface with a layer of light mineral oil.
Explants representing three positions on Table 2 where the results were negative; one stage 15, eight stage 9 and eight stage 10 somites were cultured on
FCS medium covered with a layer of liquid paraffin.
This factor was found to enhance both the survival of the explants and their
ability to differentiate cartilage, as is seen in Table 3. Although the number of
samples is low here, it does demonstrate that it is possible to define conditions
under which even stage 9 somites can develop cartilage in the absence of
inducing tissue.
336
M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
DISCUSSION
These findings suggest that chick somites are determined for cartilage differentiation as early as stage 9 (only three stages from the time that the first of the
somite pairs is delimited). The observation that factors which favour the appearance of cartilage in vitro can have an additive effect supports this, suggesting that
these extrinsic factors are not acting as specific inducers, but individually or
together are enhancing a characteristic which is already present in the cells.
The effect of the increased volume of explants also supports early determination rather than in vitro 'induction'. The ability to make cartilage matrix is
gained in this case not on addition of anything new or anything absent in the
negative cultures, but simply by the development together of a larger amount
of exactly similar material.
Again, the ability to differentiate acquired when liquid paraffin is added can
hardly be due to 'induction' since the only addition to the system is of a nonspecific factor, a chemically inert substance.
These results also suggest that after determination has taken place there is a
latent phase before the actual appearance of the differentiated characteristic,
and that during this period of stabilization the ability of the cells to differentiate
can be influenced by the environment. That single stage 15 somites, for example,
can be grown under certain conditions which permit cartilage production or
others which do not (although the cells are alive in both cases), demonstrates
that the environment can have some control over the differentiation of a
characteristic.
Observations similar to these have been made in a number of differentiating
systems.
Seno & Biiyokozer (1958) demonstrated that somites grafted from chicks of
stage 9 and onward to the chick chorioallantoic membrane were able to make
cartilage in the absence of notochord and spinal cord. They also showed that the
incidence of cartilage related to the degree of isolation of the somites, i.e. grafts
of somites plus ectoderm and endoderm gave higher values than somites plus
either ectoderm or endoderm, and these in turn gave higher values than the
somites alone. This shows a dependence of the incidence of cartilage differentiation on the immediate environment of the somites.
An environmental factor influencing the differentiation of cultured limb bud
mesenchyme was noted by Umansky (1966), who plated out dispersed limb cells
in Millipore filter wells. The initial cell density was found to be directly
associated with the resulting differentiations; low densities gave no differentiation, high densities gave cartilage only, while at intermediate densities both
muscle and cartilage formed. Umansky interprets this as a determination by the
density-dependent aggregation patterns which arose. From the data which he
presents, however, it is difficult to find firm evidence in favour of determination
of cell types, rather than a selection between two available, pre-determined
Chondrogenesis
in chick embryo
337
types. Since only one culture variable was used, it is not possible to note any
additive or supplementing effect of two or more factors which could imply a
stabilization rather than a determination.
Evidence that environmental factors play a part in the expression of an existing determination has been obtained by several workers using monolayers of
dispersed cells.
Abbott & Holtzer (1966, 1968), using vertebral chondrocytes in monolayer
culture, found that the density of cells in the population and the medium which
was used influenced both the ability of the cells to make cartilage and the rate
at which it appeared.
In a detailed study of the monolayer requirements for matrix production in
cloned sternal chondrocytes, Coon (1966) found that the proportion of clones
making cartilage matrix varied with the medium (embryo extract supplement),
with the cell density, and with the growth phase of the cells. He showed that
cells which had lost all sign of their previous differentiated characteristics could
return to re-express them when the environmental conditions were suitably
manipulated.
Konigsberg (1963), using muscle cell clones, and Cahn & Cahn (1966), using
retinal pigment cells, have demonstrated that these cell types also can re-express
function according to the conditions.
The variation of the medium effective in stabilizing differentiation may be
quite small. This has been clearly demonstrated by Simpson & Cox (1967) who
used lizard tail regenerate monolayers, growing in two media which differed
only in the defined medium component. Both media supported long-term
proliferation of the cells, but whereas the Eagle's Basal-containing medium
permitted muscle differentiation, the Ham's F 10-based medium did not. The
system also showed that the latent period between determination and phenotypic expression may be very long. Cells could be grown on the non-permissive
medium for as long as 135 days without any indication of muscle, then differentiate myotubes within 7 days of transfer to Eagle's-based medium.
That cells can retain an intrinsic potential for differentiation in long-term
culture has also been shown by Main (1966). Fourteen-day embryonic mouse
tooth germs were cultured on gelatin sponges for up to 37 days, and lost all
characteristic morphology, then, 56 days after re-implantation into newborn
mice, produced almost perfectly formed incisor teeth. This could have been due
to inductive influences after re-implantation, but it seems unlikely in view of the
ectopic site of the implants.
The heritability of the determined state is strongly supported by the findings
of Hadorn (1966) on insect imaginal disc cells. These had been serially transplanted from adult to adult for over 3 years without any differentiation, yet
were able to express the functions determined in the embryonic state when
exposed to the stabilizing environment, the pupal haemolymph.
Coon (1966) observed that at least 20 cell generations elapsed between the
338
M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
loss of visible chondrocyte characteristics and their re-expression under the
conditions mentioned earlier.
All of these findings suggest the presence of a stabilization period during
differentiation, coming after determination, but before the characteristics
appear. The cells seem to require some influence of the environment so that the
overt differentiation may be achieved. At least some cell types seem able to
remain in a state of latent determination until the necessary stabilizing conditions are available.
Little is known of the cellular events taking place during this phase. Medoff
(1967), studying limb buds, and Lash (1968), using somites, have indicated an
accumulation of cartilage precursor substances well in advance of the actual
appearance of the matrix. This suggests that the stabilization phase may be
associated with the build-up of essential cell products to critical levels, a buildup actively assisted or hindered by factors of the surroundings.
In the culture system studied here, there is clear indication that somites are
determined for cartilage at early stages, and subsequently (at least up to stage 17)
can be influenced or ' stabilized' by environmental factors.
The in vitro conditions altered the relative proportions of explants achieving
overt differentiation, so that some environments could be scored as 'more
stabilizing' than others. Presumably the most favourable conditions are those
which most adequately replace the stabilization provided in vivo by the tissues
which normally surround the developing primordia. Perhaps the exceptionally
strong cartilage enhancing effect of the notochord and spinal cord observed
in vitro can be seen as the exceptionally favourable stabilizing influence provided
by the normal environmental neighbours of the somites (see Holtzer, 1968).
SUMMARY
1. The development of cartilage matrix producing cells from somite mesoderm in organ culture has been studied in an attempt to distinguish between
'induction' of differentiation and the expression of the characteristic.
2. Stages 9-17 chick embryo somites were cultured on Millipore filter rafts
on nutrient agar, and scored for metachromatic matrix production.
3. Cartilage was differentiated in explants of each of these stages although no
'inducing tissues' (spinal cord and notochord) were present.
4. The incidence of cartilage varied with the culture conditions, matrix
development being favoured by foetal calf serum rather than horse serum, by
the addition of egg to the medium, by increased volume of the somite explant
and by the addition of a layer of liquid paraffin over the explant.
5. The evidence indicated that chick somites are determined for cartilage
differentiation as early as stage 9, and that following this, at least until stage 17,
there is a period of responsiveness to environmental conditions.
6. By comparison with published findings from other differentiating systems
Chondrogenesis in chick embryo
339
it is concluded that this environment-responsive phase demonstrates a stabilization phase following determination, during which the expression of latent
characteristics is actively permitted or prevented.
RESUME
Chondrogenese de somites d'embryon de Poulet cultives in vitro
1. Etude du developpement en culture organotypique de cellules chondrogenes du mesenchyme somitique dans le but d'etablir la distinction entre
l'induction de la differenciation et ses manifestations.
2. On etudie la reaction metachromatique des somites d'embryons des stades
9 a 17 cultives sur des filtres Millipore flottant a la surface du milieu de culture
gelose.
3. Du cartilage s'est differencie dans des explants de chacun de ces stades en
l'absence de tout tissu inducteur (tube nerveux et chorde).
4. La frequence de la formation de cartilage depend des conditions de
culture. La formation de substance intercellulaire est activee: par le serum de
veau plutot que par le serum de cheval; par l'addition d'oeuf au milieu; par
l'augmentation du volume de l'explant et par la presence d'une pellicule de
paraffine liquide couvrant l'explant.
5. Les resultats montrent que la differenciation chondrogene est determinee
dans des somites aussi jeunes que ceux du stade 9 et qu'il existe, jusqu'au stade
17, une periode de latence dependant des conditions externes.
6. En comparant ces resultats a ceux publies au sujet d'autres systemes
inducteurs on conclut que cette phase de latence prouve l'existence d'une phase
de stabilisation apres la determination. Pendant cette phase l'expression des
caracteres latents est realisee ou empechee.
This investigation has been supported by grants to the Chester Beatty Research Institute
(Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council
and the British Empire Cancer Campaign for Research, and by the Public Health Service
Research Grant no. CA-03188-08 from the National Cancer Institute, U.S. Public Health
Service.
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