J. Embryol. exp. Morph. 75, 165-188 (1983)
165
Printed in Great Britain © The Company of Biologists Limited 1983
Transfilter studies on the mechanism of epitheliomesenchymal interaction leading to chondrogenic
differentiation of neural crest cells
By LINDA SMITH AND PETER THOROGOOD 1
From the Department of Biology, University of Southampton
SUMMARY
Interaction with an epithelium is a prerequisite for avian cranial neural crest (NC) cells to
differentiate into cartilage and bone (Bee & Thorogood, 1980). In order to investigate the
causal mechanism we have selected one such interaction - that between mesencephalic NC
and retinal pigmented epithelium (RPE) for further study. Premigratory NC cells were grown
transfilter to RPE explants of different developmental ages and on Nuclepore filters of different pore size which either allowed or prevented penetration by cell processes. Initial
scanning electron microscopy (SEM) observations established that pores of 0-8/zm allowed
the passage of cell processes through the filter whereas 0-2 fim pores did not.
The transfilter experiments demonstrated that chondrogenic differentiation of NC cells will
occur only if the Nuclepore filters have a pore size large enough to permit the passage of cell
processes. Furthermore SEM observations established that cell processes do traverse the
Nuclepore filter when NC and RPE are grown in transfilter combination. The results indicate
that the mechanism is not mediated by diffusable factors but rather is mediated either by direct
contact between NC cells and non-diffusable matrix closely associated with RPE or by direct
plasmalemmal contact between RPE and NC cells through discontinuities in the basement
membrane. The results of these experiments also demonstrate that younger (stage 17) RPE
is more effective at eliciting chondrogenesis from premigratory NC cells than older (stage 24)
RPE and that the interaction between RPE and NC cells is a prolonged one, taking place over
days rather than within hours. Both of these in vitro observations are compatible with the
timing of events leading to scleral cartilage formation in vivo.
INTRODUCTION
In all types of vertebrate embryo studied to date it has been found that cranial
neural crest (NC) cells migrate extensively within the developing head, give rise
to much of the mesenchyme and later differentiate into various connective
tissues. As a consequence there is a major contribution by NC-derived cells to
the cranial and facial skeleton (Le Lievre, 1978; Morriss & Thorogood, 1978;
Noden, 1978). It has been demonstrated in the avian embryo that NC cells are
not 'committed'2 to a skeletogenic fate before migration and that instead they
1
Author's address: Department of Biology, Medical and Biological Sciences Building,
Bassett Crescent East, Southampton, SO9 3TU, U.K.
2
'committed' is used in the sense that isolated neural crest in culture cannot differentiate
skeletogenically.
166
L. SMITH AND P. THOROGOOD
require tissue interactions with epithelia encountered during migration or at the
presumptive site after migration if chondrogenic or osteogenic differentiation is
to ensue (Bee & Thorogood, 1980; Thorogood, 1981). The interaction between
mesencephalic NC cells and retinal pigmented epithelium (RPE) to form the
scleral cartilage of the avian eye is one such epithelio-mesenchymal interaction
and has been extensively studied at the phenomenological level (Weiss & Amprino, 1940; Amprino, 1951; Reinbold, 1968; Newsome, 1972; Stewart &
McCallion, 1975). NC cells migrate to the optic cup by 52 h of incubation (stage
14 - Hamburger & Hamilton, 1951) and condense to form the periocular mesenchyme (Noden, 1975, 1978). This mesenchyme is committed to chondrogenic
differentiation by 3 | to 4 days (Newsome, 1972) but the actual onset of chondrogenesis is not until day 7 (Romanoff, 1960) indicating that the RPE influence
is not required to maintain the chondrogenic potential of the ectomesenchyme.
Newsome analysed the mechanism of interaction between RPE and NC and
concluded that the mechanism was matrix-mediated (Newsome, 1976). This
conclusion was based on the fact that NC or periocular mesenchyme cells formed
cartilage when grown on niters bearing a distilled water lysate of cultured RPE
cells which, it was claimed, was composed principally of extracellular matrix
(ECM). However, in addition to matrix this lysate retained cytoskeletal components and membrane fragments (see fig. 4, Newsome, 1976) and hence these
experiments only demonstrate that living RPE cells need not be present for the
interaction to occur. However, there are at least three mechanisms (Saxen,
Ekblom & Thesleff, 1980) through which epithelio-mesenchyme interactions
could be mediated:
1. Transmission of signal substances by long range diffusion.
2. Action of extracellular matrix materials
i) If the morphogenetically active substance can move away from the matrix
and the basement membrane of the epithelium then this would be an example
of free diffusion of matrix components in the extracellular compartment,
ii) If the active molecules are a structural part of the matrix and are unable
to detach from it direct contact is required between the extracellular matrix
and the responding cells.
3. Interaction mediated by direct cell to cell contact, i.e. direct plasmalemmal
contact of epithelium cells with mesenchyme cells. The basement membrane of
the epithelium must be discontinuous for direct cell contact of the two cell
populations to occur.
To investigate more thoroughly the mechanism of interaction between RPE
and NC we adopted the transfilter culture technique which has been used to
analyse tissue interactions in amphibian primary induction (Toivonen & Wartiovaara, 1976; Toivonen, Tarin & Saxen, 1976); avian limb bud chondrogenesis
(Gumpel-Pinot, 1980,1981); lens induction (Karkinen-Jaaskelainen, 1978); cornea development (Hay & Meier, 1976); early mammalian tooth morphogenesis
Chondrogenic differentiation of neural crest cells
167
(Thesleff, Lehtonen, Wartiovaara & Saxen, 1977) and kidney tubule formation
(Wartiovaara, Nordling, Lehtonen & Saxen, 1974; Saxen etal. 1976). In essence
the technique involves the cultivation of the interacting tissues on opposite sides
of barrier membranes of known and constant pore size. Tissues can be combined
across such membranes whose pore size is known to permit or prevent the
passage of cell processes. Polycarbonate Nuclepore membranes are used in
which the pores are straight-through channels resembling bullet tracks and thus
if the filter is correctly orientated during sectioning it is possible to examine the
whole length of individual pores for the presence or absence of matrix components and cell processes traversing the filter.
In order to study the mechanism of tissue interaction in the present system
premigratory NC cells were grown transfilter to RPE explants of different
developmental ages and on different pore size filters. Scanning electron microscopy observations of the undersurface of Nuclepore membranes on which NC
or RPE explants had been grown for 24 or 48 h established which pore sizes
allowed penetration of cell processes. Pores of 0-8/an were found to allow the
passage of cell processes through the filter whereas 0-2 (jm pores did not allow
such penetration. It was assumed that both pore sizes allow soluble ECM components to diffuse through the filter. If chondrogenesis occurred in transfilter
cultures grown on Nuclepore filters that do not allow penetration of cell
processes, this would indicate that the mechanism is mediated by free diffusion
of extracellular matrix components or other signal substances. However, if differentiation occurred only in cultures grown on filters with pore sizes that allow
penetration by cell processes, this would demonstrate that either direct plasmalemmal contact of RPE and NC cells or direct contact between RPE nondiffusable matrix components and NC cells is necessary for the interaction to
occur.
MATERIALS AND METHODS
i) Source of material
All embryos used in this study were from eggs of the domestic fowl {Gallus
domesticus), White Leghorn x Rhode Island Red, obtained locally (Ross Poultry, U.K.) or from eggs of the Japanese quail {Coturnix coturnix japonica). Eggs
were incubated in a humid atmosphere in a forced draught incubator and at a
temperature of 38 ± 0-5 °C; all embryos were staged according to the developmental table of Hamburger & Hamilton (1951).
Initial dissections were carried out in sterile Dulbecco 'A' phosphate-buffered
saline (PBS) (Oxoid Ltd., England). To obtain neural crest tissue, stage-9+
embryos were removed from the vitelline membrane, placed in 'alpha' Eagle's
minimal essential medium («MEM) supplemented with 10 % foetal calf serum
(FCS), and the mesencephalic neural folds dissected out according to Bee &
Thorogood (1980) (and see fig. 3 in Thorogood, 1981). Stage-18 periocular
168
L. SMITH AND P. THOROGOOD
mesenchyme was dissected from the medial aspect of the optic cup of stage-18
embryos. Stage-17 presumptive RPE and stage-24 RPE and associated neural
retina and periocular mesenchyme were dissected from the medial aspect of the
optic cup (stage 17) or eye (stage 24) and incubated in sterile 0-25 % trypsin/
1-25 % pancreatin in calcium- and magnesium-free Tyrodes solution for 30min
at 4°C. Further enzyme activity was then blocked by adding an equal volume of
FCS and the RPE was then dissociated from the adjacent tissues by mechanical
separation.
ii) Culture techniques
Tissues were grown on a Nuclepore filter (marketed as 'Unipore' membrane
filters by Bio-Rad Laboratories, U.K.) supported by a stainless steel grid platform with a central hole to facilitate observation. This assembly was contained
within a 30 mm plastic culture dish containing 2-5 ml aMEM + 10%
FCS+ 100 units/ml penicillin and 100/ig/ml streptomycin. To maintain humidity the culture dishes were placed inside a glass bacteriological dish, the bottom
of which was covered with filter paper in which holes had been cut to accommodate two of the smaller dishes. The filter paper of the larger dish was then
saturated with sterile distilled water containing 1 % Fungizone (Gibco, U.K.).
To establish the standard transfilter cultures pieces of stage-24 RPE or stage17 RPE were placed individually on the Nuclepore filter and cultured for 48 h.
A drop of agar (1 % in aMEM) was then put on the RPE with a micropipette,
and the filter inverted. A fragment of premigratory mesencephalic NC or stage18 periocular mesenchyme was placed transfilter to the RPE and the combination culture cultured for 12 days at 37 °C in an atmosphere of 5 % CO2 in air, with
a complete medium change every 3-4 days. After 11 days of culture a drop of 1 %
agar in aMEM was put on the NC component to prevent the tissue being dislodged during processing. The culture was then fixed at 12 days and embedded
in paraffin wax for histology. In transfilter cultures chick stage-9 NC or stage-18
periocular mesenchyme was grown in combination with quail RPE so that if
necessary in later experiments the characteristic nucleolar marker of the quail
cells (Le Douarin, 1973) could be employed as a marker for the cells of the quail
tissue component. The apparently normal development of experimental chick
chimaeras testifies to the absence of any species barrier to normal development
in heterospecific combinations (Noden, 1975; Le Lievre & Le Douarin, 1975).
iii) Histology
Transfilter culture and control explants were fixed in 80 % ethanol, stained
with 1 % eosin in 80 % ethanol and dehydrated in an ethanol series, cleared in
toluene, embedded in 56°C m.p. paraffin wax and serially sectioned at 5-6(xm.
Sections were stained with alcian blue and durazol red; Hansen's haematoxylin
was used as a nuclear stain. Any cartilage or bone was identified by the blue- and
red-stained properties of their matrices and their characteristic morphology.
Chondrogenic differentiation of neural crest cells
169
Sections were photographed with a Zeiss Olympus photomicroscope using Ilford
PanF.
iv) Scanning electron microscopy
(a) Control explants
After 24 or 48 h of culture explants of neural crest, stage-18 periocular mesenchyme or stage-17 RPE grown alone on 0-8 /mi or 0-2 jum Nuclepore filters were
washed gently and briefly in sterile PBS and fixed for 45 min in 2-5 % glutaraldehyde in 0-1 M-sodium cacodylate buffer pH7-3 at room temperature. In order
to preserve extracellular matrix (ECM) components 0-5 % w/v of cetyl
pyridinium chloride (CPC) was added to the fixative when fixing RPE. The
explants were then washed in three changes of 0-2 M-buffer pH 7-3, post fixed for
1 h in 1 % osmic acid in 0-1 M-cacodylate buffer pH 7-3, washed in distilled water
and dehydrated through a graded series of ethanol (Gumpel-Pinot, 1980). The
specimens were critical-point dried using liquid CO2 and mounted on stubs so
that either the tissue or the underside of the filter opposite to the tissue was
visible, coated with gold and palladium in an SEM-PREP sputter coater and
observed in a JEOL JSM-P15 Scanning Electron Microscope.
(b) 10\xm sections through transfdter cultures
Transfilter combinations that had been cultured for 5 days were washed gently
in PBS, then fixed for 2h in 2-5% glutaraldehyde in 0-1 M-cacodylate
buffer + 0-5 % CPC pH7-3 at room temperature.
Specimens were then washed and dehydrated as described above, cleared in
toluene and embedded in paraffin wax (Kaprio, 1977). The cultures were then
sectioned at lOjum. Alternate sequences of 10-20 sections were then mounted
on slides for standard histological analysis or attached to 11 mm small round glass
cover slips (for SEM analysis) with a 1 % gelatin solution. Sections for SEM
analysis were then dewaxed in three changes of toluene, dehydrated in a 1:1
toluene: ethanol mixture followed by three changes of absolute ethanol, critical
point dried and coated with gold and palladium as described above.
RESULTS
A) Control cultures
i) Histology and light microscopy
Single explants of premigratory mesencephalic NC, stage-18 and stage-24
periocular mesenchyme and stage-17 and stage-24 RPE were each grown alone
in organ culture for 12 days (see Table 1). Cultures of isolated NC and stage-18
periocular mesenchyme consisted predominantly of small patches of unpigmented mesenchyme cells; patches of melanocytes were sometimes present in NC
cultures. In a small number of NC cultures some mesenchyme cells had an
170
L. SMITH AND P. THOROGOOD
Table 1. Percentage incidence of cartilage formation in retinal pigmented
epithelium (RPE), neural crest (NC) andperiocularmesenchyme control cultures.
Culture type
Nuclepore filter
pore size (/im)
Number of
cultures set up
Cartilage
% Cartilage
NC
NC
Stage-18 perioc. mes.
Stage-24 perioc. mes.
Stage-17 RPE
Stage-24 RPE
Stage-24 RPE
0-8
0-2
0-8
0-8
0-8
0-8
0-2
16
8
5
23
10
18
8
0
0
0
22
0
0
0
0
0
0
96
0
0
0
.
epithelioid morphology and were organized into vesicular structures; the lumen
of such vesicles usually contained material which stained weakly with alcian blue.
Stage-24 periocular mesenchyme control cultures grew into large masses of tissue
containing a range of differentiated cell types, namely cartilage with an incidence
of 96 %, bone - 65 %, melanocytes - 100 %, and muscle - 52 % (see Fig. 1).
Stage-17 RPE cultures consisted of flattened masses of unpigmented, undifferentiated cells whereas stage-24 RPE cultures consisted of large masses of
pigmented and unpigmented cells. The RPE of both developmental ages
sometimes contained neural retina (NR). This had arisen during the culture
period as care had been taken to explant RPE alone and it is well established that
RPE is able to transdifferentiate into NR in culture (Tsunematsu & Coulombre,
1981). ECM was sometimes present on the underside of 0-8/im filters but never
when the tissue was grown on 0-2/im filters (see later).
These experiments demonstrate that premigratory NC, stage-18 periocular
mesenchyme, stage-17 and stage-24 RPE are incapable of forming cartilage
when grown alone in organ culture. However cultures of stage-24 periocular
mesenchyme, which has been shown to be committed to chondrogenesis
(Newsome, 1972), regularly formed cartilage thus demonstrating that the culture
conditions used in this study are permissive for chondrogenic differentiation.
ii) Scanning electron microscopy
a) In order to establish whether there are differences in the behaviour of
neural crest cells grown on 0-8/im and 0-2 /im filters, single NC explants were
cultured on 0-8/im and 0-2/im Nuclepore filters for 24 or 48 h and the lower or
upper surfaces of these filters were then examined using SEM.
Observations on the upper side of 0-8/im and 0-2/im filters on which NC had
been cultured revealed a layer of loosely associated cells which were often sperical and had many filopodia extending from them (see Fig. 2). Examination of the
upper side of 0-8/im filters revealed filopodia from crest cells extending down
through the pores of the filter (see Fig. 3). Sometimes many cell processes were
Chondrogenic differentiation of neural crest cells
111
Fig. 1. Stage-24 periocular mesenchyme control culture grown on a 0Nuclepore filter for 12 days. Cartilage (c) has formed at a distance from the filter;
Melanocytes (m); Nuclepore filter (n). Bar = 20/mi.
Fig. 2. A scanning electron micrograph of a single explant of premigratory NC
grown for 48 h on a 0-8 jum Nuclepore filter. Bar = 10 ;um.
Fig. 3. A scanning electron micrograph showing filopodia (f) from NC cells grown
on a 0-8 jum Nuclepore filter for 48 h extending down into the pores of the filter.
Bar =
Fig. 4. A scanning electron micrograph showing thatfilopodiaextended by NC cells
grown on 0-2 jumfiltersfor 48 h do not extend into the pores; instead the ends of the
filopodia are club shaped (arrows). Bar = 2jum.
172
L. SMITH AND P. THOROGOOD
Figs 5-7.
Chondrogenic differentiation of neural crest cells
173
observed extending from a single cell. Identical filopodia were extended by cells
grown on 0-2/im filters but were never observed extending into the smaller
pores, instead the ends of the filopodia rounded up (see Fig. 4).
Predictably, cell processes were never observed projecting out of the pores on
the underside of 0-2 jm\ filters (see Fig. 5) but were regularly observed on the
underside of 0-8 //m filters on which NC had been cultured for 24 h or 48 h (Fig.
6). The density of cell processes observed appeared to be the same at these two
time intervals. Typically they comprised a filopodium which could be seen within
the pore and which then bulged to form a bulbous end on the undersurface of the
filter (see Fig. 7).
b) In order to establish whether stage-17 RPE deposited different amounts
of ECM through the pores of 0-8 or 0-2 /im Nuclepore filters single stage-17 RPE
explants were cultured on these filters for 24 h or 48 h. The lower surfaces of
these filters were then examined noting the relative abundance and organization
of any extracellular matrix. The experimental technique was the same as that
used for other control cultures, with the addition of CPC to the glutaraldehyde
fixative in order to preserve ECM components.
Neither cell processes nor extracellular matrix were observed on the lower
surface of 0-2/im filters on which RPE had been cultured for 24 or 48 h. However, since ECM was observed between the RPE and the upper surface of the
filter in such cultures (not shown) it is possible that soluble ECM components do
pass through the pores of the filter but in the absence of cell processes may diffuse
into the culture medium rather than consolidate into a matrix. Small quantities
of ECM associated with a few cell processes were present on the lower surface
of 0-8 jum filters on which RPE had been cultured for 24 h. Larger quantities of
Figs 5-10. Scanning Electron Micrographs.
Fig. 5. The underside of a 0-2//m filter on which NC has been cultured for 48 h
showing the absence of cell processes projecting out of the pores (compare with Fig.
5). Bar = ljum.
Fig. 6. Cell process on the underside of a 0- 8 jumfilteron which NC has been cultured
for 24h. Bar = 4jum.
Fig. 7. Each cell process consists of afilopodiumwhich can be seen within the pore
and which then bulges to form a bulbous end on the undersurface of the filter.
Bar = 0-5jUm.
Fig. 8. Large quantities of matrix including sheet-like (s), granular (g) and fibrous
components (arrows) on the lower surface of 0-8 jumfilterson which stage-17 RPE
had been cultured for 48 h and fixed in the presence of CPC. Bar = 5 jitm.
Fig. 9. Similar specimen to that shown in Fig. 8. All types of matrix component are
usually associated with bulbous cell processes (cp). Bar = 2 ^ .
Fig. 10. The underside of a 0-8/an filter on which stage-17 RPE has been cultured
for 48 h and fixed in the absence of CPC; far less ECM is retained and consequently
cell processes are easier to observe. These vary in morphology from individual
processes to groups of closely associated, large, bulbous structures often covered
with small vesicles (v). Any matrix that is retained is associated with these groups of
cell processes. Bar = 4jum.
174
L. SMITH AND P. THOROGOOD
VI
8
**•*
EO
Figs 8-10. For legend see p. 173.
Chondrogenic differentiation of neural crest cells
175
matrix including sheet-like, granular and fibrous components were observed at
48 h and all types of matrix components were usually associated with cell
processes (Fig. 8). The ECM was localized in the region of the tissue and was
often well organized (see Fig. 9). The unexpected presence of cell processes in
such cultures is probably the result of the temporary removal of the basement
membrane during tissue dissociation and prior to its regeneration in culture.
If the tissue is fixed without CPC included in the fixative, far less ECM is
retained on the underside of the filter and consequently cell processes are far
easier to observe. With RPE cultured on 0-2 panfiltersthere was little difference
due to the relative paucity of matrix in such cultures but with cultures on 0-8 pan
filters the removal of much of the abundant matrix revealed the presence of many
cell processes. These varied in morphology from individual processes to groups
of closely associated large bulbous structures often covered with small vesicles
(see Fig. 10). Any matrix that was retained was associated with these groups of
cell processes. The association of vesicles and ECM with cell processes may
reflect the involvement of such processes in the synthesis and organization of
matrix.
B) Transfiltercultures
Mesencephalic NC and stage-17 or stage-24 RPE were cultured in transfilter
combination on OS pan or 0-2 pan Nuclepore filters for 12 days to establish
whether the interaction between NC and RPE is mediated by diffusable factors
or cell contact.
i) Histology and light microscopy
Regardless of the developmental age of the RPE at the outset of the experiment there was virtually no cartilage formation in transfilter cultures grown on
0-2 pan Nuclepore filters (see Fig. 11 and Table 2). In marked contrast chondrogenesis did occur in transfilter cultures on 0-8 panfilters(see Figs 12 and 13 and
Table 2) but the incidence of differentiation in the NC component was related
Table 2. Percentage incidence of cartilage formation in RPE/NC transfilter cultures using stage-17 or stage-24 RPE and grown on nuclepore filters of0-8 pan or
0-2 \im pore size.
Culture type
Stage-17 RPE/NC
Stage-17 RPE/NC
Stage-24 RPE/NC
Stage-24 RPE/NC
Nuclepore filter Number of
pore size (jum)
cultures set up
0-8
0-2
0-8
0-2
20
12
43
17
Cartilage*
% Cartilage
12
0
9
1
60
0
21
6
*Any cultures which were found to have epithelial contamination in the NC component
were discounted and are excluded from the above table.
176
L. SMITH AND P. THOROGOOD
nc
rpe
!"
i »*-
nf^ai1
13
Figs 11-13.
Chondrogenic differentiation of neural crest cells
111
to the age of RPE used. When stage-17 RPE was used as the epithelial component a dramatically improved growth of NC and higher incidence of cartilage
formation compared to that in stage-24 RPE/NC transfilter cultures was observed (see Figs 12 and 13). These results suggest firstly that stage-17 RPE is a more
potent stimulator of chondrogenesis than stage-24 RPE and secondly that the
mechanism of interaction between RPE and NC is not diffusion-mediated and
is instead mediated either by contact of NC cells with a non-diffusable matrix
component of the RPE or by direct plasmalemmal contact of NC cells with RPE
cells (see Discussion). The RPE component in transfilter cultures had the same
histological appearance as RPE controls. Both stage-17 RPE and stage-24 RPE
always grew well and sometimes contained NR. Cartilage was found in cultures
containing both RPE and NR and also in cultures in which the neural tissue was
absent. Since cartilage was seen to develop in the absence of NR it is clear that
the development of cartilage is related to the presence of RPE.
Cartilage always formed against the filter in transfilter cultures (see Figs 12
and 13) which suggests that NC formed cartilage in response to RPE on the other
side of the filter. In contrast the cartilage that formed in stage-24 periocular
mesenchyme did not always form adjacent to the filter (see Fig. 1). Examination
of serial sections revealed that in both types of culture cartilage always formed
in plates or nodules - never in rods. The growth of the crest component was
variable. Occasionally in both stage-17 RPE/NC and stage-24 RPE/NC cultures
the NC grew very well but remained undifferentiated (Fig. 14), whereas in other
cultures the NC grew far less but nevertheless differentiated into cartilage.
Therefore, ectomesenchymal mass appears to be unrelated to whether or not
cartilage formation occurs although cell packing density may be.
ii) SEM of sections through transfilter cultures
This experiment was designed to investigate the contact relationships between
NC and stage-17 RPE cells when grown together in transfilter culture on 0-8/im
filters. Standard transfilter cultures were fixed at 5 days for SEM, embedded in
wax and sectioned at lO^um; dewaxed sections were then prepared for SEM to
allow examination of the interface between the two tissues within the filter.
Most of the pores of the filter in the region of the tissues contained cellular
and/or extracellular material (see Figs 15, 16,17). Thin cell processes, similar
in appearance to the filopodia observed in control culture, were frequently
Fig. 11. Stage-24 RPE/NC transfilter culture grown on a 0-2 jum filter for 12 days.
Note absence of cartilage formation and poor growth of NC. Neural crest (nc);
Retinal pigmented epithelium (rpe); nucleopore filter (n). Alcian Blue/Durazol
Red and Hansen's haematoxylin. Bar = 20jum.
Fig. 12. Stage-17 RPE/NC transfilter culture grown on a 0-8 panfilterfor 12 days.
A large plate of cartilage (c) has formed in NC adjacent to the filter. Bar = 20 pan.
Fig. 13. Stage-24 RPE/NC transfilter culture grown on a 0-8pan filter for 12 days.
A nodule of cartilage (c) has formed adjacent to the filter. Bar = 20pirn.
178
L. SMITH AND P. THOROGOOD
<,
rpe
Fig. 14. Stage-25 RPE/NC transfilter culture grown on a 0-8/im filter (n) for 12
days. The NC has grown very well but remains undifferentiated. Neural crest (nc);
Retinal pigmented epithelium (rpe). Bar = 20/an.
observed in the pores (compare Figs 3, 17). Cell processes were occasionally
observed traversing the whole width of the filter. However the incidence with
which they were observed was reduced by the fact that the probability of a single
section having a cut pore in it that traverses the whole width of the filter and
contains a cell process is low. It is probable that most cell processes extended by
the cells into the pores traverse the filter. Cell processes from NC cells were
frequently seen projecting into pores and occasionally cell processes were
associated with RPE cells but it was not possible to tell whether or not they were
Fig. 15. Scanning electron micrograph of a lOjum section of a stage-17 RPE/NC
transfilter culture grown on a 0-8 jum filter for 5 days. Note that some pores are
sectioned obliquely (large arrow) and others are sectioned along their entire length
(small arrow). Neural crest (nc); Retinal pigmented epithelium (rpe). Bar = 4^m.
Fig. 16. Scanning electron micrograph of a 10 pan section of a stage-17 RPE/NC
transfilter culture grown on a 0-8 panfilterfor 5 days. Note ECM within the pores of
the filter (arrows). Neural crest (nc). Bar = 2jum.
Fig. 17. Scanning electron micrograph of a lO^um section of a stage-17 RPE/NC
transfilter culture grown on a 0-8 panfilterfor 5 days. Note cell processes within pore
traversing the width of the filter (arrows). In bottom right, note RPE is detached
from filter and a cell process has been broken during processing of the specimen.
Neural crest (nc). Bar =:
Chondrogenic differentiation of neural crest cells
rpe
15
nc
16
nc
V.
Figs 15-17.
179
180
L. SMITH AND P. THOROGOOD
being extended by these cells. Sometimes NC cell processes were observed
extending through a pore in the filter and spreading out on the surface of an RPE
cell. It was not possible using this technique to establish whether direct plasmalemmal contact occurred through discontinuities in the basement membrane or
alternatively if NC cells made contact with RPE-associated matrix. However
these observations do provide evidence that a close association between NC and
RPE cells is established during their interaction in a transfilter culture and is
achieved by the passage of cell processes across the filter. Evidence from this
study suggests that NC cell processes pass through the filter to contact the RPE
but we cannot rule out the possibility that RPE cell processes may also participate in the interaction.
C) 'Timing' experiment
This experiment was designed to establish the minimum period of time necessary for the interaction to take place across a 0-8 jum filter. Neural crest was
grown transfilter to stage-17 RPE for different lengths of time (7 days, 4 days,
2 days, 24 h). After these time intervals the RPE was removed from the transfilter cultures and the neural crest grown alone until a total culture period of 14 days
had elapsed. When the duration of co-culture was 7 days the % incidence of
cartilage formation was comparable to that found in standard transfilter cultures
(see previous section). However, as the length of time of co-culture was
decreased the % incidence of cartilage formation decreased rapidly (see Table
3). These results suggest that the interaction between RPE and NC is prolonged
and is not completed in a short time.
When cartilage developed in these cultures it always formed against the filter
and the amount that formed was generally less than in standard transfilter cultures. When NC was exposed to RPE for 7 days the cartilage that formed was
fairly well organized but as the duration of co-culture decreased the cartilage
Table 3. Percentage incidence of cartilage formation in NC component cultured
in RPE/NC transfilter combination for different durations (7 days, 4 days, 2 days,
24 h) and then grown alone until a total culture period of 14 days had elapsed.
Duration of RPE/NC
transfilter culture
7 days
4 days
2 days
24 h
Number of
cultures
Number of cultures
forming cartilagef
% number of cultures
forming cartilage
9 (14)*
12 (15)*
12 (14)*
16 (19)*
5
2
1
1
56
17
* The number of cultures in brackets includes those which were subsequently found to have
small patches of RPE remaining on the other side of thefilter- these cultures are considered
invalid and are not included in the estimation of % cartilage formation.
t As for Table 2 any cultures in which the NC component was found to have epithelial
contamination were discounted and are excluded from the above table.
Chondrogenic differentiation of neural crest cells
181
Table 4. Percentage incidence of cartilage formation in NC grown on diff usable
matrix previously deposited by stage-17 RPE.
Number of cultures
Nuclepore filter
pore size (jum)
Cartilage
% Cartilage
16
0-8
0
0
which did form was less well organized. This observation suggests that continuous presence of the epithelium may be required for well-organized cartilage
to form.
D) 'Matrix' experiment
As described earlier when RPE was grown alone on a 0-8/xm Nuclepore filter
extracellular matrix was present on the underside of the filter - it was visible with
light microscopy after haematoxylin, alcian blue and durazol red staining and
SEM observations on RPE controls grown for 24 or 48 h on 0-8/im filters
revealed that this ECM consisted of sheet-like, granular and fibrous components
(see Fig. 9). Furthermore, ECM was never observed on the underside of the
filter with both light microscopy and SEM when RPE was grown on 0-2 /jm
filters. The experiment was designed to investigate whether this matrix, components of which had diffused through from the epithelium on the other side of
the filter, was capable of 'inducing' NC cells to differentiate into cartilage.
Individual explants of stage-17 RPE were grown on 0-8 fjaa Nuclepore filters for
approximately 2 weeks and the position of the RPE was marked on the filters
with insoluble ink. The RPE was then removed and the filter inverted. Individual
explants of NC were placed on the filters using the reference points to locate the
former position of the RPE; i. e. the NC was put on top of the matrix that the RPE
had deposited through the filter (see Table 4).
The results of this experiment demonstrated that the ECM deposited through
a 0-8/im filter by stage-17 explants cultured alone for 12-14 days is not capable
of 'inducing' NC cells to form cartilage. The majority of the cultures had a
flattened pigmented outgrowth which often had discrete loci of darkly pigmented
cells present in it. These loci have never been observed in NC control cultures
and the incidence of melanogenesis was considerably higher than in NC control
cultures. Possibly a component of this matrix induces melanocyte differentiation
as extracellular materials have been shown to influence trunk neural crest cell
differentiation in vitro (Loring, Glimelius & Weston, 1982). The lack of chondrogenesis provides further evidence that the interaction between stage-17 RPE
and NC is not mediated by a diff usable matrix component. However, it should
be noted that the matrix to which the NC is exposed has accumulated over a 12to 14-day culture period and may not necessarily be identical in composition to
182
L. SMITH AND P. THOROGOOD
that which the NC cells would encounter at the appropriate developmental time
in vivo or indeed at the outset of the standard transfilter cultures described
earlier.
DISCUSSION
Previous work has established that NC cells are relatively non-specific with
respect to the type of epithelium that will promote chondrogenesis (Bee &
Thorogood, 1980). A similar lack of specificity has also been demonstrated in the
formation of mandibular membrane bone from ectomesenchyme (Hall, 1981;
Tyler & McCobb, 1980). This lack of specificity in the nature of the epithelium
in these interactions suggests that they may be 'permissive' (Wessells, 1977) and
this interpretation is supported by the fact that such epithelia do not elicit chondrogenesis from tissues other than cranial NC or ectomesenchyme derived from
it. For example, RPE does not 'induce' cartilage formation in chorioallantoic
mesenchyme (Stewart & McCallion, 1975); if the interaction was 'instructive'
RPE would direct the extraembryonic mesenchyme to form cartilage. The
results of the present experiments clearly demonstrate that a permissive interaction leading to the chondrogenic differentiation of NC cells will occur only if
the Nuclepore filters have a pore size large enough to permit the passage of cell
processes. Furthermore, SEM observations have established that cell processes
do actually traverse the Nuclepore filter when NC and RPE are grown in transfilter combination. We conclude that the mechanism is not mediated by diffusable
factors but rather is mediated either by direct contact between NC cells and nondiffusable matrix closely associated with RPE or by direct plasmalemmal contact
between RPE and NC cells through discontinuities in the basement membrane.
In the light of this conclusion the claim (Newsome, 1976) that the interaction
between the two tissues is simply 'matrix-mediated' is not tenable. Either that
interpretation will need to be further qualified as we have proposed here (i.e.
non-diffusable matrix) or the alternative possibility considered, that the
contamination of the isolated matrix by cellular debris (see fig. 4 Newsome,
1976) led to a false positive result.
We have also demonstrated that younger (stage-17) RPE is more effective at
eliciting chondrogenesis from premigratory NC cells than older (stage-24) RPE,
in agreement with previous observations (Newsome, 1976). These observations
are compatible with the sequence of events leading to scleral cartilage formation
in vivo. NC cells arrive at the periocular region at stage 14 and are committed
for chondrogenesis by stage 19 (Newsome, 1972). Hence, we would expect RPE
to be most 'inductively' active between stage 14-19. The apparent loss of inductive ability in the older RPE could be the expression of two possible control
mechanisms. Firstly as the RPE becomes older and differentiates it loses its
inductive ability: for instance it may produce a qualitatively different matrix that
is less efficient at stimulating chondrogenesis. Alternatively as the epithelium
Chondrogenic differentiation of neural crest cells
183
grows older it may acquire inhibitory powers (Solursh, Singley & Reiter, 1981).
The two proposed mechanisms are not mutually exclusive and could operate
simultaneously.
The possibility of inhibition of cartilage formation may explain the apparent
'induction at a distance' which characterizes all epithelio-mesenchyme interactions leading to skeletogenesis. The differentiated tissue, be it cartilage (Bee
& Thorogood, 1980; Gumpel-Pinot, 1981) or bone (Hall, 1981) arises at a distance from the epithelium within the mesenchyme, e.g. in vivo scleral cartilage
is separated from the RPE by mesenchyme which becomes the fibrous choroid
coat of the eye. Either the interface between epithelium and mesenchyme
becomes occupied by another (migratory?) cell population after the interaction
has been completed or the acquisition of short-range chondrogenesis-inhibiting
ability by the epithelium might prevent adjacent mesenchyme cells from completing their differentiation into cartilage; a flbroblastic phenotype would result
at the interface.
RPE matrix components which are freely diffusable and can pass through a
0-8 /im filter and consolidate to form a matrix on the opposite side (see Figs 8 and
9) cannot elicit a chondrogenic response from NC cells (see Table 4). If the
interaction is matrix-mediated rather than cell contact-mediated then clearly
matrix components which are not mobile in this way are involved. We have
referred to these putative factors as 'non-diffusable matrix components' and it
is not obvious which molecules might be involved. RPE is unusual in that in vitro
it produces a matrix of fibrillar, banded collagen in addition to its basement
membrane (Newsome & Kenyon, 1973). As we can see no reason why
procollagen would not pass freely through a Nuclepore filter and self-assemble
in a matrix (see Fig. 9) and as such a matrix has been shown to be inactive, we
feel that the most likely candidate is the basement membrane which characteristically remains associated with the basal surface of an epithelium both in vivo and
(after regeneration) in vitro (Newsome & Kenyon, 1973). Indeed the importance
of mesenchymal cell contacts with the epithelial basement membrane has been
established in early tooth morphogenesis (Thesleff, Lehtonen & Saxen, 1978)
and in corneal epithelial differentiation (Hay & Meier, 1976). It has been
proposed, but not yet established, that a number of other epithelio-mesenchymal
interactions are mediated via mesenchymal cell contact with epithelial basement
membrane; e.g. cartilage formation in the avian limb bud (Gumpel-Pinot, 1981)
and mandibular membrane bone formation (Hall & Van Exan, 1982). Precisely
which component of the basement membrane is involved in any such interaction
is not known and it is gradually being acknowledged that isolated purified matrix
components cannot necessarily substitute for intact ECM. Newsome (1976) was
unable to induce NC cell chondrogenesis with type I collagen and although
sclerotomal cells will respond to chondroitin-4-sulphate, chondroitin-6-sulphate,
type II collagen or proteoglycan by increased synthesis of matrix (Kosher, Lash
& Minor, 1973; Kosher & Church, 1975; Lash & Vasan, 1978) it has not yet been
184
L. SMITH AND P. THOROGOOD
possible to stimulate chondrogenesis from sclerotomal cells not already displaying limited synthesis of matrix components. Thus it appears that a normally
organized and naturally synthesized matrix is a prerequisite if an interaction is
to occur; in fact a matrix may only display morphogenetic potential in the
presence of the living cells which have produced it and not when isolated on a
filter. However it should be noted that the present results do not enable us to
distinguish between the mechanism being mediated by the basement membrane
or by actual plasmalemmal contact between the two cell populations.
The results presented also indicate that the interaction between RPE and NC
cells is a prolonged one (see Table 3) and these in vitro observations are not
incompatible with the timing of events leading to scleral cartilage formation in
vivo. In the avian embryo NC cells start migrating from the tips of the neural
folds at approximately stage 10 (36 h incubation), arrive at the periocular region
at stage 14 (50-53 h) (Noden, 1975) and the periocular mesenchyme cells are
committed to forming cartilage by stage 19 ( 3 - 3 | days) (Newsome, 1972). As the
NC cells used in these experiments are premigratory NC cells it could be predicted that the interaction in vitro would be completed in approximately 2-3 days.
However it was observed that the transfilter presence of RPE was needed for 7
days before the incidence of chondrogenesis was comparable to that found with
the standard transfilter cultures. This longer period is probably artefactual and
may result from either trauma during tissue isolation or a general retardation of
growth and differentiation by the culture conditions. It should be noted that the
RPE is isolated by an enzyme treatment which removes the basement membrane
and as a consequence this experiment may, to some extent, measure the ability
of the epithelium to regenerate its basement membrane. In addition, we do not
know the effects of the migratory environment that the NC cells would normally
experience 'en route' to the periocular region. Nevertheless, we predict that the
interaction between RPE and NC in vivo is a fairly prolonged interaction of the
order of days and not hours as in some systems such as the neuralizing effect of
the dorsal mesoderm on the presumptive neurectoderm of the urodele gastrula
which is completed after 10 h (Toivonen & Wartiovaara, 1976).
The cartilage formed in these experiments generally displayed a tissue-specific
morphogenesis. In control cultures of stage-24 periocular mesenchyme, a tissue
that is already committed to chondrogenesis having undergone an interaction in
vivo, cartilage formed in plates or nodules away from the filter (see Fig. 1). In
transfilter cultures of NC and RPE, where the interaction takes place in vitro, the
cartilage likewise formed in plates or nodules, never in rods, and always formed
adjacent to the filter (see Figs 12, 13). Morphologically, the scleral cartilage in
the chick consists of a thin, curved plate of cartilage and our results and previous
observations suggest that the basic form of cartilage is intrinsically determined
(Weiss & Moscona, 1958; Newsome, 1972). Similarly cultured limb mesenchyme
forms cartilage in rods (Weiss & Moscona, 1958; Gumpel-Pinot, 1981) resembling the long bone primordia which would have formed in vivo. We suggest that
Chondrogenic differentiation of neural crest cells
185
epithelio-mesenchyme interactions underlying skeletogenesis not only determine the differentiative fate of the responding cells but endow the tissue with
morphogenetic autonomy. In fact the results of the 'timing' experiment indicate
that the presence of an epithelium is required for well-organized cartilage to form
because as the duration of co-culture of RPE and NC decreased, the cartilage
which did form was increasingly less organized. The presence of an epithelium
may be necessary for a critical time in order to organize the cells of the blastema
in some characteristic way which is later expressed as a tissue-specific morphogenesis (Thorogood, 1982).
Observations on epithelio-mesenchymal interactions indicate that the means
of transmitting the 'inductive' stimulus and the nature of that stimulus vary
considerably. Nevertheless, in the only other transfilter analysis of an interaction causing chondrogenic differentiation (limb-bud chondrogenesis in chick
embryos (Gumpel-Pinot, 1980, 1981)) it was established that close contact between epithelium and mesenchyme was necessary for cartilage formation to
occur. In spite of apparent differences at the tissue and cell level of organization
between somite chondrogenesis, limb chondrogenesis and cranial NC chondrogenesis it is possible that all are effected by the same molecular mechanism.
However, given the very different developmental histories of the mesenchymes
involved and our rudimentary understanding of most tissue interactions at the
molecular level, it would be unwise at this stage to assume that the causal
mechanisms in the different chondrogenic systems are necessarily identical or
even similar.
This work has been supported by the Medical Research Council (U.K.). Chris Hawkins'
help enabled us to do the scanning electron microscopy, Norman Sylvester gave advice on the
Appendix and David Johnston read the manuscript. Thank you.
REFERENCES
R. (1951). Developmental correlations between the eye and associated structures.
/. exp. Zool. 118, 71-99.
BEE, J. A. & THOROGOOD, P. V. (1980). The role of tissue interactions in the skeletogenic
differentiation of avian neural crest cells. Devi Biol. 78, 47-62.
GUMPEL-PINOT, M. (1980). Ectoderm and mesoderm interactions in the limb bud of the chick
embryo studied by transfilter cultures, cartilage differentiation and ultrastructural observations. /. Embryol. exp. Morph. 59, 157-173.
GUMPEL-PINOT, M. (1981). Ectoderm-mesoderm interactions in relation to limb bud chondrogenesis in the chick embryo: transfilter culture and ultrastructural studies. /. Embryol. exp.
Morph. 65, 73-87.
HALL, B. K. (1981). The induction of neural crest derived cartilage and bone by embryonic
epithelia. An analysis of the mode of action of an epithelial-mesenchymal interaction. /.
Embryol. exp. Morph. 64, 305-310.
HALL, B. K. & VAN EXAN, R. J. (1982). Induction of bone by epithelial cell products. J.
Embryol. exp. Morph. 69, 37-46.
HAY, E. D. & MEIER, S. (1976). Stimulation of corneal differentiation by interaction between
cell surface and extracellular matrix. II. Further studies on the nature and site of transfilter
induction. Devi Biol. 52, 141-157.
AMPRINO,
186
L. SMITH AND P. THOROGOOD
V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morphol. 88, 49-92.
KAPRIO, E. A. (1977). Ectoderm-mesenchymal interspace during the formation of the chick
leg bud. A Scanning and Transmission Electron Microscope study. Wilhelm Roux' Arch.
devlBiol. 182, 213-225.
KARKINEN-JAASKELAINEN, M. (1978). Transfilter lens induction in avian embryos. Differentiation 12, 31-37.
KOSHER, R. A., LASH, J. W. & MINOR, R. R. (1973). Environmental enhancement of in vitro
chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein. Devi Biol. 35, 210-220.
KOSHER, R. A. & CHURCH, R. L. (1975). Stimulation of in vitro somite chondrogenesis by
procollagen and collagen. Nature 258, 327-329.
LASH, J. W. & VASAN, N. S. (1978). Somite chondrogenesis in vitro stimulation by exogenous
extracellular matrix components. DevlBiol. 66, 151-171.
LE DOUARIN, N. M. (1973). A feulgen positive nucleolus. Expl Cell Res. 77, 459-468.
LE LIEVRE, C. S. & LE DOUARIN, N. (1975). Mesenchymal derivatives of the neural crest:
analysis of chimaeric quail and chick embryos. /. Embryol. exp. Morph. 34, 125-154.
LE LIEVRE, C. S. (1978). Participation of neural crest-derived cells in the genesis of the skull
in birds. J. Embryol. exp. Morph. 47, 17-37.
LORING, J., GLIMELIUS, B. & WESTON, J. A. (1982). Extracellular matrix materials influence
quail neural crest cell differentiation in vitro. Devi Biol. 90, 165-174.
MORRISS, G. M. & THOROGOOD, P. V. (1978). An approach to cranial neural crest migration
and differentiation in mammalian embryos. In 'Development in Mammals' 3, 363-412, (ed.
M. H. Johnson). Amsterdam: Elsevier North-Holland Biomedical Press.
NEWSOME, D. A. (1972). Cartilage induction by retinal pigmented epithelium of chick embryo. Devi Biol. 27, 575-579.
NEWSOME, D. A. (1976). In vitro stimulation of cartilage in embryonic chick neural crest cells
by products of retinal pigmented epithelium. Devi Biol. 49, 497-507.
NEWSOME, D. A. & KENYON, K. R. (1973). Collagen production in vitro by the retinal pigmented epithelium of the chick embryo. Devi Biol. 32, 387-400.
NODEN, D. (1975). An analysis of the migratory behaviour of avian cephalic neural crest cells.
Devi Biol. 42, 106-130.
NODEN, D. (1978). The control of avian cephalic neural crest cyto-differentiation. I. Skeletal
and connective tissues. Devi Biol. 69, 296-312.
REINBOLD, R. (1968). Role du tapetum dans la differentiation de la sclerotique chez l'embryon de poulet. /. Embryol. exp. Morph. 19, 43-47.
ROMANOFF, A. L. (1960). The Avian Embryo. New York: Macmillan.
HAMBURGER,
SAXEN, L., LEHTONEN, E., KARKINEN-JAASKELAINEN, M., NORDLING, S. & WARTIOVAARA, J.
(1976). Are morphogenetic tissue interactions mediated by transmissible signal substances
or through cell contacts? Nature 259, 662-663.
SAXEN, L., EKBLOM, P. & THESLEFF, I. (1980). Mechanisms of morphogenetic cell interactions. In 'Development in Mammals' 4, 161-202 (ed. M. H. Johnson). Amsterdam:
Elsevier, North Holland, Biomedical Press.
SOLURSH, M., SINGLEY, C. T. & REITER, R. S. (1981). The influence of epithelia on cartilage
and loose connective tissue formation by limb mesenchyme cultures. Devi Biol. 86,
471-482.
STEWART, P. A. & MCCALLION, D. J. (1975). Establishment of the scleral cartilage in the chick.
Devi Biol. 46, 383-389.
THESLEFF, I., LEHTONEN, E. & SAXEN, L. (1978). Basement membrane formation in transfilter
tooth culture and its relation to odontoblast differentiation. Differentiation 10, 71-79.
THESLEFF, I., LEHTONEN, E., WARTIOVAARA, J. & SAXEN, L. (1977). Interference of tooth
differentiation with interposed filters. DevlBiol. 58, 197-203.
THOROGOOD, P. V. (1981). Neural crest cells and skeletogenesis in vertebrate embryos. Histochem. J. 13, 631-642.
THOROGOOD, P. V. (1982). The morphogenesis of cartilage. In The Biology of Cartilage. Vol.
II (ed. B. K. Hall). New York: Academic Press. In Press.
Chondrogenic differentiation of neural crest cells
187
TOIVONEN, S., TARIN, D.
& SAXEN, L. (1976). The transmission of morphogenetic signals from
amphibian mesoderm to ectoderm in primary induction. Differentiation 5, 49-55.
TOIVONEN, S. & WARTIOVAARA, J. (1976). Mechanisms of cell interaction during primary
embryonic induction studied in transfilter experiments. Differentiation 5, 61-66.
TSUNEMATSU, Y. & COULOMBRE, A. J. (1981). Demonstrations of transdifferentiation of
neural retina from pigmented retina in Culture. Devi Growth and Diff. 23, (4), 297-311.
M. S. & MCCOBB, D. P. (1980). The genesis of membrane bone in embryonic chick
mandible; epithelial-mesenchymal tissue recombination studies. /. Embryol. exp. Morph.
56, 269-281.
WARTIOVAARA, J., NORDLING, S., LEHTONEN, E. & SAXEN, L. (1974). Transfilter induction of
kidney tubules - correlation with cytoplasmic penetration into nucleopore niters. /. Embryol. exp. Morph. 31, (3), 667-682.
WEISS, P. & AMPRINO, R. (1940). The effect of mechanical stress on the differentiation of
scleral cartilage in vitro and in the embryo. Growth 4, 245-248.
WEISS, P. & MOSCONA, A. (1958). Type-specific morphogenesis of cartilages developed from
dissociated limb and scleral mesenchyme in vitro. J. Embryol. exp. Morph. 6, 238-247.
WESSELLS, N. K. (1977). Tissue Interactions and Development. U.S.A.: Benjamin/Cummings.
TYLER,
{Accepted 17 December 1982)
Appendix
A number of the physical parameters of the Nuclepore filters were measured
during the course of the work and compared, where possible, with specifications
provided by Bio-Rad Laboratories (U.K.) All measurements and counts were
made on enlarged scanning electron micrographs of nuclepore filters, taken at
calibrated magnifications.
(i) frequency distributions of pore diameters in Nuclepore filters
In both filter types the diameters of approximately 80-180 pores were
measured (see Figs 18 & 19) and the average diameter calculated. The
average pore diameter in nominally 0-8 jum filters was found to be 0-698 /mi
(n = 79) and in nominally O^jum filters, 0-142/im (n = 189). The nominal
sizes have been used throughout the text of the paper.
(ii) comparison of pore densities (number of pores/cm 2 )
A B
— x — = total area of nuclepore filter in cm2
MM
(where A = length of enlarged micrograph
B = width of enlarged micrograph
M = total magnification factor)
no. of pores (N) in micrograph
,
*—^
5—-—-— = pore density
real area in cm
for 0-8 jum filters,
pore density = 2-75 x 107 pores/cm 2
(manufacturer's figure = 3 x 107 pores cm2)
188
L. SMITH AND P. THOROGOOD
100 r
100 r
18
-
19
50
§• 50
-
1
-
•6
-7 -8
diameter (jum
•05
•15
Fig. 18. Histogram to show the frequency distribution of pore diameters in a nominally 0-8 /an nuclepore filter.
Fig. 19. Histogram to show the frequency distribution of pore diameters in a nominally 0-2 jum nuclepore filter.
for 0-2 jitm filters,
pore density = 2-06 x 108 pores/cm 2
(manufacturer's figure = 3 x 108 pores/cm2)
(iii) ratio of pore to non-pore in the filters
This ratio represents the potential transfilter interface between two cultured
tissues.
(a) total micrograph area = A x B cm2
(b) total pore area = N X j c m 2
(c) non-pore area = (a) - (b)
For 0-8 jam filters, pore: non-pore = 0-118.
For 0-2[im filters, pore: non-pore = 0-033.
The fact that the ratio of a 0-8 /im filter is approximately four times greater than
that of a 0-2 ^m filter means that there is less potential interface between the
interacting tissues in transfilter cultures grown on 0-2 ^m filters than in similar
cultures grown on 0-8 fjm filters. To achieve the same area of interface between
the tissues would require a 0-2/^m filter with approximately four times the pore
density given in (ii). Such filters are not available. Although area of potential
interface may be of secondary or even negligible importance, until that has been
demonstrated the difference in ratio should not be overlooked when interpreting
data from nuclepore transfilter cultures.