/. Embryol. exp. Morph. Vol. 33, 2, pp. 37J-386, 1975
Printed in Great Britain
371
In vitro chondrogenesis of limb mesoderm from
normal and brachypod mouse embryos
WILLIAM A. ELMER 1 AND DAVID K. SELLECK
From the Department of Biology, Emory University
SUMMARY
This paper describes the cytodifferentiation of hind limb mesodermal cells from 12-day-old
normal and brachypod (bpH) mouse embryos grown in vitro at high densities. Over a 3-day
culture period normal cells underwent aggregation, nodule formation, and coalescence of
nodules into large masses of cartilage. This was associated at the biochemical level with a
cessation of cell division, with a concomitant increase in metachromatic matrix, and synthesis
of collagen. Under the described culture conditions the collagen synthesized by 48 h cultures
was predominantly of cartilage type with an al :<x2 ratio of 9:1. A change in the collagen
synthetic program was observed when the entire medium was replaced after 48 h incubation
with fresh, serum-free medium. Under these conditions the al :a2 ratio was 4:1.
In contrast, brachypod cells plated at the same density appeared large, flattened, and
stellate. Upon aggregation, normal nodule morphology was only rarely observed. More often
large, irregular clusters formed from suspended cells loosely attaching to the surface aggregates. Concomitant with the marked changes in the morphology of the mutant cells was a
linear increase in DNA synthesis and the appearance of many mitoticfigures.A biochemical
transformation in matrix synthesis was not observed, however. After a 24 h delay, mutant
matrix accumulated and stained intensely with toluidine blue. Collagen was synthesized at
approximately the normal rate and was of the cartilage type with an al :a2 ratio of 9:1.
When incubated in fresh, serum-free medium, the response of collagen subunit synthesis was
identical to the normal cultures. In view of these results the possible manner in which brachypodism causes developmental anomalies of the limb skeleton is suggested.
INTRODUCTION
As a prelude to the formation of the skeletal limb elements, dense areas,
commonly referred to as mesodermat condensations, appear in the limb
mesoderm. It appears that these areas do not result from a local stimulation of
cell division (Janners & Searls, 1970) but from a relative increase in cell packing
density; whether this arises by aggregation towards the center of the condensations as suggested by Ede & Agerbak (1968) and as shown by Thorogood &
Hinchliffe (1974) or by absence of centrifugal movement while cells outside of
the condensations become more dispersed, as suggested by Gould, Day &
Wolpert (1972), is not entirely clear. In either event, one can consider the
1
Author's address: Department of Biology, Emory University, Atlanta, Georgia 30322
U.S.A.
24
EMB 33
372
W. A. ELMER AND D. K. SELLECK
mechanisms which regulate cell contact, cell movement, and cell density in vitro
(Kohn & Fuchs, 1970) as possible in vivo control mechanisms involved in the
formation of limb mesodermal condensations.
The importance of cell density in cartilage differentiation and pattern formation during limb development has been shown from studies on the talpid3
mutation in chickens (Ede, 1971). In their recent study on brachypodism, an
inherited limb anomaly in the mouse, Griineberg & Lee (1973) proposed that
the skeletal malformations could be ascribed to a reduction in the size of the
mesodermal condensations. This finding was in agreement with earlier histochemical investigations of Milaire (1965), who indicated that a delay in matrix
metachromicity in the brachypod limbs was related to an anomalous formation
of the precartilaginous regions.
Over the past few years we have been investigating the abnormal development
of the brachypod limb skeleton as a means of providing further insight into
those processes which regulate normal limb development. In view of the above
observations on the role of cell-cell interactions during early limb development
as exemplified in both normal and mutant conditions, we decided to study the
differentiation of 12-day-old normal and brachypod hind limb mesodermal
cells cultured in vitro under conditions conducive to cartilage expression. This
report describes some morphological and biochemical changes, which normal
and mutant cells undergo during their cytodifferentiation into chondrocytes.
MATERIALS AND METHODS
Embryos were obtained from matings between parents of a Swiss albino
mouse strain homozygous for the bpH allele. The controls were from the same
strain, but homozygous for the normal allele ( + / + ) at the brachypod locus.
Staging of embryos was carried out as previously described (Krotoski &
Elmer, 1973).
Cell dissociation and culture conditions
Hind limb-buds from 12-day-old embryos were washed with Ca2+- and
Mg2+-free Tyrode's solution (CMF) and left undisturbed for an additional
5 min. The CMF was removed and an enzyme solution (2-25 % trypsin-0-75 %
pancreatin in CMF, pH 8-0) was added for 8-5 min. After enzyme treatment,
the ectoderm was peeled away in Tyrode's and the mesoblasts were collected in
CMF solution. Approximately 30-40 min later the CMF solution was removed
and the cells were dissociated in culture medium by gentle flushing with a
capillary pipette. The culture medium consisted of Eagle's BME containing
0-2 % L-glutamine (Grand Island Biological Co., Grand Island, N.Y.), with
the addition of 50 units/ml of penicillin-streptomycin, and 10 % horse serum.
The cell concentration was determined by haemocytometer counting. Cell
viability was tested by either trypan blue or erythrosine B exclusion. Viability
was between 96-98 % for both brachypod and normal cells. For culturing, cells
Brachypodism effect on chondrogenesis
373
2
were plated at a density of 4500 cells/mm in a 30 ml plastic T-flask (Falcon
Plastics) containing 4 ml of media. This concentration has been shown to be
conducive to cartilage formation by mouse limb-bud mesenchyme in vitro
(Umansky, 1966). The flasks were gassed with a 5 % CO2-95 % air mixture at
a pH of 7-0 and incubated at 37 °C. The medium was renewed after 48 h by
removing 2 ml and replacing it with 2 ml of fresh medium.
Histochemical and biochemical analyses
Cells were fixed with 10 % buffered formalin. Toluidine blue or alcian blue
were used to stain for mucopolysaccharides. Other cells were stained with
haematoxylin and counterstained with either eosin or orange G.
DNA analysis was carried out by homogenizing cells at 2 °C in a 0-1 M TrisHC1 buffer, pH 7-2, containing 0-1 % sodium dodecyl sulfate. The homogenate
was precipitated upon addition of an equal volume of cold 20 % tricholoroacetic
acid (TCA) and the DNA was extracted from the precipitate with 5 % perchloric
acid at 90 °C. DNA assays were carried out by the method of Burton (1956)
as modified by Giles & Myers (1965).
The amount of polypeptide-bound hydroxyproline in the cells plus the
medium over the 3-day culture period was used as a measurement of collagen
synthesis. Medium which had not been used for culturing provided the blank.
The medium and cells were collected and exhaustively dialyzed against running
tap water. After dialysis the samples were lyophilized to dryness, hydrolyzed
in 6 N-HC1, and assayed for hydroxyproline by the method of Switzer &
Summer (1971). The Student t test was used to determine significance of the
differences.
To determine the type of collagen being synthesized, cells were incubated
for 2 days under normal culture conditions. In one series of experiments, half
of the medium was removed from the cultures and replaced with fresh medium
plus 2/iCi/ml of L-[U-14C]proline (sp. act. 255 mCi/mmole, New England
Nuclear Corp.), 100/tg/ml of /?-aminoproprionitrile (to inhibit cross-linking in
collagen), and 50 /.ig of ascorbic acid. The cultures were incubated for an
additional 24 h.
Collagen was extracted from the medium at 4 °C with 1-OM-NaCl in a
0-05 M Tris-HCl buffer, pH 7-5, as described by Miller & Matukas (1969). Skin
collagen (15 mg) from lathyritic rats was added to the extract to aid in the
precipitation of the radioactive collagen during dialysis against 0-05 N-Na2HPO4.
The precipitates were collected, purified twice by dissolving in 0-5 M acetic acid
and precipitation with 5 % NaCl (w/v), before dialysis against starting buffer
(0-04 M sodium acetate, 1 M urea, pH 4-8) for chromatography on a carboxymethyl cellulose (CMC) column. To ensure complete denaturation of the triple
helix, the collagen solutions were warmed to 43 °C for 30 min before applying
them to a 1-5x 100cm column of CMC (Whatman, microgranular CM-32,
capacity 1-0 m-equiv/g). The column was maintained at a temperature of 42 °C
24-2
374
W. A. ELMER AND D. K. SELLECK
and the collagen subunits were eluted with a linear salt gradient (0-0-0-1 M-NaCl).
Five ml fractions were collected and 0-5 ml aliquots from each fraction were
counted with 10 ml of a scintillation fluid consisting of 0-4 % (w/v)' Omnifluor'
(New England Nuclear Corp.) in toluene, and 10 % (v/v) of Beckman 'Biosolv
BSS-3'.
In another series of experiments the entire medium was replaced after 48 h
of incubation with fresh medium as described in the preceding experiment
except that it was serum-free and the radio-isotope used to label the collagen
in the normal cultures was 5 /tCi/ml of [14C]glycine (sp. act. 102 mCi/mmole,
New England Nuclear Corp.) and in the mutant cultures it was 10 /*Ci/ml of
L-[5-3H]proline (sp. act. 20 Ci/mmole, Schwartz-Mann). The cultures were
incubated for an additional 16 h.
In these experiments the normal and brachypod media were pooled together,
15 mg of rat skin collagen was added, and the medium was then treated with
pepsin as described by Layman, McGoodwin & Martin (1971). After inactivation
of the pepsin, the medium was dialyzed against starting buffer used for the
CMC column and the collagen subunits were separated and assayed for radioactivity as previously described.
RESULTS
Cytodifferentiation of normal and brachypod hind limb mesoderm in vitro
Normal cultures
In 12-day-old mouse embryos the mesodermal condensations in the hind limbs
are apparent; however, histological differences between the cells are not yet
recognizable (Milaire, 1963). Freshly dissociated cells were spherical and of
equal size. They contained large nuclei, prominent nucleoli, and a relatively
small amount of cytoplasm.
When placed into culture the cells began to take on a variety of shapes
within the first 6 h. A few remained rounded, some developed long, tapering,
cytoplasmic projections, while many became tear-shaped (Fig. 1). As incubation
continued the cells moved along the surface and upon coming into contact
with each other in areas of high density, tended to aggregate into compact,
multicellular clusters. By 24 h many of these clusters assumed a rounded shape
and were comprised of cells actively secreting metachromatic matrix material
(Fig. 2).
Within the next 24 h the clusters grew considerably in size with the cells
Figs. 1-3. Phase-contrast micrographs of nodule formation in aggregating 12-day
normal mouse hind limb-bud mesodermal cells after 6, 24, and 48 h of culture. A
few spindle-shaped cells (arrow) could be seen overgrowing the monolayer sheets
by 48 h of incubation, x 163.
Fig. 4. Light micrograph of a normal 72 h culture in which nodules have coalesced
to form a ridge of cartilage with a large conglomerate projecting from it. x 20.
Brachypodism effect on chondrogenesis
mm.
375
376
W. A. ELMER AND D. K. SELLECK
Brachypodism effect on chondrogenesis
311
piling upon each other to form small mounds or nodules of cartilage (Fig. 3).
These nodules were of approximately 0-5 mm in diameter. They contained
large, rounded masses of cartilage-secreting cells bordered by a double layer
of oblong cells reminiscent of a perichondrium. Overlying the cells surrounding
the nodules were a few spindle-shaped cells which may have been destined to
form muscle cells. Incubations were never long enough to determine the extent
of myogenesis in these cultures.
By 72 h of culture the nodules increased considerably in number and in many
cases fused together to form ridges of cartilage with large amorphous masses
projecting from them (Fig. 4). With only slight movement of the culture flask,
the multilayered conglomerate of cartilage often detached from the ridge and
floated free in the medium.
Brachypod cultures
Tn 12-day-old brachypod mouse embryos there is no apparent morphological
difference in the hind limbs from their normal counterparts (Milaire, 1965;
Griineberg & Lee, 1973). Histologically the mesoderm cells also have a normal
morphology. When placed into culture, however, they deviated strikingly from
the shapes assumed by the normal cells. First of all those which attached to the
surface of the culture flask tended to flatten out rather quickly. Many, however,
did not attach and remained floating in the culture medium. In areas of low
density the cells were large, flattened, and stellate with conspicuous pseudopodia
(Fig. 5). In fact these cells had a morphological appearance much like chondroblasts that have been cultured in the presence of 5-bromo-2'-deoxyuridine
(Holtzer & Abbott, 1968; Schiltz, Mayne & Holtzer, 1973). As they moved
toward centers of aggregation where there was increasing cell contact, they
seemed to become 'flbroblastic-like' before rounding up as they became
positioned within the aggregate. The 24 h aggregates did not stain positive with
either toluidine blue or alcian blue.
During the next 48 h the cell aggregates increased in size and on rare occasions
would assume the typical normal nodule morphology. However, the most
predominant observation was the formation of large, irregular clusters of cells
(Figs. 6, 7). These clusters appeared to form from the loose attachment between
the cells suspended in the culture medium and the surface aggregates. By the
second day of incubation staining with toluidine blue and alcian blue was as
Figs. 5-7. Phase-contrast micrographs of nodule formation in aggregating 12-day
brachypod mouse hind limb-bud mesodermal cells after 24, 48 and 72 h of culture.
Note the expanded, flattened, stellate appearance of the cells and the presence of
many 'floaters' (arrow), x 163.
Fig. 8. A selected area of a 48 h brachypod culture showing the presence of many
spindle-shaped cells overgrowing the monolayer sheet. Also observe the cells in
mitosis (arrows) within the monolayer. x 163.
378
W. A. ELMER AND D. K. SELLECK
60 r
^
50
1 40
)
3 30
<
Q 20
O
O
10
0
1
2
Days in culture
3
Fig. 9. DNA content in cell cultures of normal and brachypod 12-day embryonic
hind limb-bud mesoderm during 72 h of incubation. O
O, Normal; •
•,
brachypod.
intense as the normal cultures. Although ridges of cartilage could be seen in
3-day cultures, the large amorphous masses projecting from them were not
observed.
In some areas of the culture flask the cells would form compact monolayer
sheets with many spindle-shaped cells overlying them (Fig. 8). The cells within
the sheets would often show mitotic figures. In normal cultures the cells in dense
monolayers contained comparatively fewer mitotic figures.
Biochemical
analyses
Morphological changes in the cell surface have been correlated with changes
in nucleic acid and protein biosynthesis (Todaro, Lazar & Green, 1965;
Pardee, 1971). To determine whether these processes were also being regulated
in the brachypod cultures, measurements of DNA content over time were made
as an indicator of cell proliferation and quantitative and qualitative assays of
collagen were carried out since it comprises the major protein component of
the cartilage matrix. The qualitative assay of collagen provided information
concerning the question of whether the cytological changes in the brachypod
cultures could be correlated with a biochemical transformation in the subunit
composition of the collagen being synthesized (Layman, Sokoloff & Miller,
1972; Schiltz et al 1973). Collagen of the type [al(II)] 3 has been shown to be
specified by the chondrocyte genome (Miller & Matukas, 1969; Trelstad, Kang,
Igarashi & Gross, 1970), whereas, fibroblastic type is designated as [al(I)]2a2
(Piez, Eigner & Lewis, 1963).
Fig. 9 shows the DNA content of normal cultures increased by 50 % during
the first 24 h of incubation. Between 24-72 h no further increase in DNA
content was observed (Fig. 9). This seems to reflect the cytological observations
which showed during this same period an increase in nodule formation surrounded by large areas of dense sheets of cells (see Fig. 3). Only rarely were
mitotic figures observed in these regions.
Brachypodism effect on chondrogenesis
379
90 r
80
IT 70
/
60
50
40
S
30
20
10
0
1
2
Days in culture
3
Fig. 10. Collagen content in cells plus medium from 12-day normal and brachypod
embryonic hind limb-bud mesodermal cultures. O
O> Normal; #
#,
brachypod.
In brachypod cultures the DNA content increased linearly over the 3-day
period (Fig. 9). This supports the observation of mitotic figures in both areas
of high and low density during the entire culture period. Both attached and
floating cells were collected for DNA analyses. If the attached cells only
represented the population of surviving cells, a linear increase in DNA content
should not have been observed (Caplan & Stoolmiller, 1973). It is not understood why many of the mutant cells did not attach to the surface of the
culture flask, but it is known from autoradiographic experiments that these
'floaters' can incorporate [3H]thymidine into their DNA (Elmer, unpublished
observations).
In the experiments carried out to determine the amount of collagen synthesized
with increasing time of incubation, values were corrected for a small amount
of non-dialyzable hydroxyproline that was present in the horse serum. Freshly
dissociated limb mesodermal cells from normal and brachypod 12-day-old
embryos contained a small, but similar amount of collagen (Fig. 10). Both cell
types also synthesized the same amount of collagen during the first 24 h of
culture. Between the first and third days the production of collagen was slightly
greater in the normal cultures (Fig. 10). The difference examined by the f-test
was found to be insignificant (P > 0-2). The observations indicate, therefore,
that (1) brachypodism has no dramatic effect on the relative amount of collagen
synthesized by limb mesodermal cells grown in vitro, and (2) the amount of
collagen synthesized does not seem to play a major role in the histotypical
expression of the cells. The latter finding agrees with the studies of Schiltz et al.
(1973) on chondroblast transformation in vitro.
380
W. A. ELMER AND D. K. SELLECK
2
0-3 -
01 -
20
30
40
Fraction number
50
60
70
Fig. 11. Carboxymethylcellulose chromatogram of collagen subunits synthesized by
normal and brachypod 12-day embryonic hind limb-bud mesodermal cultures. After
48 h of incubation, cultures were labeled for 24 h with L-[14C]proline in complete
medium (see Materials and Methods for details). Absorbancy was read at 230 nm.
, Rat skin collagen, optical density; O
O, normal cultures, radioactivity;
•
• , brachypod, radioactivity.
Although the quantitative data on collagen synthesis is useful, information
on the type of collagen being synthesized is required to determine the nature
of the cells that we are dealing with (Levitt & Dorfman, 1974). We characterized
the composition of the collagen subunits synthesized by the cells under two
very different sets of conditions. In one series of experiments we replaced half
of the culture medium (2 ml) with fresh medium plus [14C]proline, ascorbic
acid and /?-aminoproprionitrile. In another series of experiments the entire
culture medium (4 ml) was replaced with the above medium except that it was
serum-free and contained [14C]glycine in the normal cultures and [5-3H]proline
in the brachypod cultures. By using the serum-free medium and the double label
we were able to isolate and characterize the collagen subunits from both cultures
under identical conditions without going through the salt extraction. Furthermore, the two experiments allowed us to determine whether the normal and
brachypod cells would respond similarly to a modulating influence the culture
medium may have on the type of collagen subunits being synthesized.
From the elution profile of the radioactive collagen synthesized by the cells
in cultures containing serum, it is clear that both the normal and mutant cells
synthesized predominantly al type collagen (Fig. 11). The difference in the
elution position of the radioactive al chains from the rat skin carrier al chains
Brachypodism effect on chondrogenesis
381
0-6 -
10
20
30
40
Fraction number
50
70
Fig. 12. Carboxymethylcellulose chromatogram of collagen subunits synthesized by
normal and brachypod 12-day embryonic hind limb-bud mesodermal cultures.
After 48 h of incubation, normal cultures were labeled with [14C]glycine and
brachypod cultures with L-[5-3H]proline for 16 h in fresh, serum-free medium.
, Rat skin collagen, optical density; O
O, normal cultures, radioactivity;
#
# , brachypod, radioactivity.
can be attributed to their amino acid composition. A small amount of radioactivity was also observed in the ocl region which suggests the presence of some
fibroblast cells in the cultures (Fig. 11). However, the al/a2 ratio was 9:1, a
value more characteristic of cartilage collagen. These data show, therefore, that
even though the brachypod cells are not histotypically comparable to their
normal counterpart, they can on the basis of the type of collagen synthesized
still be called chondrocytes.
In the second series of experiments an increase in the radioactivity of the a2
peak was observed (Fig. 12). This indicates that the change in the culture
conditions was sufficient to cause a modulation in the type of collagen being
synthesized. An al/a2 ratio of 4:1 was obtained within the short labeling
period (16 h). This suggests that these cells have not become very stabilized
within the 48 h prior to the change in the culture media. Taken together then
the two experiments indicate that even though normal and brachypod cells
show a morphologically differential response to growth in vitro, they both
respond similarly at the transcriptional and translational levels with respect to
collagen synthesis.
382
W. A. ELMER AND D. K. SELLECK
DISCUSSION
Isolated hind limb mesodermal cells from normal and brachypod embryos
are morphologically indistinguishable. However, in situ studies have revealed
that before the onset of chondrogenesis a spatial heterogeneity in the expression
of the brachypod gene can be detected in the organization of the limb mesoderm.
The blastemata of the most severely affected limb elements have been found to
be reduced in size and delayed in their production of a metachromatic matrix
(Milaire, 1965; Griineberg & Lee, 1973). The use of the entire limb mesoderm
notwithstanding, the present studies showed that the determinants of the
brachypod effect in situ can also act on the sequence of events required for the
morphological transition of mesodermal cells to chondrocytes in vitro. This
system provides us, therefore, with a means of probing into the ultimate nature
of the disturbances brought about by the brachypodism gene.
The typical sequence of events at both the morphological and biochemical
levels that has been described by other investigators in the cytodifferentiation
of mesodermal cells into chondrocytes was also observed in our normal cultures
(Moscona & Moscona, 1952; Umansky, 1966; Caplan, 1970). At the cellular
level there was an initial aggregation into compact clusters which soon differentiated into cartilage nodules followed by a fusion of these nodules into ridges
and large conglomerates of cartilage. It should be stressed that clusters did not
develop merely as a consequence of overcrowding since many were seen in
areas which were not covered entirely with cells. The biochemical events which
paralleled the morphological changes were a cessation of cell division with a
concomitant increase in mucopolysaccharide staining, and the specific synthesis
of cartilage-type collagen.
In contrast, the brachypod cells demonstrated deviation from culture inception. They immediately flattened, forming large stellate cells with a large number
remaining suspended in the media. Many of these 'floaters' attached loosely
to the aggregates of cells on the surface of the flask giving the appearance of
nodule formation. These nodules did not illustrate the normal morphology,
being composed of large-sized and irregularly shaped clusters of cells. Furthermore, even though ridges of cartilage could be observed, the large amorphous
masses were not observed.
In many areas of the brachypod cultures, dense monolayers composed of
epithelioid cells could be seen containing many cells in mitosis. The appearance
of nodules was noticeably absent. In addition, a metachromatic matrix was only
faintly apparent. Similar results have been obtained when monodispersed
cultures of chondroblasts were grown on a plasma clot (Holtzer, Abbott, Lash
& Holtzer, 1960; Abbott & Holtzer, 1966) or in the presence of histotypically
different cells (Abbott & Holtzer, 1964). From data such as these, the suggestion
has been made that the initial steps in transforming the cell's synthetic activities
involves an alteration of the cell membrane (Holtzer & Abbott, 1968).
Brachypodism effect on chondrogenesis
383
Recently Schiltz et ah (1973) showed that environmentally induced changes
in the morphology of cultured chondroblasts can be correlated with a biochemical transformation in the type of matrix components synthesized. Normally chondroblasts synthesize chondroitin sulfate A, C, and [al(II)] 3 type
collagen. When cultured in BUDR (5-bromodeoxyuridine) or embryo extract
they synthesize predominantly hyaluronic acid and [al(I)]2a2 type collagen,
suggesting a transformation into fibroblasts. In the brachypod cultures, however, the situation does not seem to be as clearly defined. With respect to collagen
synthesis a concomitant transformation at the biochemical level does not take
place. Actively dividing brachypod cells were still able to synthesize predominantly [al(II)]3-type collagen.
Because analyses of mucopolysaccharide accumulation was followed by
metachromatic staining, conclusions concerning a possible transformation in
the type being synthesized cannot be reached. The histochemical analyses did
demonstrate a 24 h delay in metachromatic staining, a finding similar to what
occurs in situ prior to cartilage formation (Milaire, 1965). After this delay, the
staining intensity in the bpH cultures was as great as in the normal cultures.
In membranous bone development a high chondroitin sulfate to collagen ratio
has been shown to favor chondrogenesis (Hall, 1970). Therefore, instead of a
qualitative difference in mucopolysaccharide synthesis, it may have been
quantitatively reduced enough to seriously affect chondrogenesis in the brachypod cultures. This possibility is presently being investigated.
A number of environmental factors have been shown to affect chondrogenic
expression in vitro (Levitt & Dorfman, 1974). One example has been the
demonstration of conditioned-medium factors in both limb-bud mesodermal
cultures (Schacter, 1970 a, b) and embryonic chondrocyte cultures (Solurish &
Meier, 1973). These factors have been reported to influence the synthesis of
both chondroitin sulfate and collagen. Solurish, Meier & Vaerewyck (1973),
proposed that the modulation in the production of the extracellular matrix
results from a positive feedback mechanism on the synthetic machinery of the
chondrogenic cell.
There is evidence of two different hereditary skeletal mutants which produce
factors that influence the growth of embryonic long bones in vitro. One is the
Creeper mutation in chickens (Elmer, 1968) and the other is brachypodism
(Konyukhov & Bugrilova, 1968). It has been reported that extract from
13-day-old embryos homozygous for bpH significantly reduced the growth of
normal 13-day limb elements in organ culture (Ginter & Konyukhov, 1966).
Evidence that the synthesis of the growth inhibitor was under the control of
the mutant gene was produced when extract from 13-day-old embryos heterozygous for the bpH allele suppressed the growth in vitro of normal tibiae by
approximately half as much as extract from homozygotes (Bugrilova &
Konyukhov, 1971). Recently it was reported that this factor has been purified
400-fold and preliminary characterization suggests it is a protein with a
384
W. A. ELMER AND D. K. SELLECK
molecular weight of 76000 (Pleskova, Rodionov, Bugrilova & Konyukhov,
1974). It has been proposed that the brachypodism gene is exerting its effect on
the cytodifferentiation of limb mesodermal cells through the production of this
extracellular protein.
With the available information it seems possible to come to some general
conclusions about the manner in which brachypodism is causing disturbances in
the development of the limb skeleton. First, the mutant gene is acting on
processes independent of those which control the synthesis of the specific types
of collagen and possibly mucopolysaccharides characteristic of the cartilage
phenotype. This is supported by the facts that (1) limb mesodermal cells grown
in vitro synthesize [al(II)] 3 type collagen and after a short delay stain intensely
with toluidine blue, (2) normal and mutant mesodermal cells show the same
identical shift in their genetic programming for collagen synthesis when grown
in fresh, serum-free medium, and (3) the bpH neonatal fibula, which is one of
the more histologically abnormal limb elements, synthesizes [al(II)] 3 collagen
and stains metachromatically with toluidine blue (Rhodes & Elmer, 1973).
Secondly, brachypodism is acting at the site of the cell membrane which is
causing an interference with cell functions peculiar to the developmental
program of the skeletal limb elements. Support for this contention are the
observations that (1) brachypodism disturbs only appendicular skeletal development (Landauer, 1952), (2) the anomalies of the limb skeleton are related to a
possible misallocation of material between blastemata, suggesting a disturbance
in cell adhesion (Griineberg & Lee, 1973), (3) the formation of large, flattened,
stellate cells in vitro reflect a change in the cell membrane, (4) in culture bpH cells
loosely attach to each other as well as the substrate, and (5) there is a continued
synthesis of DNA and the appearance of mitotic figures in vitro which in other
culture systems has been correlated with membrane disturbances (Todaro,
Lazar & Green, 1965; Castor, 1969).
Thirdly, brachypodism is mediated through the production of a growth
factor. Support for this hypothesis comes from the investigations of Konyukhov
and his collaborators who have presented strong evidence that the bpB gene
directs the synthesis of a growth-inhibitory factor. In addition, we have preliminary evidence which suggests that mutant cells release a factor into the
culture medium which can reduce nodule formation in normal cultures. We are
presently determining whether this factor is analogous to the protein component
prepared by Pleskova et ah (1974) from 13-day brachypod embryo extract.
A preliminary report of thefindingsin this paper was presented at the American Zoologist
Meeting, Houston, Texas, December 1973.
Brachypodism effect on chondrogenesis
385
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(Received 4 June 1974)