/. Embryol. exp. Morph. Vol. 42, pp. 209-217, 1977
209
Printed in Great Britain © Company of Biologists Limited 1977
Effect of the brachypod
mutation on cell adhesion and chondrogenesis
in aggregates of mouse limb mesenchyme
By JACKIE DUKE 1 AND WILLIAM A. ELMER 1
From the Department of Biology, Emory University, Atlanta
SUMMARY
Twelve-day normal and brachypod mouse limb mesenchyme was studied in rotation
culture. Over a 3£ h period the rate of decline of single cells was significantly greater in
mutant than in normal cultures, probably because the brachypod cells were more adhesive.
However, the final size of the aggregates and their cell densities were the same by 24 h of
incubation. On the other hand their pattern of chondrogenesis was different. Normal aggregates contained condensations with typical histotypic cartilage by 24 h of incubation, and
were entirely chondrifled by 4 days in culture. The condensations in brachypod aggregates
were fewer, smaller, and delayed in their chondrogenesis. Never more than 50% of the
brachypod aggregate exhibited chondrogenesis. The importance of cell contact and cell
density to the chondrogenic process are discussed.
INTRODUCTION
Recent studies of normal and abnormal vertebrate limb development have
demonstrated that cell-surface-related events play a critical role in the differentiation and morphogenesis of the limb skeleton. In the normal limb, the formation
of prechondrogenic blastemata and their subsequent differentiation into
cartilage is accompanied by a decrease in mitosis, an increase in cell density,
and an increase in areas of broad surface contact between cells (Janners &
Searls, 1970; Gould, Day & Wolpert, 1972; Ede, Flint & Teague, 1975; Thorogood & Hinchliffe, 1975). In the chick limb skeletal mutant, talpids, the failure
of the mesenchyme to separate properly has been attributed to an increase in
cell adhesion, reduced cell movement, and continued mitosis - all possible
consequences of surface abnormalities (Ede & Agerbak, 1968; Ede & Flint,
1915a, b; Ede etal. 1975).
Studies on brachypod mice suggest that the aberrant limb development found
in this mutation is also related to a dysfunction of the cell surface of prechondrogenic mesenchyme. Chondrogenesis is delayed in brachypod limbs, and this
delay has been correlated with an anomalous formation of the blastemata of
12-day embryos (Milaire, 1965; Griineberg & Lee, 1973). In monolayer culture,
1
Authors' address: Department of Biology, Emory University, Atalanta Georgia 30322, U.S.A.
210
J. DUKE AND W. A. ELMER
brachypod limb mesenchyme continues to synthesize DNA and undergo cell
division, forms large flattened stellate cells, and aggregates into irregularly
shaped clusters rather than distinct cartilage nodules (Elmer & Selleck, 1975).
Experiments using the plant lectin Con A have shown that Con A reactivity
of both normal and mutant limb mesenchyme cells decreases between 11 and 13
days of development (Hewitt & Elmer, 1976). The decrease, however, is delayed
by 24 h in the mutant, suggesting that an effect on the topography of the cell
surface is occurring in the brachypod limb mesenchyme.
In view of the above observations, further investigations were initiated to
determine the effect of the brachypod mutation on 12-day embryonic limb
mesenchymal cells by studying (1) cell-cell interaction and cytodifferentiation in
rotation reaggregation cultures, (2) tissue interactions during fragment fusion,
and (3) cell-cell contacts, matrix production, and intracellular events at the
ultrastructural level. This paper reports the results from the first part of these
investigations. The data show that 12-day brachypod mesenchymal cells
aggregate more rapidly than normal cells. However, even under the same
rotation culture conditions chondrogenesis in mutant aggregates is delayed and
is less extensive than in normal aggregates.
MATERIALS AND METHODS
Embryos of the 12-day stage were obtained from matings between mice
homozygous for either the bp H allele or the normal allele at the same locus.
The time of vaginal plug appearance as well as morphological characteristics
of the embryos were used for age determination (Krotoski & Elmer, 1973).
Hind limb-buds were collected under sterile conditions, the ectoderm removed, and single cell suspensions prepared from the mesoblast treated with
Ca2+- and Mg2+-free Tyrode's solution as previously described (Elmer &
Selleck, 1975). Cell viability was assessed by trypan blue exclusion, and cell
counts were made with a hemocytometer.
Cells at a concentration of 1 x 106/ml were placed in 10 ml siliconized Erlenmeyer flasks in a total of 3 ml of medium [Eagle's BME with 2% glutamine
(Gibco), 10 % horse serum (Gibco), and 50 /*g/ml Gentocin (Schering)]. Culture
flasks were rotated at 70 rev./min on a rotating platform with a 1-9 cm armature
in an incubator maintained at 37 °C. Cells remaining in suspension were
estimated by removing a small aliquot of medium for counting at 15 min
intervals thereafter until the number of single cells remained constant. Statistical
analysis of the data obtained from rotation culture was carried out by using a
computer program designed to fit regression lines to the reciprocals of the data
points (Dixon & Massey, 1969).
Other cultures were incubated for 1-7 days, with half the medium being
replaced every 48 h. In one study, aggregates were labeled for 24 h prior to
fixation with 10 {id/flask of Na235SO4 (carrier-free, Amersham Radiochemical
Aggregation of mouse limb mesenchyme
0
15
30
45
60
75
120
90
150
180
211
210
Time (min)
Fig. 1. Loss of single cells from reaggregating cultures. • , Normal; 23, brachypod.
Brackets represent standard deviations.
Table 1. Statistical analysis on data from regression analyses
Genotype
a (intercept)
95%
confidence
interval of
a
+/+
bp*lbp*
0149
0150
0118-0179
0067-0-234
b (slope)
95%
confidence
interval of
b
r2
00022
00038
000195-000247
00031 -00045
0-88
0-75
Laboratory). Labeled and unlabeled aggregates were fixed in 10% buffered
neutral formalin, embedded in paraffin, and sectioned at 6 jam. Labeled sections
were placed on gelatin-coated slides, and after hydration, coated with Kodak
NTB-2 emulsion (dilution, 1 emulsion :1 dw). The sections were exposed for 3
weeks at 4 °C, then developed in Dektol (Kodak), and fixed in 30 % sodium
thiosulfate. All sections were stained with 0-1 % toluidine blue.
RESULTS
Aggregation of normal and brachypod cells in rotation culture
At the light microscopy level, freshly dissociated mesenchymal cells from
the hind limbs of 12-day brachypod embryos are indistinguishable from their
normal counterparts (Elmer & Selleck, 1975). However, when these cells are
placed in rotation culture, a difference appears (Fig. 1). Over a 3 |-h period, the
number of single cells remaining in suspension at each sampling time was
212
J. DUKE AND W. A. ELMER
01
0-2
0-3
0-4
0-5
0-6
0-7
0-8
0-9
10
15
30
45 60
75
90
120
150
180
210
Time (min)
Fig. 2. Loss of single cells from reaggregating cultures. Regression lines fitted to
the reciprocals of the means in Fig. 1. # — # , Normal; O—O, brachypod.
Table 2. Comparisons of normal and brachypod aggregates
Brachypod
Normal
One large; 6-7 small
2 mm; 0-2 mm
2-13 xlO5
6-6
183/tm
24 h
Typically present
Aggregates/culture
Diameter of aggregates
Cells/large aggregate
Mean no. condensations/aggregate
Mean diameter of condensations
Appearance of cartilage
Perichondrial-like zone
One large; 6-7 small
2 mm; 0-2 mm
215 xlO5
2-3
80 fim
48 h
Typically absent
consistently less in the mutant cultures than in the controls. Figure 2 shows
regression lines fitted to the reciprocals of the data in Fig. 1. The intercepts
of the two lines are the same, each culture having started with the same number
of cells, but the slope of the line for brachypod cells is greater (Table 1). Since
the 95 % confidence intervals for the slopes of the two lines do not overlap,
the rate of decline of single cells in mutant cultures is significantly greater than
in normal cultures. This suggests that the brachypod cells display a surface
topography more conducive to adhesion than normal cells of the same embryonic age.
It has been suggested that aggregate size is related to the degree of cell
adhesiveness (Moscona, 1962; Ede & Agerbak, 1968), but no distinct differences were seen in the pattern of reaggregation in regard to shape, size, and
number of aggregates formed in the brachypod and normal cultures (Table 2).
Cultures incubated for several days contained typically one large, slightly oval
aggregate 2-0 mm in diameter and six to seven smaller spherical aggregates
Aggregation of mouse limb mesenchyme
213
approximately 0-2 mm in diameter (Fig. 3 A and Table 2). The possibility that
the bp11 cells could be more tightly packed in the aggregates was ruled out
since no distinct differences in cell packing density could be seen in histological sections and the number of cells enzymically released from the large
aggregates was similar (Table 2).
Chondrogenesis within aggregates
The similar size and cell number of the normal and mutant aggregates was
of particular interest since the delay in chondrogenesis in brachypod limbs
has been ascribed to a reduction in size of the prechondrogenic blastemata
(Milaire, 1965; Griineberg & Lee, 1973). This system provided us, therefore,
with an excellent opportunity to discern if the delay in chondrogenesis in brachypod limbs is related solely to the observed decrease in the size of the prechondrogenic blastemata (Griineberg & Lee, 1973) and if the bp H cells within the
aggregate can produce the pattern of chondrogenesis found in normal aggregates
under similar culture conditions.
Sections through normal aggregates after 24 h of culture show several
metachromatically staining areas in various stages of cartilage development.
In areas which do not stain intensely, the cells assume no particular orientation
and are indistinguishable histologically from cells of surrounding metachromatically negative regions. In intensely staining areas, cells are closer together,
oriented in a circular manner (Fig. 3C) and surrounded by a perichondrial-like
layer of spindle-shaped cells (see arrow, Fig. 3 A). These clearly defined regions
are termed condensations. Within condensations matrix accumulation is
greatest in the center with a decreasing amount towards the periphery (Fig. 3 C),
so that the central tissue assumes a distinct histotypical appearance of cartilage.
In other areas of the aggregate where staining is not as intense, cells appear to be
oriented in a longitudinal fashion as though they are in a transient stage in the
formation of a condensation. These prechondrogenic regions also show a low
incorporation of 35S-sulfate as compared to condensation centers which show
dense silver grains (Fig. 3 A).
After 24 h of incubation, new condensations within the aggregate are not
observed. Instead, increase in the size of each condensation appears to result
from a combination of an expansion through continued deposition of matrix
and an incorporation of cells lying between condensations, leading to a fusion
of adjacent condensations. As a consequence, after 4 days in culture an aggregate consists of a single condensation with less than 10 % of the original nonmetachromatic region remaining. Examination of serial sections shows that
after 24 h normal aggregates have an average of 6-6 condensations per aggregate.
Condensations have an average diameter of 183 /mi (inset, Fig. 3 A and Table 2)
and tend to be located centrally. With further incubation the condensations
extend toward the periphery and some appear to be exposed on the surface of
the aggregate.
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J. DUKE AND W. A. ELMER
(A) Autoradiograph showing 35S incorporation in condensation within normal
aggregate after 24 h incubation. x400. Inset: shows several condensations, x 100.
(B) Autoradiograph showing 35S incorporation in condensation within brachypod
aggregate after 24 h incubation, x 400.
(C) Light micrograph of condensation in normal aggregate stained with toluidine
blue. x400.
(D) Light micrograph of condensation in brachypod aggregate stained with toluidine
blue. x400.
Aggregation of mouse limb mesenchyme
215
Some very striking and significant differences in the pattern of chondrogenesis appear in the brachypod aggregates as compared to their normal
counterparts. One of the first observations is the reduction in size and number
of condensations per aggregate. The average number of condensations is 2-3
with an average diameter of 80 /tm (Table 2). The small number and size is
probably a reflexion of the fewer number of cells which are capable of entering
into the chondrogenic pathway. Even after 7 days of incubation, 50 % of the
brachypod aggregate still remains non-metachromatic and shows no 35S
incorporation.
The cartilage tissue seen in the central regions of 24-h normal condensations
is not observed in brachypod condensations until 48 h of incubation. Even
though metachromatic staining and 35S incorporation is observed by the end
of 24 h (Fig. 3B), the cells within the precartilaginous areas have not yet
assumed a circular appearance with the typical spindle-like cells circumscribing them (Fig. 3D). When distinct condensations are recognizable,
the slow expansion of their cartilaginous regions, their small numbers,
and their random distribution, separated by large areas of undifferentiated
cells, makes fusion with neighboring condensations unlikely. As a result,
the total area of brachypod aggregates expressing a cartilage phenotype is
always less than in normal aggregates.
DISCUSSION
The loss of single cells in rotation suggests that 12-day brachypod limb
mesodermal cells display a surface topography that is more favorable for
adhesion than do normal cells of the same age. The more rapid decline in the
number of single cells in the culture suggests that either the molecules involved
in adhesion are in greater numbers on the surface of brachypod cells or they
are arrayed in a manner which is more conducive to initiation and maintenance
of cell-cell adhesive contacts. It is unlikely that the difference in adhesiveness
between the normal and mutant cells is due to a difference in cell size, differential
cell lysis during culture, or a differential effect of the dissociation treatment on
the surface of the cells. Brachypod and normal limb mesodermal cells are of
similar size (Hewitt & Elmer, 1976). It is also improbable that differential cell
lysis occurred. At each sampling time, aliquots of cells of each genotype were
examined by phase microscopy and tested for trypan blue exclusion. No
increase in the number of broken cells was observed nor was there any dramatic
change in viability. Likewise, there is evidence which suggests that dissociation
with Ca2+-/Mg2+-free Tyrode's solution does not affect these cells differentially.
When cells of each genotype are placed in monolayer culture, the rate of attachment to the culture flask is the same (Elmer, unpublished observation). In
addition, both cell types have been shown to bind the same amount of 125I in the
presence of lactoperoxidase, indicating that the number of surface proteins is
216
J. DUKE AND W. A. ELMER
similar in the two genotypes (Hewitt, unpublished observation). The evidence,
therefore, suggests that the greater adhesiveness of the mutant cells is a true
manifestation of the display of their surface molecules at this stage of their
differentiation.
Other studies on cell aggregation (Ede & Agerbak, 1968; Morris, 1976)
have shown that under conditions favoring rapid aggregation the size of the
aggregate was small. However, in our study we did not observe an inverse
relationship between degree of adhesiveness and aggregate size. For both
genotypes the aggregates were of the same size and contained similar numbers
of cells, yet the brachypod single cells were removed from culture significantly
faster than normal. Loss of single cells not only reflects the rate of successful
collisions of single cells with each other or with small groups of cells, but
also the force required to keep the cells adherent to each other. The final
size of the aggregate depends not only on this factor, but also on the force
required to hold small groups of cells adherent to other small groups. The
more rapid loss of single cells in the brachypod cultures could suggest that
less force is required to maintain adhesion of brachypod cells to each other or
to aggregates, whereas normal and mutant cells may require similar forces
to aggregate small groups of cells together so that they increase in size at the
same rate.
Even though the final size of the aggregates was the same by the end of 24 h,
condensations within the brachypod aggregate were fewer in number and smaller
in size. This could be related to a sorting out phenomenon, i.e. cells destined to
become chondrocytes at the time of dissociation sort out within the aggregate,
forming areas of cells able to incorporate 35S and to produce metachromatic
matrix. In the mutant aggregates chondrogenesis may be impaired because the
more adhesive brachypod cells do not sort out, and because the brachypod
hind limb-buds contain fewer cells capable of differentiating into chondrocytes.
Studies by Ede & Flint (1972) with the talpid3 mutant in chicks tend to argue
against this possibility. They observed no evidence of sorting out within aggregates between cells from chondrogenic portions of the limb and cells from nonchondrogenic portions. However, the dissociation treatment of the talpid3 cells
included a trypsin treatment, whereas in this study Ca2+- and Mg2+-free Tyrode's
solution was used.
Another possible explanation of the chondrogenic pattern is related to the
recruitment of adjacent non-chondrogenic cells into the chondrogenic core.
Ede & Flint (1972) have suggested that recruitment of chondrogenic cells
occurs by an attraction of the peripheral perichondrial-like cells surrounding
the condensations. Furthermore, incorporation of sulfate, production of matrix,
and formation of histotypic cartilage by chondrocytes in vitro does not occur
if contact is not made with other chondrocytes (Abbott & Holtzer, 1964;
Holtzer & Abbott, 1968; Moscona & Garber, 1968). The delay in chondrogenesis and the absence of a perichondrial-like layer surrounding the brachypod
Aggregation of mouse limb mesenchyme
217
condensations may explain why they fail to increase in size sufficiently to
occupy the entire aggregate, as eventually seen with normal tissue.
Not enough information is available at present to interpret the mechanism
of action of the bp H allele. The results of the rotational studies show that while
the ability to form stable cell-cell attachments may be necessary to the chondrogenic process, it is not in itself sufficient to cause chondrogenesis. Electron
microscopy studies are in progress to see whether other membrane-related
properties are required for the differentiation of chondrocytes.
A preliminary report of the findings in this paper was presented at the American Zoologist
Meeting, New Orleans, June 1976.
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{Received 12 April 1977, revised 1 June 1977)