/. Embryol. exp. Morph. Vol. 48, pp. 161-168, 1978
Printed in Great Britain © Company of Biologists Limited 1978
Cell adhesion and chondrogenesis
in brachypod mouse limb mesenchyme:
fragment fusion studies
By JACKIE DUKE 1 AND WILLIAM A. ELMER 2
From the Department of Biology, Emory University, Atlanta
SUMMARY
This study is a continuing investigation of the effect of the brachypod mouse mutation on
cell interactions and chondrogenesis during early limb development. In this report, cell
adhesiveness was assessed in fused fragments of brachypod and normal limb-bud mesenchyme.
Examination of the interface of fused distal postaxial limb fragments show brachypod limb
mesenchyme to be more adhesive than normal limb mesenchyme. Chondrogenesis within
brachypod fragments is delayed and less extensive than in normal fragments. In addition,
chondrogenesis within normal fragments is not affected by the juxtaposition of the brachypod
fragment, and vice versa.
INTRODUCTION
Brachypodism is a limb anomaly of the mouse which severely affects the
development of those limb elements derived from the postaxial mesenchyme. It
has been suggested that the mutation acts by interfering with the mesodermal
condensation process (Griineberg & Lee, 1973). It was reported that the disturbance in the formation of the blastemata could be related to the abnormal
behavior observed when mutant cells were grown in monolayer cultures (Elmer
& Selleck, 1975). An effect of the mutation on cell-cell interactions was recently
demonstrated when 12-day brachypod limb mesenchymal cells were placed in
rotation culture. Under these conditions it was shown that the mutant cells
aggregate more rapidly than normal cells, suggesting that the brachypod cells
are more adhesive (Duke & Elmer, 1977).
Although the more rapid loss of single cells in the mutant rotation reaggregation cultures suggested that these cells were more adhesive, other factors that
could affect the cell's adhesive properties could not be ruled out conclusively.
For example, the apparent differences in adhesiveness between the two cell
types could have been attributed to a difference in cell size, differential cell lysis
1
Author's address: Department of Pediatrics, Grady Memorial Hospital, Atlanta, Ga.
30303, U.S.A.
2
Author's address (for reprints): Department of Biology, Emory University, Atlanta,
Georgia 30322, U.S.A.
162
J. DUKE AND W. A. ELMER
during culture, or a differential effect of the dissociation treatment on the surface
of the cells. All of the evidence available suggests, however, that these factors
did not play a role in the above system since (I) no differences in cell size have
been observed (Hewitt & Elmer, 1976), (2) there was no observable increase in
broken cells in the brachypod cultures, and (3) both the rate of attachment of
these cells to culture flasks and the amount of [125I] bound to the surface proteins
is the same for both cell types under the dissociation conditions that were
described (Hewitt & Elmer, 1978).
Nevertheless, it is important to assess adhesiveness by another method. It is
well known in fusion studies employing unlike tissues, such as heart and liver,
liver and limb-bud (Holtfreter, 1943; Steinberg, 1962; Wiseman, Steinberg &
Phillips, 1972), differently treated tissues (Morris, 1976; Phillips, Wiseman &
Steinberg, \911 a), or tissues of different embryonic ages (Lesseps, 1973), that
one fragment may totally envelope the other. When the interface between two
fragments is straight, the tissues are assumed to be equally adhesive (Niederman
& Armstrong, 1972; Moyer & Steinberg, 1976). However, if the interface is
curved, the fragment exhibiting a convex boundary in the region of apposition
is regarded as being more adhesive than the fragment with a concave boundary
(Moyer & Steinberg, 1976). The configuration of the interface is therefore a
reflexion of and precursor to the equilibrium configuration assumed by combinations of tissue fragments in which the more adhesive tissue becomes
enveloped by the less adhesive tissue.
This paper reports observations made on the fusion pattern of blocks of
tissue removed from the postaxial region of normal and brachypod hindlimbs.
The data show that the brachypod fragment exhibits a convex boundary,
indicating it is more adhesive than the normal fragment. In addition, differences
observed in the pattern of chondrogenesis within the fragments during fusion
further confirm the abnormal differences observed in the rotation reaggregation
studies (Duke & Elmer, 1977).
MATERIALS AND METHODS
Twelve-day-stage embryos were obtained from timed matings of homozygous
normal ( + / + ) or brachypod (bpH/bpH) mice. The time of vaginal plug
appearance as well as morphological characteristics of the embryos were used
for age determination (Krotoski & Elmer, 1973).
Distal fragments cut from ectoderm-free postaxial halves of the hindlimbs
(Duke & Elmer, 1977) were washed twice in Tyrode's solution and then transferred to 60 mm glass Petri dishes containing 3 ml of BGJ medium (Difco),
with 25 % fetal calf serum (Gibco) and 50 /*g/ml Gentocin (Schering). Twenty
fragments were cultured per dish and allowed to round up overnight at 37 °C
in a humidified atmosphere of 95 % air: 5 % CO2. One-half of the cultures of
each genotype was labeled with 10 /tCi/dish of methyl-14C-thymidine (Amer-
Cell adhesion and chondrogenesis
163
sham Radiochemical Laboratory, specific activity 56 mCi/mmol) at the
beginning of the culture period. After 24 h of culture the fragments were washed
thoroughly with isotope-free medium, then placed in additional isotope-free
medium and allowed to stand for 30 min to permit free isotope to diffuse out.
After a final wash the fragments were placed in hanging-drop cultures suspended
from tops of siliconized 60 mm Petri dishes in the following combinations:
bpH/bpH* and + / + , bpH/bpB* and bp"/bpH, + / + * and bpH/bpH, + / + *
and + / + (asterisk refers to fragment containing radioactivity). Additional
medium was placed in the bottom of the dish to retard evaporation from the
drops, and the cultures were incubated at 37 °C in a humidified atmosphere of
95 % air: 5 % CO2. Fused fragments were removed at 24, 30, and 72 h, washed
with Tyrode's solution, fixed in 10 % buffered formalin, paraffin embedded and
sectioned at 6 /tm. Slides were hydrated, coated with NTB-2 emulsion (diluted
1:1 with water), stored for 2 weeks in light-tight boxes, developed in Dektol
(Kodak) and stained with 0-1 % toluidine blue as previously described (Duke &
Elmer, 1977).
RESULTS
A total of 16 genotypically like (bpH/bpH*:bpH/bpH
or + / + * : + / + ) and
16 genotypically different (bpu/bpH*: +/+ or + / + *: bpH/bpH) fragments were
placed in contact in hanging-drop cultures for a period of 3 days. At the end of
three time-periods (24, 30, and 72 h) tissue combinations were removed, fixed,
sectioned, and examined in serial autoradiographs to ascertain whether cells of
one tissue migrated over the surface of the second tissue to envelop it. It can be
seen in Fig. 1A, B, and C and Table 1 that the cells within the brachypod
fragments are more adhesive. In 13 of the 16 unlike combinations (81-25 %)
the brachypod fragments exhibited a convex boundary (Table 1). In the remaining three combinations (18-75 %), the boundary between the tissues was
straight (Table 1). Over the 3-day culture period a reversal of adhesiveness was
never observed; that is, none of the genotypically different combinations showed
a brachypod fragment enveloping a normal fragment.
With all of the 16 genotypically like combinations, a straight interface along
the boundary of fusion was observed (Fig. 1D, Table 1). Migration of labeled
cells into unlabeled regions was not seen and the interface between fragments
remained sharp, in accordance with the results obtained by other investigators
(Steinberg, 1962; Weston & Abercrombie, 1965). There were in a few cases
slight amounts of silver grains scattered over the unlabeled tissue in both like
and unlike combinations. This could possibly be due to the presence of nonincorporated [14C-]thymidine not removed during the extensive wash and/or the
incorporation of the labeled precursor released by autolysis of the cultured cells
(Saunders, 1966; Niederman & Armstrong, 1972).
The chondrogenic differences which were reported in the rotation reaggregation study (Duke & Elmer, 1977) were also observed in the fragment fusion
164
J. DUKE AND W. A. ELMER
D
Fig. 1. (A) Autoradiograph of labeled normal fragment (N) fused for 24 h with
unlabeled brachypod fragment (Bp). Condensation can be easily recognized in
normal fragment, x 100. Arrow points to interface showing the normal fragment
enveloping the mutant fragment. (B) Autoradiograph of labeled mutant fragment
(Bp) fused for 30 h with unlabeled normal fragment, x 100. (C) Autroradiograph
of labeled mutant fragment (Bp) fused for 72 h with unlabeled normal fragment.
Note extensive cartilage formation in normal fragment, x 100. (D) Autoradiograph
of labeled mutant fragment (Bp) fused for 30 h with unlabeled mutant fragment
(Bp). x 100. Arrow points to straight interface.
Cell adhesion and chondrogenesis
165
Table 1. Analyses offragment fusions between genotypically like
and genotypically unlike mouse postaxial hindlimb tissue
Relative adhesiveness based on shape of interface
Experimental combinations*
Control ccombinations*!
A
Fusion time
(h)
24
30
72
BpH/bpH
>
H
Bp /Bp
6
4
3
0
0
0
H
+H/+ H=
+ /+ =
+/+
BpH/BpH =
BpH/BpH
2
1
0
4
3
2
4
2
1
Bp /Bp
* Fragments were allowed to round up in culture 24 h prior to fusion either in the presence
or absence of [14C-]thymidine (see Methods and Materials),
f All combination of genotypically like tissues exhibited a straight interface.
study. After 24 h of fusion, normal fragments typically had 4-5 areas of condensation, whereas, brachypod fragments had only 1-2 areas of condensation
(Fig. 1 A). Over the 3-day culture period, however, condensations within normal
fragments appeared to expand and fuse. In brachypod fragments the extent of
expansion and fusion was less and in some cases new condensations were
initiated. The initiation of new condensations within fragments is in contrast
to the results obtained in the rotation reaggregation cultures. In the latter case,
new condensations were not initiated in either genotype after the first 24 h of
culture (Duke & Elmer, 1977). This difference is probably related to differences
in the two culture systems employed.
The delay in differentiation of brachypod limb mesodermal cells seen in vivo
(Milaire, 1965) in limb explants (Duke & Elmer, 1976), in monolayer cultures
(Elmer & Selleck, 1975), and in aggregates (Duke & Elmer, 1977) was also
observed in the tissue fragments. After 24 and 30 h of fusion (48-54 total hours
of culture) histotypic regions of cartilage are seen in normal fragments, but not
in brachypod fragments. After 3 days of fusion, brachypod fragments contain
cartilaginous areas, but the amount of cartilage is less extensive than in normal
fragments (Fig. 1C).
It was also observed that limb tissue of one genotype failed to influence the
differentiation of the limb tissue of the other genotype. This is of interest, since it
has been reported that brachypod cartilaginous limb anlagen contain a diffusable
proteinaceous inhibitor that influences the growth and differentiation of normal
cartilaginous limb anlagen in organ culture (Konyukhov & Ginter, 1966;
Konyukhov & Bugrilova, 1968; Pleskova, Rodionov, Bugrilova & Konyukhov,
1974). In the present study no difference was observed in the amount of cartilage
present in normal fragments combined with either each other or brachypod
tissue. Likewise, brachypod fragments combined with either each other or
166
J. DUKE AND W. A. ELMER
normal tissue differentiated at the same rate and contained the same number
and size of cartilaginous regions.
DISCUSSION
Fragment fusion studies employing labeled and unlabeled tissue masses from
normal and brachypod 12-day limb-buds confirm that brachypod limb mesoderm is more adhesive than normal limb mesoderm. Complete envelopment of
one fragment by another did not occur, indicating that the difference in adhesiveness between the two genotypes is relatively small when compared, for
example, with the difference between chick precartilage and embryonic heart.
In the fusion studies between fragments of normal and brachypod limbs, it is
probable that the partial envelopment observed is indeed an equilibrium
situation, since prolonged culture did not lead to complete envelopment, and no
reversal of tissue assembly occurred. Reversal of assembly patterns within
fused fragments is attributed to changes in cohesiveness during the culture
(Niederman & Armstrong, 1972; Wiseman et ah 1972; Phillips et ah 1977a).
These changes are thought to be due to in vitro maintenance of tissue, since they
do not occur as part of the normal development sequence (Niederman &
Armstrong, 1972; Phillips et ah 1977a). The lack of tissue reversal in our
system is another indication that brachypod tissue is more adhesive than normal
tissue. As the fragments develop in culture, the cells become increasingly
immobilized as matrix is laid down (Burdick, 1970) and are unable to slide
past one another as required for tissue reorganization (Phillips, Steinberg &
Lipton, 1977&). However, matrix production in brachypod fragments is minimal
during the early phases of culture and the lack of spreading of brachypod
tissues on to normal fragments must be due primarily to the strength of brachypod cell-cell adhesions, not to increasing rigidity of the tissue.
Chondrogenic differences apparent in rotation culture were also noted in
fragment fusion studies. The 4-5 condensations in normal fragments after 24 h
of fusion probably represent portions of digital, tarsal, metatarsal, and fibular
precartilage condensations. Brachypod fragments have only 1-2 condensations
after 24 h of fusion, although the number of condensations increases over the
3-day culture period. The delay in formation of condensations in the distal
region of the brachypod limb was also evident in vivo (Griineberg & Lee, 1973).
Precartilage condensations in brachypod aggregates were also smaller than
normal, but in the aggregate system, no new condensations were initiated after
24 h of culture. If the formation of precartilage condensations within aggregates
is due to a sorting out of precartilage cells within the aggregates, or perhaps a
selective reassociation of cells, precartilage cells that were unable to sort out,
and are surrounded by non-chondrogenic cells would probably not differentiate
into cartilage. Precartilage cells in fragments, however, are not displaced from
their contacts with other precartilage cells, and can go on to differentiate.
Cell adhesion and chondrogenesis
167
Condensations within normal fragments increased greatly in size, with resultant
fusion of adjacent condensations, but in brachypod fragments little expansion
of pre-existing condensations occurred and the amount of cartilage formed was
less. Lack of condensation expansion was also observed in brachypod aggregates,
and in both systems may be due to a lack of chondrogenic cells within brachypod
limbs, and/or a lack of recruitment of cells from surrounding mesenchyme.
Other disturbances in cell movement and adhesion related to abnormal limb
morphogenesis have been observed in the chick mutant, talpid3 (Ede, 1976).
Time-lapse cinematography studies of wing mesenchyme fragments of normal
and talpid3 chick embryos explanted in plastic petri dishes showed the mutant
cells moved much more slowly than normal cells (Ede & Flint, 1975). The
difference in rate of movement appeared to be related to differences in cell
morphology. The mutant cells were more flattened and during movement small
spiky microvilli all around the cell periphery were observed, rather than the
long cyloplasmic filopodia seen in the elongated normal cells (Ede & Flint,
1975). In a different study, Niederman & Armstrong (1972) reported that they
were unable to detect adhesive differences by cell sorting or fragment fusion
between limb mesenchyme from normal and taplid2 chick embryos, a mutant
which has a similar phenotype as talpid3 (Abbott, Talylor & Abplanalp, 1959).
Since different techniques may measure different aspects of cell adhesion, it is
possible that (1) fragment fusion techniques will not detect cell adhesion
differences between the talpid mutants and control mesenchymes and/or (2)
the property of cell adhesion is affected differently by the talpid3 allele than it is
by the talpid2 allele. It is clear that the studies on the talpid and brachypod
mutants only serve to underline the complexity of the cell surface in terms of
the mechanics of cell adhesion.
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(Recieved 25 April 1978, revised 3 July 1978)