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Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge.

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THE ANATOMICAL RECORD 233~467-477 (1992)
Insulin Improves Survival but Does Not Maintain Function of
Cultured Chick Wing Bud Apical Ectodermal Ridge
Department of Anatomy, University of California-SF, San Francisco, California
94143-0452 (E.L.B.); Department of Anatomy, University of Wisconsin, Madison,
Wisconsin 53706 (J.F.F.)
Previously we demonstrated that high levels of insulin (5 kg/ml)
permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and
Fallon, Dev. Biol., 104:lll-116, 1984). Here we address whether lower levels of
insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with
ridge survival. Neither IGF I nor IGF I1 (100 ng/ml) decreased ridge cell death;
however, cell death was significantly decreased with 50 ng/ml insulin. No further
improvement was obtained in the presence of both IGF I and insulin. These data
suggest that insulin improved the survival of the isolated apical ectodermal ridge
by binding its own receptor. To test for the maintenance of function, stage 20 ridges
were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 cg/ml or 5 ng/ml)
and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more
produced fewer outgrowths and these were rarely distally complete. The medium
in which the ridges had been cultured did not influence ridge activity, despite the
major differences in cell survival. Recombinants made with ridges cultured with
limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly
isolated ridges, but most of the limbs that did form were distally complete. These
results suggest that the decline in function of cultured, isolated apical ectodermal
ridge was not due merely to ridge cell death but rather, at least in part, to its
separation from limb mesoderm. o 1992 WiIey-Liss, Inc.
The apical ectodermal ridge is a morphologically distinct region of the amniote limb ectoderm located a t the
distal tip of the developing limb bud. Several experiments have demonstrated that reciprocal interactions
between this specialized epithelium and the underlying limb mesoderm are essential for the proper development of the limb (Saunders, 1948; Zwilling, 1961;
Fallon et al., 1986). Thus, in the developing chick, the
apical ectodermal ridge is required for the survival of
the distal wing mesoderm through stage 21 (Rowe et
al., 1982) and for the specification of the limb elements
through stage 29 (Saunders, 1948; Summerbell, 1974;
Rowe and Fallon, 1982).Both the induction of the ridge
in competent ectoderm (Kieny, 1960; Carrington and
Fallon, 1984a) and the maintenance of its pseudostratified (Saunders, 1948) morphology (Saunders, 1949;
Zwilling, 1956) are dependent on limb mesoderm. Furthermore, while normally there is a low level of cell
necrosis in the ridge (Boutin and Fallon, 1984; Todt
and Fallon, 1986),this becomes extensive if the ridge is
cultured in the absence of limb mesoderm (Searls and
Zwilling, 1964; Cairns, 1975; Boutin and Fallon, 1984).
At least some of the interactions occurring between
limb mesoderm and limb ectoderm appear to involve
diffusible factors (Cairns, 1975; Jorquera et al., 19791,
and the presence of a continuous basement membrane
between these two tissues (Kaprio, 1977) suggests that
most, if not all, exchanges may be mediated by diffusible or extracellular substances.
0 1992 WILEY-LISS,
The ability of the limb mesoderm to maintain the
morphology and the activity of the apical ectodermal
ridge has been attributed to limb maintenance factor
(Zwilling and Hansborough, 1956). Its presence was
postulated based on the phenotypes of limb mutants
(Zwilling and Hansborough, 1956); however, certain in
vitro data also support its existence. For example,
Jorquera and coworkers (1979) reported that ridge ectoderm cultured for 24 hr in limb mesodermal extract
remains viable and retains the ability to induce outgrowths, while Feinberg and Saunders (1979) found
that isolated ridges cultured for more than 12 hr in
serum-containing medium lacking mesodermal factors
could not promote limb development. The latter authors, however, did not examine the fate of the ridge
cells in culture, and since it has been reported that
isolated limb ectoderm dies when cultured in serumsupplemented medium (Searls and Zwilling 1964; Boutin and Fallon 1984), the loss of ridge function in Feinberg and Saunders’ cultures may have been caused by
cell death rather than the absence of mesodermal
maintenance activity.
We have demonstrated previously that the addition
of high levels of insulin (5 pg/ml) to serum-containing
medium will permit the survival of ridge ectoderm for
Received October 18, 1991; accepted December 24, 1991.
a t least 24 h r in vitro (Boutin and Fallon, 1984). This
observation raises the questions of whether insulin improves the survival of ridge cells by binding its own
receptor or that of an insulin-like growth factor (IGF)
and whether ridge function is maintained along with
cell survival in cultured ridges. At supraphysiological
levels (5 pg/ml), insulin can bind both insulin and insulin-like growth factor (IGF) receptors (for reviews,
see Czech, 1982; Jacobs and Cuatrecasas, 1983), but at
lower levels (50 ng/ml), insulin binds primarily to its
own receptor (Massague and Czech, 1982). Although a
functioning pancreas is not present at the early limb
bud stages we examined (Benzo and Green, 1974), insulin is present in early chick embryos (De Pablo et al.,
1982), and IGFs are synthesized by limb mesoderm
(D’Ercole et al., 1980; Engstrom et al., 1987; Stylianopoulou et al., 1988; Romanus et al., 1988; Bondy et al.,
1990) and are localized in the peripheral limb mesenchyme (Ralphs et al., 1990). Furthermore, receptors for
insulin and IGF I have been detected in the limb buds
of 4-day chick embryos (Bassas et al., 1985)and receptors for IGF I and I1 in the limb buds of embryonic mice
(Bhaumick and Bala, 1987; Bondy et al., 1990). Hence,
the apical ectodermal ridge may be exposed to these
molecules during early limb development; any of which
might influence ridge cell survival or function in ovo as
well a s in vitro. Therefore, we have examined the ability of IGFs and lower levels of insulin (50 ng/ml) to
permit ridge cell survival. Furthermore, we have
tested the ability of isolated apical ectodermal ridge to
induce limb outgrowths following culture in medium
with or without added insulin to see if ridge cell survival is accompanied by ridge cell function.
Ridge Isolation and Culture Conditions
Fertile White Leghorn eggs were incubated a t 37”C,
windowed, and staged according to the Hamburger and
Hamilton (1951) series. Apical ectodermal ridges were
isolated from stage 20 wing buds by cutting just proximal to the ridge and then separating ectoderm from
mesoderm by a 3.5 min, 37°C incubation in 0.15%
trypsin (Gibco; 1:300)/Simm’s balanced salt solution
(BSS). The ectoderms were rinsed three times in the
appropriate fetal bovine serum (Gibco) containing medium or in Tyrode’s solution containing 1% glucose
(Sigma) and 1%bovine serum albumin (Sigma) and
then either used immediately to make recombinant
limbs or cultured.
The ectoderms to be cultured were transferred to a
tissue culture dish in drops of medium. The epithelia
were straightened, oriented with their basal surfaces
down, and gently pressed against the surface of the
dish at their anterior and posterior ends. The anteroposterior axis of each ridge was marked on the bottom
of the dish. The tissues were incubated for 2 h r at 37°C
and 5% CO, in the drops of medium to allow the tissues
time to attach to the surface of the dish before 2 ml
medium was added. The ectoderms were cultured for a
total of 6,12,18, or 24 hr. Normally, the cultures were
checked 1 hr after the start of incubation, and any tissues that had detached from the dish were restraightened and pressed to the dish. Only those tissues that
were still attached to the dish a t the end of the culture
period were analyzed further.
Normally tissues were cultured on uncoated tissue
culture dishes. In other experiments, ridges were incubated in control or insulin-supplemented medium on
plates coated with rat tail collagen, which had been
extracted according to the procedure of Michalopoulos
and Pitot (1975) and dried on the surface of the plate.
These ectoderms were analyzed only for cell survival.
Some tissues to be tested for ridge cell function were
cultured on a layer of Matrigel (Collaborative
Research), 0.1 ml per 35 mm tissue culture dish. Since
fluids do not bead on this surface, tissues were transferred to it and excess medium withdrawn. The tissues
were straightened but could not be pressed to the surface due to the flexibility of the gel. Nevertheless, the
tissues remained flat for they were unable to curl up off
the surface in the thin layer of fluid. After 2 hr, 2 ml
medium was added.
Control medium consisted of Ham’s F12X (Marzullo
and Lash, 1970; prepared a s in Boutin and Fallon,
1984) plus 10% fetal bovine serum (Gibco). The amount
of insulin in serum varies but normally does not exceed
0.6 ng/ml (data supplied by Gibco). Therefore, the control serum contained at most 0.06 ng/ml insulin. Supplements added to the control medium included insulin
[5 Fg/ml (high-insulin cultures) or 50 ng/ml (lowinsulin cultures); Collaborative Research], multiplication stimulating factor (rat IGF 11, 100 ng/ml; Collaborative Research), and met-IGF I (100 ng/ml; Kabi
Vitrum AB, Stockholm, Sweden). In addition, initially
bacitracin (800 pglml; Sigma) was added to these media to inhibit the extracellular degradation of insulin
(Roth, 1981), but because bacitracin can also modify
the intracellular processing of insulin (Peavy et al.,
1985), it was omitted from the later cultures. Most of
the cultures analyzed for cell survival had bacitracin in
the medium; however, bacitracin did not modify the
amount of cell death, so these results are not reported
separately. Fresh medium was added a t 6-9 hr intervals in the control and low-insulin- or IGF-supplemented cultures. Medium was not changed in the highinsulin cultures.
To test the ability of mesodermal factors to maintain
ridge survival and function, mesodermal extract was
prepared according to the procedure of Jorquera et al.
(1979). Stage 21 and 22 wing and leg buds were homogenized in Tyrode’s plus 1% glucose (one part limb buds
to two parts Tyrode’s) by brief sonication. The homogenate was centrifuged a t 10,OOOg for 20 min. Enough
x 100 antibiotic-antimycotic solution (Gibco) was
added to the supernatant to make the extract x 1.5.
Due to the small amount of extract obtained, ridges
were cultured either individually in microtiter plates
or en masse in a drop of extract in the center of a 35 mm
plate surrounded by drops of Tyrode’s solution to prevent desiccation.
As a n alternate way to expose the cultured ridges to
diffusible mesodermal factors, ridges were cultured in
high-insulin medium in the center of a 35 mm tissue
culture dish. Distal mesoderm that had been separated
from ectoderm by trypsinization was placed around the
outer portion of the dish. Two rings were scratched on
the surface of the plate to keep the cells of the two
tissues separated. The tissues were incubated for 2 hr
in separate, small amounts of medium to promote adhesion, prior to the addition of 2 ml medium, which was
STAGE 18-19
STRGE 22-23
Fig. 1 . Diagram of the procedure used to make recombinant limbs.
allowed to cross the rings. The tissues were incubated
for a total of 18 hr, and the ridges were used to make
recombinant limbs.
In other experiments, ridges were not separated from
distal limb mesoderm prior to culture. The ridge and
distal mesoderm were pressed to the culture dish with
the cut surface down and cultured for 18 hr in control
or high-insulin medium. Following culture, the ectoderm was isolated from the mesoderm by an 8 min,
37°C incubation in 1%ethylenediaminetetraaceticacid
(disodium salt, EDTA; Sigma) (Errick and Saunders,
1976) in double-strength calcium-magnesium free Tyrode’s solution (Kato, 1969). The isolated ectoderms
were tested for functional activity.
was discarded. The remaining grafts were allowed to
develop for a total of 8 days. At this time, the embryos
were fixed in 10% formalin and stained for cartilage
with Victoria blue.
Some limb recombinants were prepared with quail
(Coturnix coturnix japonica) back ectoderm rather
than chick. These recombinants were fixed after 2 or 3
days in 70% ethanol, 40% formaldehyde, and glacial
acetic acid in a ratio of 17:2:1. The buds were stained en
bloc with the Feulgen reagent (Carrington and Fallon,
1984a) to detect the heterochromatin clumps characteristic of quail nuclei (LeDouarin and Barq, 1969),
embedded in paraffin, and sectioned.
In those recombinants allowed to develop for 8 days,
ridge activity was rated according to the most distal
limb element formed as 0 (nodules or small rods of
cartilage), 1 (humerus or long bone), 2 (radius and/or
ulna), or 3 (digital elements). Distal elements were
never observed without proximal elements. Grafts that
yielded any digital elements were considered distally
complete, even if the elements themselves were incomplete. The average quality of the outgrowths was calculated by summing the ratings for each recombinant
that produced an outgrowth and dividing by the number of outgrowths obtained (recombinants rated as 0
were not included in this calculation); an average of 3
would be obtained if all outgrowths were distally complete. Statistical differences were determined using a
logistic regression analysis. A P value of 0.01 was used
to determine significant differences.
Recombinant Limbs
Cell Death Analysis
Following culture, the apical ridges were rinsed in
Simm’s BSS and used to make recombinant limbs using a modification of the procedure of Errick and Saunders (1976). The procedure is shown in Figure 1. Mesodermal cores were isolated from right and left wing
buds of stage 18,19, or 20 embryos (normally stage 18)
by an 8 min, 37°C incubation in 1%EDTMCMF Tyrode’s. Stage 22-24 back ectoderm (normally stage 22),
which overlay the neural tube and somites, was isolated from dissected embryos using the same procedure. [At these stages, body ectoderm is not competent
to respond to the inductive signals of early limb mesoderm and will not form a ridge (Carrington and Fallon,
1984a)l.Both ectoderm and mesoderm were held on ice
in 10% horse serum (Gibco) in Simm’s BSS until recombined.
A piece of back ectoderm that had been trimmed to
an appropriate size was placed flat on a tissue culture
dish. A cultured or freshly isolated ridge was applied to
approximately the center of the back ectoderm so that
its basal surface faced up. A wing bud mesodermal core
was placed on top of the back ectoderm such that the
distal edge abutted the ridge along its anteroposterior
axis. The back ectoderm was then folded over and its
cut edges pinched together. The recombinant limb was
allowed to heal a t room temperature (RT) for at least
30 min, excess ectoderm was trimmed away, and the
bud was grafted to the somites of a stage 21 or 22 host.
Two drops of x 5 penicillin-streptomycin solution
(Gibco) were added at the time of grafting and 24 and
48 hr later. After healing for 30 min a t RT, the embryos
were returned to the incubator. Any recombinant that
had evidence of pooled blood or necrosis the next day
Tissues to be analyzed for cell survival were fixed
and embedded in Embed 812. The entire tissue was
sectioned in 1 pm increments and the number of live
and dead cells counted in every tenth section as previously described (Boutin and Fallon, 1984). These data
were analyzed using a logistic regression analysis and
the method of Williams (1982) to adjust for betweentissue variability. The mean percentage necrosis and
the standard deviation for each group are reported in
the tables. A P value of 0.01 was used to determine
significant differences.
Cell Survival in Cultured Ridges
General observations
The isolated ridges were thin and elongate. An
opaque region was visible along the anteroposterior
axis. This area was assumed to be the apical ectoderma1 ridge whose greater cell density and height would
reflect more light than the thinner adjacent dorsal and
ventral limb ectoderms. No major change in the gross
morphology of the ectoderms was detected after 6 hr in
culture. At 12 hr, the tissues were still elongate and
thin, but many of the tissues had begun to spread
slightly. Spreading was more extensive after 18hr. The
well-spread tissues remained elongate but were much
wider than a t the earlier times. In addition, the opaque
region was less well defined. In cross sections, spread
tissues exhibited a thin layer of cells, two to four cells
deep, flanking a thicker stratified epithelium. A peridermal layer was still evident over both regions.
Spreading was even more extensive in the 24 hr cul-
TABLE 1. Influence of 50 ng/ml insulin and of a
collagen-coated substratum on cell death in cultured
apical ectodermal ridges
Hours in
TC dish3
TC dish
TC dish
5 2 2 (10)
6 2 3 (7)
35 f. 16 (19)
35 11(6)
42 2 8 (10)
28 2 12 (7)
5 t 2 (9)
5 t l(10)
13 2 9 (18)
9 2 6 (7)
17 t 6 (10)
15 t 4 (6)
‘Data are expressed as the average percentage necrosis 2 the standard deviation. The numbers in parentheses are the number of ridges
2The data for cell death at 0 hr were taken from Boutin and Fallon
‘TC dish, tissue culture plate; collagen, collagen-coated plate.
tures that were not examined for cell survival but were
tested for ridge cell function. Although no effort was
made to quantitate the degree of spreading, in general,
tissues grown on collagen-coated plates spread more
than those grown on uncoated plates and ridges grown
in insulin-supplemented media spread more than those
in control cultures. Two of the six tissues grown on
collagen in the presence of insulin showed extreme
spreading. The tissues were very broad and were only
a few cells thick. They showed little evidence of a thick,
stratified ridge. The edges of these tissues were two to
three cells thick, while the central region was only
three to four cells deep. The cells in these tissues were
squamous, in contrast to the cuboidal to columnar morphology normally seen in vivo.
Control Medium
The pattern of cell death obtained in the control cultures supplemented with 10% fetal bovine serum was
similar to that reported in our earlier experiments
(Boutin and Fallon, 1984). By 12 hr, the amount of cell
death had increased significantly over that present in
freshly isolated tissues (Table 1; P < 0.001) with a similar amount of necrosis evident at 18 h r (Fig. 2). Although the percentages of necrotic cells were somewhat
lower than previously reported (6% +- 2% a t 6 hr, 47%
2 12% a t 12 hr, and 52% -t 11% a t 18 hr; Boutin and
Fallon, 1984), the data were not significantly different
(0.1 < P < 0.2). In addition, while the presence of a
collagen substratum has been demonstrated to affect
the function of other cell types (Hauschka and Konigsberg, 1966; Gospodarowicz et al., 19801, there was no
statistically significant decrease in the amount of cell
death in the presence of a collagen substratum (P >
0.51, despite the lower average detected a t 18 h r (Table
1).This average was skewed by the inclusion of a few
tissues with minimal necrosis and very low total cell
counts (less than one-half the average total cell count).
It seems likely that in these cases ridge cells had died
but had been eliminated from the tissue prior to harvesting, resulting in data that artificially lowered the
average necrosis.
Fig. 2. Section through a stage 20 ridge cultured for 18 hr in Ham’s
F12X plus 10% fetal bovine serum. Numerous necrotic cells are
present (arrowheads). x 600.
Fig. 3. Section through a stage 20 ridge cultured for 18 hr in Ham’s
F12X plus 10% fetal bovine serum and 50 ngiml insulin. A few dead
cells are present (arrowheads), but most cells appear healthy. x 538.
Insulin (50 ng/ml) and IGF I or I1 (100 ng/ml)
After 12 h r of culture in insulin-containing media
(50 ng/ml), the amount of cell death was increased over
that of the 0 h r level (Table 1; P < 0.001; Fig. 3); however, the amount of cell death was also significantly
less than that obtained in the control cultures (P <
0.001), indicating that insulin did enhance the survival
of the apical ectodermal ridge a t this concentration.
The survival of the ridge in the presence of 50 nglml of
insulin was not, however, a s good as it had been in the
presence of 5 pglml insulin (P < 0.001; average necrosis 2 standard deviation was 4% 2% a t 12 h r and 5%
2 2% at 18 hr; Boutin and Fallon, 1984). There was no
further improvement in the survival of the ridge in the
presence of a collagen substratum (P > 0.5).
The addition of rat IGF I1 (100 ng/ml) or IGF I (100
ng/ml) to the control medium did not change the percentage of necrotic cells in cultured ridges (P > 0.5;
Table 2). Furthermore, the presence of both 50 ng/ml
insulin and 100 ng/ml IGF I did not improve the rate of
cell survival over that found in the presence of insulin
(50 ng/ml) alone (0.2 < P < 0.5).
Additional information
Although detailed cell counts were not tallied, representative examples of ridges cultured for 18 h r in
TABLE 2. Influence of insulin-like growth factors on
cell death in cultured apical ectodermal ridges
Hours in
Supplements (%)
IGF 111
IGF I + insulin'
36 t 10 (11)3 38 t 8 (19)
18 t 9 (11)
35 t 10 (10)
23 7 ( 7)
'IGF I and I1 100 ng/ml.
'IGF I 100 ngiml, insulin 50 ngiml.
3Data are expressed as the average percentage necrosis 2 the standard deviation. The numbers in parentheses are the number of ridges
high-insulin medium on Matrigel or in mesodermal extracts were examined histologically. Ridges cultured in
insulin medium on Matrigel remained healthy but lost
their elongated morphology and did not spread. This
appeared to arise from a contraction of the gel by the
tissue and resulted in a more compact piece of ectoderm.
Ridges cultured for 18 hr in limb mesodermal extract
prepared according to Jorquera and coworkers (1979)
tended to lose their attachment to the dish and round
up. Cross sections of representative ridges exhibited
massive necrosis (not shown).
Ridge Function in Cultured Ridges
Control experiments: Noncultured ridges
Data from two groups of control experiments were
collected. One group consisted of grafting isolated limb
mesoderm in back ectoderm without an apical ectoderma1 ridge. The majority were made with stage 18 mesoderms, but four were constructed with stage 19 mesoderms and two with stage 20 mesoderms. None gave
a positive outgrowth (Table 3). The most that was obtained was a small rod or nodule of cartilage covered by
a shoulder girdle pattern of feathers (Fig. 4).
The other control group included grafts of limb mesoderms and freshly isolated stage 20 ridges in back
ectoderm. Fourteen operations survived the additional
8 days to day 11, and all produced outgrowths. The
average level of outgrowth was 2.5. Eight outgrowths
were distally complete, three of which contained at
least portions of all limb elements, the humerus, radius, ulna, and digits 2, 3, and 4 (Fig. 5).
To examine the fate of the grafted ridges, three recombinants were made with a freshly isolated, chick
apical ridge and quail back ectoderm and fixed 3 days
after grafting. Two limb buds (67%)had chick ectoderm
distally and both of these demonstrated ridge morphology. Chick cells were always located at the center of the
ridge, but quail cells often populated the edges of the
ridge and could be found basally. In some sections, it
appeared as if the quail cells were displacing the remaining chick cells. If no chick cells were present in a
particular section, ridge morphology was not evident;
the quail cells at the tip of the limb bud formed a simple, columnar epithelium capped by a squamous periderm. The third recombinant limb contained no chick
ectodermal cells distally and lacked a ridge. A small
amount of cell death was present in its distal mesoderm. Mesodermal cell death is consistent with a lack
of ridge function (Rowe et al., 1982).
Ridges cultured without mesoderm
General observations. Compared with the activity of
freshly isolated ridges, cultured ridges demonstrated a
significant decrease in ridge function with increased
time in culture. This was reflected both in the number
(P < 0.001) and the quality (P < 0.01) of outgrowths
obtained. The medium in which the ridges had been
cultured did not make a significant difference in the
quality or quantity of the outgrowths obtained (P >
0.5) with two exceptions. Therefore, except where differences were found, the results for all media are combined in the text but are presented individually in Table 3.
Six hour cultures. Seventeen recombinants were analyzed for limb morphogenesis; 13, or 76%,produced outgrowths, including seven distally complete limbs (Fig.
6). The average level of distal completeness was 2.2.
While both the percentage of outgrowths and the quality of the outgrowths were lower than those obtained
with freshly isolated ridges, neither was significantly
different (0.05 < P < 0.1 and 0.1 < P < 0.25, respectively).
Eight grafts were made with quail back ectoderm
and chick ridges that had been cultured for 6 hr in
control medium; five were fixed 2 days after grafting
and three after 3 days. Five recombinants (62%)
showed evidence of ridge morphology a t the apex of the
sectioned wing bud. All five had chick cells in the ridge
(Fig. 7). In fact, thick ridge was always associated with
most or all of the ridge cells being of chick origin, suggesting, as was reported by Saunders et al. (1976), that
ridge function was attributable to the grafted ridge and
was not assumed by the flank ectoderm. When the
distal ectoderm was populated with mainly quail cells,
the ridge became thinner. The two limb buds fixed after 2 days that lacked ridge morphology had evidence
of cell death in the distal mesoderm. This is consistent
with a lack of ridge function (Rowe et al., 1982). The
third bud lacking a ridge was fixed 3 days after grafting and did not have cell death in its distal mesoderm.
Twelve hour cultures. The 25 surviving recombinants
gave outgrowths in 56% of the cases, with an average
quality of outgrowth of 1.6. These were significantly
different from the 0 hr results (P < 0.005 and P <
0.005, respectively). Only one recombinant from the
low-insulin group yielded a distally complete limb (Fig.
8). Many of the outgrowths that were rated as 2 consisted of two long bones fused in tandem, but this was
not seen in grafts constructed with ridges that had
been cultured for 12 hr in the high-insulin medium.
Eighteen hour cultures. As was mentioned above, after
18 hr in culture, the epithelial sheets had spread considerably and the thickening of the ridge was less well
defined. Ridges cultured in control, low-insulin, or
high-insulin medium without bacitracin gave outgrowths at a lower rate than the 12 hr cultures (7121 or
33%,P < 0.001), but the average level of completeness
of those that did develop, 1.7, was essentially the same
(P > 0.5). The three outgrowths rated as 2 contained
two long bones fused in tandem. Only one graft that
had received a ridge cultured in control medium produced digital elements (Fig. 9).
Twenty-one recombinants received ridges which had
been cultured in medium containing bacitracin; none of
TABLE 3. Outgrowths induced by ridges cultured in the absence of mesoderm
Hours in
With ridge
No ridge
Low insulin
High insulin
Low insulin
High insulin
Low insulin
High insulin
High insulin
Insulin + matrigel
Low insulin
High insulin
High insulin
Level of outgrowth
'Two control groups a t 0 hr consisted of recombinants made with freshly isolated ridges or recombinants
made without ridges.
'F indicates that the long bones were fused in tandem.
these gave outgrowths (P < 0.001). This group included
eight ridges that had been cultured in control medium
and 13 that had been cultured in high-insulin medium.
Prior to this time, bacitracin did not have any inhibitory effects on either the number or the quality of the
outgrowths obtained (P > 0.5).
Of seven ridges cultured for 18 hr in high-insulin
medium on the basement membrane substratum Matrigel, none produced any evidence of limb growth. This
was in contrast t o other systems in which Matrigel
enhances function (Hadley et al., 1985; Kleinman et
al., 1986).The decrease in inductive activity may have
been due to a direct inhibitory effect of Matrigel or to
the shorter area that the limb mesoderm was in contact
with the limb ectoderm since the ridges contracted on
the gel. Alternately, since the ectoderm and gel were
separated only by mechanical means, an intervening
layer of Matrigel may have remained and inhibited the
transfer of inductive influences between the ridge and
the limb mesoderm. A layer of nonridge ectoderm delays the response of the mesoderm to the ridge (Murphy
et al., 1983); a nonliving barrier might also be inhibitory. In that we did not investigate the action of Matrigel further, we could not distinguish between these
Twenty-four hour cultures. As in the 18 hr cultures,
eight ridges cultured in the presence of bacitracin and
high insulin were unable to induce outgrowths. No outgrowths were obtained from 29 recombinants made
with ridges incubated in the control or low-insulin medium lacking bacitracin, while ridges cultured in highinsulin medium gave evidence of ridge function in two
of five cases (P < 0.001). Both positive high-insulin
recombinants were rated as two fused long bones (Fig.
Ridges cultured with limb extract or limb mesoderm
Of 11 grafts made with ridges cultured in limb mesodermal extracts prepared according to Jorquera and
coworkers (19791, only one produced an outgrowth
(Table 4).This graft yielded two long bones, the others
small rods or nodules of cartilage.
As in the cultures without mesoderm, isolated ridges
cultured for 18hr in high-insulin medium with isolated
pieces of limb mesoderm spread on the surface of the
dish. When used in recombinants, they did not exhibit
increased function over ridges cultured without mesoderm (11%vs. 33%). Only one outgrowth was obtained
from nine grafts. A long bone developed in this graft.
When intact distal tips were cultured, both the mesoderm and the ectoderm exhibited some spreading;
however, this was minimal compared with that observed with isolated ridge ectoderm. In addition, a distinct ridge capped the mesodermal mass. Eighteen recombinants were made with ridges that had been
cultured while in contact with limb mesoderm. Outgrowths were obtained in 56% of the grafts. While the
percentage of outgrowths was lower (P < 0.005), the
average level of completeness, 2.1, was comparable to
that obtained with freshly isolated ridges (P > 0.5).
Four of the limbs were distally complete (Fig. 11).
Ridges in control and high-insulin medium yielded
comparable results (P > 0.5).
These studies addressed two questions regarding the
fate of the apical ectodermal ridge in culture. The first
was since high, nonphysiological levels of insulin (5
pg/ml) increased the survival of cultured apical ectodermal ridge (Boutin and Fallon, 1984), could lower
levels of insulin or IGFs produce the same effect. We
Fig. 4. Outgrowth obtained from a control recombinant limb lacking
an apical ridge. A nodule of cartilage (arrow) covered by a shoulder
pattern of feathers has developed from the stage 18 wing mesoderm.
Fig. 6. Outgrowth obtained from a stage 19 wing mesoderm and a
stage 20 ridge cultured for 6 hr in high-insulin medium. A humerus,
radius, ulna, and digits 2 (shortened), 3, and 4 have developed.
Fig. 5. Outgrowth obtained from a stage 18 (33 somite) wing mesoderm and a freshly isolated stage 20 ridge wrapped in stage 22 back
ectoderm. A humerus, radius, ulna, and parts of digits 2,3, and 4 have
Fig. 7. Section through the tip of a 3-day-old recombinant limb made
from chick ridge cultured for 6 hr in control medium and quail back
ectoderm. The heterochromatin clumps characteristic of quail nuclei
(arrows) are evident in the dorsal and ventral limb ectoderm; however, the apical ectodermal ridge is primarily of chick origin. x 550.
have found that 50 ng/ml of insulin significantly decreased the amount of cell death in cultured, isolated
apical ectodermal ridges but that 100 ng/ml of IGF I or
IGF I1 was ineffective. These data suggest that insulin
improves ridge cell survival by acting via its own receptor. The second set of experiments addressed
whether conditions that maintain ridge cell survival
also maintain the ability of the ridge to induce limb
outgrowth. While apical ectodermal ridges cultured in
association with limb mesoderm for 18 hr retained the
ability to induce distally complete limbs, isolated
ridges rapidly lost this ability irrespective of whether
ridge viability was maintained. This suggests that
more than ridge cell survival is required to maintain
full ridge function.
The fact that 50 nglml insulin improves ridge cell
survival suggests that the apical ectodermal ridge contains insulin receptors. Although the embryonic chick
does not appear to possess distinct receptors for IGF I1
(Bassas et al., 1987, 19881, receptors for insulin and
IGF I are present in the early chick limb bud (Bassas et
al., 1987). These have not been localized to ectodermal
or mesodermal limb components, but interestingly, in
prelimb bud stage embryos, insulin and IGF receptors
are richest in ectodermal specializations (Girbau et al.,
Since insulin improves the survival of cultured apical ectodermal ridge, this raises the possibility that
insulin plays a vital role in limb development in ovo.
Insulin is present in the chick embryo from day 2 of
development (De Pablo et al., 1982), prior to the development of the pancreas (Benzo and Green, 1976; Serran0 et al., 1989), when it may be obtained from the
yolk (De Pablo et al., 1982) or synthesized at extrapancreatic sites (Serrano et al., 1989). Thus insulin is accessible to ridge cells in ovo. Limb mesoderm produces
IGF I1 (Engstrom et al., 1987; Stylianopoulou et al.,
1988; Romanus et al., 1988; Bondy et al., 1990) and IGF
I (D’Ercoleet al., 1980), and these are localized to the
peripheral limb bud mesenchyme (Ralphs et al., 1990);
however, neither IGF alone nor IGF I in concert with
insulin improved ridge cell survival in culture.
We do not know whether insulin increased ridge cell
survival through a specific influence on the differentiated state of the ridge cells or through a general metabolic effect such as an increase in glucose or amino
acid uptake (Strauss, 1984). Antibodies to insulin (De
Pablo et al., 1985) o r the insulin receptor (Girbau et al.,
Fig. 8. Outgrowth obtained from a stage 18 (34 somite) wing mesoderm and a stage 20 ridge cultured for 12 hr in low-insulin medium.
A humerus, radius, ulna, and parts of digits 2 and 3 have developed.
Fig. 9. Outgrowth obtained from a stage 18 (33 somite) wing mesoderm and a stage 20 ridge cultured for 18 hr in control medium. A
humerus, radius, ulna, and part of an unidentifiable digit have developed.
Fig. 10. Outgrowth obtained from a stage 18 (33 somite) wing mesoderm and a stage 20 ridge cultured for 24 hr in high-insulin medium. Two long bones have developed. Two small fleshly outgrowths
(arrowheads) also are evident.
Fig. 11. Outgrowth obtained from a stage 18 (33 somite) wing mesoderm and a stage 20 ridge cultured for 18 hr in contact with limb
mesoderm in high-insulin medium. A humerus, ulna, and digits 2 , 3 ,
and 4 have developed.
1984), a process believed to mimic that occurring during limb development. While insulin has profound effects on the metabolic activity of the regenerating
Level of outmowth
blastema (Vethamany-Globus et al., 19841,it is unclear
Type of mesoderm
3 whether any of this is due to effects mediated via the
wound epidermis, a structure analogous to the apical
Mesodermal extract
Mesodermal pieces
ectodermal ridge.
Tips in control medium
Despite the fact that insulin improves the survival of
2 cultured apical ectodermal ridge, insulin did not sigTips in high-insulin medium
nificantly improve the ability of cultured ridges to induce distally complete limbs. The function of stage 20
1988) significantly retard the development of 2 day apical ectodermal ridges declined with increasing time
chick embryos, indicating that insulin may have a gen- in culture irrespective of whether the medium coneral growth promoting activity very early in develop- tained insulin. This indicates that more than the mainment. However, in other systems, insulin appears to tenance of viability was necessary for ridge function
have specific effects on differentiation (Chapman et al., and that neither insulin nor the IGFs was sufficient to
1984;Wolinsky et al., 19851, at least some of which are maintain full ridge function in vitro.
independent of its growth promoting activity (Nicholas
It is unlikely that the spreading of the ridge ectoet al., 1983).It is interesting to note that serum insulin derms in culture contributed to the decline in ridge
levels rise during the initial stage of limb regeneration function and that limb mesoderm was able to prevent
in Xenopus laevis (Liversage et al., 1987) and that in- this decrease when intact distal tips were cultured
sulin appears to be an essential component for limb purely by acting in a structural manner to maintain
regeneration (Vethamany-Globus and Liversage, 1973; compact ridge morphology. Decreased ridge function
Vethamany-Globus, 1987; Vethamany-Globus et al., was evident prior to any major spreading of the culTABLE 4. Outgrowths induced by ridges cultured in
the presence of mesodermal factors for 18 hr
tured ridges, and there is evidence that neither the
flattening (Rubin and Saunders, 1972) nor the gross
rearrangement (Errick and Saunders, 1976) of ridge
cells correlates with its potential to induce limb outgrowths.
Furthermore, the decline in ridge function probably
was not related merely to normal aging of the ridge
during culture. A stage 20 ridge in situ continues to
induce outgrowth for an additional 3 days (Summerbell, 1974) and has the capacity to maintain this function even longer when combined with competent mesoderm (Rubin and Saunders, 1972).Thus, if the cultured
ridges merely had aged on schedule, ridges cultured for
24 hr still should have been able to induce digital elements (Summerbell, 1974; Rubin and Saunders, 1972).
However, digital elements rarely developed in grafts
made with ridges cultured for more than 6 hr, indicating that the cultured ridges had not maintained full
functional capacity.
Ridges cultured in association with distal limb mesoderm were able to induce the same level of limb outgrowth as freshly isolated ridges, although the incidence of outgrowths was lower. Hence, limb mesoderm
appeared to prevent or a t least slow the loss of ridge
function, supporting the concept of a limb mesoderm
maintenance factor.
While experiments performed with wingless and
polydactylous mutants support the existence of mesodermal factors which maintain ridge function (Zwilling, 1956; Zwilling and Hansborough, 1956; Carrington and Fallon, 1984b), the in vitro evidence for
mesodermal maintenance activity is more ambiguous.
Jorquera and coworkers (1979) reported that apical
ridges cultured for 24 hr in limb mesodermal extracts
remained viable and capable of inducing distally complete limbs. In contrast, Feinberg and Saunders (1979)
found that isolated apical ectodermal ridges cultured in
serum-containing medium for more than 12 hr lost
ridge function. However, since under these culture conditions isolated apical ectodermal ridges die (Searls
and Zwilling, 1965; Boutin and Fallon, 1984; this report), it is unclear if the loss of ridge function in Feinberg and Saunders’ experiments (1979) was due t o the
absence of limb factors or to ridge cell death. Furthermore, we were unable to repeat the results of Jorquera
et al. (1979); limb extracts did not maintain the viability, morphology, or function of cultured ridges. We
do not know why we obtained such disparate results.
Nevertheless, our data demonstrate that ridge function
can be diminished in the absence of ridge cell death and
that limb mesoderm supports the maintenance of ridge
The fact that bacitracin led to a precocious loss of
ridge function is intriguing. Ridges cultured in highinsulin medium containing bacitracin are viable at 18
and 24 hr (data not shown), so the loss of function was
not due to accelerated cell death. Bacitracin inhibits
some degradative enzymes (Roth, 1981; Peavy et al.,
1985),perhaps some of these are essential for the maintenance of ridge function. If it could be determined how
bacitracin accelerates the loss of ridge function, valuable insight might be gained on how function is normally maintained.
While it is not known what molecules or factors are
required in ovo for maintaining the function of limb
ectoderm, our data demonstrate that ridge cell survival
did not guarantee the retention of full ridge function,
S O , a t least under these conditions, ridge cell survival
and ridge cell function could be uncoupled. Obviously,
function can be lost in a nonviable tissue even if functional maintenance activity is present. Thus it will be
important t o distinguish between factors that regulate
function and those that influence cell survival, if we
are to understand what is required for maintaining full
ridge function.
A number of growth factors are produced or at least
present in limb mesoderm (Bell, 1986; Goedert, 1986;
Engstrom et al., 1987; Heine et al., 1987; Seed et al.,
1988; McLachlan et al., 1988; Munaim et al., 1988;
Joseph-Silverstein et al., 1989). It remains to be addressed whether these growth factors are instrumental
in maintaining the ability of the apical ectodermal
ridge to induce distally complete limb outgrowth. Since
we have established conditions that permit ridge cell
survival, it is now possible to test whether the addition
of other growth factors will permit the retention of
ridge cell function in the absence of limb mesoderm.
This would permit a more biochemical approach to be
taken in investigating the complex interactions required for limb development.
We thank Diane Widegran a t KabiVitrum AB for
the generous gift of met-IGF I. We appreciate the helpful discussions with Drs. Robert DeHaan, Arnold
Ruoho, and Michael Shannahan. We thank Kay
Simandl and Tamara Bucci for technical assistance.
This work was funded by NIH grant T32HD07118,
NSF grant PCM8406338, and NIH grant POlHD20743.
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