The function of the ectodermal apical ridge and distinctive characteristics of adjacent distal ...

15 Pages·2008·1.85 MB·English

J. Embryol. exp. Morph. Vol. 34, 1, pp. 155-169, 1975 J 55
Printed in Great Britain
The function of the ectodermal
apical ridge and distinctive characteristics of
adjacent distal mesoderm in the avian
wing-bud
By JOHN M. CAIRNS1
From the Springville Laboratories of Rosweil Park Memorial Institute,
New York State Department of Health,
Springville, New York
SUMMARY
Blocks of mesoderm about 01 mm in diameter were isolated from various regions of
chick wing-buds of stages 17 through 22 and cultured individually, or sometimes in pairs,
in microtest plate wells. Cell deaths had occurred after 10 h of culture in those explants that
had come from the region associated with the thickest part of the ectodermal ridge, and
after 11-12 h in all other mesoderm. When the adjacent ectodermal ridge was left attached
to the mesodermal block there were almost no cell deaths for up to 24 h of culture. When
the dorsal ectoderm immediately proximal to the apical ridge was left attached, but no
ridge was present, cell deaths occurred just as they did in mesoderm with no ectoderm. When
a number (usually six) of complete ridges were suspended in a wire basket at the top of a well,
cell deaths did not occur in a test mesodermal block at the bottom of the well (six of eight
cases). These experiments support previous evidence for a special function of the ectodermal
apical ridge in limb morphogenesis, and indicate that there is a chemical messenger.
The cells that migrated from distal mesodermal explants (the band up to 015 mm from
the apical ridge) differed sharply in morphology and behavior from those coming from
explants from any more proximal region. Within the proximal mesoderm there was a less
striking variation along the antero-posterior axis. These observations reveal that there is
present even at early stages a detailed pattern within the mesoderm of the limb-bud. The
particularly striking and distinctive characteristics of that mesoderm closest to the apical
ectodermal ridge provide new possibilities for the understanding of the function of the ridge
in limb morphogenesis.
INTRODUCTION
The striking elongation of the limb-buds of the chick embryo during the
fourth day of incubation is dependent on some action by the slender thickening
in the ectoderm at the distal tip, the apical ridge. In the responding mesoderm
the cellular activities that contribute to the patterned growth are still obscure
(Ede, 1971; Faber, 1971). Surgical removal of the apical ridge from the wing-
bud in ovo is followed within a few hours by the death of many cells in a narrow
1 Author's address: Springville Laboratories, Springville, New York 14141, U.S.A.
156 JOHN M. CAIRNS
band of the subjacent mesoderm (Barasa, 1960; Cairns, 1975). The first deaths
occur anteriorly, and the process then spreads posteriorly. This temporal
sequence in the onset of cell death in the denuded mesoderm is apparently
influenced by more proximal mesoderm since small blocks of central distal
mesoderm transplanted to proximal sites distant from the host ridge undergo
cell death near the anterior end of the host bud, but not at the posterior end.
Both an apical ridge factor and an effect of proximal mesoderm on distal
mesoderm are involved in the events in the distal mesoderm.
The occurrence of cell death in mesodermal cells removed from association
with the apical ridge and the absence of any cell deaths among mesodermal
cells associated with the apical ridge suggests an approach to detecting ridge
function. In the experiments to be described, blocks of mesoderm from the
wing-bud were cultured either without any ectoderm, or with ectoderm not
including any apical ridge, or with ectoderm including a portion of the apical
ridge. Cell deaths occurred in all explants except those that had apical ridge
present. In addition to this evidence for apical ridge function both morphologi-
cal and behavioral characteristics of the cells from the slender strip of mesoderm
associated with the apical ridge differed from those of more proximal cells.
These characteristics of the most distal mesodermal cells suggest new possibilities
for the interpretation of apical ridge function.
MATERIALS AND METHODS
The large majority of embryos used as a source of tissue for culture were of
the H and N 'Nick' strain obtained from a nearby commercial producer.
Others were normal embryos from a stock maintained at this laboratory carry-
ing the gene 'talpid-2'. This stock has been outcrossed to H and N birds in the
past. In addition some Japanese quail and some Muscovy duck embryo tissues
have been used.
Microtest plates (Falcon Plastics, no. 3034) were used. These dishes contain
60 wells each, and each well holds about 15 /A. of medium. F-12 with 10 % fetal
calf serum was the only medium used. For most of the experiments incubation
was in a CO incubator with 3-5 % CO to control pH, and close to 100 %
2 2
humidity. More recently HEPEs buffer has been used, without CO added in
2
the incubator. No difference in the behavior of the explants was detected
when parallel experiments with CO and HEPEs were run in separate incubators.
2
Tissues were dissected from donor wing-buds in ovo using glass needles. As
pieces were isolated they were transferred to the culture dish containing medium.
During the dissections, which in extreme cases continued for 2-3 h, the culture
dish was kept on ice so that all explants would begin development in the culture
vessel at the same time-i.e. when incubation began. To test for possible
effects of the low temperature on the explants three were kept for 6 h on ice
and then implanted on host wing-buds. The growth and fate of such transplants
Ridge function and mesoderm 157
did not differ from control direct transplants. However, when similar isolates
were kept in cold medium for 4, 2, and \ h the initial migration of cells was
slightly later from those explants that had been cold for longer times.
The cultures were photographed at intervals with a Galileo inverted phase-
contrast microscope. Time-lapse films were made of some cultures, with the
assistance of Dr Eric Mayhew. Cultures were fixed in a mixture of alcohol,
formalin and acetic acid (85:10:15) after flushing away the medium with a
saline solution. The explants and cells that had migrated were stained in the
culture dish with Wenger's hematoxylin (Wenger, 1951). As much of the
explant mass as adhered together was freed from the bottom of the well with
a nylon fiber, and embedded in parafin for sectioning. Sections were cut at 6 jLtm.
To test the behavior of the migrating cells on a different substrate, and to
retain the migrating cells with the explant in sections, some explants were
placed on small fragments of Millipore niters inserted into the wells. A further
advantage of this procedure was that explants from a single vessel could be
fixed at successive time intervals. Some explants were also cultured either on
glass cover-slips placed in small Petri dishes, with only a small drop of medium
on the region containing the explanted fragments, or in a long narrow trough
prepared by attaching strips of glass to the surface of a microscope slide. These
explants grown on glass surfaces were stained and then mounted as permanent
preparations.
RESULTS
Blocks of tissue about 0-1 mm in diameter dissected from wing-buds of
stages 17-23 and placed in culture showed a sequence of changes that began
during the first hour of culture. Initially the blocks were rather irregular. They
rounded into more or less spheroidal masses during the first 2-3 h. Cells
appeared around the periphery, migrating over the substrate. A little later the
explants began to flatten out, and still later small particles appeared in the
interior. After sectioning, these particles were found to be debris from cells that
had died. The time course of these events and their occurrence in an explant
varied with the original location of the explant in the donor wing-bud, and
with the specific tissue included. In addition there were minor variations asso-
ciated with the processing and the medium. Each individual experiment included
in a single culture vessel two and sometimes more different kinds of explant.
The differences between explants described in the following sections were those
observed consistently in single experiments. However, for brevity the tables
will not include the specific pairing.
Distal and proximal mesoderm
After removal of the apical ridge from the wing-bud in situ cell deaths occur
in a band of distal mesoderm. Explants were dissected from the central portion
of this band, and for comparison pieces of proximal mesoderm from a location
158 JOHN M. CAIRNS
adjacent to the somite border and central along the antero-posterior axis were
also isolated.
Both kinds of mesoderm rounded up similarly. Cells began to migrate first
from proximal explants, generally after 2-3 h of culture, and about 1-2 h later
from the distal explants. The time of first cell migration varied with the fetal
calf serum. When a vial was first thawed parallel cultures with serum from the
preceding vial were made. In general cells in medium with serum that had been
thawed for some weeks migrated as much as 1 h before migrating cells appeared
in the medium with recently thawed serum. There were greater differences when
the sera were of different lots. The first appearance of migrating cells was always
from proximal explants, and varied from about 1 h to about 5 h after initiation
of the culture.
After cells were migrating from both distal and proximal explants obvious
differences in the colonies were apparent. Proximal colonies included some
number of bipolar elongate cells, while distal colonies had only multipolar,
stellate cells. In the time-lapse films a single cell changed from bipolar to
stellate to spheroidal and then to bipolar again during a short time interval.
The stellate cells in distal colonies appeared in general to be somewhat larger
(area) than the stellate cells in proximal colonies. Individual cells in the proximal
colonies moved to some distance from any neighbor, while in distal colonies
the cells remained in contact with one or more adjacent cells. After about 6 h
in culture distal explants began to spread and flatten, with the result that the
sharply defined boundary between migrating cells and the explant proper
disappeared. Proximal explants, in contrast, remained spheroidal masses with
a distinct boundary for at least 10-12 h. The distinctive characteristics of the
colonies were stable in culture for at least 43 h (Fig. 1). Each of the three
species examined, chick, Japanese quail and Muscovy duck, showed the same
features in distal and proximal colonies.
Beginning at about 10 h in culture distal explants contained small particles.
The number of these increased with further culture, and after 11-12 h proximal
explants also had similar particles. When such explants were sectioned they
were found to contain debris of cells that had died.
Other cells were in various stages of mitosis. The appearance of cell deaths
in distal explants before they occurred in proximal explants was also observed
when a single distal and a single proximal explant were both placed in one well
in contact. The characteristic features that distinguish each kind of explant
were not altered by the presence of the other, and served to identify the explants
when both were in one well (Fig. 2). For the most careful comparison of the
time of onset of cell death in distal and proximal explants seven pairs were
cultured on Millipore filter pieces in a single experiment. Two pairs were fixed
after 10 h, two after 11 h, and three after 12 h of culture. At 10 h a small amount
of debris was present in one of the distal explants and none in the other distal
nor the two proximal explants. After 11 h there was a moderate amount of
Ridge function and mesoderm 159
FIGURE 1
Explants of central distal mesoderm (A, B, C) and central proximal mesoderm
(D, E, F) in culture for 3 (A, D), 8 (B, E) and 43 (C, F) h. Proximal cells have migrated
at 3 h, but no distal cells are out; some cells from the proximal explant are long
bipolar cells and migrate individually to some distance from any other cell, while
distal cells are stellate and are more closely arranged; after 8 h in culture the distal
explant has spread while the proximal explant is still a discrete mass; and after
43 h the two colonies still have their distinctive characteristics, x 114.
II EMB34
160
JOHNM. CAIRNS
#
Fig. 2. Central distal (above) and central proximal mesodermal explants and
colonies after 7 h of culture on Millipore filter. Fixed and stained, x 80.
Fig. 3. Central distal (left) and central proximal (right) explants that were cultured
in contact on Millipore filter for 11 h. There is a moderate accumulation of debris
in the distal explant, and a trace in the proximal, x 240.
Table 1. Onset and extent of cell death in explants of mesoderm from different
parts of the wing-bud
Time in Amount of debris*
Origin of culture r— A —>,
mesoderm (h) 0 1 2 3 Subtotals Totals
Central distal 10 9 2 — U)
10-12 — 5 22 5 32- 59
12 1 2 13 16j
Central proximal 10 10 — — 101
10-12 14 14 4 3 35 60
12 — 2 13 — isj
* Serial sections weres scanned and scored as having no debris (0),a little debris (1), some
debris (2), or much debris (3).
debris in both distal explants, one proximal explant contained a small amount,
and the other had none (Fig. 3) (Table 1). After 12 h of culture there was no
difference between the distal and proximal explants in the amount of debris.
The pattern of variation throughout the wing-bud
The transition between the patterns described for distal and proximal wing-
bud mesoderm and the variation within these two regions were examined by
dissecting long strips of mesoderm into individual blocks, each about 0-1 mm
in diameter, and placing the blocks in wells in sequence. The change from distal
to proximal was observed in a strip extending from the apex to the somite
border and midway along the antero-posterior length of the bud, the variation
Ridge function and mesoderm 161
../.. F(10)
Fig. 4. Variation in the distal mesoderm from anterior to posterior. The entire distal
strip of mesoderm from the anterior to the posterior borders was cut into 10 pieces
and each cultured. The first (A), third (B), fifth (C), seventh (D), ninth (E) and tenth
(F) pieces, from anterior to posterior, are illustrated. See text for details, x 114.
162 JOHN M. CAIRNS
within the distal mesoderm was examined by taking the entire band from the
anterior to the posterior limits of the bud, and that in the proximal mesoderm
similarly by taking a strip from the base and extending the entire antero-
posterior length of the bud.
The change from distal to proximal patterns occurred in a short distance.
As described, the distal mesoderm, to a depth of about 0-1 mm produced no
bipolar cells. The next proximal explant, located in the range of 0-1-0-2 mm
from the apical ridge, produced at least a few bipolar cells, the stellate cells
were somewhat smaller (area), and there were more open spaces among the
cells. The third explant, from the region 0-2-0-3 mm from the apical ridge,
was entirely proximal in its characteristics. This complete change within about
0-25 mm from the ridge was observed in sets of explants from stage 17 through
22. The band of mesoderm with distal quality is about the same proximo-distal
thickness through all these stages.
The distal mesoderm was examined in sets of explants that were taken from
the entire antero-posterior length of the wing-bud. The 'distal' characteristics
as described were most pronounced in the mesoderm that was associated with
the thickest part of the apical ridge - i.e. a length somewhat posterior from
the center. Toward both the anterior and the posterior ends there was a gradual
change. At the anterior end the pattern was similar to that for proximal explants.
The first cells to migrate appeared from the most anterior of the explants,
next from the most posterior. The cells migrating from the anterior explant
included a number of bipolar cells, the adjacent explants had fewer bipolar
cells, and the 4th to 5th explant had none at all. Bipolar cells were not seen in
the most posterior explants. The spreading of explants began in those from a
little posterior to the center, and showed a spread with time toward both the
anterior and posterior ends (Fig. 4). Cell deaths were first seen after 10 h of
culture in explants from the central region, and 1-2 h later in those from the
anterior and posterior ends.
The proximal mesoderm also showed a consistent variation along the antero-
posterior length. The most anterior explants produced a smaller proportion of
the long bipolar cells, and these did not move as far from the explants. After
about 6 h of culture the bipolar cells from the posterior explant had migrated
nearly twice the distance compared with those migrating from the most anterior
explant. The change from anterior to posterior was gradual and uniform.
The effect of ectoderm on the mesoderm
The purpose of the experiments included in this section was to determine if
the apical ridge or any other ectoderm would prevent the occurrence of cell
deaths in mesoderm cultured in contact with the ectoderm. With a few excep-
tions all the experiments of this kind were made by dissecting the mesoderm
with its overlying ectoderm attached. To include apical ridge the tissue was
taken from the apical zone, including the ridge, a short width of ventral ecto-
Ridge function and mesoderm 163
Fig. 5. Scheme of operations to compare the influence of the apical ridge
with that of other ectoderm.
derm, and a more extensive sheet of the dorsal ectoderm. The principal
comparison was with blocks of distal mesoderm that included the dorsal
ectoderm, but without any apical ridge (Fig. 5). During culture the ectoderm
in both kinds of explant developed into a more or less hemispherical cap of
ectoderm. The free edge contracted, penetrating slightly into the mesoderm.
The part of the explant that was enclosed by the ectodermal cap was somewhat
less than half the total and although less ectoderm was included when no ridge
was taken, the enclosed part after culture was similar to that with ridge. After
staining and with glancing illumination the ectoderm was distinguishable by
its smooth surface, while the mesoderm was somewhat rough (Fig. 6). Some
attempts were made to obtain central proximal mesoderm with its ectoderm.
On these pieces the ectoderm was nearly planar, and it curled most frequently
away from the mesoderm, so that it changed into a small sphere that sank into
the mesoderm and formed a cyst. In a few instances the ectoderm did develop
into a cap that enclosed some of the mesoderm.
164 JOHN M. CAIRNS
B
Fig. 6. Surface view of explants with ectoderm, after fixing and staining: (A) an
explant with apical ridge surrounded by the colony of migrating cells; (B) a similar
explant tipped over to show the constriction at the free edge of the ectoderm, x 80.
Table 2. The effect of ectoderm on cell death in mesoderm after more than 10 h
in culture
Amount of debris*
0 1 2 3 Tot;
Distal mesoderm with apical ridge 39 10 6 1 56
Distal mesoderm with ectoderm, no ridge 1 5 12 7 25
Proximal mesoderm with ectoderm, no ridge — 1 1 1 3
Mesoderm at bottom, 6 ridges suspended at top 6 1 1 8
* Serial sections were scanned and scored as having no debris (0), a little debris (1), some
debris (2), or much debris (3).
The explants with ectoderm were paired in various ways in individual experi-
ments, including distal mesoderm with apical ridge paired with distal mesoderm
with no ectoderm; distal mesoderm with apical ridge paired with distal meso-
derm with dorsal ectoderm but no ridge; distal mesoderm with dorsal ectoderm
(no ridge) paired with proximal mesoderm with ectoderm. These were intended
specifically to test the possible effect of the ectoderm. In addition there were a
small number of other experiments which included explants of distal mesoderm
with apical ridge. The results will be presented without specifying the details
of pairing.
The distal mesoderm when cultured in contact with a portion of apical ridge
had no cell debris in the large majority of explants cultured from a little more
than 10 h up to 24 h (the longest period covered) (Table 2). In contrast, similar
masses of distal mesoderm enclosed to the same extent in dorsal ectoderm, but
with no apical ridge present, had cell debris present in almost every instance