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/. Embryol. exp. Morplt. Vol. 60, pp. 189-199, 1980
Printed in Great Britain © Company of Biologists Limited 1980
Differential localization of [l!5S] sulfate
within ectodermal basement membrane in relation
to initiation of chick limb buds
DENNIS M. LUNT 1 AND ROBERT E. SEEGMILLER 1
From the Department of Zoology, Brigham Young University
Provo, Utah
SUMMARY
This study examined the possible developmental relationship between differential amounts
of glycosaminoglycans (GAG) within the ectodermal basement membrane and initiation of
limb outgrowth. Chick embryos at stages 11-20 were labeled in ovo with 35SO4 and processed
for autoradiography. After exposure to sulfate for 15 min, the label was localized within the
surface ectoderm whereas after 3 h the label was localized within the subjacent basement
membrane. The label was sensitive to chondroitinase ABC. These experiments suggest that
the labeled material was ectodermally derived chondroitin sulfate. At all stages examined,
intense labeling of the basement membrane was associated with relatively undifferentiated,
mitotically active tissue, e.g. Jimb-bud mesoderm. The labeling was less intense in regions with
decreased mitotic activity, e.g. the flank. Thus, the labeling pattern of the basement membrane
correlated with differential mitotic rates presumed to be associated with limb outgrowth. These
observations support the hypothesis that communication between tissues of different origin
resulting in altered mitotic behavior of limb and flank mesenchymal cells is facilitated by
the ectodermal basement membrane.
INTRODUCTION
The well known epithelial-mesenchymal interactions of the limb and. trunk
appear to be mediated by the ectodermal basement membrane. Indirect evidence for this comes from the observation that mesenchymal cells do not
directly contact cells of the ectoderm but only the intervening basement membrane (Ede, Bellairs & Bancroft, 1974; Kelly & Bluemink, 1974; Smith, Searls &
Hilfer, 1975; Kaprio, 1977; TheslerT, Lehtonen & Saxen, 1978). Additional
evidence is provided by the talpid3 chick embryo in which the limb reduction
anomaly is associated with fewer than normal contacts between mesenchymal
cells and basement membrane (Ede et ah 1974). Since compositional changes of
the basement membrane have been observed in developing tissues (Bernfield,
Cohn & Banerjee, 1973; TheslerT, Stenman, Vaheri & Timpl, 1979), it may be
that the basement membrane is more than passively involved in the signaling of
1
Authors' address: Department of Zoology, 593 WIDB, Brigham Young University,
Provo, Utah 84602, U.S.A.
13
EMB 60
190
D. M. LUNT AND R. E. SEEGMILLER
new developmental events. Although our understanding of the synthetic function of the ectoderm and composition of the ectodermal basement membrane of
early avian embryos has increased (Fisher & Solursh, 1977;Pintar, 1978; Sanders,
1979; Solursh, Fisher & Singley, 1979), the significance of the overall spatial
and temporal alterations of the membrane has yet to be determined.
The present study makes use of the autoradiographic technique to describe
the overall pattern of radiosulfate incorporation into the ectodermal basement
membrane of chick embryos during stages 11-20. Specific interruptions of the
general labeling pattern were observed which were temporally and spatially
associated with the initiation of limb outgrowth.
MATERIALS AND METHODS
Fertile eggs of the Babcock strain, obtained locally, were incubated at 37 °C.
Embryos, stages 11-20 inclusive (Hamburger & Hamilton, 1951), were labeled
in ovo with 10 /tCi Na235SO4 (New England Nuclear) for 3 h or with 100 /tCi for
15 min. After labeling, the embryos were removed from the yolk, fixed in
Carnoy's solution containing 0-5% cetylpyridinium chloride, embedded in
paraffin and serially sectioned at 7 jttm in transverse and sagittal planes. Random
sections were exposed for 1 h to chondroitinase ABC (Miles Laboratories) in
enriched tris buffer according to Saito, Yamagata & Suzuki. (1968), to buffer
alone or left untreated. The slides were dipped in Kodak NTB-2 nuclear track
emulsion, stored in the dark at 4 °C for 10 days, developed in Dektol and stained
with nuclear fast red. Embryos thus processed were photographed and examined
for differential labeling of the ectodermal basement membrane.
RESULTS
35
After labeling for 15 min with SO4=, the isotope was localized within the
ectoderm but not the basement membrane of stage-11 to stage-20 chick embryos.
The intensity of the label for different regions of the embryo depended on the
stage examined. The general labeling pattern was similar in chick embryos
exposed to the isotope for 3 h except that the label was localized predominantly
within the basement membrane (Figs. 1-4). This suggests that basement membrane labeled materials originated from the ectoderm (Solursh et al. 1979).
Labeled materials were sensitive to chondroitinase ABC treatment.
During stages 11-13, prior to limb initiation, the ectodermal basement
membrane overlying axial and lateral plate mesoderm labeled differentially with
35
SO4=. Relative to anterior regions (Fig. la), labeling of the basement membrane in posterior regions (e.g. segmental plate and most posterior somites,
Fig. 1 b) was more intense. The anteroposterior gradient of increasing labeling
intensity was also observed in parasagittal sections (Fig. lc). This general
Basement membrane GAG and limb outgrowth
191
a x i a l EBM
Fig. 1. Stage 11. The ectodermal basement membrane (EBM) overlying the fifth
pair of somites (a) did not label intensely relative to the more posterior EBM overlying the segmental plate (b). In sagittal section (c) an anteroposterior gradient of
increased labeling intensity is shown, x 132.
13-2
192
JKC
D. M. LUNT AND R. E. SEEGMILLER
v Xf^'^"^:" •
Sap-*"
Fig. 2. Stage 14. The axial EBM overlying the tenth pair of somites (a) did not label
intensely relative to the more posterior EBM overlying the sixteenth pair of somites,
or presumptive wing region (b). Relative to the lateral plate EBM in anterior regions
of the embryo (a) the lateral plate EBM of the wing (b) was intensely labeled. Labeling
was intense within both the lateral plate and axial EBM at the segmental plate, or
flank region (c). Note the cell-free space (arrow) in the flank (c) but not in the wing
(b) region, x 132.
Basement membrane GAG and limb outgrowth
193
Fig. 3. Stage 16. Labeling of the lateral plate EBM anterior to the wing bud (a) was
light, whereas labeling of the wing EBM (b) was intense. Labeling of the axial EBM
was light in these two regions. Relative to that of the flank (c) labeling of axial and
lateral plate EBM at the leg level was intense (d). x 132.
194
D. M. LUNT AND R. E. SEEGMILLER
Basement membrane GAG and limb outgrowth
195
!<>?
Fig. 4. Stage 18. Wing (a) and leg EBM (c) labeled intensely, particularly adjacent to
the AER, relative to flank lateral plate EBM (b). The axial EBM was lightly labeled
throughout all regions, x 132.
labeling pattern was similar throughout stages 11-13, i.e. decreased labeling
intensity of the ectodermal basement membrane was observed in more mature,
anterior regions.
At stage 14, during the initial outgrowth of the wing bud (Searls & Janners,
1971), the labeling intensity of the basement membrane overlying axial mesoderm was light anterior to the presumptive wing region (somites 1-14, Fig. 2a),
moderate at the presumptive wing region (somites 15-20, Fig. 2b) and intense
at the presumptive flank and leg regions (somites 21-22 and segmental plate,
Fig. 2c). Labeling of lateral plate basement membrane in the posterior regions
of the embryo (wing and flank, Fig. 2b, c) was intense relative to more anterior
regions (Fig. 2a).
Outgrowth of the wing bud continued and initial outgrowth of the leg bud
occurred during stages 15-16. The segmental plate had given rise to somites in
the flank and anterior part of the leg regions. The labeling pattern of the basement membrane overlying the embryonic axis was similar to that observed in
younger embryos, i.e. light anterior to the wing region (Fig. 3a), moderate
within the wing and flank region (Fig. 3 b, c), and intense within the leg region
196
D. M. LUNT AND R. E. SEEGMILLER
Stage:
11
16
Somites:
13
28
I I
Anterior
Somite
Segmental
plate
W
Posterior
Lat. plate
EBM
Fig. 5. The changing 35SO4~ labeling pattern of the ectodermal basement membrane
(EBM) overlying axial and lateral plate mesoderm as the lateral plate differentiates
into wing (W), flank (F), and leg (L) tissues. Intense labeling is indicated by a solid
line; less intense labeling by a broken line.
(Fig. 3d). The basement membrane overlying lateral plate mesoderm was similarly labeled except that the labeling intensity of the wing basement membrane
was not decreased from that of stage 14 (Fig. 3 b).
During stages 17-20, outgrowth of the wing and leg buds continued and the
apical ectodermal ridge (AER) developed. The labeling intensity of the axial
basement membrane overlying the somites of the wing, flank and leg region was
light (Fig. 4a-c). The basement membranes of the wing and leg, formerly lateral
plate, were intensely labeled, particularly in the vicinity of the AER (Fig. 4a, c).
In the flank region (Fig. 4b), the basement membrane overlying lateral plate
mesoderm was lightly labeled.
To summarize these observations, at all stages examined the ectodermal basement membrane overlying the posterior, less differentiated axial regions of the
embryo, e.g. segmental plate and newly formed somites, labeled more intensely
with 35SO4= than the more mature anterior regions (Fig. 5). The labeling pattern
of the lateral plate basement membrane was similar to that of the embryonic
axis except that within fore and hind limb regions the basement membrane
continued to label intensely as the embryo progressed from stage 11-20.
DISCUSSION
The observations presented in this paper suggest that mitotically active,
relatively undifferentiated mesoderm of chick embryos is associated with
Basement membrane GAG and limb outgrowth
197
accumulation of sulfate-containing materials, presumably chondroitin sulfate
(Cohn, Banerjee & Bernfield, 1977; Fisher & Solursh, 1977), into the ectodermally derived, expanding basement membrane. As each embryonic region
matures in an anteroposterior direction, the accumulation of labeled products
within the overlying basement membrane appears to decrease. A similar observation was made in younger embryos where the ectoderm at the level of the
heart labeled more intensely with radiosulfate at stage 8 than at stage 12
(Manasek, 1970). Whether the distribution of isotope observed in the present
study can be attributed to differences in rates of synthesis, pool sizes, or rates of
degradation is not clear.
At stage 14 the axial basement membrane overlying somites 15-20 (level of the
wing, Figs. 2b, 5) did not label as intensely with sulfate as it did earlier (Fig. 1 b,
5). The time of onset of decreased labeling coincides with increased production
of matrix hyaluronate and initiation of neural-crest-cell migration at the wing
level (Pintar, 1978). Also at the wing level, cells of somite origin begin migrating
at this stage (Chevallier, 1978; Jacob, Christ & Jacob, 1978; Chevallier, 1979) to
give rise to the myogenic cells of the limb and flank (Chevallier, Kieny &
Mauger, 1977). To what extent the observed compositional changes of the
ectodermal basement membrane of the embryonic axis are associated with the
initiation of such cellular behavior is not known.
A pattern of labeling (anterior-posterior) similar to that observed within the
basement membrane overlying axial mesoderm was also observed within the
basement membrane overlying the lateral plate mesoderm. An important
difference, however, was that labeling of wing and leg basement membranes did
not decrease as the wave of decreasing activity proceeded in a posterior direction.
Rather, as mesodermal cells accumulated and became intimately associated with
the ectoderm of the presumptive wing (Fig. 2b), the ectodermal basement
membrane of this region continued to label intensely as in younger embryos.
The difference between limb and flank regions in this regard confirms observations by Smith et al. (1975) that the composition of the subectodermal matrix
in the limb differs from that of the flank. Furthermore, intense labeling of the
limb basement membrane corresponds with the continued high mitotic activity
of the wing mesoderm (Searls & Janners, 1971) associated with early limb outgrowth. Both mitotic activity and basement membrane labeling of the wing
(Searls, 1965) decrease after stage 22. The decreased labeling of the flank basement membrane at stages 15-16 corresponds with decreasing mitotic activity of
flank mesoderm (Searls & Janners, 1971).
In conclusion, our observations suggest the active participation of the basement membrane in limb and trunk development, that compositional alterations
of the membrane and subjacent matrix may signal differing mesodermal activities such as mitosis or migration. It is conceivable that signals originating at the
basement membrane-mesoderm interface may spread throughout the mesoderm by way of junctional complexes which are present in the mesoderm
198
D. M. LUNT AND R. E. SEEGMILLER
(Kelley & Fallon, 1978). On the other hand, our observations may merely
reflect the necessary remodeling of the basement membrane during expansion of
the ectoderm in mitotically active areas. It has been suggested that the expanding
ectoderm of stage 23-25 limbs may promote the high mitotic activity within the
distal mesoderm by providing space into which cells proliferate (Summerbell &
Wolpert, 1972). Further work is needed to more clearly elucidate the functional
role of the basement membrane in limb development.
This work was supported in part by a grant from the March of Dimes Birth Defects
Foundation.
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(Received 26 November 1979, revised 28 April 1980)