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MICROSCOPY RESEARCH AND TECHNIQUE 40:446–454 (1998)
Cytochemical Localization of Adenylate Cyclase
in the Limb Buds of Bufo bufo
SIMONETTA TEI, DANIELA VAGNETTI, MONICA DI GIACOMO, AND ROSALBA M. FARNESI*
Institute of Comparative Anatomy, University of Perugia, v. A. Pascoli, Italy
KEY WORDS
cAMP; cytochemical and ultrastructural analysis; limb bud; Amphibia
ABSTRACT
The importance of cyclic nucleotides in the regulation of the processes of differentiation and embryonic development is known. The possible role that cyclic adenosine monophosphate
(cAMP) plays during the development of the posterior limb of Bufo bufo is studied by the
cytochemical localization of adenylate cyclase (AC), an enzyme that catalyzes the synthesis of the
cyclic nucleotide. The method is based on the reaction between the enzyme AC and its specific
substrate AMP-PNP (58-adenylylimidodiphosphate) in the presence of lead. The lead precipitates
that form as secondary reaction products are evidence of enzymatic activity. Reaction products are
present only at the epithelial level in the limb bud; initially, such products are visible only at the
base of the bud, particularly on the epithelial fascia located at the boundary with the body. During
successive elongation and toe formation, AC activity is only present on the cells of the proximal
portion of each new segment. Enzymatic activity is never present in correspondence to the
ectodermal apical crest. cAMP is probably not involved in the processes of cellular proliferation but,
rather, in the processes of inducing differentiation of the internal mesenchymal cells. Microsc. Res.
Tech. 40:446–454, 1998. r 1998 Wiley-Liss, Inc.
INTRODUCTION
In embryonic vertebrate limb development, the complex interactions between the ectoderm and the underlying mesenchyme are essential for the normal morphogenesis of this structure. These relationships, present
during the entire period of limb development, are
represented by the formation of the apical ectodermal
ridge (AER) (Laufer, 1993), with the induction by AER
of the expression of some HodX complex genes in the
mesenchymal cells (Vogel et al., 1995), and with the
regulation by the ectoderm of the extracellular matrix
composition in the subepidermal mesoderm (Knudson
et al., 1995).
The formation of the AER, upon the induction of the
apical mesenchyme of the limb bud, occurs very early
and the structure looks like a thickened ectodermal
hood covering the bud apex (Balinsky, 1972). The
mesoderm not only induces the formation of the AER
but also maintains it; in fact, when non-limb mesoderm
is transplanted under the AER, the crest shrinks and
limb development ceases (Gilbert, 1988).
The AER interacts with the mesenchyme and is
essential for limb growth (Zwilling, 1955; Saunders et
al., 1957; Saunders, 1972). It permits the polarized
development of the limb along the proximal–distal axis
(Bodemer, 1968; Kosher and Savage, 1980) stimulating
the proliferation of the underlying mesenchymal cells
(Bodemer, 1968; Laufer, 1993). The control of cellular
proliferation should occur as the result of the antagonistic action of two proteins, FGF-4 and BMP-2, the genes
of which are expressed in the AER (Niswander and
Martin, 1993). In addition, the AER keeps the immediately underlying mesenchymal cells in an undifferentiated state (Kosher et al., 1979; Kosher and Savage,
1980) by a gradient-like diffusion of particular inhibir 1998 WILEY-LISS, INC.
tory molecules; the concentration of these molecules is,
however, greater in the subridge mesenchymal cells.
When these subridge cells move away from this zone, as
a result of polarized proximal to distal outgrowth, they
respond less to the action of these molecules, lose the
labile state, and differentiate into chondrogenic cells.
Kosher and Savage (1980) also observed that in the
presence of cyclic adenosine monophosphate (cAMP)
derivatives, limb explants, formed from subcrest mesoderm covered by AER and surrounded by dorsal-ventral
ectoderm, failed to undergo polarized outgrowth and
characteristic contour changes. The cessation of morphogenesis is accompanied by the precocious chondrogenic
differentiation of the mesenchymal cells. These results,
according to the authors, indicate that a greater cAMP
content in the subcrestal mesenchymal cells enables
them to overcome the negative influences of cytodifferentiation and the positive influences of morphogenesis
being imposed upon them by the AER. It therefore
seems that cAMP plays a fundamental role in morphogenesis and limb differentiation (Kosher and Savage,
1980).
In order to understand more about cAMP action in
the processes of embryonic development, a cytochemical study was carried out to localize adenylate cyclase
(AC), an enzyme directly involved in cAMP synthesis,
in the epithelium of the hind limb of Bufo bufo during
some of the earliest, most significant stages of development. The results of the cytochemical test suggest that
cAMP may be involved in the differentiation processes
and limb morphogenesis.
*Correspondence to: Rosalba M. Farnesi, Istituto di Anatomia Comparata via
A. Pascoli 06100 Italy.
Received 22 July 1996; accepted in revised form 15 November 1996.
ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO
MATERIALS AND METHODS
Bufo bufo embryos were kept in aquaria (10°C, pH
6.5, natural light) until the desired stages were reached
(embryonic stages 23 and 25; larval stages I and V
(Rossi, 1959)) (Fig. 1). Thirty embryos in each stage
were prefixed for 30 minutes in 1% glutaraldehyde
buffered with 0.1 M sodium cacodylate (pH 7.4) with 4%
sucrose. For the cytochemical assay, the samples were
incubated according to the method of Howell and
Whitfield (1972), modified by Cutler and Christian
(1980), in 80 mM Tris-maleate buffer (pH 7.4), 8%
glucose, 2 mM theophylline, 2 mM magnesium sulphate, 4 mM lead citrate, 10 mM sodium fluoride, and
0.5 mM 58adenylyl-imidodiphophate (AMP-PNP) (Boehringer Mannheim, Mannheim, Germany).
The control samples of corresponding stages were
randomly selected and incubated in a substrate-free
medium. All specimens were incubated at 30°C for 45
minutes, with gentle shaking.
After the incubation, all samples were rinsed in 80
mM Tris-maleate buffer (pH 7.4) and then in 0.1 M
cacodylate buffer (pH 7.4). The material was then
postfixed for 1 hour at 4°C in 1% osmium tetroxide and
processed for electron microscopy following standard
procedures (Mollenhauer, 1964). After a brief staining
in a saturated solution of uranyl acetate in 50% ethanol, thin sections were examined with a Philips EM 300
at 60 Kv.
RESULTS
At embryonic stage 23, the hind limb bud is a small
subspherical lateral bump located at the boundary
between the body and the tail. Ultrastructural examination showed that it has an external two-layered epithelium and mesenchymal cells to the inside. The epithelial cells are flattened with numerous electron-dense
pigments throughout the cytoplasm and abundant mucous secretions at the cell tip. The intercellular spaces
between the contiguous and underlying epithelial cells
are enlarged (Fig. 2A). At the level of the fold which
marks the border between the body and the limb, the
body epithelium is cuboidal and ciliated, while that of
the limb bud is non-ciliated and rich in secretions (Fig.
2A); the epithelium is multilayered in the fold region.
The mesenchyme is made up of undifferentiated cells
with a large nucleus and nucleolus; they have less
cytoplasm and have scarce organelles. There is a noticeable distance between the cells, which have irregular
borders and long cytoplasmic strands that sometimes
connect them. At the bud base, the cells are more
abundant, closer together, and have the long shape
typical of movement cells. The extracellular matrix is
very transparent (Fig. 2B).
In this precocious stage of limb formation, only some
of the superficial epithelial cells located at the border
between the body and limb are positive for the AC
reaction (Figs. 2A,C). The reaction products are only
found along the lateral and basal membranes.
At embryonic stage 25, the epithelium of the bud
apex becomes multilayered with cube-like elements
separated by large intercellular spaces (Fig. 3A). Based
on this characteristic appearance and its location, this
epithelial region is regarded as the presumptive AER.
In the mesenchymal portion, the cells are more numer-
447
Fig. 1. (a) Embryo of Bufo bufo at stage 23, in which the limb bud is
visible at the base of the tail. (b–e) Limb bud at the embryonic and
larval stages considered.
ous and closer together than in the preceding stage. A
large blood vessel is present in the subcrestal mesenchyme (Fig. 3B). Also at this stage, the enzymatic
reaction is observed only on lateral-basal membranes of
the superficial cells located near the fold at the boundary with the body (Figs. 3C,D).
At larval stage I, the limb bud is longer and has a
subcylindrical shape. More layers of epithelial cells can
be seen at the fold; the innermost cells represent the
germinative layer and show slightly differentiated electron-dense cytoplasm with mitotic patterns (Figs. 4A,B).
The cells of the upper layers are well differentiated,
with abundant pigment-rich cytoplasm containing numerous organelles and lipid drops (Fig. 4A). The epithelium again becomes two-layered along the lateral surfaces of the limb bud (Fig. 4C).
The enzymatic reaction product is present at the fold
of the border with the body, along the lateral and basal
membranes of the outermost cell layer (Fig. 4A). The
reaction is lacking in a short region that follows (Fig.
4C) and then reappears, more distally on the basallateral membranes of the epithelial cells of the outer
layer (Fig. 4D). Reaction products were not observed on
the AER, which was still present on the bud apex. The
subcrestal mesenchymal cells are still undifferentiated,
while the more internal ones show signs of differentiation; among these elements it is possible to see clusters
of nervous layers and blood vessels.
At larval stage V the limb bud is flattened, apically
enlarged with two barely visible curvatures: one between the stylopodium and zeugopodium and the other
between the zeugopodium and the autopodium. In the
bud apical zone, the lateral protuberances of the 3rd,
5th, and 4th digits can be seen; the latter is more
accentuated and centrally located. The epithelium is
multilayered at the two-curvatures level, and twolayered along the rest of the surface. The AER is no
longer visible.
Within the limb, with the exception of the nervous
and vascular components (Fig. 5A), definite tissues are
still not visible, even though aggregations of cellular
Fig. 2. Hind limb bud of Bufo bufo at embryonic stage 23.
(A) Separation fold between ciliated epithelium of the body and that of
the limb; the latter has flattened cells with mucous secretions (m) and
pigments (p). The AC reaction products (arrows) are present along the
lateral-basal membranes of some superficial cells; (c) cilia. 5,300x.
(B) Elongated mesenchymal cells (mc) that seem to penetrate to the
interior of the limb; the cells are distant from each other and the
extracellular matrix is transparent; (e) epithelium of limb bud. 3,200x.
(C) Localization of reaction products (arrows) in the epithelial cells
near the fold between the body and limb. 8,400x.
ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO
Fig. 3. Hind limb bud of Bufo bufo at embryonic stage 25.
(A) Multilayered apical ectodermic ridge (aer); mesenchmyal cells (mc)
can be seen below. 2,550x. (B) Subridge blood vessel (v) surrounded by
449
as yet undifferentiated mesenchymal cells. 2,700x. (C, D) Localization
of enzymatic reaction products in the multilayer epithelium in the
region of the limb fold. 4,500x; 6,800x.
Fig. 4. Hind limb bud of Bufo bufo at larval stage I. (A) Multilayered epithelium in the fold region: the outermost cells are differentiated, the deeper cells, which have a high nucleus/cytoplasm ratio,
make up the germinative layer. The reaction products (arrows) are
localized only on the basal-lateral membranes of a few superficial cells.
4,500x. (B) Particular of the germinative layer of the epithelium
showing a cell in mitosis. 3,600x. (C) Two-layered epithelium of the
proximal region of the limb bud; no reaction products can be seen.
5,700x. (D) Two-layered epithelium of the intermediate and distal
regions of the limb. The reaction products (arrows) are visible along
the cell membranes in the superficial layer. 3,600x.
Fig. 5. Hind limb bud of Bufo bufo at larval stage V. (A) Nervous
processes (arrows) and blood vessel (v) between differentiating mesenchymal cells. 2,000x. (B) Clustering of mesenchymal cells around the
chondrogenic centers; (v) blood vessel. 2,100x. (C) Differentiating
mesenchymal cells; in the cytoplasm myofibrils in formation can be
seen (arrows). 5,400x. (D) Particular of intercellular substance between the differentiating mesenchymal cells; numerous collagen fibrils
can be seen. 18,500x.
Fig. 6. Hind limb bud of Bufo bufo at larval stage V. (A, B)
Multilayered epithelium at the level of the curvature between stylopodium and zeugopodium (A) and the curvature between the zeugopodium and autopodium (B). Abundant reaction products (arrows) can
be seen along the cellular membrane. 4,500x; 2,800x. (C) Two-layered
epithelium located at the boundary between the basal zone in which
reaction products are present and the apical zone of the 4th digit, in
which the reaction is lacking. 2,200x. (D) Epithelium of the apical
region of the 4th digit; reaction products are lacking. 2,800x. (E)
Epithelial region of the limb bud in which the cells clearly show signs
of degeneration: the cytoplasm is very transparent and the mitochondria and rough reticulum are fragmented. 6,500x.
ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO
elements can be seen that delimit centers of tissue
condensation (Fig. 5B) and some cellular elements have
myofibrils being formed (Fig. 5C). Noticeable accumulations of collagen fibrils are present in the matrix, which
is now denser than before (Fig. 5D).
The reaction products can be seen in the epithelium
of the curvatures (Figs. 6A,B) and at the base of the 4th
(middle) digit (Fig. 6C), while they are lacking at the tip
of the 4th digit (Figs. 6C,D). At this stage some welldefined epithelial areas with some degenerating cells
can be seen (Fig.6E).
DISCUSSION
The morphologic and cytochemical data obtained
enable us to make some observations. In the first stages
of hind limb development in Bufo bufo, there are a few
undifferentiated mesenchymal elements; those located
at the boundary between the body and limb seem to
migrate from inside the body, as reported in the literature (Balinsky, 1972). Mitosis is rare, if not completely
lacking, in both the epithelium and in the mesenchyme,
even though the bud is increasing in size. This can
probably be explained for the mesenchyme by the
continual migration of cellular elements from within
the body, while for the epithelium, by the flattening and
spreading of the cells.
In the mid-apical portion of the limb bud the AER is
present. This structure has been described in the limb
bud of vertebrates but its presence has been a matter of
discussion in amphibians (Tschumi, 1957; Bodemer,
1968; Dober and Tschumi, 1971; Tarin and Sturdee,
1969; Balinsky, 1972).
In the more advanced developmental stages (larval
stages I and V), the external morphology of the limb
bud begins to be delineated, while there is still no
internal tissue differentiation. The presence of areas of
cell degeneration observed at larval stage V could be
related precisely to the modelling of the limb shape.
The results of the cytochemical tests indicate that AC
activity is constant during limb development at the
epithelial level. The reaction products are localized only
on the lateral and basal membranes of the cells in the
outermost layer or in the most external layers in the
multilayered epithelial zones. The reaction is never
present in the basal germinative layer, which confirms
that cAMP is not associated with cellular proliferation
events (Otte et al., 1989, 1990; Hadden et al., 1972;
Kram and Tomkins, 1973; Rudland et al., 1974a,b).
The enzymatic activity of stage 23, at the epithelial
cell level, is localized along the original fold of the limb
and could be correlated with the dynamic activity of
this epithelial zone at this particular moment of development.
During the successive stages, the lead deposits are
not uniformly distributed along the entire epithelium;
rather, they are always localized at the base of each new
segment. The reaction products are never present in
the AER cells and this suggests a possible role of cAMP
during limb development. The fact that the apical
ridge, which may have the function of maintaining the
immediately underlying mesenchymal cells in an undifferentiated state (Kosher et al., 1979; Kosher and
Savage, 1980), lacked enzymatic activity suggests that
cAMP is not involved in the functions of the AER.
453
Kosher and Savage (1980) observed that in the presence of cAMP derivatives, explants of subcrestal mesenchymal cells overcome the negative influences on cytodifferentiation and the positive influences on their
morphogenesis imposed by the ridge. These authors
suggested that the increase of cAMP may either block
the mesenchymal cell response to the influence of the
AER or reduce the function of the ridge itself.
In the epithelial zone of the limb bud in which
enzymatic activity can be found, the cAMP may have,
on the contrary, a positive effect on cytodifferentiation.
Such an influence could be expressed through a mechanism that involves the production of particular diffusible molecules that induce the underlying mesenchymal cells to differentiate.
According to Kosher et al. (1979), the peripheral
mesenchymal cells of the dorsal and ventral proximal
regions of the limb differentiate into muscle and connective tissues. It can therefore be hypothesized that cAMP
at the base of each segment of the limb bud is involved
in the differentiation of the peripheral mesenchymal
cells into muscle and connective tissues. However,
there could also be a positive involvement in the
differentiation of the deeper-lying mesenchymal cells
into chondrogenic tissue as the cells move away from
the AER and cytodifferentiation is no longer inhibited.
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