/. Embryol. exp. Morph. Vol. 34, 1, pp. 1-18, 1975
Printed in Great Britain
Ultrastructural identification of
extracellular matrix and cell surface components
during limb morphogenesis in man
By ROBERT O. KELLEY 1
From the Department of Anatomy,
The University of New Mexico School of Medicine
SUMMARY
Development of the human hand plate (stages 16-17) has been analyzed with emphasis
on differentiation of elements within the extracellular matrix and the composition of the
mesenchymal cell surface. The epithelial-mesenchymal interface contains a basal lamina
and a sublaminar matrix exhibiting: (a) collagen fibrils with characteristic 63-64 nm banding; (b) non-banded filaments, 10-15 nm in diameter; (c) ruthenium red-positive particles,
12-15 nm in diameter; and (d) attenuated threads, 3-5-5-0 nm in diameter which interconnect particles, fibrils, filaments and the basal lamina. Processes of mesenchymal cells
penetrate this matrix network. In addition to staining with ruthenium red, components of
basal laminae bind to ferritin-conjugated Concanavalin A, greatest binding being localized
on the mesenchymal surface of the lamina. Asymmetry of binding is removed by incubation
of exposed laminae with trypsin (5 /tg/ml). Regional differences in these staining and binding
characteristics within the subepithelial matrix have not been observed in the hand plate.
However, precartilaginous extracellular zones deep within the plate are notably unstructured
in comparison to the sublaminar region. Ruthenium red-positive materials at mesenchymal
cell surfaces display sensitivity to testicular hyaluronidase, Pronase and trypsin but resist
removal with neuraminidase and EDTA. These features of the substrate in situ may be
important in the regulation of mesenchymal cell behavior during limb morphogenesis in
man.
INTRODUCTION
Growth of the vertebrate limb is predominantly distal (Hornbruch & Wolpert,
1970; Janners & Searls, 1970; Searls & Janners, 1971; Summerbell & Wolpert,
1972) and interactions of epithelium, mesenchyme and the surface-associated
extracellular matrix are fundamental to its morphogenesis and cytodifferentiation (see reviews by Hay, 1965; Milaire, 1965; Saunders & Gasseling, 1968;
Faber, 1971). Unfortunately, mechanisms of matrix interaction (Grobstein,
1967) are poorly understood.
At least two components of matrices, the collagens and acid glycosaminoglycans, are known to be important in developmental interactions (Bernfield,
Cohn & Bannerjee, 1973; Hay, 1973; Meier & Hay, 1973). Konigsberg &
1
Author's address: Department of Anatomy, The University of New Mexico School of
Medicine, Albuquerque, New Mexico 87131.
2
R. O. KELLEY
Hauschka (1965) were among the first to demonstrate that collagen influences
the development of skeletal muscle cells in vitro. Toole (1973) associated the
synthesis and degradation of the acid glycosaminoglycan, hyaluronate, with
proliferation and differentiation, respectively, of limb mesenchymal cells in vitro
and in vivo (Toole & Gross, 1971; Toole, Jackson & Gross, 1972). Moscona
(1971) suggested that other cell-surface carbohydrates (detected with plant
lectins) have a role in the mediation of cell social behavior during development.
In order to understand factors which permit and direct growth of limb
mesenchymal cells we need more information about the substrate in which cells
proliferate, accumulate and differentiate. The present investigation was designed
to reveal ultrastructure, and to detect regional differences in distribution, if
present, of collagens and complex protein-carbohydrates of extracellular
matrices during a period of human limb development exhibiting both morphogenesis and cyto-differentiation (Zwilling, 1968).
The following events occur concurrently during stage 17 (see O'Rahilly,
Gardner & Gray, 1956, and inset, Fig. 1): (a) changes in shape of epithelium
foretell future digital and interdigital form; (b) mesenchymal cells proliferate at
digital tips to establish growth blastemata; and (c) core mesenchymal cells
exhibit features of future digital cartilage (Milaire, 1965; Kelley, 1973). This
communication reports the ultrastructure and distribution of (a) collagen;
(b) acid mucopolysaccharide and phospholipid, detected by staining with the
inorganic dye ruthenium red (Luft, 1971a, b); and (c) other carbohydrate residues, monitored by affinity binding with Concanavalin A, at the epithelial—
mesenchymal interface and surfaces of mesenchymal cells during early development of the human hand plate. Mesenchymal cells beneath apical epithelium
(and later the epithelium at digital tips) reside in an enriched matrix of complex
protein-carbohydrate that is not found in the core of the hand plate. In addition,
limb mesenchymal cells possess a prominant surface coat which in cells immediately below the epithelium is structurally continuous with elements of both
basal lamina and sublaminar matrix.
MATERIALS AND METHODS
Limb-buds for this investigation were dissected from human embryos following therapeutic interruption of pregnancy. Developmental stage was determined
by matching structures with corresponding descriptions of O'Rahilly, et al.
(1956) and the developmental horizons of Streeter (1948). The term 'Horizon'
and 'Stage' will be synonyms in this report. Fifteen specimens in stage 16 and
eighteen in stage 17 were studied (approximately the sixth to seventh postfertilization weeks).
Extracellular matrix during limb morphogenesis in man
3
Electron microscopy
Ruthenium red
Material was immersed in either 3 % glutaraldehyde in 0-15 M cacodylate/HCl
buffer (pH 7-3) or the modified aldehyde fixative (PAFG) of Ito & Karnovsky
(1968), both at room temperature for 2-4 h. Specimens from each stage were
postfixed for 3 h (room temperature) in 2% osmium tetroxide in cacodylate
buffer either with or without the addition of ruthenium red (approximately
2000 ppm; Luft, 1971 a), rinsed in buffer, dehydrated through increasing concentrations of ethanol to propylene oxide, and embedded in Epon 812. Other
specimens were dissected and incubated (30 min at 37 °C) in a glucose-potassium-sodium solution (GKN; Kelley, Baker, Crissman & Henderson, 1973)
containing testicular hyaluronidase (300 i.u./ml; Nutritional Biochemical
Corpn., Cleveland) prior to fixation in aldehyde and postfixation in osmium
tetroxide containing ruthenium red. Thin sections (mounted on uncoated grids)
were stained for 30 min in saturated aqueous uranyl acetate, for 5 min in alkaline
lead citrate (both at room temperature) and examined in an Hitachi H U - l l C
electron microscope.
Concanavalin A
Other specimens were stabilized with 1-5% glutaraldehyde in 0-15 M cacodylate/HCl buffer (pH 7-3) for 30 min at 4 °C, embedded in 4-6% agar and sectioned (20-40/tm) on a Sorval TC-2 tissue sectioner prior to incubation in
ferritin-conjugated Concanavalin A (Fer-Con A). In addition, some tissues were
embedded in paraffin after aldehyde fixation and incubated in Con A conjugated
to fluorescein isothiocyanate (FJTC; Smith & Hollers, 1970).
Ferritin (60mg/ml; 2x crystallized, cadmium free, Nutritional Biochemical
Corpn., Cleveland) was covalently coupled to Concanavalin A (45mg/ml; 2x
crystallized, Nutritional Biochemical Corpn., Cleveland) by techniques of Stobo
& Rosenthal (1972). This procedure uses ligand protection of Con A-binding
activity by saturation with a-methyl-D-glucopyranoside (aMG; Sigma, St Louis)
prior to glutaraldehyde cross-linking with ferritin. The coupling reaction was
stopped by dialysis in 0-1 M phosphate buffered saline (PBS) containing glycine
(2 mg/ml). Conjugate was purified by chromatography on Sephadex G-200 (bed
dimensions 1 x 50 cm) and Sepharose 2B (bed dimensions 2-5 x 35 cm).
Localization of Con A binding sites was studied by incubating embryonic
tissue in Hanks's balanced salt solution (BSS) containing Fer-Con A (approximately 100/tg/ml) for 30 min at 4 °C. Control specimens were incubated (a) at
4 °C for 30 min in BSS containing 25 mg/ml of non-conjugated ferritin; (b) at
4 °C for 30 min in BSS containing Fer-Con A (100 /^g/ml) previously saturated
with aMG; and (c) at 4 °C for 10 min in BSS containing aMG (250/tg/ml),
rinsed with BSS and placed in saline containing Fer-Con A as described. To
expose basal laminae, epithelial and mesenchymal cells in some specimens were
4
R. O. KELLEY
lysed by sonication in distilled water for 2 min at room temperature. Areas of
lamina still in association with cells permitted determination of epithelial and
mesenchymal surfaces of the lamina. Fragments of laminae were isolated by the
methods reported by Kefalides (1970). Electron microscopic examination of the
fragments after further incubation in BSS containing lOO^g/ml of trypsin
(37 °C, 5 min) revealed that laminae were free of cellular debris. After incubation, all specimens were rinsed three to five times with BSS and prepared for
electron microscopy.
Enzyme dissection of cell surface
To examine the nature of cell surface materials, fragments of mesenchymal
tissues were dissected from hand plates and incubated for 30 min at 37 °C in
GKN containing either (a) testicular hyaluronidase (300i.u./ml; protein free,
chromatographically pure, Nutritional Biochemicals Corp., Cleveland);
(b) neuraminidase (Vibrio cholerae, 50 i.u./ml, Schwarz/Mann, Orangeburg,
N.Y.); (c) Pronase (500/*g/ml; K & K Laboratories, Plainview, N.Y.); (d)
EDTA (500/jg/ml; K & K Laboratories); or (d) trypsin (500/tg/ml; K & K
Laboratories). Control groups were incubated in GKN without enzyme. These
concentrations alter the surface coat without disrupting cell ultrastructure (see
also Huet & Herzberg, 1973). Mesenchymal fragments and dispersed cells were
pelleted by centrifugation (200 g), rinsed in GKN, fixed as described, postfixed
in osmium containing ruthenium red, and prepared for analysis in the electron
microscope. Additional specimens were stained with colloidal thorium for the
presence of acid mucopolysaccharide after the method of Revel (1964).
FIGURES 1-3
Fig. 1. Electron micrograph of the epithelial-mesenchyma] interface during stage
16 (area enclosed by square in inset B); material postfixed with OsO4/ruthenium
red to demonstrate acid glycosaminoglycan. Note enhanced electron density of
basal lamina, elements of the sublaminar matrix and mesenchymal cell surfaces.
e, Epithelium; //, lamina lucida; bl, lamina densa (basal lamina); slm, sublaminar
matrix; m, mesenchyme. Inset A illustrates stages 16, 17 and 18 of human limb
development. Inset B is a fluorescence micrograph of a section incubated in
fluorescein-isothiocyanate conjugated to Concanavalin A (FITC-Con A), x 28000;
inset, x 115.
Fig. 2. Higher magnification of the epithelial-mesenchymal interface illustrated in
Fig. 1. Processes of mesenchymal cells penetrate the sublaminar matrix to establish
intimate association with the epithelial basal lamina. Ruthenium red stains glycosaminoglycan-containing elements in the sublaminar matrix (arrows) and mesenchymal cell surface (mcs). x 77000.
Fig. 3. Epithelial-mesenchymal interface incubated in testicular hyaluronidase
(300 i.u./ml; 37 °C; 30 min) prior to preparation with OsOJruthenium red.
Binding of ruthenium red is diminished at the basal lamina (bl), sublaminar matrix
(slm) and cell surface (mcs), but structural integrity is maintained, x 70000.
Extracellular matrix during limb morphogenesis in man
6
R. O. KELLEY
RESULTS
By stage 16 the human limb-bud is subdivided into a distal hand plate and a
proximal arm and shoulder region (see inset A, Fig. 1). The foot plate is not yet
distinct. Blastemal condensations of future cartilages are present at stage 16
(inset B, Fig. 1) but digital rays will not become apparent until stage 17. The
ectodermal ridge diminishes by stage 18 (O'Rahilly et al. 1956) but growth of
digits continues at distal tips of the blastemata. To facilitate examination of the
epithelial-mesenchymal interface, three areas will be distinguished: (a) the basal
lamina; (b) the sublaminar matrix; and (c) the mesenchymal cell surface.
Cytochemical features of the epithelio-mesenchymal interface
The basal lamina
Since ultrastructural features of the epithelial-basal lamina-mesenchymal
complex in man have been reported previously (Kelley, 1973), only the major
features will be summarized here using Fig. 1 for orientation. At low magnification, processes of mesenchymal cells extend through their investing matrix to
establish intimate association with neighboring mesenchymal cells and the basal
lamina subtending the overlying epithelium. The electron-dense portion of the
basal lamina (lamina densa) is a continuous, filamentous structure, 35-50 nm in
thickness, separated from the epithelial cell membrane by a less dense zone, the
lamina lucida.
FIGURES 4-9
Fig. 4. Subepithelial matrix of limb sections incubated in ferritin-conjugated
Concanavalin A (Fer-Con A) to demonstrate terminal non-reducing carbohydrate
moieties. Ferritin (as single particles, small arrows; and clumps, larger arrows) is
distributed in the subepithelial compartment (sec), the basal lamina (bl) and the
sublaminar matrix (slm). x 77000.
Fig. 5. Basal lamina divested of adjacent cells by sonication before incubation in
Fer-Con A (see methods). Note predominance of ferritin marker on mesenchymal
surface and meager binding to lamina densa. x 77 000.
Fig. 6. Basal lamina isolated by sonication and brief trypsinization (see Methods)
prior to incubation in Fer-Con A. Marker is heavily distributed along the lamina
densa. x 77000.
Fig. 7. Isolated basal lamina after incubation in Fer-Con A/a-methyl-D-glucopyranoside (control). Note absence of Fer-Con A along basal lamina, x 77000.
Fig. 8. Sublaminar matrix containing collagen fibrils (cf) exhibiting a 63-64 nm
periodicity; ruthenium red-positive particles (rpp), 12-15 nm in diameter; filaments
(/), 10—15 nm in diameter; and attenuated threads (arrows), 3-5-5-0 nm in diameter,
which interconnect particles, fibrils, filaments and the basal lamina (bl). Note
mesenchymal cell process penetrating the sublaminar matrix, x 73000.
Fig. 9. Epithelial-mesenchymal interface at low magnification to illustrate presence
of structural elements at the interface and paucity of similar features in extra
cellular areas deep to the zone, x 11 500.
Extracellular matrix during limb morphogenesis in man
8
R. O. KELLEY
By stage 16, mesenchymal cells in the distal portion of the limb (a zone
approximately 60-70 /im in width immediately below the epithelium) become
more closely associated in comparison to other non-blastemal mesenchyme
within the bud (inset B, Fig. 1). In addition, the epithelial-mesenchymal interface
in tissues prepared with ruthenium red displays a striking degree of complexity
in comparison to conventional preparations. Electron density of the basal
lamina, the sublaminar matrix, intercellular elements and epithelial and
mesenchymal cell surfaces is markedly increased. Ruthenium red labels the
lamina densa most heavily whereas the lamina lucida and the matrix on the
mesenchymal surface of the lamina densa are less electron-dense. The affinity for
ruthenium red is uniform in all areas of the basal lamina throughout stages 16
and 17, and is neither unique nor localized to the apical tip of limb-buds.
Processes of mesenchymal cells abut the lamina densa (Fig. 2) and exhibit a coat
of ruthenium red-positive particulate material at the surface of the cell membrane. Incubation of tissues in testicular hyaluronidase prior to staining with
ruthenium red reduces the affinity of matrix elements for the dye (Fig. 3).
Sections of tissue incubated in ferritin-conjugated Concanavalin A reveal
binding activity along both epithelial and mesenchymal surfaces of the basal
lamina (arrows, Fig. 4). Electron-dense clusters (larger arrows) in addition to
single particles of ferritin (smaller arrows) are apparent. Unfortunately,
determination of the extent of Con A binding to the basal lamina is difficult in
intact tissue preparations.
To explore further the affinity binding of Con A to the lamina, epithelial and
mesenchymal elements were removed by sonication to expose surfaces of the
lamina to incubation medium containing Fer-Con A. Fig. 5 illustrates binding of
Con A to carbohydrate moieties which are predominant on the mesenchymal
surface of the lamina. Few particles of ferritin can be seen within the interstices
of the lamina densa. After an exposed lamina is incubated in trypsin (50 /^g/ml
in GKN) the sublaminar material is removed and Fer-Con A is visualized binding directly to the lamina densa (Fig. 6). Fig. 7 depicts the appearance of an
isolated lamina incubated in Fer-Con A which has been previously bound to the
ligand, a-methyl-D-glucopyranoside. Binding of the ferritin-labeled conjugate
is notably diminished. Aside from the asymmetry in Con A binding illustrated in
FIGURES 10 AND 11
Fig. 10. Mesenchymal cells in limb periphery (area enclosed by square in inset)
after incubation in Fer-Con A. Dense patches of ferritin particles are present on
some cell surfaces (arrows) whereas other areas of membrane exhibit little affinity
for Con A (brackets). Inset illustrates cell and matrix dense zone immediately
beneath apical epithelium. FITC-Con A preparation, x 15000; inset, x 200.
Fig. 11. Mesenchymal cells in core of limb adjacent to a capillary (similar to area
enclosed by square in inset). Surfaces of cells which contribute to capillary endothelium are heavily invested with labeled Con A (arrows) as are mesenchymal cells
abutting the vascular structure, x 15000; inset, x 200.
Extracellular matrix during limb morphogenesis in man
mm*
10
R. O. KELLEY
Fig. 5, no differences in pattern or regional distribution of Con A have been
detected at the epithelial-mesenchymal interface in the stages examined. However, sections of tissues incubated in fluorescein isothiocyanate-conjugated Con
A exhibit greater affinity binding in the zone of mesenchyme immediately below
the epithelium, in contrast to core regions of the limb (insets, Figs. 10 and 11).
Whether this difference is due to matrix elements or cell density is, at present,
uncertain.
The sublaminar matrix
The extracellular zone between basal laminae and mesenchymal cell surfaces,
after preparation with ruthenium red (Fig. 8) contains (a) collagen fibrils
exhibiting a 63-64 nm periodicity; (b) filaments, 10-15 nm in diameter; (c)
ruthenium red-positive particles 12-15 nm in diameter; and (d) attenuated
threads, 3-5-5-0 nm in diameter, which interconnect particles, fibrils, filaments
and the basal lamina. Fibrils which can be structurally identified as collagen
are sparsely distributed throughout the sublaminar matrix in all stages examined, and are rarely observed in zones of mesenchyme which are not blastemata of future cartilage. The non-striated filaments are more prevalent and
exhibit an apparent random distribution in intercellular zones throughout the
developing limb. In Fig. 8, ruthenium red-positive particles decorate the
collagen fibril and fine threads extend from that structure to the basal lamina
and to other particles and filaments deeper within the matrix. Consequently,
the sublaminar zone exhibits features of a web of collagen-mucopolysaccharide
into which processes of mesenchymal cells extend. This network is unique to the
immediate sublaminar zone, however, as Fig. 9 illustrates the paucity of similar
structure adjacent to the deep surface of cells abutting the basal lamina.
FIGURES
12-18
Fig. 12. Mesenchymal cell surface (mcs) after postfixation with OsO4/rutheniurn
red. x 110000.
Fig. 13. Mesenchymal cell surfaces after staining with colloidal thorium, x 27000.
Fig. .14. Mesenchymal cell surface after incubation in testicular hyaluronidase
(300 i.u./ml) prior to staining with ruthenium red. Note absence of cell surface
elements, x 96000.
Fig. 15. Mesenchymal cell surface after incubation in neuraminidase (50 i.u./ml)
prior to staining with ruthenium red. Structural elements which are stained with
ruthenium red remain on the cell surface, x 100000.
Fig. 16. Mesenchymal cell surface after incubation in Pronase (500/tg/ml) prior to
staining with ruthenium red. Some stainable material remains on the cell membrane
(arrows), x 96000.
Fig. 17. Mesenchymal cell surface after incubation in EDTA (500/tg/ml) prior to
staining with ruthenium red. Cell surface elements remain intact, x 96000.
Fig. 18. Mesenchymal cell surface after incubation in trypsin (500/tg/ml) prior to
staining with ruthenium red. Some materials remain on surfaces of cell membranes (arrows), x 100000.
Extracellular matrix during limb morphogenesis in man
11
12
R. O. KELLEY
Processes extending into intercellular zones several microns distant from the
sublaminar area do not encounter a matrix exhibiting similar ultrastructural or
cytochemical characteristics.
The mesenchymal cell surface
Preparation of material with ruthenium red (Figs. 1,2, 12) reveals a uniform
coat of material (approximately 15-20 nm in thickness) investing all mesenchymal cells within limb-buds during the stages examined. Distribution of
binding sites for Con A, however, exhibit subtle differences within limb regions.
Fig. 10 illustrates mesenchymal elements in the cell-dense zone beneath apical
epithelium (see inset). After incubation of material in ferritin-conjugated Con A,
patches of ferritin particles can be visualized on some cell surfaces (arrows)
whereas other areas of membrane exhibit relatively little affinity for Con A
(brackets). A similar distribution of binding sites is present on mesenchymal
cells deep within the limb (i.e. those elements not in the cell dense, subepithelial
zones; see inset, Fig. 11). The only notable variation of this binding pattern is
observed on surfaces of mesenchymal cells which contribute to (or are in the
vicinity of) a vascular structure within the limb. Fig. 11 illustrates profiles of
cells surrounding a vascular channel on the periphery of a blastema of cartilage.
Surfaces of cells which contribute to the capillary endothelium are heavily
invested with labeled Con A as are mesenchymal cells abutting the vascular
element.
The presence of acid mucopolysaccharide in the mesenchymal cell surface coat
is also revealed by its ability to bind colloidal thorium (Fig. 13), in addition to
staining with ruthenium red. Incubation in testicular hyaluronidase removes
most of the ruthenium red-positive material (Fig. 14) from the cell surface,
whereas neuraminidase (50 i.u./ml) has little apparent effect on the morphology
of the surface coat (Fig. 15). Pronase (500/tg/ml) also removes elements which
exhibit affinity for ruthenium red, but careful examination of Fig. 16 reveals a
residue of electron-dense material on the outer leaflet of the cell membrane.
Fig. 17 illustrates the minimal effect of EDTA (500/tg/ml) in altering the fine
structure of the cell surface, whereas trypsin (500 /tg/ml; Fig. 18) has an effect on
the surface similar to that of Pronase. The 'unit' membrane is divested of its
ruthenium red-positive material in some areas (arrows) but not in others.
DISCUSSION
The present results reveal two structurally distinct areas of matrix within
developing human hand plates (stages 16-17): (a) a region immediately below
the distal epithelium which includes basal lamina and sublaminar elements; and
(b) material which invests mesenchymal cells in the core of the bud. This discussion will consider first the organization of some cytochemical features of these
matrices, and secondly the fine structural nature of the mesenchymal cell
surface.
Extracellular matrix during limb morphogenesis in man
13
Organization of subepithelial and core matrices
Complex carbohydrate
The inorganic dye, ruthenium red, has proved to be a useful probe in the
ultrastructural identification of acid mucopolysaccharide and phospholipids in
tissues (Luft, 1971 a, b). In the present investigation, the OsO4/ruthenium red
complex is predominantly bound to the basal lamina (Figs. 1, 2), both epithelial
and mesenchymal cell surfaces (Fig. 1), and those extracellular structures visible
in the sublaminar zone (Fig. 8). Incubation of materials in testicular hyaluronidase decreases electron density resulting from postfixation in OsO4/ruthenium
red (Fig. 3) and suggests that hyaluronate, chondroitin and chondroitin sulphate
may be principal acidic glycosaminoglycans in these extracellular elements (see
Meier & Hay, 1973; Lash, Saxen & Kosher, 1974). The distribution of ruthenium
red-reacting material is uniform along the basal lamina (Figs. 1, 2) in all regions
of the limb. However, the absence of stained intercellular materials in deeper
regions of the bud in contrast to the prevalence of ruthenium red-bound elements in the immediate sublaminar zone (Fig. 9) is notable.
The specificity of interaction between polysaccharides and Concanavalin A
has also been thoroughly documented (see review of Sharon & Lis, 1972).
Precipitates form with terminal, non-reducing a-D-glucopyranosyl, a-Dmannopyranosyl, a-D-fructofuranosyl and a-D-arabinofuranosyl residues. By
attaching a visible marker to the Con A molecule and by using the ligandbinding properties of the conjugate, the distribution of these carbohydrate
residues within tissues can be analyzed. A differential distribution of carbohydrate is apparent in FITC-Con A preparations (insets, Figs. 1, 10, 11), the
greater fluorescence being localized in peripheral mesenchyme. Unfortunately,
the difference in cell density between subepithelial and core regions of the limb
makes quantitative interpretation of matrix differences difficult at this level of
resolution.
Ferritin-conjugated Con A binds uniformly to the basal lamina subjacent to
apical epithelium and to electron-dense materials visible in the sublaminar
matrix (Fig. 4). Differences in distribution of Fer-Con A binding to basal
laminae have not been detected throughout stages 16-17, nor have variations
been observed in different regions of the developing limb (namely laminae
below either presumptive digital or interdigital zones of the hand plate). However, when an effort is made to examine the distribution of Con A binding to an
exposed lamina, the carbohydrate-containing components are found to be
asymmetrically dense on the mesenchymal surface of that structure (Fig. 5). It is
thought that these electron-lucent, carbohydrate-containing elements are normal
structural components of the basal lamina in that they remain with the lamina
during experimental manipulation. Additional Con A-binding residues are
associated with trypsin-sensitive elements within the inner structure of the
14
R. O. KELLEY
lamina densa and are inaccessible for affinity binding to Con A in a native
(i.e. enzymically undissected) state.
Collagen
At least three genetically distinct types of collagen have been identified in
human cartilage and skin by Miller, Epstein & Piez (1971) and, more recently,
Linsenmayer, Toole & Trelstad (1973) have discovered that differing types of
collagen are synthesized in well-defined spatial and temporal patterns during
limb development in the chick. In the latter report, early (homogeneous) limb
mesenchyme produces an (al) 2 a2 type of collagen (type I) whereas, during later
stages, the core (pre-cartilaginous) portion of the limb contains a new and different (al) 3 type of collagen. The latter is thought to represent cartilage-type
collagen (known as type II, designated [al(II)] 3 ; Miller, Woodall & Vail, 1973),
whereas the former is synthesized by mesenchymal cells in the periphery of the
limb (for a concise summary of collagen heterogeneity, see Trelstad, 1974).
Ultrastructural features of collagen are known to differ within single limbbuds (Kelley & Bluemink, 1974) but the correlation between ultrastructure and
molecular type is not clear. It may be useful to note that fibrils with banding
patterns characteristic of 'native' collagen are present in both subepithelial and
core matrices during the stages examined (16-17), whereas nonstriated fibrils,
approximately 10-15 nm in diameter, are restricted to subepithelial zones.
Figure 8 illustrates a single striated fibril which is decorated with ruthenium-redpositive particles. These, in turn, are interconnected by fine threads to the basal
lamina and other elements deep within the matrix. Based on the report of
Linsenmayer et al. (1973), structure present in this collagen fiber may represent
an (al) 2 a2 type configuration. It is of interest to note that the ultrastructure of
collagen fibrils in embryonic cartilage matrix (e.g. in the chick; Matukas, Panner
& Orbison, 1967; and man; Kelley, 1974), exhibits striated profiles characteristic
of type I collagen, but may be of the [al(Il)] 3 type characteristic of cartilage
(which usually forms thin fibrils in vivo; Bruns, Trelstad & Gross, 1973). The
majority of filaments present in the subepithelial matrix are of lesser diameter
(approximately 10-15 nm) and are not visibly striated (Fig. 8). These elements
may reflect the structural organization of the [al(IV)]3 type collagen characteristic of basal laminae, but such a suggestion demands further investigation.
Mesenchymal cell surface
Complex carbohydrates (i.e. glycoproteins, glycolipids and glycosaminoglycans) are important molecules in biological membranes and cells both in
vitro and in vivo synthesize them in considerable variety (see Rambourg, 1971;
Kraemer, 1972, for reviews). In addition, they are significant components of cell
surface antigens (Slavkin, et al. 1974), receptors (Cuatrecasas & Tell, 1973) and
enzymes (Perrone & Blostein, 1973) and may confer to the cell surface a high
degree of specificity. Short-range cellular interactions are generally held to be
Extracellular matrix during limb morphogenesis in man
15
primary determinants of cytodifferentiation and the complex carbohydrates,
because of their peripheral position on cell borders and immediate microenvironments, are favored candidates for mechanistic explanations of these
events.
The affinity of mesenchymal cell surfaces in situ for ruthenium red (Figs. 1, 2,
12), Concanavalin A (Figs. 10, 11) and colloidal thorium dioxide (Fig. 13)
demonstrates the presence of acid mucopolysaccharide and phospholipid (Luft,
1911a, b), a-D-mannopyranosyl-like residues (Agrawal & Goldstein, 1967) and
acidic carbohydrate (Revel, 1964), respectively, within the molecular structure
of the cell surface. The sensitivity of ruthenium-red stainable material to dissection with testicular hyaluronidase (Fig. 14) suggests that hyaluronate, chondroitin and chondroitin sulphate may be significant elements of the surface,
whereas removal of sialic acid residues with neuraminidase (Fig. 15) has little
effect on the ultrastructural appearance of electron-dense reaction product.
Proteases (Pronase and trypsin) alter the morphology of the cell surface (Figs.
16, 18) whereas chelation of divalent cations does not alter the presence of
ruthenium-red binding elements.
Carbohydrate residues which have an affinity for Con-A are distributed over
most of the mesenchymal cell surface (Figs. 10, 11), although regions of cell
membrane are notably lacking Fer-Con A (brackets, Fig. 10). In addition, cells
contributing to vascular channels within the limb are heavily invested with
Con-A binding elements (Fig. 11). Edidin & Weiss (1974) and others have
reported that agglutinin-binding sites on membrane surfaces are free to move in
the plane of the membrane, but the phenomenon is inhibited by aldehyde
fixation. Presumably, binding loci are also free to move in mesenchymal cell
membranes during morphogenesis, although the present information from fixed
material precludes such an hypothesis.
In addition, Grinnell (1973) has demonstrated that cell surface glycoproteins are involved in the aggregation of baby hamster kidney (BHK) cells and
that the ligand-binding capability of Con A increases the adhesiveness of BHK
cells to their in vitro substratum. Implicit in this report is the functional importance of Con A binding loci in the events of cell migration and adhesion.
Possibly carbohydrate residues on mesenchymal cell surfaces in vivo are important in the ability of a cell to acquire and change its position within a
developing limb (see Wolpert, 1972).
Since apical epithelium influences the organization of proliferating mesenchyme in developing vertebrate limbs (Saunders & Gasseling, 1968), it may be
that mesenchymal cells obtain developmental information from the association
of cell products from both participating tissue layers. Interactions between
carbohydrates and collagens in these extracellular matrices are likely to be
among the molecular complexes predicted by Grobstein (1967). The present
observations suggest that the enriched matrix of acid mucopolysaccharide and
collagen in the sublaminar zone of the human limb provides a substratum which
2
EMB 34
16
R. O. KELLEY
is important in the regulation of mesenchymal cell movement and proliferation.
It is not clear what information a cell derives from its presence in the subepithelial matrix (in contrast to the less structured matrix of the core), but
increasing evidence suggests that surface-associated hyaluronate is integral in
mediating the response of cells to the microenvironment (Toole, 1973).
Grateful acknowledgment is made to Dr Robert E. Waterman and Prof. A. J. Ladman
for helpful discussions and critical reading of the manuscript, to Mrs Rita Lauer for technical assistance, to Ms Anita Kimbrell for assistance in preparation of the manuscript, and
to the United States Public Health Service for grant support through HD 06177 and a
Research Career Development Award (HD 70407).
REFERENCES
B. B. L. & GOLDSTEIN, I. J. (1967). Physical and chemical characterization of
Concanavalin A, the hemagglutinin from Jack Bean (Canavalia ensiformis). Biochim.
biophys. Ada 133, 376-379.
AGRAWAL,
BERNFIELD, M. R., COHN, R. H. & BANNERJEE, S. D. (1973). Glycosaminoglycans and
epithelial organ formation. Am. Zool. 13, 1067-1084.
R. R., TRELSTAD, R.I. & GROSS, J. (1973). Cartilage collagen: a staggered substructure in reconstituted fibrils. Science, N.Y. 181, 269-271.
CUATRECASAS, P. & TELL, G. P. E. (1973). Insulin-like activity of Concanavalin A and
wheat germ agglutinin - direct interactions with insulin receptors. Proc. natn. Acad. Sci.
U.S.A. 70, 485-489.
EDIDIN, M. & WEISS, A. (1974). Restriction of antigen mobility in the plasma membranes
of some cultured fibroblasts. In Control of Proliferation in Animal Cells vol. I (ed. B.
Clarkson & R. Baserga), pp. 213-219. Cold Spring Harbor Laboratory. Cold Spring
Harbor Conferences on Cell Proliferation.
FABER, J. (1971). Vertebrate limb ontogeny and limb regeneration: morphogenetic parallels.
Adv. Morphogen. 9, 127-147.
GRINNELL, F. (1973). Concanavalin A increases the strength of baby hamster kidney cell
attachment to a substratum. /. Cell Biol. 58, 602-607.
GROBSTEIN, C. (1967). Mechanism of organogenetic tissue interaction. Natn. Cancer Inst.
Monogr. 26, 279-299.
HAY, E. D. (1965). Metabolic patterns in limb development and regeneration. In Organogenesis (ed. R. L. DeHaan & H. Ursprung), pp. 315-336. New York: Holt, Rinehart and
Winston.
HAY, E. D. (1973). Origin and role of collagen in the embryo. Am. Zool. 13, 1085-1108.
HORNBRUCH, A. & WOLPERT, L. (1970). Cell division in the early growth and morphogenesis of the chick limb. Nature, Lond. 226, 764-766.
HUET, C. L. & HERZBERG, M. (1973). Effects of enzymes and EDTA on ruthenium red and
Concanavalin A labeling of the cell surface. /. Ultrastruct. Res. 42, 186-199.
ITO, S. & KARNOVSKY, M. J. (1968). Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. / . Cell Biol. 39, 168a-169a.
JANNERS, M. & SEARLS, R. L. (1970). Change in rate of cellular proliferation during the
differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing.
Devi Biol. 23, 136-168.
KEFALIDES, M. A. (1970). Comparative biochemistry of mammalian basement membranes.
In Chemistry and Molecular Biology of the Intercellular Matrix (ed. E. A. Balazs), pp.
535-573. London, New York: Academic Press.
KELLEY, R. O. (1973). Fine structure of the apical rim-mesenchyme complex during limb
morphogenesis in man. /. Embryol. exp. Morph. 29, 117-131.
KELLEY, R. O. (1974). Ultrastructural features of chondrogenesis in the human hand plate:
a cytochemical and autoradiographic study. J. Embryol. exp. Morph. 33, 387-401.
BRUNS,
Extracellular matrix during limb morphogenesis in man
17
R. O., BAKER, T. I., CRISSMAN, H. A. & HENDERSON, C. A. (1973). Ultrastructure
and growth of human limb mesenchyme (HLM15) in vitro. Anat. Rec. 175, 657-672.
KELLEY, R. O. & BLUEMINK, J. G. (1974). An ultrastructural analysis of cell and matrix
differentiation during early limb development in Xenopus laevis. Devi Biol. 37, 1-17.
KONIGSBERG, I. R. & HAUSCHKA, S. D. (1965). Cell and tissue interactions in the reproduction of cell type. In Reproduction: Molecular, Subcellular and Cellular (ed. M. Locke),
pp. 243-290. New York: Academic Press.
KRAEMER, P. M. (1972). Complex carbohydrates of mammalian cells in culture. In Growth,
Nutrition and Metabolism of Cells in Culture, vol. i (ed. G. H. Rothblat & V. J. Cristofalo),
pp. 371-426. New York, London: Academic Press.
LASH, J. W., SAXEN, L. & KOSHER, R. A. (1974). Human chondrogenesis: glycosaminoglycan
content of embryonic human cartilage. /. exp. Zool. 189, 127-131.
LINSENMAYER, T. F., TOOLE, B. P. & TRELSTAD, R. L. (1973). Temporal and spatial transitions in collagen types during embryonic chick limb development. Devi Biol. 35, 232239.
LUFT, J. H. (1971a). Ruthenium red and violet. I. Chemistry, purification, methods of use
for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368.
LUFT, J. H. (19716). Ruthenium red and violet. II. Fine structural localization in animal
tissues. Anat. Rec. 171, 369-416.
MATUKAS, V. J., PANNER, B. J. & ORBISON, J. L. (1967). Studies on ultrastructural identification and distribution of protein-polysaccharide in cartilage matrix. /. Cell Biol. 32,
365-377.
MEIER, S. & HAY, E. D. (1973). Synthesis of sulfated glycosaminoglycans by embryonic
corneal epithelium. Devi Biol. 35, 318-331.
MEIER, S. & HAY, E. D. (1974). Control of corneal differentiation by extracellular materials.
Collagen as a promoter and stabilizer of epithelial stroma production. Devi Biol. 38,
249-270.
MILAIRE, J. (1965). Aspects of limb morphogenesis in mammals. In Organogenesis (ed. R.
DeHaan & H. Ursprung), pp. 283-300. New York: Holt, Rinehart and Winston.
MILLER, E. J., EPSTEIN, E. H. Jr. & PIEZ, K. A. (1971). Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage
collagen. Biochem. biophys. Res. Commun. 42, 1024-1029.
MILLER, E. J., WOODALL, D. L. & VAIL, M. S. (1973). Biosynthesis of cartilage collagen.
/. biol. Chem. 248, 1666-1671.
MOSCONA, A. A. (1971). Embryonic and neoplastic cell surfaces: availability of receptors
for Concanavalin A and wheat germ agglutinin. Science, N. Y. 171, 905-907.
O'RAHILLY, R., GARDNER, E. & GRAY, D. J. (1956). The ectodermal thickening and ridge
in the limbs of staged human embryos. /. Embryol. exp. Morph. 4, 254-264.
PERRONE, J. R. & BLOSTEIN, R. (1973). Asymmetric interaction of inside-out and right-side
out erythrocyte vesicles with ouabain. Biochim. biophys. Acta 291, 680-689.
RAMBOURG, A. (1971). Morphological and histochemical aspects of glycoproteins at the
surface of animal cells. Int. Rev. Cytol. 31, 57-114.
REVEL, J. P. (1964). A stain for the ultrastructural localization of acid mucopolysaccharides.
/. Microscopie 3, 535-544.
SAUNDERS, J. W. & GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the
origin of limb symmetry. In Epithelial-Mesenchymal Interactions (ed. R. Fleischmajer &
R. Billingham), pp. 78-97. Baltimore, Maryland: Williams and Wilkins.
SEARLS, R. L. & JANNERS, M. Y. (1971). The initiation of limb bud outgrowth in the
embryonic chick. Devi Biol. 24, 198-213.
SHARON, N. & Lis, H. (1972). Lectins: Cell agglutinating and sugar-specific proteins.
Science, N. Y. 177, 949-959.
SLAVKIN, H. C, TRUMP, G. N., MANSOUR, V., MATOSIAN, P. & MINO, W. G. (1974).
Localization of H-2 histocompatibility alloantigens on mouse embryonic tooth epithelial
and mesenchymal cell surfaces. /. Biol. 60, 795-801.
SMITH, C. W. & HOLLERS, J. C. (1970). The pattern of binding of fluorescein-labeled Concanavalin A to the motile lymphocyte. J. Reticuloendothelial Soc. 8, 458-464.
KELLEY,
18
R. O. KELLEY
J. D. & ROSENTHAL, A. S. (1972). Biologically active Concanavalin A complexes
suitable for light and electron microscopy. Expl Cell Res. 70, 443-447.
STREETER, G.L. (1948). Developmental horizons in human embryos. Description of age
groups XV, XVI, XVII and XVIII being the third issue of a survey of the Carnegie
collection. Contr. Embryol. 32, 133-204.
SUMMERBELL, D. & WOLPERT, L. (1972). Cell density and cell division in the early morphogenesis of the chick wing. Nature, New Biology 238, 24-26.
TOOLE, B. P. (1973). Hyaluronate and hyaluronidase in morphogenesis and differentiation.
Am. Zool. 13, 1061-1066.
TOOLE, B. P. & GROSS, J. (1971). The extracellular matrix of the regenerating newt limb:
synthesis and removal of hyaluronate prior to differentiation. Devi Biol. 25, 57-77.
TOOLE, B. P., JACKSON, G. & GROSS, J. (1972). Hyaluronate in morphogenesis: Inhibition
of chondrogenesis in vitro. Proc. natn. Acad. Sci. U.S.A. 69, 1384-1386.
TRELSTAD, R. L. (1974). Vertebrate collagen heterogeneity. Devi Biol. 38, f 13-fl6.
WOLPERT, L. (1972). The concept of positional information and pattern formation. In:
Towards a Theoretical Biology (ed. C. H. Waddington), pp. 83-94. Edinburgh: Edinburgh
University Press.
ZWILLING, E. (1968). Morphogenetic phases in development. Devi Biol. (Suppl.) 2, 184-207.
STOBO,
(Received 17 October 1974, revised 24 January 1975)