California State University, Northridge
IMMUNOFLUORESCENCE LOCALIZATION OF EXTRACELLULAR
MATRIX COMPONENT(S) IN TWO SPECIES OF SEA URCHIN
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Mina Alikani
August, 1985
The Thesis of Mina Alikani is approved:
(.~()Yep
B. Maxwell, Ph.D.
(ChaJ.r
California State University, Northridge
ii
ACKNOWLEDGMENTS
The preparation of this thesis would have been an impossible task without the guidance of my advising committee
and the encouragement of my family and friends.
I would like to express my sincere appreciation to Dr.
Joyce B. Maxwell and Dr. Kenneth C. Jones for accepting to
serve in my committee and carefully reviewing my work.
I am especially grateful to Dr. Steven B. Oppenheimer
who provided me with the opportunity to conduct this
research in his laboratory.
His support and his enthusiasm
made the initiation and completion of this project
possible.
His friendship and his unique sense of humor,
on the other hand, made my experience in his laboratory
pleasantly unforgettable.
Very special thanks to James T. Meyer who has taught
me much of what I know about research.
His friendship is
invaluable, and his critical evaluation of this thesis is
much appreciated.
I wish to thank Stanley D. Liang for providing me with
the essential component of this project, the anti-S-2
antibody. ·
iii
I also wish to thank my sisters, Vida and Sirna
Alikani, and my very special friend, Yolanda Sinisgallo,
for their friendship and encouragement.
Last but not least, I would like to thank my very dear
friend, Hassan Danesh, for his patience and understanding
during the course of this study.
iv
DEDICATION
This thesis is dedicated to my parents, Ebrahim and
Parichehr Alikani, on their thirtieth wedding anniversary,
in appreciation of their unlimited love and support during
the past twenty-four years.
v
TABLE OF CONTENTS
ACKNOWLEDGMENTS .
iii
DEDICATION
v
LIST OF PLATES
vii
LIST OF ABBREVIATIONS
ix
ABSTRACT
X
INTRODUCTION
1
MATERIALS AND METHODS .
7
Procurement of gametes .
7
Removal of the fertilization membrane and
growth of embryos . . . . .
. . .
7
Indirect immunofluorescence
8
Immunofluorescence microscopy
9
RESULTS
10
PLATES
13
DISCUSSION
22
BIBLIOGRAPHY
27
vi
LIST OF PLATES
PLATE I
Figure 1.
Figure 2.
Light micrograph, S. purpuratus
fertilized egg . . -. . .
. .
13
Fluorescence micrograph, S.
purpuratus fertilized eggincubated with AS-2 and
labeled with FITC-GR . . . . . . . . 13
PLATE II
Figure 3.
Figure 4.
Light micrograph, S. purpuratus
2~cell embryo
. . -.
. . 15
Fluorescence micrograph, S.
purpuratus 2-cell embryo incubated with AS-2 and labeled
with FITC-GR
. . . . . . • . . . . 15
PLATE III
Figure 5.
Figure 6.
Light micrograph, S. purpuratus
4-cell embryo . . -. . .
. . 17
Fluorescence micrograph, S.
purpuratus 4-cell embryo
incubated with AS-2 and
labeled with FITC-GR . . . • . • . . 17
PLATE IV
Figure 7.
Figure 8.
Light micrograph, S. purpuratus
8-cell embryo . . -. . •
. . 19
Fluorescence micrograph, s.
purpuratus 8-cell embryo
incubated with AS-2 and labeled
with FITC-GR
. . . . . • . . . . . 19
vii
LIST OF PLATES - continued
PLATE V
Figure 9.
Figure 10.
Fluorescence micrograph, L. pictus
fertilized egg incubated with AS-2
and labeled with FITC-GR . . . • .
21
Fluorescence micrograph, L. pictus
2-cell embryo incubated w1th AS-2
and labeled with FITC-GR . . . . .
21
viii
LIST OF ABBREVIATIONS
AS-2
anti S-2 antibody
ATA
3-amino-1,2,4-triazole
BSA
bovine serum albumin
CMF-SW
calcium-magnesium-free sea water
DS
dissociation supernatant
ECM
extracellular matrix
FITC-GR
fluorescein isothiocyanate conjugated goat
anti-rabbit antibody
MFSW
Millipore filtered sea water
NRS
normal rabbit serum
S-2
supernatant-2
ix
ABSTRACT
IMMUNOFLUORESCENCE LOCALIZATION OF EXTRACELLULAR MATRIX
COMPONENT(S) IN TWO SPECIES OF SEA URCHIN
by
Mina Alikani
Master of Science in Biology
August, 1985
An indirect immunofluorescence method was used to
localize and trace the expression of an aggregation-promoting factor(s)
(S-2) in the early stage embryos (1, 2, 4,
8, and 16-cell) of two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus.
The S-2
antigen(s) was isolated from the supernatant of calciummagnesium-free sea water dissociated S. purpuratus
blastulae.
Whole embryos from which fertilization membranes had
been removed were incubated with purified anti-S-2 antibody (AS-2).
Bound AS-2 was labeled with a second antibody
(goat anti-rabbit IgG) that had been conjugated with
fluorescein isothiocyanate.
Immunofluorescence was
X
assessed and photographed using a fluorescence microscope.
The fertilized eggs and early stage embryos of both
species of urchin displayed peripheral fluorescence.
The
antibody did not bind to the surfaces of unfertilized eggs
nor did it penetrate the hardened fertilization membrane of
embryos.
Embryos incubated with preimmune rabbit serum
showed no staining.
These results suggest that at least one of the aggregation-promoting factors present in the
~·
purpuratus
blastula appears immediately after fertilization and persists throughout the early developmental stages.
The S-2
antigen(s) seems to be similar in the two species of urchin
during their early development.
The pattern of fluores-
cence is suggestive of the localization of the S-2
antigen(s) in the extracellular matrix.
Therefore, S-2
may be a participant in the morphogenetic process as a
mediator of cellular adhesion.
xi
INTRODUCTION
The greatest challenge of developmental biology has
long been to determine the molecular basis of morphogenesis
or the development of form in embryos.
This task is
enormous since morphogenesis is not a single event but
rather a cs>}-lection of remarkab:Ly complex events which are
interrelated.
If this puzzle is to be solved, morpho-
genesis must be reduced to its primary components-cellular
adhes~on,
migration, and differentiation.
Each of
these components must be explored individually at the
molecular level and then analyzed in relation to the
other~
In recent years, cellular adhesion has been the subject of intensive investigation as an absolute necessity
for the establishment and maintenance of form in embryos.
At every stage of the development of an embryo, the cells
must adhere to one another and any cellular rearrangement
occurs, at least in part, as a result of alterations in
cellular adhesiveness.
Adhesion is believed to be mediated
by specific macromolecules.
this area is to (1)
these molecules,
The goal of most research in
~solate,
characterize, and localize
(2) find the mechanisms by which they
1
2
mediate adhesion of cells, and (3) establish their role in
the process of morphogenesis.
The pioneering experiments on cellular adhesion were
performed by H.V. vJilson, who selected a simple animal
system, the sponge, for his experiments (Wilson, 1907).
Different species of sponge were mechanically dissociated
and the single cells were found to selectively reaggregate
to form new sponges when allowed to settle in sea water.
These observations remained virtually
unexplained until a
series of classic experiments by Moscona and Humphreys
revealed the presence of specific aggregation promoting
factors in the supernatant of the dissociated sponge cells
(Moscona, 1963; Humphreys, 1963).
Using a gyratory shaker
technique, these investigators found that addition of the
dissociation supernatant from one species to the dissociated cells of the same species caused their reaggregation
but this supernatant had no aggregation promoting effect
when added to the dissociated cells of another species.
~
Therefore, the conclusion was made that this factor was
/
species specific and it mediated, through an unknown
mechanism, the selective adhesion of the sponge cells.
Subsequent experiments by these investigators showed that
the sponge adhesion factor was a high molecular weight
glycoprotein (Moscona, 1968).
Selective reaggregation has since been investigated in
embryonic systems where. specific adhesive recognition
similar to that in the sponge system has been observed.
3
The molecular mechanisms controlling selective cell associations are largely
unknow~.
-
---··
~
···-
---
However, various hypotheses
have been postulated that attempt to explain the adhesive
behavior of cells during development.
These hypotheses are
based on models that support the importance of the role
that the cell surface plays in mediating cellular
adhesion.
.
I
~
One interesting model, proposed by Roseman,.
suggests the
,.
adhesion or separation of neighboring cell,s to be a result
of the interaction of
g~ycosyl
transferases and carbo-
hydrate chains of glycoproteins, <Jlycolipids or polysaccharides (Roseman, 1974).
The specificity of each
glycosyl transferase for a cell surface receptor and for
the carbohydrate molecule that it transfers can explain the
observed specificity of cell-cell associations.
If the
transferase and the carbohydrate molecule "match 11
,
cells
will adhere to one another, if they do not match, no
~/
adhesion will take place.
Release of a carbohydrate chain
by a transferase, _<:m the other hand, causes the !:)eparation
or
11
de-adhesion" of cells.
·'
Several lines of evidence support the validity of this
model and the role of cell surface protein-carbohydrate
complexes in controlling cellular adhesion.
If sugar
molecules are indeed involved in this process, their manipulation should alter the mode of adhesion of cells.
Such
alterations are in fact observed when embryonic cells are
treated with glycosidases--enzymes that facilitate the
re!lloval of sugar residues from carbohydrates (Roth, 1971).
4
Additional evidence for this model is provided by the isolation of protein molecules from embryonic cells, that are
found to have special affinity for carbohydrates (Oppenheimer and Meyer, 1982b).
Embryos of various organisms can be dissociated into
single cells by treatment with proteolytic enzymes, alkaline solutions, or calcium-magnesium-free solutions.
Sea
urchin embryos are particularly suitable for the study of
cell-cell interactions since these embryos can be dissociated easily and completely by treatment with calciummagnesium-free sea water (Herbst, 1900); enzymatic treatment which may alter cell surface molecules is not
necessary.
Dissociated embryonic sea urchin cells are
found to have the capacity to spontaneously reaggregate and
form morula-like structures which can develop to further
stages (Giudice, 1962).
Some components of the extra7
cellular matrix and aggregation promoting factors from
embryonic cell surfaces have been investigated as possible
mediators of sea urchin embryo cellular adhesion.
Kondo and Sakai (1971) have isolated an aggregation
enhancing substance from a urea-EDTA extract of dissociated sea urchin morulae or early blastulae.
This sub-
stance, called ovacquenin, is believed to be a glycoprotein.
Tonegawa (1973) has reported the isolation of a
particulate aggregation factor (PAF) from the calciummagnesium-free sea water dissociation extract of sea urchin
blastulae.
Similar to ovacquenin and the sponge factor,
5
PAF is believed to be a mucopolysaccharide-protein complex.
In other experiments by Noll and his colleagues (1981),
reaggregation of dissociated sea urchin embryonic cells has
been shown to be promoted by a butanol extract of cell
membranes.
It is speculated that the proteins present in
this extract directly mediate cell contact since reaggregation is completely inhibited by antibodies against
these components.
Oppenheimer and Meyer (1982a) recently found that the
supernatant obtained from dissociation of live Strongylocentrotus purpuratus blastulae in calcium-magnesium-free
sea water promoted reaggregation of (1) live and glutaraldehyde-fixed blastula single cells of
~-
purpuratus
and (2) glutaraldehyde-fixed embryonic cells of three other
species of sea urchin
esculentus).
(~.
pictus,
~·
variegatus and T.
However, reaggregation in the other species
was not as dramatic as that seen in
~·
purpuratus.
Upon
incubation, the aggregatesof live blastula cells formed
in the presence of the dissociation supernatant (DS) gave
rise to vi§lble swimming embryoids suggesting that the components present in DS play a role in mediating cell
adhesion in the living embryo.
The aggregation promoting
/
activity was shown to be s:eegies and developmental stage
specific and it apparently resulted from the binding of
the components of DS to specific carbohydrate containing
cell surface receptor sites (Oppenheimer and Meyer, 1982b).
An attempt was made to purify the adhesion com-
/
6
ponent(s) from the dissociation supernatant using its cell
binding properties.
Partial purification was achieved when
the adhesive component(s) was adsorbed by and released from
glutaraldehyde-fixed
~-
purpuratus blastula cells.
The
partially purified product, referred to as S-2, displayed
one major and two minor bands on polyacrylamide, silver
stained slab gels compared to
crude DS.
~6
bands demonstrated by
Polyclonal antibodies were raised in rabbits
against S-2 to be used in subsequent antigen localization
~
and characterization studies.
In this study, the S-2 antigen{s) was localized and
its expression was traced in the early developmental stages
of both
~-
purpuratus and L. pictus using indirect immuno-
fluorescence.
The anti-S-2 rabbit antibody bound to the
cells and was localized with FITC goat anti-rabbit IgG.
The S-2 antigen(s) is discussed as a component of the
extracellular matrix of sea urchin embryos.
A comparison
is made between S-2 and some other previously isolated
extracellular matrix components.
The possible role of this
antigen(s) as a participant in the morphogenetic process is
also discussed.
MATERIALS AND METHODS
Procurement of gametes
Two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus (purchased from Pacific BioMarine, Venice, California) were used in this study.
They
were injected intracoelomically with 1 ml of 0.55M KCl to
induce spawning (Tyler, 1949).
The male gametes were
collected by placing the animal dorsal side down over a
polystyrene Petri plate.
in a beaker of
pH 8.0.
'o. 45
The female gametes were collected
l.lm Millipore filtered sea water {MFSW),
Both male and female gametes were collected on
ice.
Removal of the fertilization membrane and growth of
embryos
The eggs were washed twice in MFSW and fertilized in
the presence of 3-amino-1,2,4-triazole (ATA, Sigma
Chemical Company) to prevent hardening of the fertilization
membrane which occurs as a result of the production of diand trityrosyl residues.
ATA inhibits the crosslinking of
tyrosyl residues--by inhibition of the enzyme ovoperoxi-
7
8
dase--and therefore prevents hardening of the fertilization
membrane (Foerder and Shapiro, 1977; Showman and Foerder,
1979).
0.5ml of
packed~-
or~-
purpuratus
pictus eggs
were suspended in 10 ml MFSW, pH 8.0, containing 10
~g
ATA.
A few drops of a diluted sperm solution were added and the
egg-sperm suspension was gently swirled for 45-60 seconds
to promote fertilization.
The suspension of zygotes was
diluted lOX with calcium-magnesium-free sea water (CMF-SW),
pH 8.0, containing 90
~g
ATA, and then incubated for 15
minutes at l5°C with gentle agitation on a magnetic
stirrer.
The zygotes were allowed to settle and they were
washed twice in CMF-SW by hand centrifugation.
To remove
the loosely formed fertilization membrane, the fertilized
eggs were gently passed through a Nitex mesh
(S.
.
- purpuratus
~----·-~
45
~m mesh,~·
pictus, 72
~m
mesh).
~
.
The CMF-SW was re-
placed with MFSW, pH 8.0, and this suspension of fertilization membrane-free zygotes was poured into polystyrene
Petri plates and incubated at l5°C.
The embryos to be
used in the experiments were removed from the incubator at
the 2, 4, 8, and 16-cell stages.
Indirect immunofluorescence
50
~l
of concentrated embryo suspension from the l, 2,
4, 8, and 16-cell stages, were placed in l ml conical tubes
and incubated on ice for 30 minutes with 50
against the isolated protein(s)
ml).
(AS-2, 500
~l
~g
of antibody
protein per
As controls, the embryos were incubated with 50
normal rabbit serum (NRS, 500
~g
~l
protein per ml) or 0.1%
of
9
bovine serum albumin in MFSW (BSA/MFSW) .
BSA was used to
detect any non-specific binding of the antibody.
NRS and
AS-2were kindly provided by Stanley Liang from Dr. S.B.
Oppenheimer's laboratory.
AS-2 was generated and purified
according to the protocol of Hjelm et al (1973).
Embryos
were washed twice for one minute in BSA/MFSW by hand
centrifugation and incubated on ice for 30 minutes with 50
~1
goat anti-rabbit antibody (Fab fragments, heavy and
light chain specific, 18.7 mg protein per ml, Cappell
Laboratories) conjugated with fluorescein isothiocyanate
(FITC-GR) .
This incubation was followed by two one-minute
washes in BSA/MFSW.
The labeled embryos were then ready to
be viewed on a fluorescence microscope.
Immunofluorescence
microscopy
Immunofluorescence was assessed and photographed using
a Zeiss fluorescence microscope equipped with an Olympus
OH-1, 35mm camera.
Exposures were for 3-4 seconds
(fluorescence photographs)
and~-~
of a second at 0.5 volt
light intensity (light photographs).
Kodak 400 ASA color
print film was used for all photography.
RESULTS
Strongylocentrotus purpuratus embryos at 1, 2, 4, 8,
and 16-cell stages of development displayed peripheral
fluorescence when treated with antibody against
~·
purpuratus blastula "adhesive" antigen(s) and labeled with
FITC-GR.
Intercellular and surface fluorescence was
evident; the pattern of fluorescence could be described as
"patchy" or discontinuous on the periphery (Figures 2, 4,
6 and 8).
The unfertilized egg of
~-
purpuratus did not stain
and was therefore included in the experiments as a negative
control.
Results with sea water controls and normal rabbit
serum controls were 100% negative; no fluorescence was
.
observed in these
~amples.
'
To test for the specificity of S-2 during the early
'I
stages of development, early stage embryos of Lytechinus
pictus were treated with AS-2 and FITC-GR as described in
the materials and methods.
As represented by figures 9 and
'/ 10, whole embryos examined at the 1, 2, 4, 8, and 16-cell
stages of development displayed fluorescence similar in
pattern and intensity to that displayed by S. purpuratus
10
v
11
embryos.
As observed in
£·
purpuratus, the antibody did not
bind to unfertilized eggs.
Sea water and preimmune serum
controls did not show any staining.
The antibody did not
bind to nor did it penetrate the hardened fertilization
membrane of the embryos of either species.
These results were found to be the same in all the
experimental runs.
Photographed embryos are representative
of 95% or more of the embryos in the samples.
Sharpness of
the photographic image was greatly limited by the spherical
shape of the embryos.
12
PLATE I
Figure 1.
Light micrograph, S. purpuratus
fertilized egg.
Figure 2.
Fluorescence micrograph,
§..
pur-
puratus fertilized egg incubated
with AS-2 and labeled with
FITC-GR.
14
PLATE II
Figure 3.
Light micrograph,
s.
purpuratus
2-cell embryo.
Figure 4.
Fluorescence micrograph,
s.
purpuratus 2-cell embryo
incubated with AS-2 and labeled
with FITC-GR.
16
Plate III
Figure 5.
Light micrograph, S. purpuratus
4-cell embryo.
Figure 6.
Fluorescence micrograph, S.
purpuratus 4-cell embryo
incubated with AS-2 and labeled
with FITC-GR.
18
PLATE IV
Figure 7.
Light micrograph, S. purpuratus
8-cell embryo.
Figure 8.
Fluorescence micrograph, S.
purpuratus 8-cell embryo
incubated with AS-2 and labeled
with FITC-GR.
20
Plate V
Figure 9.
Fluorescence micrograph, L. pictus
fertilized egg incubated with AS-2
and labeled with FITC-GR.
Figure 10.
Fluorescence micrograph,
~·
pictus
2-cell embryo incubated with AS-2
and labeled with FITC-GR.
DISCUSSION
The expression of an aggregation promoting factor(s)
(S-2) extracted from the blastulae of S. purpuratus was
traced from the unfertilized egg through the early developmental stages (1, 2, 4, 8, and 16-cell).
Peripheral
immunofluorescence is indicative of the localization of S-2
in the outer extracellular matrix (ECM) suggesting that it
may play a role in mediating some cell-cell interactions
during early development.
The outer ECM is often equated with the hyaline layer.
This layer is formed on the surface of the egg at the time
of fertilization, as a result of the release of hyalin
v
protein from the cortical granules into the previtalline
space (Endo, 1961; Stephens and Kane, 1970).
Although we
have not yet determined whether S-2 is originally housed
within the cortical granules of the unfertilized egg, it is
significant that the appearance of S-2 on the embryos is
concomitant with the appearance of the hyaline layer.
Moreover, as an essential factor in the adherence of the
blastomeres in the embryo (Dan, 1960), the hyaline layer
persists on embryonic cell surfaces and is not lost until
22
23
metamorphosis; this is in agreement with the observed persistence of S-2 on embryos throughout the early stages of
development.
A question is raised as to whether hyalin and S-2
could be the same--or very similar.
McClay and Fink (1982)
have examined hyalin by immunofluorescence using an antihyalin antibody.
Their results are very similar to the
v results obtained with S-2 in the present study.
Hyalin was
found to appear at fertilization whereas it was absent in
the
unfe~tilized
egg.
Also, the anti-hyalin antibody was
found to stain the embryo surface throughout early development.
As part of the protocol for fertilization membrane
removal in these experiments, the embryos were treated with
calcium-magnesium-free sea water containing aminotriazole.
This treatment should remove most, if not all, of the
hyalin in the hyaline layer since this protein has been
shown to dissolve in the absence of calcium ions (Kane and
Hersh, 1959).
Still other components of the hyaline layer
must have been left on the surface since the complete
removal of this layer leads to spearation of the blastomeres after division (Citkowitz, 1971, 1972).
Embryos
stripped of their hyalin at fertilization have been shown
to regenerate an appreciable fraction of the hyalin but
this regeneration apparently does not occur until the time
of blastulation (Kane, 1973) or early gastrulation (McClay
and Fink, 1982); during cleavage stages there is little, if
any, new hyalin synthesis.
S-2 could therefore be a com-
24
ponent that persists on the surface within the hyaline
layer afte! removal of hyalin.
It should be noted that ultrastructural studies do
support an apparent non-homogeneity of the hyaline layer
(Wolpert and Mercer, 1963).
Several distinct components
seem to be divided into separate layers within the hyaline
layer; it is therefore appropriate to refer to a "hyaline
complex" as the possible site of localization of S-2 since
the resolution of the immunofluorescence technique used in
this study is not sufficient to distinguish between
different possible matrices within the outer ECM.
In agreement with the ultrastructural studies, Hall
and Vacquier (1982) have recently reported the existence of
a fibrous glycoprotein network, the apical lamina, which
seems to persist around the embryos (one-hour old and
blastulae) after hyalin and presumably other proteins of
the hyaline layer have been dissolved in glycine and the
hyaline layer have been removed.
They propose that the
apical lamina is associated with but distinct from the
J;lYCI.line layer and that it may therefore mediate cellular
attachment to the "outside" layer (the hyaline layer) of
f
the outer ECM.
It is possible that the appearance of
hyaline and the apical lamina glycoproteins as separate
r
layers is a result of their differential solubilities in
glycine and that they may form one single layer in the
intact embryo.
However, either as a single layer or as
separate layers, the hyaline and the apical lamina glyco-
25
proteins seem to work in concert rather than independently
to coordinate morphogenetic events.
participant in these interactions.
S-2 may be another
It is of interest that
the estimated molecular weight of at least one of the S-2
bands displayed on polyacrylamide gels (greater than 90Kdaltons) is in the same molecular weight range as those
reported for the apical lamj,na
daltons).
mate:~;ial
(145K and 175K-
This is an indication of the similarity of these
components.
In other related studies, Evelyn Spiegel and her
colleagues have reported the presence of yet another
possible extracellular layer in early sea urchin embryos
(Spiegel et al, 1980, 1983).
The results of their immuno-
fluorescence experiments showed that fibronectin and
laminin--both high molecular weight glycoproteins--form a
continuous matrix surrounding the embryonic cells.
It is
thought that this matrix may be a link between the hyaline
layer and the basement membrane (inner ECM) , playing a role
in such events as cell adhesion and migration during
development.
In some earlier studies, Spiegel and Spiegel
(1979) had reported the presence of collagenous, fibril,----~------
-
like structures within the hyaline layer.
Comparative studies between the pattern of staining of
embryos with our anti-S-2 antibodies and the pattern obtained with antibodies against fibronectin, laminin, and
collagen (as well as other known ECM components) are
currently being undertaken in our laboratory.
Future
26
results from these comparative studies combined with the
results of the present study should help in further
characterization of S-2 as a molecule that may be responsible for regulation of morphogenetic events.
BIBLIOGRAPHY
Its isolation
Citkowitz, E. (1971). The hyaline layer:
and role in echinoderm development. Dev. Biol. 24:
348-362.
Citkowitz, E. (1972). Analysis of the isolated hyaline
layer of sea urchin embryos. Dev. Biol. 27: 494-503.
Dan, K. (1960). Cyto-embryology of echinoderms and
Int. Rev. Cytol. 9: 321-367.
amphibia.
Endo, Y. (1961). Changes in the cortical layer of sea
urchin eggs at fertilization as studied with the
electron microscope:
I. Clypeaster japonicus.
Exp. Cell. Res. 25: 383-397.
Foerder, C.A. and B.M. Shapiro (1977). Release of ovaperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc. Natl.
Acad. Sci. USA, 74: 4214-4218.
Giudice, G. (1962). Restitution of whole larvae from disaggregated cells of sea urchin embryos. Dev. Biol.
5: 402-411.
Hall, H.G., and V. Vacquier (1982). The apical lamina of
the sea urchin embryo: major glycoproteins associated
with the hyaline layer. Dev. Biol. 89: 168-178.
Herbst, C. (1900). Uber das Auseinandergehen von Forchungs und Gewebezellen in Kalkfreiem Medium.
Wilhelm Roux Arch. Entwicklungsmech. Organ. 9: 424463.
-Hjelm, H., K. Hjelm, and J. Sjoquist (1973). Protein A
from Staphylococcus aureus.
Its isolation by affinity
chromatography and its use as an immunosorbent for
isolation of immunoglobulins. Febs. Lett. 28: 73-76.
27
28
Humphreys, T. (1963). Chemical dissolution and in vitro
reconstitution of sponge cell adhesions.
I-.-Isolation
and functional demonstration of components involved.
Dev. Biol., 8: 27-47.
Kane, R.E. and R.T. Hersh (1959). The isolation and preliminary characterization of a major soluble protein
of the sea urchin egg. Exp. Cell Res. 16: 59-69.
Kane, R.E. (1973). Hyalin release during normal sea urchin
development and its replacement after removal at
fertilization.
Exp. Cell Res. 81: 301-311.
Kondo, K. and H. Sakai (1971). Demonstration and preliminary characterization of reaggregation-promoting
substances from embryonic sea urchin cells. Dev.
Growth and Differ., 13: 1-14.
McClay, D.R. and R.D. Fink (1982). Sea urchin hyalin:
Appearance and function in development. Dev. Biol.
9 2: 2 8 5-2 9 3.
Moscona, A.A. (1963). Studies on cell aggregation:
Demonstration of materials with selective cell binding
activity.
Proc. Natl. Acad. Sci. USA, 49: 742-747.
Moscona, A.A. (1968). Cell aggregation: Properties of
specific cell-ligands and their role in the formation
of multicellular systems. Dev. Biol. 18: 250-277.
Noll, H., V. Matranga, P. Palma, F. Cutrona, and L.
Vittorelli (1981). Species-specific dissociation
into single cells of live sea urchin embryos by Fab
against membrane components of Paracentrotus lividus
and Arbacia lixula. Dev. Biol., 87: 229-24t.
Oppenheimer, S.B., and JrT. Meyer (1982a).
Isolation of
species-specific and stage-specific adhesion promoting
component by disaggregation of intact sea urchin
embryo cells. Exp. Cell. Res., 137: 472-476.
Oppenheimer, S.B., and J.T. Meyer (1982b). Carbohydrate
specificity of sea urchin blastula adhesion component.
Exp. Cell. Res., 139: 451-455.
Roseman, S. (1974). The biosynthesis of complex carbohydrates and their potential role in intercellular
adhesion.
In A.A. Moscona, ed., The Cell Surface in
Development, pp. 255-272. New York: John Wiley.
29
Roth, S., E.J. McGuire, and S. Roseman (1971). Evidence
for cell-surface glycosyltransferases: Their
potential role in cellular recognition. ~ Cell
Biol. 51: 536-547.
Showman, R., and C.A. Foerder (1979). Removal of the
fertilization membrane of sea urchin embryos employing
aminotriazole. Exp. Cell. Res., 120: 253-255.
Spiegel, E. and M. Spiegel (1979). The hyaline layer is a
collagen-containing extracellular matrix in sea urchin
embryos and reaggregating cells. Exp. Cell. Res.,
123: 434-441.
Spiegel, E., M.M. Burger, and M. Spiegel (1980).
nectin in the developing sea urchin embryo.
Biol. , 87: 309-313.
FibroJ. Cell
Spiegel, M., M.M. Burger, and M. Spiegel (1983). Fibronectin and laminin in extracellular matrix and basement membrane of sea urchin embryos. Exp. Cell.
Res., 144: 47-55.
---Stephens, R.E. and R.E. Kane (1970). Some properties of
hyalin. J. Cell Biol. 44: 611-617.
Tonegawa, Y. (1973).
Isolation and characterization of a
particulate cell-aggregation factor from sea urchin
embryos. Dev. Growth and Differ., 14: 337-352.
Tyler, A. (1949). A simple, non-injurious method for
inducing spawning of sea urchins and sand dollars.
Collect. Net., 19: 19-20.
Wilson, H.V. (1907). On some phenomena of coalescence and
reaggregation in sponges. ~ Exp. Zool., 5: 245-258.
Wolpert, L. and E.H. Mercer (1963). An electron microscopic study of the development of the blastula of the
sea urchin embryo and its radial polarity. Exp.
Cell. Res. 30: 280-300.