Development 99, 155-162 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
155
Cell- and tissue-specific monoclonal antibodies in eggs and embryos of
the ascidian Halocynthia roretzi*
IZUMI MTTA-MIYAZAWA, TAKAHITO NISHIKATA and NORIYUKI SATOH
Department of Zoology, Kyoto University, Kyoto 606 and Asamushi Marine Biological Station of Tohoku University, Aomori 039-34, Japan
' All the monoclonal antibodies are available for studies of ascidian developmental biology on request from I.M.-M.
Summary
To obtain specific immunological probes for studying
molecular mechanisms involved in the early embryonic development of ascidians, we have produced
monoclonal antibodies directed against a homogenate
of larvae of the ascidian Halocynthia roretzi. Among
these, we have screened monoclonal antibodies that
specifically recognize cells and/or tissues of the
embryo. ..Characterization of six epidermis-specific
monoclonal antibodies (including larval tunic-specific
and larval fin-specific), three muscle-specific antibodies, two endoderm-specific antibodies, one
notochord-specific antibody and two monoclonal
antibodies that specifically recognize trunk-lateral
cells suggests that these monoclonal antibodies may be
useful as markers for analysing molecular mechanisms involved in specification of these cells. Seven
monoclonal antibodies characteristically stain intercellular materials of the developing embryo and may
therefore be valid for studying cellular construction of
the embryo. Furthermore, monoclonal antibodies that
recognize components of follicle cells, perivitelline
space and sperm have also been established.
Introduction
ments and acetylcholinesterase for the muscle cells,
melanin and tyrosinase for the melanocytes, vacuolate inclusions for the notochordal cells, extracellular secretions for the epidermal cells and alkaline
phosphatase for the endoderm cells. In addition,
these morphological and histochemical markers are
not always used directly in molecular studies on cell
specification. This situation therefore seems to restrict an overall understanding of mechanisms underlying differentiation in the mosaic eggs.
Immunological probes, particularly those with
monoclonal antibodies, have successfully been introduced in various fields of developmental biology. In
order to facilitate a study of the molecular mechanisms underlying early development of this mosaic egg
and also to investigate the functions of accessory cells
(i.e. follicle cells and test cells), we have attempted
to produce monoclonal antibodies that specifically
recognize cells and tissues of developing ascidian
embryos. Results obtained are described here and
discussed with respect to the validity for their use in
further investigations.
Eggs of ascidians (subphylum Urochordata, class
Ascidiacea) offer advantages for studying cellular
and molecular mechanisms involved in specification
of embryonic cells (see reviews by Reverberi, 1971;
Whittaker, 1979; Jeffery, 1984): (1) an egg develops
very rapidly into a tadpole larva which consists of
a comparatively small number (about 2500) of cells
but of several distinct types of differentiated cells;
(2) embryonic cell lineages are well known; the
developmental pattern is 'mosaic' and there is
accumulating evidence for the existence of egg cytoplasmic determinants which are differentially segregated by a determinate cleavage pattern into certain
cell lineages where they appear to play a crucial role
in programming the differentiation pathways of the
cells and (3) although a certain number of DNA
replications may be an essential prerequisite, neither
cytokinesis nor nuclear division is required for tissuespecific enzyme development.
However, despite such advantages, the number of
differentiation markers is rather limited; myofila-
Key words: ascidian embryos, monoclonal antibodies,
differentiation markers, intercellular'materials, follicle
cells, perivitelline space, sperm.
156
/. Mita-Miyazawa, T. Nishikata and N. Satoh
Materials and methods
Results
Embryos
To obtain specific immunological probes for studying
molecular mechanisms involved in cell specification
of ascidian embryos, we immunized mice with a
homogenate of newly hatched larvae, fused the
mouse spleen cells to mouse myeloma cells to establish hybridoma cell lines and screened individual
hybridoma culture fluid samples by immunofluorescence microscopy. In four series of productions,
more than 800 samples were tested, from which we
selected and cloned hybridoma cell lines that secreted
antibodies that specifically recognize certain cells.
Naturally spawned eggs of the ascidian, Hahcynthia roretzi,
were fertilized artificially with a dilute suspension of nonself
sperm and raised in filtered seawater at 13-15 °C.
Antigen preparation and immunization
Newly hatched larvae were collected by centrifugation,
washed with an ice-cold physiological saline solution (PSS)
for mouse and suspended in an equal volume of PSS. The
suspension was homogenized by several vertical strokes of
a Teflon pestle fitted into a Potter glass homogenizer and
centrifuged at 3000 revs min"1 for lOmin, then at 10000
revs min" 1 for 5min. The supernatant was used as an
antigen. Female BALB/c mice were injected intraperitoneally with 0-5 ml supernatant and boosted intraperitoneally once or twice at about 3 week intervals with 0-5 ml
injection of the immunogen.
Production of monoclonal antibodies
Three days after the last intraperitoneal hyperimmunization, the mice were killed and their spleens removed.
Spleen cells-myeloma hybridomas were generated according to the protocol of Galfre, Howe, Milstein, Butcher &
Howard (1977); about lxlO 8 spleen cells were fused with
2-5xlO7 P3U-1 myeloma cells (purchased from How Lab.)
by using 50% (w/v) polyethylene glycol 4000 (Nakarai
Chem. Co. Ltd, Kyoto). Hybridomas were raised in HAT
medium according to Littlefield (1964). The fused cells
were separated into 96 multiwells (106 cells/well) and,
when cell growth was apparent (7-10 days after fusion),
samples of supernatant of hybridoma culture medium from
each well were assayed by immunofluorescence microscopy. Hybrid cells producing antibodies of interest
were cloned once or twice by picking up a single cell with a
small pipette under an inverted microscope.
Fixation and immunofluorescence staining of eggs and
embryos
For screening the hybridoma supernatant medium, large
quantities of Halocynthia eggs and embryos at various
stages were fixed for 10 min in methanol (—20 °C), followed
by ethanol (—20 °C), and embedded in polyester wax
(Steedman, 1957; BDH Chem. Ltd). Sectioned specimens
were mounted on small coverslips. After removal of polyester wax with absolute ethanol, specimens were washed
with phosphate-buffered saline (PBS), then immersed in
100/d of hybridoma culture fluid for l h at room temperature. Coverslips were then washed in PBS at room
temperature for 30min, and each coverslip incubated for
30 min with 6 [A of fluorescein isothiocyanate-conjugated
rabbit anti-mouse IgG serum (Miles-Yeda, Ltd) diluted
1:60 in PBS. Coverslips were then washed in PBS at
room temperature for 30 min, mounted in 80% glycerol
and observed with a Nikon Labophoto equipped with an
epifluorescence optic unit (EFD). Photomicrographs were
taken with Kodak Tri-X Pan film.
Epidermis-specific, larval tunic-specific and/'or larval
fin-specific monoclonal antibodies
The outermost part of an ascidian embryo consists of
a layer of epidermal cells and a developing tadpolelike embryo is surrounded by two acellular layers, the
larval tunic and larval fin (Fig. 1). As summarized in
Table 1, six hybridoma clones, 3A7D5 (Fig. 2),
4C5B7 (Fig. 3), 5D3E8, 7H6B6, 12N1D2 and 5F1F8
(Fig. 4), were found to secrete monoclonal antibodies
which specifically recognized epidermal cells, larval
tunic, and/or larval fin. For example, the antigen
recognized by each of 3A7D5 and 4C5B7 first appears
at the early tailbud stage, initially within epidermal
cells. Progressively with developmental time, a forming larval tunic becomes stained very intensely
(Figs 2,3). 3A7D5 stains the boundary of neighbouring epidermal cells more intensely than the inside
of cells (Fig. 2), whereas the staining with 4C5B7
appears punctate and intracellular (Fig. 3). In contrast to the five other antibodies that recognize
epidermal cells and larval tunic, 5F1F8 recognizes
larval fin (Fig. 4). The antigen isfirstexpressed at the
middle tailbud stage, when formation of the larval fin
begins. In tadpole larvae of this species, fin-ray-like
striped structures running very regularly through the
larval fin are easily observed with differential interference contrast microscopy. As development proceeds, 5F1F8 actually recognizes the component of
thisfin-ray-likestructure (Fig. 4).
Muscle-specific monoclonal antibodies
A tadpole larva of H. roretzi contains 21 striated but
mononucleate muscle cells on each side of the tail
(Fig. 1). We have produced three muscle-specific
monoclonal antibodies, 6F2D3 (Fig. 5), 3M1F11
(Fig. 6) and 3M3A7 (Table 1). As clearly shown in
Figs 5 and 6, the monoclonal antibodies recognize
components exclusively present in muscle cells. The
antigenicity of each of them first appears at the early
tailbud stage, but is not detected at the neurula and
earlier stages. 3M1F11 and 3M3A7 react with muscle
Monoclonal antibodies against ascidian embryos
mu
bs
n
157
If
tic
SC
ep
mu
es
A
en
It
B
Fig. 1. Schematic representation of structures of the tailbud-stage H. roretzi embryo. (A) Midsagittal section of the
embryo; (B) sagittal section; (C) cross section through the middle part of the tail, b, brain; bs, brain stem; en,
endoderm; ep, epidermis; es, endodermal strand; //, larval fin; It, larval tunic; mch, mesenchyme; mu, muscle; n,
notochord; sc, spinal cord; tic, trunk-lateral cell.
Table 1. Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidian Halocynthia roretzi
Trunk-lateral cell-specific
5A4E1
5A4A3
5A4A6
5A11C11
+
+
+
+
tic
+
+
+
+
+
early tailbud
early tailbud
early tailbud
middle tailbud
late tailbud
middle tailbud
+
+
+
+
early tailbud
early tailbud
early tailbud
middle
middle
middle
middle
+
+
+
+
Notochord-specific
5F1D5
+
Endoderm-specific
4B3C3
6C9D1
±
±
+
+
tailbud
tailbud
tailbud
tailbud
Ciona intestinalis
+ (larval tunic)
+ (larval fin)
+ (muscle)
+ (muscle)
1 1
Muscle-specific
3M3A7
4M1F11
6F2D3
V
ep
1
Epidermis-specific
3A7D5
4C5B7
5D3E8
7H6B6
12N1D2
5F1F8
Regional distribution of antigens*
Cross reactivity
with embryonic
tissues of
1
Monoclonal
antibody
Developmental stage
at which the
antigenicity
first appears
early tailbud
-
early gastrula
fertilized egg
-
* ep, epidermis; It, larval tunic; //, larval fin; mu, muscle; tic, trunk-lateral cell; n, notochord; en, endoderm.
components of another ascidian species, Ciona intestinalis, and also recognize body wall muscle cells of
the adult animal. Therefore, the antigens may be
common components of ascidian muscle cells.
vegetal blastomeres) is marked with horseradish peroxidase at the 32-cell stage, the dorsal endoderm of
the head region and undefined cells lying lateral to
the brain, stem are stained (Nishida & Satoh, 1985).
Further analyses have revealed that the property of
Monoclonal antibodies that specifically identify trunk- the undefined cells, designated as 'trunk-lateral cells',
lateral cells
is inherited by the A7.6 cell of the 64-cell embryo; i.e.
the A7.6 cell gives rise to only trunk-lateral cells
A recent study of cell lineages in ascidian embryos
(Nishida, unpublished).
has shown that if the A6.3 cell (one of the anterior
158
/. Mita-Miyazawa, T. Nishikata and N. Satoh
Monoclonal antibodies against ascidian embryos
We have produced four monoclonal antibodies,
5A4E1 (Fig. 7), 5A4A3, 5A4A6 and 5A11C11, which
specifically identify trunk-lateral cells (Table 1). The
antigen recognized with 5A4A3 or 5A4E1 is highly
restricted to trunk-lateral cells. The antigens are
expressed from the middle tailbud stage onwards and
the entire cell, except for the nucleus, stains very
strongly with FITC-labelled monoclonal antibodies
(Fig- 7).
Notochord-specific monoclonal antibody
In a developing H. roretzi larva, 40 notochord cells
run anteroposteriorly through the centre of the tail
(Fig. 1). A monoclonal antibody 5F1D5 has been
identified to be almost specific to notochord cells
(Fig. 8), although a faint staining is sometimes seen
in endodermal cells. The antigen is expressed from
the initial tailbud stage onwards. It initially appears
punctate and intracellular but as development proceeds, the border of notochord cells or the notochordal sheath stains very strongly (Fig. 8).
Endoderm-speciflc monoclonal antibodies
The central region of the head of a developing tailbud
embryo is occupied by a group of endodermal cells
which give rise to the gut of adult animals (Fig. 1).
Two monoclonal antibodies, 4B3C3 (Fig. 9) and
6C9D1 (Fig. 10), recognize components of endodermal cells (Table 1). The antigen of 4B3C3 is first
expressed in endodermal cells at the early gastrula
Figs 2—8. Regional distribution of antigens recognized
with tissue-specific monoclonal antibodies on polyester
wax sections of middle to late tailbud-stage embryos (Figs
2, 3, 5-8) and on a whole mount of swimming tadpole
larvae (Fig. 4) of the ascidian Halocynthia roretzi. Scale
bar, 50 um.
Fig. 2. An epidermis-specific monoclonal antibody
3A7D5 stains the boundary of the epidermal cells and
larval tunic very strongly.
Fig. 3. The staining with an epidermis-specific antibody
4C5B7 appears punctate and intracellular.
Fig. 4. The antigen of an epidermis-specific 5F1F8 is
expressed in the fin-ray-like structure of the larval fin (If).
The opaque appearance of the larval body (Ib) is not due
to immunofluorescence staining.
Fig. 5. A section of a late tailbud embryo stained with a
muscle-specific 6F2D3 clearly shows that muscle cells
(mu) of each side of the elongating tail are recognized
with this antibody. B is an enlargement of A.
Fig. 6. A muscle-specific 3M1F11 stains exclusively
muscle cells.
Fig. 7. Staining with 5A4E1 indicates that the antigen
recognized with this antibody is expressed in only trunklateral cells (arrow).
Fig. 8. A monoclonal antibody 5F1D5 intensely stains
the boundary of notochord cells (n) or notochordal
sheath.
159
stage. A weaker staining is sometimes seen in notochord (Fig. 9).
Monoclonal antibodies against components of
intercellular materials
Seven monoclonal antibodies summarized in Table 2
have been found to identify intercellular materials.
The antibodies recognize components that first
appear at the neural plate (12N2D4, Fig. 11), neurula
(4B4D2, 3M2A11, 3M5A10) and early tailbud stages
(5D7E7,6A1F2), respectively. The component recognized with 6B12C3 (Fig. 12), however, is present
from the unfertilized egg through to embryogenesis.
Staining patterns of the antibodies that recognize
newly synthesized extracellular materials differ in
detail from each other (Table 2). For example, the
antigen identified with 12N2D4 is expressed at the
boundary between notochord, muscle, endodermal
strand, brain and spinal chord, but not at the border
of epidermal cells (Fig. 11), whereas 3M5A10 stains
epidermal cells and larval tunic of the head region
very strongly.
Follicle cell-specific monoclonal antibodies
An ascidian egg is enclosed by the outer follicle cells
and the inner test cells, with an acellular vitelline
membrane or the chorion separating these cells. Two
monoclonal antibodies, 4D9D3 (Fig. 13) and 5F3H2,
stain follicle cells very markedly. The antigenicity of
both monoclonal antibodies is already detected in
follicle cells of the gonad. The reactivity of 5F3H2 is
exclusively found in follicle cells, whereas a faint
staining with 4D9D3 is also detected in the yolk area
of a developing embryo (Fig. 13). These monoclonal
antibodies do not stain Ciona embryonic tissues.
Monoclonal antibodies for components of the
perivitelline space
The perivitelline space of an ascidian egg is thought to
contain not only water and inorganic substances but
also mucus-like components whose function has not
yet been defined. Three monoclonal antibodies,
3PvA6, 3PvC2 and 3F2F10, recognized components
of the perivitelline space. No crossreactivity of these
monoclonal antibodies with Ciona tissue is observed.
Sperm-specific monoclonal antibodies
Although the antigen used to immunize mice was
a homogenate of larvae, two sperm-specific monoclonal antibodies, 4D5A10 and 3M2G8 (Fig. 14),
were obtained. The antigen recognized by 4D5A10 is
seen on the overall surface of sperm including its head
and tail: It is evident that an oviform structure near
the head is intensely stained with 3M2G8 (Fig. 14). It
has been shown that an ascidian sperm contains one
large mitochondrion, which moves during activation
from its original position at the middle piece to the
160
/. Mita-Miyazawa, T. Nishikata and N. Satoh
Monoclonal antibodies against ascidian embryos
161
Table 2. Regional distribution of antigens recognized with seven monoclonal antibodies against components of
intercellular materials
Monoclonal
antibody
Regional distribution of antigens*
ep
//
sc
mu mch
12N2D4
4B4D2
3M2A11
3M5A10
5D7E7
6A1F2
6B12C3
Developmental stage
at which the
antigenicity first
appears
neural plate
neurula
neurula
neurula
early tailbud
early tailbud
unfertilized eggs
Cross reactivity
with Ciona
embryonic tissues
+ (epidermis)
'ep, epidermis; It, larval tunic; //, larval fin; b, brain; sc, spinal cord; mu, muscle; mch, mesenchyme; n, notochord; e, endoderm; es,
endodermal strand.
tip of the tail and is then finally discarded (Lambert
& Epel, 1979). Judging from its position and morphology, 3M2G8 is likely to recognize some antigen localized at this sperm mitochondrion. The
sperm-specific antibodies do not stain sperm of Ciona
intestinalis.
Discussion
The objective of this study was to establish hybridoma clones that secrete monoclonal antibodies
specific to cells or tissues of ascidian embryos. As
described, we have obtained many monoclonal antibodies fulfilling this objective. Characterization of
six epidermis-specific monoclonal antibodies, three
muscle-specific antibodies, two endoderm-specific
antibodies, one notochord-specific antibody and two
monoclonal antibodies that specifically recognize
Fig. 9. The antigen of 4B3C3 is expressed on the surface
of endoderm cells (en). This antigen is first expressed at
the early gastrula stage, although a faint staining is
sometimes seen in notochord cells. Scale bar, 50 fim.
Fig. 10. The antigen of 6C9D1 is present in fertilized
eggs. As development proceeds, the antigen becomes
mainly distributed in endoderm cells (en). Scale bar,
50/mi.
Fig. 11. 12N2D4 stains the borders of the notochord (n),
muscle (mu), endodermal strand (es), brain (b), and
spinal cord (sc), but does not stain the border of the
epidermis. A shows a midsagittal section of a late tailbudstage embryo, while B is a cross section of the middle
part of the tail of the same stage. Scale bar, 50 /an.
Fig. 12. The antigen of 6B12C3 is detected at the surface
of an unfertilized egg. As development proceeds, the
boundary of every cell stains with this antibody.
Fig. 13. A monoclonal antibody 4D9D3 specifically
recognizes follicle cells (fc). em, embryo. Scale bar,
50 /mi.
Fig. 14. A sperm-specific monoclonal antibody 3M2G8
stains mitochondria (arrows) intensely. Scale bar, 10/an.
trunk-lateral cells suggests that these monoclonal
antibodies may be useful as markers for analysing
molecular mechanisms involved in specification of
these cells. However, prior to using these monoclonal
antibodies as immunological probes, we have to
characterize antigenic polypeptides recognized by
these antibodies. Although all the monoclonal antibodies are different in their temporal appearance
and spatial localization, there remains the possibility
that two or more antibodies recognize an identical
antigenic polypeptide.
Most developmental biological studies of ascidian
embryos have focused on the specification of
embryonic cells and studies of morphogenesis or
construction of a particular form of the embryo are
rather scarce. We have produced seven monoclonal
antibodies specific to intercellular materials which
may be used as immunological probes for further
studies of mechanisms involved in the construction of
the embryonic form. In addition, we have obtained
several antibodies specific to components of follicle
cells, the perivitelline space and sperm. These antibodies may also be useful for further analysis of their
functions.
In this study we have attempted to produce monoclonal antibodies that can be used as differentiation
markers. The results obtained are not always complete, since it was not possible to produce monoclonal
antibodies specific to mesenchyme, brain or spinal
cord. However, we envisage the monoclonal antibodies established in this study as being useful as
immunological probes for further studies of ascidian
developmental biology.
We thank Dr M. Takeichi and Mr K. Hatta for their
technical advice and help during the course of monoclonal antibody production. Thanks are also due to Dr T.
Numakunai and all other members of Asamushi Marine
Biological Station for their kind hospitality. We are also
162
/. Mita-Miyazawa, T. Nishikata and N. Satoh
grateful to Dr A. Rossiter for his help in the preparation of
the manuscript. This study was supported by a Grant-inAid from the Ministry of Education, Science and Culture,
Japan (No. 60105001) and by Kudo Academic Foundation.
References
GALFRE, G., HOWE, S. C , MILSTEIN, C , BUTCHER, G. W.
& HOWARD, J. C. (1977). Antibodies to major histo-
compatibility antigens produced by hybrid cell lines.
Nature, Lond. 266, 550-552.
JEFFERY, W. R. (1984). Pattern formation by ooplasmic
segregation in ascidian eggs. Biol. Bull. mar. biol.
Lab., Woods Hole 166, 277-298.
LAMBERT, C. C. & EPEL, D. (1979). Calcium-mediated
mitochondria movement in ascidian sperm during fertilization. Devi Biol. 69, 296-304.
J. W. (1964). Selection of hybrids from
matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710.
NiSfflDA, H. & SATOH, N. (1985). Cell lineage analysis in
ascidian embryos by intracellular injection of a tracer
enzyme. II. The 16- and 32-cell stages. Devi Biol. 110,
440-454.
REVERBERI, G. (1971). Ascidians. In Experimental Embryology of Marine and Fresh-Water Invertebrates (ed. G.
Reverberi), pp. 507-550. Amsterdam: North-Holland.
STEEDMAN, H. F. (1957). Polyester wax. A new ribboning
embedding medium for histology. Nature, Lond. 179,
1345.
WHTTTAKER, J. R. (1979). Cytoplasmic determinants of
tissue differentiation in the ascidian egg. In
Determinants of Spatial Organization (ed. S. Subtelny &
I. R. Konigsberg), pp. 29-51. New York: Academic
Press.
LITTLEFIELD,
(Accepted 29 August 1986)