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995
Development 113, 995-1005 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Budding-specific lectin induced in epithelial cells is an extracellular matrix
component for stem cell aggregation in tunicates
KAZUO KAWAMURA*, SHIGEKI FUJIWARA and YASUO M. SUGINO
Department of Biology, Faculty of Science, Kochi University, Akebono-cho,
Kochi 780, Japan
* Author for correspondence
Summary
We have examined immunocytochemically the expression, localization and in vivo function of a calciumdependent and galactose-binding 14xlO3Afr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner
epithelium of a double-walled bud vesicle. Soon after the
bud entered the developmental phase, the granules were
secreted into the mesenchymal space, where the lectinpositive extracellular matrix (ECM) developed. The
lectin was also produced and secreted by granular
leucocytes during budding. Hemoblasts, pluripotent
stem cells in the blood, were often found in association
with the ECM and they aggregated with epithelial cells
to form organ rudiments. The lectin showed a high
binding affinity for hemoblast precursors. The blockage
of epithelial transformation of stem cells by galactose in
in vivo bioassay was ineffective in the presence of the
lectin. Polyclonal anti-lectin antibody prevented the
hemoblasts spreading on the ECM and moving toward
the epithelium, but it did not block the cell-cell adhesion
of hemoblasts. By three days of bud development, lectin
granules and ECM have almost disappeared from the
developing bud together with a cessation of hemoblast
aggregation. These results show that Polyandrocarpa
lectin is a component of the ECM induced specifically in
budding and suggest strongly that it plays a role in bud
morphogenesis by directing the migration of pluripotent
stem cells to the epithelium.
Introduction
the C-type lectin plays an important role in cell-cell
communication in a wide variety of animal species.
Recently, we have purified a 14X103 Afr protein from
the tunicate, Polyandrocarpa misakiensis. Its amino
acid sequencing and biochemical characterization
showed that it is a calcium-dependent, galactosebinding lectin (Suzuki et al. 1990), referred to as TC-14
(tunicate-derived C-type lectin of i^xlO 3 MT) tentatively in this work. Unlike P. misakiensis with budding
capacity (Watanabe and Tokioka, 1972; Kawamura and
Watanabe, 1983), solitary tunicates such as Styela
plicata and Ciona intestinalis did not contain TC-14
(Suzuki et al. 1990 and our unpublished data). TC-14
might be involved in asexual reproduction of budding
tunicates.
In this work, we have purified TC-14, labeled a
portion of it with fluorescein or biotin and prepared
anti-TC-14 polyclonal antibodies. They were used to
examine the expression, localization and possible role
of the TC-14 lectin in asexual development of Polyandrocarpa. This is the first report of a budding-specific
protein from tunicates. The results are discussed in the
context of the manner by which TC-14 plays a role in
Animal lectins have recently been classified into two
groups (Drickamer, 1988). One, referred to as C-type
lectin, requires calcium to exert carbohydrate-binding
activity; the other, the S-type lectin, depends on the
protection of SH group in polypeptide chains for its
activity. The C-type lectins have been extracted from
barnacle (Muramoto and Kamiya, 1986), fly (Takahashi
et al. 1985), sea urchin (Giga et al. 1987) and many
organs of mammals (Drickamer et al. 1984). They have
a characteristic carbohydrate-recognition domain
(CRD) consisting of 120-130 residues including four
invariable half-cysteines that form two intrachain
disulfide bridges (Drickamer, 1988).
Lectin in the fly, Sarcophaga, is involved in the
development of embryos or pupae (Takahashi et al.
1986) as well as defense mechanisms of instars (Komano
et al. 1983). In mammals, glycoproteins containing a
lectin domain at the N terminus serve as a lymphocytehoming receptor (Lasky et al. 1989) and platelet-vascular endothelium adhesion molecule (Johnston et al.
1989). Thus, increasing evidence strongly suggests that
Key words: lectin, extracellular matrix, stem cells,
epithelial transformation, budding, tunicate.
996
K. Kawamura and others
epithelial-mesenchymal collaboration during bud morphogenesis of Polyandrocarpa.
Materials and methods
Purification of TC-14
Colonies of Polyandrocarpa (Eusynstyela) misakiensis were
attached to glass slides and reared in the inlet near the Usa
Marine Biological Institute, Kochi University. A galactosebinding 14xl(r MT lectin (TC-14) was purified, as described
previously (Suzuki et al. 1990). In brief, about 100 g of
asexually developing animals was homogenized with 300 ml of
7.5 mM phosphate buffer (pH7.2) and extracted for 60min in
an ice bath. After centrifugation (12000g, 15min), a portion
of the supernatant containing lmM phenylmethylsulfonyl
fluoride (PMSF) was stored as crude extracts at —80°C. The
supernatant was fractionated with 40-95 % saturated ammonium sulfate, after boiling and centrifugation. The crude
lectin fraction was dialyzed against 0.1M ammonium acetate
and passed through a gel filtration column (Ultrogel AcA44;
3x100 cm) equilibrated with the same buffer. After dialysis
against 20mM Tris-HCl (pH8.0), the lectin fraction was
applied to a column (16x40mm) of DE-32 (Whatman
Biosystems, Ltd), equilibrated with the same buffer. The
column was eluted overnight with a linear gradient of 0-0.5 M
NaCl in the same buffer (400 ml). The eluate was monitored
for absorbance at 280 nm. The purity of TC-14 was estimated
from the elution profile of anion exchange chromatography
and SDS-PAGE. Its amino acid composition was determined
in an amino acid analyzer (Hitachi 8335-50), as described
previously (Suzuki et al. 1990).
SDS-PAGE and western blots
SDS-PAGE of the crude extracts and purified TC-14 was
carried out on 15 % acrylamide gel or 5-20 % gradient gel
containing 0.1% SDS in 0.375M Tris-HCl (pH8.8)
(Laemmli, 1970). The gels were transferred electrically to
nitrocellulose for 1.5 h at 30 volts.
Polyclonal antibody
Polyclonal anti-TC-14 antibodies were raised in rabbits. The
antiserum as well as non-immunized rabbit serum were
fractionated with 50% saturated ammonium sulfate. After
centrifugation (12000g, lOmin), a crude y-globulin fraction
was suspended in 20 ml of 20 mM phosphate buffer (pH7.4)
containing 0.15 M NaCl and dialyzed against the same buffer.
Total amount of proteins in the y-globulin fraction was
determined by the method of Lowry et al. (1951).
Immunohisto chemistry
Buds of various developmental stages and adult animals were
used. These animals were fixed either in Zamboni's fixative
(Zamboni and DeMartino, 1967) at 4°C for 30min or in
methanol at —20°C for 20 min followed by a rinse with ethanol
for the same duration. After dehydration, the specimens were
embedded in JB-4 plastic medium (Polysciences, Inc.) and
sectioned with glass knives at 2jan. Sections were mounted
serially on coverslips.
The blocking was carried out in 2 % dry milk or 2 % bovine
serum albumin (BSA) suspended in 20 mM Tris-buffered salt
solution (TBS containing 0.15 M NaCl, pH7.4) for 30 min.
Goat anti-rabbit IgG antibody labeled with horseradish
peroxidase was purchased from Zymed Laboratories. The
primary and secondary antibodies were diluted 2000-fold with
20 mM TBS containing 0.2 % dry milk or BSA. Sections were
reacted with the antibodies for 30min, followed by washing
with 0.1 % Tween 20. They were stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Graham and Karnovsky, 1966). As controls, sections were reacted with nonimmunized rabbit y-globulin or anti-TC-14 antibody absorbed
by antigen.
Electron microscopy
Specimens were prefixed in 2.5% glutaraldehyde in phosphate buffer (pH7.4) containing 8% sucrose for 2h in an ice
bath. After washing with the buffer, they were postfixed in
1% OsO4 solution for 2h. They were dehydrated and
embedded in Spurr low-viscosity resin (Spurr, 1969). Sections
were stained with uranyl acetate and Reynolds' lead citrate
(Reynolds, 1963), and observed with a JEOL JEM 100U
electron microscope.
Bioassay
Growing buds of 3-4 mm in length were extirpated from the
parental animal. They were incubated in Millipore-filtered sea
water (MFSW) containing 0.1 ^jg, 0.3 ^g, 1/igor lO^gml" 1 of
TC-14 and/or 5mM galactose, and were allowed to develop
for two days in the presence of streptomycin
(0.5xlO- 4 gUmr') and kanamycin (lxlO^gUmF 1 ). Bioassays were also done, using 5/zgml"1 or 10/igml"1 of antiTC-14 y-globulin fraction in the presence of antibiotics. As a
control, non-immunized rabbit y-globulin was used. Buds
treated were incubated in 1 mM colchicine for the last 12 h
before fixation in order to augment mitotic figures (Kawamura and Nakauchi, 1986a). They were fixed in Bouin's
fixative, dehydrated and embedded in paraffin. The specimens were sectioned at 5^m and stained with hematoxylin
and eosin.
Labeling of TC-14
In order to determine target cells of the lectin, lyophylized
powder of TC-14 (about 0.5 mg) was labeled with fluorescein
isothiocyanate (FITC, F-7250 Sigma) in an ice bath for 60 min
in 2 ml of a reaction mixture containing 50 mM sodium
bicarbonate buffer (pH9.0), 0.5 mg FITC, 0.1 M galactose and
0.5 mM CaCl2. The products were dialyzed against distilled
water. The lectin (0.5 mg) was also labeled with biotin. It was
dissolved in 0.5 ml of 0.1M sodium bicarbonate buffer
(pH8.5) and incubated with 0.1 mg of N-hydroxysuccinimidyl-6-(biotinamido)-hexanoate (biotin-NHS, Vector Laboratories) for 4h at room temperature. The reaction products
were dialyzed against 20mM Tris-HCl (pH7.4).
Sections were stained for 30 min with TC-14-FTTC or
biotinyl TC-14 diluted 500-fold with 20 HIM Tris-HCl (pH 7.2)
containing 10 mM CaCl2. In the case of biotinyl TC-14, they
were reacted secondarily with avidin-peroxidase (Vector
Laboratories). They were colored with DAB, as described
above. Blood smears were fixed in methanol at —20°C and
stained as above. As controls, specimens were stained in the
presence of either 10mM EDTA or 50 mM lactose.
Results
Appearance and localization of TC-14 during budding
A monomeric, 14X103 MT lectin is a major component
of Polyandrocarpa's 95 % ammonium acetate fraction
(PAM95). It shows the electrophoretic mobility equivalent to a 17 x 103 Mr protein on SDS-PAGE (Lane a of
Fig. 1A) (see Suzuki et al. 1990). After gel filtration
(Fig. IB), it was found in the fraction of PAM95-5
Budding-specific lectin in tunicates
997
kDa
0.2-
o
oo
0.1-
B
50
100
200
150
FRACTION NUMBER
Fig. 1. Purification of TC-14 from colonies of P. misakiensis (see Materials and methods). (A) Coomassie brilliant blue
staining after SDS-PAGE (a,b,d), and anti-TC-14 polyclonal antibody staining after western blotting to nitrocellulose
membrane (c,e). (a) Crude extracts; (b,c) PAM95-3 fraction after gel nitration; (d,e) TC-14 purified from PAM95-5 by
anion exchange chromatography (DE-32). (B) Gel filtration profile of PAM-95.
G
D
Fig. 2. Schematic illustration of bud formation and development in P. misakiensis. Each square corresponds to
photographs in Figs 3 and 4. (A) Bud primordium and adjacent parental mantle wall. (B) Growing bud. Parental epidermis
and atrial epithelium form the outer and inner epithelia of the bud. (C) Developing bud, one day after isolating from the
parent. Square shows the morphogenesis domain. (D) 2-day developing bud. Organ rudiments form at the proximal end
(square at the bottom). Another square shows a non-morphogenetic domain, a, atrial epithelium; b, blood cell; e,
epidermis; g, gut rudiment; i, inner epithelium; p, pharyngeal rudiment.
{Lane d of Fig. 1A). A polypeptide with the same
electrophoretic mobility was also found in PAM95-3
{Lane b of Fig. 1A). Rabbit anti-TC-14 polyclonal
antibody reacted with the respective bands from
PAM95-3 and PAM95-5 {Lanes c and e of Fig. 1A).
So, it appears that in living animals there are some
polymeric forms of TC-14 as well as the monomeric
one.
To provide the context for considering results of
immunohistochemistry, we describe briefly bud formation and development of P. misakiensis. The bud
primordium forms as an evagination of the parental
mantle wall, which consists of the epidermis and atrial
epithelium (Fig. 2A). It grows to form a double-walled
vesicle (Fig. 2B). The inner epithelial cells take a
squamous shape (Fig. 3A) and have a long Gj (Go)
998
K. Kawamura and others
Fig. 3. Cellular behaviors during the earliest stage of bud development. (A) Growing stage, the proximal end. Inner
epithelial cells are squamous. Hemoblasts cannot be recognized. Bar, 25/mi. (B) 36 h after isolation, the proximal end.
Inner epithelial cells are cuboidal and the nucleus becomes swollen. Hemoblasts are associated with the inner epithelium.
Bar, 25 fan. (C) 48 h after isolation. Arrowheads show mitotic figures. Note that the inner epithelium becomes temporarily
multilayered probably owing to hemoblast adhesion. Bar, 25 fan. (D) The proximal end of a 2-day developing bud. The gut
rudiment is established. Arrow shows hemoblast aggregation. Bar, 100/an. e, epidermis; g, gut rudiment; i, inner
epithelium; t, tunic
phase of cell cycle (Kawamura et al. 1988). After
isolation from the parent, the bud enters the developmental phase. In this work, all specimens were
encouraged to develop by extirpating them from the
parent with razor blades. The wound heals after about
10 h, the inner epithelial cells at the cut end become
thickened by 30 h (Figs 2C, 3B), and then the cells
enter the cell division cycle (Fig. 3C, Kawamura and
Nakauchi, 1986a; Kawamura et al. 1988). Together with
hemoblasts (about 6 fxm in diameter) with a prominent
nucleolus (Wright's nomenclature, 1981), they form
first the gut rudiment and then the pharyngeal rudiment
(Figs 2D, 3D, Kawamura and Nakauchi, 1991a). There
is experimental evidence showing that the DNA
replication of epithelial cells and epithelial transformation of hemoblasts are essential for establishing those
primary organ rudiments (Kawamura and Nakauchi,
1991&).
Anti-TC-14 antibody reacted with granules of the
inner epithelial cells at the primordial bud stage
(Figs 2A, 4A, 4B). Parental tissues around the bud
primordium did not have such granules (Fig. 4C). The
basal lamina was stained, but it was a non-specific
reaction, as shown later. As the bud grew, lectin-
Fig. 4. Expression of TC-14 in Polyandrocarpa buds
visualized by peroxydase-DAB (see also Fig. 1). A-D, H
and K were fixed in Zamboni's fixative and others in
alcohol. (A) Bud primordium. (B) Higher magnification of
the primordium. Arrows show positive granules in the
inner epithelium. (C) Parental tissues adjacent to the
primordium. There were no granules in the atrial (inner)
epithelium. (D) 1-day developing bud, the proximal end
(phase-contrast microscopy). Note that the granules are.
secreted in the mesenchymal space (arrows) and that the
ECM forms in situ (arrowhead). (E) 2-day developing bud,
morphogenesis domain. The ECM developed fully. Blood
cells and their periphery became lectin-positive
(arrowhead). (F) Higher magnification of blood cells,
granular leukocytes, which are characterized by large
granules in the cytoplasm. (G) An aggregate of hemoblasts
(arrow) and single hemoblasts associated with the ECM.
(H) Aggregating hemoblasts, hematoxylin-eosin staining.
(I) Non-morphogenetic domain of a 2Aiay-old bud. Only
the basal lamina was positive (arrowhead). No ECM was
observable. (J) Control staining of the morphogenesis
domain of a 2-day-old bud. The primary antibody was
absorbed by TC-14 before staining. The basal lamina still
stained (arrowhead). (K) Three-day-old bud. The granules
(arrows) were situated on the apical surface of the gut
rudiment and atrial epithelium. The ECM disappeared, a,
atrial epithelium; e, epidermis; g, gut rudiment; gr,
granular leukocyte; h, hemoblast; i, inner epithelium, mo,
morula cell; mu, muscle cell; t, tunic. Bars in A and E,
100/en. Others bars, 75 fan.
Budding-specific lectin in tunicates
inner epithelial cells began to secrete lectin granules
into the mesenchymal space at the proximal end of a
bud (Figs 2C, 4D arrows), and the extracellular matrix
(ECM) appeared (Fig. 4D arrowhead). The ECM
developed fully within two days of bud development
and extended its dendritic extremities to the epidermis
(Fig. 4E). Granular leukocytes were also stained
positively (Fig. 4E,F). This type of cell is characterized
positive granules were observed throughout the proximodistally elongated inner vesicle (not shown). Morula
cells, a kind of .vacuolated blood cell in tunicates
(Fig. 5A), reactedpositively after fixation in Zamboni's
fixative (Fig. 4B); but, not after alcohol fixation (cf.,
Fig. 4F). This was owing to endogenous peroxidase
activity (detailed account will be published elsewhere).
About one day after the onset of bud development,
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1000 K. Kawamura and others
by several acidophilic granules in the cytoplasm
(Fig. 5B, see Kawamura et al. 1988). Positive reaction
around the cell body was considered as coelomic lectin
secreted by granular leukocytes (Fig. 4F).
Hemoblasts were observed associated with the lectinpositive ECM at the morphogenesis domain (Fig. 4G).
They were identified by a prominent nucleolus in the
large nucleus (Fig. 4H). Cell boundaries of aggregated
hemoblasts could not be stained with anti-TC-14
antibody (Fig. 4G arrow), suggesting that TC-14 does
not play a role in cell-cell adhesion at this stage.
Neither the ECM nor hemoblast aggregation could be
seen at the non-morphogenesis domain of a 2-day
developing bud (Figs 2D, 41).
As controls, sections of 2-day-old buds were stained
with the antibody absorbed by TC-14 or with nonimmunized rabbit y-globulin. In both cases, no staining
could be observed in the inner epithelium, ECM and
granular leukocytes. However, the basal lamina was
still stained (Fig. 4J), showing that it was a non-specific
reaction.
From two days of bud development onward, the
granules of the inner epithelium and gut rudiment
became located at the apical surface of the cells
(Fig. 4K arrows). They were secreted into the lumen
and hardly observed in further developing buds and
zooids (see Fig. 4C). At the same time, the ECM
disappeared from the mesenchymal space (Fig. 4K).
Binding affinity of TC-14 for blood cells
Bud sections were stained with TC-14 labeled with
FTTC or biotin. Only undifferentiated blood cells
(precursors of hemoblasts, see Discussion) with diameters of 4-5 pan were stained heavily (Fig. 6A,B
arrowheads). These cells had a high nucleus: cytoplasm
ratio and had no vacuoles in the cytoplasm (Figs 5C,
6C). The plasma membrane and, especially, pseudopods showed a high affinity for TC-14 (Fig. 6C,D).
Unlike the hemoblast precursors, single or aggregated
mature hemoblasts did not bind TC-14 (Fig. 6E,F).
Galactose-binding plant lectin, peanut agglutinin
(PNA), showed a similar pattern of staining (not
shown). The binding was also found in biotinyl TC-14
(Fig. 6G). It was blocked completely by EDTA
(Fig. 6H).
Biotinyl TC-14 also stained the outline of morula cells
(Fig. 6G). This type of cell was sometimes observed in
association with the ECM or inner epithelium at the
morphogenesis domain (not shown), but their function
was unclear in the process of bud development.
Fig. 5. Electron micrographs of Polyandrocarpa blood
cells. (A) Morula cell. Vacuoles include electron-dense
materials. (B) Granular leukocyte. The cell is characterized
by several acidophilic granules and myelin figures.
(C) Hemoblast precursor. The nucleus is relatively large.
The cytoplasm is not specialized, g, granule, mi,
mitochondrion; my, myelin figure; n, nucleus; v, vacuole.
Bar, I/an.
Effect of TC-14 and anti-TC-14 antibody on bud
development
The gut rudiment is thefirstorgan to be established (cf.,
Fig. 3D). TC-14 did not accelerate the formation of this
organ rudiment (Table 1). At a concentration of
lO^gml" 1 , it reduced the average number of mitotic
figures in the morphogenesis domain by half compared
with non-treated controls (Table 1). It may result from
a non-specific effect of the protein. 1 /igml" 1 of TC-14
often enhanced the mitotic activity, although the
Budding-specific lectin in tunicates 1001
H
Fig. 6. Identification of lectin-binding cells. (A-F) Specimens were stained with TC-14 labeled with FITC. (G,H) Biotinyl
TC-14 staining followed by avidin-peroxidase and DAB. (A) Section of a growing bud, phase-contrast microscopy.
Arrowheads show precursors of hemoblast. (B) The same section as A, fluorescent microscopy. Only hemoblast precursors
emitted the fluorescence (arrowheads). (C) Hemoblast precursor smeared on glass slide, phase-contrast microscopy. It has
the large nucleus. (D) Fluorescent microscopy of the same specimen. Pseudopods were stained. (E) Morphogenesis domain
of a 2-day developing bud. (F) Fluorescent microscopy of the same section. Neither aggregated hemoblasts nor inner
epithelium emitted fluorescence. (G) Blood smear. The outline of hemoblast precursor was stained heavily (arrowhead).
(H) Blood smear stained in the presence of 10 mM EDTA. Hemoblast precursors were stained negatively (arrowheads), a,
aggregate of hemoblast; e, epidermis; i, inner epithelium; m, morula cell; n, nucleus. Bar, 25/an.
difference was not statistically significant. In most
specimens examined, the epithelial transformation of
hemoblasts was observed in the mesenchymal space.
Galactose, an inhibition sugar of the lectin, did not have
so drastic an effect on organogenesis (Table 1). The
mitotic activity was reduced, but in some cases high
activity was maintained (see 3.6±6.6 in Table 1).
Galactose blocked the epithelial transformation of
hemoblasts to large extent (Table 1, Fig. 7A). It should
be noted that the inner epithelium was single-layered,
in contrast to the untreated controls which have
transiently multilayered epithelium owing, partly, to
hemoblast adhesion (cf., Figs3B, 3C). This inhibition
became ineffective by the addition of TC-14 (Table 1,
Fig. 7B).
Buds were allowed to develop in the presence of
lO^gml" 1 of anti-TC-14 antibody (10 cases). In all
cases, small aggregates of hemoblasts were scattered in
the mesenchymal space (Fig. 7C arrows). They had no
pseudopods and were never associated with the inner
epithelium. There were no cases in which organogenesis took place. At a concentration of 5/igmF 1 , the
1002 K. Kawamura and others
Table 1. Effects of TC-14 and galactose on bud development in P. misakiensis
Treatment
Control
10/igml"1 TC-14
l/zgrnr 1 TC-14
0.3/igmP 1 TC-14
0.1/igmr 1 TC-14
5mM Gal
5mM Gal + 1/igml"1 TC-14
5mM Gal+OJ/igmP 1 TC-14
No. of
cases
% Formation
of gut rudiment
No. of mitotic
figures/section
16
10
11
10
6
10
7
5
62.5
60.0
63.6
40.0
50.0
40.0
42.9
40.0
9.1±2.8*
4.3±2.3
10.7±6.3
9.5±13.1
6.8±2.9
3.6±6.6
4.7±2.6
N.D.
% Formation of
aggregated hemoblasts
93.8
100
100
80.0
83.3
20.0
100
80.0
2-day-old buds were examined.
* The ranges show the limit of 95 %confidence.
Fig. 7. Results of bioassays in 2-day developing buds. (A) 5mM of galactose. This photograph shows an exceptional case in
which hemoblasts have aggregated. Note that the inner epithelium remains single-layered. Arrowhead shows a mitotic
figure. Bar, 25 /an. (B) 5ITLM of galactose and 0.3/igml"1 of TC-14. Some hemoblasts extend pseudopods to the inner
epithelium. Arrowheads show mitotic figures. Bar, 25 /an. (C) 10/tgmF 1 of anti-TC-14 antibody, differential interference
microscopy. Arrows show small aggregates without pseudopods. Bar, 50/an. (D) 5/igml"1 of antibody, differential
interference microscopy. Thick arrows show large aggregates with pseudopods. Thin arrow shows an aggregate without
pseudopods. Bar, 50 fim. h, hemoblast; i, inner epithelium.
antibody blocked the organogenesis in 10 cases out of
15. In the mesenchymal space of most cases, some
aggregates of hemoblasts extended pseudopods toward
the thickened epithelium at the morphogenesis domain
(Fig. 7D thick arrows), as observed in intact buds. But,
there existed another kind of aggregate, which was far
distant from the inner epithelium and had a smooth
outline without any pseudopods (Fig. 7D thin arrow).
Non-immunized rabbit y-globulin had no such deteriorative effect on bud development (not shown).
Discussion
Monomeric and polymeric forms of TC-14
Recently, a monomeric, 14xlO3A/r protein has been
purified from the hemolymph of the tunicate, Poly an-
Budding-specific lectin in tunicates 1003
drocarpa misakiensis (Suzuki et al. 1990, and our
unpublished observation). Its characterization and
amino acid sequencing have shown that it belongs to a
calcium-dependent (C-type), galactose-binding lectin
that has four conservative Cys residues forming two
intrachain disulfide bridges (cf. Drickamer, 1988).
Galactose-binding lectins of hemolymph origin have
been extracted previously from tunicates, Halocynthia
roretzi (Yokosawa et al. 1982), Phallusia mamillata
(Parrinello and Canicatti, 1983), Didemnum candidum
(Vasta et al. 1986), Ascidia malaca (Parrinello and
Arizza, 1988) and Botrylloides leachii (Schluter and Ey,
1989). Although some of them require Ca 2+ to exert
carbohydrate-binding activity, it is uncertain whether
they belong to a C-type lectin family, because the
information is lacking about the primary structure of
proteins except D. candidum lectin (DCL-1) (Vasta et
al. 1986). No reports are available for the expression,
localization and possible role of tunicate lectins in
relation to embryonic or postembryonic development.
The present work has shown for the first time that, in P.
misakiensis, the tunicate-derived C-type lectin (TC-14)
is induced specifically during budding, although there
might be a trace of lectin in the hemolymph of adult
animals (not shown).
Rabbit anti-TC-14 polyclonal antibody reacted with
granules in the bud's inner epithelium from the earliest
stage of bud formation until two days of bud
development. It was during this stage of bud development that the granules were secreted into the mesenchymal space where the ECM is formed. The
antibody also reacted with granular leukocytes, a kind
of blood cell in the hemolymph (see Wright, 1981) of a
developing bud, suggesting that the lectin is of both
epithelial and blood cell origins.
Immunoblot analyses suggested that TC-14 existed in
both monomeric and polymeric forms. It seems likely
that the humoral antigen secreted by granular leukocytes is identical to the monomeric lectin eluted as
PAM95-5 fraction in gel filtration chromatography. It
is possible that the antigen remains around the blood
cells, because there is no blood flow in a bud until it
develops into a functional animal. However, the ECM
secreted by the inner epithelium may include molecular
complexes consisting of TC-14 and other components,
as seen in the PAM95-3 fraction. Alternatively, the
ECM may consist of high molecular weight proteins(s)
with lectin domains. For example, the lymphocytehoming receptor or platelet-endothelium adhesion
molecule (GMP-140) in mammals has a C-type lectin
domain at the N terminus (Lasky et al. 1989; Johnston et
al. 1989). Fibroblast proteoglycan core protein is also
known to contain the lectin domain (Krusius et al.
1987). Now, we are trying to purify the functional ECM
from developing buds.
Cytochemically, inner epithelial cells of a bud are
different from the parental atrial epithelium, as they
carry lectin granules at the very start of budding.
Interestingly, lectin granules were also induced in the
process of regeneration (Kawamura, in preparation).
They were found only in regenerating organ rudiments
that originated from the inner (atrial) epithelium. So,
the lectin granules may be essential for both budding
and regeneration of Polyandrocarpa.
The role of TC-14 in budding
As already discussed, both monomeric and polymeric
forms of TC-14 seem to exist in Polyandrocarpa buds
and it is possible that they might exert different
physiological activities. A monomeric TC-14 bound to
small undifferentiated blood cells with high affinity in a
calcium-dependent manner. The staining pattern was
similar to that of PNA (a galactose-binding phytohemagglutinin). However, single or aggregated hemoblasts did not show such an affinity, suggesting that they
might lose galactosyl conjugate(s) from the plasma
membrane in the process of epithelial transformation.
In some budding tunicates, many kinds of somatic
tissues and germ cells differentiate from blood cells with
a prominent nucleolus, suggesting pluripotent stem
cells in the hemolymph (for review, Kawamura and
Nakauchi, 1991a). In P. misakiensis, as shown in this
work, hemoblasts (Wright's nomenclature, 1981) take
part in histogenesis at the earliest stage of bud
development. We considered them to be derived from
smaller undifferentiated blood cells. One of the
grounds for supporting this is that hemoblasts with
typical morphology were rarely observed before the
epithelial transformation stage (cf., Figs 3A, 3C).
There is a great possibility that hemoblasts may come
from other type(s) of cell through, for example,
dedifferentiation. Our autoradiographic study (Kawamura et al. 1988) has shown that, in the blood, small
undifferentiated cells incorporate [3H]thymidine actively, but we have never observed mitotic figures in
those smaller cells. We suggested that smaller and
larger undifferentiated cells might merely represent
different phases of their cell cycle (Kawamura et al.
1988). This is another reason for considering the smaller
cell as a hemoblast precursor. Of course, our grounds
are inconclusive because the classification of blood cells
is based only on morphological differences, as in other
tunicates (Wright, 1981). Molecular markers that
recognize particular subpopulations of undifferentiated
cells are required.
In developing buds, hemoblasts and their precursors
were often observed in contact with the lectin-positive
ECM. The ECM disappeared at the same stage as the
termination of hemoblast aggregation. TC-14 competed
with galactose, an inhibition sugar of the lectin, for
hemoblast aggregation. Anti-TC-14 antibody prevented
hemoblasts from forming a large aggregate, extending
pseudopods and being associated with the inner
epithelium. These results have strongly suggested that
TC-14 serves as a component of ECM to direct
hemoblast precursors to the inner epithelium. However, mature hemoblasts should interact with other
component(s) of the ECM as they had no affinity for
monomeric TC-14. The antibody did not stain cell
boundaries of aggregated hemoblasts, nor did it affect
cell aggregation as such in the bioassay, indicating that
TC-14 plays no part in cell-cell adhesion at this stage.
1004 K. Kawamura and others
The Polyandrocarpa ECM is similar to tenascin in its
pattern of expression and its possible role. Tenascin is
expressed at very limited areas during fetal development of mammals (Chiquet-Ehrismann et al. 1986). It
appears to be involved in epithelial-mesenchymal
interactions during embryogenesis (Crossin et al. 1986;
Aufderheide et al. 1987; Vainio et al. 1989) and
regeneration (Mackie et al. 1988; Arsanto et al. 1990).
We do not know whether the Polyandrocarpa ECM
contains tenascin and/or other components such as
fibronectin, laminin and vitronectin. Some of these
components have a unique amino acid sequence
essential for cell-ECM adhesion; for example, ArgGly-Asp-Ser in fibronectin (Pierschbacher and Ruoslahti, 1984) and Arg-Gly-Asp-Val in vitronectin (Suzuki
et al. 1985). TC-14 does not have such a RGDX
structure. Among C-type lectins, only echinoidin from
sea urchin has been shown to possess a single RGD
domain (Giga et al. 1987).
Mature hemoblasts show a high mitotic activity
(Kawamura and Nakauchi, 1986a, 1991a; Kawamura et
al. 1988). This activity should not be attributed to a
monomeric lectin, as the extrinsic lectin did not act as
mitogen in spite of enough time to work through the cut
surface. Of course, it is possible that polymeric TC-14
and/or other components of the ECM may trigger cell
cycling. For example, fibroblast proteoglycan core
protein contains epidermal growth factor-like repeats,
following the C-type lectin domain (Krusius et al. 1987).
The fly lectin from Sarcophaga peregrina can be
induced in instars by surgical injury (Komano et al.
1980). It plays a role in defense mechanisms (Komano et
al. 1983). Monomeric TC-14 may serve as opsonin, as
suggested in other tunicate lectins (see Vasta et al.
1986). This function is important, because Polyandrocarpa buds may possibly be attacked by foreign
materials during early development following extirpation from the parent. Interestingly, TC-14 could not
be found serologically or biochemically in Botrylloides
and Symplegma, although their colonies propagate by
means of budding (Berrill, 1940, 1941). Unlike Polyandrocarpa, their buds remain in contact with the parental
colony through vascular networks during bud development (Izzard, 1973; Kawamura and Nakauchi, 1986i>),
which may protect the buds from bacterial infection.
We thank Drs Kozoh Utsumi and Eisuke Satoh of Kochi
Medical University for helping us prepare the rabbit anti-TC14 polyclonal antibody. Thanks are also due to Mr T.
Yamamoto and Ms K. Kikuchi for their assistance throughout
the work. This work was supported in part by a Grant-in-Aid
from the Ministry of Education, Science and Culture, Japan.
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