Localization of Laminin in the Subepidermal Basal Lamina of the Planarian Dugesia japonica

6 Pages·1992·2.7 MB·English

by  I Hori

Reference: Biol. Bull. 183: 78-83. (Auaist. 1992)
Localization of Laminin in the Subepidermal Basal
Lamina of the Planarian Dugesia japonica
ISAO HORI
Department ofBiology, Kanazawa Medical University, Uchinada, Ishikawa 920-02, Japan
Abstract. The planarian subepidermal basal lamina The subepidermal basal lamina ofplanarians is struc-
consists of three structural elements: namely, an elec- turally quite different from that of higher animals. The
tronlucentzone,alimitinglayer,andamicrofibrillarlayer. planarian basal lamina is characterized by an electron-
Ultrastructural observations following ruthenium red lucent zone of irregular shape and a microfibrillar layer
staining were in agreement with previously published re- (Pedersen, 1966; Bedini and Papi, 1974; Rieger. 1981;
ports. The staining clearly revealed positive material on Tyler, 1984). Extracellular matrix components are con-
the limiting layer and the individual microfibrils. The centrated mainly in the subepidermal basal lamina. Our
limitinglayerwascontinuousand separated the electron- previous studies using electron microscopy and autora-
lucent zone from the microfibrillar layer. The distribution diography haveshown that thebasal lamina isregenerated
oflaminin. a noncollagenous basal lamina glycoprotein, by interaction between the wound epidermis and differ-
wasdetermined forthe planarian basal lamina usingvar- entiating myoblasts (Hori, 1979. 1980). Although the
ious methods of immunocytochemistry. The reactive morphological changes and behaviorofregenerative cells
substance appeared only on the limiting layer and in the havebeen studied well in planarians, cytochemical infor-
electronlucent zone alongthe limiting layer. The reactive mation about its molecular components is not readily
products on the limiting layer appeared discontinuous. available. Thisstudyexamines by immunocytochemistry
They were also present in the regenerated basal lamina, the localization of laminin, a kind of extracellular gly-
though the reactivity was a little weaker than in that of coprotein, in the planarian basal lamina and compares it
intact animals. Localization oflaminin in the planarian with the results ofruthenium red staining.
subepidermal basal lamina is discussed in comparison
with that ofthe basal lamina ofvertebrates. Materials and Methods
Animals
Introduction
The freshwater planarian Dugesia japonica was used
mm
Thebasal lamina orbasement membrane is responsible in this study. Healthy worms (10-15 in length) were
for the maintenance ofepithelial integrity, and its con- selected and maintained without food for a week in our
dition determines cell behavior during wound repair laboratory. Intact tissues from the region between theau-
(Schittny el ai, 1988). Consequently, a variety ofstudies ricles and pharynx were dissected from six worms. Re-
have been carried out with regard to each component of generates ofsix other worms were obtained by decapita-
the basal lamina. In view ofcurrent knowledge, its mo- tion. Specimenswereallowedtoregenerate for4or6days
lecularcomponentsaretype IV collagen, sulfated proteo- in tap water at 18C.
glycans, fibrono; . and laminin, and their distribution
can be associated v-ith the function ofthe epithelial cells Electron microscopyfor ruthenium redstaining
(Farquhar, 1981; La e el ai. 1982; Newgreen, 1984;
Simoelal., 1991). Forthedetection ofextracellularproteoglycans, tissues
were processed forruthenium red (RR) stainingaccording
to Luft (1971). They were fixed for 60 min in 1.2% glu-
Received 21 October 1991; 18 May 1992. taraldehyde at 4C, and postfixed for 3 h in 1% osmium
78
LOCALIZATION OF LAMININ IN PLANARIAN 79
tetroxide at roMom temperature. Both fixatives were buf- 2 h. Then they were treated for one minute with DAB.
fered with 0.1 sodium cacodylate (pH 7.4) containing Afterbeingrinsed with TBSanddistilled water, theywere
O.lr; ruthenium red (TAAB). Fixation wascarried out in observed without counterstaining.
a dark room. Specimens were dehydrated with a graded (2) PAG method. Another group ofsections were im-
ethanol series and embedded in Epon 812. Thin sections munostained by Protein A-gold (PAG) reagents according
were counterstained with uranyl acetate and lead citrate, to Roth et al. ( 1978). Thin sections were rinsed for 5 min
and then photographed with a Hitachi H-500 electron with PBS and preincubated for 20 min with PBS BSA
microscope. (1%). Theywere then incubated overnight with polyclonal
rabbit anti-laminin antibody (Sanbio)diluted 1:50-1:500
Immunohistochemistry in PBS BSA at 4C. After being rinsed with PBS, they
Tissues were fixed in Zamboni fixative (Zamboni and were incubated for 45 min with Protein A-gold colloids
DeMartino, 1967) for 4 h at 4C. After being rinsed for (Funakoshi) diluted 1:10 in PBS. They were rinsed with
30 min with cold TBS (pH 7.5), they were dehydrated PBS and distilled water and stained with uranyl acetate
with graded ethanols and embedded in paraffin. The im- and lead citrate.
munoreaction wascarried outaccording to the ABCom- Results
plex method (Hsu el a/.. 1981). Deparaffinized sections
(3-5 ^m thick) were incubated for 5 min with 3% hydro- Morphological aspects ofthe basal lamina
gen peroxideand incubated for20 min with bovine serum The general architecture of the subepidermal basal
(normal) diluted 1:5 in TBS. The sections were then in- lamina stained with ruthenium red is shown in Figure 1.
cubated overnight at 4C with rabbit anti-laminin anti- The basal lamina separates the single-layered epidermis
body (Sanbio) diluted 1:50 in TBS. Forcontrols, sections from underlying muscle fibers. It is divided into three
were incubated with TBS. All the sections were then in- structural elements; namely, an electronlucent zone sur-
cubated for 30 min with biotinylated swine anti-rabbit rounded by the basal cytoplasmic processes ofepidermal
immunoglobulins (DAKO) diluted 1:300 in TBS, and cells, a microfibrillar layer including a number ofmicro-
then again for 30 min with avidin andbiotinylated horse- fibrils, and a limiting layer separating these elements.
radish peroxidase reagents (AB Complex/HRP, DAKO). Theelectronlucent zonechanges itsshapeaccordingto
After being rinsed with TBS they were incubated for 5 basal alterations of the epidermal cells. In most areas,
min with 3.3 diaminobenzidine tetra-hydrochloride specific filaments are seen producing a meshwork within
(DAB). this zone. In the cross-sectioned basal lamina, these fila-
mentsoften run parallel to the limiting layer(Fig. 1). The
Tissueprocessingfor immunoelectron microscopy limitinglayerislinearand uniform, identical tothelamina
densa ofthe vertebrate basal lamina. The limiting layer
Tissueswere fixed for2Mh in 1% glutaraldehydeand 2% hadastrongaffinity for RR(Fig. 1), andthisdye revealed
paraformaldehyde in 0.1 phosphate buffer (pH 7.4) at the continuous nature ofthe layer. Each basal process of
0C. Tissue pieces were rinsed for 30 min in the buffer, an epidermal cell characteristically develops a hemides-
dehydrated in graded ethanols at progressively lowered mosome atitstip. andtheepidermal cellscomein contact
temperature(down to -25C), andembedded in Lowicryl with the limiting layer through such hemidesmosomes.
HM20 accordingto Carlemalm et al. (1982). The samples When RR-treated specimens were viewed at a higher
were transferred to pure resin at -35C and maintained magnification, the RR-positive material was particularly
povoelrynmiegrhit.zeCdapfsorulaetslfeialslted2w4ithhufnrdeeshrpUreVcoloilgehtdartes-in3w5eCr.e eTvhiedemnitcroatfibtrhiellhaermliaydeerscmoonsstoimtautlesrmegoisotnsof(tFhige.ba1s,alinlseatm)-.
They were then further hardened at room temperature ina. It is underlain with plasma membranes of muscle
for 2 days. Thin sections were mounted on collodion- cells. Itsthickness varies from 1 to 4 j/m. Viewed in cross
supported nickel gridsand immunostained by the follow- section, most ofmicrofibrilswerecoated with RR-positive
ing(1t)woABmeCthmoedtsh.od. The sections were preincubated for matTehreiacly(tFoipgl.as1,miicnspeto)r.tions ofsome kind ofparenchyma!
30 min with TBS containing 1% bovine serum albumin gland cells are often seen intruding into the epidermal
(BSA). Then they were incubated overnight with poly- layer. Therefore theircytoplasmic portions, including se-
clonal rabbit anti-laminin antibody(Sanbio)diluted 1:50- cretory granules, can also be seen within the microfibrillar
1:200 in TBSat4C. Forcontrols, sectionswereincubated layer (Fig. 1).
with TBS. After being rinsed with TBS, they were incu-
bated for30 min with affinity-isolated, biotinylated swine Immunohistochemical observations
anti-rabbit immunoglobulins (DAKO) diluted 1:300 in Thedistribution oflaminin was first investigated at the
TBS. followed by incubation with AB Complex/HRP for light microscopic level by indirect immunohistochem-
80 I. HORI
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x< a%-
-ii^ *il
Figure 1. Low magnification \iew to demonstrate ruthenium red-positive areas in the subepidermal
basallamina.Threestructuralelementsofthebasallaminaareevident.Arrowheadsindicatethecytoplasmic
portion ofa gland cell. Scale bar = 2 nm. Inset: High magnification view ofthe hemidesmosomal region.
Ruthenium red staining. The limiting layer is stained clearly. Arrowheads indicate positive material sur-
rounding microfibrils. Scale bar = 0.5 ^m. Abbreviations: Ep, epidermal cell; P. cytoplasmic process; E,
electronlucentzone; L. limitinglayer: M. microfibrillarlayer; Mf, muscle fiber; H. hemidesmosome.
istry. Denv its. indicating binding ofthe antibody Immunoelectron microscopic observations
to laminin. i served on the basal lamina. The de-
posits were esp prominent on its epidermal side To resolve the localization oflaminin in the basal lam-
(Fig. 2). Simila, swerealsoseen inthesame region ina, immunoelectron microscopywasapplied to Lowicryl-
ofthe 6-day regeno 4). The control sections in- embedded thin sections. The ABC method showed dense
cubated with TBS should no reactivedepositsin thebasal immunoreactivity on the limiting layer and on a part of
lamina (Fig. 3). the electronlucent zone along the limiting layer (Fig. 5).
LOCALIZATION OF LAMININ IN PLANARIAN 81
arian basal lamina may not reflect the phylogenetic po-
sition ofanimals, but ratherunique functional significance
ofthe basal lamina (Lindroos, 1991). Variations in thick-
nessmainly relatetotheextenttowhichthemicrofibrillar
layer is developed (Sluys, 1989). For example, Rhabdo-
coela have no microfibrils (Holt and Metrick, 1975),
whereas some species ofmarine triclad have well-organ-
ized microfibrillar layer (MacRae, 1965). The basal lam-
ina ofthe former is similar to that ofmammals, and the
. basal lamina ofthe latter is similar to that ofamphibian
larvae (Hay and Revel. 1963). In spite ofsuch variations,
thelimitinglayercan beseen in thebasal laminaeofmost
turbellarians.
The results of RR staining provide additional infor-
mation about the structure and chemical nature of the
basal lamina. Because wecould demonstratethe RR-pos-
itive material in the planarian basal lamina, the limiting
layer probably includes sulfated proteoglycans. This isin
agreement with the results of experiments on human
basement membranes (Horiguchi et a/., 1989). The elec-
tronlucent zone is occupied by reticular filaments. In our
Ep;
samples, these filaments appear as a meshwork running
<
paralleltothelimitinglayer. Suchameshworkis,however,
notalwaysseenin theplanarian basal lamina(Skaer, 1961;
Rieger. 1981). The variation in the structure ofreticular
filaments may depend, not only on their chemical prop-
erties, but also on methods oftissue preparation.
Laminin is one ofthe main components ofbasal lam-
inae. Its distribution has been examined in many verte-
Figures 2-4. Immunohistochemical localization oflaminin in the brates (Jacob ct al. 1991). Because the antigenic deter-
basal lamina. Reactive productsare distributed alongthe basal lamina
(arrowheads).Scalebar= 20^m. Figure2.Intact. Figure3.Intact:con- minants oflaminin are not species-specific(Foidart elal..
trol. Figure4.Six-day regenerate. Ep. epidermal cell. 1980). we have used rabbitanti-laminin antibodiestode-
tect the localization of laminin in the planarian basal
lamina. Immunohistochemica! stainingdemonstratedthat
In control sections, no reactive substances were seen in the distribution of laminin is restricted to the limiting
the basal lamina (Fig. 6). The stained material on the layerand a part ofthe electronlucent zone. In contrast to
limiting layer appeared discontinuous. In the 6-day re- the RR-positive proteoglycans. laminin is distributed on
generate, when newly formed basal lamina appeared, the thelimitinglayerdiscontinuously. Thisfindingisinaccord
reactivity of the limiting layer was also evident, though with the observations of Lindroos and Still (1988), who
the staining was slightly weak (data not shown). In both noted irregulardepositsoflaminin beneath theepithelium
cases,therewerenoreactiveproductsinthemicrofibrillar ofPolycelis nigra.
layer. The localization of laminin on vertebrate basement
The localization oflaminin in thebasal laminaofintact membranes differs from one report to the next: i.e.. it is
and regenerating worms was compared by the PAG localized in the entire region ofthe basal lamina (Inoue.
method. The labeling of laminin resulted in significant 1989); in the lamina densa (Laurie el al.. 1982); in the
deposits in the limiting layers ofboth intact and regen- lamina lucida (Foidart et al.. 1980; Madri et al.. 1980);
erated tissues(Figs. 7, 8). Fewergold particleswere in the and at thejunction between the laminadensaand lamina
microfibrillar layer than in the limiting layer. lucida (Schittny et al.. 1988). Laminin is known to play
many important roles in various phenomena, but nothing
Discussion has been reported about the significance of such varied
distributions.
The planarian subepidermal basal lamina has varying Planarian epidermal cells have no ability to proliferate
degrees ofdevelopment in different species (Bedini and mitotically so that certain parenchyma! cells migrate into
Papi, 1974). Theoccurrenceand constitution ofthe plan- the epidermis to produce its cellular succession. In my
82 I. HORI
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'-'*-''- . '.''
.
M
8
Mgures 5-6. Localization oflaminin by ABC method of Lowicnl-embedded sections. The limiting
.siid a part ofthe electronlucent zone are stained. Arrowhead indicates a discontinuous portion of
reacti\v products. Figure5. Intact. Scalebar = 0.5 /jm. Figure6.Control. Scalebar = 0.5 ^m.
Figures 7-8. Localization oflaminin by immunogold labeling of Lowicryl-embedded sections. Gold
particlesareseer associated with the limitinglayer. Figure 7. Intact. Scalebar = I j/m. Figure8. Four-day
regenerate. ScaL- bar = 0.5 ^m. E, electronlucent zone; L, limitinglayer; M, micronbrillar layer.
LOCALIZATION OF LAMININ IN PLANARIAN 83
previous study. I ascertained that rhabdite-forming cells togenyofthecoreproteinofahumansulfateproteoglycaninhuman
are differentiated from regenerative cells and contribute skin and otherbasement membranes. / Hislochem. Cytochem. 37:
961-970.
to the cellularsuccession, both in intact and regenerating Hsu, S-M., L. Raine. and II. Fanger. 1981. A comparative study of
planarians (Hori, 1978). Moreover, other kinds ofparen- theperoxidase-antiperoxidasemethodandanavidin-biotincomplex
chymal gland cells usually extend their cytoplasmic pro- methodforstudyingpolypeptidehormoneswith radioimmunoassay
cesses, which contain secretory granules, into the epider- antibodies.Am. J Clm. Palhol. 75: 734-738.
mal layer (Tyler, 1984). Thus one can assume that the Inoue,S. 1989. Infrastructureofbasementmembranes. Int.Rev. Cylol.
117: 57-98.
planarian basal lamina probably provides a microenvi- Jacob,M.,B.Christ,H.J.Jacob,and R.E.Poelmann. 1991. Therole
ronment that guidessuch cell movement from the paren- offihronectinandlaminin indevelopmentandmigrationoftheavian
chyma to the epidermis. Recent studies suggest that Wolftian duct with referencetosomitogenesis. Anal. Embryo/. 183:
changes of laminin accumulation affect various cell be- 385-395.
haviors, such as cell movement (Simo et ai, 1991), cell kubootfal,aYm.i,nHi.nKa.ndKlbeaisnemamne,ntG.mRe.mMbarratnine,ianndthTe.mJo.rLpahwolleo)gi.c1a9l88d.iffeRroelne-
differentiation (Kubota et ai, 1988). interaction between tiation ofhuman endothelial cells into capillary-like structures. J.
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is also a complex process including extracellular matrix oftype IV collagen, laminin. heparan sulfate proteoglycan, and fi-
bronectin tothebasal laminaofbasement membranes. J. CellBiol.
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tin, anotherextracellularglycoprotein, isdetectedaround l.indroos, P. 1991. Aspects on the extracellular matrix and protone-
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butthedatawerebasedonlyontheimmunocytochemistry phrl/obotliriitmdenclntiaim. Pp.6-53inThesis. AboAkademisKo-
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requirethatexperimentsbecarried out //; vitrotoexamine LindProoloysc,eliP.s,naingdraM(.TurJb.elSltailrli.a,19T8r8i.cladiEdxat)r.acFenlrltuslcahr.mZaotorli.x3c6o:mp15o7n-e1n6t2s. in
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