Extracellular matrix (mesoglea) of Hydra vulgaris

DEVELOPMENTAL

148,

BIOLOGY

481-494

(1!+91)

Extracellular

Matrix I. Isolation

(Mesoglea)

of Hydra vulgar-is

and Characterization

MICHAEL P. SARRAS, JR.,**~ MICHAEL E. MADDEN,**’ XIAOMING ZHANG,* SRIPAD GuNwAR,t JACQUELYN K. HUFF,* AND BILLY G. HUDSON?

Hydrozoans such as Hydra wlgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to d&ermine what extracellular matrix (ECM) components are associated with Hydra mesoglea Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocy-tochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of Iibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (ai subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM. c 1991 Academic Press, I~C. INTRODIJCTION

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. They are considered one of the first multicellular animal groups to develop an extracellular matrix (ECM) boundary zone between two functionally distinct tissue layers, and they have competed successfully throughout evolution with this rather simplified body structure (Field et oh, 1988; Margulis and Schwartz, 1988). Early ultrastructural studies by Fawcett (1961) indicated similarities between the mesoglea of Hydra and vertebrate basement membrane (defined as a specialized form of ECM associated with epithelium). Classification of Hydra mesoglea as a primitive basement membrane was later supported by (1) biochemical studies on isolated mesoglea (Shostak, et ul., 1965; Barzansky and Lenhoff, 1974; Barzansky et ab, 1975), (2) pulse-chase autoradiographic studies using [3H]proline as a radiotracer (Hausman and Burnett, 1971), and (3) functional studies related to the cell binding properties of isolated mesoglea (Day and 1 To whom correspondence ’ Current address: Dept. Fort Hays State University,

should be addressed. of Biological Sciences and Allied Health, 600 Park St., Hays, KS 67601-4099. 481

Lenhoff, 1981). However, except for indirect evidence suggesting the presence of collagen-like molecules in Hydra mesoglea (Shostak et al., 1965; Barzansky and Lenhoff, 1974; Barzansky et ul., 1975) and one recent symposium report regarding Anthomedusae laminin (Beck et ul., 1989), previous studies have not dcmonstrated the presence of the broad spectrum of major ECM molecules characteristic of vertebrate and higher invertebrate (e.g., sea urchins, nematodes, and arthropods) basement membranes (i.e., type IV collagen, laminin, and heparan sulfate proteoglycan) (see reviews by LeBlond and Inoue, 1989; McDonald, 1989; Timpl, 1989). In fact, analysis of mesoglea of another class of Cnidaria, Scyphozoa (jellyfish), has indicated the presence of a type V-like collagen molecule (Miura and Kimura, 1985) which is a fibril-forming type typically associated with vertebrate connective tissues (Niyibizi et al., 1984). While the epithelial origin (Hausman and Burnett, 1971) of Hydra mesoglea might suggest that this ECM is a primitive basement membrane, existing data are in fact inconclusive on this matter. Given the importance of basement membrane and ECM components in cell signaling events during development (see reviews by Ekblom et ul., 1986; McDonald, 1989; Timpl, 1989), the present study has focused on an analysis of Hydra mesoglea with the broader objective of utilizing Hydra as an in viz10 model for study of ma0012.1606191 Copyright All ngbts

$3.00

ii’ 1991 by Academic Press, Inc of reproduction I” any form reservrd.

482

DEVELOPMENTALBIOLOGY V0~~~~148,1991

trix/cell interactions. Hydra lends itself to such studies because of (1) its extensive cell turnover dynamics related to growth maintenance mechanisms (Campbell, 1967a,b; David and Campbell, 1972; Bosch and David, 1984) and (2) its great regenerative capacity (Gierer e2 ab, 1972; Javois et ah, 1988). With this in mind, the objective of the current study was to determine what ECM components are associated with Hydra mesoglea. The potential function of mesoglea components during head regeneration in Hydra is discussed in the accompanying paper of this series (Sarras et al., 1991a). An earlier report of some these findings was published in abstract form (Sarras et al., 1988). MATERIALS AND METHODS Large Scale Culture

of Hydra

For isolation of sufficient quantities of mesoglea for biochemical analyses, Hydra vulgaris (formerly classified as Hydra attenuata) were maintained in large scale cultures using procedures modified from Lenhoff (1983). Hydra medium (HM) containing 1.0 mM CaCl,, 1.0 mM NaHCO,, 0.25 mM MgCl,, and 0.01 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5, was used. These procedures involved construction of plastic racks for use in 15 gallon fish aquariums fitted with bottom and top water circulation systems. Each rack was designed to hold 22 removable vertical plates (23 X 53 cm) which were used as a substrate for attachment of growing Hydra. Racks were removed from the aquarium and placed in a plastic holding tank during feeding or when plates were to be periodically cleaned. For feeding, brine shrimp (Artemia salinas, Wards Natural Science Establishment, Inc., Rochester, NY) were hatched in a 0.2 M NaCl solution containing 40 pg/ml gentamicin using 1 liter separatory funnels (VWR Scientific, St. Louis, MO, No. 30356-802) with constant aeration. Feeding was performed every other day, although higher growth rates could be achieved with daily feeding when required. Growing plates were removed from the aquarium rack and placed in specially designed trays (25 X 55 cm) for feeding and subsequent removal of excess shrimp. After return to the holding tank, the rack was returned to the aquarium following a 6- to 8-hr period to allow for discharge of undigested material, Detached Hydra were collected from the holding tank and used for additional seeding of plates or frozen for subsequent use in biochemical extractions. Using two such aquarium systems up to 30-50 ml packed volume of Hydra can be obtained per month. Detailed procedures for the construction and use of this large scale culture system can be obtained from the author (M.P.S.) upon request.

Immunocytochemistry

Hydra mesoglea was screened for the presence of various extracellular matrix components using indirect immunocytochemistry at both the light and electron microscopic (TEM) levels. The antiserum used in this study included: rabbit anti-human plasma fibronectin (ICN ImmunoBiologicals, Lisle, IL); rabbit anti-EHS cell laminin (GIBCO Laboratories, Gaithersburg, MD); rabbit anti-mouse placental laminin (provided by Dr. M. Soares, Soares, et ab, 1988); rabbit anti-human placenta type IV collagen (Chemicon International, El Segundo, CA); rabbit anti-bovine glomerular basement membrane (GBM) type IV collagen (specific for the noncollagenous domain unique to type IV collagen) (provided by Dr. B. G. Hudson, Butkowski et ah, 1987); and anti-rat GBM heparan sulfate proteoglycan (specific for the core protein of heparan sulfate proteoglycan) (provided by Dr. Y. S. Kanwar, Makino et al., 1986). Antiserum was used for the immunocytochemical and Western blot studies described in this paper. For immunofluorescence, appropriate fluorescein isothiocyanate (FITC)coupled secondary antibodies were obtained from ZymedLaboratories (San Francisco, CA). For TEM studies, a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA)/streptavidin-gold (15 nm) (Jansen Pharmaceutics sold through Ted Pella, Inc., Tustin, CA) amplification system was used. Immunojluorescence. Indirect immunofluorescence of Hydra was performed on frozen sections and whole mounts. In both cases, Hydra were fixed with Lavdowsky’s fixative (Gurr, 1962) for 2-4 hr or overnight at 4°C prior to antibody fixative. For frozen sectioning, Hydra were immersed in OCT compound (Miles Laboratories, Elkhart, IN) and frozen in liquid nitrogen for cryostat sectioning. Eight to ten-micrometer sections were placed on glass slides and incubated with primary and FITC-coupled secondary antibodies as previous described (Sarras et al., 1986). For whole mount procedures, fixed Hydra were immediately incubated with primary and FITC-coupled secondary antibody as described by Dunne et al, (1985). For both frozen sections and whole mounts, controls were run with preimmune serum, nonimmune serum, or in the presence of FITCcoupled secondary antibody alone. Specimens were viewed on a Leitz photomicroscope fitted with an epifluorescence attachment. Transmission electron microscopy immunocytochemistry. For ultrastructural localization of ECM compo-

nents, Hydra were fixed in a 2% formaldehyde (made freshly from paraformaldehyde)/0.2% glutaraldehyde solution for 90 min at room temperature. After washing, specimens were en block stained with 0.5% uranyl magnesium acetate overnight at 4°C and then processed into

SARRASETAL.

Extracellular

Spurr’s embedding medium (Spurr, 1969). Antibody incubations were performed on ultrathin sections collected on nickel grids. To enhance antigen exposure to antibody, ultrathin sections were etched with 10% hydrogen peroxide for 15 min and then washed three times with distilled water. Following quench and blocking steps using avidin, biotin, bovine serum albumin, and goat serum, primary antibody (dilution range from 1:lO to 1:25) was added in the presence of 0.45 MNaCl, 0.3% Triton X-100, 0.02 M phosphate buffer, pH 7.4, and 10% goat serum for l-2 hr or overnight at room temperature. Grids were then washed several times with 0.1 M phosphate-buffered saline (PBS), pH 7.4, and incubated with biotinylated secondary antibody (1:lOO dilution) for 60 min at room temperature. To optimize streptavidin-gold binding, a pH change was introduced using two washes with 0.1 M Tris-buffered saline (TBS), pH 8.0. Streptavidin-gold incubations were performed at room temperature for 1 hr using a dilution of 1:lOO in TBS. Following several washes, grids were incubated with 2% glutaraldehyde in PBS for 10 min at room temperature to stabilize gold binding and then washed with distilled water prior to staining with lead citrate and uranyl acetate. Transmission Microscopy

(TEM)

and Scanning

(SEM)

Electron

For descriptive morphological studies, Hydra were fixed in an aldehyde-osmium mixture containing 0.75% formaldehyde (freshly prepared from paraformaldehyde), 0.75% glutaraldehyde, 1% OsO,, and buffered with 0.05 M sodium phosphate buffer, pH 7.4, for 1 hr on ice and processed for either TEM or SEM as described by Wood (1983a,b). As described for the immunocytochemical studies, specimens for TEM were processed into Spurr’s embedding medium (Spurr, 1969). For TEM studies a JEOL 100X or 1OOCX electron microscope was used and for SEM studies a JEOL JSM-35 electron microscope was used. Isolation

of Hydra

Mesoglea

Previous procedures described for the isolation of Hydra mesoglea involved freeze-thawing, detergent extraction, and hand pipetting steps to mechanically remove cells from the ECM (Shostak et al., 1965; Barzansky and Lenhoff, 1974; Barzansky et al., 1975). These procedures are not appropriate when large quantities (0.1-l g) of Hydra mesoglea are required for biochemical analyses. In the present study, procedures were modified from those developed for the isolation of kidney glomerular basement membrane (Carlson et al., 1978). All steps in this procedure were conducted at 4°C using a nutator (Clay Adams, Parsippany, NJ) for constant mixing except where indicated. At each step in the pro-

Matrix

of Hydra

mlguris,

I

483

cedure, material was observed with phase microscopy or processed for TEM analysis to determine degree of purity. Due to the presence of ECM proteases in Hydra, it was necessary to use a protease inhibitor mixture containing: 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 1 mM l-10 phenanthroline (PA), 50 pME-64,l mM diisopropyl fluorophosphate (DFP), and 10 mM ethylene glyco1 bis(P-aminoethyl ether)N,N,N’N’-tetraacetic acid (EGTA) in all solutions used except where indicated. In addition, all solutions were filtered through 0.45 pm nitrocellulose prior to use. A ratio of at least 1:2 (Hydra packed volume to solution volume) was used for each extraction step. For conveyance, 15 ml packed volume of Hydra were placed in 50 ml disposable screw-top centrifuge tubes for all subsequent extraction steps. Hydra were initially washed with 0.05% NaN, and centrifuged at 800g. The Hydra pellet was resuspended in 35 ml of the same solution for 1 hr. Following centrifugation as before, the pellet was resuspended in 35 ml of a 1% Triton X-100/0.02% NaN, solution for 5 hr. For all subsequent steps’ centrifugation at 30009 was necessary for pelleting of extracted material. Following the first detergent extraction, the material was centrifuged as described and the pellet was then repeatedly resuspended and repelleted in 35 ml of 95% ETOH to remove contaminating pigment granules which arose from the brine shrimp during Hydra feeding. The protease inhibitor mixture was not used with the ETOH extraction steps. ETOH extractions was performed until the pellet lost its reddish color and became white (usually five to seven extractions were necessary). After this step, 35 ml of a DNAse I (Sigma Chemicals) solution (2 mg/50 ml total volume with 0.02% NaN,) was added to the pellet and the suspension was incubated overnight at 4°C on a nutator apparatus to remove contaminating nuclei. The next day, the material was centrifuged at 3000g and the pellet was resuspended in 35 ml of a 4% deoxycholate, 0.05% NaN, solution. This second detergent extraction was performed at room temperature for 4 hr. After centrifugation, the pellet was given a final 95% ETOH extraction to remove any residual pigment granules. At this point the material was centrifuged and either (1) resuspended in distilled water and centrifuged prior to freezing and storage of the pellet at -70°C or (2) resuspended in 0.2 M carbonate-bicarbonate buffer, pH 10.5, 0.05% NaN,, 6.5 mMdithiothreito1 (DTT) and incubated overnight at room temperature on a nutator apparatus to remove contaminating nematocyst capsules (Barzansky and Lenhoff, 1974; Barzansky et al., 1975). It was found that an overnight incubation was required to ensure complete solubilization of nematocyst capsules. Following the DTT step, the material was centrifuged, washed with distilled water, and stored at -70°C as before.

484 Procedures Mesoglea

DEVELOPMENTALBIOLOGY V0~~~~148,1991

for

Characterization

of Isolated Hydra

SDS-PAGE analysis. Isolated Hydra mesoglea was analyzed by SDS-PAGE analysis using 5-20% gradient gels with the discontinuous system of Maize1 (1971) under reducing and nonreducing conditions. Enzyme linked immunosorbent assay (ELISA) analysis. Isolated Hydra mesoglea which had been lyophilized was sonicated in a solution containing 0.1 Mcarbonatebicarbonate buffer, pH 10.0 at a concentration of 1 mg/ ml final concentration of mesoglea. The mesoglea suspension was coated on 96-well microtiter plates (Falcon 3915 Pro-Bind assay plates) and analyzed with various ECM component antibodies following the procedures described for ELISA by Rennard et al. (1980). Western blot analysis. Components of isolated Hydra mesoglea were analyzed for reactivity to ECM antibodies using Western blot analysis essentially as described by Gershoni and Palade (1983). Better transfer of high molecular weight components was achieved by using a Genie transfer apparatus (Idea Scientific, Corvallis, OR) and Immobilon paper (Millipore Corp., Bedford, MA). Greater sensitivity was achieved using a biotinylated streptavidin amplification technique (Vector Laboratories, Burlingame, CA). Amino acid analysis. Amino acid analysis of both isolated Hydra mesoglea and individual mesoglea components was performed. Prior to derivatization with phenyl isothiocyanate (PITC), material was hydrolyzed for 18 hr under HCl vapors at 110°C. Phenylthiocarbamate amino acid derivatives were then analyzed using a Water’s Pica-Tag system as described by Maugh (1984). Cysteine was determined using performic acid oxidation. For analysis of individual Hydra mesoglea components, isolated mesogleal preparations were separated by SDS-PAGE and transferred to Immobilon paper as described by Matsudaira (1987). Transferred material was stained with Coomassie blue R and selected proteins were cut from the paper, acid hydrolyzed, and analyzed as described above. RESULTS 1. IN SITU IMMUNOCYTOCHEMICAL STUDIES TO IDENTIFY ECM COMPONENTSINHYDRAMESOGLEA

For purposes of structural orientation, SEM images are shown in Fig. 1. A freeze-fraction specimen of an intact adult Hydra is shown Fig. la with a higher magnification of the epithelial bilayer with the intervening mesoglea shown in Fig. lb. In frozen sections, antibodies to ECM components appear localized by indirect immunofluorescence to the mesoglea layer between the epithelial bilayer (Fig. lc) and in whole mounts, a linear sub-

ectodermal image is obtained (Fig. Id). Controls performed with nonimmune serum or in the absence of primary antiserum, lacked a mesoglea signal (Fig. Id, inset); although in some cases nematocyst capsules reacted with control serum or antibody. As shown in Table 1 mesoglea reacted positively with a number of polyclonal antibodies to various vertebrate ECM components to include: type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan (core protein of heparan sulfate proteoglycan). As a positive control, all of these antibodies were found to bind to kidney glomerular basement membrane (data not shown). Each of these antibodies has been shown to be specific for their respective matrix molecule (see references provided for each antibody under Materials and Methods or data presented in this article). When cellular localization was observed with these antibodies, it was typically punctate throughout the cytoplasm and usually associated with the ectoderm layer as indicated in Fig. 2. which shows the pattern for antibody to the NC1 domain of GBM type IV collagen. Such cellular localization was best observed with antibody to type IV collagen. Antiserum to collagen type I and entactin did not react with Hydra mesoglea. As shown in Fig. 3a, immunocytochemistry at the ultrastructural level indicated localization of all ECM components tested (type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin) throughout the entire width of Hydra mesoglea. As shown in Fig. 3a, Hydra fixed in normal strength fixative with 0~0, shows the fibrous network associated with the mesoglea matrix (see below and Davis and Haynes, 1968; Haynes et al., 1968 for further description of mesoglea ultrastructure). In the case of all antibodies, gold particles were on or adjacent to matrix fibers (Fig. 3b). In some cases, antibody binding was associated with amorphous matrix aggregates (Fig. 3b). Controls in the presence of nonimmune serum or in the absence of primary antiserum were devoid of gold particles (Fig. 3~). 2. ISOLATION AND CHARACTERIZATION OFHYDRAMESOGLEA

a. Ultrastructural

Analysis

of Isolated Mesoglea

Mesoglea was isolated from Hydra using a modification of a detergent extraction procedure originally developed for the purification of kidney glomerular basement membranes. As compared to that observed in situ (Fig. 3a), mesoglea isolated with this procedure appeared to retain normal ultrastructural characteristics (Figs. 4a and 4b). Because previous studies have extensively described the ultrastructure of Hydra mesoglea (Davis and Haynes, 1968; Haynes et al., 1968), a detailed description will not be undertaken in this article. In brief,

485

SARRAS ET AL.

FIG. 1. Structure of Hydra and immunofluorescent localization of ECM components within mesoglea. As organized as a gastric tube with a mouth (hypostome) and adjacent 5-7 tentacles (a). When the gastric cavity seen as an epithelial bilayer with intervening mesoglea (extracellular matrix, arrows) (b). In frozen sections, with antibodies raised to mammalian extracellular components such as laminin as shown in c. A linear obtained when ECM component antibodies are incubated with fixed adult Hydra (whole mounts) as shown apical border of the ectodermal cell layer. As shown in the inset of d, preimmune antibody or nonimmune mesoglea (arrow) in frozen sections, although in some cases a reaction with nematocyst capsules could be tions: a, ‘78~; b, 153x; c, 768x; d, 88~; d inset, 250x.

however, as shown in Figs. 4a and 4b, the lateral edges (that adjacent to ectoderm and endoderm cells) of isolated mesoglea contained a greater concentration of matrix material than observed centrally in the mesoglea. As previously described (Davis and Haynes, 1968; Haynes et al., 1968), isolated mesoglea was organized from an amorphous ground substance within which was embedded a number of different types of fibers and par-

viewed by SEM, Hydra vulgaris is is fractured the body wall can be the mesoglea (arrowheads) reacts subectodermal image (arrows) is in d. The arrowheads indicate the antibody fail to react with the observed (arrowhead). Magnifica-

ticulate material. As will be further described later, previous studies have indicated that (1) nematocyte capsules will coisolate with mesoglea and (2) DTT (6.5 mM) treatment can be used to solubilize this contaminating fraction from mesoglea (Barzansky and Lenhoff, 1974; Barzansky et al., 1975; Day and Lenhoff, 1981). Although DTT treatment significantly removed contaminating nematocyst capsules, it did not appear to alter meso-

486

DEVELOPMENTALBIOLOGY

V0~~~~148,1991

TABLE1 IMMUNOCYTOCHEMICALANALYSISOFHYDRA EXTRACELLULARMATRIX(MESOGLEA) Antibody

to ECM

Component

Reactivity

Type IV Collagen” Laminin* Fibronectin” Heparan Sulfate Proteoglycan core proteind

(0 to +++) ++ ++ ++ ++

“Anti-human placental type IV collagen and anti-bovine kidney glomerular basement membrane type IV collagen (NC1 domain of (Ye subunit). * Anti-EHS laminin and anit-mouse placental laminin. ‘Anti-human plasma fibronectin. ‘Anti-rat kidney glomerular basement membrane heparan sulfate proteoglycan core protein.

gleal ultrastructure (Fig. 4b). The degree of nematocyte capsule contamination of the mesoglea fraction was extensive as indicated by the fact that up to 80% of total protein in the preparation was removed by DTT treatment. b. Detection of EC34 Components by ELISA

in Isolated

Mesoglea

To determine if ECM components identified in situ were lost due to extraction during the mesogleal isolation procedure, ELBA was performed on both DTT and non-DTT-treated preparations. As shown in Fig. 5, all

FIG. 2. Immunofluorescent localization of ECM components to the ectodermal cell layer of Hydra as shown in a frozen cross section taken from the head region. Using antibody specific for the NC1 domain of GBM type IV collagen, a punctate pattern (arrows) was observed in the ectodermal layer of cells. An intense staining of the mesoglea is also observed (arrowheads). Magnification: 432~.

FIG. 3. Ultrastructural localization of ECM components within Hydra mesoglea using antibodies raised to mammalian matrix components. The normal ultrastructure of Hydra mesoglea is shown in a. In Hydra fixed with lower concentrations of glutaraldehyde (0.1%) and with no osmium tetroxide, antibodies to various ECM components could be localized throughout the width of the mesoglea and was typically associated with fibers and particulate material within the matrix. Binding of laminin is shown in b. Controls incubated with preimmune or nonimmune antibody lacked immunogold binding as shown in c. Magnifications: a, b, and c, 40,000~.

SARRASETAL.

E~tracellular

Matrix

of Hydra

m1gnri.s.

487

I

did not result in a total extraction of matrix components even though as stated above this treatment did result in loss of nematocyte capsules which represented a major contaminating component in the mesoglea preparation prior to DTT treatment. c. Am,ino Acid Com,positicm

of Isolated Mesoglea

Amino acid analysis of isolated mesoglea was performed on DTT and non-DTT-treated preparations. The results obtained from these studies are shown in Table 2 where amino acid results are compared with those reported previously for isolated Hydra mesoglea (Barzansky and Lenhoff, 1974; Barzansky et al., 1975). The values for amino acids indicative of collagens (e.g., glytine, hydroxyproline, and hydroxylysine) in the nonDTT and DTT-treated preparations (Table 2, columns 1 and 2, respectively) are lower than those reported previously (Table 2, column 3) for Hydra mesoglea isolated by a different procedure and treated with DTT (Barzansky and Lenhoff, 1974; Barzansky et al., 1975). The respective values for these amino acids were lowest in DTT-treated mesoglea (column 2, Table 2), and a relatively high value for these amino acids was found in the supernatant following DTT treatment (e.g., residues per 1000 for the DTT supernatant: glycine, 117; proline, 102; 4-hydroxyproline, 42). d. SDS-PAGE

Analysis of Isolated Mesoglea

1. SDS-PAGE patterns under reducin.y and nonreducing conditions. An electrophoretic pattern of the isolated

Hydra mesoglea is shown in Fig. 6. Under reducing conditions, the most prominent high molecular weight bands appearing with Coomassie blue staining are a

O-ONon Immune Ab @--@Anti-Human Type IV Collagen a--AAnti-Mouse Laminin Ab A--Anti-Rat HSPG Ab q -•Anti-Human Fibronectin Ab

FIG. 4. Purity

and ultrastructure of isolated mesoglea fractions. TEM analysis of isolated mesoglea was performed to assess the relative purity of the preparation. As shown at lower power (a), sheets of purified mesoglea could be observed in the preparation. At higher magnification (b), isolated mesoglea appeared to retain the ultrastructural characteristics observed in sifu (compare with Fig. 3a). Magnihcations: a, 11,250~; b, 20,000~.

antiserum that showed binding in the in situ immunocytochemical studies reacted with the isolated mesogleal fraction (results from a DTT-treated fraction shown) when compared to preimmune or nonimmune antiserum binding, indicating that the components had been retained to some degree. The fact that similar results were obtained with the mesoglea preparation prior to and after DTT treatment indicates that DTT treatment

PRIMARY

FIG. 5. Determination

ANTIBODY

Ab

DILUTION

of ECM components retained in isolated Hydra mesoglea as monitored by ELISA. As indicated by these ELISA studies, the isolated mesoglea preparation retained its reactivity to the ECM component antibodies found to bind to mesoglea it! situ.

488

DEVELOPMENTAL BIOLOGY

TABLE 2 AMINO ACID COMPOSITION OF HYDRA EXTRACELLULAR MATRIX (MESOGLEA) Amino Acid Y-OH-proline 4-OH-proline Aspartic acid Threonine Serine Glutamic acid Proline Glvcine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenyalanine Histidine OH-lysine Lysine Arginine

Residues11000 (minus DTT)

Residues/1000 (plus DTT)b

2 16

0 5

97 45 65 133 63 125 64 26 50 18 48 71 45 48 18 4 65 55

111 53 79 111 54 81 71 20 59 18 55 85 53 55 21 0 67 58

Residues0000 (as reported”) 30 both 3- and (4-OH-proline) 96 38 61 121 57 229 57 15 27 12 24 56 20 23 13 40 43 38

VOLUME 148,199l

ysis alone. With this in mind, suspected collagen molecules resolved by SDS-PAGE of the mesogleal preparation were analyzed in terms of their amino acid composition. As shown in Table 3, proteins at 165,000,190,000, and 200,000 Da had an amino acid composition indicative of collagen molecules with a high giycine, 4-hydroxyproline, and hydroxylysine content. Although high in glycine, these Hydra proteins appear to have a noncollagenous domain(s) as compared to the collagen molecules identified in the mesoglea of jellyfish (Table 3, fourth column, as reported by Miura and Kimura, 1985) and the sea anenome (Table 3, fifth column, as reported by Nordwig et al., 1973). The glycine content of the three Hydra proteins (165,190, and 200 kDa) is comparable to that observed in a type IV collagen such as the aI subunit of kidneyglomerular basement membrane (Table 3, sixth column {as reported by Dean et al., 1983)). 3. Identification of mesoglea compments by Western blot analysis. ECM components identified in the immuno-

a Mean from 8 determinations. ’ Mean from 5 determinations. ’ Barzansky et al, 1974.

doublet (Fig. 6d, arrows) whose two components were at approximately 200,000 and 190,000 Da, respectively. A band at approximately 45,000 Da (Fig. 6d, arrow) was also prominent. Less prominent proteins at 300,000 and 165,000 Da (Fig. 6d, arrows) appeared magenta in color with Coomassie blue staining which is characteristic of collagen proteins. When the sample was overloaded, as shown in lane e of Fig. 6, the magenta-colored 300,000Da protein is more easily seen, as well as at least four other proteins with molecular weights greater than 200 kDa. This same pattern under reducing conditions was observed with both the DTT- and non-DTT-treated mesogleal preparations. Under nonreducing conditions (Fig. 6c), high molecular weight bands appeared at 300,000 and 165,000 Da (arrowheads) with the 165,000Da protein being the most prominent. As before, both the 300,000- and 165,000-Da proteins appeared magenta in color with Coomassie blue staining. 2. Amino acid composition of individual components of isolated mesoglea.Unlike most proteins that require se-

quence analysis for accurate classification, the high glytine, hydroxyproline, and hydroxylysine content of collagens allows us to characterize proteins as being in the collagen family based on amino acid compositional anal-

FIG. 6. SDS-PAGE analysis of isolated Hydra mesoglea. A nonreduced (lane c) and reduced (lanes d and e) Coomassie blue staining pattern of isolated mesoglea is shown. Under nonreducing conditions (lane c), high molecular weight bands were observed at 300 and 165 kDa, as indicated by the arrowheads. These bands were magenta in color in the original gels which is reflective of collagen proteins. Under reducing conditions (lanes d and e), the 300 and 165 kDa magenta-colored bands (arrows) were present as well as other molecular weight bands higher than 200 kDa. A prominant doublet appeared near 200 kDa (200 and 190 kDa, arrows) as well as a prominant band at 45 kDa (arrow). An overloaded reduced sample is shown in lane e so that the 300.kDa band and other high molecular weight bands can be more easily seen. Molecular weight markers are shown in lane a: myosin, 200 kDa; P-galactosidase, 116.25 kDa; phosphorylase B, 92.5 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa (all indicated by arrowheads, respectively). Type I collagen standards are shown in lane b: top band (just above 200 kDa) = p subunit; lower doublet = 01subunit.

SARRAS ET AL.

AMINO

acid

165 kDa

3-OH-proline 4-OH-proline Aspartic acid Threonine Serine Glutamic acid Prolinf Gly-tine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phengalanine Histidine OH-lysine Lysine Arginine

5 32 86 40 87 122 41 260 54 10 36 13 38 58 9 32 9 13 33 46

mesoglea

digestion

mrlgur-is,

489

I

Collagens

0 32 94 44 143 139 40 274 96 8 38 16 33 79 23 30 15 10 58 51

and characterization

Jellyfishb

200 kDa 3 68 78 42 153 108 40 260 123 9 37 13 31 59 22 29 37 7 68 43

pro-a,

subunit;

cytochemical and ELISA studies described above were identified in the SDS-PAGE patterns by Western blot analysis. As shown in Fig. 7, a consistent pattern of immunoreactive bands in the high molecular weight range was obtained with antibodies to laminin (Fig. 7, lane b, i?l, = 330 and 200 k), and fibronectin (Fig. 7, lane c, M,. = 335 and 200 k). Antibody binding to these bands could be completed with incubation with 100 pg of purified rat laminin (Fig. ‘7, lane a) or human plasma fibronectin (Fig. 7, lane d). Nonimmune and secondary antibody controls were negative in this molecular weight range as shown in Fig. 7, lane e. Although immunoreactive bands were obtained with antibodies to type IV collagen, the binding pattern was variable and therefore further studies were conducted to clarify the existance of a type IV collagen in mesoglea. 4. Partial

of Hydra

proteins”

190 kDa

a Average from two determinations. ’ Mesoglea &I collagen; Miura and Kimura, 1985. ’ Mesoglea CYcollagen; Nordwig et al, 1973. ’ Glomerular basement membrane type IV collagen ’ Not reported.

of mesoglea

Type IV collagen contains a noncollagenous domain at its carboxy terminal; this domain is retained after collagenase digestion. To obtain further evidence for the presence of type IV collagen in mesoglea, partial extraction and collagenase digestion studies were performed along with Western blot analysis using antibody raised to the NC1 domain (al subunit) of GBM type IV

collagens.

Mutris

TABLE 3 ACID COMPOSITION (RESIDUES PER 1000) OF HYDRA MESOCLEA COLLAGEN-LIKE AND COMPARISON TO COLLAGENS OF OTHER SPECIES Hydra

Amino

Extrucellulctr

1 45 78 31 47 98 74 309 78 6 31 7 21 32 9 12 6 30 32 52

Dean

PROTEINS

of other

Sea anenomeC 19 81 ‘71 36 39 94 65 329 64 -e 27 10 21 29 3 8 1 24 16 67

species Bovine

GBM” 7 102 58 27 45 92 65 283 44 11 31 13 34 64 11 27 11 37 10 27

et al.. 1983.

collagen. For these experiments, mesoglea was extracted overnight at 4°C with 0.5 N acetic acid containing protease inhibitors. As monitored by SDS-PAGE, the pellet (10,OOOg) obtained following this extraction step contained mainly the 165- and SOO-kDa magentastained bands. The pellet was digested with bacterial collagenase (CLSPA; Worthington, used at 0.2% collagenase/dry wt mesoglea) at 37°C for 24 hr with stirring, and the resulting material was separated by reverse phase HPLC. Following SDS-PAGE of HPLC fractions, material was transferred to Imobilon-P paper. Western blot analysis of this material with the NC1 antiserum indicated immunoreactive bands at approximately 26 and 44 kDa (Fig. 8). which is the size range for monomer and dimer forms of the (Yesubunits of NC1 from GBM type IV collagen as previously shown (Butkowski et al., 1987). 5. Partial isolation of a Hydrajibrmectin. Because of the comigration of collagens, laminin, and fibronectin at 200 kDa, an alternate approach was taken to confirm that these molecules were present in Hydra. Given the gelatin binding properties of fibronectin, experiments were designed to determine if a fibronectin-like molecule could be isolated from whole Hydra using gelatin-

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DEVELOPMENTAL

a

b kDa c 335%

d

BIOLOGY

e

“330

-2oo-

-

LN

FN

FIG. 7. Western blot analysis of ECM components in isolated Hydra mesoglea using antibodies to mammalian laminin (LN) and fibronectin (FN). With anti-laminin antibodies (diluted 1:400), binding to high molecular weight bands at 200 and 330 kDa could be observed (lane b). This binding was eliminated in the presence of mouse laminin (lane a). With anti-fibronectin antibodies (diluted 1:500), binding to high molecular weight bands at 200 and 335 kDa could be observed (lane c). This binding was eliminated in the presence of human plasma fibronectin (lane d). Nonspecific binding of preimmune or nonimmune antibody is shown in lane e.

affinity chromatography. Hydra were homogenized in 50 mM Tris, pH 7.4, 0.5 M NaCl, and 0.05% Brij 35 containing protease inhibitors. Following 100,OOOgcentrifugation, the supernatant was loaded on a gelatin-affinity column and elutated with the same buffer containing 4 M urea. The eluated fraction was dialyzed against the homogenization buffer and concentrated using an Amicon P-10 membrane. As shown in Fig. 9 (lane a), an immunoreactive band was observed at 200 kDa using the antiserum to human fibronectin employed in the previous Elisa (Fig. 5), Western blot (Fig. 7), and immunocytochemical studies (Table 1). No reaction of the secondary antibody was observed with this gelatin-binding fraction (lane b). Reaction of this antiserum to human fibronectin is shown in lane c of Fig. 9 as a positive control. DISCUSSION

While Cnidarians such as Hydra are considered an early divergent metazoan group which did not lead to higher animal forms (Field et ab, 1988; Margulis and Schwartz, 1988), their development of an epithelium with an adherent ECM represents a fundamental tissue pattern utilized in higher organisms. Therefore, in terms of animal evolution the mesoglea of Hydra is likely one of the earliest forms of ECM to be associated with defined tissue layers (Margulis and Schwartz, 1988). Given the extensive regenerative capacity of Hy-

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dra, the molecular composition of this primitive ECM has important implications regarding the fundamental role of matrix molecules in developmental processes. With this in mind we have undertaken an analysis of Hydra mesoglea in order to better characterize its molecular composition, organization, and functional properties. The major finding of this study is the identification of type IV collagen, laminin, heparan sulfate proteoglycan core protein, and fibronectin immunoreactive molecules in Hydra mesoglea; although the structural homology of these molecules to vertebrate matrix molecules remains to be determined. The Structure of Hydra Mesoglea as Compared to That Reported f&r Vertebrate and Invertebrate ECM The identification of collagens and other matrix molecules in Hydra mesoglea is consistent with previous studies that have found vertebrate-like matrix molecules in the ECM and basement membranes of a broad spectrum of invertebrate groups including those that are considered more primitive than Hydra in their cellular organization (e.g., sponges) and those that are clearly more complex than Hydra (e.g., sea urchins, nematodes, and Drosophila). For example, proteoglycans

KDa 200-

a

b I

c

j

!

FIG. 8. Western blot analysis of noncollageneous domains of Hydra 165- and 300-kDa collagen. A enriched fraction of the 165- and 300kDa proteins of mesoglea was incubated with bacterial collagenase and the resistant noncollagen fragments were separated by HPLC and analyzed by Western blot analysis using antibodies specific for the NC1 domain (q subunit) of GBM type IV collagen. As indicated in lanes a and b, immunoreactive bands were observed at 26 kDa (arrowhead, lane a) and 44 kDa (arrowhead, lane b), which is reflective of monomers and dimers of NC1 subunits as previously shown by our laboratory (Butkowski et c/J., 1987). A nonimmune control is shown in lane c. The positions of molecular weight markers are indicated by the dashes and include: myosin (heavy chain), 200 kDa; phosphorylase B, 92.5 kDa; bovine serum albumin, 68 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; lysozyme, 14.4 kDa.

SARRAS ET AL.

abc

FIG. 9. Western blot analysis of a fihronectin-immunoreactive molecule isolated by gelatin-affinity chromatography. As indicated under Results, whole Hydra were homogenized and the resulting high speed supernatant was applied to a Sepharose 4B gelatin-affinity column (Pharmacia). After washing, elution with 4 M urea resulted in the appearance of a immunoreactive band at 200 and 335 kDa using antibody raised to human plasma fibronectin as was observed in the mesoglea preparation (Fig. 7). No bands were observed with secondary antibody as shown in lane b. Reactivity of the antibody to human plasma fibronectin is shown in lane c (arrowhead) as a positive control.

have been identified in the ECM of both sponges (Cauldet al., 1973) and Drosophila well et al., 1973; Henkart (Jackson et al., 1986; Fessler and Fessler, 1989); laminin has been identified in the ECM of sea urchins (Monte11 and (McCarthy et ab, 1987) and Drosophila Goodman, 1988); a number of collagens (fibrillar and nonfibrillar) have been identified in the ECM of sponges et al., 1986; (Garrone, 1978), sea urchins (Venkatesan et al., 1989), nematodes (Kramer et ah, 1982; D’Alessio Noelken et al., 1986; Cox et al, 1989), jellyfish (Kimura et al., 1983; Miura and Kimura et al., 1985), sea anenome (Katzman and Kang, 1972; Nordwig et al., 1973), and Drosophila (Blumberg et al., 1988; Fessler and Fessler, 1989); and fibronectin has been identified in the ECM of sponges (Labat-Robert et al., 1981), sea urchins (Spiegel et al., 1980), jellyfish (Schlage, 1988), and Drosophila (Gratecos et al., 1988). As described in the accompanying paper of this series (Sarras et al, 1991a), pulse-chase autoradiographic studies using [3H]proline (see also Hausman and Burnett, 1971) and 35S0, as general precursors for collagens and proteoglycans, respectively, indicate an incorporation of these molecules into the mesoglea within a 3- to 4-hr time frame from initial pulse. This suggests a continual synthesis of mesogleal components even during periods of relatively normal growth and maintenance of Hydra since no regenerative pro-

cesses were initiated in our autoradiographic experiments. As indicated in previous pulse-chase autoradiographic studies (Hausman and Burnett, 1971), our immunocytochemical studies indicated that a punctate fluorescent pattern could be observed with the ectoderma1 layer, thus indicating a prominent biosynthetic role of this cell layer in mesoglea formation. Because matrix proteases have been identified in Hydra (Chen et al., 1989; Sarras et al., 1991b), some degree of mesoglea breakdown may also be occurring under normal growth conditions in Hydra. At the ultrastructural level, our immunocytochemical studies indicate an even distribution of all matrix molecules across the entire width of mesoglea just as is seen with many vertebrate basement membranes (Grant and Leblond, 1988; and reviewed by Martinez-Hernadez, 1987). As with previous vertebrate studies (Martinez-Hernadez, 1987; Sawada et al., 1987), antibody binding to all molecules tested was typically associated with matrix fibers or amorphous matrix aggregates throughout the mesoglea. Taken together, the immunocytochemical and pulse-chase autoradiographic studies suggest the presence of a number of vertebrate-like ECM components in Hydra mesoglea. To obtain supportive data for the immunocytochemical and autoradiographic studies discussed above, Hydra mesoglea was isolated and biochemically characterized. In order to obtain large quantities of mesoglea, procedures were modified from methods developed for the isolation of kidney glomerular basement membrane (Carlson et al., 1978). Critical to this isolation was the necessity to include a wide spectrum of protease inhibitors. Without the use of the protease inhibitor cocktail described under Materials and Methods, inconsistent SDS-PAGE staining patterns were observed. As monitored by morphological criteria, it was also necessary to include ETOH extraction steps to remove large quantities of contaminating shrimp pigments. Using these procedures, a mesoglea fraction was obtained that retained the structural properties of Hydra mesoglea observed in situ. As previously reported, the use of a final DTT extraction step was necessary to remove contaminating nematocyst capsules as monitored by phase microscopy and TEM analysis. ELISA studies indicated that the isolation procedure did not lead to a complete extraction from the mesoglea of those ECM components studies. In identified in the in situ immunocytochemical regard to amino acid composition, Hydra mesoglea was more similar to the basement membrane of the nematode, Ascaris (Peczon et al., 1975), than that reported for bovine kidney glomerular basement membrane (West et al., 1980). The amino acid composition of Hydra mesoglea obtained in the present study is in some variance with that reported by others (Barzansky and Lenhoff, et al, 1975). For both DTT- and non1974; Barzansky

492

DEVELOPMENTALBIOLOGY

DTT-treated preparations, we report a significantly lower glycine (81-125 vs 229 residues per lOOO), hydroxyproline (5-16 vs 30 residues per lOOO), and hydroxylysine (O-4 vs 40 residues per 1000) content. These amino acids likely reflect collagen content and the variation in our results as compared to the previous study is likely due to the action of proteases during the isolation procedure. The previous study did not utilize protease inhibitors during mesoglea isolation and we have observed that in the absence of protease inhibitors, a high glycine content can be observed (data not shown). Our findings suggest that mesoglea may have a lower collagen content than previously thought. The decreased content of amino acids such as glycine and hydroxyproline in DTTtreated preparations may reflect the loss of non-mesogleal collagens that have been proposed to be structural components of nematocyst capsules (Lenhoff et aZ., 1957). These purported nematocyst collagens are likely to be smaller than ECM collagens since DTT treatment did not alter the SDS-PAGE staining pattern in the molecular weight range of 150 kDa and higher. In support of a collagen-like proteins being associated with nematocyst capsules, we did note a relatively high content of glycine, proline, and 4-hydroxyproline in the supernatant following DTT treatment of isolated mesoglea. The SDS-PAGE pattern obtained with Hydra mesoglea reflected a broad molecular weight spectrum of proteins. The detection of prominent high molecular species (150 kDa and greater) is consistent with the presence of ECM molecules such as collagen, laminin, and fibronectin which characteristically have subunits with masses in the range of 200 kDa. Lack of appearance of the disulfide-linked forms of laminin and fibronectin in nonreducing gels likely reflects low levels in the mesoglea preparation or lack of solubility under the conditions used in these studies. Western blot analysis, however, supports the presence of a laminin-like and fibronectin-like molecule being present in Hydra mesoglea although variations in apparent molecular weights exist. The presence of a fibronectin-like molecule in Hydra was corroborated by those studies indicating that a 200kDa protein that was immunoreactive to fibronectin antiserum could be isolated from Hydra using gelatin-affinity chromatography. The fibronectin-like molecule observed in Hydra is smaller than expected from other studies (see review by Ruoslahti, 1988 and Yamada, 1989). While vertebrate fibronectin (M, = 550,000) is typically made up of two subunits, each with a molecular weight of approximately 225 kDa, the fibronectin-like proteins detected in Hydra mesoglea have molecular weights of 200 and 335 kDa. In a similar vein, laminin of vertebrates and higher invertebrates is reported to have subunits with masses of approximately 400 kDa (A

V0~~~~148,1991

chain), 210 kDa (Bl chain), and 200 kDa (B2 chain) (see review by Kleinman and Weeks, 1989), while the laminin-like proteins of mesoglea have masses of 200 and 330 kDa. Although Western blot studies of isolated mesoglea were inconclusive regarding the presence of intact collagen type IV, the amino acid studies indicated that collagens exist in Hydra mesoglea. Both the 300and 165kDa proteins were suspected collagens due to their magenta color following Coomassie blue staining. In the case of the 165-kDa protein, amino acid analysis indicated a collagen-like composition due to the high glycine, 4-hydroxyproline, and hydroxylysine content. Although proteins at a molecular mass of 190 and 200 kDa also appeared to be collagen-like due their amino acid composition, this analysis is complicated by the fact that the Western blot data indicate that a number of different proteins are comigratingwith similar molecular mass in the 200-kDa range. Nevertheless, these results indicate that collagen-like proteins exist in the 200-kDa range. Further purification of these proteins will be required to clarify this point and to determine whether more than one type of collagen exists in Hydra mesoglea. If one focuses on the 165-kDa collagen-like protein which can be resolved on gradient gels, our data suggest that it contains noncollagen domains as reflected by its glycine content of 260 residues per thousand. In addition, digestion of the 165- and 300-kDa proteins with collagenase indicated the presence of a NC1 domain based on Western blot analysis using antiserum which was specific for the NC1 domain of bovine GBM type IV collagen. In contrast, the collagens of jellyfish (Miura and Kimura, 1985) and sea anenomes (Nordwig et al., 1973) which belong to separate classes of the phylum, Cnidaria, appear not to contain noncollagenous domains because their glycine content is greater than 300 residues per thousand. This suggests that differences may exist in the mesogleal collagens of Hydrozoans vs other classes in Cnidaria; although it should be noted that previous studies did not use protease inhibitors during collagen isolation and this could have resulted in loss of noncollagenous domains. Taken in concert, the morphological and biochemical approaches described in this study suggest the presence of collagen(s) and components with cross-immunoreactivity to antibodies raised against vertebrate laminin, heparan sulfate proteoglycan core protein, and fibronectin. The structural homology of these molecules to those of vertebrate and other invertebrate ECM molecules remains to be determined. These observations indicate that Hydra mesoglea has matrix components reflective of both an interstitial ECM (i.e., fibronectin) and those of an epithelial-associated ECM (i.e., basement membrane containing type IV collagen, laminin, and heparan sulfate proteoglycan). This suggests that Hydra meso-

493

SARRAS ET AL.

glea may be evolutionarily a primitive form of basement membrane which has retained some characteristics of interstitial ECMs as first developed in sponges (Henkart ef a,l., 1973; Garrone, 1978; Labat-Robert et ab, 1981). The authors thank Dr. Yashpal S. Kanwar, Northwestern Univ., for supplying the polyclonal antibody to heparan sulfate proteoglycan for use in this study. The authors express a special thanks to Dr. Hans Bode, IJniv. of California, Irvine, for supplying strains of Hgdrn vulf/rrris for initiation of these studies and for providing help and suggestions throughout the course of these studies. The authors also thank Dr. Stanley R. Nelson for critically reviewing this manuscript prior to its submission. This work was supported in part by NIH grants RR06500 (MPS) and DK38219 (BGH); funds awarded to MPS by the Juvenile Diabetes Foundation International, and funds from the University of Kansas Medical Center BRSG program awarded to M.P.S. REFERENCES BARZANSKY, B., and LENHOFF, H. M. (1974). On the chemical composition and developmental role of the mesoglea of Hgdra. Aw. Zool. 14, 575-581. BARZANSKY, B., L,ENHOFF, H. M., and BODE, H. (1975). Hydra mesoglca: Similarity of it.s amino acid and neutral sugar composition to that of vertebrate basal lamina. Cotnp Biocheru. Physiol. R. 50,419424. BECK, Ii., MCCARTHY, R. A., CHIQUET, M., MASUDA-NAKAGAWA, L.. and SCHLAGE, W. K. (1989). Structure of the basement membrane protein laminin: Variations on a theme. 172“Qtoskeletal and Extracellular Proteins in Biophysics” (IT. Aebi and J. Engel, Eds.), Vol. 3, pp. 102-105, Springer-Verlag, New York/Berlin. BLUMBERG, B., MACKRELL. A. J., and FESSLER, J. H. (1988). Drosophila hasement membrane procollagen nl(IV). II. Complete cDNA se(Iucnce, genomic structure, and general implications for supramolecular assemblies. J. Biol. C/ten/. 263, 18,32818,337. BOSCH,T. C. G., and DAVID, C. N. (1984). Growth regulation in H>-dra: Relationship between epithelial cell cgcle and growth rate. Det: Bid. 104, 161-171. BUTKOWSKI, R. J., LANGEVELD, J. P. M., WIESLANDER, J., HAMILTON, J., and HUDSON, B. G. (1987). Localization of the Goodpasture epitope to a novel chain of basement membrane collagen. J. Biol. C!)errc. 262,7874-7877. CAMPBELL, R. D. (1967a) Tissue dynamics of steady state growth in Hgdra littoralis. I. Patterns of cell division. Dev. Biol. 15, 487-502. CAMPBELL, R. D. (1967b). Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement. J. Mor/jho/. 121, 1928. CARLSON, E. C., BRENDEL, K., HJELLE, J. T., and MEEZAN, E. (1978). Ultrastructure and biochemical analyses of isolated basement membranes from kidney glomeruli and tubules and brain and retinal microvessels. .J. I’ltrtrstr~rct. Rm. 62, 26-53. CAULDWELL, C. B., HENKART, P., and HUMPHREYS, T. (1973). Physical properties of sponge aggregation factor: A unique proteoglycan complex. Biochen~ist~jj 12, 3051-3055. CHEN, J. M., KING, K. S., and NEWMAN, L. M. (1989). Identification of matrix-degrading proteases from Hydra attenuata. J. Cc/l Biol. 109, 136a (Abstract). Cox, CT.N., FIELDS, C., KRAMER, J. M., ROSENZWEIG,B., and HIRSH, D. (1989). Sequence comparisons of developmentally regulated collagen genes of Caenorhabditis elegans. &rte 76, 331-344. D’ALESSIO, M., RAMIREZ, F., SUZUKI, H. R., SOLURSH, M., and GAMBINO, R. (1989). Structure and developmental expression of a sea

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MCCARTHY, R. A., BECK, K., and BURGER, M. M. (1987). Laminin is structurally conserved in sea urchin basal lamina. EMBOJ. 6,10371043. MCDONALD, J. A. (1989). Matrix regulation of cell shape and gene expression. 17~ “Current Opinion in Cell Biology” (C. H. Damsky and M. Bernfield, Eds.), pp. 995-999. Current Science Press, PA. MIURA, S., and KIMURA, S. (1985). Jellyfish mesoglea collagen: Characterization of molecules as ala2a3 heterotrimers. J. Biol. Chem 260, 15,352-15,356. MONTELL, D. J., and GOODMAN, C. S. (1988). Drosophila substrate adhesion molecule: Sequence of laminin Bl chain reveals domains of homology with mouse. Cell 53,463-473. NIYIBIZI, C., FIETZEK, P. P., and VAN DER REST, M. (1984). Human placenta type V collagen: Evidence for the existance of an nl(V)ctB(V)c~3(V) collagen molecule. J. Biol. Chem. 259, 14,17014,174. NOELKEN, M. E., WISDOM, B. J., DEAN, D. C., HUNG, C. H., and HUDSON, B. G. (1986). Intestinal basement membrane of Ascaris suum: Molecular organization and properties of the collagen molecules. J. BioL

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