Extracellular matrix (mesoglea) of Hydra vulgaris

DEVELOPMENTAL

148,495-500

BIOLOGY

(19%)

Extracellular II. Influence

of Collagen

Matrix

(Mesoglea)

and Proteoglycan

of Hydra Vulgaris

Components

MICHAELP.SARRAS,JR.,**~DARRELMEADOR,*

on Head

Regeneration

ANDXIAOMINGZHANG*

Hydra are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. As an extension of the previous study which indicated that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial extracellular matrix, the present study was undertaken to analyze the role of mesoglea components during head regeneration in Hydra uulgaris. Studies were conducted that utilized drugs that affect collagen processing or secondary collagen structure (P-aminoproprionitrile; 2,2’dipydridyl: and cis-4-hydroxy-L-proline) and a drug that inhibits addition of glycosaminoglycan chains to proteoglycan core proteins (I.‘-nitrophenyl-P-D-xylopyranoside). These sludies indicated that alterations in the structure of collagens or proteoglycans caused blockage of head regeneration in Hydra as monitored over a 4%hr period. Blockage of head regeneration was reversible once the drugs were removed, indicating that the drugs were not having a general toxic effect on the organism. Radiotracer studies also indicated that blockage of head regeneration was not simply due to a general depression of protein synthesis hy t,he drugs. Various controls indicated that each drug was affecting mesoglea components under the conditions utilized in these studies. These observations indicate that preservation of normal IL ml Academic PESS, IX mesoglea structure is required for Hydra head regeneration to proceed.

Because basement membrane and ECM components have been shown to have a critical role in cell signaling events during development (see reviews by Ekblom et al., 1986; McDonald, 1989; Timpl, 1989), the present study has been conducted to clarify the role of mesoglea collagen and proteoglycan components during head regeneration in Hydra vulgaris. An earlier report of some of these findings was published in abstract form (Sarras et al., 1988).

INTRODUCTION

Hydra are organized with a body wall consisting of an epithelial bilayer with an intervening acellular layer termed the mesoglea. As described in the previous paper of this series (Sarras et al., 1991), the mesoglea of Hydra vulgaris is composed of a number of extracellular matrix (ECM) components which are immunoreactive to antibodies raised to mammalian: type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan core protein. While the structural homology of these mesoglea components to those present in the ECM of other invertebrates and vertebrate remains to be determined, these findings suggest that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial ECM. Pulse-chase autoradiographic studies using [3H]proline have shown that the synthesis of mesoglea components is increased during head regeneration (Hausman and Burnett, 1971) and that this regenerative process is retarded if one perturbs the normal cross-linking of collagen molecules (Barzansky and Lenhoff, 1974). More recent studies have indicated that specific cell surface binding sites exist on Hydra cells for mesoglea and ECM components such as fibronectin (Day and Lenhoff, 1981; Gonzalez-Agosti and Stidwill, 1990).

’ To whom

correspondence

should

MATERIALS

Culture

AND

METHODS

of Hydra

Hydra vulgaris (formerly classified as Hydra attenuata) were maintained as described in the previous paper of this series (Sarras et ah, 1991). Procedures for Study of ECM Component during Hydra Head Regenemtion

Functim

To determine the potential role of ECM components in head regeneration, studies were performed with various drugs and compounds that are known to perturb normal processing of matrix components. The study focused on agents that aflect collagens and proteoglycans. To test the role of collagen in head regeneration, three compounds were tested to include ,&aminoproprionitrile (APN), 2,2’-dipydridyl (DP), and cis-4-hydroxy-Lproline (cis-hydroxyproline). To test proteoglycan func-

be addressed 495

0012.1606/91 Copyright All rights

$3.00

:c) 1991 by Academic Press, Inc. of reproduction in any form reserved.

496

DEVELOPMENTALBIOLOGY

tion, p-nitrophenyl-P-D-xylopyranoside (P-xyloside) and its inactive a-isomer (a-xyloside) form were used. All of these compounds were obtained from Sigma Chemical Co. (St. Louis, MO) except for a-xyloside which was obtained from Koch-Light Ltd. (Suffolk, England). Hydra medium containing 0.05% DMSO was required for solubility of the xylosides. In the case of compounds that may not be transported well across the Hydra ectoderm (compounds which are metabolites such as cis-hydroxyproline), direct injection into the gastric cavity was performed as described by David (1983) prior to decapitation. For all experiments, Hydra were starved 1 day and decapitated as described by MaeWilliams (1983). Head regeneration was monitored over 48 hr at which time controls had developed hypostomes and tentacles. In the case of the xyloside experiments, controls in the presence of 0.05% DMSO were also monitored. Hydra were placed in hydra medium containing the drugs immediately following decapitation and monitored over 48 hr. For recovery experiments, drugs were washed out at 48 hr and any subsequent regeneration was monitored over an additional 72 hr. As will be discussed under Results, biochemical and pulse-chase autoradiographic experiments were conducted to determine if the compounds used in these studies were actually affecting the ECM molecules of interest in Hydra. Amino Acid Analysis of APN-Treated

Hydra

Amino acid analysis of control and APN-treated Hydra was conducted to monitor the effect of this drug on the extractability of 4-hydroxyproline containing components such as collagens. Fractions to be analyzed for 4-hydroxyproline content were first lyophilized. 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). Experiments to Monitor the Effect of Drugs on Hydra Protein Synthesis

To determine the effect of the drugs used in this study on general protein synthesis, control and drug-treated Hydra were pulsed for 1 hr in the presence of [35S]cysteine (100 &i/ml in Hydra medium). The drug concentrations and exposure times described for the head regeneration experiments were utilized for these radiotracer studies. The radioisotope was introduced into the gastric cavity to ensure maximal incorporation. Three Hydra were pooled after the pulse. Pooled Hydra were washed, sonicated, and precipitated in 15% TCA using bovine serum albumin as a carrier. Precipitates were trapped and washed on glass fiber discs and DPM

vOLUME148,1%1

were determined using a Packard 1900CA liquid scintillation counter. Each disc represented one assay point. Incorporation for each assay point was normalized on a protein basis (Bradford, 1976) from aliquots obtained from the original sonicate. Each final data point shown in Fig. 4 was the mean from triplicate assays from two experiments. Pulse-Chase Autoradiographic Analysis of [3HJProline and “SO4 Incorporation into Hydra Mesoglea

To determine the kinetics of incorporation of matrix proteins into Hydra mesoglea and as a control for matrix inhibitor drug studies, pulse-chase autoradiography was performed. Hydra were given a microinjection of each radiotracer (10 pCi[3H]proline/ml HM and 400 yCi 35S0,/ml HM, both obtained from New England Nuclear (Wilmington, DE) into the gastric cavity through the mouth (Campbell, 1965; David and Campbell, 1972) followed by a lo-fold excess of unlabeled tracer for chase studies. A pulse of 3 hr was performed prior to initiation of the chase phase. Hydra were fixed immediately after the end of the pulse period (T,) or at various times during the chase ( T3,,*, T16hr, and T%hr). Fixation, processing, and use of film emulsions for autoradiography was performed as described previously (Sarras et al, 1985). Pulse-chase experiments were conducted in the presence of various drugs that affect the synthesis or assembly of matrix molecules to determine if these drugs actually perturbed synthesis of mesoglea components. RESULTS

Functional Analysis of ECM Components during Hydra Head Regeneration

To determine if mesoglea components have a functional role in Hydra head regeneration, studies were undertaken with compounds that affect the synthesis and/ or processing of collagen and proteoglycan molecules. The potential role of collagens in head regeneration was studied using agents that affect secondary or quaternary structure of collagens to include APN, DP, and cishydroxyproline, while the role, of proteoglycans was studied using P-xyloside which is an agent that inhibits addition of glycosaminoglycan (GAG) chains to the proteoglycan core molecule. In the case of proteoglycan studies, the inactive a-isomer form was used as a control. Preliminary dose-response studies were conducted to determine optimal concentrations for each compound. As shown in Table 1, drugs that affected collagen and proteoglycan synthesis inhibited head regeneration as monitored over a 4%hr period when compared to controls. While a higher than expected number of Hydra were inhibited with a-xyloside, the active isomer form,

SARRAS,

MEADOR,

TABLE 1 INHIBITORY EFFECT OF DRUGS WHICH PERTURB COLLAGEN OR PROTEOGLYCAN SECONDARY STRUCTURE OR PROCESSING ON HEAD REGENERATION IN HYDRA”

Treatment Control Control 0.4 m&f 0.1 mM 1.0 mM 5.0 mJ1 5.0 mlZl

Percentage of blockage of head regeneration

groups

HM* HM + 0.05% APNd DP’ cis-OH-proline” 13.Xylosideg n-Xylosiden

DMSO’

497

AND ZHANG

8 0 75 85 54 90 50

(Y/Ill)’ (O/21) (45/60) (51/60) (61/113) (44/49) (22/44)

a Decapitated Hydra were incubated with drugs for 48 hr and monitored for head regeneration using a Wild dissecting microscope. The percentage of Hydra which did not regenerate a head structure (hypostome and tentacles) within 48 hr is indicated for control and each drug tested. h (:ontrols for drugs that perturbed collagen secondary structure or processing (APN, DP, and cis-OH-proline) were tested in Hgdra medium (HM) which was the solvent used for these compounds. ’ Controls for drugs that perturbed proteoglycan secondary structure or processing (/+xgloside and wxyloside) were tested in Hgdra medium containing 0.05’%, DMSP which was the solvent used for these compounds. ’ +Aminoproprionitrile. ’ 2.2’-Dipsridyl. .r c,is-4-Hvdroxy-L-proline. ” ,I-Nitrc;phenyl+D-xylopyranoside. h I)-Nitrophengl-tu-I)-xylopyranoside. ’ Number of Hydra which u-ere inhibited from head regeneration per total number of Hydra decapitated are in parentheses.

fi-xyloside, had a significantly higher inhibitory effect (Table 1). To determine if the compounds were having a nonspecific toxic effect on Hydra, recovery experiments were performed following wash-out of the drugs. As shown in Table 2 head regeneration was reinitiated and was completed over a 72-hr period in a majority of the specimens once the compounds were removed from the medium.

Control Experiments to Determine the Efect of Each Drug on Mesoglea Formation and Hydra Protein Synthesis Eflect of APN on extractability of i-hydroxyproline containing proteins. A number of positive controls were conducted to determine if the compounds used were indeed affecting matrix components in Hydra. The lathyritic agents APN and DP are both reported to inhibit lysine-derived cross-links of collagen molecules (Page and Benditt, 1972; Berg and Prockop, 1973). As a result of this inhibition, collagens are more easily extracted from the matrix (Levene and Gross, 1959). To determine if collagens of Hydra were affected in this way following

treatment with lathyritic agents, a crude collagen extraction of control and APN-treated Hydra cultures was conducted. Briefly, Hydra were treated with 0.05 mM APN for 2 weeks. Control and APN-treated groups were then collected, and sonicated in a 3.4 M NaCl solution containing all proteases used in the mesoglea isolation procedure. The sonicate was centrifuged at 100,OOOg for 1 hr to obtain a pellet of all insoluble material. The pellet was then sonicated in 2 M guanidine-HCl with protease inhibitors and the resulting sonicate was again centrifuged at 100,OOOg for 1 hr. Collagen content was followed by determining the 4-hydroxyproline content in each supernatant and pellet fraction. The content for all fractions (normalized on a protein or mass basis) was summed and a percentage of distribution was expressed for each fraction. Under this crude isolation scheme, collagens would normally pellet after the first high salt extraction and then be at least partially extracted from this pellet by 2 M guanidine-HCl. In contrast, lathyritic agents should make collagens more susceptible to extraction at each step in the procedure. Our results indicated that Hydra treated with APN had the majority of their collagen content (91%) removed by simple high salt extraction as compared to 0% in controls. The remaining collagen content was then totally extracted by 2 Mguanidine-HCl in APN-treated specimens; whereas, only 70% was extracted from the high salt pellet in controls under these same conditions. Effects ofeuclz drug on Hydra protein synthesis. The effect of each drug on protein synthesis was monitored using [35S]cysteine as a precursor. Under the conditions

TABLE 2 RECOVERY OF HEAD REGENERATION IN SPECIMENS WASHED DRUGSWHICHPERTURBEDCOLLAGENORPROTEOGLYCANSECONDARY STRUCTURE OR PROCESSING”

Treatment 0.4 0.1 1.0 5.0

groups

mM APN” mM DP’ rnhl cis-OH-proline” rnnf @-Xyloside’

Percentage of specimens recovered completel!: after 82 86 87 87

FREE OF

which blockage

(37/45)f (44/51) (53161) (39/44)

’ At 48 hr of blockage, for each drug tested, Hydra were washed free of the drug and then observed for an additional 72 hr. At the end of this period, the number of Hydra which recovered with complete head regeneration were noted as indicated above. Nonrecovered Hydra were observed to either survive without recoverg or die during the recovery observation period. ’ ij-Aminoproprionitrile. ’ 2,2’-Dipyridyl. ’ cis-4-Hydroxy-L-proline. ’ p-Nitrophenpl-fi-D-xylopyranoside. “Number of recovered Hydra per total number of originally blocked Hydra are in parentheses.

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DEVELOPMENTALBIOLOGY

V0~~~~148,1991

should metabolically compete for the radiotracer and a reduced number of grains should be seen over the mesoglea. As shown in Fig. 2d, cis-hydroxyproline-treated Hydra had a significantly reduced number of grains over the mesoglea as compared to controls that were given a [3H]proline pulse in the absence of the analog (Fig. 2~). DISCUSSION

Control

,5-Xyloside

APN

cis-OH-pro1

OP

FIG. 1. The effect of drugs on general protein synthesis in Hydra. As described under Materials and Methods, Hydra were exposed to the drugs under the same conditions (concentrations and incubation times) used in the head regeneration studies. Following a 1-hr pulse with [3SS]cysteine, TCA precipitable counts were determined for control and drug-treated Hydra. Counts were expressed as DPM and normalized per microgram protein. Each point represents the mean from triplicate determinations with standard error bars shown. No statistically significant differences were observed between control and drugtreated groups (P < 0.05 using an ANOVA test). p-Xyloside (5.0 mM p-nitophenyi-p-D-xylopyranoside); APN (0.4 mM fl-aminoproprionitrile); &s-OH-pro1 (1 mM cis-4-hydroxy-L-proline); DP (0.1 mM 2,2’dipydridyl).

As indicated by immunocytochemical and biochemical studies (Sarras et aZ.,1991), the mesoglea of Hydra is a primitive basement membrane that has retained some of the characteristics of interstitial ECM. Given the importance of basement membrane and ECM components in cell signaling during development, it is important to determine the role of mesoglea components during Hydra developmental processes. With this in mind, the current study has focused on the role of mesoglea collagen and proteoglycan components during head regeneration in Hydra vulgaris. The Role of Mesoglea Regeneration

Components

during Hydra Head

In regard to functional considerations, our work expands the earlier studies of Barzansky and Lenhoff (1974) and indicates that both collagens and proteoglycans have a role during head regeneration in Hydra. Alteration of collagen secondary structure through the Puke-chase autoradiographic studies to determine the use of the proline analog, cis-hydroxyproline (Uito et al., of enzymes that promote cross-linkeflects of cis-hydroxyproline and P-xyloside on mesoglea 1972), or inhibition formation. As a positive control for cis-hydroxyproline ing of collagen molecules through the use of APN or DP and P-xyloside, a pulse-chase autoradiographic ap- (Page and Benditt, 1972; Berg and Prockop, 1973) causes proach was taken. While some grains were present over a blockage of head regeneration at drug concentrations the mesoglea immediately following the pulse at !Z’,,(3- that are reversible and therefore not toxic. Positive conhr pulse) using either [3H]proline or 35S0, as radiotrols for the action of these drugs indicated (1) lathyritic tracers, maximal labeling was not observed until 24 hr agents such as APN and DP reduced cross-linking of of chase (Figs. 2a and 2~). In the case of P-xyloside, HyHydra collagens and (2) the proline analog, cis-hydroxydra were given a pulse of 35S0, and incubated during the proline, was incorporated in mesoglea components. Simi24-hr chase period with /3-xyloside at the optimal con- larly, P-xyloside reversibly blocked head regeneration centrations used in the head regeneration studies. If p- and appeared to inhibit the normal flow of sulfated xyloside is functioning as expected, fewer grains (sul- GAG chains to the mesoglea. This is in line with studies fated GAG chains) should be assembled in the mesoglea in other developing systems where it has been shown that P-xyloside blocks elongation of GAG chains on the of Hydra treated with the compound. As shown in Fig. 2b, /3-xyloside-treated Hydra had a significantly re- proteoglycan cores and thereby causes abnormal histogenesis (Lelongt et al., 1988; Bansal et ah, 1989). The acduced number of grains over the mesoglea as compared to controls (Fig. Za). cis-Hydroxyproline functions is an tion of these drugs indicates that the incorporation of analog of proline that causes disruption of normal collaabnormally structured components into the mesoglea gen secondary structure (Uito et ah, 1972). To determine retards normal head regeneration. Therefore, in these if cis-hydroxyproline was being incorporated into me- experiments blockage of head regeneration did not resoglea collagens, Hydra were given a pulse of [3H]proline sult from inhibition of synthesis of mesoglea compoin the presence of 1 mM cis-hydroxyproline and chased nents, but rather from formation of an abnormally configured matrix. Previous studies have clearly estabas normally with unlabeled proline. If cis-hydroxyproline is an effective analog of proline in Hydra, then it lished the importance of ECM and basement membrane used in the head regeneration experiments, no significant reductions (P < 0.05 using an ANOVA test) in TCA precipitable counts were observed with any of the drugs used as compared to untreated Hydra controls (Fig. 1).

SARRAS,

MEADOR,

AND ZHANG

Extrucdlular

~~utris

of’Hyth

dguris.

II

499

FIG. 2. Effect of /Gxyloside and cis-hydroxyproline on pulse-chase autoradiography of %O, and [3HJproline incorporation into Hydra mesoglea. (a and b) These show the results from %GO, autoradiography and c and d show the results from [3H]proline autoradiography. In Hydra treated with 5.0 mAf)!Gxyloside (b) a reduction of grains over the mesoglea is observed at 24 hr of chase as compared to controls not treated with the drug (a). In Hydra treated with 1.0 mMcis-OH-proline during the initial pulse with [“Hlproline, a reduction of grains over the mesoglea is observed at 24 hr of chase as compared to controls not treated with the proline analog (c). Magnifications: a-d, 615x.

components in a wide variety of developmental processes (see reviews by Ekblom et al., 1986; McDonald, 1989; Timpl, 1989). The results obtained in the present study do not distinguish whether blockage of head regeneration was due to (1) drug-induced changes in the normal three-dimensional structure of mesoglea or (2) drug-induced abnormalities in collagen and proteoglycan structure which affected epitheliaimatrix interactions via specific cell surface matrix receptor systems (Akiyama et al., 1990). In regard to Hydra matrix receptor systems, it should be noted that dissociated Hydra cells do specifically bind to isolated mesoglea (Day and Lenhoff, 1981) and the migratory behavior of cultured

Hydra nematocytes can vary depending on whether one coats the substrate with isolated mesoglea, laminin, fibronectin, or type IV collagen (Gonzalez-Agosti and Stidwill, 1990). Similar cell/mesoglea binding studies have been reported for jellyfish (Schmid and Bally, 1988), which represent another class of the phylum, Cnidarium, to which Hydra belong. Preliminary studies in our laboratory indicate that dissociated Hydra cells specifically bind rhodaminated human plasma fibronectin. Taken together, these studies imply that cell surface matrix receptors are involved with Hydra cell function. Studies are currently underway to characterize the precise structure of mesoglea components and to determine

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which domains of these molecules are critical during Hydra development. In this regard, Hydra offers the advantage that it is a well-characterized experimental model in which matrix/cell interactions can be studied under in vivo conditions in terms of a number of specific processes such as cell proliferation, cell migration, cell positioning, and cell differentiation. The authors thank Adra Witherspoon and Conley J. Lynch for their technical assistance during the early phases of this study. The authors also thank Dr. Yashpal S. Kanwar, Northwestern Univ., for supplying initial samples of cu-xyloside. The authors express a special thanks to Dr. Hans Bode, Univ. of California, Irvine, for supplying strains of Hydra vulgaris for initiation of these studies and for providing help and suggestions throughout the course of these studies. The authors thank Dr. Stanley R. Nelson for critically reviewing this manuscript prior to its submission. This work was supported in part by NIH grants RR06500 (M.P.S.); funds were awarded to M.P.S. by the Juvenile Diabetes Foundation International; and funds from the University of Kansas Medical Center BRSG program were awarded to M.P.S. Funds supporting the salary of D. Meador were awarded from the American Society of Biological Chemists through their “High School Teacher Fellowship Program.” REFERENCES AKIYAMA, S. K., NAGATA, K., and YAMADA, K. M. (1990). Cell surface receptors for extracellular matrix components. Biochim. Biophys. Acta 1031,91-110. BANSAL, M., Ross, A. S. A., and BARD, J. B. L. (1989). Does chondroitin sulfate have a role to play in the morphogenesis of the chick primary cornea1 stroma. Dev. Biol. 133, 185-195. BARZANSKY, B., and LENHOFF, H. M. (1974). On the chemical composition and developmental role of the mesoglea of Hydra. Am. 2001. 14, 575-581. BERG, R. A., and PROCKOP, D. J. (1973). The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. Biophys. Res. Commun. 52, 115-120. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Bioch,em. 72,248-254. CAMPBELL, R. D. (1965). Cell proliferation in Hydra: An autoradiographic approach. Science 148,1231-1232. DAVID, C. N. (1983). Incorporation 3H-thymidine into Hydra by microinjection. In “Hydra: Research Methods” (H. M. Lenhoff, Ed.) pp. 189-191. Plenum Press, New York. DAVID, C. N., and CAMPBELL, R. D. (1972). Cell cycle kinetics and devel-

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opment in Hydra attenuata. I. Epithelial cells. J. Cell Sci. 11, 557568. DAY, R. M., and LENHOFF, H. M. (1981). Hydra mesoglea: A model for investigating epithelial cell-basement membrane interactions, Science 211,291-294. EKBLOM, P., VESTWEBER, D., and KEMLER, R. (1986). Cell-matrix interactions and cell adhesion during development. In “Annual Review of Cell Biology” (G. E. Palade, B. M. Alberts, and J. A. Spudich, Eds.), pp. 27-47. Annual Reviews, Inc., Palo Alto, CA. GONZALEZ-AGOSTI, C., and STIDWILL, R. P. (1990). h vitro migratory behavior of isolated Hydra nematocytes on their natural extracellular matrix (ECM) and on artificial substrates coated with ECM components. J. Cell Biol. 111,290a (abstract). HAUSMAN, R. E., and BURNETT, A. L. (1971). The mesoglea of Hydra: IV. A quantitative radiographic study of the protein component, J. Exp. Zool. 177,435-446. LELONGT, B., MAKINO, H., DALECKI, T. M., and KANWAR, Y. S. (1988). Role of proteoglycans in renal development. Dev. Biol. 128,256-276. LEVENE, C. L., and GROSS, J. (1959). Alterations in state of molecular aggregation of collagen induced in chick embryos by P-aminopropionitile (Lathyrus factor). J. Exp. Med. 110,771-790. MACWILLIAMS, H. K. (1983). Grafting: A rapid method for transplanting tissues. 17~ “Hydra: Research Methods” (H. M. Lenhoff, Ed.), pp. 225-232. Plenum Press, New York. MAUGH, T. H., II. (1984). New tool for amino acid analysis. Science 225, 42. MCDONALD, J. A. (1989). Matrix regulation of cell shape and gene expression. In “Current Opinion in Cell Biology” (C. H. Damsky and M. Bernfield, Eds.), pp. 995-999, Current Science Press, PA. PAGE, R. C., and BENDITT, E. P. (1972). Diseases of connective and vascular tissues. IV. The molecular basis for lathyrism. Lab. Invest. 26,22-26. SARRAS, M. P., JR., MADDEN, M. E., MEADOR, D. T., and HUDSON, B. G. (1988). Isolation, characterization, and functional analysis of Hydra basement membrane (mesoglea). J. Cell Biol. 107,597a (Abstract). SARRAS, M. P., JR., MADDEN, M. E., ZHANG, X., GUNWAR, S., HUFF, J. K., and HUDSON, B. G. (1991). Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization. Dev. Biol. 148, SARRAS, M. P., JR., ROSENZWEIG, L. J., ADDIS, J. S., and HOSSLER, F. E. (1985). Plasma membrane biogenesis in the avian salt gland: A biochemical and quantitative electron microscopic autoradiographic study. Am. J Anaf. 174.45-60. SCHMID, V., and BALLY, A. (1988). Species specificity in cell-substrate interactions in medusae. Dev. Biol. 129, 573-581. TIMPL, R. (1989). Structure and biological activity of basement membrane proteins. Eur. J B&hem. 180,487-502. UITTO, J., DEHM, P., and PROCKOP, D. J. (1972). Incorporation of cis-hydroxyproline into collagen by tendon cells. Failure of the intracellular collagen to assume a triple-helical conformation. Biochim. Biophys. Actu 278, 601-605.