Mesenchyme Cells Degrade Epithelial Basal Lamina Glycosaminoglycan R. LANE SMITHYAND Department
School of Medicine,
Received March 22, 1982; accepted in reuised form July 21, 1982 We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGS are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.
enchyme. In the absence of mesenchyme, the epithelia can regenerate a new basal lamina which has the same characteristic ultrastructure as the lamina present in intact glands (Bernfield et al., 1977). Organ culture studies suggest that mesenchyme may be involved in basal lamina turnover (Banerjee et al., 1977). The mesenchyme appears to be deleterious to recovery of the epithelium from enzymatic removal of its lamina. It was proposed that the mesenchyme degrades the basal lamina during normal morphogenesis. We have developed an assay to establish whether mesenchyme is able to degrade the basal lamina. Epithelia are prelabeled and histologically prepared to preserve the surface label and to eliminate any endogenous enzymatic activity. When the epithelia contain labeled surface GAG, recombination with living mesenchyme stimulates the release of GAG label. The type of material released is specific for living mesenchyme and the enhanced release requires close association of the tissues. Thus, one mechanism of this morphogenetic tissue interaction may involve basal laminar GAG degradation.
Morphogenesis is a complex process involving diverse cell-cell and cell-matrix interactions within specialized extracellular environments (see reviews by Hay, 1979; Bernfield, 1980). The importance of the extracellular matrix and its turnover in organogenesis are becoming increasingly apparent through investigations seeking to characterize or to modify the pericellular environment. During mouse submandibular salivary gland morphogenesis, the epithelium requires a specialized extracellular matrix, the basal lamina, for maintenance of lobular morphology (Bernfield et al., 1972; 1973; Banerjee et al., 1977). The submandibular epithelial basal lamina contains glycosaminoglycans (GAG). After brief labeling, one-half of the newly synthesized GAG is in hyaluronic acid, 40% in chondroitin sulfate, with the remainder likely in heparan sulfate (Cohn et al., 1977). Ultrastructural studies show that this GAG is in highly ordered arrays intimately associated with the plasmalemma (Cohn et al., 1977). The surrounding salivary mesenchyme cells are essential for continued epithelial morphogenesis (Grobstein, 1953), but the synthesis, deposition, and organization of the basal lamina are independent of the mes-
Preparation 1 To whom correspondence should be addressed: Orthopedic Research Laboratory, Division of Orthopedic Surgery, R171, Stanford University, Stanford, Calif. 94305. 0012-1606/82/120378-13$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
of Fixed, Labeled Epithelia
Dissection and culture techniques. Submandibular glands were obtained from 13-day Swiss-Webster hybrid 378
SMITH AND BERNFIELD
embryos (Day 0 is the clay of the vaginal plug), the stage when the epithelial are first multilobular. The mesenthyme surrounding ealch epithelial rudiment was removed by microdissection and placed in Eagle’s basal medium (BME-Gransd Island Biological Co.) without serum at room temperature and 5% CO, in air for 1 to 2 hr. Epithelia were freed of adherent mesenchymal cells and other extracellular materials, including the basal lamina, by treatment with a freshly prepared trypsinpancreatin solution [2.1 mg trypsin, 1:300 (Nutritional Biochemicals), 9 mg pancreatin (Difco) in 10 ml Tyrode’s solution preincubated at 37°C for 10 min and filter sterilized]. Approximately 10 epithelia were washed free of serum by suspension in 20 ml of pregassed Tyrode’s. The epithelia vvere transferred to the trypsinpancreatin solution (10 ml) and incubated at 37°C in 5% COZ in air for 7 min. After incubation and under a dissection microscope, the adherent extracellular materials were removed from the epithelia by gentle pipetting through the polished tip of a finely drawn glass pipet. After each epithelium was denuded, it was placed in a solution of 1:l Tyrode’s:horse serum and maintained in 5% COZ in air prior to labeling. Labeling and fixation. Isolated epithelia were labeled with [3H]glucosamine (20.7 Ci/mmole; New England Nuclear) or [3H]glucose (5.8 Ci/mmole; New England Nuclear) at 40 pCi/ml in glucose-free BME containing 10% dialyzed horse serum, 2 mA4 glutamine, 0.1 mM pyruvate, two times the standard concentrations of BME amino acids and vitamins, and 1% antibiotic-antimycotic mixture. Labeling with 14C-amino acids (207 mCi/mmole; New England Nuclear) was done in the absence of supplemental amino acids. After washing three times with 500 ~1 labeling medium without radioactive precursor, approximately 25 denuded epithelia were labeled in 200 ~1 media. Labeling time was limited to 2.5 hr to ensure sufficient labeling without loss of viability or alterations of the epithelial surface (Cohn et al., 1977). After removal of the labeling solution and washing (500 ~1) with pregassed Tyrode’s solution, labeled epithelia were put into Carnoy’s fixative. Care was taken to gently agitate the glass dish during fixation to prevent the epithelia from sticking to it. Fixed epithelia were stored at 4°C in Carnoy’s prior to use. Carnoy’s solution wals used because it allowed retention of both lamina materials, as observed ultrastructurally, and enzymatilc susceptibility of the labeled GAGS. Other fixatives, ;such as glutaraldehyde, provided good tissue preservation but minimal radioactive material was released with either trypsin or hyaluronidase. Alcohol fixation resulted in good enzymatic release of label but tissue preservation following the rehydration was very poor. In contrast, Carnoy’s provided excellent
tissue preservation, good stability of the radioactive label during rehydration, and susceptibility of the label to either trypsin or hyaluronidase at concentrations effective with unfixed epithelia (Banerjee et al., 1977). Testicular hyaluronidase was obtained from Sigma, St. Louis, Missouri (Type VI), and from Leo Pharmaceutical Company, Helsingborg, Sweden. Crystalline trypsin was from Sigma. Autoradiography and electron microscopy. Autoradiography was as previously described (Bernfield and Banerjee, 1972), and electron microscopy was as described by Cohn et al., 1977. Assay of mesenchyme. Fixed epithelia were removed from Carnoy’s and gently flushed in 10 ml of ethanol/ water (30% v/v) for 5 min to remove the fixative. The epithelia were then transferred to microtiter dishes having beveled bottoms (0.2 ml). The 30% ethanol was removed and the epithelia were washed three times with 200 ~110% ethanol. A final wash with 200 ~1 of pregassed Tyrode’s was used to remove the remaining ethanolic solution. The rehydrated epithelia were preincubated for 2 hr at 37°C (5% CO2 in air) in 200 ~1 of recombination medium (serum-free BME supplemented with 2 mM glutamine, and two times the standard concentration of BME amino acids and vitamins and 1% antibiotic-antimycotic mixture). The epithelia were washed twice with 200 ~1 of recombination medium and left in 50 ~1 of medium before adding mesenchyme. Serum-free medium was used; inclusion of serum released epithelial label in the absence of mesenchyme. Mesenchyme and epithelia were recombined by pipetting mesenchyme pieces (two per epithelium) into the microtiter wells. After transferring the mesenchyme, all liquid was removed from the well. Recombination medium (200 ~1) was added dropwise with care to ensure that both epithelium and mesenchyme remain at the bottom of the well. Using a finely drawn pipet, epithelia were positioned into contact with mesenchyme pieces taking care not to touch the mesenchyme which adheres to glass. Control epithelia that were not recombined with mesenchyme were washed with the media in which the mesenchyme pieces were stored and were manipulated in the same manner as epithelia recombined with mesenchyme. To assess the release of radioactive material, a 20~1 aliquot was removed immediately following recombination (the initial or zero time) and lo-p1 aliquots were removed at 30-min intervals during the subsequent 150-min incubation. The amount of total epithelial radioactivity in the assay was determined after solubilizing the tissues in 1% sodium dodecyl sulfate, removing the solubilized material from wells and washing with Tyrode’s solution (2 X 0.5 ml). For biochemical analyses, sufficient released radio-
active material was obtained by large-scale preparations (15-20 epithelia) incubated for 180 min with 30-40 pieces of mesenchyme. The release of epithelial label in these experiments was greater (B-12% of the total radioactivity) than that seen in the kinetic experiments due to greater number of epithelia being handled and to better packing of mesenchyme around the epithelia. At the beginning and end of the incubation period, aliquots were taken for determining radioactivity released. Analysis
Gel filtration chromatography. The material released by the mesenchyme was analyzed by gel filtration on a Sephadex G-200 column (0.9 X 30 cm) and a Sephadex G-10 column (1.5 X 90 cm). Columns were equilibrated with 0.05 Tris-HCl buffer, pH 7.5, containing 1 M LiCl, conditions which allowed 90% recovery of applied radioactivity. For counting, aliquots (0.1 ml) of each column fraction (0.5 ml) were diluted with 0.5 ml of Hz0 and then 10 ml of Beckman Ready-Solv was added. Samples were counted in a Beckman LS-250 scintillation counter at an efficiency of 40% for 3H and 86% for 14C. Desalting procedures. To remove salts for subsequent analysis, pooled eluates were passed first through Dowex 50 and then through Dowex 1 columns with an overall recovery of 76%. The Dowex 1 column was omitted prior to P-glucuronidase treatment (100% recovery). High-voltage electrophoresis. Separation of N-acetylated glucosamine and galactosamine was done on Whatman 3 MM paper strips (10 X 56 cm) pretreated with 0.03 M sodium tetraborate, pH 9.2, after application of the samples. Electrophoresis was carried out in 0.05 M sodium tetraborate, pH 9.2, buffer and Varsol for 90 min. Voltage was constant at 50 V/cm (total 2750) (Roseman et al., 1966; Schapira et al., 1968). Standards were identified by Elson-Morgan spray reagent. Radioactivity was determined after elution of the label from the paper by overnight agitation in HzO. Thin-layer chromatography. Thin-layer analysis on cellulose was carried out by a modification of the procedure of Gunther and Schweiger (1965) in a solvent system consisting of pyridine, ethyl acetate, acetic acid, and Hz0 (36:36:7:21). Each strip was run for 2 hr and the mobilities of N-acetylhexosamine and hexosamine were determined by silver nitrate staining of the standards. Silica gel chromatography was carried out in two systems. Using the solvent system of Gal (1968), each strip was run for 7.5 hr in n-propanol and Hz0 (7:l) and hexosamine standards located by applying ammonium bisulfate and heating at 140°C for 30 min.
In a second procedure N-acetylated hexosamines and hexosamines were completely separated by using the affinity of vicinal hydroxyl groups on N-acetylgalactosamine and galactosamine for the borate ion to alter their chromatographic properties. Silica gel sheets were first pretreated with 0.03 M sodium tetraborate, pH 9.2, and then heated of 100°C for 30 min. The sugars were then resolved in a solvent system of acetone, butanol, and sodium tetraborate, 0.003 M, pH 9.2 (140:30:30). The R, values (*SD) obtained were galactosamine, 0.082 + 0.019; glucosamine, 0.146 + 0.025; N-acetylgalactosamine, 0.320 f 0.043; N-acetylglucosamine, 0.495 + 0.039. Enzymatic digestions. Chondroitin ABC lyase treatment (Miles Biochemicals) was done on pooled column fractions in 0.05 M Tris-HCl, pH 7.5, 1 M LiCl, at an enzyme concentration of 1 unit/ml of sample. These conditions were found adequate to digest authentic carrier chondroitin sulfate. After enzyme treatment the sample was concentrated by rotary evaporation for rechromatography. Prior to treatment with bovine hepatic glucuronidase (Sigma), all samples were desalted using Dowex 50. Following desalting, the samples were concentrated by lyophilization, subsequently resolubilized in buffer, and the pH was adjusted when necessary. Glucuronidase digestion of the pooled material was carried out in sodium acetate buffer (0.10 M, pH 4.5), under conditions of enzyme excess. When excess N-acetylhexosaminidase was added to the glucuronidase reaction, the conditions were altered to decrease the amount of acetate ion (0.02 M). Reaction conditions were optimized using hyaluronate tetrasaccharide as substrate. Reaction rates were determined by measuring increases in reducing sugar (Park and Johnson, 1949) and by the Morgon-Elson reaction (Reissig et al., 1955). RESULTS
To determine whether mesenchyme cells degrade epithelial basal lamina GAGS, we developed an assay relevant to epithelial-mesenchymal interactions; submandibular epithelia with prelabeled basal lamina GAGS were incubated in contact with submandibular mesenthyme. To distinguish between epithelial and mesenchymal activities, epithelia were fixed after labeling with radioactive precursors. Preparation of Fixed, Prelabeled Epithelia Basal Lamina
Salivary epithelia were isolated, labeled, fixed, rehydrated, and analyzed by electron microscopy as described under Materials and Methods. Cellular organization is maintained and the periphery of the rudi-
SMITH AND BERNFIELD
FIG. 1. Microscopy of fixed, rehydrated epithelia. Epithelia were freed of mesenchyme, fixed, and rehydrated as and Methods, prior to refixation and processing for light and electron microscopy. (a) A light micrograph showing cellular organization of the rehydrated epithelium is intact (X250). (b) An electron micrograph showing basal lamina across the boundary of two adjacent cells (X15,225). Bar is 1.0 Fm. (c) A higher-power view of the basal lamina filamentous bundles (F) und.erlying the plasma membrane (X90,188). Bar is 0.2 pm.
ment remains intact (Figs. la,b) in such rehydrated epithelia although some intracellular material appears to be lost. Higher magnification (Fig. lc) shows ruthenium red-positive material at basal epithelial cell surfaces; however, no organization of the stain is seen. Autoradiography (Fig. 2) shows localization of glucosamine label around the epithelial periphery, indicating that radioactive label is retained during rehydration. To characterize the fixed rehydrated epithelia as substrates, they were treated with enzymes using conditions and quantities of enzyme shown previously to remove GAGS and basal lamina from living epithelia (Banerjee et al., 1977). With epithelia fixed in Carnoy’s solution, release of radioactivity by trypsin and hyaluronidase was complete in 30 min. Materials released by trypsin are large in size and susceptible to hyaluronidase. Materials released by hya.luronidase are the size of oligosaccharides (Fig. 3). These studies demonstrate that the basal lamina is retained after fixation and rehydration and that glucosamine label is released from fixed epithelia under virtually identical conditions as those which remove the lamina and release GAGS from living epithelia.
described under Materials that the morphology and materials (BL) extending (BL) showing cytoplasmic
Release of Epithelial Label by Mesenchyme To assess the effects of mesenchymal cells, fixed, rehydrated, prelabeled epithelia were recombined with pieces of salivary mesenchyme. With contact between the mesenchyme and the epithelia, the pieces adhere; during the incubation period, the mesenchyme spreads around the epithelia (Fig. 4). Serum-free medium was used because the inclusion of serum in the medium released epithelial label in the absence of mesenchyme. The reaction was initiated by adding mesenchyme to the epithelia. The kinetics of release of epithelial label into the medium was measured by removing aliquots of the incubation medium at zero time and at 30-min intervals for 150 min. To normalize the data for variations in number or size of epithelia, the label released into the medium is expressed as a percentage of the total epithelial radioactivity present in the assay. The kinetic experiments were conducted with epithelia prelabeled with either [3H]glucosamine or a mixture of 14C-amino acids (Fig. 5). In the absence of mesenchyme, there was a linear increase in the proportion of epithelial glucosamine label that was released into
the medium. This rate increased nearly threefold when the epithelia were recombined with mesenchyme. In contrast, little label was released and no difference between the presence and absence of mesenchyme was seen when the epithelia were labeled with amino acids (Fig. 5). The release of epithelial label was assessed further in reactions where the state of the mesenchyme and the conditions of recombination were varied (Fig. 6). For each parameter examined, both plus and minus mesenchyme were run simultaneously. Each experiment was done in triplicate and repeated twice. Culture media conditioned by preincubation with mesenchyme for 150 min, the same period used in the kinetic experiments, do not enhance the release of epithelial label (Fig. 6A). This result, which suggests that the release of epithelial label requires the presence of tissue, allowed us to test whether close proximity of the tissues is involved in the release of label. Millipore filter discs (pore size 0.45 pm) were used to separate the tissues. Mesenchyme was either placed on top of the filter with epithelia below or epithelia were on top of the filter with the mesenchyme below. Additionally, the discs were scalloped to allow a free exchange of medium between both surfaces. The intervening filter completely
VOLUME 94, 1982 80
I I TRYPSIN o AFTER HYALURONIOASE
40 20 0
FIG. 3. Enzyme treatment of fixed, rehydrated epithelia. Rehydrated [3H]glucosamine-labeled epithelia were treated with crystalline trypsin (10 fig/ml) or highly purified hyaluronidase (70 rig/ml). Material released by trypsin was subsequently treated by hyaluronidase after the trypsin was inactivated. Released radioactivity was chromatographed on Sephadex G-200 in 0.05 M Tris-HCl buffer, pH 7.5, containing 1 M LiCl. The column was 0.9 X 30 cm; void volume at 6.5 ml, total volume at 19 ml.
abolished the effect of the mesenchyme (Fig. 6B), suggesting that close apposition of mesenchyme is involved in the release of epithelial label. To assess how the mesenchyme might be involved, frozen-thawed mesenchyme and a mesenchyme homogenate were tested. The cellular mass remaining after freeze-thaw did not increase the release of epithelial label. However, the medium recovered from the freezing
FIG. 2. Autoradiogram of a fixed, rehydrated epithelium. Epithelia were freed of mesenchyme and basal lamina, labeled in [sH]glucosamine during deposition of new lamina, fixed, and rehydrated, prior to refixation and processing for autoradiography. The [sH]glucosamine label (arrows) is present as a dense band surrounding the epithelium, corresponding to the location of the newly replaced basal lamina (X530).
FIG. 4. Recombination of mesenchymal tissue with a fixed, rehydrated epithelium. Following rehydration, the epithelium was recombined with mesenchyme as described under Materials and Methods. After 150 min incubation, the epithelium and adherent mesenchyme were removed for photography (X150). The mesenchymal cells have adhered to the epithelium and appear to have migrated around the epithelial bud.
SMITH AND BERNFIELD 6
MES it; l o 8 0
yielding a profile similar to that shown by the material released by trypsin treatment (cf Fig. 3). Thus, the material released by intact living mesenchyme is distinct from that produced by the disrupted tissue or by trypsin. Incubation of isolated epithelia with [3H]glucose also labels the basal lamina (Banerjee et al., 1977). To test
[3H] GLUCOSAMINE [14Cl AMINO ACIDS
5 _ o CONDITIONED MEDIA 0 NO MES 0. MILLIPORE
FIG. 5. Mesenchymal release of epithelial glucosamine and amino acid label. Fixed, rehydrated epithelia were recombined with salivary mesenchyme; aliquots of the recombination medium were removed for determination of radioactivity at the times indicated. Each epithelium prelabeled with [3H]glucosamine or with a mixture of “‘C-amino acids contained approximately 1500 cpm; three to four epithelia were used for each recombination. The data for [3H]glucosamine are from 12 experiments with mesenchyme (0) and 11 experiments without mesenchyme (0). The amino acid experiments were done in triplicate. Data points represent the mean and standard error of the mean.
and thawing and the mesenchymal homogenate did stimulate the release of label, although at a lower rate than living mesenchyme (Fig. 6B). Analysis
of the Epithelial
The medium from epithelial incubations with and without mesenchyme was analyzed for released GAG radioactivity by cetylpyridinium chloride (CPC) precipitation (Table 1). In the presence of mesenchyme nearly twice as much CPC-precipitable radioactivity, presumably GAG, was released; but over lo-fold more CPCsoluble material was released. Subsequent analysis focused on the nature of the CPC-soluble material. When the medium from epithelia incubated alone was analyzed by gel filtration, a substantial proportion of the label elutes in the void volume (Fig. 7A). The remainder of the radioactivity elutes in a broad included peak. Label released in the presence of mesenchyme contains this large-molecular-sized material but the major proportion of the mesenchyme-released material (65%) elutes near the tlotal volume. This latter profile is similar to that seen when epithelia are treated with hyaluronidase (cf Fig. 3). The epithelial label released by the eluate of frozen-thawed mesenchyme is predominantly in high-molecular-weight materials (Fig. 7B)
MES A MES HOMOGENATE l ELUATE FREEZE-THAW
0 NO MES q FREEZE-THAW MES
,a 3 2
FIG. 6. Release of epithelial label under several conditions. Experiments were done in triplicate as described in the legend to Fig. 5 and repeated at least once. A positive control with mesenchyme (0) and a negative control without mesenchyme (0) were done simultaneously for each condition tested. (A) Effect of conditioned medium and cell contact. Mesenchyme (6-8 pieces) was incubated in medium for 150 min as in a recombination experiment. This conditioned medium was transferred to dish containing three to four [3H]glucosamine-labeled epithelia to assess the release of epithelial label (0). Mesenchyme was separated from epithelia by Millipore filters (THWP, 0.45 wm) cut to allow exchange of media (A). No differences in epithelial label released were found when epithelia were above or below the filter. (B) Effect of disrupting mesenchymal cells. Mesenchyme was either homogenized in a Teflon-glass homogenizer (A) or disrupted by repeated freezethawing. The soluble fractions (w) and the residual mesenchymal mass (0) from the freeze-thaw treatment were tested independently.
VOLUME 94, 1982 1.4
whether a similar profile of epithelial label is produced by mesenchyme, epithelia prelabeled with [3H]glucose were incubated with mesenchyme and the released label analyzed by G-200 gel filtration (Fig. 7C). For this prucursor, the materials released eluted similarly to those for [3H] glucosamine.
. MES 0 NO MES
1.2 1.0 0.8
Recombinations were carried out with mesenchyme dissected from lung, jaw, and trachea, and, to control for the presence of living tissue, with living salivary epithelia (Fig. 8). Each type of mesenchyme released epithelial label at a similar rate, equivalent to that with salivary mesenchyme. In contrast, recombination with living salivary epithelia had little effect on the release of label, nor did similar recombinations with aggregates of living chick neural retina cells (data not shown). These data indicate that release of label is not a consequence of recombination with living tissue but appears to be due to the presence of mesenchymal tissue. Living epithelia and neural retina aggregates, unlike the mesenchyme, did not exhibit the strong physical attraction seen between the mesenchymal tissue and the fixed epithelia.
0.4 0 ii< Y E
To assess the nature of the released epithelial label, the included volume of the Sephadex G-200 columns was rechromatographed on Sephadex G-10 columns (Fig. 9). Although all of the labeled material released from control epithelia eluted in the void volume, more than 40% of the label released by mesenchyme eluted in the included volume, with nearly 20% eluting as a monosaccharide (Fig. 9A). Data from several preparative experiments are sum-
0.2 0 0.6
; E ;
zl s E
Nature of the Mesenchyme-Released
0.1 0 1.0 I 0.8
Epithelia alone Epithelia with mesenchyme
label released CPC precipitable
Note. Aliquots of released material were removed, boiled for 3 min, and digested with thermolysin for 3 hr at 37°C. Carrier chondroitin sulfate was added and the glycosaminoglycans precipitated with cetylpyridinium chloride. Samples were filtered on Whatman glass-fiber filters after incubation at 37°C for 30 min. Averages were of duplicate samples.
0.6 0.4 0.2 0
TABLE 1 CETYLPYRIDINIUMCHLORIDE(CPC) PRECIPITATIONOFMATERIAL RELEASEDBYMESENCHYME
FIG. 7. Analysis of material released by mesenchyme. Epithelial radioactivity released during 150 min incubation was chromatographed on Sephadex G-200 as in Fig. 3. The radioactivity in each elution profile (ca. 10,000 cpm with mesenchyme or extract, 5000 cpm without mesenchyme) is plotted as the percentage of epithelial label released during the incubation. (A) Radioactivity released during recombination of [3H]glucosamine-labeled epithelia with (0) and without (0) mesenchyme. (B) Radioactivity released during incubation of [3H]glucosamine-labeled epithelia with mesenchyme killed by repeated freeze-thaw cycles (m). (C) Radioactivity released during recombination of [3H]glucose-labeled epithelia with (0) and without (0) mesenchyme.
marized in Table 2. The percentage epithelial label released is greater than that in the kinetic experiments because larger numbers of epithelia were handled simultaneously. In these experiments mesenchyme re-
SMITH AND BERNFIELD
SALIVARY LUNG A JAW v TRACHEA l l
0 NO ME!S A EPITHELIA
revealed label at the origin and at mobilities corresponding to glucosamine, possibly produced after Dowex chromatography, and N-acetylglucosamine (Fig. 11A). To confirm the composition, desalted material was treated with 1 M HCL at 110°C overnight prior to TLC on silica gel. This deacetylated material migrated exclusively as glucosamine (Fig. 11B). Thus, the labeled monosaccharide released is N-acetylglucosamine. 400
FIG. 8. Release of epithelial label by different tissues. Recombination experiments were performed as in Fig. 5 with 13%day salivary (B), 11-day lung (a), 131/r-dayjaw (A), and 11-day trachea (V) mesenchyme as well as with 13Wday salivary epithelium (A) and no tissue (0). The amounts of mesenchyme were adjusted so that the tissue mass surrounding the labeled epithelia was visually comparable to that for salivary mesenchyme. Two living epithelia were recombined with each labeled epithelium. All recombinations were tested in triplicate and repeated at least once. Only the means of the mesenchymal recombinations are presented.
leased more than twice the epithelial label than controls (Table 2A). This difference was due exclusively to the three-fold greater label eluting in the included volume of G-200 chromatograms (Table 2B). When these G200-included materials were chromatographed on G-10, none of the label released from control incubations eluted in the included volume (Table 2C). In contrast, the bulk of the label relseased by mesenchyme eluted in the included volume. Identification and Characterization Mesenchyme-Releasea! Material
To identify the material eluting as a monosaccharide on Sephadex G-10 (cf. Fig. 9A) this fraction was pooled, aliquots were lyophilized for high-voltage electrophoresis (HVE) and desalted on a Dowex mixed-bed ionexchange column prior to lyophilization for analysis by thin-layer chromatography (TLC). The label in the monosaccharide fraction migrated exclusively as Nacetylglucosamine on HVE (Fig. 10). Silica gel TLC
FIG. 9. Enzymatic treatment of released epithelial label. The material eluting in the included volume of Sephadex G-200 chromatograms (Fig. 7) was fractionated on a Sephadex G-10 column equilibrated with Tris-HCl buffer, 0.05 M, pH 7.5, containing 1 M LiCl. The column was 1.5 X 87 cm; void volume at 54 ml. The arrow designates the elution position of sucrose. (A) The epithelial label included on the G-200 column is distributed in three principal fractions on the G-10 column. A significant proportion of material elutes at the void volume. A second class of material, more heterogeneous in nature, elutes between 55 and 75 ml. Finally there is homogeneous material eluting after 75 ml. (B) Material present in the shaded region of A was pooled, treated with chondroitin ABC lyase overnight, and rechromatographed. (C) Material present in the shaded region of A was pooled, desalted on Dowex 50, incubated overnight with P-glucuronidase which contained hexosaminidase activity, and then rechromatographed.
To assess the nature of the label eluting in the included volume on G-10 (cf. Fig. 9) the materials (shaded fraction, Fig. 9A) were pooled. Treatment with chondroitin ABC lyase and rechromatography on Sephadex G-10 (Fig. 9B) produced little change in the profile, indicating either that the material did not contain susceptible linkages, the resolution of the G-10 column was inadequate to detect any cleavages, or the material was sufficiently heterogeneous to obscure any cleavages. To evaluate whether GAG oligosaccharides might be in this heterogeneously eluting material, we investigated whether it contained glucuronic acid in glycosidic linkage, a characteristic of authentic GAGS. The plan was to treat with a mixture of P-glucuronidase and N-ace-
Percentage of epithelial label released
Sample A. Recombination
B. Gel filtration of recombination medium on Sephadex G-200’ 1. Void volume 2. Included volume C. Gel filtration of G-200included volume on Sephadex G-10d 1. Void volume 2. Included volume
5.61 f 0.70 (71
12.07 f 1.13 (I51
2.46 f 0.50 2.72 IL 0.45 (4)
2.48 f 0.28 8.56 f 0.49
3.50 f 0.52 5.04 * 0.54 (4)
“8765432111234567 Origin DISTANCE
FIG. 10. High-voltage electrophoresis. The radioactive material eluting after 75 ml on Sephadex G-10 chromatography (Fig. 9A) was lyophilized, dissolved in water, and applied to Whatman 3 MM paper (10 X 56 cm). The paper was wet with 0.03 M sodium tetraborate, pH 9.2. Electrophoresis was carried out in 0.05 M sodium tetraborate, pH 9.2, at 2750 V for 90 min.
TABLE 2 EPITHELIAL LABEL RELEASED BY MESENCHYME
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a Standard errors of the mean are presented. The number of trials for each sample is given in the parentheses. * For analysis of epithelial label released, large-scale incubations were prepared as described under Materials and Methods. All epithelia were prelabeled with [3H]glucosamine. The incubation period was 180 min. ’ The recombination medium radioactivity was fractionated into void volume and included volume. Radioactivity in each fraction is expressed as the percentage of epithelial label released. In a typical scale-up experiment for analysis of radioactivity, the total epithelial radioactivity was 28,825 cpm and 21,553 for minus and plus mesenthyme, respectively. Released radioactivity was 884 cpm (3.1%) without mesenchyme and 2720 cpm (12.6%) in the presence of mesenthyme. d The radioactivity in the pooled included fractions from G-200 chromatography was applied to a Sephadex G-10 column. All of the radioactivity from incubations without mesenchyme eluted in the void volume.
tylhexosaminidase, to assay for release of amino sugar, and to assess whether this release was dependent on glucuronidase activity. Treatment of the heterogeneously eluting material with a glucuronidase preparation containing hexosaminidase activity converted much of the material to a peak eluting as a monosaccharide (Fig. 9C). Production of monosaccharide by this treatment was further assessed with added hexosaminidase in the presence and absence of a glucuronidase inhibitor (Lewy, 1952). Conditions were established in which the glucuronidase was completely inhibited by saccharic acid, 1,4-lactone. Using authentic tetrasaccharide prepared from hyaluronic acid, we could show by Morgan-Elson determinations that these conditions prevented the release of N-acetylglucosamine by the dual enzyme mixture. The heterogeneously eluting material was treated with the dual enzyme mixture with and without the inhibitor. The reaction mixtures were chromatographed on a Dowex 1 column which removes the heterogeneously eluting material but does not retard N-acetylhexosamine (Table 3). In the absence of the enzymes, no radioactivity was eluted. In the presence of the enzyme about 30% of the sample appeared as N-acetylhexosamine and this value was significantly reduced by the inhibitor. Thus, inhibition of glucuronidase decreased the release of N-acetylglucosamine by P-hexosaminidase. Consequently, the material must contain GAG oligosaccharides. Mesenchymal
Release of Label from Live Epithdia
The identification of N-acetylglucosamine as a product of the epithelial-mesenchymal interaction made
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1 2 3 4 5 6 7 IB 9 1011121314151617181920 MIGRATION
FIG. 11. Thin-layer chromatography. (A) Radioactive material obtained, as in Fig. 10, was desalted, lyophilized, and applied to activated silica gel previously wet with 3 mM sodium tetraborate, pH 9.2, and chromatographed in acetone:butanol:0.003 M sodium tetraborate, pH 9.2 (7:1.5:1.5, by volume). (B) Radioactive material obtained as in Fig. 10 was desalted, lyophilized, and deacetylated by heating in 1 A4 HCl at 100°C for 3 hr, dried, and chromatographed as above.
possible a test of whether living epithelia could replace the fixed, prelabeled ep.ithelia as substrates. Live epithelia was isolated, labeled for 2.5 hr, and recombined with mesenchyme. The release of epithelial label was followed by passing the medium through a mixed-bed ion-exchange column. Mesenchyme stimulates the release from the live epithelia of neutral-labeled material, presumably N-acetylglucosamine (Fig. 12). Thus, mesenchyme catalyzes the release of epithelial label from living, as well as fixed epithelia.
mesenchyme in morphogenesis likely includes matic remodeling of the extracellular matrix. An Effect of Mesenchyme
Glandular epithelia require mesenchyme for their morphogenesis (Grobstein, 1967; Hodges, 1969; Kratochwil, 1972; Masters, 1976). The mesenchyme requirement includes essential soluble factors (Wessells and Cohn, 1967; Ronzio and Rutter, 1973). Varying degrees of mesenchymal specificities have been demonstrated (Grobstein, 1967; Cunha, 1972; Lawson, 1974; Ball, 1974; Sakakura et al., 1976; Kratochwil and Schwartz, 1976; Durnberger and Kratochwil, 1980). Submandibular salivary epithelial morphogenesis depends, in part, on the specialized extracellular matrix synthesized by the epithelia, the basal lamina (Bernfield et al., 1972). This basal lamina contains GAGS (Banerjee et al., 1977; Cohn et al., 1977) which are continuously and rapidly turning over (Bernfield and Banerjee, 1982). To evaluate whether the mesenchyme is involved in this turnover, we prepared epithelia as substrates and assessed whether mesenchyme catalyzed the release of GAG label. Our autoradiographic results show that the radioactive label from glucosamine persisted at the epithelial surface after rehydration. Furthermore, this epithelial label was susceptible to exogenous enzymes at levels identical to those that remove the basal lamina from living epithelia. Analysis of the epithelial label released by trypsin and by testicular hyaluronidase showed the TABLE 3 ENZYMATICDEGRADATIONOFHETEROGENEOUSLY ELUTINC RADIOACTIVITY Percentage of sample counts per minute as N-acetylhexosamine
Experimentally induced loss and replacement of the submandibular embyronic basal lamina alters epithelial morphology (Bernfield et al., 1972; Banerjee et al., 1977). The basal lamina is continuously degraded and replaced with the site of greatest turnover coinciding with the areas of maximal morphological change (Bernfield and Banerjee, 1982). We examined the possible role of the mesenchyme in basal lamina turnover by recombination experiments using as substrate fixed, prelabeled epithelia which retain basal lamina GAGS. Kinetic analysis of the release of epithelial label by mesenchyme and analysis of the products of the mesenchyme-epithelial recombinations establish that the mesenchyme degrades epithelial surface GAG. Therefore, the epithelial requirement for
Enzyme mixture + inhibitor 19 17
Note. Enzymatic reactions were carried out in 70.~1 reaction mixtures in 0.03 M sodium acetate, pH 4.5, containing 50 pg of 1:l enzyme mixture of fl-glucuronidase and N-acetylhexosaminidase. Reactions were run at 37’C overnight, placed on Dowex 1 (acetate), and the free N-acetylhexosamine was eluted and counted. The inhibition of p-glucuronidase was carried out at a final concentration of 0.02 M saccharic acid 1,4-lactone. The inhibition was complete as shown by MorgenElson and reducing sugar assays using authentic hyaluronic acid tetrasaccharide under identical conditions to those for the digests. NAcetylhexosaminidase was shown to be unaffected by this concentration of inhibitor when tested with susceptible substrate. o Average of duplicate samples. b Average of triplicate samples.
expected products. Together, the data suggest that the epithelia and epithelial labels are suitable substrates for recombination experiments with living mesenchyme. During 150 min incubation of rehydrated epithelia, there was little loss of radioactivity, reaffirming that the state of the fixed tissue prior to recombination was good. In the presence of mesenchyme, release of epithelial radioactivity did not occur when amino acid precursors were used; however, with labeled sugars, augmented release was linear with respect to time over 150 min. Addition of mesenchyme to rehydrated epithelia had two consequences: (a) the mesenchyme bound to and spread over the epithelial rudiment; and (b) the release of epithelial radioactivity was enhanced. Mesenchymal
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Release of Epithelial
The augmented release of radioactivity was abolished when close association between epithelia and intact living mesenchyme was prevented. We do not know whether direct tissue contact is required or whether this result is a consequence of membrane-linked enzymes, local enzyme concentration, pH, or other factors. Disruption of the mesenchymal tissue did release radioactive label but, as shown, did not yield products characteristic of the label released by intact mesenchyme. The recombinations were done in the absence of serum, indicating that no serum-derived factor is involved. The presence of living mesenchymal cells is required for release of epithelial label. Neither living epithelia nor neural retina aggregates produced significantly enhanced release of label. Lack of tissue specificity for mesenchyme was shown in the kinetics of epithelial label release. No differences in mesenchymal affinities for the epithelia were seen, and the stimulation of release was similar for each tissue. These findings suggest that the degradative influence of the mesenchyme is not specific to a morphogenetic pattern and may not be limited to submandibular development. Analysis of the released epithelial radioactivity by cetylpyridinium chloride precipitation showed that glycosaminoglycan was released by mesenchyme. However, the greatest proportion of material released by mesenthyme was not precipitable. These findings dictated further analysis of the nonprecipitable radioactivity to determine the size and chemical nature of the degradation products. Our results show that these products are low molecular weight and heterogeneous, and, consist in part, of N-acetylglucosamine. The heterogeneous material was susceptible to further degradation by exogenous glucuronidase and hexosaminidase, but this was markedly reduced in the presence of a specific inhibitor of glucuronidase. Therefore, because uranic acid in glycosidic linkages is unique to GAG, a portion of the het-
LIVE EPITHELIA l MES 0 NO MES
TIME (minutes) FIG. 12. Mesenchymal release of label from living epithelia. Epithelia were freed of mesenchyme, labeled with [3H]glucosamine, washed, and then, without fixation, immediately recombined with mesenchyme as described in Fig. 5. Aliquots of the recombination media were taken at the indicated times and applied to 0.5 X 4-cm columns consisting of a layer each of Dowex 50 and Dowex 1. The columns were eluted with water and the eluate was counted. Recombinations were carried out in triplicate and repeated. The means and the standard error of the means are plotted.
erogeneous material must be in GAG oligosaccharides. The nature of the remaining material is unknown, but may include hyalobiuronate which is known to be resistant to glucuronidase (Linker et al., 1955; Aronson and de Duve, 1968). The mesenchyme also released N-acetylglucosamine from living epithelia, thus confirming the results obtained with fixed epithelia. Because the GAG on the living epithelia is in the basal lamina (Banerjee et al., 1977), this finding implies that mesenchymal degradation of basal laminar GAG occurs physiologically. Moreover, the ion-exchange technique used may allow a facile assay for investigating mesenchymal degradation in other systems. Enzymatic
Basis for GAG Degradation
The basis of the mesenchymal degradative activity demonstrated here is unknown. During the embryogenesis of several tissues, there is temporal correlation in the appearance of lysosomal hyaluronidase and the loss of hyaluronic acid, suggesting that lysosomal hyaluronidases may be responsible for extracellular matrix degradation (Toole and Trelstad, 1971; Toole, 1972; Orkin and Toole, 1980). Studies on mouse salivary mesenthyme indicate that in addition to lysosomal activity, nonlysosomal hyaluronidases active at near-neutral pH exist and may also be involved in matrix degradation (Banerjee and Bernfield, 1979). Of particular interest here is that the size of the major
SMITH AND BERNFIELD
proportion of mesenchyme products are tetrasaccharide or smaller. Available evidence (Hayashi, 1977; Hayashi, 1978; Hayashi et al., 1979; Ingmar and Wasteson, 1979) suggests that lysosomal glycosidases can sequentially degrade glycosaminoglycans to hyalobiuronate and monosaccharides. Indeed, most of the heterogeneous material seen on Sephadex G-10 corresponds to trisaccharide, and the most prominent degradation product was N-acetylglucosamine. Although these products suggest that mesenchymal glycosidases are involved in the degradation, it is not known whether they are lysosomal in origin and able to act on endocytosed epithelial GAG, or, whether they are at or near the cell surface. Evidence exits for cell-surface-associated N-acetylglucosaminidase and glucuronidase activities but their physiologic significance is unclear (Bach and Geiger, 1978; Willcox, 1978; Von Figura, 1978; Von Figura and Voss, 1979). The degradation products released by salivary mesenchyme in recombination with epithelia provide evidence for a matrix degrading function for embryonic mesenchyme. It is our h,ypothesis that the catabolic activities demonstrated here are involved in the tissue interactions leading to the acquisition of organ morphology. Rapid turnover of extracellular matrix must occur during morphogenesis. The control of this turnover and the underlying mechanisms by which it occurs are fundamental to an understanding of normal development and pathogenesis alike. We thank Ms. Margareta !Svensson-Rosenberg, Suzanne Tharpe, and Patty Glennon for their assistance and acknowledge contributions of Drs. Shib Banerjee and Bernadette van der Schueren. We would also like to thank Dr. Klaus Kratochwil for helpful discussions. This work was supported by NIH Grant HD 06763. R. Lane Smith was a PHS postdoctoral fellow, Grant GM 07026.
RElFERENCES ARONSON, N. N., JR., and DE DUVE, C. (1968). Digestive activity of lysosomes. II. The digestion of macromolecular carbohydrates by extracts of rat liver lysosomes. J. Biol. Chem. 243, 4564-4573. BACH, G., and GEIGER, B. (1978). Human placental N-acetyl-&D-hexosaminidase isozymes-activity toward native hyaluronic acid. Arch. Biochem. Biophys. 189, 37-43. BALL, W. D. (1974). Development of the rat salivary glands. III. Mesenchymal specificity in the morphogenesis of the embryonic submaxillary and sublingual glands of the rat. J. Ezp. Zool. 188, 277288. BANERJEE, S. D., and BERNFIELD, M. (1979). Developmentally regu lated neutral hyaluronidase activity during epithelial-mesenchymal interaction. J. Cell Biol. 83., 469a. BANERJEE, S. D., COHN, R. H., and BERNFIELD, M. (1977). The basal lamina of embryonic salivary epithelia: Production by the epithelium and role in maintaining lobular morphology. J. Cell Biol. 73, 445463. BERNFIELD, M. (1980). Organization and Remodeling of the Extracellular Matrix in Morphogenesis. In “Morphogenesis and Pattern Formation: Implications for Normal and Abnormal Development.”
(L. L. Brinkley, B. M. Carlson, and T. G. Connelly, eds.). Raven Press, New York. BERNFIELD, M., and BANERTEE, S. D. (1972). Acid mucopolysaccharide (glycosaminoglycan) at the epithelial-mesenchymal interface of mouse embryo salivary glands. J. Cell Biol. 52, 664-673. BERNFIELD, M., and BANERJEE, S. D. (1982). The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis. Dev. Biol. 90, 291-305. BERNFIELD, M., BANERJEE, S. D., and COHN, R. H. (1972). Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. J. Cell Biol. 52, 674-689. BERNFIELD, M., COHN, R. H., and BANERJEE, S. D. (1973). Glycosaminoglycans and epithelial organ formation. Amer. Zool. 13,10671083. COHN, R. H., BANERTEE, S. D., and BERNFIELD, M. R. (1977). The basal lamina of embryonic salivary epithelia: Nature of glycosaminoglycan and organization of extracellular materials. J. Cell Biol.
73, 464-478. CUNHA, G. R. (1972). Support of normal salivary gland morphogenesis by mesenchyme derived from accessory sexual glands of embryonic mice. Anat. Rec. 173, 205. DURNBERGER, H., and KRATOCHWIL, K. (1980). Specificity of tissue interaction and origin of mesenchymal cells in the androgen response of the embryonic mammary gland. Cell 19, 465-471. GAL, A. E. (1968). Separation and identification of monosaccharides from biological materials by thin layer chromatography. Anal. Biochem. 24, 4522461. GROBSTEIN, C. (1953). Epithelial-mesenchymal specificity in the morphogenesis of mouse submandibular rudiments in vitro. J. Ezp. Zool.
124,383-404. GROBSTEIN, C. (1967). Mechanism of organogenetic tissue interaction. Nat. Cancer Inst. Monogr. 26, 279-299. GUNTHER, M., and SCHWEIGER, H. (1965). Dunnschichtchromatographie von Aminozuckern auf cellulosepulver. J. Chromatogr. 17,602. HAY, E. D. (1979). “Cell-matrix Interaction in Embryonic Induction. International Cell Biology, 1976-1977.” Papers presented at the First International Congress on Cell Biology, Boston, 1976. (B. R. Brinkley and K. R. Porter, eds.). Rockefeller University Press, New York, 1977. HAYASHI, S. (1977). Study on the degradation of glycosaminoglycans by canine liver lysosomal enzymes I. The mode of contribution of hyaluronidase, fi-glucuronidase, and P-N-acetylhexosaminidase on hyaluronic acid. J. Biochem. 82, 1287-1295. HAYASHI, S. (1978). Study on the degradation by canine liver lysosomal enzymes II. The uronidase, /3-glucuronidase, sulfatase, and dase in the case of chondroitin 4sulfate. J.
of glycosaminoglycans contributions of hyalfi-N-acetylhexosaminiBiochem. 83, 149-157.
HAYASHI, S., KIMURA, A., and TSURUMI, K. (1979). Contribution of P-glucuronidase to the degradation of chondroitin 4-sulfate by canine liver lysosomal enzymes. Tohoku J. Exp. Med. 127, 317-326. HODGES, G. (1969). Stromal-epithelial interactions. In “Biology of the Periodontium” (T. Melcher, ed.), pp. 27-52. Academic Press, London. INGMAR, B., and WASTESON, A. (1979). Sequential degradation of a chondroitin sulfate trisaccharide by lysosomal enzymes from embryonic-chick epiphyseal cartilage. Biochem. J. 179, 7-13. LEVVY, G. A. (1952). The preparation and properties of fl-glucuronidase. 4. Inhibition by sugar acids and their lactones. Biochem. J.
52,464-472. LINKER, A., MEYER, K., and WEISSMAN, B. (1955). Enzymatic formation of monosaccharides from hyaluronate. J. Biol. Chem. 213,
LAWSON, K. A. (1974). Mesenchyme specificity in rodent salivary gland development. The response of salivary epithelium to lung mesenchyme in vitro. J. Embryol. Exp. Morphol. 32, 469-493. MASTERS, J. R. W. (1976). Epithelial-mesenchymal interaction during lung development: The effect of mesenchymal mass. Deu. Biol. 51,
98-108. R. W., and TOOLE, B. P. (1980). Isolation and characterization of hyaluronidase from cultures of chick embryo skin and muscle derived fibroblasts. J. Biol. Chem. 255, 1036-1042. PARK, J. T., and JOHNSON, M. S. (1949). A submicrodetermination of glucose. J. Biol. Chem. 181, 149-151. REISSIG, J. L., STROMINGER, J. L., and LELOIR, L. F. (1955). A modified calorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217, 959-966. ROSEMAN, S., CARLSON, D. M., JOURDIAN, G. W., MCGUIRE, E. J., KAUFMAN, B., BASU, S., and BARTHOLOMEW, J. (1966). Animal sialic acid transferases (sialyltransferases). In “Methods in Enzymology, Vol VIII, Complex Carbohydrates” (E. F. Neufeld and V. Ginsberg, eds.), pp. 354-372. Academic Press, New York. RONZIO, R. A., and RUTTER, W. J. (1973). Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia. Deu. Biol. 30, 307-320. ORKIN,
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SCHAPIRA, G., ROSA, J., MALEKNIA, N., and PADIEU, P. (1968). Methods of identification of peptides during hemoglobin biosynthesis and measurement of their sequential synthesis. In “Methods in Enzymology, Vol. XIIB, Nucleic Acids” (L. Grossman, and K. Moldave, eds.), pp. 757-758. Academic Press, New York. SAKAKURA, T., NISHIZUKA, Y., and DAME, C. J. (1976). Mesenchymedepende It morphogenesis and epithelium-specific cytodifferentiation in n ouse mammary gland. Science 1439-1441. TOOLE, B. P. (1972). Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton. Deu. Biol. 29, 321329. TOOLE, B. P., and TRELSTAD, R. L. (1971). Hyaluronate production and removal during cornea1 development in the chick. Dev. Biol.
26, 28-35. VON FIGURA, K. (1978). Secretion of P-hexosaminidase human skin fibroblasts. Exp. Cell Res. 111, 15-21.
VON FIGURA, K., and Voss, B. (1979). Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts. Exp. Cell Res.
121,267-276. WILLCOX, P. (1978). Secretion of P-N-acetylglucosaminidase isoenzymes by normal human fibroblasts. Biochem. J. 173, 433-439.