Embed Size (px)
Citation preview
J. Cell Sci. 25, 335-354 0977) 335Printed in Great Britain
EXTRACELLULAR MATRIX SYNTHESIS IN
BLASTULA AND GASTRULA STAGES OF
NORMAL AND HYBRID FROG EMBRYOS
III. CHARACTERIZATION OF GALACTOSE- ANDGLUCOSAMINE-LABELLED MATERIALS
KURT E. JOHNSON*Box 3011, Department of Anatomy, Duke University Medical Center, Durham,North Carolina 27710, U.S.A.
SUMMARYNormal Rana pipiens gastrulae show more incorporation of isotopically-labelled galactose and
glucosamine into TCA-insoluble materials than blastulae. Interspecific hybrid embryos whichundergo developmental arrest at the onset of gastrulation often synthesize reduced amounts ofgalactose- and glucosamine-labelled materials. These materials are high-molecular weight, butnot collagen. After pronase digestion, labelled materials elute in the void volume of SephadexG-50. Labelled materials migrate slowly on cellulose acetate, bind to several kinds of anionexchangers and elute at low ionic strength, are precipitated by cetyl pyridinium chloride (CPC)when mixed with carrier compounds, and are not degraded by Clostridium neuraminidase orStreptomyces, leech, and bovine testicular hyaluronidases.
INTRODUCTION
Rana pipiens embryos show an increase in the amount of toluidine blue metachro-matic material (TBMM) in the extracellular matrix during gastrulation. Differentkinds of arrested hybrid embryos do not show the same increase in amount ofTBMM (Johnson, 1976, 1977a). Autoradiographic studies, using [3H]galactose and[3H]glucosamine as precursors, have revealed that normal embryos synthesize andsecrete extracellular matrix components during gastrulation and that one arrestedhybrid appears to be partially defective in the secretion of presumptive matrix com-ponents (Johnson, 1976, 19776). In the present study, details of the characterizationof galactose and glucosamine-labelled materials made by different kinds of normaland hybrid embryos will be presented.
MATERIALS AND METHODS
Frogs and embryos were obtained as described previously (Johnson, 1977a).Short-term incorporation studies. For several of the isotope incorporation experiments reported
below, radioactive sugars were dried in small sterile vessels and redissolved in sterile 50 %Steinberg's solution with penicillin (200 I.U./ml), streptomycin (200 /tg/ml), and sulphadiazine(saturated) added; 10 fi\ of the label-containing medium were deposited in a well of a FalconMicrotest Tissue Culture Plate (no. 3034). One embryo was transferred to the well on the tipsof Dumont no. 5 forceps, submerged and bisected. After incubation, 10 /tl of incubation
* Reprint requests to Department of Anatomy, George Washington University School ofMedicine, 2300 Eye Street N.W. Washington D.C. 20037, U.S.A.
336 K. E. Johnson
medium without labelled sugars were added to the well and the 2 fragments of the embryowere briefly dispersed by sucking them up and down in the tip of a io-/tl automatic pipette.The dispersed embryo was then transferred to a 23-mm disk of Whatman 3MM filter paper andextracted with trichloroacetic acid (TCA), ethanol: ether, and ether according to Mans &Novelli (1961) except that the hot TCA wash was omitted. After drying, disks were placed inscintillation vials with 7 ml of toluene-Liquifluor (New England Nuclear) and counted in aBeckman LS-100 liquid scintillation counter. Using this technique, a number of differentsugars at a concentration of 250 fiM were screened as precursors to study glycoprotein andglycosaminoglycan synthesis. To see if there were changes in amount of sugar incorporationwith development, different developmental stages of normal and hybrid embryos were incu-bated for the same length of time at different developmental stages. Protein synthesis wasmonitored by incubating embryos in [3H]leucine and extracting dispersed embryos with TCAon filter paper disks exactly as described by Mans & Novelli (1961).
Production of galactose- and glucosamine-labelled materials for analytical studies has beendescribed previously (Johnson, 19776). Groups of 10-50 bisected embryos were incubated in50 % Steinberg's solution (5 /tl/embryo) containing isotopically-labelled galactose and glucos-amine at different concentrations and specific activities (see below). Embryos were incubatedfor 2-12 h at 20—24 °C, flooded with 50% Steinberg's solution, and collected in homogenizersfor further processing.
Agarose-acrylamide gel electrophoresis was performed according to Peacock & Dingham(1968) using O'S % agarose-i'5 % acrylamide. Preliminary experiments showed that galactose-labelled materials failed to enter 6 % acrylamide gels prepared according to Fairbanks, Steck &Wallach (1971). In one experiment, 27-h pipiens-pipiens late gastrulae were incubated in 1 mMPHjglucosamine (636 Ci/mivi) and 2 mM ["CJgalactose (50-4 mCi/mM) and homogenized in500 /tl 50 mM Tris-HCl pH 7 2 with 3 % sodium dodecyl sulphate (SDS) and 1 % /S-mercapto-ethanol. The homogenizer was then plunged in a boiling water bath and left for 10 min. Next,samples were centrifuged for 10 min at 1000 g. The supernatant was collected and the pellet wasresuspended in 500 fi\ Tris-SDS-A-mercaptoethanol. Both fractions were dialysed for 48 hagainst 50 mM Tris-HCl with o - i % S D S and /?-mercaptoethanol, after which samples con-tained no TCA-soluble radioactivity. After dialysis, io/tl of retentates were counted in 10 mlscintillation fluid (Anderson & McClure, 1973). The supernatant contained 90-98% of thetotal radioactivity in the sample. Supernatant fractions were prepared for electrophoresis bythe addition of SDS, /?-mercaptoethancl, pyronin Y, and glycerol (Fairbanks et al. 1971).Following electrophoresis on a 3-mm thick slab gel run at 85-100 mA for 8-14 h, gels weresliced into i-mm segments, solubilized with Protosol (New England Nuclear), mixed with8 ml toluene-Liquifluor (New England Nuclear) and counted.
Neutral sugar analysis was performed on galactose-labelled materials which had been pre-pared as described above, except that they were dialysed against deionized water rather than50 mM Tris. Retentates were collected in screw cap vials, made 2'O N with trifluoracetic acid,sealed, and hydrolysed for 2 h at 100 °C. Next, the hydrolysates were dried in a stream of nitro-gen, and redissolved in 100—200 fil of a standard solution of 10 mM D-mannose, L-fucose,D-galactose, D-xylose, and D-glucose. After brief centrifugation to remove bits of flocculentmaterial, 50 /tl of the sugar solution were chromatographed on an anion exchange resin elutedwith a borate buffer gradient, essentially as described by Lee, Johnson, White & Scocca (1971)except that the anion exchange resin used was Aminex A-25 (Biorad) and the gradient wasformed from 70 ml of 0-35 M sodium borate (pH 7-00) and 70 ml of 040 M sodium borate(pH 1000). Column effluent was collected into scintillation vials in i'5O-ml fractions. Fromeach vial, 0-30 ml was removed and tested for sugar by the addition of 255 ml of orcinolreagent made by mixing 1 vol. of 1 6 % aqueous orcinol (K & K Laboratories) with 7-5 vol. of6 0 % aqueous sulphuric acid, heating at 80 °C for 15 min, and reading the optical density at420 nm. The remainder of each fraction was counted for radioactivity after addition of 7 ml ofscintillation fluid.
Analysis of galactose- and glucosamine-labelled material synthesized during gastrulation wascarried out as diagrammed in Fig. 1 and described below. Groups of 50 18-h embryos werebisected and incubated in 25O-/tl drops of Steinberg's solution containing [3H]glucosamine and[l4C]galactose. At the beginning of the incubation, normal embryos were early gastrulae(Shumway, 1940, Stage 10) with only a trace of a blastopore. Hybrid embryos did not show any
Frog gastrula extracellular matrix, III 337
indication of the initiation of gastrulation. Incubation was carried out for 10-12 h at 22-24 °C.At the end of the incubation, normal control embryos had completed gastrulation and hybridcontrol embryos were arrested early gastrulae. Embryos were collected into a homogenizer,disrupted in 125 ml of 1 % Triton X-100 and frozen. Samples were later thawed, mixed with1-25 ml of 040 M Tris-HCl, pH 8-o, homogenized, and boiled to denature protein. After a24-48 h pronase digestion (DeLa Haba & Holtzer, 1965), samples were chilled on ice, andmade 5 % in TCA with ice-cold 50 % TCA. After 30 min on ice, samples were spun at 12000 g,4 °C, for 15 min. The resultant pellets contained no radioactivity. The supernatants were
50°,, Steinberg's solution with[iHJglucosamme and[14C]galaccoic added
Supernatant
Embryos homogenizedin pH 8 Tris-HCl and 'bo.led
Lipid extraction with A volchloroform methanol, 2 1
Pronate digestion48 h at 50 C
5% TCA
PelletCount anddiscard
Upper phase.Dialysc and lyophilize
I
Lower phase,Discard
Sephadex G-50chromatography
T4
Collect Voand lyophilize
chondroitin sulphate
Cellulose acetateelcctrophoresis
Dowex-1 (Cl )chromatography,elute with NaCI
(g)
DEAE-celluloscchromatography,elute with 0 - 1 MNaCI gradient
Add hyaluronatc
CPC-cellulosechromatography
Enzyme
Add hyaluronate
Tcmcular. Sireptomycesor leech hyaluromdase
ihromatography
cpm and O D «Tor turbidity
cpm Uronic acid
Fig. 1. Schematic representation of the preparation and analysis of galactose- andglucosamine-labelled material.
extracted with 4 vol. of chloroform:methanol (2:1) and the upper phase after this extractionwas collected and dialysed for several days against deionized water at 4 °C. The retentates fromdialysis were lyophilized, redissolved in 10 ml deionized water plus 0-50 ml glycerol, applied toa 2-5 x 60 cm column of Sephadex G-5O, and eluted with o-io M ammonium acetate in 20%(v/v) cthanol. For further analysis, void volume fractions were pooled, lyophilized, and re-dissolved in deionized water. Portions of these samples were then: (1) subjected to electro-phoresis on cellulose acetate strips (Roden, Baker, Cifonelli & Mathews, 1972); (2) applied toBiorad AG1X2 anion exchange resin and eluted step-wise with NaCI (Schiller, Slover 8cDorfman, 1961); (3) applied to Whatman DE-52 DEAE-cellulose equilibrated with 50 mM Tris-HCl and eluted with a 0-1 M NaCI gradient (Orkin, Pratt & Martin, 1976); (4) applied to
338 K. E. Johnson
cellulose columns and precipitated with i % cetyl pyridinium chloride (CPC) followed byelution with increasing concentrations of MgCl2 in 0-05 % CPC (Roden, Baker, Cifonelli &Mathews, 1972); (5) treated with Clostridium neuraminidase; or (6) treated with varioushyaluronidase preparations. Sialic acid was determined according to Warren (1959) and uronicacids determined according to Blumenkrantz & Asboe-Hansen (1973). The details of theseprocedures are presented in the appropriate Tables and figure legends below.
RESULTS
Preliminary experiments
The results of incubating pipiens-pipiens mid-gastrulae in [14C]galactose containingmedium under different conditions (Table 1) show that frog embryos have limitedincorporation unless there is at least a hole punctured in the embryo, presumablybecause they have limited permeability to sugars. There is a dramatic reduction inamount of incorporation if embryos are boiled before incubation, if embryos are
Table 1. Amount of galactose incorporation into macromolecules ofpipiens-pipiens under different conditions*
Experimental conditions
Intact embryo intact vitelline membraneHole in roof of blastocoelBisected, vitelline membrane not in wellBisected, vitelline membrane in well
(Control)Embryo cut into 4 fragmentsEmbryo dispersed at beginning ofincubation
10 mM cold galactose in mediumBoiled embryosLive embryos at 0 °CNo embryos during all of i-hincubation
Embryos bisected in well and removedat beginning of incubation
cpm [14C]galactoseincorporated + s.D.
70 ± 13648! 172664±i16
552 ±54
499 ±!°5198 + 71
27 ±5i8±424 ±328 ±3
22 ±2
% Controlvaluej
[14C]galactose
13117
1 2 0
1 0 0
9 0
36
539S
4
• Pipiens-pipiens 24-h mid-gastrulae (Shumway Stage n ) were incubated for 1 h in 500 fiMD-[i — 14C]galactose (529 mCi/mM).
f Values are the average of 6 determinations and standard deviation (s.D.) is indicated.% The % control value is the ratio of each value to the value obtained for bisected embryos
with the vitelline membrane (VM) left in the well during the incubation x 100 %.
incubated at o °C, if embryos are omitted, or if embryos are bisected in wells andimmediately removed from incubation medium. Incorporation of labelled galactoseshows isotope dilution with unlabelled galactose added to the incubation medium.There is also little difference in the amount of incorporation observed with or withoutthe vitelline membrane left in the incubation well. Similar results were obtained forglucosamine incorporation. Other preliminary experiments show that galactose and
Frog gastrula extracellular matrix, III 339
glucosamine incorporation increase with increasing time of incubation and increasinglabel concentration at constant specific activity.
Relative amounts of incorporation of different sugars
Normal pipiens-pipiens and arrested hybrid pipiens-catesbeiana (Stage 10-ioi)embryos were incubated for 2 h in different sugars and amino sugars at 250 /<M anddifferent specific activities. At the end of the incubation, embryos were dispersed,placed on filter paper disks, and extracted with cold TCA, etc., as described above.
Table 2. Sugar incorporation by pipiens-pipiens (normal) andpipiens-catesbeiana (hybrid) embryos*
Embryosf
NH
NH
NH
NH
NH
NH
SugarJ
D- [1 — 14C]galactoseD-[I — 14C]galactose
D-[I — 14C]mannoseD-[I — 14C]mannose
D-[I - 14C]glucoseD-[i-14C]glucose
L-[I — 14C]fucoseL-[i-14C]fucose
D-[U — 14C]mannosamineD-[U — 14C]mannosamine
D-[I — 14C]glucosamineD-[I — "Clglucosamine
cpm/embryo + S.D.§
370 ±61292 ±78
426 ± 92216 ±82
4i2±883i6±99
84 ±1223±7
1604±1821548 + 208
126 ± 1446 ±9
pM incorporated/embryo ||
1428n-3717.228.73
I7-4S13.38
3°5084
I4-751423
4-471-23
* Pipiens-pipiens 24-h mid-gastrulae (Shumvvay Stage 10J) and pipiens-catesbeiana 24 harrested early gastrulae were incubated for 2 h in 250 /wnolar sugar of different specificactivities.
f N = normal; H = hybrid.J The sp. act. in mCi/mM, were: galactose, 529; mannose, 505; glucose, 48-2; fucose,
56-2; mannosamine, 222; and glucosamine, 575.§ Values are the average of 6 determinations + S.D.|| This value was calculated to normalize for differences in label sp. act. from knowing the
cpm, counting efficiency, and sp. act. and assuming a negligible precursor pool.
The results are presented in Table 2. Subsequent experiments have been performedwith all precursors except mannosamine, but only results for galactose and glucos-amine will be reported in this paper. Autoradiographic experiments conducted withmannose, glucose, and fucose show that these precursors label components of theextracellular matrix and several cytoplasmic structures (Johnson, unpublishedobservations).
Amount of incorporation in different developmental stages
Developmental curves were constructed by determining the amount of galactoseand glucosamine incorporated during a 1 -h incubation period at different times duringdevelopment. For example, a determination was made for 6 embryos for the period
34° K. E. Johnson
covering 12-13 n °^ development and this value was plotted as 12 h. The same thingwas done for the period 14-15 h, and so forth. Results of a typical experiment forglucosamine incorporation are shown in Fig. 2. There is a low incorporation inblastula stage embryos (Stage 8-9). Just prior to the onset of gastrulation at 18 h(Stage 10), there appears to be an increase in the amount of incorporation whichincreases steadily in pipiens-pipiens and pipiens-catesbeiana but which increases onlyslightly in pipiens-esculenta. Similar results have been obtained for the incorporationof galactose, mannose, fucose, and glucose. In addition, galactose incorporation inanother arrested hybrid, pipiens-sylvatica is very similar to pipiens-esculenta. Finally,galactose incorporation in a viable hybrid, pipiens-palustris, is similar to pipiens-pipiens.These differences in amount of incorporation in different kinds of hybrid embryos
xEo.
10
8
6
4
2
-
A /
• /
L TN/ l
V>-~^=&^~~~& 5 g § £__£
1 1 1 1 1 1 1 1 1 1.12
8148
169
1810
2010
2210
2411
2611
28 30 h12 12 stage
Fig. 2. Glucosamine incorporation during a i-h pulse at different developmental stagesin pipiens-pipiens (O O), pipiens-catesbeiana ( • # ) , and pipiens-esculenta(A A)- Arrow indicates the beginning of gastrulation. Bisected embryos incubatedin 0-5 mM D-[6-3H]glucosamine-HCl, 1-97 Ci/mM in 50% Steinberg's solution.Points are average of 6 determinations and standard deviations are indicated byvertical bars.
might be due to a defect in the synthesis of protein acceptors. There are no significantdifferences in the amount of protein synthesis, measured by [3H]leucine incorporationand the Mans & Novelli (1961) filter paper assay, in different developmental stages ofnormal and hybrid embryos. There is a constant, high level of leucine incorporationinto TCA-insoluble material, from the early blastula to late gastrula stage in bothnormal and hybrid embryos. Also, the SDS-acrylamide gel patterns (Fairbanks et al.1971) for [3H]leucine- and [3H]proline-labelled materials from different developmentalstages of normal and arrested hybrid embryos show only minor differences.
Frog gastrula extracellular matrix, III 341
Gel electrophoresis
The galactose- and glucosamine-labelled materials made by pipiens-pipiens gastrulaeand pipiens-catesbeiana arrested gastrulae migrate slowly as a single broad peak in an°"S% agarose-i-5% acrylamide gel (Fig. 3). There are no striking differences in therate of migration of labelled materials made by gastrula (or arrest) stage normal andhybrid embryos. In the gel shown in Fig. 3, calf skin collagen and human transferrin(Sigma), applied to adjacent channels in the same gel and stained with Coomassieblue, migrate 70-75 mm. This suggests that labelled materials are not collagen.
10 20 30 40 50 60 70Migration, mm
Fig. 3. Electrophoretic behaviour of l3H]glucosamine- ( ) and [14C]galactose-( ) labelled material from 27-h pipiens-pipiens late gastrulae.
Identification of neutral sugars in labelled product
When whole embryos are incubated in neutral sugars, there are many possiblemetabolic interconversions of the label that might occur in the embryo betweentransport and final incorporation into a product. Therefore, acid hydrolysis andneutral sugar analysis of hydrolysates was performed on galactose-labelled materialfrom different developmental stages of normal and hybrid embryos.
Preliminary experiments showed that samples had no TCA-soluble counts beforehydrolysis and that all counts were TCA-soluble after hydrolysis. Furthermore, 94 %of the counts in the sample prior to hydrolysis could be recovered in the materialwhich eluted from the column. Results from typical experiments are shown in Figs. 4and 5 and Table 3.
There was commonly a small peak of radioactivity which elutes from the column in10-20 min. Amino sugars do not form borate complexes and pass through the column
342 K. E. Johnson
rapidly, e.g. glucosamine comes off the column in 16 min. The small peaks that eluteahead of mannose probably represent amino sugars. In most experiments, 90-95 %of the counts eluting from the column are in the neutral sugar peaks. When pipiens-pipiens and pipiens-palustris (a viable interspecific hybrid) embryos of different
QO
- 6 I
- 4
- 2
40 60 80
Time, min
100 120
Fig. 4. Chromatogram of hydrolysate of galactose-labelled material from 30-hpipiens-pipiens embryos. The solid line is the O.D.42o (orcinol-positive material) andthe broken line is 3H-cpm.
developmental stages were incubated in galactose, most of the neutral sugar countsremained as galactose. In most experiments, pipiens-catesbeiana embryos showed thesame pattern but in a few, substantial counts were found in mannose, fucose, galactose,and glucose. Pipiens-sylvatica embryos (an arrested hybrid) similarly show inter-conversion of galactose to mannose and glucose.
Pronase digestion and gel filtration
Following extensive pronase digestion of homogenates of galactose- and gluco-samine-labelled materials, 10-15% of the radioactive material is lost during dialysis.Of the 85-90% which is not lost during dialysis, more than 95% of the labelledmaterial elutes in the void volume of a long Sephadex G-50 column. A typical resultfor pipiens-catesbeiana embryos is shown in Fig. 6. Similar results were obtained with
Frog gastrula extracellular matrix, III 343
pipiens-pipiens, pipiens-palustris, pipiens-esculenta, esculenta-esculenta, esculenta-pipiemand esculenta-catesbeiana embryos. Pronase digests were subjected to SDS-acrylamidegel electrophoresis (Fairbanks et al. 1971). All Coomassie blue bands migratedrapidly in these gels, immediately behind the tracking dye, indicating that extensiveproteolysis had occurred during pronase digestion. Void volume fractions were thencollected, lyophilized, and subjected to further analysis. The total amount of radio-activity present in pooled G-50 void volume fractions varied in different normal andhybrid embryos (Table 4).
qO
0020 40 60 80 100
Time, mm
Fig. 5. Chromatogram of hydrolysate of galactose-labelled material from 30-h pipiens-catesbeiana embryos. The solid line is the O.D.420 and the broken line is the 3H-cpm.
Cellulose acetate electrophoresis
The bulk of the galactose- and glucosamine-labelled material from normal andarrested hybrid embryos of different sorts has an electrophoretic mobility similar tothat shown by unsulphated glycosaminoglycans such as hyaluronic acid and chond-roitin (Fig. 7). In the experiment shown in Fig. 7, equal volumes from pooled voidvolume fractions from the Sephadex G-50 step were applied to cellulose acetate strips.The relatively large amounts of material made in pipiens-pipiens and pipiens-cates-beiana and the relatively small amounts made in pipiens-esculenta correlate with thehistochemical observations presented in the first paper of this series (Johnson, 1977 a)
344 K. E. Johnson
Table 3. Relative areas* of neutral sugar peaks on chromatograms\
of hydrolysates of normal and hybrid embryos
Embryo
pipiens-pipienspipiens-catesbeiana
pipiens-pipiens
pipiens-pipienspipiens-pipienspipiens-pipienspipiens-catesbeianapipiens-catesbeianapipiens-catesbeiana
pipiens-pipienspipiens-catesbeiana
pipiens-pipienspipiens-catesbeiana
pipiens-pipienspipiens-palustrispipiens-catesbeianapipiens-sylvatica
pipiens-pipienspipiens-catesbeianapipiens-catesbeiana
Age, h
1818
18
2424242424
24
2424
2626
3°3°303°
30303°
Sugarlabel
GalactoseGalactose
Galactose
GalactoseGalactoseGalactoseGalactoseGalactoseGalactose
GalactoseGalactose
GalactoseGalactose
GalactoseGalactoseGalactoseGalactose
GalactoseGalactoseGalactose
Labelconcen-tration,
mM
i - 4
4-0
1'41-4
404-0
070-7
9 9
9 9N
N
N
N
4-0
4O
Pre-mannosej
34
3
1
522
41 1
44
89
2433
334
Man-nose
83
13
121723121017
912
1910
2211
1320
1299
Fu-cose
00
17
500
50
2512
14
148
000
12
769
Galac-tose
8445
67
7978
798638
7469
5958
70786026
767876
Xy-lose
00
0
002000
00
00
< 1< 1< 1< 1
000
Glu-cose
548
1
202208
01
016
68
2439
2
52
* The relative areas were determined by measuring the area of each sugar peak and then determining whatfraction as a percentage of the total area in all peaks was in any particular peak. The area of each peak wasdetermined on chromatograms by taking the product of the height and the width at half height. The productsfor each peak were then summed and the product for an individual peak was then calculated as a percentage ofthe total. This percentage is displayed in the table.
f Hydrolysate was dried in a stream of nitrogen and redissolved in 100-200 /tl of a standard solution of10 mM D-mannose, L-fucose, D-galactose, D-xylose, and D-glucose. After brief centrifugation to remove smallbits of flocculent material, 50 /tl of solution were injected with a sample injector valve (Glenco) on to an o-6 x24 cm water-jacketed column (Glenco) maintained at 55 °C with a circulating water bath (Lauda). The anionexchange resin was Biorad Aminex A-25. The column was eluted with a gradient formed from 70 ml of 0̂ 35 Msodium borate (pH 700) and 70 ml of 040 M sodium borate (pH io-oo) in a 2-chamber gradient maker(Glenco). The gradient was pumped at 0-75 ml/min, 103-171 kN m~2, by a Milton-Roy instrument mini-pump.
X The ' pre-mannose' peak eluted in 10-20 min after sample injection. From the flow rate on the columnand the length of tubing between sample injector valve and fraction collector, it is clear that this materialinteracts very weakly with the column and therefore is not a neutral sugar. Glucosamine elutes in 16 min,suggesting that the 'pre-mannose' peak is an unresolved mixture of amino sugars.
and with the developmental profile for galactose- and glucosamine-incorporation
described earlier in this paper. Normal esculenta-esculenta embryos and various arrested
hybrids formed from esculenta eggs do not form as much labelled material as normal
pipiens-pipiens embryos, in part because esculenta eggs are smaller than pipiens eggs
(see Discussion).
Frog gastrula extracellular matrix, III 345
10 20 30
Fraction no.
Fig. 6. Gel nitration of pronase digest of [3H]glucosamine- ( ) and [14C]galactose-( ) labelled material from 18-30-h pipiens-catesbeiana arrested hybrid embryos.Sephadex G-50, 2.5 x 60 cm, o-i M ammonium acetate in 20% ethanol. Collected4 min, 80-ml fractions, and 0-5 ml were counted for radioactivity in 10-ml cocktail.In this experiment, more than 95 % of the radioactivity was in void volume fractions.Similar results were obtained with pipiens-pipiens, esculenta-esculenta, and variousother arrested hybrid embryos.
Table 4. Relative amounts of [3H]galactose and ^C\glucosamine-labelled materialmade during gastrulation {or arrest) in several different Rana embryos
Gametecombination
pipiens-pipienspipiens-catesbeianapipiens-escidentaesculenta-esculentaesculenta-catesbeianaescidenta-pipiens
Descrip-tion
NormalArrestArrestNormalArrestArrest
Total cpm in Sephadex G-50void volume pool*
[3H]galactose
1 986 1441 622 832
147 120
597 9°4503 872246 928
[14C]glucos-amine
102 400
94302
10 528
42 480
S4S6o21 920
% of pipiens-pipiensvalue
[3H]galac-tose
1 0 0
82
73°251 2
[14C]gluCO3-amine
1 0 0
92
1 0
4 1
53t3 i
* Total cpm was calculated by summing cpm in 05 ml of 8-ml pooled fractions, subtractinga background value, and making a volume correction. The counting efficiency in all sampleswas the same.
t In this experiment, the total amount of [14C]glucosamine-labelled material made byesculenta-catesbeiana appears to be more than esculenta-esculenta. In other experiments,pipiens-catesbeiana made more galactose-labelled material than pipiens-pipiens.
346 K. E. Johnson
Q origin ©
Fig. 7. Cellulose acetate electrophoresis of galactose-labelled material from normaland hybrid embryos. A to F respectively show: pipiens-pipiens; pipiens-catesbeiawa;pipiens-esculenta; esculenta-esadenta; esculenta-catesbeiana; and esculenta-pipiens.One milligramme of hyaluronic acid and chondroitin sulphate were added to a samplewhich was applied to a 25 x 15 cm strip (Sepraphore III) moistened with 25 mMpyridine-formate, pH 3-0. The buffer was also used for electrophoresis for 50 min at17 V/cm. Duplicate strips were run for each sample. One was stained with 1 % alcianblue in 1 % acetic acid and the other was cut into o-5-cm strips and counted in 5 mlof scintillation fluid.
Anton exchange chromatography
Galactose- and glucosamine-labelled materials from normal and hybrid embryosbind extensively to Dowex-i and elute from this resin in 0-5 M NaCl (Table 5).Traces of labelled material also elute at higher salt concentrations. Labelled materialsalso bind to DEAE-cellulose equilibrated with 50 mM Tris-HCl and elute in a0-1 M NaCl gradient at a salt concentration slightly lower than the elution concen-
Frog gastrula extracellular matrix, III 347
tration of hyaluronic acid (Fig. 8). When DEAE-cellulose is equilibrated with 50 railphosphate buffer and eluted with a 50-250 mM phosphate buffer gradient, galactose-and glucosamine-labelled materials from pipiens-pipiens and pipiens-catesbeiana elutetogether as a single peak (Fig. 9). Under these conditions, hyaluronic acid will remainbound to the column but can be recovered by elution with 500 mM phosphate buffer.If the same column is eluted with a 50-100 mM phosphate buffer gradient, labelledmaterials remain bound. Recovery of input radioactivity from DEAE-cellulosecolumns was 95-100% in all experiments.
Table 5. Relative amounts of galactose and glucosamine-labelled material eluting fromonion exchange resin* at different NaCl concentrations, % of total radioactivity elutingfrom column with each particular eluant^
Eluant
H2ONaCl, M
0-50i-oo1-251-502'OO
2-SO3-00
3-5°4-00
pipiens-pipiens
Galact-ose
i - 3
90-06 0
i - o
o-So-So-So- io- io- i
Glucos-amine
2-4
9°-5O-2
0-42-7
o-32 - 1
O-2
1-2
o- i
pipiens-catesbeiana
Galact-ose
0-3
9492-8
o-8o-60 4o- i
o-oo- i
o-o
Glucos-amine
2 9
78-78 0
o-o0-44-2
3 - i1-20-9
o-S
pipiens-esculentaA
Galact-ose
3-9
84-13 0
2-73-So-80-7o - i
o-8o-S
Glucos-amine
1-5
90-12 - 1
0-3O-2
0-42 - 1
1-3i - oi - o
* An o-6 x 13 cm column of Biorad AG1-X2 (Cl~, 200-400 mesh) was eluted with deionizedwater and the indicated NaCl solutions until no more radioactivity eluted with each eluant.The volume of each eluant was recorded and 2 ml were counted in 10 ml scintillation fluid;this allowed a calculation of amount of radioactivity eluting.
f In different experiments, different amounts of radioactive material were applied to thecolumn. Thus, the total amounts of radioactivity eluting with a particular elution varied, whiletheir relative amounts as percentages of the total radioactivity eluting were constant. Therecovery of input radioactivity was calculated to be 59 % in one experiment.
CPC-cellulose chromatography
When galactose- and glucosamine-labelled materials are mixed with carrierhyaluronate and applied to a cellulose column equilibrated with 1% CPC, about90% of the radioactive material precipitates. The precipitated material elutes almostexclusively with 0-15 M MgCl2 (82-88%) with traces eluting at higher MgCl2 concen-trations (Table 6). There is 95-100% recovery of input radioactivity from thesecolumns.
Neuraminidase digestion studies
Neuraminidase (Nase) treatment of galactose- and glucosamine-labelled materialdoes not alter its chromatographic behaviour on DEAE-cellulose (Fig. 10). The
348 K. E. Johnson
electrophoretic mobility on cellulose acetate strips is also unchanged by Nase treat-ment. The experiments were performed initially without bovine submaxillary mucin(BSM, Sigma) added to labelled material. When negative results were obtained, Nase,BSM and labelled material were mixed in one tube and incubated. The Nase releasedneuraminic acid from BSM as measured by Warren's assay (1959) but did not alter thechromatographic or electrophoretic behaviour of labelled materials.
4 r
xEa.
do
U
o
X
• i
cpm
min
e
ro0
CM)
x1
4
3
2
1
0
-
IAI
-III1
|
: •
i '**.i \ f\^ _ W \
I i i
- 0-3
- 0-2
- 0 1
§rid
10 20 30
Fraction no.
40
Fig. 8. Chromatogram of PHJglucosamine- (O O) and [14C]galactose-labelled(• •) material from pipiens-pipiens embryos on DEAE-cellulose. A portion of thepooled void volume fractions from the Sephadex G-50 run was mixed with i-6 mg ofhyaluronic acid (HA) and applied to a 0-9 x 15 cm DEAE-cellulose (DE-52, What-man) column equilibrated with 50 mM Tris-HCl and eluted with a 0-1 M NaClgradient beginning at the end of fraction 5. Four-minute, 6-ml fractions were col-lected and assayed for uronic acid (O.D.520) dotted line) and radioactivity.
Hyaluronidase digestion studies
Hyaluronidase (Hyase) treatment of labelled materials does not alter chromato-graphic behaviour on Sephadex G-50 or electrophoretic behaviour on celluloseacetate. The experiments were performed initially without hyaluronate added to thelabelled material-Hyase mixture. When there was no change in the chromatographicor electrophoretic behaviour of labelled material, hyaluronate was added to theincubation mixture. The results of these experiments show that Hyase preparationsdegraded hyaluronate into fragments which elute in the included volume of G-50but did not change the chromatographic behaviour of labelled material (Table 7).These results show that the enzymes used were active and that samples of labelledmaterial did not contain inhibitors of Hyase activity.
Frog gastrula extracellular matrix, III 349
10 20 30
Fraction no.
Fig. 9. Chromatogram of [3H]glucosamine- ( ) and [14C]galactose-labelled ( )material from pipiens-catesbeiana embryos on DEAE-cellulose. A part of the pooledvoid volume fractions from the Sephadex G-50 run was applied to an 0-9 x 15 cmDEAE-cellulose (DE-52, Whatman) column equilibrated with 50 mM phosphatebuffer, pH 7-6 and eluted with a 50-250 mM phosphate buffer gradient beginningat the end of fraction 5. Four-minute, 6-ml fractions were collected and assayed forradioactivity.
Table 6. Relative amounts of galactose- and glucosamine-labelled material elutingfrom CPC-cellulose column* at different MgCl2 concentrations
Eluant
CPC, 1 %MgCl2, M
0-15
°'5°i-oo1-50
pipiens-pipiens1
Galact-ose
io-if
8 7 8
i -3o-60 2
Glucos-amine
1 0 7
8761 0
o-6o- i
pipiens-catesbeiana1
Galact-ose
9 9
8 8 00 7I - I
O-2
Glucos-amine
14-9
8 3 4I - I
0 3
0 4
pipiens-esculenta_j.
1
Galact-ose
I O I
8 2 0
5'42 0
^
Glucos-amine
9 1
8 2 0
4 44 . 0
* One milligramme of hyaluronic acid was added to the sample which was applied to an0-9 x 12 cm column of Whatman CF11 cellulose powder which had been equilibrated with1 % cetylpyridinium chloride (CPC). Then the column was eluted with 5-10 vol. of 1 % CPC,0-15 M MgCl2 in 0-05 % CPC, etc.
t In different experiments, different amounts of radioactive material were applied to thecolumn. Thus, the total amounts of radioactivity eluting with a particular elution varied, whiletheir relative amounts as a percentage of the total radioactivity eluting were constant. Therecovery of input radioactivity was calculated to be 100 % in one experiment.
23 c 1:1- 25
35° K. E. Johnson
15
O
x 10
cpm
0
gala
ct
I
n
-
i\l/ll
I U.
i !j \
/ \
• i i
B
1
10 20 30
Fraction no.
10 20 30
Fraction no.
40
Fig. io. Chromatogram of [3H]glucosamine-labelled material from pipiens-pipiensembryos (A), and [3H]galactose-labelled material from pipiens-catesbeiana (B) onDEAE-cellulose as described for Fig. 9. Labelled material was dissolved in o-i Msodium acetate buffer, pH 5-0 and mixed with o-oi units (with bovine submaxillarymucin as a standard) of Sigma Clostridium neuraminidase (Type VI) in buffer andincubated for 6 h at 37 °C ( ). Buffer controls were incubated in buffer withoutenzyme ( ). At the end of incubation, samples were boiled and applied to DEAE-cellulose as described for Fig. 9. Samples containing labelled material were mixed with0-4 mg of bovine submaxillary mucin in buffer and enzyme and incubated as above.The enzyme released 4-5 fig of neuraminic acid as measured by the Warren (1959)assay using iV-acetyl-neuraminic acid as a standard, but had no effect on the behaviourof labelled material on DEAE-cellulose. This indicates that the neuraminidiase wasactive and that samples of labelled material contained no inhibitors of neuraminidaseactivity. Other experiments show that there is no change in the electrophoreticbehaviour of labelled material on cellulose acetate under conditions described inFig. 7.
DISCUSSION
The results of this study show that normal gastrulating embryos are activelyengaged in the synthesis of a polyanionic material which behaves in many respectslike an unsulphated glycosaminoglycan or a sialic acid-containing glycopeptide (or amixture of both). In contrast, arrested hybrid embryos show apparent disruptions inthe synthesis of these materials of moderate (pipiens-catesbeina, and esculenta-cates-beiana) or extreme (pipiens-esculenta and esculenta-pipiens) severity.
One question of considerable interest is the exact nature of the labelled materials.The PHJglucosamine-labelled material might be the glycosaminoglycan hyaluronicacid. Manasek (1975) has shown that gastrula-stage chick embryos are making a[3H]glucosamine-labelled material which migrates like hyaluronate on celluloseacetate, contains radioactivity as [3H]glucosamine in the macromolecule (rather thanas [3H]galactosamine), and is sensitive to testicular hyaluronidase. These criteriaindicate that [3H]glucosamine-labelled material is hyaluronate. Similar results havebeen reported by Solursh (1976). The [3H]glucosamine-labelled material made byamphibian embryos has an electrophoretic mobility like hyaluronate but neither its
Frog gastrula extracellular matrix, HI 351
Table 7. Hyaluronidase digestion of hyaluronic acid (HA) and galactose-labelledmaterial from normal (pipiens-pipiens) and hybrid (pipiens-catesbeiana) embryos^
Chromatographic behaviour on Sephadex G—50
Sample Carrier HA, O.D.5
Galactose-labelled material,
CPM
mg HA + Gal *'pipiens-pipiens
+ testicular hyase J+ Streptomyces hyase§
+ leech hyase||, +buffer
/ + testicular hyaseI + Streptomyces hyase
1 mg HA + Ga[*pipiens-catesbeiana II + leech hyaseI + buffer
Included fractionsSome in void fractions,most in included fractions
Included fractionsVoid fractionsIncluded fractionsSome in void fractions,most in included fractions
Included fractionsVoid fractions
Void fractionsVoid fractions
Void fractionsVoid fractionsVoid fractionsVoid fractions
Void fractionsVoid fractions
f After pronase digestion and Sephadex G-50 chromatography, galactose-labelled material was lyophilizedand redissolved in a small volume of 0-02 M sodium acetate buffer, pH 5-0, with 015 M NaCl added.
J Bovine testicular hyaluronidase (Sigma H-2Ooi), [i mg = 460 turbidity reducing units (TRU) dissolvedin 250 /tl acetate buffer]. Incubation 20 h at 37 °C.
§ Streptomyces hyaluronidase (Calbiochem 389561), 12-5 TRU in 250/tl acetate buffer added at time o, 2, 4,and 6 h of a 20-h incubation at 37 °C.
|| Leech hyaluronidase (Biotrics), 87-5 fig in 250 /tl acetate buffer added at time o, 2, 4, and 6 h of a 20-hincubation at 37 °C.
chromatographic behaviour on Sephadex G-50 nor its electrophoretic mobility isaltered by treatment with testicular, leech, or Streptomyces hyaluronidase. Thiscould mean that [3H]glucosamine-labelled material is not hyaluronate or that it isassociated in some way with other molecules which alter both ion exchange behaviourand susceptibility to enzymes. Further studies are necessary to identify [3H]glucos-amine-labelled materials. The galactose- and glucosamine-labelled materials mightalso be sialic acid-containing glycopeptides of a mucin-like component of the ex-tracellular matrix. If their polyanionic nature was due to neuraminic acid, one mightexpect that the chromatographic and electrophoretic behaviour would be altered byNase treatment. No Nase effect was observed. This enzymic study is not definitivebecause the amphibian material may be insensitive to Clostridium neuraminidase andstill contain neuraminic acid. Mild acid hydrolysis might release neuraminic acid andalter behaviour of labelled material. Such an experiment is planned.
The biochemical results presented here correlate closely with earlier histochemicaland autoradiographic results (Johnson, 1976, 19776). For example, pipiens-pipiensembryos have a bulk accumulation of toluidine blue metachromatic material (TBMM)in their extracellular spaces during gastrulation. At the same time, they are manu-facturing and secreting galactose- and glucosamine-labelled materials at an increasingrate into the extracellular matrix. They first show intense synthesis of these materials
23-2
352 K. E. Johnson
at the early gastrula stage just above the dorsal lip of the blastopore. This is alsowhere there is a bulk accumulation of TBMM. Pipiens-catesbeiana makes nearly asmuch labelled material as pipiens-pipiens but shows defective accumulation of TBMMin the extracellular matrix, probably because presumptive matrix components aremanufactured but not expelled from cells into the extracellular matrix. Pipiens-esculenta shows little accumulation of TBMM and makes little galactose- and glucos-amine-labelled material. In 2 different kinds of arrested hybrids, there are 2 differentkinds of defects in accumulation of components of the extracellular matrix anddefective morphogenetic cell movements as well. This suggests that changes in theextracellular matrix are somehow involved in gastrulation. Esculenta-esculenta embryosdo show an increase in the amount of TBMM during gastrulation, but the increase isnot as dramatic as that seen in pipiens-pipiens. Esculenta-esculenta embryos are alsoless active in the synthesis of galactose- and glucosamine-labelled materials, even ifallowance is made for the 2-fold differences in egg and embryonic volume.
The differences in synthesis of matrix components could be due to many causes.They are not simply due to differences in rate of cell division, since normal and hybridembryos have equal numbers of cells (Johnson, 1970) and equal amounts of DNA/em-bryo (Gregg & Lovtrup, i960) at comparable ages. All arrested hybrid embryos areeventually going to die, but defects in matrix synthesis are not trivial consequences ofdying. For example, arrested pipiens-esculenta have severe matrix defects but do notdie for 5-7 days after arrest. The rates of cell division, DNA synthesis, and proteinsynthesis are not abnormal in hybrids at the time when matrix synthesis becomesdefective.
The changes in amount of incorporation during development could be explained inseveral ways. There might be a dramatic decrease in the free sugar, sugar-phosphate,or nucleotide-sugar pools with development, which would give an increase in amountof label incorporation without any change in rate of synthesis. This possibility cannotbe eliminated without information on pool sizes. Changes in amount of incorporationcould also be due to changes in permeability to sugars. It would be difficult to ruleout such a possibility in this system. Uptake of label could be measured by incubatingembryos in label, washing in cold medium, and counting embryos without TCAextraction. Such an experiment would not distinguish between sugars which hadbecome intracellular during transport; sugars which were extracellular but not easilywashed out, i.e. in pinocytotic or phagocytic vesicles (such vesicles are numerous inamphibian embryos (Baker, 1965)); and sugars which were in the archenteron and lesseasily washed out. However, it seems unlikely that the permeability of frog embryosto sugar would change at all. During normal development, frog embryos are neitherexposed to free sugars nor are they very permeable to free sugars. Finally, differencesin amount of incorporation could reflect developmental changes in the activities of theenzymes for the synthesis of high molecular weight carbohydrate-containing com-pounds (Roseman, 1970; Levitt & Dorfman, 1974). Further experiments are necessaryto resolve these complex problems.
What could be the function of new matrix components? During gastrulation, cellsin the embryo become actively migratory. For their locomotion, they require some
Frog gastrula extracellular matrix, III 353
kind of a substratum. Newly synthesized extracellular materials and cell surfaceconstitutents may represent the formation of a new substratum for adhesion andmigration. The migration of chick corneal fibroblasts seems to occur during thesynthesis of hyaluronic acid (Toole & Trelstad, 1971) as does mesenchymal cellmigration in a regeneration blastema (Toole & Gross, 1971) and neural crest cellmigration (Pratt, Larsen & Johnston, 1975). Solursh (1976) has suggested thathyaluronate synthesis in chick gastrulae provides an extracellular matrix conducive tocell migration. The present results, where there is a correlation between defects incell movements and bulk matrix accumulation, also suggest that changes in the matrixare involved somehow in cell movements of gastrulation.
Dr Mark Adelman made helpful contributions to this research effort by giving advice,criticism, and access to equipment in his laboratory. Drs S. J. Counce, S. Roth, D. McClay,B. Kaufman, and B. Toole criticized the manuscript. Research support came from NIH GrantHD-07082, United Health Services of North Carolina, General Research Support Grants,Duke University Research Council, and Sigma Xi. The author wishes to express his gratitudefor this help and support.
REFERENCES
ANDERSON, L. E. & MCCLURE, W. O. (1973). An improved scintillation cocktail of high-solubilizing power. Analyt. Biochem. 51, 173-179.
BAKER, P. C. (1965). Fine structure and morphogenetic movements in the gastrula of the treefrog, Hyla regilla. J. Cell Biol. 24, 95-116.
BLUMENKRANTZ, N. & ASBOE-HANSEN, G. (1973). New method for quantitative determinationof uronic acids. Analyt. Biochem. 54, 484-489.
D E L A HABA, G. & HOLTZER, H. (1965). Chondroitin sulfate: inhibition of synthesis by puro-mycin. Science, N.Y. 149, 1263-1265.
FAIRBANKS, G., STECK, T. L. & WALLACH, D. F. H. (1971). Electrophoretic analysis of themajor polypeptides of the human erythrocyte membrane. Biochemistry, N.Y. 10, 2606-2617.
GREGG, J. R. & LOVTRUP, S. (i960). A reinvestigation of DNA synthesis in lethal amphibianhybrids. Expl Cell Res. 19, 621-623.
JOHNSON, K. E. (1970). The role of changes in cell contact behavior in amphibian gastrulation.J. exp. Zool. 175, 391-428.
JOHNSON, K. E. (1976). Extracellular matrix synthesis in normal and arrested hybrid frogembryos. (Abstract). J. gen. Physiol. 68, A7.
JOHNSON, K. E. (1977 a). Extracellular matrix synthesis in blastula and gastrula stages ofnormal and hybrid frog embryos. I. Toluidine blue and lanthanum staining. J. Cell Sci.25. 313-322.
JOHNSON, K. E. (19776). Extracellular matrix synthesis in blastula and gastrula stages of normaland hybrid frog embryos. II . Autoradiographic observations on the sites of synthesis andmode of transport of galactose- and glucosamine-labelled materials. J. Cell Sci. 25, 323-334.
LEE, Y. C , JOHNSON, G. S., WHITE, B. & SCOCCA, J. (1971). An accelerated system for analysisof neutral sugars in complex carbohydrates. Analyt. Biochem. 43, 640—643.
LEVITT, D. & DORFMAN, A. (1974). Concepts and mechanisms of cartilage differentiation. Curr.Top. dev. Biol. 8, 103-149.
MANASEK, F. J. (1975). The extracellular matrix: A dynamic component of the developingembryo. Curr. Top. dev. Biol. 10, 35-102.
MANS, R. J. & NOVELLI, G. D. (1961). Measurement of the incorporation of radioactive aminoacids into protein by a filter-paper disk method. Archs Biochem. Biophys. 94, 48-53.
ORKIN, R. W., PRATT, R. M. & MARTIN, G. R. (1976). Undersulfated chondroitin sulfate inthe cartilage matrix of Brachymorphic mice. Devi Biol. 50, 82-94.
354 K. E. Johnson
PEACOCK, A. C. & DINGHAM, C. W. (1968). Molecular weight estimation and separation ofribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry, N. Y.7, 668-674.
PRATT, R. M., LARSEN, M. A. & JOHNSTON, M. C. (1975). Migration of cranial neural crest cellsin cell-free hyaluroate-rich matrix. Devi Biol. 44, 298-305.
RODEN, L., BAKER, J. R., CIFONELLI, J. A. & MATHEWS, M. B. (1972). Isolation and characteriz-ation of connective tissue polysaccharides. Meth. Ensym. 28, 73-104.
ROSEMAN, S. (1970). The synthesis of complex carbohydrates by multiglycosyltransferasesystems and their potential function in intercellular adhesion. Chem. Phys. Lipids 5, 270-297.
SCHILLER, S., SLOVER, G. A. & DORFMAN, A. (1961). A method for the separation of acidmucopolysaccharides: its application to the isolation of heparin from the skin of rats. J. biol.Chem. 236, 983-987.
SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. Anat. Rec. 78, 139-H7-
SOLURSH, M. (1976). Glycosaminoglycan synthesis in the chick gastrula. Devi Biol. 50, 525-530.TOOLE, B. P. & GROSS, J. (1971). The extracellular matrix of the regenerating newt limb:
synthesis and removal of hyaluronate prior to differentiation. Devi Biol. 25, 57-77.TOOLE, B. P. & TRELSTAD, R. L. (1971). Hyaluronate production and removal during corneal
development in the chick. Devi Biol. 26, 28-35.WARREN, L. (1959). The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975.
{Received 4 November 1976)
