DEYELOPMENTAL
BIOLOGY
Notochordal Before
42,362-378
(1975)
Stimulation
and After
of in Vitro Somite
Enzymatic
Removal
Chondrogenesis
of Perinotochordal
Materials1 A.
ROBERT Department
of Anatomy,
University
KOSHER’
AND
of Pennsylvania
Accepted
School October
JAMES
W.
of Medicine,
LASH Philadelphia,
Pennsylvania
19174
29, 1974
In the present investigation, evidence is presented directly implicating proteoglycans produced by the embryonic notochord in the ccMro1 of somite chondrogenesis. It has been demonstrated by several histochemical techniques that during the period of its interaction with somites, the notochord synthesizes perinotochordal proteoglycans, and these proteoglycans have been shown to contain chondroitin 4-sulfate(40%), chondroitin g-sulfate (40%). and heparan sulfate (20%). Dissection of notochords from embryos with the aid of a brief treatment with trypsin results in the removal of perinotochordal extracellular matrix materials including proteoglycans, while dissection of notochords without the aid ofenzyme treatment or with a low concentration of collagenase results in their retention. There is a considerable increase in the rate and amount of cartilage formation and a corresponding 2 to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated by somites cultured in association with notochords dissected under conditions in which perinotochordal materials are retained. Treatment of collagenase-dissected or freely dissected notochords with highly purified enzymes (chondroitinase ABC, AC, and testicular hyaluronidase) which specifically degrade proteoglycans causes a loss of histochemically detectable perinotochordal proteoglycans. These notochords are considerably impaired in their ability to support in vitro somite chondrogenesis. In addition, when trypsin-treated notochords are cultured (“precultured”) for 24 hr on nutrient agar (in the absence of somites), perinotochordal material reaccumulates. Somites cultured in association with such “precultured” notochords exhibit considerable increase in the amount of cartilage formed and a 2- to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated as compared to somites cultured in association with trypsin-treated notochords which have not been “precultured.” This observation indicates that trypsin-treated notochords reacquire their ability to maximally stimulate in vitro somite chondrogenesis by resynthesizing and accumulating perinotochordal material. Finally, “precultured” notochords treated with chondroitinase to remove perinotochordal proteoglycans are considerably impaired in their ability to support uz vitro somite chondrogenesis. These observations are consonant with the concept that proteoglycans produced by the embryonic notochord play an important role in somite chondrogenesis. INTRODUCTION
The possibility that extracellular matrix components (i.e., proteoglycans and collagen) mediate tissue interactions has received much attention in recent years (Slavkin, 1972; Nevo and Dorfman, 1972; Solursh and Meier, 1973; Kosher et al., 1973; Meier and Hay, 1974a,b). One sys1 Supported by Grant HD-00380 from the U.S. Public Health Service (JWL) and a U.S.P.H.S. postdoctoral fellowship (RAK). * Current address: Department of Anatomy, University of Connecticut Health Center, Farmington, Conn 06032. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
362
tern in which extracellular materials have been implicated is the interaction between the embryonic notochord and somites, which results in the formation of vertebral cartilage. It has long been known that the embryonic notochord stimulates or induces chondrogenesis of somitic mesoderm in vim and in uitro (see Lash, 1963; 1968a,b,c for reviews). During the period of interaction between the notochord and somites m uiuo, the notochord synthesizes and secretes proteoglycans and collagen (FrancoBrowder et al., 1963; Marzullo and Lash, 1967; Lash, 1968a,b,c; Kvist and Finnegan,
KOSHER
AND LASH
Notochordal proteoglycans and chondrogenesis
1970a,b; Strudel, 1971; Ruggeri, 1972; Bazin and Strudel, 1972; Minor, 1973; Linsenmayer et al., 1973; Hay and Meier, 1974). These extracellular materials, which initially accumulate in the perinotochordal sheath, diffuse away from the notochord and become distributed among adjacent somitic sclerotomal cells (Ruggeri, 1972). The overt differentiation of sclerotomal cells into cartilage occurs after these extracellular materials have become distributed among them (Ruggeri, 1972). As a result of these correlative observations, we have been investigating the possibility that proteoglycans produced by the embryonic notochord mediate the conversion of somitic sclerotomal cells into cartilage. Recently it has indeed been demonstrated that proteoglycan complex (consisting predominantly of proteochondroitin 4- and 6-sulfate) extracted from embryonic chick cartilage greatly stimulates in vitro somite chondrogenesis, i.e., exogenous proteoglycan complex can effectively substitute for the notochord as a promoter of in vitro somite chondrogenesis (Kosher et al., 1973). The latter observation does not, however, provide direct experimental evidence that proteoglycans produced by the notochord itself stimulate chondrogenesis, since the exogenous proteoglycan was obtained from cartilage tissue. In view of the obvious difficulties involved in obtaining an adequate amount of purified proteoglycan from embryonic notochords, we have undertaken an alternate approach to demonstrating the stimulatory role of proteoglycans and other extracellular materials produced by the notochord itself on somite chondrogenesis. We have confirmed that during the period of its interaction with somites the notochord synthesizes proteoglycans that accumulate in the perinotochordal sheath, and these proteoglycans have been shown to contain chondroitin 4-sulfate (40%), chondroitin 6-sulfate (40%), and heparan sulfate (20%). Notochords from which perinotochordal extra-
363
cellular materials have been removed by trypsin or from which perinotochordal proteoglycans have been selectively removed by highly purified enzymes that specifically degrade proteoglycans, are considerably impaired in their ability to support in vitro somite chondrogenesis compared to notochords which retain perinotochordal proteoglycans and other extracellular components of the perinotochordal sheath. MATERIALS
AND METHODS
Culture Techniques Preparation of control cultures. Somites and notochords were dissected from stage 17 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks after treatment of embryonic trunks with trypsin as previously described (Ellison and Lash, 1971). Explants, consisting of 8-10 somites either in the presence or absence of notochordal tissue, were cultured on nutrient agar containing Simms’ balanced salt solution (SBSS), fetal calf serum (FSC), and the nutrient supplement F12X in the proportions 2:2:1 (Gordon and Lash, 1974). Somites were derived from the region of the embryonic trunk between the anterior border of the forelimb bud and posterior border of the hindlimb bud. Explants were fed with liquid nutrient medium containing SBSS, FCS, and F12X (2:2:1). To label the glycosaminoglycans, the nutrient agar and liquid nutrient feeding medium contained either 5.0 pCi/ml of NaYSO, (carrier-free; Amersham/Searle) or 5.0 pCi/ml of [3H]glucosamine (New England Nuclear). Cultures were examined at daily intervals for visual signs of chondrogenesis. Dissection of notochords without the aid of enzyme
treatment
or with
the aid of were dissected embryonic trunks without the aid of enzyme treatment. Notochords were dissected from embryos after treatof embryonic trunks for 10 min at temperature with freshly prepared gassed (5% CO, in air) collagenase
collagenase. Notochords
from any also ment room and
364
DEVELOPMENTAL
BIOLOGY
(code no. CLSPA; Worthington Biochemical Corp.) in calcium- and magnesium-free Simms’ balanced salt solution (CMF). The concentration of the collagenase solution was 0.2 mg/ml. The freely dissected (no enzyme treatment) and collagenasetreated notochords were placed upon nutrient agar and supplied with somites dissected from trypsin-treated embryonic trunks. Enzymatic treatment of collagenase-dissected and freely dissected notochords. Notochords dissected with the aid of collagenase were subjected to a second enzyme treatment prior to being cultured in association with somites. Freely dissected notochords were also subjected to enzyme treatment. All of these second-stage enzyme treatments were done for 15 min in pregassed CMF at room temperature. The enzymes were chondroitinase ABC (0.1 unit/ml; Miles Laboratories), chondroitinase AC (0.1 unit/ml; Miles Laboratories) and testicular hyaluronidase (0.003 mg/ml; Type I Sigma). Preculturing of notochords. Notochords which had been dissected from embryos after treatment with trypsin were cultured on nonradioactive nutrient agar for 24 hr in the absence of somites. These “precultured” notochords were then transferred to radioactive nutrient agar and supplied with somites which were freshly prepared from embryos after trypsin treatment. In addition, notochords which had been “precultured” for 24 hr were treated with chondroitinase ABC, chondroitinase AC, and testicular hyaluronidase prior to being transferred to radioactive medium and supplied with somites. Histochemical
Analyses
Several histochemical procedures were used to examine the accumulation of acidic glycosaminoglycans (proteoglycans) in the perinotochordal region of notochords (prepared by the various procedures detailed in the previous section); and to examine the
VOLUME 42,1975
accumulation of cartilaginous matrix by somite explants. Notochords and explants were fixed in Carnoy’s solution (absolute ethanol:acetic acid, 3:1), dehydrated, embedded in paraffin, and sectioned at 5 pm. Sections were stained by the Alcian Blue/ hematoxylin procedure described by Ellison and Lash (1971), and with Alcian Blue at various MgCl, concentrations as described by Quintarelli and Dellovo (1965). The two-step PAS procedure of Scott and Dorling (1969) was used to demonstrate uranic acid-containing glycosaminoglycans. Staining with aprotinin (Trasylol; FBA Pharmaceuticals) conjugated with fluorescein isothiocyanate (FITC) was performed as described by Kiernan and Stoddart (1973). Fluorescence was detected with a Reichert “Binolux” microscope with excitation by blue-violet (350-450 nm) light. Biochemical
Analyses
Incorporation of radioactive precursors into glycosaminoglycans. Glycosaminoglycans were extracted from somite explants and notochords continuously exposed to either [YS]sulfate or [3H]glucosamine by procedures previously described (Kosher et al., 1973). Briefly, tissues were sonicated, then digested with Pronase (Calbiochem), treated with cold TCA, and dialyzed against Na,SO, and distilled water. The dialysates were lyophilized, dissolved in a minimal amount of distilled water, and aliquots were supplied with scintillation fluid (0.6% PPO and 0.3% POPOP in toluene) containing 5% BIO-Solv (Beckman) for determination of radioactivity by means of Intertechnic liquid scintillation spectrometer. The remainder of the dissolved lyophilisate (glycosaminoglycan) was utilized for further characterization. Characterization of [35S]sulfate-labeled glycosaminoglycan. The relative amounts of chondroitin 4-sulfate and chondroitin 6-sulfate synthesized by tissues incubated in the presence of (35S]sulfate were deter-
KOSHER
AND LASH
Notochordal
mined by the enzymatic method of Saito et al. (1968) using chondroitinase ABC and AC as previously described (Kosher and Searls, 1973). The relative amount of [%]sulfate-labeled heparan sulfate was determined as described by Kosher and Searls (1973) utilizing nitrous acid which specifically degrades the N-sulfated GAG heparan sulfate (Lagunoff and Warren, 1962; Cifonelli, 1968a,b; Lindahl, 1970; Kraemer, 1971a,b). Characterization of [3H]glucosaminelabeled glycosaminoglycans. The amount of [3H]glucosamine-labeled hyaluronic acid was determined as described by Dan iel et al. (1973) utilizing leech hyaluronidase (hyaluronic acid- 1,3 hydrolase; Biotries, Inc.), an enzyme which specifically degrades hyaluronic acid (see also Meier and Hay, 1973b). Briefly, after treatment of [3H]glucosamine-labeled glycosaminoglycan samples with leech hyaluronidase, the amount of radioactivity associated with leech hyaluronidase-sensitive and -resistant material was determined after precipitation of undegraded GAG with cetylpyridinium chloride (CPC) (see Daniel et al., 1973 for details). Similarly, the amount of [3H]glucosamine-labeled chondroitin sulfate was determined as described by Daniel et al. (1973). Aliquots of the glycosaminoglycan samples were treated with chondroitinase ABC, and the amount of radioactivity associated with chondroitinase-sensitive and -resistant material was determined after precipitation with CPC.
proteoglycans
and chondrogenesis
365
2 M-day chick embryos exposed for 24 hr to [3H]tryptophane. Collagenase activity was determined as described by Mandl et al. (1953) and as described by Peterkofsky and Diegelmann (1971) using total labeled protein extracted from 15-day embryonic chick chondrocytes exposed for 24 hr to [3H]glycine on the fifth day of culture. RNase and DNase activities were assayed as described by Zimmerman and Sandeen (1965).
DNA Analysis DNA was determined by the Santoianni and Ayala (1965) modification of the procedure of Kissane and Robins (1958), except that fluorescence was determined in 1 N HCl (Hinegardner, 1971). RESULTS
During the period of its interaction with somites in vivo, the embryonic notochord synthesizes proteoglycans and other extracellular matrix materials which accumulate predominantly in the perinotochordal sheath (see for example, Ruggeri, 1972). In preparation for organ culture, notochords are routinely dissected from embryos after a brief treatment with trypsin (see Lash et al., 1964; Lash, 1967). As a result of this treatment, it appeared likely that extracellular materials which had accumulated along the periphery of notochords would be destroyed. If products of the notochord are involved in stimulating somite chondrogenesis, then notochords which retain these extracellular materials should stimulate in vitro somite chondrogenesis to a greater extent than notochords which iniDegradative Activities of Enzymes tially lack these components (e.g., trypsinThe enzymes chondroitinase ABC, AC, treated notochords). Accordingly, two proand testicular hyaluronidase were tested cedures have been utilized to prepare notofor protease, RNase, DNase, and collagen- chords which retain perinotochordal matease activities by the following procedures. rials. The first procedure involves dissecNonspecific protease activity was assayed tion of notochords without the aid of any as described by Davis and Smith (1955) enzyme treatment, and the second involves using casein as substrate and as described dissection of notochords from embryos with by Peterkofsky and Diegelmann (1971) the aid of a low concentration of collagenusing total labeled protein extracted from ase (rather than trypsin).
366
DEVELOPMENTAL
BIOLOGY
Histochemical evidence of proteoglycans along the perinotochordal sheath. Cross sections of notochords dissected from embryos without the aid of enzyme treatment
VOLUME 42,1975
and with collagenase are shown in Figs. 1 and 3. Note in Figs. la and 3a, the intense accumulation of Alcian Blue-positive material along the periphery of notochords
Pm. 1. Cross sections of notochords dissected without the aid of enzyme treatment. x 1000. Note the accumulation of perinotochordal material as demonstrated by (1A) the stain Alcian Blue, (1B) Schiff-positive material after the two-step PAS procedure, (1C) intense fluorescence after treatment with FITC-labeled aprotinin. FIG. 2. Cross sections of notochords dissected with the aid of brief treatment with trypsin. x 1000. Note the absence of perinotochordal material after (2A) staining with Alcian Blue, (ZB) the two-step PAS procedure, (LX) treatment with FITC-labeled aprotinin. FIG. 3. Cross sections of notochords dissected with the aid of collagenase. x 1000. Note the accumulation of perinotochordal material as demonstrated by (3A) the stain Alcian Blue, (3B) Schiff-positive material after the two-step PAS procedure, (3C) intense fluorescence after treatment with FITC-labeled aprotinin.
KOSHER
AND LASH
Notochordal
prepared by these two procedures. No perinotochordal Alcian Blue-positive material could be detected at a Mg2+ concentration of 0.9 M; a Mg2+ concentration at which Alcian Blue is characteristically displaced from polyanionic glycosaminoglycans (Quintarelli and Dellovo, 1965). Perinotochordal Schiff-positive material could also be detected around notochords (Figs. lb and 3b) dissected by these two procedures after treatment of sections for 15 min with periodate followed by sodium borohydride reduction and a 24-hr periodate oxidation (the two-step PAS procedure of Scott and Dorling, 1969). This stain indicates the presence of uranic acid-containing compounds. Finally, staining with aprotinin conjugated with fluorescein isothiocyanate (FITC) revealed an intense fluorescence along the periphery of notochords dissected with no enzyme treatment and collagenase (Figs. lc and 3~). FITC-labeled aprotinin is a specific fluorochrome for acidic glycosaminoglycans (Kiernan and Stoddart, 1973). These results indicate that notochords dissected without the aid of enzyme treatment or with the aid of a low concentration of collagenase retain perinotochorda1 proteoglycans. These procedures, however, undoubtedly result in the retention of other perinotochordal extracellular materials as well as proteoglycans. In contrast, notochords dissected with the aid of trypsin did not posesses perinotochordal Alcian Blue-positive material (Fig. 2a) or Schiff-positive material after the two-step PAS procedure (Fig. 2b). There was a complete absence of fluorescence along the periphery of trypsin-treated notochords stained with FITC-labeled aprotinin (Fig. 2~). It should be added that trypsin treatment does not reduce the viability of notochords. Notochords dissected by the three methods described all initially possess the same amount of DNA and accumulate DNA in culture at identical rates. Characterization of the glycosaminogly-
proteogl.ycans
367
and chondrogenesis
cans synthesized by notochords. To confirm the histochemical indications of the accumulation of perinotochordal glycosaminoglycans, notochords dissected with no enzyme treatment, with trypsin, or with collagenase, were cultured on nutrient agar for 24 hr in the presence of either [35S]~u1fate or [3H]glucosamine, and the glycosaminoglycans (GAG) synthesized were subjected to biochemical analyses. Analysis with chondroitinase ABC and AC indicates that the [35S]sulfate-labeled GAG synthesized by notochords consist of 40% chondroitin 4-sulfate, 40% chondroitin 6sulfate, and 20% chondroitinase-resistant material (Table 1). The chondroitinaseresistant material has been characterized as heparan sulfate by virtue of the fact that it can be degraded by nitrous acid (Table 1). Of the [3H]glucosamine-labeled GAG synthesized by notochords, 78% was chondroitin sulfate (chondroitinase-sensitive material) and 22% was chondroitinaseresistant (i.e., heparan sulfate) confirming the analysis of the [35S]sulfate-labeled TABLE
1
RELATIVE AMOUNTS OF THE VARIOUS [?~]SULFATE-LABELED GLYCOSAMINOGLYCANS SYNTHESIZED BY EMBRYONIC NOTOCHORDS AS DETERMINED BY CHONDROITINASE ANALYSIS AND NITROUS ACID DEGRADATION Glycosaminoglycan Chondroitin &sulfate’ Chondroitin 4-sulfated Heparan sulfate’
w 40 40 20
n The amount of [%]sulfate-labeled GAG present in the samples utilized for these characterizations ranged between 1000 and 3000 dpm/Fg DNA, and the amount of DNA ranged from 1.5-2.5 pg. b Each value is the average of three determinations. Standard deviation = * 3-4 in each case. c Determined as the relative amount of radioactivity associated with 6-sulfated disaccharide residues after chondroitinase ABC and AC treatment. ’ Relative amount of radioactivity associated with I-sulfated disaccharide residues after chondroitinase ABC and AC treatment. e [YS] sulfate-labeled GAG which is not degraded by chondroitinase ABC or AC. This material can be degraded (i.e., rendered CPC soluble) by nitrous acid.
368
DEVELOPMENTAL
BIOLOGY
GAG (Table 2). Analysis of the [3H]glucosamine-labeled GAG synthesized by notochords with leech hyaluronidase (an enzyme which specifically degrades hyaluronic acid) indicates that notochords do not produce detectable amounts of hyaluronic acid (i.e., leech hyaluronidase-sensitive material) (Table 2). Although no difference could be detected in the types of GAG synthesized by either trypsin-dissected, collagenase-dissected, or freely dissected notochords, the amount synthesized by trypsin-dissected notochords (5120 dpmlpg DNA) was approximately 50% greater than the amount synthesized by collagenase-dissected (3780 dpm/kg DNA) or freely dissected notochords (3693 dpm/pg DNA). The greater synthesis of GAG by trypsin-treated notochords is undoubtedly a result of the fact that these notochords are replacing perinotochordal proteoglycans (see below). Chondrogenesis of somites in the presence of notochords dissected with no enzyme treatment, the aid of collagenase or trypsin. There have been repeated demonTABLE
2
RELATIVE AMOUNTS OF THE VARIOUS [3H]G~u~~~~~~~~-~~~~~~~ GLYCOSAMINOGLYCANS SYNTHESIZED BY EMBRYONIC NOTOCHORDS” Glycosaminoglycan
9c
Chondroitin sulfateb Heparan sulfate’ Hyaluronic acidd
78 22
BIOLOGY
Notochordal Before
42,362-378
(1975)
Stimulation
and After
of in Vitro Somite
Enzymatic
Removal
Chondrogenesis
of Perinotochordal
Materials1 A.
ROBERT Department
of Anatomy,
University
KOSHER’
AND
of Pennsylvania
Accepted
School October
JAMES
W.
of Medicine,
LASH Philadelphia,
Pennsylvania
19174
29, 1974
In the present investigation, evidence is presented directly implicating proteoglycans produced by the embryonic notochord in the ccMro1 of somite chondrogenesis. It has been demonstrated by several histochemical techniques that during the period of its interaction with somites, the notochord synthesizes perinotochordal proteoglycans, and these proteoglycans have been shown to contain chondroitin 4-sulfate(40%), chondroitin g-sulfate (40%). and heparan sulfate (20%). Dissection of notochords from embryos with the aid of a brief treatment with trypsin results in the removal of perinotochordal extracellular matrix materials including proteoglycans, while dissection of notochords without the aid ofenzyme treatment or with a low concentration of collagenase results in their retention. There is a considerable increase in the rate and amount of cartilage formation and a corresponding 2 to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated by somites cultured in association with notochords dissected under conditions in which perinotochordal materials are retained. Treatment of collagenase-dissected or freely dissected notochords with highly purified enzymes (chondroitinase ABC, AC, and testicular hyaluronidase) which specifically degrade proteoglycans causes a loss of histochemically detectable perinotochordal proteoglycans. These notochords are considerably impaired in their ability to support in vitro somite chondrogenesis. In addition, when trypsin-treated notochords are cultured (“precultured”) for 24 hr on nutrient agar (in the absence of somites), perinotochordal material reaccumulates. Somites cultured in association with such “precultured” notochords exhibit considerable increase in the amount of cartilage formed and a 2- to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated as compared to somites cultured in association with trypsin-treated notochords which have not been “precultured.” This observation indicates that trypsin-treated notochords reacquire their ability to maximally stimulate in vitro somite chondrogenesis by resynthesizing and accumulating perinotochordal material. Finally, “precultured” notochords treated with chondroitinase to remove perinotochordal proteoglycans are considerably impaired in their ability to support uz vitro somite chondrogenesis. These observations are consonant with the concept that proteoglycans produced by the embryonic notochord play an important role in somite chondrogenesis. INTRODUCTION
The possibility that extracellular matrix components (i.e., proteoglycans and collagen) mediate tissue interactions has received much attention in recent years (Slavkin, 1972; Nevo and Dorfman, 1972; Solursh and Meier, 1973; Kosher et al., 1973; Meier and Hay, 1974a,b). One sys1 Supported by Grant HD-00380 from the U.S. Public Health Service (JWL) and a U.S.P.H.S. postdoctoral fellowship (RAK). * Current address: Department of Anatomy, University of Connecticut Health Center, Farmington, Conn 06032. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
362
tern in which extracellular materials have been implicated is the interaction between the embryonic notochord and somites, which results in the formation of vertebral cartilage. It has long been known that the embryonic notochord stimulates or induces chondrogenesis of somitic mesoderm in vim and in uitro (see Lash, 1963; 1968a,b,c for reviews). During the period of interaction between the notochord and somites m uiuo, the notochord synthesizes and secretes proteoglycans and collagen (FrancoBrowder et al., 1963; Marzullo and Lash, 1967; Lash, 1968a,b,c; Kvist and Finnegan,
KOSHER
AND LASH
Notochordal proteoglycans and chondrogenesis
1970a,b; Strudel, 1971; Ruggeri, 1972; Bazin and Strudel, 1972; Minor, 1973; Linsenmayer et al., 1973; Hay and Meier, 1974). These extracellular materials, which initially accumulate in the perinotochordal sheath, diffuse away from the notochord and become distributed among adjacent somitic sclerotomal cells (Ruggeri, 1972). The overt differentiation of sclerotomal cells into cartilage occurs after these extracellular materials have become distributed among them (Ruggeri, 1972). As a result of these correlative observations, we have been investigating the possibility that proteoglycans produced by the embryonic notochord mediate the conversion of somitic sclerotomal cells into cartilage. Recently it has indeed been demonstrated that proteoglycan complex (consisting predominantly of proteochondroitin 4- and 6-sulfate) extracted from embryonic chick cartilage greatly stimulates in vitro somite chondrogenesis, i.e., exogenous proteoglycan complex can effectively substitute for the notochord as a promoter of in vitro somite chondrogenesis (Kosher et al., 1973). The latter observation does not, however, provide direct experimental evidence that proteoglycans produced by the notochord itself stimulate chondrogenesis, since the exogenous proteoglycan was obtained from cartilage tissue. In view of the obvious difficulties involved in obtaining an adequate amount of purified proteoglycan from embryonic notochords, we have undertaken an alternate approach to demonstrating the stimulatory role of proteoglycans and other extracellular materials produced by the notochord itself on somite chondrogenesis. We have confirmed that during the period of its interaction with somites the notochord synthesizes proteoglycans that accumulate in the perinotochordal sheath, and these proteoglycans have been shown to contain chondroitin 4-sulfate (40%), chondroitin 6-sulfate (40%), and heparan sulfate (20%). Notochords from which perinotochordal extra-
363
cellular materials have been removed by trypsin or from which perinotochordal proteoglycans have been selectively removed by highly purified enzymes that specifically degrade proteoglycans, are considerably impaired in their ability to support in vitro somite chondrogenesis compared to notochords which retain perinotochordal proteoglycans and other extracellular components of the perinotochordal sheath. MATERIALS
AND METHODS
Culture Techniques Preparation of control cultures. Somites and notochords were dissected from stage 17 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks after treatment of embryonic trunks with trypsin as previously described (Ellison and Lash, 1971). Explants, consisting of 8-10 somites either in the presence or absence of notochordal tissue, were cultured on nutrient agar containing Simms’ balanced salt solution (SBSS), fetal calf serum (FSC), and the nutrient supplement F12X in the proportions 2:2:1 (Gordon and Lash, 1974). Somites were derived from the region of the embryonic trunk between the anterior border of the forelimb bud and posterior border of the hindlimb bud. Explants were fed with liquid nutrient medium containing SBSS, FCS, and F12X (2:2:1). To label the glycosaminoglycans, the nutrient agar and liquid nutrient feeding medium contained either 5.0 pCi/ml of NaYSO, (carrier-free; Amersham/Searle) or 5.0 pCi/ml of [3H]glucosamine (New England Nuclear). Cultures were examined at daily intervals for visual signs of chondrogenesis. Dissection of notochords without the aid of enzyme
treatment
or with
the aid of were dissected embryonic trunks without the aid of enzyme treatment. Notochords were dissected from embryos after treatof embryonic trunks for 10 min at temperature with freshly prepared gassed (5% CO, in air) collagenase
collagenase. Notochords
from any also ment room and
364
DEVELOPMENTAL
BIOLOGY
(code no. CLSPA; Worthington Biochemical Corp.) in calcium- and magnesium-free Simms’ balanced salt solution (CMF). The concentration of the collagenase solution was 0.2 mg/ml. The freely dissected (no enzyme treatment) and collagenasetreated notochords were placed upon nutrient agar and supplied with somites dissected from trypsin-treated embryonic trunks. Enzymatic treatment of collagenase-dissected and freely dissected notochords. Notochords dissected with the aid of collagenase were subjected to a second enzyme treatment prior to being cultured in association with somites. Freely dissected notochords were also subjected to enzyme treatment. All of these second-stage enzyme treatments were done for 15 min in pregassed CMF at room temperature. The enzymes were chondroitinase ABC (0.1 unit/ml; Miles Laboratories), chondroitinase AC (0.1 unit/ml; Miles Laboratories) and testicular hyaluronidase (0.003 mg/ml; Type I Sigma). Preculturing of notochords. Notochords which had been dissected from embryos after treatment with trypsin were cultured on nonradioactive nutrient agar for 24 hr in the absence of somites. These “precultured” notochords were then transferred to radioactive nutrient agar and supplied with somites which were freshly prepared from embryos after trypsin treatment. In addition, notochords which had been “precultured” for 24 hr were treated with chondroitinase ABC, chondroitinase AC, and testicular hyaluronidase prior to being transferred to radioactive medium and supplied with somites. Histochemical
Analyses
Several histochemical procedures were used to examine the accumulation of acidic glycosaminoglycans (proteoglycans) in the perinotochordal region of notochords (prepared by the various procedures detailed in the previous section); and to examine the
VOLUME 42,1975
accumulation of cartilaginous matrix by somite explants. Notochords and explants were fixed in Carnoy’s solution (absolute ethanol:acetic acid, 3:1), dehydrated, embedded in paraffin, and sectioned at 5 pm. Sections were stained by the Alcian Blue/ hematoxylin procedure described by Ellison and Lash (1971), and with Alcian Blue at various MgCl, concentrations as described by Quintarelli and Dellovo (1965). The two-step PAS procedure of Scott and Dorling (1969) was used to demonstrate uranic acid-containing glycosaminoglycans. Staining with aprotinin (Trasylol; FBA Pharmaceuticals) conjugated with fluorescein isothiocyanate (FITC) was performed as described by Kiernan and Stoddart (1973). Fluorescence was detected with a Reichert “Binolux” microscope with excitation by blue-violet (350-450 nm) light. Biochemical
Analyses
Incorporation of radioactive precursors into glycosaminoglycans. Glycosaminoglycans were extracted from somite explants and notochords continuously exposed to either [YS]sulfate or [3H]glucosamine by procedures previously described (Kosher et al., 1973). Briefly, tissues were sonicated, then digested with Pronase (Calbiochem), treated with cold TCA, and dialyzed against Na,SO, and distilled water. The dialysates were lyophilized, dissolved in a minimal amount of distilled water, and aliquots were supplied with scintillation fluid (0.6% PPO and 0.3% POPOP in toluene) containing 5% BIO-Solv (Beckman) for determination of radioactivity by means of Intertechnic liquid scintillation spectrometer. The remainder of the dissolved lyophilisate (glycosaminoglycan) was utilized for further characterization. Characterization of [35S]sulfate-labeled glycosaminoglycan. The relative amounts of chondroitin 4-sulfate and chondroitin 6-sulfate synthesized by tissues incubated in the presence of (35S]sulfate were deter-
KOSHER
AND LASH
Notochordal
mined by the enzymatic method of Saito et al. (1968) using chondroitinase ABC and AC as previously described (Kosher and Searls, 1973). The relative amount of [%]sulfate-labeled heparan sulfate was determined as described by Kosher and Searls (1973) utilizing nitrous acid which specifically degrades the N-sulfated GAG heparan sulfate (Lagunoff and Warren, 1962; Cifonelli, 1968a,b; Lindahl, 1970; Kraemer, 1971a,b). Characterization of [3H]glucosaminelabeled glycosaminoglycans. The amount of [3H]glucosamine-labeled hyaluronic acid was determined as described by Dan iel et al. (1973) utilizing leech hyaluronidase (hyaluronic acid- 1,3 hydrolase; Biotries, Inc.), an enzyme which specifically degrades hyaluronic acid (see also Meier and Hay, 1973b). Briefly, after treatment of [3H]glucosamine-labeled glycosaminoglycan samples with leech hyaluronidase, the amount of radioactivity associated with leech hyaluronidase-sensitive and -resistant material was determined after precipitation of undegraded GAG with cetylpyridinium chloride (CPC) (see Daniel et al., 1973 for details). Similarly, the amount of [3H]glucosamine-labeled chondroitin sulfate was determined as described by Daniel et al. (1973). Aliquots of the glycosaminoglycan samples were treated with chondroitinase ABC, and the amount of radioactivity associated with chondroitinase-sensitive and -resistant material was determined after precipitation with CPC.
proteoglycans
and chondrogenesis
365
2 M-day chick embryos exposed for 24 hr to [3H]tryptophane. Collagenase activity was determined as described by Mandl et al. (1953) and as described by Peterkofsky and Diegelmann (1971) using total labeled protein extracted from 15-day embryonic chick chondrocytes exposed for 24 hr to [3H]glycine on the fifth day of culture. RNase and DNase activities were assayed as described by Zimmerman and Sandeen (1965).
DNA Analysis DNA was determined by the Santoianni and Ayala (1965) modification of the procedure of Kissane and Robins (1958), except that fluorescence was determined in 1 N HCl (Hinegardner, 1971). RESULTS
During the period of its interaction with somites in vivo, the embryonic notochord synthesizes proteoglycans and other extracellular matrix materials which accumulate predominantly in the perinotochordal sheath (see for example, Ruggeri, 1972). In preparation for organ culture, notochords are routinely dissected from embryos after a brief treatment with trypsin (see Lash et al., 1964; Lash, 1967). As a result of this treatment, it appeared likely that extracellular materials which had accumulated along the periphery of notochords would be destroyed. If products of the notochord are involved in stimulating somite chondrogenesis, then notochords which retain these extracellular materials should stimulate in vitro somite chondrogenesis to a greater extent than notochords which iniDegradative Activities of Enzymes tially lack these components (e.g., trypsinThe enzymes chondroitinase ABC, AC, treated notochords). Accordingly, two proand testicular hyaluronidase were tested cedures have been utilized to prepare notofor protease, RNase, DNase, and collagen- chords which retain perinotochordal matease activities by the following procedures. rials. The first procedure involves dissecNonspecific protease activity was assayed tion of notochords without the aid of any as described by Davis and Smith (1955) enzyme treatment, and the second involves using casein as substrate and as described dissection of notochords from embryos with by Peterkofsky and Diegelmann (1971) the aid of a low concentration of collagenusing total labeled protein extracted from ase (rather than trypsin).
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Histochemical evidence of proteoglycans along the perinotochordal sheath. Cross sections of notochords dissected from embryos without the aid of enzyme treatment
VOLUME 42,1975
and with collagenase are shown in Figs. 1 and 3. Note in Figs. la and 3a, the intense accumulation of Alcian Blue-positive material along the periphery of notochords
Pm. 1. Cross sections of notochords dissected without the aid of enzyme treatment. x 1000. Note the accumulation of perinotochordal material as demonstrated by (1A) the stain Alcian Blue, (1B) Schiff-positive material after the two-step PAS procedure, (1C) intense fluorescence after treatment with FITC-labeled aprotinin. FIG. 2. Cross sections of notochords dissected with the aid of brief treatment with trypsin. x 1000. Note the absence of perinotochordal material after (2A) staining with Alcian Blue, (ZB) the two-step PAS procedure, (LX) treatment with FITC-labeled aprotinin. FIG. 3. Cross sections of notochords dissected with the aid of collagenase. x 1000. Note the accumulation of perinotochordal material as demonstrated by (3A) the stain Alcian Blue, (3B) Schiff-positive material after the two-step PAS procedure, (3C) intense fluorescence after treatment with FITC-labeled aprotinin.
KOSHER
AND LASH
Notochordal
prepared by these two procedures. No perinotochordal Alcian Blue-positive material could be detected at a Mg2+ concentration of 0.9 M; a Mg2+ concentration at which Alcian Blue is characteristically displaced from polyanionic glycosaminoglycans (Quintarelli and Dellovo, 1965). Perinotochordal Schiff-positive material could also be detected around notochords (Figs. lb and 3b) dissected by these two procedures after treatment of sections for 15 min with periodate followed by sodium borohydride reduction and a 24-hr periodate oxidation (the two-step PAS procedure of Scott and Dorling, 1969). This stain indicates the presence of uranic acid-containing compounds. Finally, staining with aprotinin conjugated with fluorescein isothiocyanate (FITC) revealed an intense fluorescence along the periphery of notochords dissected with no enzyme treatment and collagenase (Figs. lc and 3~). FITC-labeled aprotinin is a specific fluorochrome for acidic glycosaminoglycans (Kiernan and Stoddart, 1973). These results indicate that notochords dissected without the aid of enzyme treatment or with the aid of a low concentration of collagenase retain perinotochorda1 proteoglycans. These procedures, however, undoubtedly result in the retention of other perinotochordal extracellular materials as well as proteoglycans. In contrast, notochords dissected with the aid of trypsin did not posesses perinotochordal Alcian Blue-positive material (Fig. 2a) or Schiff-positive material after the two-step PAS procedure (Fig. 2b). There was a complete absence of fluorescence along the periphery of trypsin-treated notochords stained with FITC-labeled aprotinin (Fig. 2~). It should be added that trypsin treatment does not reduce the viability of notochords. Notochords dissected by the three methods described all initially possess the same amount of DNA and accumulate DNA in culture at identical rates. Characterization of the glycosaminogly-
proteogl.ycans
367
and chondrogenesis
cans synthesized by notochords. To confirm the histochemical indications of the accumulation of perinotochordal glycosaminoglycans, notochords dissected with no enzyme treatment, with trypsin, or with collagenase, were cultured on nutrient agar for 24 hr in the presence of either [35S]~u1fate or [3H]glucosamine, and the glycosaminoglycans (GAG) synthesized were subjected to biochemical analyses. Analysis with chondroitinase ABC and AC indicates that the [35S]sulfate-labeled GAG synthesized by notochords consist of 40% chondroitin 4-sulfate, 40% chondroitin 6sulfate, and 20% chondroitinase-resistant material (Table 1). The chondroitinaseresistant material has been characterized as heparan sulfate by virtue of the fact that it can be degraded by nitrous acid (Table 1). Of the [3H]glucosamine-labeled GAG synthesized by notochords, 78% was chondroitin sulfate (chondroitinase-sensitive material) and 22% was chondroitinaseresistant (i.e., heparan sulfate) confirming the analysis of the [35S]sulfate-labeled TABLE
1
RELATIVE AMOUNTS OF THE VARIOUS [?~]SULFATE-LABELED GLYCOSAMINOGLYCANS SYNTHESIZED BY EMBRYONIC NOTOCHORDS AS DETERMINED BY CHONDROITINASE ANALYSIS AND NITROUS ACID DEGRADATION Glycosaminoglycan Chondroitin &sulfate’ Chondroitin 4-sulfated Heparan sulfate’
w 40 40 20
n The amount of [%]sulfate-labeled GAG present in the samples utilized for these characterizations ranged between 1000 and 3000 dpm/Fg DNA, and the amount of DNA ranged from 1.5-2.5 pg. b Each value is the average of three determinations. Standard deviation = * 3-4 in each case. c Determined as the relative amount of radioactivity associated with 6-sulfated disaccharide residues after chondroitinase ABC and AC treatment. ’ Relative amount of radioactivity associated with I-sulfated disaccharide residues after chondroitinase ABC and AC treatment. e [YS] sulfate-labeled GAG which is not degraded by chondroitinase ABC or AC. This material can be degraded (i.e., rendered CPC soluble) by nitrous acid.
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DEVELOPMENTAL
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GAG (Table 2). Analysis of the [3H]glucosamine-labeled GAG synthesized by notochords with leech hyaluronidase (an enzyme which specifically degrades hyaluronic acid) indicates that notochords do not produce detectable amounts of hyaluronic acid (i.e., leech hyaluronidase-sensitive material) (Table 2). Although no difference could be detected in the types of GAG synthesized by either trypsin-dissected, collagenase-dissected, or freely dissected notochords, the amount synthesized by trypsin-dissected notochords (5120 dpmlpg DNA) was approximately 50% greater than the amount synthesized by collagenase-dissected (3780 dpm/kg DNA) or freely dissected notochords (3693 dpm/pg DNA). The greater synthesis of GAG by trypsin-treated notochords is undoubtedly a result of the fact that these notochords are replacing perinotochordal proteoglycans (see below). Chondrogenesis of somites in the presence of notochords dissected with no enzyme treatment, the aid of collagenase or trypsin. There have been repeated demonTABLE
2
RELATIVE AMOUNTS OF THE VARIOUS [3H]G~u~~~~~~~~-~~~~~~~ GLYCOSAMINOGLYCANS SYNTHESIZED BY EMBRYONIC NOTOCHORDS” Glycosaminoglycan
9c
Chondroitin sulfateb Heparan sulfate’ Hyaluronic acidd
78 22
