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An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt
I)F:\ F:I.oI’M\~ESTAL
~IOI.O(:Y
96, 355-365 (1983)
An Immunofluorescent Study of the Distribution of Fibronectin Laminin during Limb Regeneration in the Adult Newt’ ADARSH
K. GuT,ATI,*~~ ANDREW
A. ZAI,EWSKI,*
AND
and
A. H. Renn1-t
Fibronectin and laminin arc two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation descrihrs changes in their distribution during regeneration of the newt forclimb, as determined by indirect immunofluorescence. The distrihution of fihronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myotihers corresponding to its basement memhrane; the pcrineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithrlium, and dermal glands. The cytoplasm of myotihers, axons, skin cpithelium, and hone matrix lacked fluorescence for hoth glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, hlastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the mgoblast fusion zone. Laminin was first ohserved around the regenerated myotubrs; this was followed hy the appearance of fihronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distrihution of laminin and Iibronectin was similar to that seen in normal timh. Based on the distribution pattern of these glycoproteins, it is concluded that fihroncctin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fihronectin may he important in the determination of the architecture of the regenerated limb. INTRODUCTION
After limb amputation in the adult newt, a new limb regenerates from the tissues of the remaining limb stump. During this process which is under neural and hormonal control, the following sequence of events occurs. The differentiated muscle, bone, and other tissue elements present in the remaining stump undergo a process of dedifferentiation and give rise to undifferentiated blastemal cells of mesenchymal nature. The blastemal cells proliferate, aggregate, and redifferentiate into a new limb (Butler and Schotte, 1941; Schotte and Hall, 1952; Singer, 1952; Chalkley, 1954; Hay, 1962; Iten and Bryant, 1973). Changes in the extracellular matrix also occur during limb regeneration. These include synthesis of various collagen types and proteoglycans (Revel and Hay, 1963; Schmidt, 1970; Toole and Gross, 1971; Linsenmayer and Smith, 1975, 1976; Mailman and Dresden, 1976; Smith and Linsenmayer, 1982). Fibronectin and laminin are two high-molecularweight extracellular glycoproteins present in basement I This paper was presented at the First Mid-Atlantic Conference of the Society for Developmental Biology, Bethesda, Md., May 16-18, 1982. ’ To whom all correspondence should he addressed:Building 36, Room 4D-20, National Institutes of Health, Bethesda, Md. 20205.
membranes (Timpl et al., 1979). The distribution of laminin is more restricted than fibronectin (also present in plasma and connective tissue) and it is present only in the basement membranes (Timpl et ccl., 1979; Foidart ef al., 1980; Foidart and Reddi, 1980). In recent years, considerable interest has been focused on the role of these glycoproteins during various processes of cellular development and differentiation. Fibronectin is known to play an important role in cell to cell binding; cell to substratum binding; and in cell spreading and migration (Klebe, 1974; Yamada and Olden, 1978). Changes in the expression and functional role of fibronectin have been reported during myogenesis and muscle regeneration (Chen, 1977; Furcht et al., 1978; Podleski et al., 1979; Chiquet et ab., 1981; Gulati ef al., 1982); and differentiation of cartilage and bone (Dessau et ul., 1981; Weiss and Reddi, 1980; 1981a; Silver et al., 1981). Since laminin is an important component of all basement membranes, it plays a role in maintaining the architecture and integrity of various tissues. In addition, laminin has been described as a specific attachment glycoprotein for epithelial cells (Terranova et ul., 1980). Since both fibronectin and laminin are newly identified extracellular glycoproteins, the present study describes their distribution in normal limb tissues of the adult newt. Indirect immunofluorescence technique, 355 OOlZ-1606/83 $3.00
FIG;. 1. Distribution of laminin in a cross section of normal newt limb. Laminin is seen in endomysium fibers (M). In addition, laminin is seen in the region corresponding to basement membrane (arrows) epithelium (E). Similar distribution is also seen for fibronectin. Outside the skin epithelium is the muscle. 120X. FIG. 2. Distribution of laminin in a cross section of normal newt limb. The perineurium of the nerve (arrow). The sarcoplasm of the muscle (M) and the matrix of bone (B) are devoid of laminin. Similar nectin. 140X. 356
(pericellular) of the skelett mu scle of the dermal glands (G) and skin fluorescence seen in the supporting (N) possesses a thin rim of laminin distribution is also seen for fibro-
employing specific antibodies against fibronectin and laminin, is applied to determine the localization of these glycoproteins. In addition, alterations in the expression of these glycoproteins during dedifferentiation, blastema formation, and finally redifferentiation and morphogenesis are described. MATERIALS
AND
METHODS
Male and female adult newts (Notophthdmus viridescens), obtained from Lee’s Newt Farm in Oak Ridge, Tennessee, were used. The animals were kept in dechlorinated tap water at room temperature (20-25°C). After anesthesia with 0.1% tricaine (MS-222, Sigma), animals were placed in amphibian Ringer’s solution (pH 7.2) and their forelimbs were amputated bilaterally using a pair of scissors. The amputation site in all animals was through the middle of radius and ulna; the amputation site was then flattened by trimming the protruding bones. After surgery, the animals were placed in a container with a moist gauze and kept at lo-12°C overnight to recover and later transferred to conditioned tap water. Animals showing signs of fungal infection were discarded. At 2, 3, 7, 14, 21,28, and 42 days after amputation, the animals were anesthetized, and the blastema regenerates were harvested by reamputation at the elbow. The limb regenerates were placed between slabs of skeletal muscle (to facilitate sectioning) and immediately frozen in liquid nitrogen. At least six regenerated limbs were analyzed at each time interval; in addition, several normal limbs were analyzed. Longitudinal or cross-frozen sections of 6 pm thickness were cut serially in a cryostat at -20°C and mounted on multiple glass slides. A few of these slides were stained with periodic acid-Schiff (PAS) hematoxylin for histological analysis of the regenerating limb. The remaining slides were stored in a freezer at -20°C until immunofluorescent staining was performed. Preparatimz
bronectin antibodies were then isolated from immunized rabbit serum by fibronectin-Sepharose affinity chromatography. Laminin was purified from the EHS murine sarcoma, which is known to produce large amounts of basement membrane components grown in lathyritic mice (Orkin et ul., 1977; Timpl et al., 1979; Foidart and Reddi, 1980). Laminin was dissolved in phosphate-buffered saline (PBS), pH 7.4, and emulsified with an equal amount of Freund’s complete adjuvant and injected into rabbits. Blood was drawn from the rabbits and antibodies against laminin were isolated by cross immunoadsorption. The specificity of the antibodies to laminin and fibronectin has been reported earlier (Foidart and Reddi, 1980; Weiss and Reddi, 1981b). Indirect
ImmunoJuorescence
The purified antibodies to fibronectin and laminin were applied to adjacent tissue sections and incubated at room temperature in a moist chamber for 30 min. The protein concentration of both the antibody solutions was 20 pg/ ml. After incubation, the slides were washed in three changes of PBS for 5 min each. The sections were then incubated with fluorescein isothiocyanate-conjugated goat antibodies to rabbit IgG, diluted 1:20 with PBS for 30 min (Miles Laboratories). The slides were again washed in three changes of PBS and mounted in 90% glycerol and 10% PBS solution. A few slides were treated with testicular hyaluronidase (1 mg/ml, Sigma) before staining for fibronectin or laminin. All slides were viewed with a Leitz Ortholux II, epi-iluminated fluorescent microscope (E. Leitz Inc., Rockleigh, N. J.). Controls consisted of slides treated with preimmune serum, or sera in which the antibody was preabsorbed with purified antigen, or PBS as the first incubating solution, none of which yielded any fluorescence (Fig. 4).
of Antibodies
Fibronectin was isolated from rat plasma Sepharose affinity chromatography and gel resis (Weiss and Reddi, 1981a, 1981b). The band was sliced from the gel, homogenized plete Freund’s adjuvant, and injected into
Staining
RESULTS
by gelatinelectrophofibronectin in incomrabbits. Fi-
Fibronectin
und Laminin
Distribution
in Normal
Limb
The distribution of fibronectin and laminin was quite similar in normal limb tissues and was associated with their basement membranes (Figs. 1 and 2). Each myofi-
FIG. 3. Distribution of laminin in a cross section of a nerve present in the normal limb. The perineurium and endoneurium contain laminin, whereas, the axons (arrows) are devoid of it. 560X. FIG. 4. A representative control staining pattern of a normal limb, in which the primary antibody was omitted yielding no fluorescence. The bone (B) which was shattered during sectioning presents an autofluorescence artifact. 141)~. FIG:. 5. Distribution of libronectin in a cross section of muscle (M) present in the norm1 limb. Each myofiber possesses a thin, continuous rim of libronectin in the endomysium (arrow). 480x. FIG;. 6. Distribution of fibronectin in a cross section of muscle (M) in the distal portion of 2-day amputated limb stump. Thin, continuous endomysial distribution of fibronectin is not seen (compare to Fig. 5). 560X. FIG. 7. Distribution of laminin in a cross section of the distal portion of :&day amputated limb stump. Laminin distribution is irregular especially in the endomysial region of degenerating skeletal muscle CM) (compare to Fig. 1). 120x.
FIG. 8. Longitudinal section of the limb regenerate 14 days after amputation stained with PAS-hematoxylin. Large numbers of undifferentiated blastemal cells (BL) are seen in the distal tip of the limb regenerate. 30X. FIG. 9. Distribution of fibronectin in the distal tip of the 14-day limb regenerate. Fibronectin (mostly extracellular) is seen in the region containing blastemal cells (BL); however, it is not present in the skin epithelium (E). 140X. FIG. 10. Distribution of fibronectin in a cross section of the 14-day limb regenerate proximal to the distal tip. Fibronectin is again expressed in the region of undifferentiated blastemal cells which lie between the skin epithelium (E) and the bone (B). 140X. FIG. 11. Distribution of laminin in a tissue section adjacent to Fig. 10. Laminin is absent in the region of undifferentiated blastemal cells (compare to Fig. 10). The arrow points to the basement membrane underlying the skin epithelium (E). 140X. 358
ber possessed a thin, continuous layer of fibronectin (Fig. 5) and laminin in the region corresponding to the basement membrane (pericellular, referred to as endomysial throughout the present discussion). The sarcoplasm of the myofibers was, however, devoid of both glycoproteins. The nerves present in the limb contained fibronectin and laminin in the perineurium and endoneurium; the axoplasm of the nerve fibers did not contain any of these glycoproteins (Figs. 2, 3). All blood vessels stained intensely for fibronectin and laminin, especially in the region of the endothelial basement membrane. The basement membrane underlying the skin epithelium was also positive for fibronectin and laminin, whereas the epidermis was negative for both glycoproteins (Fig. 1). Some fibronectin but not laminin was found under the basement membrane of the epidermis in the connective tissue of the dermis. The basement membrane of the dermal mucous glands also contained fibronectin and laminin as a thin and continuous layer (Fig. 1). The extracellular matrix of the bones in the limb was devoid of fibronectin and laminin; however, the lining of the lacunae which contains osteocytes did exhibit a thin rim of fibronectin and laminin (Fig. 2).
Regmeruting
Limb
The histological progression of limb regeneration after amputation in the present study was similar to the earlier description of Iten and Bryant (1973). There was some variation in the extent of regeneration at each interval studied; however, the basic events were similar. Iten and Bryant (1973) described three major phases during limb regeneration. The first phase includes the healing of the amputation site and dedifferentiation of the remaining stump tissues. This is followed by the formation of the blastema and blastema growth. During the final phase, redifferentiation and morphogenesis occur resulting in the formation of a complete limb regenerate. In the following description, changes in fibronectin and laminin during various stages of limb regeneration are presented.
Within 2 days after amputation, the amputation site was covered with a thin layer of epidermis which had
migrated from the edges of the epithelium of the remaining limb stump. This migrated epidermis lacked a basement membrane since no laminin or fibronectin was seen in the corresponding region. Previous electron microscopic studies have also shown similar absence of basement membrane under the migrated skin (Salpeter and Singer, 1960a; Hay, 1962). In PAS-hematoxylin stained slides, the various tissue components in the limb stump did not show any changes initially. However, the distribution of fibronectin in the endomysium of the distal muscle fibers decreased and became patchy (compare Fig. 5 and 6). Expression of laminin did not change initially but as the degeneration of the distal muscles progressed, it also became irregular (Fig. 7). The distribution of fibronectin, as well as laminin, did not change in the more proximal region of the remaining limb stump, and was similar to the normal limb distribution.
At 7 days, the end of the limb had a round appearance and dedifferentiation of the muscle and bone was apparent in the remaining stump.Small numbers of undifferentiated blastemal cells were present between the apical epidermis and dedifferentiating stump tissues. The presence of fibronectin was seen primarily in the extracellular region of the blastema; laminin was not seen at this time in the blastema. At 14 days (Fig. 9), the expression of fibronectin increased in the blastema region as compared to 7 days. The number of blastemal cells increased and dense cellular aggregations were also visible in some regions of the blastema (Fig. 8). The blastema was still devoid of laminin (compare Fig. 10 and Fig. 11). Regeneration of skeletal muscle independent of the blastema (called tissue mode regeneration) (see Carlson, 1979) was prominent in the region proximal to the blastema. This regeneration occurs through the tissue mode and is a result of local injury to the skeletal muscle caused by amputation (Carlson, 1979). No expression of fibronectin and laminin was seen in the region of local muscle regeneration until the formation of myotubes. These newly formed myotubes were easily distinguishable from the nonregenerating original myofibers (Fig. 12). The original myofibers were larger and possessed a continuous endomysial ring of laminin and
Frc:. 12. Distribution of laminin in a cross section of the proximal region of the remaining stump 14 days after amputation; in this region, skeletal muscle regeneration (caused by injury) through the tissue mode is occurring. The original muscle (OM) and the regenerated muscle (RM) are easily distinguishable. Original muscle fibers arc large as compared to smaller, round shaped regenerated myofibers. The arrows point to the basement membrane of the glands and epidermis. 120X. FIG;. 13. Distribution of fibronectin in a tissue section adjacent to Fig. 12 for comparison. Few of the regenerated myofibers (RM) have fibronectin around them. 120X.
FIG. 14. Longitudinal section of the limb regenerate 21 days after amputation stained with PAS-hematoxylin. Aggregates of blastemal cells (BL) are seen in the distal tip; however, in the proximal region blastemal cells are aligning for redifferentiation of cartilage (precartilage, PC) and muscle (premyogenic, PM). 30x. FIG. 15. Longitudinal section of the distal (digit) limb regenerate 42 days after amputation stained with PAS-hematoxylin. A few blastemal cells (BL) are still present in the distal tip. Regenerated cartilage (C) and muscle (M) are also present in the regenerated limb. 48X. FIG. 16. Distribution of fibronectin in a longitudinal section of the limb regenerate 42 days after amputation. Extensive presence of fibronectin is seen in the distal tip (contains blastemal cells, BL); however, its expression decreases proximally, forming a gradient. The cartilage matrix (arrow) is devoid of fibronectin but the chondrocytes and the lacunar lining continue to be positive for it. 120X. FIG. 17. Distribution of laminin in a tissue section adjacent to Fig. 16. Laminin is seen in the endomysial region of regenerated muscle (M). The arrow points to the basement membrane of the skin epithelium (E). Weak fluorescence for the presence of laminin is seen in the lacunar lining of the developing cartilage. 120X.
FIG. 1X. Distribution of fibronectin in the precartilage region of a 42day limb regenerate. Extensive presence of fibronectin is seen in the perichondrial (P) region where the blastemal cells are aggregating and aligning. As the chondrocytes mature and deposit cartilage specific matrix, the latter becomes devoid of fibronectin. The chondrocgtcs, however, show presence of fibronectin (arrow). 560X. FIG. 19. Distribution of fibronectin in the cartilage matrix after its unmasking by hyaluronidase treatment in a 42.day regenerate. Fibronectin is seen throughout the cartilage matrix; however, the fibronectin associated with the chondrocytes is lost (arrows) after hyaluronidase treatment (compare to arrow in Fig. 18). 560X. FIG. 20. Distribution of fibronectin in the premyogenic region of the blastema of a 21-day limb regenerate. Longitudinal strands (arrows) of fibronectin are seen around the aligning blastcmal cells destined to muscle formation. 560X.
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362
DEVELOPMENTAL
BIOLOGY
fibronectin. The regenerated myotubes were small and round, and all of them had a complete circular ring of laminin; however, only a few of these myotubes showed the presence of fibronectin (compare Fig. 12 and Fig. 13). No sign of redifferentiation of the blastemal cells was visible in any of the limbs studied at this interval. Blastema Growth, Morphogenesis
Rediflerentiation, and Phase (21, 28, and 4%’Days)
The limb regenerate at 21 days after amputation was cone shaped and flattened at the distal end. The blastema had grown further and the blastemal cells in the central axis of the limb were closely packed; and initiation of the redifferentiation process was apparent (Fig. 14). There was an extensive distribution of fibronectin in the blastema region but laminin was not seen there. Redifferentiation marked by chondrogenesis and myogenesis was clearly seen at 28 and 42 days after amputation (Fig. 15). The blastemal cells elongated and aligned themselves in the center of the regenerate. These cells possessed intense extracellular fibronectin expression (Fig. 16). Chondrogenesis began proximally and extended distally. The chondrocytes derived from blastemal cells contain fibronectin (Figs. 16 and 18); however, as they matured and started forming their matrix, fibronectin slowly disappeared from the matrix. Intense immunofluorescence for fibronectin was seen in the perichondrial region, as well as in the blastemal cell aggregates located in the distal tip of the chondrogenic site (Fig. 18). As chondrogenesis progressed, the hypertrophic chondrocytes became distant from each other because of the deposition of extracellular matrix which was now devoid of staining for fibronectin. As a result of chondrogenesis, the length of the limb regenerate increased, and at 42 days a normal looking limb with distinct digits was seen. Chondrocytes continued to show the presence of fibronectin in their cytoplasm and in the lacunar lining. Laminin, which was not seen earlier, was codistributed with fibronectin in the lacunar lining of the developing cartilage and persisted in the lacunar lining of the bone (Fig. 17). The matrices of cartilage and bone, however, were completely devoid of both fibronectin and laminin. To determine whether these glycoproteins disappeared from the matrix or were being masked by cartilage proteoglycan, a few slides were treated with testicular hyaluronidase before staining for fibronectin and laminin. Intense fluorescence for the presence of fibronectin (Fig. 19) and not laminin was seen throughout the cartilage and bone matrix after proteoglycan digestion. The chondrocyte-associated fibronectin was, however, lost after hyaluronidase treatment (Fig. 19). Redifferentiation of skeletal muscle occurred simultaneously with chondrogenesis. Blastemal cells destined
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for myogenesis elongated and arranged themselves parallel to the long axis of the regenerate (Fig. 14). Longitudinal strands of fibronectin were seen around these aligning blastemal cells (Fig. 20). Myogenesis also proceeded proximally to distally. At this time, a reverse gradient of fibronectin was seen, i.e., intense fibronectin in the distal blastema region which decreased proximally (Fig. 16). Fusion of blastemal cells resulted in formation of myotubes (called epimorphic mode regeneration, dependent on blastemal cells) (Carlson, 1979). The myotubes and myofibers were small initially (Figs. 15 and 17); however, they eventually grew in size and became quite similar to the original myofibers. Again, laminin was seen before fibronectin around these regenerated myotubes (not shown, similar to Figs. 12 and 13). The nerves and blood vessels from the remaining stump grew into the limb regenerate during the redifferentiation of cartilage and muscle. The regeneration process was now almost complete since the major components of the limb were formed and what remained was the physical growth. DISCUSSION
In the present study, fibronectin and laminin were localized in normal and regenerating newt limbs. The distribution of these glycoproteins in various normal tissues of the limb was similar to that described in previous reports (Stenman and Vaheri, 19’78; Linder et al., 1978; Foidart et al., 1980; Weiss and Reddi, 1981a; Foidart and Reddi, 1980; Rapesh et al., 1981; Gulati et al., 1982). During wound healing after limb amputation, fibronectin disappears from the muscles present in the distal region of the remaining limb stump. In dedifferentiating limbs, the injured ends of the muscle, bone, and other tissue components are broken down. These changes have been described as degenerative and differentiated tissue components (cytoplasm, basement membrane, etc.) disappear from the distal stump (Manner, 1953; Bodemer, 1958; Hay, 1962). During this degeneration, first fibronectin and later laminin disappears from the distal portion of the remaining limb stump. However, normal distribution of these glycoproteins is seen in the more proximal region. The disappearance of fibronectin before laminin in the distal portion of the stump suggests that the fibronectin is more sensitive to injury. Similar selective disappearance of endomysial fibronectin is seen in the ischemic myofibers of rat skeletal muscle autotransplants (Gulati et al., 1982; Gulati and Zalewski, 1982). As the blastema formed, fibronectin appeared in the extracellular matrix. The blastemal cells proliferated and as the blastema grew, increased fluorescence for fibronectin was present in the blastemal region. Since
blastemal cells are metabolically very active and synthesize DNA and proteins (Bodemer and Everett, 1959; Riddiford, 1960; O’Steen, 1960; Hay and Fischman, 1961), it is possible that these cells also synthesize fibronectin. Cultures of fibroblasts, Schwann, epithelial, and endothelial cells can also synthesize fibronectin and secrete it into the extracellular medium (Yamada et ul., 19’77; Kurkinen and Alitalo, 1979; Chen et al., 197’7; Jaffe and Mosher, 1978). The presence of fibronectin, as seen in the blastema, has been recently demonstrated in undifferentiated mesenchymal cells of the developing chick limb bud (Dessau et al., 1980; Tomasek et al., 1982) and in developing mouse limb and palate (Silver et al., 1981). Since fibronectin is a major component of the early matrix of the blastema, it may be involved in blastemal cell adhesion, migration, and alignment necessary for redifferentiation. In contrast to the accumulation of fibronectin, laminin was not seen in the blastema. Because of its distribution, fibronectin also seems to play a role during early stages of redifferentiation. Redifferentiation of cartilage and muscle occurred simultaneously and progressed in a proximal to distal direction. A reverse gradient of fibronectin distribution was seen within the limb; the maximum amount of fibronectin (mostly extracellular) was seen in the distal undifferentiated region as compared to the more proximal region undergoing redifferentiation. This would mean that fibronectin was important in initial aggregation of blastemal cells; however, it slowly disappeared from the matrix at the onset of cartilage and muscle redifferentiation. During cartilage redifferentiation when blastemal cells aggregate and align themselves in the precartilage region, extensive presence of fibronectin was seen around them. As the precartilage cells (chondrocytes) mature and synthesize extracellular matrix, fibronectin begins to disappear from the matrix. Since intense fluorescence for fibronectin was seen at the site of cartilage differentiation, it is concluded that fibronectin somehow is important for initiating chondrogenesis. This role of fibronectin has also been proposed by others in different experimental models for chondrogenesis (Newman and Frish, 1979; Dessau et crl., 1980; Silver et al., 1981; Weiss and Reddi, 1981a; Tomasek et al., 1982). As chondrogenesis advances, the matrix becomes completely devoid of detectable fibronectin. That the loss of immunofluorescence for fibronectin from the cartilage and finally bone matrix is due to the masking of fibronectin by cartilage proteoglycans and not its actual disappearance was confirmed by pretreating limb tissue sections with hyaluronidase prior to fibronectin staining (Weiss and Reddi, 1980, 1981a; Silver et al., 1981). The cartilage matrix was completely devoid of laminin at all stages of chondrogenesis, except for some weak fluorescence in the chondrocytes and within the lacunar
spaces of the cartilage and bone. This presence of laminin is not explainable at present since it is known that both cartilage and bone cells lack basement membranes. Blastemal cells destined to undergo myogenesis (myoblasts) align themselves along the long axis of the limb regenerate. Fibronectin disappears as these cells align and undergo fusion to form myotubes. This suggests that fibronectin is not required for actual fusion of the myoblasts; however, it may be required for cell aggregation and alignment prior to fusion. It has been reported in several in vitro studies that fibronectin is not required for myoblast fusion (Chen, 1977; Furcht et ul., 1978; Puri et ul., 1979; Podleski et al., 1979). The disappearance of fibronectin in redifferentiating muscle and cartilage results in a decreasing gradient of fibronectin toward the more proximal regions. Laminin is seen for the first time around the newly regenerated myotubes in the region corresponding to their basement membrane. Fibronectin was also seen around the newly regenerated myotubes, but it appeared later than laminin. Based on these findings, it can be concluded that blastemal cells and myoblasts lack basement membranes (or its important components). The blastemal cells are known to lack basement membranes as previously described in ultrastructural studies (Salpeter and Singer, 1960b; Hay, 1962). However, after fusion of these cells and formation of myotubes, a new basement membrane is synthesized which persists throughout maturation. In addition to muscle differentiation from blastemal cells, muscle regeneration by the tissue mode occurs in the proximal region of the remaining limb stump. Laminin also appears before fibronectin in the basement membrane region of these myotubes. This would mean that various components of basement membranes are formed in sequence. Similar sequential appearance of various basement membrane components (type IV collagen, laminin, and fibronectin) is also seen in regenerating skeletal muscle of rats (Gulati et al., 1982; Gulati and Zalewski, 1982) and during embryonic kidney development (Ekblom, 1981). The redifferentiation process is essentially complete within 42 days; however, further growth of the limb regenerate continues. Distribution of fibronectin and laminin in regenerated limb tissues is similar to the distribution seen in normal limb tissues. It is interesting to note that although the distribution of both fibronectin and laminin was similar in normal limb tissues, this similarity was not present during limb regeneration. This suggests that these two glycoproteins may have different functional roles. Fibronectin seems to be more important in blastema formation, aggregation of blastemal cells, and initiating redifferentiation; whereas, laminin may play a role in determining the architecture of the regenerating limb after redifferentiation.
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The authors wish to thank Drs. Randall W. Reyer and Richard G. Frederickson for helpful comments and to Mrs. Lois V. Trigg for skillful preparation of the manuscript. REFERENCES BODEMER, C. W. (1958). The development of nerve-induced supernumerary limbs in the adult newt, ‘Z’ritum~s viridescent. J. Morph&. 102, 555-582. BODEMER, C. W., and EVERETT, N. B. (1959). Localization of newly synthesized proteins in regenerating newt limb as determined by radioautographic localization of injected methionine-S%. Dev. Biol. 1, 327-342. BUTLER, E. G., and SCHOTTE, 0. E. (1941). Histological alterations in denervated nonregenerating limbs of urodele larvae. J. Exp. Zool. 88, 307-341. CARLSON, B. M. (1979). Relationship between tissue and epimorphic regeneration of skeletal muscle. In “Muscle Regeneration” (A. Mauro, ed.), pp. 57-71. Raven Press, New York. CHALKEY, D. T. (1954). A quantitative histological analysis of forelimb regeneration in Triturus viridescens J. Morphol. 94, 21-70. CHEN, L. B. (1977). Alteration in cell surface LET protein during myogenesis. Cell, 10, 393-400. CHEN, L. B., MAITLAND, N., GALLIMORE, P. H., and MCDOUGALL, J. K. (1977). Detection of the large external transformation-sensitive protein on some epithelial cells. E;cp. Cell Res. 106, 39-49. CHIQUET, M., EPPENBERGER, H. M., and TURNER, D. C. (1981). Muscle morphogenesis: Evidence for an organizing function of exogenous fibronectin. Dev. Biol. 88, 220-235. DESSAU, W., VERTEL, B. M., VON DER MARK, H., and VON DER MARK, K. (1981). Extracellular matrix formation by chondrocytes in monolayer culture. J. Cell Biol. 90, 78-83. EKBLOM, P. (1981). Formation of basement membrane in the embryonic kidney: An immunohistological study. J. Cell Biol. 91, l-10. FOIDART, J. M., BERE, E. W., YAAR, M., RENNARD, S. I., GULLINO, M., MARTIN, G. R., and KATZ, S. I. (1980). Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Lab. Invest. 42, 336-342. FOIDART, J. M., and REDDI, A. H. (1980). Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone. Dev. Biol. 75, 130-136. FIIRCHT, L. T., MOSHER, D. F., and WENDELSCHAFER-CRABB, G. (1978). Immunocytochemical localization of fibronectin (LETS protein) on the surface of L6 myoblasts: Light and electron microscopic studies. Cell. 13, 263-271.
GTJLATI, A. K., RE~~I, A. H., and ZALEWSKI, A. A. (1982). Distribution of fibronectin in normal and regenerating skeletal muscle. Anut. Rec. 204, 175-183. GULATI, A. K., and ZALEWSKI, A. A. (1982). Renewal of the basement membrane during skeletal muscle regeneration. J. Cell Biol. 95,365a. HAY, E. D. (1962). Cytological studies of dedifferentiation and differentiation in regenerating amphibian limbs. Zn “Regeneration” (D. Rudnick, ed.), pp. 177-210. Ronald Press, New York. HAY, E. D., and FISCHMAN, D. A. (1961). Origin of the blastema in regenerating limbs of the newt, Triturus viridescens. An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev. Biol. 3, 26-59. ITEN, L. E., and BRYANT, S. V. (1973). Forelimb regeneration from different levels of amputation in the newt, Notophthalmus viridestens: Length, rate, and stages. Wilhelm Roux’s Arch. 173, 263-282. JAFFE, E. A., and MOSHER, D. F. (1978). Synthesis of fibronectin by cultured human endothelial cells. Ann. N. Y Acad. Sci. 312, 122131.
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KLEBE, R. J. (1974). Isolation of a collagen dependent cell attachment factor. Nature (London) 250, 248-251. KURKINEN, M., and ALITALO, K. (1979). Fibronectin and procollagen produced by a clonal line of Schwann cells. FEBS Lett. 102, 64-68. LINDER, E., STENMAN, S., LEHTO, V. P., and VAHERI, A. (1978). Distribution of fibronectin in human tissues and relationship to other connective tissue components. Ann. N. K Acad. Sci 312, 151-159. LINSENMAYER, T. F., and SMITH, G. N., JR. (1975). The biosynthesis of type II collagen (cartilage) during limb morphogenesis and regeneration. In “Extracellular Matrix Influences on Gene Expression” (H. C. Slavkin and R. C. Grevlich, eds.), pp. 303-310. Academic Press, New York. LINSENMAYER, T. F., and SMITH, G. N., JR. (1976). The biosynthesis of cartilage type collagen during limb regeneration in the larval salamander. Dew. Biol. 52, 19-30. MAILMAN, M. L., and DRESDEN, M. H. (1976). Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation. Dev. Biol. 50, 378-394. MANNER, H. W. (1953). The origin of the blastema and of new tissues in regenerating forelimb of adult Triturus viridescens. J Exp. 2001. 122, 229-257.
NEWMAN, S. A., and FRISCH, H. L. (1979). Dynamics of skeletal pattern formation in the developing chick limb. Science 205, 662-668. ORKIN,
R. W., GEHRON,
P., MCGOODWIN,
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ENTINE, R., and SWARM, R. (1977). A murine tumor producing a matrix of basement membrane. .Z E;cp. Med. 145, 204-220. O’STEEN, N. K. (1960). Radioautographic studies of cell proliferation in normal and regenerating tissues of the adult newt. Anat. Rec. 136, 253-254.
PODLESKI, T. R., GREENBERG, I., SCHLESSINGER, J., and YAMADA, K. M. (1979). Fibronectin delays the fusion of L6 myoblasts. Exp. Cell Res. 122, 317-326. P~JRI, E. C., CHIQUET, M., and TIJRNER, D. C. (1979). Fibronectin-independent myoblast fusion in suspension cultures. Biochem Bio phys. Res. Commun. 90, 883-889. RAPESH, L. A., FURCHT, L. T., and SMITH, D. (1981). Immunocytochemical localization of fibronectin in limb tissues of the adult newt. Notophthalmus viridescent. .I Histochem. Cytochem. 29, 937-945. REVEL, J. P., and HAY, E. D. (1963). An autoradiographic and electronmicroscopic study- of collagen synthesis in differentiating cartilage. Z. Zellforsch. 61, 110-144. RIDDIFORL), L. M. (1960). Autoradiographic studies on tritiated thymidine infused into the blastema of the early regenerate in the adult newt, Triturus. J. Exp. Zool. 144, 25-32. SALPETER, M. M., and SINGER, M. (1960a). Differentiation of the submicroscopic adepidermal membrane during limb regeneration in adult Triturus, including a note on the use of the term basement membrane. An&. Rec. 136, 27-40. SALPETER, M. M., and SINGER, M. (1960b). The fine structure of mesenchymatous cells in the regenerating forelimb of the adult newt Triturus. Dev. Biol. 2, 516-534. SCHMIDT, A. J. (1970). Collagen and its synthesis in regenerating limbs of the adult newt, Diemictylus viridescens. Amer. Zool. 10, 57. SCHOTTE, 0. E., and HALL, A. B. (1952). Effects of hypophysectomy upon phases of regeneration in progress (Triturus viridescwa). J. Exp. Zool. 121, 521-560. SILVER, M. H., FOIDART, J. M., and PRATT, R. M. (1981). Distribution of fibronectin and collagen during mouse limb and palate development. Diflewntiation 18, 141-149. SINGER, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. &art. Rev. Biol. 27, 169-200. SMITH, G. N., JR., and LINSENMAYER, T. F. (1982). Collagens for the skin and cartilage of the larval salamander. Ambystoma tigrinum. J. Ezp. Zool. 220, 243-250.
QTENMAN, S., and VAI~ERI, A. (197X). Distribution of a major connective tissue protein, fibronectin, in normal human tissues. J. lC.rp. iWed. 147, 1054-1064. TERRANOVA, V. P., RO~IRUAUI, D. H., and MARTIN, G. R. (1980). Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen. Cell 22, 719-726. TIMPL, R., Rorinti, H., GERHON ROREY, P., RENNARn, S. I., FOIUART, J. M., and MARTIN, G. R. (1979). Laminin-A glycoprotein from basement membranes. J. Bid. Chwn. 254, 9933-9937. TOMASEK, J. J., MAZURKIEWICZ, J. E., and NEWMAN, S. A. (1982). Nonuniform distribution of fibronectin during avian limb development. I)cu. Biol 90, 118-126. TOOLE, B. P., and GROSS, J. (1971). The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. L)Pv. Bid. 25, 57-77.
R. E., and REI)L)I, A. H. (1980). Synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentiation of cartilage and bone irr r.ivo. Proc. Nut.
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WEISS, R. E., and REI)I)I, A. H. (1981a). Appearance of fibronectin during the differentiation of cartilage, bone and bone marrow. J. Cell Bid.
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WEISS, R. E., and REnnr, A. I-I. (1981b). Isolation and characterization .J, 197, 529-534. of rat plasma fibronectin. Biochrm. YAMADA, K. M., and OL~XN, K. (1978). Fibronectin-adhesive glycoproteins of cell surface and blood. Nolure (Lodm) 275, 179-184. YAMADA, K. M., YAMAHA, S. S., and PASTAN, I. (1977). Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. J. Cd/ Bid.
74, 649-654.
~IOI.O(:Y
96, 355-365 (1983)
An Immunofluorescent Study of the Distribution of Fibronectin Laminin during Limb Regeneration in the Adult Newt’ ADARSH
K. GuT,ATI,*~~ ANDREW
A. ZAI,EWSKI,*
AND
and
A. H. Renn1-t
Fibronectin and laminin arc two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation descrihrs changes in their distribution during regeneration of the newt forclimb, as determined by indirect immunofluorescence. The distrihution of fihronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myotihers corresponding to its basement memhrane; the pcrineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithrlium, and dermal glands. The cytoplasm of myotihers, axons, skin cpithelium, and hone matrix lacked fluorescence for hoth glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, hlastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the mgoblast fusion zone. Laminin was first ohserved around the regenerated myotubrs; this was followed hy the appearance of fihronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distrihution of laminin and Iibronectin was similar to that seen in normal timh. Based on the distribution pattern of these glycoproteins, it is concluded that fihroncctin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fihronectin may he important in the determination of the architecture of the regenerated limb. INTRODUCTION
After limb amputation in the adult newt, a new limb regenerates from the tissues of the remaining limb stump. During this process which is under neural and hormonal control, the following sequence of events occurs. The differentiated muscle, bone, and other tissue elements present in the remaining stump undergo a process of dedifferentiation and give rise to undifferentiated blastemal cells of mesenchymal nature. The blastemal cells proliferate, aggregate, and redifferentiate into a new limb (Butler and Schotte, 1941; Schotte and Hall, 1952; Singer, 1952; Chalkley, 1954; Hay, 1962; Iten and Bryant, 1973). Changes in the extracellular matrix also occur during limb regeneration. These include synthesis of various collagen types and proteoglycans (Revel and Hay, 1963; Schmidt, 1970; Toole and Gross, 1971; Linsenmayer and Smith, 1975, 1976; Mailman and Dresden, 1976; Smith and Linsenmayer, 1982). Fibronectin and laminin are two high-molecularweight extracellular glycoproteins present in basement I This paper was presented at the First Mid-Atlantic Conference of the Society for Developmental Biology, Bethesda, Md., May 16-18, 1982. ’ To whom all correspondence should he addressed:Building 36, Room 4D-20, National Institutes of Health, Bethesda, Md. 20205.
membranes (Timpl et al., 1979). The distribution of laminin is more restricted than fibronectin (also present in plasma and connective tissue) and it is present only in the basement membranes (Timpl et ccl., 1979; Foidart ef al., 1980; Foidart and Reddi, 1980). In recent years, considerable interest has been focused on the role of these glycoproteins during various processes of cellular development and differentiation. Fibronectin is known to play an important role in cell to cell binding; cell to substratum binding; and in cell spreading and migration (Klebe, 1974; Yamada and Olden, 1978). Changes in the expression and functional role of fibronectin have been reported during myogenesis and muscle regeneration (Chen, 1977; Furcht et al., 1978; Podleski et al., 1979; Chiquet et ab., 1981; Gulati ef al., 1982); and differentiation of cartilage and bone (Dessau et ul., 1981; Weiss and Reddi, 1980; 1981a; Silver et al., 1981). Since laminin is an important component of all basement membranes, it plays a role in maintaining the architecture and integrity of various tissues. In addition, laminin has been described as a specific attachment glycoprotein for epithelial cells (Terranova et ul., 1980). Since both fibronectin and laminin are newly identified extracellular glycoproteins, the present study describes their distribution in normal limb tissues of the adult newt. Indirect immunofluorescence technique, 355 OOlZ-1606/83 $3.00
FIG;. 1. Distribution of laminin in a cross section of normal newt limb. Laminin is seen in endomysium fibers (M). In addition, laminin is seen in the region corresponding to basement membrane (arrows) epithelium (E). Similar distribution is also seen for fibronectin. Outside the skin epithelium is the muscle. 120X. FIG. 2. Distribution of laminin in a cross section of normal newt limb. The perineurium of the nerve (arrow). The sarcoplasm of the muscle (M) and the matrix of bone (B) are devoid of laminin. Similar nectin. 140X. 356
(pericellular) of the skelett mu scle of the dermal glands (G) and skin fluorescence seen in the supporting (N) possesses a thin rim of laminin distribution is also seen for fibro-
employing specific antibodies against fibronectin and laminin, is applied to determine the localization of these glycoproteins. In addition, alterations in the expression of these glycoproteins during dedifferentiation, blastema formation, and finally redifferentiation and morphogenesis are described. MATERIALS
AND
METHODS
Male and female adult newts (Notophthdmus viridescens), obtained from Lee’s Newt Farm in Oak Ridge, Tennessee, were used. The animals were kept in dechlorinated tap water at room temperature (20-25°C). After anesthesia with 0.1% tricaine (MS-222, Sigma), animals were placed in amphibian Ringer’s solution (pH 7.2) and their forelimbs were amputated bilaterally using a pair of scissors. The amputation site in all animals was through the middle of radius and ulna; the amputation site was then flattened by trimming the protruding bones. After surgery, the animals were placed in a container with a moist gauze and kept at lo-12°C overnight to recover and later transferred to conditioned tap water. Animals showing signs of fungal infection were discarded. At 2, 3, 7, 14, 21,28, and 42 days after amputation, the animals were anesthetized, and the blastema regenerates were harvested by reamputation at the elbow. The limb regenerates were placed between slabs of skeletal muscle (to facilitate sectioning) and immediately frozen in liquid nitrogen. At least six regenerated limbs were analyzed at each time interval; in addition, several normal limbs were analyzed. Longitudinal or cross-frozen sections of 6 pm thickness were cut serially in a cryostat at -20°C and mounted on multiple glass slides. A few of these slides were stained with periodic acid-Schiff (PAS) hematoxylin for histological analysis of the regenerating limb. The remaining slides were stored in a freezer at -20°C until immunofluorescent staining was performed. Preparatimz
bronectin antibodies were then isolated from immunized rabbit serum by fibronectin-Sepharose affinity chromatography. Laminin was purified from the EHS murine sarcoma, which is known to produce large amounts of basement membrane components grown in lathyritic mice (Orkin et ul., 1977; Timpl et al., 1979; Foidart and Reddi, 1980). Laminin was dissolved in phosphate-buffered saline (PBS), pH 7.4, and emulsified with an equal amount of Freund’s complete adjuvant and injected into rabbits. Blood was drawn from the rabbits and antibodies against laminin were isolated by cross immunoadsorption. The specificity of the antibodies to laminin and fibronectin has been reported earlier (Foidart and Reddi, 1980; Weiss and Reddi, 1981b). Indirect
ImmunoJuorescence
The purified antibodies to fibronectin and laminin were applied to adjacent tissue sections and incubated at room temperature in a moist chamber for 30 min. The protein concentration of both the antibody solutions was 20 pg/ ml. After incubation, the slides were washed in three changes of PBS for 5 min each. The sections were then incubated with fluorescein isothiocyanate-conjugated goat antibodies to rabbit IgG, diluted 1:20 with PBS for 30 min (Miles Laboratories). The slides were again washed in three changes of PBS and mounted in 90% glycerol and 10% PBS solution. A few slides were treated with testicular hyaluronidase (1 mg/ml, Sigma) before staining for fibronectin or laminin. All slides were viewed with a Leitz Ortholux II, epi-iluminated fluorescent microscope (E. Leitz Inc., Rockleigh, N. J.). Controls consisted of slides treated with preimmune serum, or sera in which the antibody was preabsorbed with purified antigen, or PBS as the first incubating solution, none of which yielded any fluorescence (Fig. 4).
of Antibodies
Fibronectin was isolated from rat plasma Sepharose affinity chromatography and gel resis (Weiss and Reddi, 1981a, 1981b). The band was sliced from the gel, homogenized plete Freund’s adjuvant, and injected into
Staining
RESULTS
by gelatinelectrophofibronectin in incomrabbits. Fi-
Fibronectin
und Laminin
Distribution
in Normal
Limb
The distribution of fibronectin and laminin was quite similar in normal limb tissues and was associated with their basement membranes (Figs. 1 and 2). Each myofi-
FIG. 3. Distribution of laminin in a cross section of a nerve present in the normal limb. The perineurium and endoneurium contain laminin, whereas, the axons (arrows) are devoid of it. 560X. FIG. 4. A representative control staining pattern of a normal limb, in which the primary antibody was omitted yielding no fluorescence. The bone (B) which was shattered during sectioning presents an autofluorescence artifact. 141)~. FIG:. 5. Distribution of libronectin in a cross section of muscle (M) present in the norm1 limb. Each myofiber possesses a thin, continuous rim of libronectin in the endomysium (arrow). 480x. FIG;. 6. Distribution of fibronectin in a cross section of muscle (M) in the distal portion of 2-day amputated limb stump. Thin, continuous endomysial distribution of fibronectin is not seen (compare to Fig. 5). 560X. FIG. 7. Distribution of laminin in a cross section of the distal portion of :&day amputated limb stump. Laminin distribution is irregular especially in the endomysial region of degenerating skeletal muscle CM) (compare to Fig. 1). 120x.
FIG. 8. Longitudinal section of the limb regenerate 14 days after amputation stained with PAS-hematoxylin. Large numbers of undifferentiated blastemal cells (BL) are seen in the distal tip of the limb regenerate. 30X. FIG. 9. Distribution of fibronectin in the distal tip of the 14-day limb regenerate. Fibronectin (mostly extracellular) is seen in the region containing blastemal cells (BL); however, it is not present in the skin epithelium (E). 140X. FIG. 10. Distribution of fibronectin in a cross section of the 14-day limb regenerate proximal to the distal tip. Fibronectin is again expressed in the region of undifferentiated blastemal cells which lie between the skin epithelium (E) and the bone (B). 140X. FIG. 11. Distribution of laminin in a tissue section adjacent to Fig. 10. Laminin is absent in the region of undifferentiated blastemal cells (compare to Fig. 10). The arrow points to the basement membrane underlying the skin epithelium (E). 140X. 358
ber possessed a thin, continuous layer of fibronectin (Fig. 5) and laminin in the region corresponding to the basement membrane (pericellular, referred to as endomysial throughout the present discussion). The sarcoplasm of the myofibers was, however, devoid of both glycoproteins. The nerves present in the limb contained fibronectin and laminin in the perineurium and endoneurium; the axoplasm of the nerve fibers did not contain any of these glycoproteins (Figs. 2, 3). All blood vessels stained intensely for fibronectin and laminin, especially in the region of the endothelial basement membrane. The basement membrane underlying the skin epithelium was also positive for fibronectin and laminin, whereas the epidermis was negative for both glycoproteins (Fig. 1). Some fibronectin but not laminin was found under the basement membrane of the epidermis in the connective tissue of the dermis. The basement membrane of the dermal mucous glands also contained fibronectin and laminin as a thin and continuous layer (Fig. 1). The extracellular matrix of the bones in the limb was devoid of fibronectin and laminin; however, the lining of the lacunae which contains osteocytes did exhibit a thin rim of fibronectin and laminin (Fig. 2).
Regmeruting
Limb
The histological progression of limb regeneration after amputation in the present study was similar to the earlier description of Iten and Bryant (1973). There was some variation in the extent of regeneration at each interval studied; however, the basic events were similar. Iten and Bryant (1973) described three major phases during limb regeneration. The first phase includes the healing of the amputation site and dedifferentiation of the remaining stump tissues. This is followed by the formation of the blastema and blastema growth. During the final phase, redifferentiation and morphogenesis occur resulting in the formation of a complete limb regenerate. In the following description, changes in fibronectin and laminin during various stages of limb regeneration are presented.
Within 2 days after amputation, the amputation site was covered with a thin layer of epidermis which had
migrated from the edges of the epithelium of the remaining limb stump. This migrated epidermis lacked a basement membrane since no laminin or fibronectin was seen in the corresponding region. Previous electron microscopic studies have also shown similar absence of basement membrane under the migrated skin (Salpeter and Singer, 1960a; Hay, 1962). In PAS-hematoxylin stained slides, the various tissue components in the limb stump did not show any changes initially. However, the distribution of fibronectin in the endomysium of the distal muscle fibers decreased and became patchy (compare Fig. 5 and 6). Expression of laminin did not change initially but as the degeneration of the distal muscles progressed, it also became irregular (Fig. 7). The distribution of fibronectin, as well as laminin, did not change in the more proximal region of the remaining limb stump, and was similar to the normal limb distribution.
At 7 days, the end of the limb had a round appearance and dedifferentiation of the muscle and bone was apparent in the remaining stump.Small numbers of undifferentiated blastemal cells were present between the apical epidermis and dedifferentiating stump tissues. The presence of fibronectin was seen primarily in the extracellular region of the blastema; laminin was not seen at this time in the blastema. At 14 days (Fig. 9), the expression of fibronectin increased in the blastema region as compared to 7 days. The number of blastemal cells increased and dense cellular aggregations were also visible in some regions of the blastema (Fig. 8). The blastema was still devoid of laminin (compare Fig. 10 and Fig. 11). Regeneration of skeletal muscle independent of the blastema (called tissue mode regeneration) (see Carlson, 1979) was prominent in the region proximal to the blastema. This regeneration occurs through the tissue mode and is a result of local injury to the skeletal muscle caused by amputation (Carlson, 1979). No expression of fibronectin and laminin was seen in the region of local muscle regeneration until the formation of myotubes. These newly formed myotubes were easily distinguishable from the nonregenerating original myofibers (Fig. 12). The original myofibers were larger and possessed a continuous endomysial ring of laminin and
Frc:. 12. Distribution of laminin in a cross section of the proximal region of the remaining stump 14 days after amputation; in this region, skeletal muscle regeneration (caused by injury) through the tissue mode is occurring. The original muscle (OM) and the regenerated muscle (RM) are easily distinguishable. Original muscle fibers arc large as compared to smaller, round shaped regenerated myofibers. The arrows point to the basement membrane of the glands and epidermis. 120X. FIG;. 13. Distribution of fibronectin in a tissue section adjacent to Fig. 12 for comparison. Few of the regenerated myofibers (RM) have fibronectin around them. 120X.
FIG. 14. Longitudinal section of the limb regenerate 21 days after amputation stained with PAS-hematoxylin. Aggregates of blastemal cells (BL) are seen in the distal tip; however, in the proximal region blastemal cells are aligning for redifferentiation of cartilage (precartilage, PC) and muscle (premyogenic, PM). 30x. FIG. 15. Longitudinal section of the distal (digit) limb regenerate 42 days after amputation stained with PAS-hematoxylin. A few blastemal cells (BL) are still present in the distal tip. Regenerated cartilage (C) and muscle (M) are also present in the regenerated limb. 48X. FIG. 16. Distribution of fibronectin in a longitudinal section of the limb regenerate 42 days after amputation. Extensive presence of fibronectin is seen in the distal tip (contains blastemal cells, BL); however, its expression decreases proximally, forming a gradient. The cartilage matrix (arrow) is devoid of fibronectin but the chondrocytes and the lacunar lining continue to be positive for it. 120X. FIG. 17. Distribution of laminin in a tissue section adjacent to Fig. 16. Laminin is seen in the endomysial region of regenerated muscle (M). The arrow points to the basement membrane of the skin epithelium (E). Weak fluorescence for the presence of laminin is seen in the lacunar lining of the developing cartilage. 120X.
FIG. 1X. Distribution of fibronectin in the precartilage region of a 42day limb regenerate. Extensive presence of fibronectin is seen in the perichondrial (P) region where the blastemal cells are aggregating and aligning. As the chondrocytes mature and deposit cartilage specific matrix, the latter becomes devoid of fibronectin. The chondrocgtcs, however, show presence of fibronectin (arrow). 560X. FIG. 19. Distribution of fibronectin in the cartilage matrix after its unmasking by hyaluronidase treatment in a 42.day regenerate. Fibronectin is seen throughout the cartilage matrix; however, the fibronectin associated with the chondrocytes is lost (arrows) after hyaluronidase treatment (compare to arrow in Fig. 18). 560X. FIG. 20. Distribution of fibronectin in the premyogenic region of the blastema of a 21-day limb regenerate. Longitudinal strands (arrows) of fibronectin are seen around the aligning blastcmal cells destined to muscle formation. 560X.
t-. 3 c ti 2 m $ 2 5’
s.
362
DEVELOPMENTAL
BIOLOGY
fibronectin. The regenerated myotubes were small and round, and all of them had a complete circular ring of laminin; however, only a few of these myotubes showed the presence of fibronectin (compare Fig. 12 and Fig. 13). No sign of redifferentiation of the blastemal cells was visible in any of the limbs studied at this interval. Blastema Growth, Morphogenesis
Rediflerentiation, and Phase (21, 28, and 4%’Days)
The limb regenerate at 21 days after amputation was cone shaped and flattened at the distal end. The blastema had grown further and the blastemal cells in the central axis of the limb were closely packed; and initiation of the redifferentiation process was apparent (Fig. 14). There was an extensive distribution of fibronectin in the blastema region but laminin was not seen there. Redifferentiation marked by chondrogenesis and myogenesis was clearly seen at 28 and 42 days after amputation (Fig. 15). The blastemal cells elongated and aligned themselves in the center of the regenerate. These cells possessed intense extracellular fibronectin expression (Fig. 16). Chondrogenesis began proximally and extended distally. The chondrocytes derived from blastemal cells contain fibronectin (Figs. 16 and 18); however, as they matured and started forming their matrix, fibronectin slowly disappeared from the matrix. Intense immunofluorescence for fibronectin was seen in the perichondrial region, as well as in the blastemal cell aggregates located in the distal tip of the chondrogenic site (Fig. 18). As chondrogenesis progressed, the hypertrophic chondrocytes became distant from each other because of the deposition of extracellular matrix which was now devoid of staining for fibronectin. As a result of chondrogenesis, the length of the limb regenerate increased, and at 42 days a normal looking limb with distinct digits was seen. Chondrocytes continued to show the presence of fibronectin in their cytoplasm and in the lacunar lining. Laminin, which was not seen earlier, was codistributed with fibronectin in the lacunar lining of the developing cartilage and persisted in the lacunar lining of the bone (Fig. 17). The matrices of cartilage and bone, however, were completely devoid of both fibronectin and laminin. To determine whether these glycoproteins disappeared from the matrix or were being masked by cartilage proteoglycan, a few slides were treated with testicular hyaluronidase before staining for fibronectin and laminin. Intense fluorescence for the presence of fibronectin (Fig. 19) and not laminin was seen throughout the cartilage and bone matrix after proteoglycan digestion. The chondrocyte-associated fibronectin was, however, lost after hyaluronidase treatment (Fig. 19). Redifferentiation of skeletal muscle occurred simultaneously with chondrogenesis. Blastemal cells destined
VOLIJME
96, 1983
for myogenesis elongated and arranged themselves parallel to the long axis of the regenerate (Fig. 14). Longitudinal strands of fibronectin were seen around these aligning blastemal cells (Fig. 20). Myogenesis also proceeded proximally to distally. At this time, a reverse gradient of fibronectin was seen, i.e., intense fibronectin in the distal blastema region which decreased proximally (Fig. 16). Fusion of blastemal cells resulted in formation of myotubes (called epimorphic mode regeneration, dependent on blastemal cells) (Carlson, 1979). The myotubes and myofibers were small initially (Figs. 15 and 17); however, they eventually grew in size and became quite similar to the original myofibers. Again, laminin was seen before fibronectin around these regenerated myotubes (not shown, similar to Figs. 12 and 13). The nerves and blood vessels from the remaining stump grew into the limb regenerate during the redifferentiation of cartilage and muscle. The regeneration process was now almost complete since the major components of the limb were formed and what remained was the physical growth. DISCUSSION
In the present study, fibronectin and laminin were localized in normal and regenerating newt limbs. The distribution of these glycoproteins in various normal tissues of the limb was similar to that described in previous reports (Stenman and Vaheri, 19’78; Linder et al., 1978; Foidart et al., 1980; Weiss and Reddi, 1981a; Foidart and Reddi, 1980; Rapesh et al., 1981; Gulati et al., 1982). During wound healing after limb amputation, fibronectin disappears from the muscles present in the distal region of the remaining limb stump. In dedifferentiating limbs, the injured ends of the muscle, bone, and other tissue components are broken down. These changes have been described as degenerative and differentiated tissue components (cytoplasm, basement membrane, etc.) disappear from the distal stump (Manner, 1953; Bodemer, 1958; Hay, 1962). During this degeneration, first fibronectin and later laminin disappears from the distal portion of the remaining limb stump. However, normal distribution of these glycoproteins is seen in the more proximal region. The disappearance of fibronectin before laminin in the distal portion of the stump suggests that the fibronectin is more sensitive to injury. Similar selective disappearance of endomysial fibronectin is seen in the ischemic myofibers of rat skeletal muscle autotransplants (Gulati et al., 1982; Gulati and Zalewski, 1982). As the blastema formed, fibronectin appeared in the extracellular matrix. The blastemal cells proliferated and as the blastema grew, increased fluorescence for fibronectin was present in the blastemal region. Since
blastemal cells are metabolically very active and synthesize DNA and proteins (Bodemer and Everett, 1959; Riddiford, 1960; O’Steen, 1960; Hay and Fischman, 1961), it is possible that these cells also synthesize fibronectin. Cultures of fibroblasts, Schwann, epithelial, and endothelial cells can also synthesize fibronectin and secrete it into the extracellular medium (Yamada et ul., 19’77; Kurkinen and Alitalo, 1979; Chen et al., 197’7; Jaffe and Mosher, 1978). The presence of fibronectin, as seen in the blastema, has been recently demonstrated in undifferentiated mesenchymal cells of the developing chick limb bud (Dessau et al., 1980; Tomasek et al., 1982) and in developing mouse limb and palate (Silver et al., 1981). Since fibronectin is a major component of the early matrix of the blastema, it may be involved in blastemal cell adhesion, migration, and alignment necessary for redifferentiation. In contrast to the accumulation of fibronectin, laminin was not seen in the blastema. Because of its distribution, fibronectin also seems to play a role during early stages of redifferentiation. Redifferentiation of cartilage and muscle occurred simultaneously and progressed in a proximal to distal direction. A reverse gradient of fibronectin distribution was seen within the limb; the maximum amount of fibronectin (mostly extracellular) was seen in the distal undifferentiated region as compared to the more proximal region undergoing redifferentiation. This would mean that fibronectin was important in initial aggregation of blastemal cells; however, it slowly disappeared from the matrix at the onset of cartilage and muscle redifferentiation. During cartilage redifferentiation when blastemal cells aggregate and align themselves in the precartilage region, extensive presence of fibronectin was seen around them. As the precartilage cells (chondrocytes) mature and synthesize extracellular matrix, fibronectin begins to disappear from the matrix. Since intense fluorescence for fibronectin was seen at the site of cartilage differentiation, it is concluded that fibronectin somehow is important for initiating chondrogenesis. This role of fibronectin has also been proposed by others in different experimental models for chondrogenesis (Newman and Frish, 1979; Dessau et crl., 1980; Silver et al., 1981; Weiss and Reddi, 1981a; Tomasek et al., 1982). As chondrogenesis advances, the matrix becomes completely devoid of detectable fibronectin. That the loss of immunofluorescence for fibronectin from the cartilage and finally bone matrix is due to the masking of fibronectin by cartilage proteoglycans and not its actual disappearance was confirmed by pretreating limb tissue sections with hyaluronidase prior to fibronectin staining (Weiss and Reddi, 1980, 1981a; Silver et al., 1981). The cartilage matrix was completely devoid of laminin at all stages of chondrogenesis, except for some weak fluorescence in the chondrocytes and within the lacunar
spaces of the cartilage and bone. This presence of laminin is not explainable at present since it is known that both cartilage and bone cells lack basement membranes. Blastemal cells destined to undergo myogenesis (myoblasts) align themselves along the long axis of the limb regenerate. Fibronectin disappears as these cells align and undergo fusion to form myotubes. This suggests that fibronectin is not required for actual fusion of the myoblasts; however, it may be required for cell aggregation and alignment prior to fusion. It has been reported in several in vitro studies that fibronectin is not required for myoblast fusion (Chen, 1977; Furcht et ul., 1978; Puri et ul., 1979; Podleski et al., 1979). The disappearance of fibronectin in redifferentiating muscle and cartilage results in a decreasing gradient of fibronectin toward the more proximal regions. Laminin is seen for the first time around the newly regenerated myotubes in the region corresponding to their basement membrane. Fibronectin was also seen around the newly regenerated myotubes, but it appeared later than laminin. Based on these findings, it can be concluded that blastemal cells and myoblasts lack basement membranes (or its important components). The blastemal cells are known to lack basement membranes as previously described in ultrastructural studies (Salpeter and Singer, 1960b; Hay, 1962). However, after fusion of these cells and formation of myotubes, a new basement membrane is synthesized which persists throughout maturation. In addition to muscle differentiation from blastemal cells, muscle regeneration by the tissue mode occurs in the proximal region of the remaining limb stump. Laminin also appears before fibronectin in the basement membrane region of these myotubes. This would mean that various components of basement membranes are formed in sequence. Similar sequential appearance of various basement membrane components (type IV collagen, laminin, and fibronectin) is also seen in regenerating skeletal muscle of rats (Gulati et al., 1982; Gulati and Zalewski, 1982) and during embryonic kidney development (Ekblom, 1981). The redifferentiation process is essentially complete within 42 days; however, further growth of the limb regenerate continues. Distribution of fibronectin and laminin in regenerated limb tissues is similar to the distribution seen in normal limb tissues. It is interesting to note that although the distribution of both fibronectin and laminin was similar in normal limb tissues, this similarity was not present during limb regeneration. This suggests that these two glycoproteins may have different functional roles. Fibronectin seems to be more important in blastema formation, aggregation of blastemal cells, and initiating redifferentiation; whereas, laminin may play a role in determining the architecture of the regenerating limb after redifferentiation.
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