THE AMERICAN JOURNAL OF ANATOMY 187232-246 (1990)
Distribution of Type IV Collagen, Laminin, and Fibronectin During Maxillary Process Formation in the Chick Embryo ZENGLU XU, SUSAN B. PARKER, AND ROBERT MINKOFF Department of Orthodontics, Dental Branch, The University of Texas Health Science Center at Houston, Houston, Texas 77225
ABSTRACT The presence and distribution of laminin, type IV collagen, and fibronectin were analyzed in the facial primordia and developing primary palates of chick embryos from stages of development corresponding to maxillary process formation and primary palate closure. Frozen sections through the maxillary process and roof of the stomodeum were prepared for indirect immunofluorescence employing a biotin-avidin system using monoclonal antibodies against laminin, type IV collagen, and fibronectin. Light microscopic examination of sections stained with antibodies against type IV collagen revealed a much stronger fluorescent signal in the roof of the stomodeum than in the maxillary process at all stages examined. Regional differences in signal intensity and staining patterns were noted within the maxillary process; for example, the lateral surface of the maxillary process displayed a much less intense signal at most stages examined than the inferior and medial surfaces. The signal from sections of the maxillary process stained with laminin was much stronger than the signal from the same tissues stained with collagen. Regional differences in signal intensity within the maxillary process were minimal in sections stained with antibodies to laminin, in contrast to the differences seen in sections stained with antibodies to type IV collagen. Differences in signal intensity between the maxillary process and the roof of the stomodeum with laminin were slight. Sections stained with antibody to fibronectin displayed intense staining throughout the mesenchyme in both the maxillary process and the roof of the stomodeum. From comparison of the data of type IV collagen and laminin, the following hypothesis is proposed. In structures which undergo rapid change in form, such as the facial primordia, collagen distribution and/or organization is altered to a much greater extent than laminin, which is more uniformly distributed and which may be required for structural support of other developmentally regulated macromolecules. Where tissue morphology must be maintained, such as the roof of the stomodeum, the concentration and organization of type IV collagen is maintained in a manner that confers stability to these regions. 0 1990 WILEY-LISS, INC.
INTRODUCTION
The significance of extracellular matrix (ECM) components to the regulation of cellular activities during embryogenesis has been a matter of considerable interest in recent years (for reviews, see Trelstad, 1984; Ekblom et al., 1986; and Kleinman et al., 1987). In particular, the basement membrane has been implicated in developmental events associated with morphogenesis and differentiation. The basement membrane has been shown to contain numerous components including type IV collagen, laminin, entactin, and heparan sulfate proteoglycan (Timpl, 1985; Tuckett and MorrissKay, 1986; Kitten et al., 1987; Martin and Timpl, 1987; Furthmayr, 1988). Another glycoprotein, fibronectin, is associated with stromal ECM as well as being present in some basement membranes (Yamada et al., 1984; Akiyama and Yamada, 1987). Prior studies have implicated the basement membrane in events associated with directed cell migration, morphogenetic change, as a medium by which inductive interactions are mediated, as well as other developmental phenomena (Meier and Hay, 1974; Wakely and England, 1979; Ekblom et al., 1980; Bernfield et al., 1984; Dziadek and Timpl, 1985; Sternberg and Kimber, 1986; Duband and Thiery, 1987; Kitten et al., 1987). In the present study, the distribution of a group of macromolecules associated with the basement membrane in the developing primary palate was analyzed. We attempted to determine whether regional or temporal changes occurred in the distribution of these macromolecules and whether these changes might be associated with developmental mechanisms that underlie the formation of facial primordia. Our results indicate that temporal and spatial differences in the distribution of basement-membrane components arise in the maxillary processes and the roof of the stomodeum during primary palate formation. Regional differences within the maxillary process and between the maxillary process and adjacent regions such as the roof of the stomodeum lead us to believe that maxillary process outgrowth may be associated with
Received May 5, 1989. Accepted September 21, 1989. Address reprint requests to Dr. Robert Minkoff, The University of Texas Health Science Center at Houston, Dental Branch, Department of Orthodontics, P.O. Box 20068, Houston, Texas 77225. Dr. Xu’s present address is Department of Histology and Embryology, Faculty of Basic Medicine, Peking Union Medical College, Beijing, China.
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developmentally regulated changes in the presence and distribution of type IV collagen. Laminin, in contrast, appears to be distributed more uniformly among the regions analyzed. MATERIALS AND METHODS Preparation of Specimens
Fertile white Leghorn eggs were incubated at 37°C in a humidified atmosphere to yield normal embryos ranging between stages 22 and 31 (Hamburger and Hamilton, 1951). Embryo heads were embedded in Tissue-Tek OCT compound (Miles Scientific, Naperville, IL) and rapidly frozen by immersion in liquid nitrogen. Ten-micrometer cryostat sections were mounted on gelatinized slides. The slides were stored a t -20°C until stained (refer to Fig. 1 for orientation and plane of sectioning). Monoclonal Antibodies
Anti-chick type IV collagen Three monoclonal antibodies against type IV collagen were used. Each had previously been demonstrated to be specific for only one of three pepsin-resistant fragments of the molecule (Mayne and Zettergren, 1980; Fitch et al., 1982; Mayne et al., 1982,1983).Antibodies that specifically recognized each of the three native fragments were called, respectively, IA8, IIb12, and ID2. Electron microscopic mapping of the epitopes after rotary shadowing indicated that monoclonal antibody IA8 recognized a n epitope located in the center of a fragment a t a distance of 288 nm from the region of overlap of four type IV molecules (referred to a s the “7s” domain). Monoclonal antibody IIBl2 recognized a n epitope located in a n adjacent fragment 73 nm from the region of overlap. The epitope for antibody ID2 was located within the region of overlap of the 7 s domain (Linsenmayer et al., 1984; Mayne et al., 1984). These three monoclonal antibodies were gifts from Dr. Richard Mayne and Dr. Thomas Linsenmayer.
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alternative technique was also employed in which the sections were incubated for 20 min with 10% goat serumiPBS (to avoid possible non-specific staining), and then incubated overnight a t 4°C with a 1:lOO dilution of the primary monoclonal antibodies described above. The slides were then washed with PBS and incubated for 1h r with 10 pgiml fluorescein-conjugated goat anti-mouse IgG (Calbiochem, San Diego, CAI, re-washed, and mounted a s described above. Specimens were then examined with a Nikon Optiphot microscope equipped for epifluorescence. Photographs were prepared using Tri-X pan ASA 400 film. The number of embryos and sections examined by developmental stage and type of antibody employed is displayed in Table 1. RESULTS Type IV Collagen Distribution
The most striking observation derived from this study was the difference in staining intensity of type IV collagen between the maxillary process and the roof of the stomodeum (refer to Fig. 1 for orientation). A much stronger signal emanated from the roof of the stomodeum compared to that observed in all regions of the maxillary process (Figs. 2-4). These differences were most noticeable at stages 22 through 28 and somewhat less a t stage 31. A second significant observation was the difference in staining intensity observed in various regions of the maxillary process itself. The most noticeable difference was the intense signal emanating from the inferior and medial regions of the maxillary process compared to the signal derived from the lateral surface of the maxillary process, especially a t stage 25 (Figs. 3, 4) and stage 28. In those regions where the signal was intense, it tended to be more pronounced either in the epithelium or, in other locations, within the basement membrane itself (e.g., compare Fig. 3c with d). In the roof of the stomodeum, stain appeared to be Anti-chick laminin and fibronectin located predominantly in the basement membrane on The anti-chick laminin monoclonal antibody was a the lateral surfaces; while in the central portion of the gift from Dr. Douglas M. Fambrough. The preparation roof, the stain tended to be located predominately in and characterization of this antibody had been de- the epithelial cells themselves (Figs. 2, 3). In certain scribed previously (Bayne et al., 1984). The anti-chick instances, the stain was concentrated in the basal epfibronectin monoclonal antibody was also a gift from ithelial layer, while the superficial layers of the epiDr. Fambrough and was described by Gardner and thelium appeared unstained. When primary antibody Fambrough (1983). was omitted from the protocol, control slides were unstained and a negative result was obtained in all cases lmmunofluorescence Staining tested (Fins. 2d, 3e). Tissue sections were incubated for 20 min with 10% The findings described above were confirmed by the horse serum in phosphate-buffered saline (PBS) to use of two additional monoclonal antibodies to type IV block non-specific binding. After overnight incubation collagen (Fig. 4). Each of these (IIB12 and IA8) had at 4°C with monoclonal antibodies against laminin, been demonstrated to be specific for regions of the coltype IV collagen, or fibronectin (1:lO-1:600 dilution), lagen molecule widely divergent from the region in the slides were washed in PBS and incubated for 1 h r which the principal antibody (ID21 employed in these with a 7.5 pgiml biotinylated horse anti-mouse IgG studies had been obtained. When stained sections (Vector Laboratories, Burlingame, CA) at 4°C. The slides were then washed and incubated for 1h r with 10 kg/ml fluorescein-Avidin DCS (Vector Laboratories, Burlingame, CA) a t 4°C. Primary antibodies were Abbreviations omitted in negative controls. The slides were washed, epithelium coverslipped, and mounted with glycerol containing Epi Mes mesenchyme 10% PBS and 1%P-phenylenediamine to reduce pho- MxP maxillary process tobleaching (Johnson and Nogueira Araujo, 1981). An RS roof of stomodeum
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Fig. 1, a: Scanning electron micrograph of a chick embryo (approximately 5 days) illustrating the facial primordia including the maxillary process (MxP), lateral nasal process (LNP), medial nasal process (MNP), and the roof of the stomodeum (RS). The region representing the primary palate includes the MNP and the medial parts of the LNP and MxP. The solid line represents the plane of
sectioning principally employed, while the dotted lines represent the most anterior and posterior sections used. Photograph courtesy of Dr. K.K. Sulik. b: Phase-contrast photograph of a cryostat section through the MxP and RS. c , d Higher-power phase-contrast photographs of the MxP and RS, respectively. Arrows outline the epithelium.
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TABLE 1. Number of embryos and sections examined by developmental stage and type of antibody
Sections examined No. of
Stages 22 25 28 31
Total
embryos 26 40 27 25 118
ID2 50 52 48 58 208
Type IV Collagen IA8 8 16 8 8 40
which were adjacent or closely approximate were examined and compared, each of the monoclonal antibodies displayed identical staining intensities; that is, the lateral surface of the maxillary process was less intensely stained than the inferior surface (compare Fig. 4a with b; d with e), and both surfaces of the maxillary process were less intensely stained than the roof of the stomodeum (compare Fig. 4a, b with c; d, e with f). The signal observed in these regions displayed the same pattern of staining as well. That is, all three monoclonal antibodies produced a very strong signal uniformly distributed in the epithelium of the roof of the stomodeum, a very weak signal uniformly throughout the epithelium of the lateral surface of the maxillary process, and a signal concentrated predominantly in the basement-membrane region of the inferior surface of the maxillary process (compare Fig. 4 with Fig. 3b,c,e). Laminin Distribution
When tissue sections were stained with monoclonal antibody to laminin, differences in signal intensity between the maxillary process and the roof of the stomodeum were negligible in comparison to the differences observed when the same tissues were stained with antibodies to type IV collagen (Figs. 5-7). In addition, regional differences in signal intensity within the maxillary process were slight. This was in marked contrast to the differences observed when these tissues were stained with antibodies to type IV collagen. Some regional differences were noted, however, with regard to the distribution of stain. For example, staining of the basement membrane was seen on the inferior surface of the maxillary process at stages 28 and 31, whereas, on the lateral surface, uniform heavy staining of the epithelium was present (Fig. 7b,c). In the roof of the stomodeum, stain was observed predominantly in the basement membrane in the lateral regions, while the central region was characterized by stain in the epithelial cells themselves, particularly the basal epithelial cells. The superficial epithelium in the center of the roof tended to be unstained. This was seen mostly a t stages 22 and 25 (Fig. 6e). Control sections were negative at all stages examined when primary antibody to laminin was omitted from the protocol (Figs. 5d, 6e, 7e). When serial sections through the maxillary process and roof of the stomodeum were stained either with laminin or type IV collagen and then examined (Fig. l a ) , the two major observations described above were confirmed. First, the signal from sections of the maxillary process stained with laminin was much stronger than
IIB 12 8 16 8 8 40
Laminin 52 66 46 30 194
Fibronectin
Total
30 34 36 52 152
148 184 146 156 634
the signal from the same tissues stained with type IV collagen. Second, staining intensities of laminin and type IV collagen in the roof of the stomodeum were comparable. Fibronectin Distribution
When tissues of the primary palate were stained with monoclonal antibody to fibronectin, intense staining was seen throughout the mesenchyme in both the maxillary process and the roof of the stomodeum a t all stages examined (stages 22-31; Fig. 8). At later stages (e.g., 31), there appeared to be a stronger signal emanating from the subepithelial mesenchyme on the lateral surface of the maxillary process. In addition, more stain appeared to be present in the basal epithelium of the maxillary process compared to the epithelium of the roof of the stomodeum. As in the case for laminin and type IV collagen, controls in which primary antibody was omitted from the protocol were negative and staining for fibronectin could not be detected (Figs. 8eh). Laminin and Type IV Collagen in the Palatal Shelf at Stage 31
At stage 31, sections of the maxillary process and roof of the stomodeum contain regions of the emerging secondary palatal shelf. When sections containing the palatal shelves were stained with monoclonal antibodies to laminin, intense signal was observed in the epithelium. In addition, a t both the oral and nasal junction of the palatal shelf, a multilayered epithelium delineated the boundaries of the palatal shelf, separating i t from the roof of the stomodeum on one side and the remainder of the maxillary process on the other (Fig. 9a). In these regions a strong signal emanated from the basement membrane or the basal epithelial cell layer; however, the multilayered epithelium was unstained. In contrast, the epithelium of the palatal shelf itself contained regions of heavy laminin staining. When the palatal shelf was stained with antibodies to type IV collagen, a similar picture emerged a t the boundaries of the palatal shelf; that is, a t both the oral and nasal boundaries a multilayered epithelium was found in which a stained basement membrane (or basal cell layer) could be observed, but the overlying multilayered epithelium itself was completely unstained. Within the palatal shelf, the epithelium appeared weakly stained and it was difficult to distinguish a basement membrane. A comparison of staining intensities between laminin and collagen in the epithelium of the palatal shelf revealed striking differences (Fig.
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inferior
Fig. 2. Immunofluorescent photomicrographs of frozen sections of a stage 22 chick embryo stained with monoclonal antibody (ID2) against type IV collagen. a: A schematic diagram indicating the locations in the MxP and the RS in which fluorescence photographs were taken. Compare intensity of signal on the lateral (b)and inferior
9b,c). These differences were comparable to those observed a t earlier stages in the maxillary process when a much stronger signal was observed in sections stained for laminin than in those stained for type IV collagen.
(c)surfaces of the maxillary process with that from the roof of the
stomodeum (d). A control section through the RS in which primary antibody was omitted is shown as an insert in d. Magnification of b-d is shown in b. The epithelium is indicated by arrows in c and d.
DISCUSSION
The most striking observations in this study were, first, the regional differences in staining patterns in the maxillary process, and second, the marked differences in the intensity of signal between the roof of the
DISTRIBUTION OF ECM I N THE MAXILLARY PROCESS
3a
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RS
inferior
Fig. 3. Immunofluorescent photomicrograph of frozen sections of a stage 25 chick embryo stained with monoclonal antibody (ID2) against type IV collagen. a: Schematic diagram showing location of fluorescence photographs. Compare intensity of signal on the lateral surface (b) with that of the inferior ( c )and medial surfaces (d) of the
maxillary process and with that from the roof of the stomodeum (e).A control section through the roof of the stomodeum in which primary antibody was omitted is shown in the insert in e. Magnification of b e is shown in b.
Fig. 4. Immunofluorescence photomicrographs of frozen sections of a stage 25 chick embryo stained with different monoclonal antibodies against type IV collagen than that used in the previous examples. a-c: Stained with antibody IA8. d-f: Stained with antibody IIB12. Control sections through the RS in which primary antibody was omit-
ted are shown a s inserts in c and f. Compare the intensity and pattern of staining obtained with these two alternative monoclonal antibodies to type IV collagen with the results obtained with the first monoclonal antibody (Fig. 3b,c,e).
DISTRIBUTION O F ECM I N THE MAXILLARY PROCESS
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Fig. 5. Immunofluorescence photomicrographs of frozen sections of a stage 22 chick embryo stained with monoclonal antibody against laminin. Locations of photographs in b-d are indicated in a. A control
section through the RS in which the primary antibody was omitted is shown in the insert in d. Compare with Figure 2, in which comparable regions were stained with antibody against type IV collagen.
stomodeum and the maxillary process when tissues were stained with antibodies to type IV collagen. In contrast, the differences in staining intensity among these regions were much less in tissues stained with monoclonal antibodies to laminin. The similarities and differences in staining patterns suggest that these patterns may be associated with the morphogenetic events of primary palate formation, including outgrowth of the maxillary process itself. The observations lead us to believe that, during primary
palate formation, regions in which contours are to be maintained in their original configurations contain greater amounts (or other characteristics related to turnover, stability, etc.) of type IV collagen. In regions undergoing extensive morphogenetic change, however, regional differences appear in the amount, organization, or turnover of type IV collagen. Differences in laminin in these regions remain minimal. As a working hypothesis, i t is proposed that the morphogenetic alterations that occur during outgrowth of a primordium
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6a
RS
inferior
Fig. 6. Immunofluorescence photomicrographs offrozen sections of a stage 25 embryo stained with monoclonal antibody against laminin. Location of photographs b-e is indicated in a. A control section
through the RS in which primary antibody was omitted is shown in the insert in e. Compare Figure 6 (laminin)with comparable regions stained with antibody against type IV collagen (Fig. 3).
may be related directly to the presence and distribution of type IV collagen. In those regions undergoing rapid change in form, collagen distribution may be altered to
a much greater extent than that of laminin, which may serve principally as a structural support by which other ECM macromolecules are modulated.
DISTRIUUTION O F ECM I N THE MAXILLARY PROCESS
7a
RS
1-I
inferior
Fig. 7. Immunofluorescence photomicrographs of frozen sections of a stage 28 embryo stained with monoclonal antibody against laminin. Location of photographs in b-e is indicated in a. A control section through the RS in which primary antibody was omitted is shown in the insert in e.
24 1
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Fig. 8. Representative frozen sections through the MxP and RS of chick embryos at stages 22 (a,e), 25 (b,fl, 28 (c,g), and 31, (d,h), stained with antibody against fibronectin. Extensive staining is seen
throughout the mesenchyme of both the RS (a-d) and the MxP (e-h). A control section in which primary antibody has been omitted is shown in the inserts in e-h.
DISTRIBUTION OF ECM I N THE MAXILLARY PROCESS
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Observations of staining patterns in the emerging secondary palatal shelves a t stage 31 also revealed marked differences between laminin and type IV collagen. In regions of outgrowth of the palatal shelf, a much stronger signal was observed in tissues stained for laminin than in adjacent sections stained for type IV collagen. A strong and uniform staining pattern for laminin was seen throughout the palatal shelf, including the areas of attachment in the oral and nasal transition zones where a multilayered epithelium was found. A multilayered epithelium at the oral and nasal boundaries has been postulated by Brinkley (1984) to represent a buttress by which outgrowth of the palatal shelf is directed. Differences in type IV collagen and laminin concentrations andlor organization may also play a role in directed outgrowth of the secondary palatal shelf and may augment the buttressing effect of the junctional epithelium postulated by Brinkley. As a control for the validity of our observations, serial sections were examined throughout the maxillary process and the roof of the stomodeum. In general, the observations from serial-section analysis were consistent, although slight differences were noted. For example, when the basement membrane was stained with laminin, it tended to be more continuous and longer in posterior sections of the maxillary process. The observations of spatial and temporal differences in the presence and distribution of laminin and type IV collagen described in this report, therefore, were confirmed by serial-section analysis as well a s by the use of different monoclonal antibodies to type IV collagen. There did not appear to be a n association between fibronectin distribution or concentration and the observations described above. Fibronectin was found distributed throughout the mesenchyme at all stages; and, in general, there was a tendency for the appearance of increased staining in subepithelial mesenchyme. Differences were noted in fibronectin staining in the basal epithelium of the maxillary process compared to the epithelium of the roof of the stomodeum which appeared unstained. However, since fibronectin staining of the subepithelial mesenchyme appeared to be more concentrated in the maxillary process, it may have obscured the appearance of the basal epithelium in these regions. The presence of a cell process meshwork (Sulik et al., 1979; Igawa et al., 1986) may be associated with increased fibronectin staining in these regions of the maxillary process and could account for these observations. In general, however, staining patterns with fibronectin did not appear to be correlated in any direct fashion with outgrowth of the maxillary process and did not appear to be correlated with the staining patterns observed for laminin and type IV collagen. It is well documented that basement membrane components such as laminin and type IV collagen have patterns of distribution that are altered during organogenesis. Changing patterns of distribution of basement membrane components have been reported during ocular development (Fitch and Linsenmayer, 1983; Fitch et al., 19831, salivary gland morphogenesis (Bernfield et al., 19841, in the developing kidney (Ekblom et al., 1981; Mounier e t al., 1986), in the developing genitourinary system (Ikawa et al., 1984), during neural crest development (Duband and Thiery, 1987),
in the developing lung (Jaskoll and Slavkin, 1984), during tooth development (Thesleff et al., 1981; Thesleff and Hurmerinta, 1981), in the developing caudal neural tube (O’Shea, 1987), and in other tissues (Laurie e t al., 1983; Wan et al., 1984). For example, during tooth development, contact between preodontoblasts and the basement membrane appears to be required for odontoblast differentiation (Thesleff and Hurmerinta, 1981). During submandibular gland morphogenesis, the basement membrane becomes thinned and regionally discontinuous, and direct contacts between epithelial and mesenchymal cells appear (Cutler and Chaudhry, 1973). Other studies demonstrated that basement-membrane components within interlobular clefts were stable but were transiently lost from lobules. Type I collagen was deposited a t sites of morphologic stability, while type IV collagen was lost from the basement membrane a t sites of rapid cell proliferation (Bernfield et al., 1984). In the developing kidney, the distribution of type IV collagen is considered to have an important role in nephron morphogenesis and to be a critical structural component of the definitive semipermeable glomerular filter. The developing glomerular capillary and mesangium contained type IV collagen a t all stages of development (Michael e t al., 1983). Mesangial immunofluorescence intensity increased progressively with glomerular maturation (Mounier et al., 1986). Changing patterns of distribution of the collagens and laminin have also been observed during neural crest cell migration and subsequent morphogenetic events. For example, both laminin and type IV collagen disappear from the basal surface of the neural tube at sites where neural crest cells emerge (Duband and Thiery, 1987). Although type IV collagen and laminin were found on the basal surfaces of epithelia lining neural crest migration pathways, these components were rarely seen on migrating neural crest cells themselves. Termination of migration of neural crest cells and aggregation into ganglia was then observed by Duband and Thiery to be accompanied by the subsequent reappearance of type IV collagen and laminin within the neural crest cell population. Perhaps the most relevant examples of basement membrane alteration during organogenesis were those observed by Fitch, Linsenmayer, and co-workers during ocular development (Fitch and Linsenmayer, 1983; Fitch et al., 1983; Linsenmayer et al., 1984). For example, during development of the morphologically continuous avian lens capsule, staining with all three monoclonal antibodies to type IV collagen used in this study (ID2, IIB12, and IA8) displayed a uniform pattern of stain in tissue sections of lenses of 4- to 8-dayold chick embryos. By 7 to 8 days of development, a gradient of staining intensity developed which was fully established by 11-12 days of development. At this time striking differences in the staining pattern of the basement membrane of the lens capsule were seen. Bright fluorescence was seen in the anterior lens capsule, while the posterior lens appeared relatively unstained. In a control study, antibodies against non-collagenous components of the lens capsule basement membrane were prepared. Staining of the lens capsule with these antibodies was uniform and intense with no indication of the regional differences that were ob-
DISTRIBUTION OF ECM IN THE MAXILLARY PROCESS
served a t later developmental stages when antibodies to type IV collagen were employed. These examples illustrate the modulation of basement-membrane components associated with organogenesis and tissue differentiation. The present report proposes that specific alterations of basement membrane components is associated with morphogenesis of external body form a s well. ACKNOWLEDGMENTS
This study was supported by NIH grant DE-07674 from the National Institute for Dental Research. The authors wish to thank Mrs. J a n e Laine and Barbara Barton for their expert secretarial assistance in the preparation of this manuscript. They also wish to thank Drs. Richard Mayne, Thomas Linsenmayer, and Douglas Fambrough for providing the antibodies to type IV collagen, laminin, and fibronectin used in this study. Finally, they wish to thank Dr. Richard Mayne for the advice, helpful discussion, and insight he provided to u s during the course of this project. LITERATURE CITED Akiyama, S.K., and K.M. Yamada 1987 Fibronectin. Adv. Enzymol., 59:l-57. Bayne, E.K., M.J. Anderson, and D.M. Fambrough 1984 Extracellular matrix organization in developing muscle: Correlation with acetylcholine receptor aggregates. J. Cell. Biol., 99:1486-1501. Bernfield, M., S.D. Banerjee, J.E. Koda, and A.C. Rapraeger 1984 Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: The Role of Extracellular Matrix in Development. R.L. Trelstad, ed. Alan R. Liss, Inc., New York, pp. 545-572. Brinhley, L.L. 1984 Changes in cell distribution during mouse secondary palate closure in uiuo and in uitro. Dev. Biol., 102:216227. Cutler, L.S., and A.P. Chaudhry 1973 Intercellular contacts a t the epithelial-mesenchymal interface during the prenatal development of the rat sub-mandibular gland. Dev. Biol., 33229-240. Duband, J.L., and J.P. Thiery 1987 Distribution of laminin and collagens during avian neural crest development. Development, 101:461-478. Dziadek, M., and R. Timpl 1985 Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol., I11:372-382. Ekblom, P., K. Alitalo, A. Vaheri, R. Timpl, and L. Saxen 1980 Induction of basement membrane glycoprotein in embryonic kidney: Possible role of laminin in morphogenesis. Proc. Natl. Acad. Sci. USA, 77t485-489. Ekblom, P., E. Lehtonen, L. Saxen, and R. Timpl 1981 Shift in collagen type as an early response to induction of the metanephric mesenchyme. J. Cell Biol., 89:276-283. Ekblom, P., D. Vestweber, and R. Kemler 1986 Cell-matrix interactions and cell adhesion during development. Ann. Rev. Cell Biol., 2:27-47. Fitch, J.M., and T.F. Linsenmayer 1983 Monoclonal antibody analysis of ocular basement membranes during development. Dev. Biol., 95:137-153. Fitch, J.M., E. Gibney, R.D. Sanderson, R. Mayne, and T.F. Linsenmayer 1982 Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen. J. Cell Biol., 95t641-647. Fitch, J.M., R. Mayne, and T.F. Linsenmayer 1983 Developmental acquisition of basement membrane heterogeneity: Type IV collagen in the avian lens capsule. J . Cell Biol., 97:940-943. Furthmayr, H. 1988 Assembly of basement membrane macromolecules. In: Self-Assembling Architecture, J.E. Varner, ed. Alan R. Liss, Inc., New York, pp. 43-59. Gardner, J.M., and D.M. Fambrough 1983 Fibronectin expression during myogenesis. J. Cell Biol., 96:474-485. Hamburger, V., and H.L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morphol., 88r49-92. Igawa, H.H., M. Yasuda, H. Nakamura, and T. Ohura 1986 Changes in the subepithelial mesenchymal cell process meshwork in de-
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veloping facial prominences in mouse embryos. J. Craniofac. Genet. Dev. Biol., 6:27-39. Ikawa, H., R.L. Trelstad, J.M. Hutson, T.F. Manganaro, and P.K. Donahoe 1984 Changing patterns of fibronectin, laminin, type IV collagen, and a basement membrane proteoglycan during rat Mullerian duct regression. Dev. Biol., 102;260-263. Jaskoll, T.F., and H.C. Slavkin 1984 Ultrastructural and immunofluorescence studies of basal-lamina alterations during mouselung morphogenesis. Differentiation, 28:36-48. Johnson, G.D., and G.M. de C. Nogueira Araujo 1981 A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth., 43:349-350. Kitten, G.T., R.R. Markwald, and D.L. Bolender 1987 Distribution of basement membrane antigens in cryopreserved early embryonic hearts. Anat. Rec., 21 7:379-390. Kleinman, H.K., J. Graf, Y. Iwamoto, G.T. Kitten, R.C. Ogle, M. Sasaki, Y. Yamada, G.R. Martin, and L. Luckenbill-Edds 1987 Role of basement membranes in cell differentiation. Annals NY Acad. Sci., 513:134-145. Laurie, G.W., C.P. Leblond, and G.R. Martin 1983 Light microscopic immunolocalization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes of a variety of rat organs. Am. J. Anat., 167:71-82. Linsenmayer, T.F., J.M. Fitch, and R. Mayne 1984 Basement membrane structure and assembly: Inferences from immunological studies with monoclonal antibodies. In: The Role of Extracellular Matrix in Development, R.L. Trelstad, ed. Alan R. Liss, Inc., New York, pp. 145-172. Martin, G.R., and R. Timpl 1987 Laminin and other basement membrane components. Ann. Rev. Cell Biol., 3.57-85. Mayne, R., and J.G. Zettergren 1980 Type IV collagen from chicken muscular tissues. Isolation and characterization of the pepsinresistant fragments. Biochemistry, I9:4065-4072. Mayne, R., H. Wiedemann, W. Dessau, K. von der Mark, and P. Bruckner 1982 Structural and immunological characterization of type IV collagen isolated from chicken tissue. Eur. J . Biochem., 126:417-423. Mayne, R., R.D. Sanderson, H. Wiedemann, J.M. Fitch, and T.F. Linsenmayer 1983 The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies. J. Biol. Chem., 2585794-5797. Mayne, R., H. Wiedemann, M.H. Irwin, R.D. Sanderson, J.M. Fitch, T.F. Linsenmayer, and K. Kuhn 1984 Monoclonal antibodies against chicken type IV and V collagen: Electron microscopic mapping of the epitopes after rotary shadowing. J . Cell Biol., 98: 1637-1644. Meier, S., and E.D. Hay 1974 Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production. Dev. Biol., 38.249-270. Michael, A.F.,J.Y. Yang, R.J. Falk, M.J. Bennington, J.I. Scheinman, R.L. Vernier. and A.J. Fish 1983 Monoclonal antibodies to human renal basement membranes: heterogenic and ontogenic changes. Kidney Int., 24:74-86. Mounier, F., J.M. Foidart, and M.C. Gubler 1986 Distribution of extracellular matrix glycoproteins during normal development of human kidney-an immunohistochemical study. Lab. Invest., 54: 394-401. O’Shea, K.S. 1987 Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo. Development, 99:509-519. Sternberg, J., and S.J. Kimber 1986 Distribution of fibronectin, laminin and entactin in the environment of migrating neural crest cells in early mouse embryos. J. Embryol. Exp. Morphol., 91: 267-282. Sulik, K.K., M.C. Johnston, L.J.H. Ambrose, and D. Dorgan 1979 Phenytoin (di1antin)binduced cleft lip: A scanning and transmission electron microscopic study. Anat. Rec., 195243-256. Thesleff, I., and K. Hurmerinta 1981 Tissue interactions in tooth development. Differentiation, 18t75-88. Thesleff, I., H.J. Barrach, J.M. Foidart, A. Vaheri, R.M. Pratt, and G.R. Martin 1981 Changes in the distribution of type IV collagen, laminin, proteoglycan, and fibronectin during mouse tooth develooment. Dev. Biol.. 81:182-192. Timpi, R. 1985 Molecular aspects of basement membrane structure. Prog. Clin. Biol. Res., 171:63-74. Trelstad, R.L. 1984 The Role of Extracellular Matrix in Development. Alan R. Liss, Inc., New York. Tuckett. F., and G.M. Morriss-Kav 1986 The distribution of fibronectin, laminin and entactin in the neurulating rat embryo studied by indirect immunofluorescence. J. Embryol. Exp. Morphol., 94: 95-112.
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Wakely, J., and M. England 1979 Scanning electron microscopal and histochemical study of the structure and function of basement membranes in early chick embryo. Proc. R. Sac. Land. (Biol.), 206t329-352.
Wan, Y.J., T.C. Wu, A.E. Chung, and I. Damjanov 1984 Monoclonal antibodies to laminin reveal the heterogeneity of basement mem-
branes in the developing and adult mouse tissues. J. Cell. Biol., 98,971-979, Yamada, K.M., M. Hayashi, H. Hirano, and S.K. Akiyama 1984 Fibronectin and cell surface interactions. In: The Role of Extracellular Matrix in Development, R.L. Trelstad, ed. Alan R. Liss, Inc., New York, pp. 89-121.
Distribution of Type IV Collagen, Laminin, and Fibronectin During Maxillary Process Formation in the Chick Embryo ZENGLU XU, SUSAN B. PARKER, AND ROBERT MINKOFF Department of Orthodontics, Dental Branch, The University of Texas Health Science Center at Houston, Houston, Texas 77225
ABSTRACT The presence and distribution of laminin, type IV collagen, and fibronectin were analyzed in the facial primordia and developing primary palates of chick embryos from stages of development corresponding to maxillary process formation and primary palate closure. Frozen sections through the maxillary process and roof of the stomodeum were prepared for indirect immunofluorescence employing a biotin-avidin system using monoclonal antibodies against laminin, type IV collagen, and fibronectin. Light microscopic examination of sections stained with antibodies against type IV collagen revealed a much stronger fluorescent signal in the roof of the stomodeum than in the maxillary process at all stages examined. Regional differences in signal intensity and staining patterns were noted within the maxillary process; for example, the lateral surface of the maxillary process displayed a much less intense signal at most stages examined than the inferior and medial surfaces. The signal from sections of the maxillary process stained with laminin was much stronger than the signal from the same tissues stained with collagen. Regional differences in signal intensity within the maxillary process were minimal in sections stained with antibodies to laminin, in contrast to the differences seen in sections stained with antibodies to type IV collagen. Differences in signal intensity between the maxillary process and the roof of the stomodeum with laminin were slight. Sections stained with antibody to fibronectin displayed intense staining throughout the mesenchyme in both the maxillary process and the roof of the stomodeum. From comparison of the data of type IV collagen and laminin, the following hypothesis is proposed. In structures which undergo rapid change in form, such as the facial primordia, collagen distribution and/or organization is altered to a much greater extent than laminin, which is more uniformly distributed and which may be required for structural support of other developmentally regulated macromolecules. Where tissue morphology must be maintained, such as the roof of the stomodeum, the concentration and organization of type IV collagen is maintained in a manner that confers stability to these regions. 0 1990 WILEY-LISS, INC.
INTRODUCTION
The significance of extracellular matrix (ECM) components to the regulation of cellular activities during embryogenesis has been a matter of considerable interest in recent years (for reviews, see Trelstad, 1984; Ekblom et al., 1986; and Kleinman et al., 1987). In particular, the basement membrane has been implicated in developmental events associated with morphogenesis and differentiation. The basement membrane has been shown to contain numerous components including type IV collagen, laminin, entactin, and heparan sulfate proteoglycan (Timpl, 1985; Tuckett and MorrissKay, 1986; Kitten et al., 1987; Martin and Timpl, 1987; Furthmayr, 1988). Another glycoprotein, fibronectin, is associated with stromal ECM as well as being present in some basement membranes (Yamada et al., 1984; Akiyama and Yamada, 1987). Prior studies have implicated the basement membrane in events associated with directed cell migration, morphogenetic change, as a medium by which inductive interactions are mediated, as well as other developmental phenomena (Meier and Hay, 1974; Wakely and England, 1979; Ekblom et al., 1980; Bernfield et al., 1984; Dziadek and Timpl, 1985; Sternberg and Kimber, 1986; Duband and Thiery, 1987; Kitten et al., 1987). In the present study, the distribution of a group of macromolecules associated with the basement membrane in the developing primary palate was analyzed. We attempted to determine whether regional or temporal changes occurred in the distribution of these macromolecules and whether these changes might be associated with developmental mechanisms that underlie the formation of facial primordia. Our results indicate that temporal and spatial differences in the distribution of basement-membrane components arise in the maxillary processes and the roof of the stomodeum during primary palate formation. Regional differences within the maxillary process and between the maxillary process and adjacent regions such as the roof of the stomodeum lead us to believe that maxillary process outgrowth may be associated with
Received May 5, 1989. Accepted September 21, 1989. Address reprint requests to Dr. Robert Minkoff, The University of Texas Health Science Center at Houston, Dental Branch, Department of Orthodontics, P.O. Box 20068, Houston, Texas 77225. Dr. Xu’s present address is Department of Histology and Embryology, Faculty of Basic Medicine, Peking Union Medical College, Beijing, China.
DISTRIBLJTION OF ECM I N THE MAXILLARY PROCESS
developmentally regulated changes in the presence and distribution of type IV collagen. Laminin, in contrast, appears to be distributed more uniformly among the regions analyzed. MATERIALS AND METHODS Preparation of Specimens
Fertile white Leghorn eggs were incubated at 37°C in a humidified atmosphere to yield normal embryos ranging between stages 22 and 31 (Hamburger and Hamilton, 1951). Embryo heads were embedded in Tissue-Tek OCT compound (Miles Scientific, Naperville, IL) and rapidly frozen by immersion in liquid nitrogen. Ten-micrometer cryostat sections were mounted on gelatinized slides. The slides were stored a t -20°C until stained (refer to Fig. 1 for orientation and plane of sectioning). Monoclonal Antibodies
Anti-chick type IV collagen Three monoclonal antibodies against type IV collagen were used. Each had previously been demonstrated to be specific for only one of three pepsin-resistant fragments of the molecule (Mayne and Zettergren, 1980; Fitch et al., 1982; Mayne et al., 1982,1983).Antibodies that specifically recognized each of the three native fragments were called, respectively, IA8, IIb12, and ID2. Electron microscopic mapping of the epitopes after rotary shadowing indicated that monoclonal antibody IA8 recognized a n epitope located in the center of a fragment a t a distance of 288 nm from the region of overlap of four type IV molecules (referred to a s the “7s” domain). Monoclonal antibody IIBl2 recognized a n epitope located in a n adjacent fragment 73 nm from the region of overlap. The epitope for antibody ID2 was located within the region of overlap of the 7 s domain (Linsenmayer et al., 1984; Mayne et al., 1984). These three monoclonal antibodies were gifts from Dr. Richard Mayne and Dr. Thomas Linsenmayer.
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alternative technique was also employed in which the sections were incubated for 20 min with 10% goat serumiPBS (to avoid possible non-specific staining), and then incubated overnight a t 4°C with a 1:lOO dilution of the primary monoclonal antibodies described above. The slides were then washed with PBS and incubated for 1h r with 10 pgiml fluorescein-conjugated goat anti-mouse IgG (Calbiochem, San Diego, CAI, re-washed, and mounted a s described above. Specimens were then examined with a Nikon Optiphot microscope equipped for epifluorescence. Photographs were prepared using Tri-X pan ASA 400 film. The number of embryos and sections examined by developmental stage and type of antibody employed is displayed in Table 1. RESULTS Type IV Collagen Distribution
The most striking observation derived from this study was the difference in staining intensity of type IV collagen between the maxillary process and the roof of the stomodeum (refer to Fig. 1 for orientation). A much stronger signal emanated from the roof of the stomodeum compared to that observed in all regions of the maxillary process (Figs. 2-4). These differences were most noticeable at stages 22 through 28 and somewhat less a t stage 31. A second significant observation was the difference in staining intensity observed in various regions of the maxillary process itself. The most noticeable difference was the intense signal emanating from the inferior and medial regions of the maxillary process compared to the signal derived from the lateral surface of the maxillary process, especially a t stage 25 (Figs. 3, 4) and stage 28. In those regions where the signal was intense, it tended to be more pronounced either in the epithelium or, in other locations, within the basement membrane itself (e.g., compare Fig. 3c with d). In the roof of the stomodeum, stain appeared to be Anti-chick laminin and fibronectin located predominantly in the basement membrane on The anti-chick laminin monoclonal antibody was a the lateral surfaces; while in the central portion of the gift from Dr. Douglas M. Fambrough. The preparation roof, the stain tended to be located predominately in and characterization of this antibody had been de- the epithelial cells themselves (Figs. 2, 3). In certain scribed previously (Bayne et al., 1984). The anti-chick instances, the stain was concentrated in the basal epfibronectin monoclonal antibody was also a gift from ithelial layer, while the superficial layers of the epiDr. Fambrough and was described by Gardner and thelium appeared unstained. When primary antibody Fambrough (1983). was omitted from the protocol, control slides were unstained and a negative result was obtained in all cases lmmunofluorescence Staining tested (Fins. 2d, 3e). Tissue sections were incubated for 20 min with 10% The findings described above were confirmed by the horse serum in phosphate-buffered saline (PBS) to use of two additional monoclonal antibodies to type IV block non-specific binding. After overnight incubation collagen (Fig. 4). Each of these (IIB12 and IA8) had at 4°C with monoclonal antibodies against laminin, been demonstrated to be specific for regions of the coltype IV collagen, or fibronectin (1:lO-1:600 dilution), lagen molecule widely divergent from the region in the slides were washed in PBS and incubated for 1 h r which the principal antibody (ID21 employed in these with a 7.5 pgiml biotinylated horse anti-mouse IgG studies had been obtained. When stained sections (Vector Laboratories, Burlingame, CA) at 4°C. The slides were then washed and incubated for 1h r with 10 kg/ml fluorescein-Avidin DCS (Vector Laboratories, Burlingame, CA) a t 4°C. Primary antibodies were Abbreviations omitted in negative controls. The slides were washed, epithelium coverslipped, and mounted with glycerol containing Epi Mes mesenchyme 10% PBS and 1%P-phenylenediamine to reduce pho- MxP maxillary process tobleaching (Johnson and Nogueira Araujo, 1981). An RS roof of stomodeum
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Fig. 1, a: Scanning electron micrograph of a chick embryo (approximately 5 days) illustrating the facial primordia including the maxillary process (MxP), lateral nasal process (LNP), medial nasal process (MNP), and the roof of the stomodeum (RS). The region representing the primary palate includes the MNP and the medial parts of the LNP and MxP. The solid line represents the plane of
sectioning principally employed, while the dotted lines represent the most anterior and posterior sections used. Photograph courtesy of Dr. K.K. Sulik. b: Phase-contrast photograph of a cryostat section through the MxP and RS. c , d Higher-power phase-contrast photographs of the MxP and RS, respectively. Arrows outline the epithelium.
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DISTRIBUTION OF ECM IN THE MAXILLARY PROCESS
TABLE 1. Number of embryos and sections examined by developmental stage and type of antibody
Sections examined No. of
Stages 22 25 28 31
Total
embryos 26 40 27 25 118
ID2 50 52 48 58 208
Type IV Collagen IA8 8 16 8 8 40
which were adjacent or closely approximate were examined and compared, each of the monoclonal antibodies displayed identical staining intensities; that is, the lateral surface of the maxillary process was less intensely stained than the inferior surface (compare Fig. 4a with b; d with e), and both surfaces of the maxillary process were less intensely stained than the roof of the stomodeum (compare Fig. 4a, b with c; d, e with f). The signal observed in these regions displayed the same pattern of staining as well. That is, all three monoclonal antibodies produced a very strong signal uniformly distributed in the epithelium of the roof of the stomodeum, a very weak signal uniformly throughout the epithelium of the lateral surface of the maxillary process, and a signal concentrated predominantly in the basement-membrane region of the inferior surface of the maxillary process (compare Fig. 4 with Fig. 3b,c,e). Laminin Distribution
When tissue sections were stained with monoclonal antibody to laminin, differences in signal intensity between the maxillary process and the roof of the stomodeum were negligible in comparison to the differences observed when the same tissues were stained with antibodies to type IV collagen (Figs. 5-7). In addition, regional differences in signal intensity within the maxillary process were slight. This was in marked contrast to the differences observed when these tissues were stained with antibodies to type IV collagen. Some regional differences were noted, however, with regard to the distribution of stain. For example, staining of the basement membrane was seen on the inferior surface of the maxillary process at stages 28 and 31, whereas, on the lateral surface, uniform heavy staining of the epithelium was present (Fig. 7b,c). In the roof of the stomodeum, stain was observed predominantly in the basement membrane in the lateral regions, while the central region was characterized by stain in the epithelial cells themselves, particularly the basal epithelial cells. The superficial epithelium in the center of the roof tended to be unstained. This was seen mostly a t stages 22 and 25 (Fig. 6e). Control sections were negative at all stages examined when primary antibody to laminin was omitted from the protocol (Figs. 5d, 6e, 7e). When serial sections through the maxillary process and roof of the stomodeum were stained either with laminin or type IV collagen and then examined (Fig. l a ) , the two major observations described above were confirmed. First, the signal from sections of the maxillary process stained with laminin was much stronger than
IIB 12 8 16 8 8 40
Laminin 52 66 46 30 194
Fibronectin
Total
30 34 36 52 152
148 184 146 156 634
the signal from the same tissues stained with type IV collagen. Second, staining intensities of laminin and type IV collagen in the roof of the stomodeum were comparable. Fibronectin Distribution
When tissues of the primary palate were stained with monoclonal antibody to fibronectin, intense staining was seen throughout the mesenchyme in both the maxillary process and the roof of the stomodeum a t all stages examined (stages 22-31; Fig. 8). At later stages (e.g., 31), there appeared to be a stronger signal emanating from the subepithelial mesenchyme on the lateral surface of the maxillary process. In addition, more stain appeared to be present in the basal epithelium of the maxillary process compared to the epithelium of the roof of the stomodeum. As in the case for laminin and type IV collagen, controls in which primary antibody was omitted from the protocol were negative and staining for fibronectin could not be detected (Figs. 8eh). Laminin and Type IV Collagen in the Palatal Shelf at Stage 31
At stage 31, sections of the maxillary process and roof of the stomodeum contain regions of the emerging secondary palatal shelf. When sections containing the palatal shelves were stained with monoclonal antibodies to laminin, intense signal was observed in the epithelium. In addition, a t both the oral and nasal junction of the palatal shelf, a multilayered epithelium delineated the boundaries of the palatal shelf, separating i t from the roof of the stomodeum on one side and the remainder of the maxillary process on the other (Fig. 9a). In these regions a strong signal emanated from the basement membrane or the basal epithelial cell layer; however, the multilayered epithelium was unstained. In contrast, the epithelium of the palatal shelf itself contained regions of heavy laminin staining. When the palatal shelf was stained with antibodies to type IV collagen, a similar picture emerged a t the boundaries of the palatal shelf; that is, a t both the oral and nasal boundaries a multilayered epithelium was found in which a stained basement membrane (or basal cell layer) could be observed, but the overlying multilayered epithelium itself was completely unstained. Within the palatal shelf, the epithelium appeared weakly stained and it was difficult to distinguish a basement membrane. A comparison of staining intensities between laminin and collagen in the epithelium of the palatal shelf revealed striking differences (Fig.
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inferior
Fig. 2. Immunofluorescent photomicrographs of frozen sections of a stage 22 chick embryo stained with monoclonal antibody (ID2) against type IV collagen. a: A schematic diagram indicating the locations in the MxP and the RS in which fluorescence photographs were taken. Compare intensity of signal on the lateral (b)and inferior
9b,c). These differences were comparable to those observed a t earlier stages in the maxillary process when a much stronger signal was observed in sections stained for laminin than in those stained for type IV collagen.
(c)surfaces of the maxillary process with that from the roof of the
stomodeum (d). A control section through the RS in which primary antibody was omitted is shown as an insert in d. Magnification of b-d is shown in b. The epithelium is indicated by arrows in c and d.
DISCUSSION
The most striking observations in this study were, first, the regional differences in staining patterns in the maxillary process, and second, the marked differences in the intensity of signal between the roof of the
DISTRIBUTION OF ECM I N THE MAXILLARY PROCESS
3a
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RS
inferior
Fig. 3. Immunofluorescent photomicrograph of frozen sections of a stage 25 chick embryo stained with monoclonal antibody (ID2) against type IV collagen. a: Schematic diagram showing location of fluorescence photographs. Compare intensity of signal on the lateral surface (b) with that of the inferior ( c )and medial surfaces (d) of the
maxillary process and with that from the roof of the stomodeum (e).A control section through the roof of the stomodeum in which primary antibody was omitted is shown in the insert in e. Magnification of b e is shown in b.
Fig. 4. Immunofluorescence photomicrographs of frozen sections of a stage 25 chick embryo stained with different monoclonal antibodies against type IV collagen than that used in the previous examples. a-c: Stained with antibody IA8. d-f: Stained with antibody IIB12. Control sections through the RS in which primary antibody was omit-
ted are shown a s inserts in c and f. Compare the intensity and pattern of staining obtained with these two alternative monoclonal antibodies to type IV collagen with the results obtained with the first monoclonal antibody (Fig. 3b,c,e).
DISTRIBUTION O F ECM I N THE MAXILLARY PROCESS
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Fig. 5. Immunofluorescence photomicrographs of frozen sections of a stage 22 chick embryo stained with monoclonal antibody against laminin. Locations of photographs in b-d are indicated in a. A control
section through the RS in which the primary antibody was omitted is shown in the insert in d. Compare with Figure 2, in which comparable regions were stained with antibody against type IV collagen.
stomodeum and the maxillary process when tissues were stained with antibodies to type IV collagen. In contrast, the differences in staining intensity among these regions were much less in tissues stained with monoclonal antibodies to laminin. The similarities and differences in staining patterns suggest that these patterns may be associated with the morphogenetic events of primary palate formation, including outgrowth of the maxillary process itself. The observations lead us to believe that, during primary
palate formation, regions in which contours are to be maintained in their original configurations contain greater amounts (or other characteristics related to turnover, stability, etc.) of type IV collagen. In regions undergoing extensive morphogenetic change, however, regional differences appear in the amount, organization, or turnover of type IV collagen. Differences in laminin in these regions remain minimal. As a working hypothesis, i t is proposed that the morphogenetic alterations that occur during outgrowth of a primordium
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6a
RS
inferior
Fig. 6. Immunofluorescence photomicrographs offrozen sections of a stage 25 embryo stained with monoclonal antibody against laminin. Location of photographs b-e is indicated in a. A control section
through the RS in which primary antibody was omitted is shown in the insert in e. Compare Figure 6 (laminin)with comparable regions stained with antibody against type IV collagen (Fig. 3).
may be related directly to the presence and distribution of type IV collagen. In those regions undergoing rapid change in form, collagen distribution may be altered to
a much greater extent than that of laminin, which may serve principally as a structural support by which other ECM macromolecules are modulated.
DISTRIUUTION O F ECM I N THE MAXILLARY PROCESS
7a
RS
1-I
inferior
Fig. 7. Immunofluorescence photomicrographs of frozen sections of a stage 28 embryo stained with monoclonal antibody against laminin. Location of photographs in b-e is indicated in a. A control section through the RS in which primary antibody was omitted is shown in the insert in e.
24 1
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Fig. 8. Representative frozen sections through the MxP and RS of chick embryos at stages 22 (a,e), 25 (b,fl, 28 (c,g), and 31, (d,h), stained with antibody against fibronectin. Extensive staining is seen
throughout the mesenchyme of both the RS (a-d) and the MxP (e-h). A control section in which primary antibody has been omitted is shown in the inserts in e-h.
DISTRIBUTION OF ECM I N THE MAXILLARY PROCESS
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Observations of staining patterns in the emerging secondary palatal shelves a t stage 31 also revealed marked differences between laminin and type IV collagen. In regions of outgrowth of the palatal shelf, a much stronger signal was observed in tissues stained for laminin than in adjacent sections stained for type IV collagen. A strong and uniform staining pattern for laminin was seen throughout the palatal shelf, including the areas of attachment in the oral and nasal transition zones where a multilayered epithelium was found. A multilayered epithelium at the oral and nasal boundaries has been postulated by Brinkley (1984) to represent a buttress by which outgrowth of the palatal shelf is directed. Differences in type IV collagen and laminin concentrations andlor organization may also play a role in directed outgrowth of the secondary palatal shelf and may augment the buttressing effect of the junctional epithelium postulated by Brinkley. As a control for the validity of our observations, serial sections were examined throughout the maxillary process and the roof of the stomodeum. In general, the observations from serial-section analysis were consistent, although slight differences were noted. For example, when the basement membrane was stained with laminin, it tended to be more continuous and longer in posterior sections of the maxillary process. The observations of spatial and temporal differences in the presence and distribution of laminin and type IV collagen described in this report, therefore, were confirmed by serial-section analysis as well a s by the use of different monoclonal antibodies to type IV collagen. There did not appear to be a n association between fibronectin distribution or concentration and the observations described above. Fibronectin was found distributed throughout the mesenchyme at all stages; and, in general, there was a tendency for the appearance of increased staining in subepithelial mesenchyme. Differences were noted in fibronectin staining in the basal epithelium of the maxillary process compared to the epithelium of the roof of the stomodeum which appeared unstained. However, since fibronectin staining of the subepithelial mesenchyme appeared to be more concentrated in the maxillary process, it may have obscured the appearance of the basal epithelium in these regions. The presence of a cell process meshwork (Sulik et al., 1979; Igawa et al., 1986) may be associated with increased fibronectin staining in these regions of the maxillary process and could account for these observations. In general, however, staining patterns with fibronectin did not appear to be correlated in any direct fashion with outgrowth of the maxillary process and did not appear to be correlated with the staining patterns observed for laminin and type IV collagen. It is well documented that basement membrane components such as laminin and type IV collagen have patterns of distribution that are altered during organogenesis. Changing patterns of distribution of basement membrane components have been reported during ocular development (Fitch and Linsenmayer, 1983; Fitch et al., 19831, salivary gland morphogenesis (Bernfield et al., 19841, in the developing kidney (Ekblom et al., 1981; Mounier e t al., 1986), in the developing genitourinary system (Ikawa et al., 1984), during neural crest development (Duband and Thiery, 1987),
in the developing lung (Jaskoll and Slavkin, 1984), during tooth development (Thesleff et al., 1981; Thesleff and Hurmerinta, 1981), in the developing caudal neural tube (O’Shea, 1987), and in other tissues (Laurie e t al., 1983; Wan et al., 1984). For example, during tooth development, contact between preodontoblasts and the basement membrane appears to be required for odontoblast differentiation (Thesleff and Hurmerinta, 1981). During submandibular gland morphogenesis, the basement membrane becomes thinned and regionally discontinuous, and direct contacts between epithelial and mesenchymal cells appear (Cutler and Chaudhry, 1973). Other studies demonstrated that basement-membrane components within interlobular clefts were stable but were transiently lost from lobules. Type I collagen was deposited a t sites of morphologic stability, while type IV collagen was lost from the basement membrane a t sites of rapid cell proliferation (Bernfield et al., 1984). In the developing kidney, the distribution of type IV collagen is considered to have an important role in nephron morphogenesis and to be a critical structural component of the definitive semipermeable glomerular filter. The developing glomerular capillary and mesangium contained type IV collagen a t all stages of development (Michael e t al., 1983). Mesangial immunofluorescence intensity increased progressively with glomerular maturation (Mounier et al., 1986). Changing patterns of distribution of the collagens and laminin have also been observed during neural crest cell migration and subsequent morphogenetic events. For example, both laminin and type IV collagen disappear from the basal surface of the neural tube at sites where neural crest cells emerge (Duband and Thiery, 1987). Although type IV collagen and laminin were found on the basal surfaces of epithelia lining neural crest migration pathways, these components were rarely seen on migrating neural crest cells themselves. Termination of migration of neural crest cells and aggregation into ganglia was then observed by Duband and Thiery to be accompanied by the subsequent reappearance of type IV collagen and laminin within the neural crest cell population. Perhaps the most relevant examples of basement membrane alteration during organogenesis were those observed by Fitch, Linsenmayer, and co-workers during ocular development (Fitch and Linsenmayer, 1983; Fitch et al., 1983; Linsenmayer et al., 1984). For example, during development of the morphologically continuous avian lens capsule, staining with all three monoclonal antibodies to type IV collagen used in this study (ID2, IIB12, and IA8) displayed a uniform pattern of stain in tissue sections of lenses of 4- to 8-dayold chick embryos. By 7 to 8 days of development, a gradient of staining intensity developed which was fully established by 11-12 days of development. At this time striking differences in the staining pattern of the basement membrane of the lens capsule were seen. Bright fluorescence was seen in the anterior lens capsule, while the posterior lens appeared relatively unstained. In a control study, antibodies against non-collagenous components of the lens capsule basement membrane were prepared. Staining of the lens capsule with these antibodies was uniform and intense with no indication of the regional differences that were ob-
DISTRIBUTION OF ECM IN THE MAXILLARY PROCESS
served a t later developmental stages when antibodies to type IV collagen were employed. These examples illustrate the modulation of basement-membrane components associated with organogenesis and tissue differentiation. The present report proposes that specific alterations of basement membrane components is associated with morphogenesis of external body form a s well. ACKNOWLEDGMENTS
This study was supported by NIH grant DE-07674 from the National Institute for Dental Research. The authors wish to thank Mrs. J a n e Laine and Barbara Barton for their expert secretarial assistance in the preparation of this manuscript. They also wish to thank Drs. Richard Mayne, Thomas Linsenmayer, and Douglas Fambrough for providing the antibodies to type IV collagen, laminin, and fibronectin used in this study. Finally, they wish to thank Dr. Richard Mayne for the advice, helpful discussion, and insight he provided to u s during the course of this project. LITERATURE CITED Akiyama, S.K., and K.M. Yamada 1987 Fibronectin. Adv. Enzymol., 59:l-57. Bayne, E.K., M.J. Anderson, and D.M. Fambrough 1984 Extracellular matrix organization in developing muscle: Correlation with acetylcholine receptor aggregates. J. Cell. Biol., 99:1486-1501. Bernfield, M., S.D. Banerjee, J.E. Koda, and A.C. Rapraeger 1984 Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: The Role of Extracellular Matrix in Development. R.L. Trelstad, ed. Alan R. Liss, Inc., New York, pp. 545-572. Brinhley, L.L. 1984 Changes in cell distribution during mouse secondary palate closure in uiuo and in uitro. Dev. Biol., 102:216227. Cutler, L.S., and A.P. Chaudhry 1973 Intercellular contacts a t the epithelial-mesenchymal interface during the prenatal development of the rat sub-mandibular gland. Dev. Biol., 33229-240. Duband, J.L., and J.P. Thiery 1987 Distribution of laminin and collagens during avian neural crest development. Development, 101:461-478. Dziadek, M., and R. Timpl 1985 Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol., I11:372-382. Ekblom, P., K. Alitalo, A. Vaheri, R. Timpl, and L. Saxen 1980 Induction of basement membrane glycoprotein in embryonic kidney: Possible role of laminin in morphogenesis. Proc. Natl. Acad. Sci. USA, 77t485-489. Ekblom, P., E. Lehtonen, L. Saxen, and R. Timpl 1981 Shift in collagen type as an early response to induction of the metanephric mesenchyme. J. Cell Biol., 89:276-283. Ekblom, P., D. Vestweber, and R. Kemler 1986 Cell-matrix interactions and cell adhesion during development. Ann. Rev. Cell Biol., 2:27-47. Fitch, J.M., and T.F. Linsenmayer 1983 Monoclonal antibody analysis of ocular basement membranes during development. Dev. Biol., 95:137-153. Fitch, J.M., E. Gibney, R.D. Sanderson, R. Mayne, and T.F. Linsenmayer 1982 Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen. J. Cell Biol., 95t641-647. Fitch, J.M., R. Mayne, and T.F. Linsenmayer 1983 Developmental acquisition of basement membrane heterogeneity: Type IV collagen in the avian lens capsule. J . Cell Biol., 97:940-943. Furthmayr, H. 1988 Assembly of basement membrane macromolecules. In: Self-Assembling Architecture, J.E. Varner, ed. Alan R. Liss, Inc., New York, pp. 43-59. Gardner, J.M., and D.M. Fambrough 1983 Fibronectin expression during myogenesis. J. Cell Biol., 96:474-485. Hamburger, V., and H.L. Hamilton 1951 A series of normal stages in the development of the chick embryo. J. Morphol., 88r49-92. Igawa, H.H., M. Yasuda, H. Nakamura, and T. Ohura 1986 Changes in the subepithelial mesenchymal cell process meshwork in de-
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