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Fibronectin expression during the processes leading to axis formation in the chick embryo
DEVELOPMENTAL
BIOLOGY
91,
197-201 (1982)
BRIEF NOTES Fibronectini
Expression during the Processes Leading to Axis Formation in the Chick Embryo E. MITRANI AND A. FARBEROV
Embryology
Sectimz, Department
of Zoology, The Hebrew
University
of Jerusalem,
Received October 6, 1981; accepted in revised form November
Jerusalem
91904,
Israel
23, 1981
Fibronectin expression was studied and found not to be present during the shedding process of stage VII chick embryos which indicates that fibronectin is not relevant during the implementation of the gravity-determined process of symmetrization. Fibronectin was detected, however, at the later stage XIII just prior to streak formation as a thin fluorescent sheet on the epiblastic side facing the hypoblast suggesting that it might be involved in the specific interactions that occur between epiblast and hypoblast and that lead to axis formation. Cultures of either epiblastic or hypoblastic chick cells indicate that both types of cells are capable of autonomous expression of fibronectin under in vitro conditions.
INTRODUCTION
Fibronectin has been identified on the surface of various cell types (Yamada and Olden, 1978) and it has been suggested that because it may mediate cell-cell and cell-substrate interactions in viva, a likely place to perform these functions would be in the developing embryo (Zetter and Martin, 1978). In the mouse, early development fibronectin was first found on the inner cell mass at the time differentiation of this area into endoderm and ectoderm begins (Zetter and Martin, 19’78) and in the chick, fibronectin has been detected in association with the ectoderm during gastrulation, and it has been suggested thLat it may provide a guidance system utilized by mesodermal and primordial germ cells (Critchley et al., 19’79). Two earlier developmental processes in the chick, involved with the determi.nation and implementation of the embryonic axis, have been recently elucidated in considerable detail. The first process is concerned with symmetrization which in the chick is determined in utero by the action of gravity (Kochav and Eyal-Giladi, 1971; Eyal-Giladi and Fabian, 1980). Gravity causes an ordered shedding of cells which invariably starts at the highest point of the stage VI blastodisc’ (future posterior aspect) and ends at the lowest point (future anterior side of the chick embryo) (Eyal-Giladi and Kochav, 1976; Kochav et al., 1980). The second. stage is the critical time at which the two layers of the stage XIII blastoderm, the ‘Throughout this work, developmental stages are classified cording to Eyal-Giladi and Ko’chav’s (1976) normal table.
ac-
epiblast and the hypoblast, interact to bring about the immediate formation of the primitive streak and subsequently the embryonic axis (Azar and Eyal-Giladi, 1981). In this study we examine the expression of fibronectin by early chick embryos and by cells in vitro from the cleavage stages up to the appearance of the embryonic axis as manifested by the primitive streak. MATERIALS
AND METHODS
Cell cultures, Cells were obtained
from whole chick embryos from stage VIII up to stage XII. Hypoblastic cells were obtained from whole hypoblasts removed microsurgically from stage XIII blastoderms. Epiblastic cells were obtained only from the epiblastic region of the area pellucida of stage XIII blastoderms which is covered by a hypoblast. No area opaca or marginal zone areas were included as source of epiblastic cells. The cell cultures were prepared as described elsewhere (Mitrani and Eyal-Giladi, 1982). The tissues were incubated for 10 min in trypsin-EDTA solution and carefully transferred to the culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% horse serum, penicillin (100 u/ml), and streptomycin (100 pg/ml). Once in the culture medium the tissue fragments were gently pipetted and dissociated into single cells. The cells were then grown on 12-mm glass coverslips in the culture medium at a concentration of lo5 cells/ml in 5% COz, 95% air at 38”C, for at least 48 hr. Whole embryos. Embryos at stages X, XIII, and primitive-streak stage were processed in toto for immunofluorescence studies as described by Critchley et al. 197 0012-1606/82/050197-05$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
198
FIG. 1. Montage through the whole length of a median sagittal section from a young stage XIII blastoderm. At the interface between the epiblast (e) and hypoblast (h) the fibronectin immunofluorescent staining can be seen as a thin band; (P) posterior (X225). In the central region the hypoblast has become separated from the epiblast during fixation. FIG. 2. Median sagittal section from part of an older stage XIII blastoderm showing a more defined fluorescent band between the epiblast (e) and the hypoblast (h) (x400). FIG. 3. Sagittal section through a peripheral area of the same stage XIII blastoderm as shown in Fig. 2 (x400). (e) Epiblast, (h) hypoblast.
BRIEF NOTES
(1979). Only at stage X is the blastoderm sufficiently thin for observation in toto of either the ventral or dorsal aspect of the cellular sheet. At stages XIII or older the hypoblast (or endoderm) was microsurgically separated from the epiblast (or ectoderm) and each sheet was processed for immunofluorescence and examined separately as described below. Parafj%n sections. Paraflfin sections were preferred to cryostat sections because of our intention to look not only for the presence of fibronectin but also for some sort of pattern. By using paraffin sections it was possible to obtain serial sections, select some areas for immunofluorescence and if possible to go back to the same specimen and to repeat thie immunofluorescence test on other areas. Embryos from the various stages studied were removed from the egg. For Istages III to IX the eggs had to be aborted from the hlen (Kochav and Eyal-Giladi, 1971). The posterior side of each embryo was carefully marked with carbon. The embryos were then fixed in 10% formalin in PBS for 3 hr at 4”C, rinsed overnight in various changes of PBS, and processed for paraffin sections (Sainte-Marie, 1962). Sagittal sections were obtained from embryos at each developmental stage. Slides were prepared so that they would contain a medium sagittal section of an embryo at a particular stage, one sagittal section from each side from a more peripheral area, plus a sagittal section from the median area of ;a primitive-streak stage embryo which would serve as a positive control. Once the slides were deparaffinized they were extensively rinsed in PBS and processed for immunofluorescence as described below. ImmunoJuorescence. Monospecific goat antifibronectin IgG was a gift from K.. Yamada. The properties of this monospecific antiserum have been described by Yamada (1978). Cells previously grown on coverslips, whole embryos, or histological sections were incubated with antifibronectin IgG at a concentration of 100 yg/ ml for 30 min at 3i’“C, extensively rinsed in PBS, then incubated for 30 min at 37°C with FITC-conjugated rabbit anti-goat IgG (1:lOO) (Miles, Yeda, Israel). For the control experiments the antifibronectin IgG was replaced by normal pooled goat IgG (500 pug/ml) in specimens from all tissues being examined. The coverslips were rinsed again and mounted on slides (or coverslips) in 9:1, glycerol:PBS and examined using a Reichart Zetopan microscope equipped1 with an HBO-200 high-pres-
sure mercury-vapor ter combination.
bulb and an FITC interference
fil-
RESULTS
No fibronectin was detected by indirect immunofluorescence in any of the stages III to XII embryos. At stage XIII by the time the second layer, the hypoblast, is completed, on medial sagittal sections a thin fluorescent band was found on the epiblast at the interface with the hypoblast. This fluorescent band was very weak in younger stage XIII embryos (XIII-) and sometimes could only be detected at the posterior end of the embryo (Fig. 1). The fluorescence became progressively stronger in older embryos even before the appearance of the primitive streak (XIII+) and the whole length of the interface between the epiblast and the hypoblast was delineated by a fluorescent band (Fig. 2). Sagittal sections cut at various distances from the midline of the blastoderm were also found to show positive fibronectin immunofluorescent staining (Fig. 3) and perhaps with the exception of the anterior side, which seemed to be stained more weakly by the antifibronectin antibodies in young stage XIII embryos, the whole interface between the epiblast and the hypoblast expressed fibronectin as detected by the immunofluorescence technique. Fibronectin was only detected in areas covered by the hypoblast. No fibronectin expression was detected in the area of the marginal zone or of the area opaca. When whole epiblasts from stage XIII blastoderms were examined in toto, the fibronectin immunofluorescence staining was difficult to examine as it appeared as thin broken threads with no signs of organization. Presumably the fibronectin network that is being formed at this stage is disrupted when the hypoblast is surgically removed from the epiblast. Similar sagittal sections of primitive-streak-stage embryos showed a strong fluorescent band located at the lower ectodermal side which seemed to concentrate at both the posterior and anterior ends of the area pellucida (Figs. 4 and 5). All cell cultures studied, that is, cells derived from whole stage VIII up to stage XII embryos, epiblastic cells and hypoblastic cells when examined with the indirect immunofluorescence technique, expressed fibronectin in vitro. The fluorescence pattern was not ubiquitous on the surface of the cells but it was apparent in “denuded” areas within the monolayers as thin fibers interconnecting isolated groups of cells (Fig. 6). Closer
FIG. 4. Fibronectin immunofluorescent staining of a montage through the whole length of a median sagittal section anterior to the primitive streak of a young primitive-streak-stage blastoderm (X225). The posterior side is to the left. FIG. 5. Fibronectin immunofluorescent staining of a median sagittal section from part of an older primitive-streak-stage blastoderm (X400). FIG. 6. Fibronectin immunofluorescence staining of cultured hypoblastic cells (hc) (x400).
200
DEVELOPMENTALBIOLOGYVOLUME91,1982
examination indicated, however, that in these cell cultures of early chick embryonic cells, fibronectin was deposited between the cells and the substrate as a network whieh can be considered as sparse if compared to that secreted by ordinary fibroblasts in culture Yamada (1978). All control experiments performed were negative throughout. DISCUSSION Kochav and Eyal-Giladi (1971) demonstrated that bilateral symmetry of chick blastodiscs is determined by the earth’s gravity. Further work indicated that following axis determination by gravity, an extensive central area of the five- to six-cell-layer blastoderm of stage VI loses most of its lower cells through a gradual process of cell shedding starting at the future posterior side and spreading toward the anterior side (Kochav et al., 1980). Because fibronectin has been shown to play a role in cell-cell contact and cell-substrate interactions, we considered it a likely candidate to act as a mediator in this morphogenetic shedding process. It now becomes apparent that fibronectin is absent up to and during the shedding process and is therefore not instrumental in the implementation of the gravity-determined process of symmetrization. The primitive streak develops from one of the two layers of cells of the stage XIII blastoderm: the epiblast. We have recently shown that the other layer of cells, the hypoblast is indeed necessary for the induction and not only for determining the direction of the streak (Mitrani and Eyal-Giladi, 1981). In normal conditions the hypoblast also determines the orientation of the embryonic axis since rotation of the hypoblast by 90” is followed by a similar rotation of the axis (Azar and Eyal-Giladi, 1981). The direction of the axis can, however, be determined by the epiblast and therefore determination of the direction of the axis is not a necessary function of the hypoblast (Mitrani and EyalGiladi, 1981). Although the nature of the induction process performed by the hypoblast is not known we have presented evidence which indicates that the hypoblast’s function is not just one of a physical layer that provides some sort of cover to an otherwise denuded epiblast and/or provides nonspecific anchorage or guidance for the movement of mesodermal cells (Mitrani and EyalGiladi, 1981). Within this framework the possibility exists that as a result of a specific interaction between the epiblast and the hypoblast a particular extracellular network is produced between the two layers and that this network is necessary for axial development to occur. Primitive streak formation follows immediately from stage XIII and is a process which essentially en&ails fissive migration of cells from the epiblast to the
lower layer and into the space between the two original layers. The number of cellular changes required to bring about the process of streak formation is bound to be large. Extracellular networks are believed to affect the organization of intercellular cytoskeletal elements in viva (Wessels, 1977). In vitro, effects of the extracellular matrix on growth (through alteration of cell shapej (Ben-Zeev et al., 1980), on intercellular metabolism (Gospodarowicz and Ill, 1980), on the organization of the microfilaments in the cytoskeleton (Sugrue and Hay, 1980), and on cellular differentiation (West et al., 1979) have been reported (for a thorough review see Hynes (1982)). The fact that fibronectin was found after the completion of hypoblast formation and not during earlier developmental stages, and the fact that fibronectin was found between the two layers is an indication that an extracellular network does indeed exist between the epiblast and the hypoblast and suggests that it might be of some relevance during the immediately following process of streak formation. The actual role the extracellular matrix plays in bringing about the primitive streak is not yet known but it seems likely that it works at least as a migration pathway for the invaginating epiblastic cells. As development proceeds and the primitive streak progresses, the fibronectin fluorescence staining becomes much stronger. It has been suggested that at these stages a different fibronectin pattern is being formed which might be involved in supporting migration of other types of cells (Critchley et al, 1979). It is worth pointing out that in spite of the gross difference in cell number, size, and overall shape between the chick blastula and the mouse blastula, the earliest stage at which we observed fibronectin expression in the chick embryo (stage XIII) precisely coincides with the first stage in which fibronectin was detected in the mouse embryo. In the mouse, fibronectin was found at the inner cell mass stage, and not at earlier stages, between the epiblast and the primary endoderm or hypoblast. Studies in the mouse were undecisive with respect to whether fibronectin is produced by the epiblast, hypoblast, or as a result of an interaction between both (Zetter and Martin, 1978). For the chick we have recently developed a technique to grow the various populations of pregastrulation cells in vitro (Mitrani and Eyal-Giladi, 1982). We found that cells derived from whole embryos which were taken from stages VIII to XII can synthesize fibronectin under the conditions encountered in the in vitro situation although they normally do not express fibronectin in vivo. Examinations of primary cultures of only epiblastic or hypoblastic cells indicate that both
BRIEF NOTES
types of cells can synthesize fibronectin and that they can do it, at least in vitro, without the need of the two populations of cells to interact with each other. The fact that at the full hypoblast stage (stage XIII), fibronectin is being expressed in vivo but not by the hypoblastic cells and that these cells if cultured in vitro express fibronectin, suggests that synthesis of fibronectin is probably triggered by the culture conditions and not by the developmental stage achieved in culture. We believe the same type of response is involved in those cells from earlier stages (stages VIII to XII) which were also found to express fibronectin only in vitro. Of the two processes that have been examined which are concerned with axialization, the first one, which is one of the few in vivo processes known to require vigorous organized cell shedding, does not involve fibronectin either during its preliminary stages or during its occurrence. The second process which involves a very special type of interaction between two layers of cells, the epiblast and the hypoblast, to bring about organized movement of large cellular masses, may be mediated by fibronectin.
We are grateful to K. Yamada (NIH Bethesda, Md.) for providing fibronectin antibody and to Professor Eyal-Giladi for reading the manuscript. REFERENCES AZAR, Y., and EYAL-GILADI, H:. (1981). Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments. J. Embryo1 Exp. Morph01 61, 133-144. BEN-ZEEV, A., FARMER, S. R., and PENMAN, S. (1980). Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage dependent fibroblasts. Cell 21, 365-372. CRITCHLEY,D. R., ENGLAND, M. A., WAKELY, J., and HYNES, R. 0. (1979). Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature (London) 180, 498-500.
201
EYAL-GILADI, H., and KOCHAV,S. (1976). From cleavage to primitive stage formation: A complementary normal table and a new look at the first stages of the development of the chick. 1. General morphology. Develop. Biol. 49, 321-337. EYAL-GILADI, H., and FABIAN, B. C. (1980). Axis determination in uterine chick blastodiscs under changing spatial positions during the sensitive period for polarity. Develop. Biol. 77, 228-232. GOSPODAROWICZ, D., and ILL, C. R. (1980). Do plasma and serum have different abilities to promote cell growth? Proc. Nat. Acad. Ski. USA 77, 2’726-2730. HYNES, R. 0. (1982). Fibronectin and its relation to cellular structure and behaviour. In “Cell Biology of the Extracellular Matrix” (E. D. Hay, ed.), pp. 295-333. Plenum, New York. KOCHAV,S., and EYAL-GILADI, H. (1971). Bilateral symmetry in chick embryo, determination by gravity. Science 171, 1027-1029. KOCHAV, S., GINSBURG,M., and EVAL-GILADI, H. (1980). From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. II. Microscopic anatomy and cell population dynamics. Develop. Biol. 79, 296-308.
MITRANI, E., and EYAL-GILADI, H. (1981). Hypoblastic cells can form a disc inducing an embryonic axis in the chick epiblast. Nature (London) 189,800-801. MITRANI, E., and EYAL-GILADI, H. (1982). Cells from early chick embryos in culture. Difjeerentiation, in press. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem Cytochem. 10, 250256. SUGRUE,S. P., and HAY, E. D. (1980). Response of basal epithelial cell surface and cytoskeleton to solubilized extracellular matrix molecules. J. Cell. Biol. 87, 126. WESSELS,N. K. (1977). Tissue Interactions and Development. Benjamin, Menlo Park, Calif. WEST,C. M., LANZA, R., ROSEMBLOOM, J., LOWE,M., HOLTZER,H., and AVDALOVIC,N. (1979). Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts. Cell 17, 491-501. YAMADA, K. M. (1978). Immunological characterization of a major transformation-sensitive fibroblast cell surface protein. J. Cell Biol. 78, 520-541. YAMADA, K. M., and OLDEN, K. (1978). Fibronectins-Adhesive glycoproteins of cell surface and blood. Nature (London) 275.179-184. ZETTER,B. R., and MARTIN, G. R. (1978). Expression of a high molecular weight cell surface glycoprotein (LETS protein) by preimplantation mouse embryos and teratocarcinoma stem cells. Proc. Nat. Acad. Sci. USA 75, 2324-2328.
BIOLOGY
91,
197-201 (1982)
BRIEF NOTES Fibronectini
Expression during the Processes Leading to Axis Formation in the Chick Embryo E. MITRANI AND A. FARBEROV
Embryology
Sectimz, Department
of Zoology, The Hebrew
University
of Jerusalem,
Received October 6, 1981; accepted in revised form November
Jerusalem
91904,
Israel
23, 1981
Fibronectin expression was studied and found not to be present during the shedding process of stage VII chick embryos which indicates that fibronectin is not relevant during the implementation of the gravity-determined process of symmetrization. Fibronectin was detected, however, at the later stage XIII just prior to streak formation as a thin fluorescent sheet on the epiblastic side facing the hypoblast suggesting that it might be involved in the specific interactions that occur between epiblast and hypoblast and that lead to axis formation. Cultures of either epiblastic or hypoblastic chick cells indicate that both types of cells are capable of autonomous expression of fibronectin under in vitro conditions.
INTRODUCTION
Fibronectin has been identified on the surface of various cell types (Yamada and Olden, 1978) and it has been suggested that because it may mediate cell-cell and cell-substrate interactions in viva, a likely place to perform these functions would be in the developing embryo (Zetter and Martin, 1978). In the mouse, early development fibronectin was first found on the inner cell mass at the time differentiation of this area into endoderm and ectoderm begins (Zetter and Martin, 19’78) and in the chick, fibronectin has been detected in association with the ectoderm during gastrulation, and it has been suggested thLat it may provide a guidance system utilized by mesodermal and primordial germ cells (Critchley et al., 19’79). Two earlier developmental processes in the chick, involved with the determi.nation and implementation of the embryonic axis, have been recently elucidated in considerable detail. The first process is concerned with symmetrization which in the chick is determined in utero by the action of gravity (Kochav and Eyal-Giladi, 1971; Eyal-Giladi and Fabian, 1980). Gravity causes an ordered shedding of cells which invariably starts at the highest point of the stage VI blastodisc’ (future posterior aspect) and ends at the lowest point (future anterior side of the chick embryo) (Eyal-Giladi and Kochav, 1976; Kochav et al., 1980). The second. stage is the critical time at which the two layers of the stage XIII blastoderm, the ‘Throughout this work, developmental stages are classified cording to Eyal-Giladi and Ko’chav’s (1976) normal table.
ac-
epiblast and the hypoblast, interact to bring about the immediate formation of the primitive streak and subsequently the embryonic axis (Azar and Eyal-Giladi, 1981). In this study we examine the expression of fibronectin by early chick embryos and by cells in vitro from the cleavage stages up to the appearance of the embryonic axis as manifested by the primitive streak. MATERIALS
AND METHODS
Cell cultures, Cells were obtained
from whole chick embryos from stage VIII up to stage XII. Hypoblastic cells were obtained from whole hypoblasts removed microsurgically from stage XIII blastoderms. Epiblastic cells were obtained only from the epiblastic region of the area pellucida of stage XIII blastoderms which is covered by a hypoblast. No area opaca or marginal zone areas were included as source of epiblastic cells. The cell cultures were prepared as described elsewhere (Mitrani and Eyal-Giladi, 1982). The tissues were incubated for 10 min in trypsin-EDTA solution and carefully transferred to the culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% horse serum, penicillin (100 u/ml), and streptomycin (100 pg/ml). Once in the culture medium the tissue fragments were gently pipetted and dissociated into single cells. The cells were then grown on 12-mm glass coverslips in the culture medium at a concentration of lo5 cells/ml in 5% COz, 95% air at 38”C, for at least 48 hr. Whole embryos. Embryos at stages X, XIII, and primitive-streak stage were processed in toto for immunofluorescence studies as described by Critchley et al. 197 0012-1606/82/050197-05$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
198
FIG. 1. Montage through the whole length of a median sagittal section from a young stage XIII blastoderm. At the interface between the epiblast (e) and hypoblast (h) the fibronectin immunofluorescent staining can be seen as a thin band; (P) posterior (X225). In the central region the hypoblast has become separated from the epiblast during fixation. FIG. 2. Median sagittal section from part of an older stage XIII blastoderm showing a more defined fluorescent band between the epiblast (e) and the hypoblast (h) (x400). FIG. 3. Sagittal section through a peripheral area of the same stage XIII blastoderm as shown in Fig. 2 (x400). (e) Epiblast, (h) hypoblast.
BRIEF NOTES
(1979). Only at stage X is the blastoderm sufficiently thin for observation in toto of either the ventral or dorsal aspect of the cellular sheet. At stages XIII or older the hypoblast (or endoderm) was microsurgically separated from the epiblast (or ectoderm) and each sheet was processed for immunofluorescence and examined separately as described below. Parafj%n sections. Paraflfin sections were preferred to cryostat sections because of our intention to look not only for the presence of fibronectin but also for some sort of pattern. By using paraffin sections it was possible to obtain serial sections, select some areas for immunofluorescence and if possible to go back to the same specimen and to repeat thie immunofluorescence test on other areas. Embryos from the various stages studied were removed from the egg. For Istages III to IX the eggs had to be aborted from the hlen (Kochav and Eyal-Giladi, 1971). The posterior side of each embryo was carefully marked with carbon. The embryos were then fixed in 10% formalin in PBS for 3 hr at 4”C, rinsed overnight in various changes of PBS, and processed for paraffin sections (Sainte-Marie, 1962). Sagittal sections were obtained from embryos at each developmental stage. Slides were prepared so that they would contain a medium sagittal section of an embryo at a particular stage, one sagittal section from each side from a more peripheral area, plus a sagittal section from the median area of ;a primitive-streak stage embryo which would serve as a positive control. Once the slides were deparaffinized they were extensively rinsed in PBS and processed for immunofluorescence as described below. ImmunoJuorescence. Monospecific goat antifibronectin IgG was a gift from K.. Yamada. The properties of this monospecific antiserum have been described by Yamada (1978). Cells previously grown on coverslips, whole embryos, or histological sections were incubated with antifibronectin IgG at a concentration of 100 yg/ ml for 30 min at 3i’“C, extensively rinsed in PBS, then incubated for 30 min at 37°C with FITC-conjugated rabbit anti-goat IgG (1:lOO) (Miles, Yeda, Israel). For the control experiments the antifibronectin IgG was replaced by normal pooled goat IgG (500 pug/ml) in specimens from all tissues being examined. The coverslips were rinsed again and mounted on slides (or coverslips) in 9:1, glycerol:PBS and examined using a Reichart Zetopan microscope equipped1 with an HBO-200 high-pres-
sure mercury-vapor ter combination.
bulb and an FITC interference
fil-
RESULTS
No fibronectin was detected by indirect immunofluorescence in any of the stages III to XII embryos. At stage XIII by the time the second layer, the hypoblast, is completed, on medial sagittal sections a thin fluorescent band was found on the epiblast at the interface with the hypoblast. This fluorescent band was very weak in younger stage XIII embryos (XIII-) and sometimes could only be detected at the posterior end of the embryo (Fig. 1). The fluorescence became progressively stronger in older embryos even before the appearance of the primitive streak (XIII+) and the whole length of the interface between the epiblast and the hypoblast was delineated by a fluorescent band (Fig. 2). Sagittal sections cut at various distances from the midline of the blastoderm were also found to show positive fibronectin immunofluorescent staining (Fig. 3) and perhaps with the exception of the anterior side, which seemed to be stained more weakly by the antifibronectin antibodies in young stage XIII embryos, the whole interface between the epiblast and the hypoblast expressed fibronectin as detected by the immunofluorescence technique. Fibronectin was only detected in areas covered by the hypoblast. No fibronectin expression was detected in the area of the marginal zone or of the area opaca. When whole epiblasts from stage XIII blastoderms were examined in toto, the fibronectin immunofluorescence staining was difficult to examine as it appeared as thin broken threads with no signs of organization. Presumably the fibronectin network that is being formed at this stage is disrupted when the hypoblast is surgically removed from the epiblast. Similar sagittal sections of primitive-streak-stage embryos showed a strong fluorescent band located at the lower ectodermal side which seemed to concentrate at both the posterior and anterior ends of the area pellucida (Figs. 4 and 5). All cell cultures studied, that is, cells derived from whole stage VIII up to stage XII embryos, epiblastic cells and hypoblastic cells when examined with the indirect immunofluorescence technique, expressed fibronectin in vitro. The fluorescence pattern was not ubiquitous on the surface of the cells but it was apparent in “denuded” areas within the monolayers as thin fibers interconnecting isolated groups of cells (Fig. 6). Closer
FIG. 4. Fibronectin immunofluorescent staining of a montage through the whole length of a median sagittal section anterior to the primitive streak of a young primitive-streak-stage blastoderm (X225). The posterior side is to the left. FIG. 5. Fibronectin immunofluorescent staining of a median sagittal section from part of an older primitive-streak-stage blastoderm (X400). FIG. 6. Fibronectin immunofluorescence staining of cultured hypoblastic cells (hc) (x400).
200
DEVELOPMENTALBIOLOGYVOLUME91,1982
examination indicated, however, that in these cell cultures of early chick embryonic cells, fibronectin was deposited between the cells and the substrate as a network whieh can be considered as sparse if compared to that secreted by ordinary fibroblasts in culture Yamada (1978). All control experiments performed were negative throughout. DISCUSSION Kochav and Eyal-Giladi (1971) demonstrated that bilateral symmetry of chick blastodiscs is determined by the earth’s gravity. Further work indicated that following axis determination by gravity, an extensive central area of the five- to six-cell-layer blastoderm of stage VI loses most of its lower cells through a gradual process of cell shedding starting at the future posterior side and spreading toward the anterior side (Kochav et al., 1980). Because fibronectin has been shown to play a role in cell-cell contact and cell-substrate interactions, we considered it a likely candidate to act as a mediator in this morphogenetic shedding process. It now becomes apparent that fibronectin is absent up to and during the shedding process and is therefore not instrumental in the implementation of the gravity-determined process of symmetrization. The primitive streak develops from one of the two layers of cells of the stage XIII blastoderm: the epiblast. We have recently shown that the other layer of cells, the hypoblast is indeed necessary for the induction and not only for determining the direction of the streak (Mitrani and Eyal-Giladi, 1981). In normal conditions the hypoblast also determines the orientation of the embryonic axis since rotation of the hypoblast by 90” is followed by a similar rotation of the axis (Azar and Eyal-Giladi, 1981). The direction of the axis can, however, be determined by the epiblast and therefore determination of the direction of the axis is not a necessary function of the hypoblast (Mitrani and EyalGiladi, 1981). Although the nature of the induction process performed by the hypoblast is not known we have presented evidence which indicates that the hypoblast’s function is not just one of a physical layer that provides some sort of cover to an otherwise denuded epiblast and/or provides nonspecific anchorage or guidance for the movement of mesodermal cells (Mitrani and EyalGiladi, 1981). Within this framework the possibility exists that as a result of a specific interaction between the epiblast and the hypoblast a particular extracellular network is produced between the two layers and that this network is necessary for axial development to occur. Primitive streak formation follows immediately from stage XIII and is a process which essentially en&ails fissive migration of cells from the epiblast to the
lower layer and into the space between the two original layers. The number of cellular changes required to bring about the process of streak formation is bound to be large. Extracellular networks are believed to affect the organization of intercellular cytoskeletal elements in viva (Wessels, 1977). In vitro, effects of the extracellular matrix on growth (through alteration of cell shapej (Ben-Zeev et al., 1980), on intercellular metabolism (Gospodarowicz and Ill, 1980), on the organization of the microfilaments in the cytoskeleton (Sugrue and Hay, 1980), and on cellular differentiation (West et al., 1979) have been reported (for a thorough review see Hynes (1982)). The fact that fibronectin was found after the completion of hypoblast formation and not during earlier developmental stages, and the fact that fibronectin was found between the two layers is an indication that an extracellular network does indeed exist between the epiblast and the hypoblast and suggests that it might be of some relevance during the immediately following process of streak formation. The actual role the extracellular matrix plays in bringing about the primitive streak is not yet known but it seems likely that it works at least as a migration pathway for the invaginating epiblastic cells. As development proceeds and the primitive streak progresses, the fibronectin fluorescence staining becomes much stronger. It has been suggested that at these stages a different fibronectin pattern is being formed which might be involved in supporting migration of other types of cells (Critchley et al, 1979). It is worth pointing out that in spite of the gross difference in cell number, size, and overall shape between the chick blastula and the mouse blastula, the earliest stage at which we observed fibronectin expression in the chick embryo (stage XIII) precisely coincides with the first stage in which fibronectin was detected in the mouse embryo. In the mouse, fibronectin was found at the inner cell mass stage, and not at earlier stages, between the epiblast and the primary endoderm or hypoblast. Studies in the mouse were undecisive with respect to whether fibronectin is produced by the epiblast, hypoblast, or as a result of an interaction between both (Zetter and Martin, 1978). For the chick we have recently developed a technique to grow the various populations of pregastrulation cells in vitro (Mitrani and Eyal-Giladi, 1982). We found that cells derived from whole embryos which were taken from stages VIII to XII can synthesize fibronectin under the conditions encountered in the in vitro situation although they normally do not express fibronectin in vivo. Examinations of primary cultures of only epiblastic or hypoblastic cells indicate that both
BRIEF NOTES
types of cells can synthesize fibronectin and that they can do it, at least in vitro, without the need of the two populations of cells to interact with each other. The fact that at the full hypoblast stage (stage XIII), fibronectin is being expressed in vivo but not by the hypoblastic cells and that these cells if cultured in vitro express fibronectin, suggests that synthesis of fibronectin is probably triggered by the culture conditions and not by the developmental stage achieved in culture. We believe the same type of response is involved in those cells from earlier stages (stages VIII to XII) which were also found to express fibronectin only in vitro. Of the two processes that have been examined which are concerned with axialization, the first one, which is one of the few in vivo processes known to require vigorous organized cell shedding, does not involve fibronectin either during its preliminary stages or during its occurrence. The second process which involves a very special type of interaction between two layers of cells, the epiblast and the hypoblast, to bring about organized movement of large cellular masses, may be mediated by fibronectin.
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