The ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the chick embryo.код для вставкиСкачать
THE ANATOMICAL RECORD 197: 441-470 (1980) The Ultrastructure of Oral (Buccopharyngeal) Membrane Formation and Rupture in the Chick Embryo ROBERT E. WATERMAN AND GARY C. SCHOENWOLF Department of Anatomy, University of New Mexico,School of Medicine, Albuquerque, New Mexico 87131 (RB.W.,G.C.S.) ABSTRACT The ultrastructure of the oral (buccopharyngeal) membrane was examined by transmission and scanning electron microscopy (SEMI from its initial formation (stage 8) to its complete disappearance (stage 20) in the chick embryo. Thinning of the oral membrane prior t o rupture occurs in large measure by increased interdigitation between cells of the stomodeal ectoderm and foregut endoderm coincident with a decrease in the width of the intervening extracellular space. Large numbers of necrotic cells were not observed. Interdigitation of ectodermal and endodermal cells makes it increasingly dif?icult t o discern two discrete epithelia, and no evidence that one germ layer disappears prior to the other was observed. Changes occurred in the fine structure of the extracellular matrix during formation and rupture of the oral membrane, and the organization of this material within the oral membrane differed from that in regions immediately lateral to it. Copious amounts of amorphous, flocculant (“lamina-like”) material are present within the oral membrane a t all stages. The basal lamina of the ectoderm exhibits small loops or folds at early stages. These decrease in number as the basal lamina becomes discontinuous prior to establishment of direct intercellular contact between cells of the ectoderm and endoderm across the intervening extracellular compartment. Initial perforations of the oral membrane are preceeded by clefts between cells on both sides of this structure, and SEM observations suggest that cells of the oral membrane continue to interdigitate, elongate, and change relative positions during the rupture process. The oral (buccopharyngeal, pharyngeal, oropharyngeal) membrane is composed of a region of close apposition between stomodeal ectoderm and foregut endoderm. It forms a temporary barrier between the stomodeal cavity and the lumen of the foregut during early embryonic development of apparently all vertebrates. The oral membrane thins and eventually ruptures to create a patent passage between the external embryonic environment and the cranial end of the developing digestive tract. Rupture of the oral membrane is vital to the survival of the individual, since an opening from the external environment into the gastrointestinal tract is required to meet the organism’s eventual nutritional needs. An opening into the pharynx is also essential for respiration in those vertebrates, such as fish, which possess internal gills but no internal nares. The oral membrane is illustrated and mentioned incidently in numerous descriptions of vertebrate cranial development, but detailed information regarding its formation 0003-276X/80/1974-0441$05.00 1980 ALAN R. LISS, INC. and demise is rare, and is usually found only in histologic studies of adjacent structures such as Rathke’s pouch, the prechordal region, or the heart (Parker, 1917; Adelmann, 1922; Davis, 1923, 1927; Schwind, 1928; Aasar, 1931; Brahms, 1932; Gilbert, 1934, 1935; Kerr, 1946; Gilbert, 1957; Blechschmidt, 1961; Hillman and Hillman, 1965; Hammond, 1974; Betz and Jarskar, 1974; Fremont and Ferrand, 1978; Jacobson et al., 1979). Intimate contact between ectoderm and endoderm in the region of the oral membrane prior t o its disappearance presumably prevents the migration of mesenchymal cells across the ventral midline, thus preventing a situation which might preclude or interfere All correspondence and reprint requests should be sent to: Robert E. Waterman, Ph.D.,Department of Anatomy, The University of New Mexico, School of Medicine, Albuquerque, NM 87131. Gary Schoenwolfs present address is: Department of Anatomy, University of Utah, College of Medicine, Salt Lake City, UT 84132 USA. Fkceived November 15. 1979; accepted March 7, 1980. 441 442 R.E. WATERMAN AND with formation of a future mouth opening. This applies to the mesodermal cells of the prechordal plate and head process as well as to neural crest cells which migrate into the craniofacial region. The presence of the oral membrane appears to create a physical barrier which aids in directing the expansion of these cell populations, as shown in the avian embryo in which the migrations of mesodermal and cranial neural crest cells are known in considerable detail (Johnston, 1966; Noden, 1973, 1975, 1978). This is particularly important during migration of neural crest cells into the mandibular and maxillary processes which grow forward around the margins of the oral membrane to enlarge the stomodeum, and rupture of the oral membrane does not occur until these processes a r e well developed. Adhesion between the ectoderm and endoderm of the oral membrane a t early stages may also serve to “anchor” the cranial end of the foregut as it elongates (Stalsberg and DeHaan, 1968). Mechanisms responsible for the rupture and eventual disappearance of the oral membrane a r e largely unknown. An ultrastructural study of the oral membrane in the hamster embryo (Waterman, 1977) revealed that thinning of this structure prior to its rupture is due in part to increased intermingling of ectodermal and endodermal cells accompanied by apparent phagocytosis of intervening extracellular material, but evidence of extensive cell death during initial perforation of the oral membrane was not observed. Several histologic studies of the cranial regions of avian embryos suggest that sequential disappearance of the epithelial layers forming the oral membrane may occur in some birds, although both ectodermal (Rex, 1897; Nicolas and Weber, 1901) and endodermal (Manno, 1903) cells have been reported to disappear initially. The present study was undertaken to clarify, by means of techniques affording greater resolution than the light microscopic procedures used previously, the morphologic changes exhibited by components of the oral membrane during development and rupture of this structure in the chick embryo. MATERIALS AND METHODS Several dozen fertile White Leghorn eggs were incubated in a forced-draft incubator a t 38°C until embryos reached stages 8-21 (Hamburger and Hamilton, 1951). Eggs were then opened into a finger bowl containing warm 0.9% saline, and the blastoderms were G.C. SCHOENWOLF rapidly cut away from the yolk, washed in fresh saline, and immediately fixed and processed for light or electron microscopy. Processing for scanning electron microscopy (SEMI Blastoderms were immersed for 2 to 3 hours in 2% glutaraldehyde in 0.1 M cacodylate buffer a t pH 7.2 (Sabatini et al., 1963). Fixed embryos were then dissected free of surrounding membranes, pooled according to their stage of development, washed in several changes of buffer, secondarily fixed with 0.1 M cacodylate buffered 1% osmium tetroxide for 1 hour, dehydrated in a graded ethanol series, and dried by the critical-point method using liquid CO,. The oral membrane in more advanced stages was exposed by carefully cross-sectioning the pharyngeal region at the level of the first pharyngeal groove with razor blades prior to dehydration and critical-point drying. Dried embryos were affixed to aluminum stubs with their ventral surface facing upwards by means of double-stick adhesive tape and conductive silver paint. The ectoderm covering the ventral surface of the head, including the ectodermal component of the oral membrane, was removed in some younger specimens by gently applying a small piece of double adhesive cellophane tape to the apical surface of the ectoderm and lifting it upwards. The tape with the adhering ectoderm was then inverted and mounted on the stub adjacent to the remainder of the specimen to permit examination of complimentary surfaces. Some samples were dissected by means of small pieces of broken razor blades held by forceps either prior to or following critical-point drying. Specimens were then coated with a thin layer of go1d:palladium (60:40) in a Hummer I sputter coater, and examined with an ETEC Autoscan SEM operated a t 10 kV. Some dried embryos were sequentially dissected with adhesive tape; the exposed surfaces being recoated with go1d:palladium and rephotographed prior to succeeding manipulations. Stereopairs of micrographs were recorded at a relative tilt angle of 10”. Processing for light microscopy (LM) and transmission electron microscopy (TEM) Blastoderms were briefly immersed (approximately 30 seconds) in cold (4°C) cacodylate buffered (0.1 M at pH 7.2) 2% glutaraldehyde (plus 0.05% calcium chloride and 0.1 M su- CHICK ORAL MEMBRANE crose) while small blocks of tissue containing the desired regions of the embryos were dissected out. These portions of the embryos were then immediately fixed on ice for one hour with a cacodylate buffered (0.1 M a t pH 7.2) mixture containing a final concentration of 2% glutaraldehyde/l% osmium tetroxide (plus 0.05% calcium chloride). Dissected regions were then dehydrated with ethanol, transferred to propylene oxide, and embedded in Epon 812 (Luft, 1961). Thick (1Fm) and thin (100 nm) sections were cut with diamond knives. Thick sections were stained with methylene blue/azure I1 (Richardson et al., 1960) and mounted on glass slides for examination by light microscopy (LM).Thin sections were supported on uncoated copper grids, stained with uranyl acetate and lead citrate (Reynolds, 1963), and examined with a Hitachi HS-7S TEM operated a t 50 kV. OBSERVATIONS Stages 8-9 At stage 8 (4 somite paris; 26-29 hr), the foregut is a broad, flattened pouch, the roof of which is in close proximity t o the ventral surface of the neuroepithelium (Fig. 1).The floor of the foregut is thickened near the ventral midline and will form the endodermal component of the oral membrane, although the precise limits of the membrane are difficult t o establish at this early stage. A considerable extracellular space separates the endoderm of the floor of the foregut and the ectoderm covering the ventral surface of the head. The opposed basal surfaces of both ectoderm and endoderm are somewhat irregular in the region of the presumptive oral membrane. The basal ends of some ectodermal cells project toward the endoderm, appearing as “peaks” in histological sections. The primary mesenchyme is sparse in the cephalic region a t this stage. By stage 9 (7 somite paris; 29-33 hr), the oral membrane consists of a rather precisely delimited region of close approximation between the thickened endodermal floor of the foregut and the ectoderm near the ventral midline (Fig. 2). A large, relatively cell-free, extracellular space exists immediately beneath the surface ectoderm on each side of the head, causing a bulging of the ventrolateral aspects of the cranium. The oral membrane consequently forms the floor of a shallow midline depression along the ventral surface of 443 the head cranial to the reflection of the body fold. This depression consitutes the beginning of the stomodeum. The developing ventral aortae appear as small capillaries subjacent to the floor of the foregut just lateral to the oral membrane. Neural crest cells have not yet migrated extensively into the cranial region, and mesenchymal cells are only slightly more numerous than at stage 8. The tip of the notochord consists of a poorly circumscribed mass of cells wedged between the neuroepithelium and the roof of the foregut. The ectoderm of the oral membrane is slightly thicker than the ectoderm covering the remainder of the ventrolateral aspects of the head. The ectodermal cells of the oral membrane are connected by small intercellular junctions a t both their apical and basal ends, and the ectoderm is underlain by a continuous basal lamina which is thrown a t intervals into small folds or looping projections (Fig. 3a, b). Numerous patches of fibrillar and flocculent extracellular material are associated with the extracellular surface of the basal lamina. These are more prevalent in regions where the basal lamina is folded than in regions where it is flattened. A few profiles of cellular processes are present in the extracellular space between the ectoderm and endoderm (Fig. 3c). Some of these come into close proximity to the basal lamina and associated flocculent material beneath the ectoderm, but direct cell-to-cell contact through the basal lamina was not observed. The basal surface of the endoderm of the oral membrane is more irregular than that of the ectodermal component. Cells of the foregut endoderm are connected by small intercellular junctions a t their apical ends facing the lumen of the foregut, but the basal ends of the endodermal cells in the oral membrane are more widely separated (Fig. 3a). There is no continuous basal lamina beneath the endoderm, but small patches of electron dense, flocculent material are associated with the basal surfaces of many endodermal cells. The apical surfaces of the ectodermal cells covering the ventral aspects of the head a t these stages exhibit no discernible regional differences in surface topography when viewed with the SEM (Fig. 4). Removal of the ectoderm exposes the basal surface of the thickened endoderm in the region of the developing oral membrane as well as the mesenchymal cells surrounding the foregut (Fig. 5). The basal aspects of the loosely organized endod- 444 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 1. Cross section through the cranial region of a stage 8 chick embryo. The thickened floor of the foregut in the ventral midline, together with the overlying ectoderm, will form the oral membrane, although this structure is not clearly delimited at this stage. The ectoderm and endoderm of the presumptive oral membrane are separated by a distinct intercellular space. The neural folds are unfwed, and few mesenchymal cells are present at this level. x 102. Fig. 2. Cross section of a stage 9 embryo at the level of the mesencephalon. The neural folds are approximated, but neural crest cells have not yet begun to migrate into the large, cell-free extracellular spaces (*) at the lateral and ventral sides of the head. The oral membrane is now recognizable as an area of close approximation between the thickened endodermal floor of the foregut and overlying ectoderm separated by a narrow extracellular space. The oral membrane forms the floor of the developing stomodeum. x 107. Fig. 3a-c. The ultrastructure of the apposed basal surfaces of endoderm and ectoderm in the oral membrane at stage 9 is illustrated in a section nearly adjacent to that in Figure 2. (3a) The basal lamina underlying the ectoderm is associated with accumulations of fibrillar and flocculent extracellular material, and exhibits small folds or “pleats” at various intervals. Small patches of fibrillar extracellular material are associated with the basal ends of many endodermal cells (arrows), but a continuous basal lamina is absent beneath the endoderm. x 8,684. (3b) A portion of the basal lamina and associated extracellular material from the region indicated by the asterisk in Fig. 3a is seen at higher magnification. X 23,827. (3c) A cell process (arrow) within the extracellular space of the oral membrane is associated with a patch of extracellular material. x 10,085. CHICK ORAL MEMBRANE 445 446 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 4. Stereopair of scanning electron micrographs illustrating ventral aspect of cranial region of stage 8+ embryo. The anterior end of the neural groove, reflection of the body fold (cut), and anterior intestinal portal are seen. (This, and all subsequent stereopairs, are mounted for viewing with stereoscopic glasses.) x 249. Fig. 5. Stereopair of the same stage 8+ embryo shown in Figure 4, but with ectoderm removed from the ventral surface of the head to reveal the basal surface of the foregut and associated mesenchyme. The basal surface of the endodenn in the region of the presumptive oral membrane (arrow) is not as flattened as that more caudally (*) at the level of the pericardial ccelom and dorsal mesocardium. x 249. Fig. 6. Stereopair of micrographs illustrating the morphology of the ventral surface of the cranial region of a stage 11embryo. The cranial ends of the neural folds are approximated, but not completely fused, in the region of the anterior neuropore. The lateral and ventral aspects of the cranial region bulge outward caudal to the outlines of the laterally projecting optic vesicles. The oral membrane (arrow) forms a portion of the floor of the forming stomodeum. A section of the oral membrane in the region indicated by the arrow is shown in Figure 7. x 158. CHICK ORAL MEMBRANE 447 448 R.E. WATERMAN AND G.C. SCHOENWOLF ermal cells are connected by short strands which probably represent both cellular processes and extracellular material. This is in sharp contrast to the smoother basal surface of the foregut lateral to the oral membrane. Longitudinally oriented strands of flattened cells on either side of the future oral membrane comprise the developing ventral aortae which join the developing heart caudally. lateral borders of adjacent endodermal cells. The ectodermal cells remain attached by intercellular junctions a t both apical and basal ends, but few small junctions are present between the basal ends of the endodermal cells. It was not possible to further characterize these embryonic junctions in the conventional TEM preparations examined, although it appears that no desmosomes are present between cells of either epithelium a t this stage. When the ectoderm is carefully removed from the ventral surfaces of dried specimens and inverted, the extracellular material associated with its basal surface can be seen to form strands which are aligned in the craniocaudal direction in the region of the oral membrane (Figs. 12, 131, in contrast to the more random organization of extracellular material laterally (Fig. 13). Views of the complimentary endodermal surface reveal what in stereopairs of micrographs appear to be longitudinally oriented profiles of extracellular material, and fractured specimens indicate that this material is interposed between the basal ends of the endodermal cells of the oral membrane (Fig. 14). The amount of aligned material associated with both the ectoderm and endoderm is most prominent near the ventral midline. With the ectoderm removed, the basal surface of the foregut in the midline between the developing ventral aortae can be seen to differ craniocaudally. Caudally, near the bifurcation of the aortae and caudal to the oral membrane, the endodermal cells are tightly compacted and covered by a blanket of extracellular matrix (Fig. 12). More cranially, endodermal cells within the oral membrane become increasingly rounded and separated. Short cellular processes connect adjacent endodermal cells of the oral membrane and some extracellular material is present, but a uniform extracellular coating is lacking. Stages 10-1 1 At stage 10 (10 somite pairs; 33-38 hr), the heart is beginning to loop to the right, the optic vesicles are not yet constricted, and the cranial flexure is beginning. By stage 11 (13 somite pairs-not counting the first pair which has dispersed; 40-45 hr), the heart is bent prominently to the right and the bases of the optic vesicles are constricted. The anterior neuropore closes during these stages, although complete union between all components of the neural folds in this region is not complete until stages 13-14 (Schoenwolf, 1979). There is no clear indication of a demarcation on the ventral surface of the cranium between the ectoderm of the oral membrane and that which will become Rathke’s pouch (Fig. 6), and no striking differences in the apical morphology of the ectodermal cells of the oral membrane relative to that of the ectodermal cells lateral to the oral membrane were observed by SEM. The appearance of the oral membrane in histologic sections is similar to that a t previous stages, except for an increase in the amount of extracellular material between ectoderm and endoderm. Strands of material visible even with the light microscope extend between the irregular basal surfaces of endodenn and ectoderm (Fig. 7). The basal lamina of the ectoderm of the oral membrane is extensively folded or pleated and is associated with accumulations of flocculent material (Figs. 8, 10, 11). This is in sharp distinction to the continuous basal lamina Stages 12-15 beneath the ectoderm lateral to the oral membrane, which does not exhibit prominent folds As development proceeds, the endoderm and and is associated primarily with small round- ectoderm of the oral membrane become ined profiles termed “interstitial bodies” by Low creasingly apposed. By stage 13 (19 somite (1970) (Fig. 9). A basal lamina is forming pairs; 48-52 hr), the cranial and cervical flexalong the dorsal and lateral walls of the fore- ures are broad curves and the head is turning gut, but is largely discontinuous in the region onto its left side. Migrating cranial neural of the oral membrane. Patches of flocculent crest cells have reached the lateral margins of material are numerous within the oral mem- the oral membrane (Fig. 15). Rathke’s pouch brane, where they are located primarily a t the appears as a small depression caudal to the basal surfaces of the endodermal cells (Fig. closing anterior neuropore a t about stage 14 10).Similar material is only occasionally pres- (22 somite pairs; 50-53 hr), and the ectoderm ent in the intercellular spaces between the near the midline caudal to Rathke’s pouch and CHICK ORAL MEMBRANE cranial to the small mandibular processes may now be recognized as belonging exclusively to the oral membrane. The extracellular space between the endoderm and ectoderm of the oral membrane contains numerous profiles of cellular processes and copious amounts of fibrillar and flocculent extracellular material at stage 13 (Fig. 16). Some extracellular fibrils exhibit the characteristic striated appearance of collagen (Fig. 17). The basal lamina of the ectoderm no longer displays folds or pleats, and instead exhibits occasional discontinuities which in some places are associated with wide gaps between the bases of the overlying ectodermal cells (Fig. 16). The basal contour of the ectoderm is more irregular than in previous stages, and appears more similar to that of the endoderm. Profiles of basal lamina are present along the basal surfaces of some endodermal cells, but a continuous basal lamina is still not observed beneath the endoderm. By stage 14, direct cell-to-cell contact between ectodermal and endodermal cells has progressed to a point where extensive regions of close apposition between cells are seen (Fig. 18). The intervening extracellular space is obliterated a t such points. Interdigitation between ectodermal and endodermal cells makes it difficult to discern the germ-layer origin of some cells within the oral membrane. Dense bodies are present, but never numerous, in some cells e s p e c i a l l y those clearly recognizable as endodermal. Increases in the degree of cranial flexure and continued forward growth of the mandibular processes transform the originally flat oral membrane into a curved, largely bilaminar, epithelial structure lying between the mandibular processes and the deepening Rathke’s pouch by stage 15 (ca. 50-55 hr) (Figs. 19, 21). The apical profiles of the ectoderm and endoderm of the oral membrane appear similar when viewed with the SEM, and many cells of both sides are elongated transversely across the oral membrane (Fig. 23). Stages 16-18 The cells of the oral membrane continue to interdigitate, making it increasingly difficult to identify two distinct epithelial layers in sections (Fig. 22). Small holes appear in the oral membrane at about stage 16 (ca. 51-56 hr). These gradually enlarge during stages 17 (ca. 52-64 hr) and 18 (ca. 3 days) (Figs. 20, 24, 25). The initial perforations often form 449 near the lateral margins of the oral membrane (Fig. 25), but a constant pattern of rupture was not detected. The early perforations appear to be slit-like openings between adjacent cells, which may be preceeded by deep clefts between cells on either surface of the oral membrane. The appearance of the apical surface of both the ectoderm and endoderm becomes increasingly irregular. Regions of obvious cell lysis were not observed within the oral membrane, although some debris of unknown origin was sometimes present. As the gaps in the oral membrane enlarge, the intervening strands become increasingly thin (Figs. 26-28). Points along some strands are eventually formed by a single cell or a small number of cell processes (Fig. 28). The intercellular boundaries between adjacent cells of the strands are distinct and the cell surfaces appear intact when viewed with the SEM. The cells of the oral membrane are tightly organized in solid epithelial cords a t these stages, showing little extracellular space between adjacent cells (Fig. 29). Some cells extend across the entire thickness of the oral membrane (Fig. 30). The cross-sectional areas of cellular profiles seen in thin sections become increasingly heterogeneous. Many very small profiles are seen, and presumably correspond to very thin cellular processes observed with the SEM (Fig. 35). Numerous junctions, including desmosomes and small focal junctions, occur between cells of the oral membrane. Junctions are present not only between adjacent cells of the same germ layer, but between ectodenn and endoderm cells as well. Pools of extracellular material associated with patches of basal lamina beneath the surfaces of adjacent cells are present a t intervals within the oral membrane as the extent of interdigitation between ectodermal and endodermal cells increases (Figs. 29, 32,331. Some smaller aggregations of extracellular material are partially encircled by cell processes and may be undergoing phagocytosis. Some cells of the oral membrane contain dense bodies resembling phagosomes, but these are not numerous. At later stages of perforation, the bases of the remaining strands of oral membrane are primarily conical, while the center of the strands becomes more attenuated (Figs. 31, 34, 36, 37). Small groups of cells are seen for a time along the junction between ectoderm and endoderm and presumably represent remnants of strands which have ruptured and 450 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 7. Cross section of the oral membrane of a stage 11 embryo. The basal surface of the endoderm (top) is more irregular than that of the ectcderm (bottom).The ultrastructure of a portion of the oral membrane indicated by the rectangle is seen in Figure 8. x 312. Fig. 8. A portion of the lateral aspect of the oral membrane indicated by the rectangle in Figure 7 is seen in this transmission electron micrograph of a nearly adjacent section. The basal surfaces of the endodermal cells are not underlain by a continuous basal lamina. The basal lamina beneath the ectoderm exhibits numerous folds or loops which project into the extracellular space and are associated with a flocculent extracellular material. The morphology of the ectcdermal b a d lamina within the oral membrane differs from that of the basal lamina beneath the ectoderm of the head immediately lateral to the oral membrane (*I which does not exhibit numerous loops. x 14,442). Fig. 9. Portion of the basal lamina and associated “interstitial bodies” (arrows) beneath the edoderm lateral to the oral membrane at stage 11. x 16,867. Fig. 10. A portion of the extracellular space and apposed basal surfaces of the ectoderm and endoderm of the oral membrane illustrating the difference in extracellular materials associated with these two epithelia at stage 11. Numerous patches of fibrillar and flocculent extracellular material (*), hut no continuous basal lamina, are commonly located beneath the endodermal cells. The basal lamina of the ectderm is folded and associated with similar flocculent extracellular material. x 13,006. Fig. 11. h p s or folds of edodermal basal lamina within oral membrane are shown at higher magnification. x 39,509. CHICK ORAL MEMBRANE 451 452 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 12. Stereopair of micrographs of a stage 11 embryo. The ectodenn has been removed from the ventral aspect of the head. The basal surfaces of the enddermal cells in the region of the oral membrane (arrow) are irregular. The ventral aortae (*I are present at the lateral margins of the oral membrane. x 249. Fig. 13. Stereopair of micrographs illustrating the basal surface of the ectodem removed from the stage 11 embryo shown in Figure 12. Extracellular material tends to be aligned craniocaudally in the region of the oral membrane, particularly near the ventral midline (arrow). Strands of fibrillar extracellular material are more randomly organized lateral to the oral membrane (toward bottom of micrographs). X 839. Fig. 14. A fracture through the oral membrane of a stage 11 embryo reveals linear arrays of extracellular material (arrow) between the basal surfaces of the ectoderm and endoderm. x 2,177. Fig. 15. One-micron section through the cranial region of a stage 13 embryo. Mesenchymal cells, presumably of cranial neural crest origin, have migrated to the lateral aspect of the ventral aortae (VA) and oral membrane on each side of the head. x 102. CHICK ORAL MEMBRANE 453 454 R.E. WATERMAN A N D G.C. SCHOENWOLF Fig. 16. Portion of the extracellular space within the oral membrane of a stage 13 embryo. Discontinuities are now present in the basal lamina of the ectoderm (*I. Cell processes (arrows) project into the extracellular space near points where the basal lamina is absent. Flocculent and fibrillar material is present between the basal surfaces of the ectoderm and endoderm. The basal lamina beneath the endoderm is discontinuous. X 12,880. Fig. 17. Extracellular fibril exhibiting striation characteristic of collagen from the extracellular space of the oral membrane from a stage 13 embryo. x 67,442. Fig. 18. Portion of the extracellular space within the oral membrane of a stage 14 embryo. Direct cellular contacts between cells of the e d e r m and endoderm are now present across the extracellular space (*). Basal laminae of both edoderm and endoderm are discontinuous. Cells of both edoderm and endodem contain pleomorphic dense bodies characteristic of lysosomes or phagosomes (arrows). x 11,586. CHICK ORAL MEMBRANE 455 456 R.E. WATERMAN AND G.C. SCHOENWOLF by components of the intervening extracellular matrix, particularly at early stages prior to formation of direct intercellular junctions between the apposed epithelia. The fibrillar and flocculent materials observed within the extracellular space of the oral membrane during this study may represent substances responsible for such adhesion. The fact that this matrix does not permit mesenchymal cell migration, and failure of the extracellular space within the oral membrane to expand at a time when an increased extracellular space containing a hyaluronate-rich matrix forms lateral to it during neural crest migration (Pratt et al., 19751, further suggest that the composition of the extracellular matrix of the oral membrane may differ from that lateral to it. The ultrastructural differences of these matrices observed during this study are at least consistent with this suggestion, although, except for obviously striated collagen fibrils, the DISCUSSION composition of this material has not been The histologic and ultrastructural features determined. Folding of the basal lamina subjacent t o the of oral membrane formation in the chick embryo are essentially identical to those previ- ectoderm of the oral membrane, and the cranously described in the hamster (Waterman, iocaudal alignment of extracellular material 1977). Thinning of the oral membrane prior within the oral membrane at early stages (ca. to rupture, in both species, results from in- 9-11], were striking features not previously creased interdigitation between cells of the reported. This coincides with a period of rapid stomodeal ectoderm and foregut endoderm elongation of the foregut (Seidl and Steding, coincident with a decrease in the width of the 19781, and may reflect mechanical alterations intervening extracellular space. This in- caused by positional changes of the endodercreased cellular intermingling produces dis- ma1 cells during this elongation (Bellairs, continuities in the extracellular compartment, 1953; Rosenquist, 1966). Details of the morand the resulting pools of extracellular mate- phogenetic movements resulting in formation rial are apparently reduced in size, at least in of the foregut remain incompletely underpart, through phagocytosis of material by the stood, however. Folding or duplication of basal cells of the oral membrane. As in the hamster, lamina material has also been described in the interdigitation between ectodermal and regions where epithelial cells are undergoing endodermal cells makes it increasingly diffi- a functional transformation or are losing their cult to discern two discrete epithelia, and no integrity as an epithelium (Parakkal, 1969; evidence that one germ layer degenerates Byskov, 1978; Yamada et al., 1978; Bride and Gomot, 19781, and in certain pathologic conprior t o the other was observed. A significant degree of adhesion between ditions in a variety of tissues and organs ectoderm and endoderm in the region of the (Birks et al., 1959; Hay, 1970; Vrako, 1974; oral membrane is often assumed, although McNutt, 1976). The presence of folded basal direct evidence demonstrating this is lacking. lamina within the oral membrane may, thereIndirect evidence, such as the failure of ce- fore, reflect a functional change in the ectophalic mesoderm to migrate into the oral dermal cells during the initial stages of oral membrane, or of cranial neural crest cells to membrane formation which in turn may be penetrate across the ventral midline as they somehow related to the establishment of a migrate around the foregut and enter the close association between ectoderm and endodpharyngeal arches and facial processes (John- erm. A small region of pleated basal lamina beston, 1966; Noden, 1975, 19781, suggests the existence of a strong local adhesion between neath the epiblast associated with increased the epithelial components of the oral mem- amounts of flocculent extracellular material brane. Such adhesion is presumably mediated has been described at the advancing margin retracted laterally (Fig. 37). The irregular contours and blebbing of some cells within these clusters viewed with the SEM may represent cell lysis (Fig. 38). The portions of the epithelial lining of the pharynx and oral cavity derived from endoderm and ectoderm blend imperceptibly between such remnants, and some cells at the bases of the attachments may be incorporated into the wall of the oral cavity. Large cellular masses are occasionally present in the preoral gut (Seessel’s Pouch) and may represent remnants of the oral membrane which were sloughed prior to fixation or were trapped during specimen preparation. Only a few strands of the oral membrane are present in late stage 18 embryos (Fig. 371, and they are largely absent by stage 19 (ca. 3-3% days). The oral membrane is completely absent in stage 20 and older embryos (Fig. 39). CHICK ORAL MEMBRANE of the mesenchyme (“edge cells”)just distal to the terminal sinus of the expanding area vasculosa of stage 12 chick embryos (Bellairs, 1963; Mayer and Packard, 1978). It was suggested (Mayer and Packard, 1978) that the pleated basal lamina and associated extracellular material may participate in the adhesion between the mesoderm of the area vasculosa and the epiblast known to exist in this region (Augustin 1970). The presence of glycosaminoglycans (GAG) in this same region has been demonstrated histochemically (Mayer and Packard, 19781, although the ultrastructural localization of these components is not known. The fine structure of the flocculent material associated with the pleated basal lamina in the area vasculosa is similar to that of extracellular material between the ectoderm and endoderm of the oral membrane a t similar stages in specimens fixed and processed under similar conditions. It also resembles “laminalike” (Cohen and Hay, 1971) material containing glycoprotein (Manasek et al., 1973; Manasek, 1976) associated with the floor of the foregut in the region of the dorsal mesocardium during early stages of cardiogenesis in the chick (Shain et al., 1972; Johnson et al., 1974). The SEM observations reported in the present study suggest that a continuous band of “lamina-like” material may exist beneath the ventral midline of the foregut during stages 9- 11,extending from the cardiac region through the oral membrane. Making an analogy with the characterization of this material in the developing heart, this material may be composed in large part of glycoprotein, which may serve a variety of functions, including cellular adhesion. While the ultrastructure of “lamina-like” material within the oral membrane may resemble that seen elsewhere, however, caution must be used in ascribing specific functions to extracellular material within the oral membrane without confirming data. The interdigitation of ectodermal and endcdermal cells within the oral membrane is preceeded by the appearance of discontinuities in the ectodermal basal lamina. These are first observed about stage 13, and may result from changes in the secretion, or possible enzymatic digestion, of basal lamina material. The basal lamina is largely secreted by its associated epithelium (Kefalides, 1973; Briggaman and Wheeler, 1975). Its function as a scaffold necessary for epithelial tissue differentiation has been stressed by Vracko (1974) and by studies in which experimental removal of the basal lamina results in changes in 457 epithelial morphology and loss of normal behavior (Banerjee et al., 1977). Breakdown or absence of a basal lamina occurs during normal development in regions where cells are migrating from an epithelium (Wakely and England, 1977; Tosney, 1978; Meier, 19781, and in several normal and experimental conditions allowing contact between epithelial cell processes and extracellular matrix and/or direct heterotypic cell contacts (Morgan, 1976; Slavkin and Bringas, 1976; Peck et al., 1977; Hardy et al., 1973, 1978). In all these instances, discontinuities in the basal lamina are correlated with structural and functional changes in the overlying epithelial cells. Breakdown of basal laminae and contact between processes of apposed epithelia also occur during obliteration of the optic fissure in the developing hamster eye (Geeraets, 1976), and may reflect a feature common to other examples of fusion between the basal surfaces of localized regions, such as fusion between pharyngeal pouch endoderm and ectoderm of the corresponding pharyngeal grooves or formation of the cloaca1 membrane a t the caudal end of the embryo. However, while the histologic appearance of these regions resembles that of the oral membrane, the ultrastructure of these latter two interactions has not been reported in any vertebrate species. Subtle changes in the production of basal lamina components may not have been detected by the methods of fixation used in this study. Addition of compounds such as ruthenium red, alcian blue, and tannic acid to fixative solutions has been shown to preserve additional ultrastructurally detectable material associated with the lamina densa of the basal lamina (LuR, 19761, a t least some of which has been characterized as sulfated and non-sulfated GAG by correlated isotope labeling and enzymatic digestion (Martinez-Palomo, 1970; Trelstad et al., 1974; Hay, 1978; Sanders, 1979; Solursh et al., 1979). The observations that certain basal laminae may be removed by exposure to enzymes which presumably digest GAG components (Banerjee et al., 1977; Sanders, 1979; Solursh et al., 1979) suggests that the observed alterations in the basal laminae within the oral membrane may reflect changes in associated GAG, although the presence of GAG in the oral membrane has not yet been examined. Degradation of the basal lamina allowing direct contact between epithelial and mesenchymal cells during tooth formation in the rabbit has been tentatively attributed to collagenase and/or 458 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 19. Midsagittal section of a stage 15 embryo. The oral membrane separating the stomodeum and foregut is thin, but intact. A portion of the oral membrane is enlarged in Figure 21. x 133. Fig. 20. Midsagittal section of a stage 17 embryo. Gaps (*) are now present in the oral membrane. A portion of an oral membrane at a similar stage is shown at higher magnification in Figure 22. x 139. Fig. 21. Portion of the oral membrane of the stage 15 embryo shown in Figure 19. It is difficult to differentiate between the edoderm and endoderm with certainty. x 727. Fig. 22. A portion of the rupturing oral membrane from a stage 17 embryo. The intact region of the oral membrane is thin, and the cells are compact, with little distinct extracellular space evident at the light microscopic level. Gap in membrane (*I. x 727. Fig. 23. (SEM) of the endodermal surface of the oral membrane of a stage 15 embryo. x 1,167. Fig. 24. SEM of a stage 18 embryo fractured through the mandibular pmesses and rupturing oral membrane interposed between Seessel’s pouch at the cranial end of the foregut and the stomodeum and Rathke’s pouch. x 250. Fig. 25. Stereopair of micrographs of fractured oral membrane from a stage 16 embryo showing clefts between adjacent endodermal cells (arrows) of the oral membrane near its lateral margin. Many endodermal cells exhibit a single short cilium near the center of the apical surface. X 1,496. CHICK ORAL MEMBRANE 459 460 Fig. 26.-28 R.E. WATERMAN AND G.C. SCHOENWOLF Progressive stages of rupture of the oral membrane. Fig. 26. Oral membrane of a stage 17-18 embryo viewed fmm the ectdermal surface. The apical profiles of the extodermal cells are heterogeneous. Several small gaps are present. The tips of the mandibular processes are visible toward the right. x 752. Fig. 27. Oral membrane of a stage 17-18 embryo with more numerous perforations than in embryo shown in Figure 26. Deep clefts are present between adjacent cells of intact regions. Many cells are elongated transversely across the oral membrane. Some cells exhibit micmvilli; others have smooth surfaces. X 1,086. Fig. 28. A portion of an oral membrane from a stage 18 embryo. Strands separating gaps are variable in size, some apparently formed by processes of a single cell or a small number of cells. x 1,573. CHICK ORAL MEMBRANE 461 462 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 29. Portion of the oral membrane from a stage 17 embryo. Extensive areas of apposition between ectodermal and endodermal cells are seen. Pools of extracellular materials (*) of variable sizes are present near the center of the oral membrane. Profiles of cells vary greatly in size, with smaller ones often present near the apical surfaces (arrows). x 13,420). Fig. 30. Portion of an intact oral membrane from a stage 17 embryo. The oral membrane is almost entirely cellular at this point, with some cells extending across the entire width of this structure (*I. It is difficult to establish the germ-layer origin of many cells. Distinct intercellular junctions are frequently observed. X 10,581. CHICK ORAL MEMBRANE 463 464 R.E. WATERMAN AND G.C. SCHOENWOLF Fig. 31. A stage 18 embryo sectioned between the first and second pharyngeal arches. The endodermal surface of the rupturing oral membrane is viewed between the mandibular processes (the tips of which were mechanically damaged during the dissection process). Nasal pit (NF').The oral membrane is shown at higher magnification in Figure 34. x 49. Fig. 32. Cells of the oral membrane from a stage 17 embryo near a perforation (*) appear similar to those more distant from the perforation. A small pool of apparent extracellular material is seen (arrow). x 11,727. Fig. 33. Portion of a large accumulation of predominantly fibrillar extracellular material within oral membrane of a stage 17 embryo. x 12,000. Fig. 34. Stereopair of micrographs of the rupturing oral membrane of the stage 18 embryo illustrated in Figure 31. The thin strand toward the left was presumably broken during preparation. x 500. CHICK ORAL MEMBRANE 465 466 R.E. WATERMAN AND G . C . SCHOENWOLF Fig. 35. Stereopair of micrographs illustrating a thin cellular process (arrow) a t the surface of a strand of rupturing oral membrane from a stage 18 embryo. ( X 1,785). Fig. 36. Stereopair of micrographs illustrating strands of oral membrane from a stage 18 embryo. ( x 403) Fig. 37. Stereopair of micrographs illustrating a terminal stage of rupture of the oral membrane in a stage 18 embryo. A single strand is viewed from the foregut surface. Several clumps of cells (arrow) presumably representing lateral attachments of ruptured strands are seen along the lateral wall of the oral opening. ( X 333) Fig. 38. A small group of cells representing the lateral attachment of a strand of oral membrane and showing signs of cellular necrosis is seen in a stage 18 embryo. ( x 1,406). Fig. 39. Stage 20 embryo cut in the midsagittal plane. No evidence of the oral membrane is visible along the lateral wall of the developing oral cavity. ( X 107). CHICK ORAL MEMBRANE 467 468 R.E. WATERMAN A N D G.C. SCHOENWOLF other protease activity (Sorgente et al., 1977), but factors responsible for degradation of basal lamina material in other systems, including the oral membrane, are largely unknown. The changes in shape and arrangement of both the ectodermal and endodermal cells prior to and during rupture of the oral membrane revealed by the SEM in the present study suggest a possible mechanism for the initiation of gaps in this structure. Many cells of the oral membrane which originally exhibit polygonal or rounded apical profiles, become elongated transversely across the oral membrane a s it thins, and long, slender cell extensions are associated with strands of the oral membrane a t advanced stages of its rupture (Figs. 28, 31, 34-36). Portions of these extensions presumably account for some of the small cellular profiles seen in thin sections, although direct correlations between SEM and TEM images of the same specimen were not attempted. It is possible that a t least some of the initial perforations in the oral membrane may arise a s spaces between cells as they interdigitate and reorient within the thinning oral membrane-perhaps in a manner analogous to formation of the foramina secunda in the interatrial septum of the chick heart (Hendrix and Morse, 1977; Morse and Hendrix, 1980).Slit-like perforations appear in the cranial end of the septum primum of the chick heart on day 4 of incubation and enlarge to create gaps separated by thin trabeculae of septa1 tissue. The initial perforations appear to be formed by processes of endocardial cells which extend into the core of the septum to join with similar cell processes from the opposite side, thereby creating slit-like perforations while at the same time maintaining the integrity of the endocardial lining (Morse, 1979). The following paradigm of oral membrane formation and rupture is proposed based on currently available evidence. Interactions between foregut endodenn and surface ectoderm are mediated initially by extracellular matrical components. Circumstantial evidence suggests that a degree of adhesion exists between ectoderm and endoderm which is perhaps mediated by GAG or glycoprotein materials. Except for ultrastructurally detectable striated collagen fibrils, however, the biochemical identity of such materials remains unknown. Direct intercellular contacts and junctions are subsequently established between cells of the endodermal and ectodermal epithelia with concomitant breakup of the initially continuous intervening extracellular compartment into pools of variable size. The protrusion of cellular processes through the basal lamina to allow establishment of direct intercellular contact across the extracellular space presumably reflects a functional change in the epithelial cells which may be related to the synthesis and/or maintenance of the basal lamina or other components of the extracellular matrix. Increased interdigitation of epithelial cells results in thinning of the oral membrane. Continuation of such cellular rearrangements, perhaps combined with dissociation of certain intercellular contacts, may create the small perforations which initially appear in the oral membrane. It is also possible that other mechanisms, such as focal cell death or rupture a t sites near large accumulations of extracellular materials, may play a role in perforation of the oral membrane. The numbers of sections and samples examined make i t unlikely that this latter event occurs with great frequency, and direct continuity between pools of extracellular material and the cavities of either the stomodeum or foregut was not observed in this study nor in the hamster (Waterman, 1977). Since dispersal of such discharged material or the absence of a complete series of serial thin sections may have prevented detection of this situation, however, the possibility that pools of extracellular material may create weak spots in the thinning oral membrane cannot be entirely ruled out. The amount of extracellular material within the oral membrane may be reduced by enzymatic degradation during thinning and rupture of the oral membrane, although the degree of reduction and whether extracellular and/or intracellular degradation occurs has not been determined. Dense intracytoplasmic bodies with ultrastructural features of lysosomes or phagocytic vacules can be seen in cells of the chick oral membrane a t this stage (Betz and Jarskar, 19741, but the presence of lytic enzyme activity associated with these organelles has not been reported. The strands of oral membrane thin progressively as the gaps in the oral membrane enlarge, and eventually consist of a single, or a small number of cell processes. These slender strands may either break or separate as cell junctions are broken, and the ends retract laterally, where they may persist for a short CHICK ORAL MEMBRANE time. Some cells of these clumps may degenerate. Others may be incorporated into the epithelial lining of the oral cavity. ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Judi DeLongo during this study. Supported i n part by a g r a n t (#lF32NS06055-01) from the NIH to Dr. Schoenwolf. Dr. R. Waterman is a recipient of United States Public Health Services Research Career Development Award No. DE 00013. LITERATURE CITED Aasar, Y.H. (1931) The history of the prochordal plate in the rabbit. J . Anat., 66:17-45. Adelmann, H.B. (1922) The significance of the prechordal plate: An interpretive study. Amer. J . Anta., 31:55-91. 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