Distribution of Surface Coat Material on Nasal Folds of Mouse Embryos as Demonstrated by Concanavalin A Binding DOROTHY BURK, T. W. SADLER AND JAN LANGMAN Department of Anatomy, Medical School, University of Virginia, Charlottesuille, Virginia 22901
ABSTRACT 3H-concanavalinA and the concanavalin A-horseradish peroxidase staining technique were used to study the distribution of surface coat material on the epithelium of the nasal folds and nasal groove of mouse embryos. In stages shortly before and during epithelial fusion concanavalan A stained or labeled material was present a t apical surfaces of epithelial cells of the nasal groove and nasal folds. Silver grains, representing bound 3H-concanavalin A, were counted in defined areas of the nasal groove and presumptive fusion area in both anterior and posterior regions of the nasal folds. For both stages examined there was a significant increase in the amount of 3H-concanavalin A bound by presumptive fusion areas in posterior regions of the nasal folds as compared with anterior regions; i.e., the amount of surface coat was greater in the region just prior to the point of contact between the nasal folds. This finding is consistent with results from investigations of palatal shelf and neural fold fusion which suggest that increased synthesis of surface coat material is associated with adhesion and fusion of epithelial folds and shelves. Contact and fusion between two epithelial folds or shelves is an important event during organogenesis of several embryonic structures. Epithelial fusion occurs during formation of the palate, neural tube, lip, heart, penis and eye and appears to involve several specific events, including a build-up of cell surface coat material. An increase in cell surface coat material has been observed on the fusing epithelial surfaces of amphibian, chick and mouse neural folds (Moran and Rice, '75; Lee et al., '76; Silver, '78; Sadler, '78) and mouse and rat palatal shelves (Green and Kochhar, '74; Souchon, '75; Pratt and Hassell, '75) prior to their initial contact. This finding has led to the suggestion that surface coat material may be an essential factor in the fusion process by providing an initial adhesiveness between contacting epithelial surfaces until more stable cell to cell attachments can be established (Greene and Pratt, '76). Support for this hypothesis was provided by an investigation in which administration of diazo-0x0-norleucine (DON), an agent which interferes with coat synthesis, prevented adhesion between palatal shelves in vitro (Greene and Pratt, '77). ANAT. REC. (1979)193: 185-196.
Formation of the lip and primary palate of mouse and rat embryos involves the fusion of the medial and lateral nasal folds which are formed as the nasal (olfactory) placode invaginates (Trader, '68; Lejour, '70; Pourtois, '72; Gaare and Langman, '77). In the mouse the process begins on the eleventh day of gestation with placode invagination and continues to the twelfth day when contact and fusion of the nasal folds occurs. Initial contact occurs between posterior ends of the nasal folds and fusion proceeds .in an anterior direction. The epithelial seam (nasal fin) which exists as the folds adhere subsequently regresses and is replaced by mesenchyme (except a t its most posterior aspect where the fin remains as the bucconasal membrane). Since nasal fold fusion is similar to fusion processes in other developing regions, the question is raised as to whether or not a cell surface coat is also important in adhesion between nasal folds. Distribution of cell surface coat material associated with surfaces of the nasal folds a t the Received May 26, '78. Accepted Sept. 9, '78. ' Supported by NIH Grant 5732 DE07037-02 for craniofacial developmental
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DOROTHY BURK, T. W. SADLER AND J A N LANGMAN
time of fusion has not been clearly defined. Smuts ('771, in an investigation of early nasal placode stages in the mouse, reported that a surface coat, as demonstrated by 3H-concanavalin A binding, was first detectable over placode primordia and in later stage embryos was present over facial epithelium (surface ectoderm) as well. Furthermore, there was some evidence that epithelial cells in the "shoulder" (presumptive fusion) region of the invaginating placode bound more 3H-concanavalin A than did placode cells. However, embryos in which fusion of the nasal folds had begun were not included in the study. Gaare and Langman ('77) used ruthenium red to demonstrate the cell surface coat over the epithelium of fusing mouse nasal folds but were unable to show conclusively that an increased amount of stained material was present in the presumptive fusion region. While ruthenium red is a general surface coat stain, the lectin concanavalin A binds only to glucose and mannose residues of the glycoproteins and glycolipids which constitute the cell surface coat (Sharon and Lis, '72; Luft, '76). Bound concanavalin A may be detected cytochemically by coupling with horseradish peroxidase (Bernhard and Avrameas, '7 1) or autoradiographically using 3Hconcanavalin A. Both of these techniques have been employed previously to study surface coat distribution (Pratt and Hassell, '75; Greene and Pratt, '76; Smuts, '77). In this investigation we have used labeled and unlabeled concanavalin A in an attempt to analyze the distribution of cell surface coat material associated with epithelium of mouse nasal folds in stages shortly before and during fusion. We have also attempted to correlate quantitative data with electron microscopic observations.
A demonstration described below are based on procedures used by Pratt and Hassell ('751, Greene and Pratt ('761, and Smuts ('77). All procedures were carried out a t room temperature unless otherwise indicated.
Electron microscopy of concanavalin A-incubated heads Following fixation, embryonic heads were rinsed extensively in 0.1 M cacodylate buffer, pH 6.9, and then incubated in a solution of 100 pg/ml concanavalin A (CON A) (Sigma, Grade IV) in cacodylate buffer for 30 minutes. After a buffer rinse, heads were placed in 50 pglml horseradish peroxidase (HRP) (Sigma, Type VI) in cacodylate buffer for 30 minutes, then rinsed and fixed again for 15 to 30 minutes. After further rinsing, heads were treated with 0.5 mg/ml diaminobenzidine (DAB) (Sigma) in 0.5 M Tris, pH 7.6, containing 0.01% H,O, for 20 minutes. Heads were then postfixed in 1%OsO, in 0.1 M cacodylate buffer, pH 7.3 for one hour a t 4"C, dehydrated, and embedded in araldite. Control heads were subjected to a similar procedure except that the CON A solution and buffer rinses contained 0.1 M 1-0Methyl-a:-D-Glucopyranoside (aMG), which competes with HRP and surface coat sugars for reactive groups of CON A (Bernhard and Avrameas, '71). Thin sections were made with glass knives on an LKB-Huxley ultramicrotome, stained with uranyl acetate and lead citrate, and examined with an RCA EMU-3H electron microscope.
Autoradiography of 3H-concanavalin A-incubated heads Following fixation and extensive buffer rinsing (0.1 M sodium cacodylate, pH 6.9) mouse embryo heads were placed in plastic vials containing 5 pCi of 3H-CONA (New England Nuclear) in 0.3 ml cacodylate buffer for MATERIALS AND METHODS 30 minutes. Heads were again rinsed thorICR/DUB mice (Flow Laboratories, Dublin, oughly in buffer and were subsequently postVirginia) were mated from 9:OO A.M. to 1:OO fixed in a solution of 1.25% glutaraldehyde and 0.5% OsO, in 0.1 M cacodylate buffer, pH P.M. and pregnancy was indicated by the presence of a vaginal plug (plug day = day 1). 7.3, for two hours a t 4°C. Control heads were Pregnant mice were sacrificed on the eleventh incubated in a "-CON A solution which also and twelfth days of gestation. Embryos were contained 0.1 M aMG (added 15 minutes prior dissected from the uterus in saline and their to addition of the tissue) and were rinsed heads were quickly removed, staged (accord- withbuffer containing 0.1 M aMG. Serial, ing to Trader, '68) and fixed for 30 minutes in 1 p m sections of araldite-embedded heads fixative containing 2% paraformaldehyde, 2% were mounted on glass slides, dipped in Kodak glutaraldehyde and 0.01% CaC12 in a 0.1 M NTB-2 emulsion, and stored a t 4°C for two sodium cacodylate buffer, pH 7.3 (modified weeks, after which time they were developed Karnovsky's). The methods for concanavalin and stained with 0.25% Azure 11. In total, 21
SURFACE COAT DISTRIBUTION ON NASAL FOLDS
embryos were processed for autoradiography, including 16 experimental and 5 control specimens. Stained sections were examined under oil (1,000 x 1 and counts were made of all silver grains within a 50 pm distance along the cell surface in four regions of the nasal area: two from the nasal groove (non-fusing surface) and two, one medial and one lateral, from the presumptive fusion areas (fusing surface) (figs. 1-41. Only silver grains a t the apical surfaces of cells were counted, i.e., grains located over cell cytoplasm or between cells were not included. For purposes of analysis, results of counts were pooled into two groups - fusing and non-fusing surfaces. In addition, since serial sections had been made through the nasal area, groups of sections from anterior and posterior nasal groove regions were also analyzed. In the anterior region of the nasal groove, nasal folds fuse late, if a t all; whereas, in the posterior region the folds are close to the point of initial contact. Analysis of variance and t tests were used to study the interaction between fusing and non-fusing surfaces and anterior versus posterior regions. RESULTS
The nasal folds of embryo obtained a t the eleventh day of development (32-35 somites) were in the deep oval or oblong configuration, as defined by Trader ('68). The nasal areas of these embryos were characterized by the presence of a nasal groove flanked by nasal folds which had not yet begun to fuse (figs. 1 , 2 ) . In contrast, nasal folds of embryos obtained from mice sacrificed on the twelfth gestational day were in the comma stage (Trader, '68) (figs. 3, 4). In this stage posterior ends of the nasal folds had contacted and fused and fusion was proceeding in an anterior direction. At both stages of development the nasal groove was lined by pseudostratified columnar epithelium with mitotic figures located a t the luminal ends of cells. Surface ectoderm in areas adjacent to the groove consisted of a basal layer of cuboidal cells covered with a squamous periderm layer. Epithelium in the transition region between the nasal groove and surface ectoderm was two to three cell layers thick. This transition region epithelium was located in areas which would a t a later stage participate in contact and fusion between nasal folds. Ultrastructurally, pseudostratified columnar epithelial cells of the nasal groove were
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long and narrow with terminal bars located near their apical ends. Numerous microvilli and some cilia were present a t the apical surfaces of these cells (fig. 5). In contrast, epithelium in presumptive fusion (transition) areas was composed of larger, rounded cells with relatively smooth surfaces (fig. 6). Microvilli were generally confined to areas near intercellular junctions. Both nasal groove and surface epithelial cells contained components characteristic of embryonic cells, i.e., mitochondria, numerous polysomes, occasional profiles of rough endoplasmic reticulum and Golgi complexes. Embryos incubated with CON A-HRP exhibited an electron dense precipitate, representing cell surface coat material, a t apical surfaces, including microvilli, of nasal groove epithelial cells (fig. 5) as well as a t the surfaces of epithelial cells in presumptive fusion regions (fig. 6). In autoradiographs of both eleventh and twelfth day embryos silver grains, indicating bound 3H-CON A, were located a t apical surfaces of nasal groove and prefusion epithelial cells (figs. 8, 10,111. Grains were also observed between surface ectoderm cells and epithelial cells in the presumptive fusion (transition) region of eleventh day embryos (fig. 8 ) . As nasal folds came into apposition in twelfth day embryos, silver grains were present between apposing epithelial surfaces, but no grains were observed a few sections posterior to contact. It was for this reason that counting of grains ended a t the point of initial union of the nasal folds. In both electron microscopic and autoradiographic investigations, binding of CON A was inhibited by preincubation with =MG, such that sections from these tissues showed no CON A-HRP staining or silver grains above background level (figs. 7, 9). The results of counts of apical silver grains from surfaces and regions of the nasal folds and groove analyzed are depicted in figure 12. With regard to eleventh day embryos, analysis of variance revealed that a significant difference existed in the amount of 3H-CON A bound by epithelial surfaces in the anterior as compared with the posterior region of the nasal folds (F = 10.02, df = 1/4, p < 0.05). Counts from both fusing and non-fusing surfaces in the posterior region were significantly higher than respective counts from the anterior nasal fold region (p < 0.05). Although counts from non-fusing surfaces were generally higher than from fusing surfaces, the difference was not statistically significant. In
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twelfth day embryos there was a significant difference (F = 9.09, df = 1/10, p < 0.02) in amount of “H-CON A bound by fusing and non-fusing surfaces (taken a s a whole), with higher counts recorded from t h e non-fusing epithelium. There was also a significant interaction ( F = 73.02, df = 1/10, p < 0.001) between surface and region of t h e nasal folds in that the difference between fusing and nonfusing surfaces was present only in t h e anterior region. Furthermore, fusing surfaces alone exhibited a significant (p < 0.01) increase in the amount of bound 3H-CONA between anterior and posterior regions of t h e nasal folds, i.e., the amount of bound 3H-CON A was greater in presumptive fusion areas located just prior to nasal fold contact. In contrast, t h e mean number of counts from t h e non-fusing nasal groove epithelium decreased from t h e anterior to t h e posterior region (p < 0.05). DISCUSSION
Localization of CON A-HRP stained material in electron micrographs and of silver grains, representing bound 3H-CON A, in autoradiographs confirms t h e fact t h a t surface coat carbohydrates are present at apical surfaces of epithelial cells of the nasal groove and nasal folds of mouse embryos in stages shortly before and during nasal fold fusion. While CON A binding was restricted to apical surfaces of nasal groove epithelial cells and presumptive fusion areas of twelfth day embryos, both cell surfaces and intercellular spaces of surface ectoderm in eleventh day embryos were often labeled with CON A. Electron microscopic observation provides support for the suggestion t h a t penetration of CON A between these cells may be due to differences in cell attachments (Smuts, ’77). Surface ectoderm cells tend to be loosely joined by desmosomes, while tight junctions are present between apical ends of pseudostratified columnar epithelial cells in t h e nasal groove. The higher silver grain counts recorded from non-fusing versus fusing surfaces of both eleventh and twelfth day embryos suggests a n increased binding of CON A by nasal groove epithelium. However, this increase may be explained on t h e basis of differences in surface morphology of epithelial cells in t h e two locations. For example, cells in t h e non-fusing nasal groove epithelium have numerous microvilli; whereas, cells in presumptive fusion areas have smoother surfaces with few projec-
tions. Therefore, for any given distance, the actual surface area of cells in t h e nasal groove is greater than for cells in t h e presumptive fusion region. Variation in surface morphology may also be responsible for differences in amount of CON A bound by anterior and posterior non-fusing regions of eleventh and twelfth day stages. However, other possibilities, such as alterations in synthesis or distribution of surface coat material, may also play a role. The observation of higher counts for nonfusing nasal groove epithelium than for epithelium from presumptive fusion regions conflicts with findings reported by Smuts (’77). Although her counting procedure was not clearly defined, Smuts found one and one-half times more silver grains (bound 3H-CON A) over cells in t h e presumptive fusion region than over epithelial cells in the nasal placode at t h e stage of late placode invagination. It is difficult to explain this difference from our study, unless Smuts has employed different counting criteria from our own. For example, at the stage employed by Smuts (our eleventh day), silver grains are found not only along cell surfaces but between cells in the presumptive fusion (“shoulder”) region. Therefore, when analyzing surface coat distribution, only grains located at t h e apical ends of cells should be counted, since inclusion of grains located over cell cytoplasm or between cells would not represent binding to surface glycoproteins. I t is also possible t h a t t h e microvilli which characterize the nasal groove epithelium of more advanced stages are not as prevalent a t earlier stages of placode differentiation with a consequent effect on counting results. Statistical analysis of counts from presumptive fusion surfaces of eleventh and twelfth day embryos revealed a significant increase in t h e amount of bound 3H-CONA between anterior and posterior regions, i.e., mean counts were greater for t h e group of sections located closer to the point of initial contact (fusion) of t h e nasal folds. Assuming t h a t the amount of bound 3H-CON A reflects t h e actual number of CON A binding sites (Collard and Temmink, ’74) and, therefore, t h e relative amount of surface coat carbohydrates present, this finding suggests t h a t there is a n increased amount of surface coat material associated with prefusion epithelium shortly before nasal fold contact. An increase in surface coat material is
SURFACE COAT DISTRIBUTION ON NASAL FOLDS
consistent with findings in the secondary palate (Greene and Kochhar, '74; Souchon, '74; Pratt and Hassell, '75) and neural tube (Moran and Rice, '75; Lee et al., '76; Silver, '78; Sadler, '78) in which an increase in surface coat material prior to fusion has also been reported. The hypothesis that the cell surface coat is associated with the ability of epithelial shelves or folds to adhere and fuse (Pourtois, '72; Greene and Pratt, '76) is reinforced by these findings and further supported by the fact that complex carbohydrates have for some time been implicated in cell aggregation and intercellular adhesion in various in vitro cell systems (Pessac and Defendi, '72; Oppenheimer, '73; Roseman, '74; Greig and Jones, '77). Since CON A binds to only two of the sugar components of the glycoproteins and glycolipids which make up the surface coat (Sharon and Lis, '72; Luft, '761, it is conceivable that in addition to changes in coat quantity there may also be qualitative changes in surface carbohydrates associated with adhesion, which might not be detected using CON A or ruthenium red techniques. Further testing, including manipulation of the cell surface coat, will be necessary in order to determine the importance of synthesis and integrity of cell surface coat material in the fusion of the nasal folds and consequent importance of the surface coat in terms of production of cleft lip and other congenital malformations. LITERATURE CITED Bernhard, W., and S. Avrameas 1971 Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp. Cell Res., 64: 232-263. Collard, J. G . , and J. H. M. Temmink 1974 Binding and cytochemical detection of cell bound concanavalin A. Exp. Cell Res., 86: 81-86. Cook, G. M. W., and R. W. Stoddart 1973 Surface Carbohydrates of t h e Eukaryotic Cell, New York, Academic Press, p. 79. Gaare, J. D., and J. Langman 1977 Fusion of nasal swellings in the mouse embryo: Surface coat and initial contact. Am. J. Anat., 150: 461-476.
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Greene, R. M., and D. M. Kochhar 1974 Surface coat on the epithelium of developing palatine shelves in the mouse as revealed by electron microscopy. J. Embryol. exp. Morph.. 31: 683-692. Greene, R. M., and R. M. P r a t t 1976 Developmental aspects of secondary palate formation. J. Embryol. exp. Morph., 36: 225-245. 1977 Inhibition by diazo-0x0-norleucine (DON) of rat palatal glycoprotein synthesis and epithelial cell adhesion in uitro. Exp. Cell Res., 105: 27-37. Greig, R. G., and M. N. Jones 1977 Mechanisms of intercellular adhesion. BioSystems, 9: 43-55. Lee, H. Y., J. B. Sheffield, R. G. Nagele and G. W. Kalmus 1976 The role of extracellular material in chick neurulation. I. Effects of concanavalin A. J. Exp. Morph., 198: 261-266. Lejour, M. 1970 Cleft lip induced in the rat. Cleft Palate J., 7: 169.186. Luft, J. H. 1976 The structure and properties of the cell surface coat. Int. Rev. Cytol., 45: 291-382. Moran, D., and R. W. Rice 1975 An ultrastructural examination of the role of cell membrane surface coat material during neurulation. J. Cell Biol., 64: 172-181. Oppenheimer, S. B. 1973 Utilization of L-glutamine in intercellular adhesion: Ascites tumor and embryonic cells. Exp. Cell Res., 77: 175-182. Pessac, B., and V. Defendi 1972 Cell aggregation: Role of acid mucopolysaccharides. Science, 175: 898.900. Pourtois, M. 1972 Morphogenesis of the primary and secondary palate. In: Developmental Aspects of Oral Biology. H. S. Slavkin and L. S. Bovetta, eds. Academic Press, New York, pp. 81-108. Pratt, R. M., and J . R. Hassell 1975 Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf. Dev. Biol., 45: 192.198. Roseman, S. 1974 Complex carbohydrates and intercellular adhesion. In: Biology and Chemistry of Eukaryotic Cell Surfaces. E. Y. C. Lee and E. E. Smith, eds. Academic Press, New York, pp. 317-354. Sadler, T. W. 1978 Distribution of surface coat material on fusing neural folds of mouse embryos during neurulation. Anat. Rec., 191: 345-350. Sharon, N., and H. Lis 1972 Lectins: Cell-agglutinating and sugar-specific proteins. Science, I 77: 949-959. Silver, M. H. 1978 Ultrastructure of neural fold fusion in chick embryos. Anat. Rec., 190: 541-542 (Abstract). Smuts, M. S. 1977 Concanavalin A binding to the epithelial surface of the mouse olfactory placode. Anat. Rec., 188: 29-38. Souchon, R. 1975 Surface coat of the palatal shelf epithelium during palatogenesis in mouse embryos. Anat. Embryol., 147: 133.142. Trader, D. G. 1968 Pathogenesis of cleft lip and its relation to embrvonic face shape in A/J and C57BL mice. Teratology, 1: 33-50.
w 0
4 Coronal l - F m section through the nasal folds and groove a t approximately the level of the ' in figure 4. Counts of silver grains, representing bound 3H-concanavalin A, were made in areas indicated by brackets (presumptive fusing surfaces) and between t h e arrows in the nasal groove (non-fusing surfaces). M, medial nasal fold; L, lateral nasal fold. X 10.
3 View of the roof of the primitive oral cavity of a twelfth day embryo (comma stage). The medial (M) and lateral (L) nasal folds have begun to fuse a t their posterior ends. Fusion proceeds in an anterior direction toward the top of the photograph.
2 Coronal l-Fm section through the nasal folds and groove a t approximately t h e level of the ' in figure 2. Counts of silver grains, representing bound 3HH-concanavalin A, were made in areas indicated by brackets (presumptive fusing surfaces) and between the arrows in the nasal groove (non-fusingsurfaces). M, medial nasal fold; L, lateral nasal fold. X 10.
1 View of the roof of the primitive oral cavity in an eleventh day embryo (oblong stage). The medial (M) and Lateral (L) nasal folds, which flank the nasal groove, have not yet contacted. R, Rathke's pocket. The arrow on the inset indicates the nasal groove of an eleventh day embryo viewed from t h e side.
EXPLANATION OF FIGURES
PLATE I
191
PLATE 2 E X P L A N A T I O N OF FIGLIKES
192
5
Electron micrograph of surfaces of pseudostratified columnar epithelial cells in t h e nasal groove of a n eleventh day embryo. Note numerous microvilli. Following incubation with CON A-HRP a n electron dense precipitate. representing cell surface coat material Iscm). I S present a t apical surfaces of cells. X 8.930.
6
Electron micrograph of surfaces of stratified squamous epithelial cells in t h e presumptive fusion r e o o n of a twelfth day embryo Note t h a t t h e surface is relatively smooth (free of projections) a s compared with t h e epithelium in figure 5 . scm. surface coat material. x 8,930.
7
Electron micrograph of surfaces of pseudostratified columnar epithelial cells in t h e nasal groove of a twelfth day embryo incubated with CON A plus a MG. S t a m i n g of surface coat material is elimited. c. cilium. X 8,930.
SURFACE COAT DISTRIBUTION ON NASAL FOLDS Dorothy Burk, T. W. Sadler and Jan Langman
PLATE 2
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PLATE 3 EXPLANATIOK OF F I G U R E S
8
Autoradiograph of a coronal 1 - p m section through t h e lateral nasal fold a n d nasal groove of a n eleventh day embryo. Silver grains are restricted to apical surfaces of epithelial cells in t h e nasal groove; whereas, grains a r e located over t h e between epithelial cells in t h e presumptive fusion (transition, region. X 40.
9
Autoradiograph of a coronal 1 - p m section through t h e lateral nasal fold and nasal groove of a n eleventh day control ( = M G ) embryo a t a level similar t o t h a t shown in figure 8. X 40.
10 Autoradiograph of a coronal 1 - p m section through t h e nasal groove of a twelfth day embryo. Silver grains a r e present only a t apical surfaces of pseudostratified columnar epithelial cells. X 40. 11 Autoradiograph of a coronal 1 - p m section through t h e presumptive fusior region of t h e nasal folds of a twelfth day embryo. The folds a r e seen prior t o contact. Silver grains a r e present a t t h e apical surfaces of epithelial cells which will make contact. X 40.
SURFACE COAT DISTRIBUTION ON NASAL FOLDS Dorothy Burk. T W Sadler and J a n Langman
PLATE 3
195
E
Q)
0
C
V
0
3
c C v)
r
20
posterior
v)
20-
.
300
EXPLANATION OF FIGURES
E
Q,
0
C
U
3 0
c C
401
anterior
J
posterior
12th DAY
1 2 Comparison of mean number of silver grains (per 50 p m length) on aplcal surfaces of fusing and non-fusing nasal epithelium. Means represent pooled counts from groups of sections in anterior and posterior regions of t h e nasal folds in two stages of development.
anterior
30L1
401
fusing
non-fusing
11th DAY
PLATE 4
ABSTRACT 3H-concanavalinA and the concanavalin A-horseradish peroxidase staining technique were used to study the distribution of surface coat material on the epithelium of the nasal folds and nasal groove of mouse embryos. In stages shortly before and during epithelial fusion concanavalan A stained or labeled material was present a t apical surfaces of epithelial cells of the nasal groove and nasal folds. Silver grains, representing bound 3H-concanavalin A, were counted in defined areas of the nasal groove and presumptive fusion area in both anterior and posterior regions of the nasal folds. For both stages examined there was a significant increase in the amount of 3H-concanavalin A bound by presumptive fusion areas in posterior regions of the nasal folds as compared with anterior regions; i.e., the amount of surface coat was greater in the region just prior to the point of contact between the nasal folds. This finding is consistent with results from investigations of palatal shelf and neural fold fusion which suggest that increased synthesis of surface coat material is associated with adhesion and fusion of epithelial folds and shelves. Contact and fusion between two epithelial folds or shelves is an important event during organogenesis of several embryonic structures. Epithelial fusion occurs during formation of the palate, neural tube, lip, heart, penis and eye and appears to involve several specific events, including a build-up of cell surface coat material. An increase in cell surface coat material has been observed on the fusing epithelial surfaces of amphibian, chick and mouse neural folds (Moran and Rice, '75; Lee et al., '76; Silver, '78; Sadler, '78) and mouse and rat palatal shelves (Green and Kochhar, '74; Souchon, '75; Pratt and Hassell, '75) prior to their initial contact. This finding has led to the suggestion that surface coat material may be an essential factor in the fusion process by providing an initial adhesiveness between contacting epithelial surfaces until more stable cell to cell attachments can be established (Greene and Pratt, '76). Support for this hypothesis was provided by an investigation in which administration of diazo-0x0-norleucine (DON), an agent which interferes with coat synthesis, prevented adhesion between palatal shelves in vitro (Greene and Pratt, '77). ANAT. REC. (1979)193: 185-196.
Formation of the lip and primary palate of mouse and rat embryos involves the fusion of the medial and lateral nasal folds which are formed as the nasal (olfactory) placode invaginates (Trader, '68; Lejour, '70; Pourtois, '72; Gaare and Langman, '77). In the mouse the process begins on the eleventh day of gestation with placode invagination and continues to the twelfth day when contact and fusion of the nasal folds occurs. Initial contact occurs between posterior ends of the nasal folds and fusion proceeds .in an anterior direction. The epithelial seam (nasal fin) which exists as the folds adhere subsequently regresses and is replaced by mesenchyme (except a t its most posterior aspect where the fin remains as the bucconasal membrane). Since nasal fold fusion is similar to fusion processes in other developing regions, the question is raised as to whether or not a cell surface coat is also important in adhesion between nasal folds. Distribution of cell surface coat material associated with surfaces of the nasal folds a t the Received May 26, '78. Accepted Sept. 9, '78. ' Supported by NIH Grant 5732 DE07037-02 for craniofacial developmental
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DOROTHY BURK, T. W. SADLER AND J A N LANGMAN
time of fusion has not been clearly defined. Smuts ('771, in an investigation of early nasal placode stages in the mouse, reported that a surface coat, as demonstrated by 3H-concanavalin A binding, was first detectable over placode primordia and in later stage embryos was present over facial epithelium (surface ectoderm) as well. Furthermore, there was some evidence that epithelial cells in the "shoulder" (presumptive fusion) region of the invaginating placode bound more 3H-concanavalin A than did placode cells. However, embryos in which fusion of the nasal folds had begun were not included in the study. Gaare and Langman ('77) used ruthenium red to demonstrate the cell surface coat over the epithelium of fusing mouse nasal folds but were unable to show conclusively that an increased amount of stained material was present in the presumptive fusion region. While ruthenium red is a general surface coat stain, the lectin concanavalin A binds only to glucose and mannose residues of the glycoproteins and glycolipids which constitute the cell surface coat (Sharon and Lis, '72; Luft, '76). Bound concanavalin A may be detected cytochemically by coupling with horseradish peroxidase (Bernhard and Avrameas, '7 1) or autoradiographically using 3Hconcanavalin A. Both of these techniques have been employed previously to study surface coat distribution (Pratt and Hassell, '75; Greene and Pratt, '76; Smuts, '77). In this investigation we have used labeled and unlabeled concanavalin A in an attempt to analyze the distribution of cell surface coat material associated with epithelium of mouse nasal folds in stages shortly before and during fusion. We have also attempted to correlate quantitative data with electron microscopic observations.
A demonstration described below are based on procedures used by Pratt and Hassell ('751, Greene and Pratt ('761, and Smuts ('77). All procedures were carried out a t room temperature unless otherwise indicated.
Electron microscopy of concanavalin A-incubated heads Following fixation, embryonic heads were rinsed extensively in 0.1 M cacodylate buffer, pH 6.9, and then incubated in a solution of 100 pg/ml concanavalin A (CON A) (Sigma, Grade IV) in cacodylate buffer for 30 minutes. After a buffer rinse, heads were placed in 50 pglml horseradish peroxidase (HRP) (Sigma, Type VI) in cacodylate buffer for 30 minutes, then rinsed and fixed again for 15 to 30 minutes. After further rinsing, heads were treated with 0.5 mg/ml diaminobenzidine (DAB) (Sigma) in 0.5 M Tris, pH 7.6, containing 0.01% H,O, for 20 minutes. Heads were then postfixed in 1%OsO, in 0.1 M cacodylate buffer, pH 7.3 for one hour a t 4"C, dehydrated, and embedded in araldite. Control heads were subjected to a similar procedure except that the CON A solution and buffer rinses contained 0.1 M 1-0Methyl-a:-D-Glucopyranoside (aMG), which competes with HRP and surface coat sugars for reactive groups of CON A (Bernhard and Avrameas, '71). Thin sections were made with glass knives on an LKB-Huxley ultramicrotome, stained with uranyl acetate and lead citrate, and examined with an RCA EMU-3H electron microscope.
Autoradiography of 3H-concanavalin A-incubated heads Following fixation and extensive buffer rinsing (0.1 M sodium cacodylate, pH 6.9) mouse embryo heads were placed in plastic vials containing 5 pCi of 3H-CONA (New England Nuclear) in 0.3 ml cacodylate buffer for MATERIALS AND METHODS 30 minutes. Heads were again rinsed thorICR/DUB mice (Flow Laboratories, Dublin, oughly in buffer and were subsequently postVirginia) were mated from 9:OO A.M. to 1:OO fixed in a solution of 1.25% glutaraldehyde and 0.5% OsO, in 0.1 M cacodylate buffer, pH P.M. and pregnancy was indicated by the presence of a vaginal plug (plug day = day 1). 7.3, for two hours a t 4°C. Control heads were Pregnant mice were sacrificed on the eleventh incubated in a "-CON A solution which also and twelfth days of gestation. Embryos were contained 0.1 M aMG (added 15 minutes prior dissected from the uterus in saline and their to addition of the tissue) and were rinsed heads were quickly removed, staged (accord- withbuffer containing 0.1 M aMG. Serial, ing to Trader, '68) and fixed for 30 minutes in 1 p m sections of araldite-embedded heads fixative containing 2% paraformaldehyde, 2% were mounted on glass slides, dipped in Kodak glutaraldehyde and 0.01% CaC12 in a 0.1 M NTB-2 emulsion, and stored a t 4°C for two sodium cacodylate buffer, pH 7.3 (modified weeks, after which time they were developed Karnovsky's). The methods for concanavalin and stained with 0.25% Azure 11. In total, 21
SURFACE COAT DISTRIBUTION ON NASAL FOLDS
embryos were processed for autoradiography, including 16 experimental and 5 control specimens. Stained sections were examined under oil (1,000 x 1 and counts were made of all silver grains within a 50 pm distance along the cell surface in four regions of the nasal area: two from the nasal groove (non-fusing surface) and two, one medial and one lateral, from the presumptive fusion areas (fusing surface) (figs. 1-41. Only silver grains a t the apical surfaces of cells were counted, i.e., grains located over cell cytoplasm or between cells were not included. For purposes of analysis, results of counts were pooled into two groups - fusing and non-fusing surfaces. In addition, since serial sections had been made through the nasal area, groups of sections from anterior and posterior nasal groove regions were also analyzed. In the anterior region of the nasal groove, nasal folds fuse late, if a t all; whereas, in the posterior region the folds are close to the point of initial contact. Analysis of variance and t tests were used to study the interaction between fusing and non-fusing surfaces and anterior versus posterior regions. RESULTS
The nasal folds of embryo obtained a t the eleventh day of development (32-35 somites) were in the deep oval or oblong configuration, as defined by Trader ('68). The nasal areas of these embryos were characterized by the presence of a nasal groove flanked by nasal folds which had not yet begun to fuse (figs. 1 , 2 ) . In contrast, nasal folds of embryos obtained from mice sacrificed on the twelfth gestational day were in the comma stage (Trader, '68) (figs. 3, 4). In this stage posterior ends of the nasal folds had contacted and fused and fusion was proceeding in an anterior direction. At both stages of development the nasal groove was lined by pseudostratified columnar epithelium with mitotic figures located a t the luminal ends of cells. Surface ectoderm in areas adjacent to the groove consisted of a basal layer of cuboidal cells covered with a squamous periderm layer. Epithelium in the transition region between the nasal groove and surface ectoderm was two to three cell layers thick. This transition region epithelium was located in areas which would a t a later stage participate in contact and fusion between nasal folds. Ultrastructurally, pseudostratified columnar epithelial cells of the nasal groove were
187
long and narrow with terminal bars located near their apical ends. Numerous microvilli and some cilia were present a t the apical surfaces of these cells (fig. 5). In contrast, epithelium in presumptive fusion (transition) areas was composed of larger, rounded cells with relatively smooth surfaces (fig. 6). Microvilli were generally confined to areas near intercellular junctions. Both nasal groove and surface epithelial cells contained components characteristic of embryonic cells, i.e., mitochondria, numerous polysomes, occasional profiles of rough endoplasmic reticulum and Golgi complexes. Embryos incubated with CON A-HRP exhibited an electron dense precipitate, representing cell surface coat material, a t apical surfaces, including microvilli, of nasal groove epithelial cells (fig. 5) as well as a t the surfaces of epithelial cells in presumptive fusion regions (fig. 6). In autoradiographs of both eleventh and twelfth day embryos silver grains, indicating bound 3H-CON A, were located a t apical surfaces of nasal groove and prefusion epithelial cells (figs. 8, 10,111. Grains were also observed between surface ectoderm cells and epithelial cells in the presumptive fusion (transition) region of eleventh day embryos (fig. 8 ) . As nasal folds came into apposition in twelfth day embryos, silver grains were present between apposing epithelial surfaces, but no grains were observed a few sections posterior to contact. It was for this reason that counting of grains ended a t the point of initial union of the nasal folds. In both electron microscopic and autoradiographic investigations, binding of CON A was inhibited by preincubation with =MG, such that sections from these tissues showed no CON A-HRP staining or silver grains above background level (figs. 7, 9). The results of counts of apical silver grains from surfaces and regions of the nasal folds and groove analyzed are depicted in figure 12. With regard to eleventh day embryos, analysis of variance revealed that a significant difference existed in the amount of 3H-CON A bound by epithelial surfaces in the anterior as compared with the posterior region of the nasal folds (F = 10.02, df = 1/4, p < 0.05). Counts from both fusing and non-fusing surfaces in the posterior region were significantly higher than respective counts from the anterior nasal fold region (p < 0.05). Although counts from non-fusing surfaces were generally higher than from fusing surfaces, the difference was not statistically significant. In
188
DOROTHY BURK, T. W. SADLER AND JAN LANGMAN
twelfth day embryos there was a significant difference (F = 9.09, df = 1/10, p < 0.02) in amount of “H-CON A bound by fusing and non-fusing surfaces (taken a s a whole), with higher counts recorded from t h e non-fusing epithelium. There was also a significant interaction ( F = 73.02, df = 1/10, p < 0.001) between surface and region of t h e nasal folds in that the difference between fusing and nonfusing surfaces was present only in t h e anterior region. Furthermore, fusing surfaces alone exhibited a significant (p < 0.01) increase in the amount of bound 3H-CONA between anterior and posterior regions of t h e nasal folds, i.e., the amount of bound 3H-CON A was greater in presumptive fusion areas located just prior to nasal fold contact. In contrast, t h e mean number of counts from t h e non-fusing nasal groove epithelium decreased from t h e anterior to t h e posterior region (p < 0.05). DISCUSSION
Localization of CON A-HRP stained material in electron micrographs and of silver grains, representing bound 3H-CON A, in autoradiographs confirms t h e fact t h a t surface coat carbohydrates are present at apical surfaces of epithelial cells of the nasal groove and nasal folds of mouse embryos in stages shortly before and during nasal fold fusion. While CON A binding was restricted to apical surfaces of nasal groove epithelial cells and presumptive fusion areas of twelfth day embryos, both cell surfaces and intercellular spaces of surface ectoderm in eleventh day embryos were often labeled with CON A. Electron microscopic observation provides support for the suggestion t h a t penetration of CON A between these cells may be due to differences in cell attachments (Smuts, ’77). Surface ectoderm cells tend to be loosely joined by desmosomes, while tight junctions are present between apical ends of pseudostratified columnar epithelial cells in t h e nasal groove. The higher silver grain counts recorded from non-fusing versus fusing surfaces of both eleventh and twelfth day embryos suggests a n increased binding of CON A by nasal groove epithelium. However, this increase may be explained on t h e basis of differences in surface morphology of epithelial cells in t h e two locations. For example, cells in t h e non-fusing nasal groove epithelium have numerous microvilli; whereas, cells in presumptive fusion areas have smoother surfaces with few projec-
tions. Therefore, for any given distance, the actual surface area of cells in t h e nasal groove is greater than for cells in t h e presumptive fusion region. Variation in surface morphology may also be responsible for differences in amount of CON A bound by anterior and posterior non-fusing regions of eleventh and twelfth day stages. However, other possibilities, such as alterations in synthesis or distribution of surface coat material, may also play a role. The observation of higher counts for nonfusing nasal groove epithelium than for epithelium from presumptive fusion regions conflicts with findings reported by Smuts (’77). Although her counting procedure was not clearly defined, Smuts found one and one-half times more silver grains (bound 3H-CON A) over cells in t h e presumptive fusion region than over epithelial cells in the nasal placode at t h e stage of late placode invagination. It is difficult to explain this difference from our study, unless Smuts has employed different counting criteria from our own. For example, at the stage employed by Smuts (our eleventh day), silver grains are found not only along cell surfaces but between cells in the presumptive fusion (“shoulder”) region. Therefore, when analyzing surface coat distribution, only grains located at t h e apical ends of cells should be counted, since inclusion of grains located over cell cytoplasm or between cells would not represent binding to surface glycoproteins. I t is also possible t h a t t h e microvilli which characterize the nasal groove epithelium of more advanced stages are not as prevalent a t earlier stages of placode differentiation with a consequent effect on counting results. Statistical analysis of counts from presumptive fusion surfaces of eleventh and twelfth day embryos revealed a significant increase in t h e amount of bound 3H-CONA between anterior and posterior regions, i.e., mean counts were greater for t h e group of sections located closer to the point of initial contact (fusion) of t h e nasal folds. Assuming t h a t the amount of bound 3H-CON A reflects t h e actual number of CON A binding sites (Collard and Temmink, ’74) and, therefore, t h e relative amount of surface coat carbohydrates present, this finding suggests t h a t there is a n increased amount of surface coat material associated with prefusion epithelium shortly before nasal fold contact. An increase in surface coat material is
SURFACE COAT DISTRIBUTION ON NASAL FOLDS
consistent with findings in the secondary palate (Greene and Kochhar, '74; Souchon, '74; Pratt and Hassell, '75) and neural tube (Moran and Rice, '75; Lee et al., '76; Silver, '78; Sadler, '78) in which an increase in surface coat material prior to fusion has also been reported. The hypothesis that the cell surface coat is associated with the ability of epithelial shelves or folds to adhere and fuse (Pourtois, '72; Greene and Pratt, '76) is reinforced by these findings and further supported by the fact that complex carbohydrates have for some time been implicated in cell aggregation and intercellular adhesion in various in vitro cell systems (Pessac and Defendi, '72; Oppenheimer, '73; Roseman, '74; Greig and Jones, '77). Since CON A binds to only two of the sugar components of the glycoproteins and glycolipids which make up the surface coat (Sharon and Lis, '72; Luft, '761, it is conceivable that in addition to changes in coat quantity there may also be qualitative changes in surface carbohydrates associated with adhesion, which might not be detected using CON A or ruthenium red techniques. Further testing, including manipulation of the cell surface coat, will be necessary in order to determine the importance of synthesis and integrity of cell surface coat material in the fusion of the nasal folds and consequent importance of the surface coat in terms of production of cleft lip and other congenital malformations. LITERATURE CITED Bernhard, W., and S. Avrameas 1971 Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp. Cell Res., 64: 232-263. Collard, J. G . , and J. H. M. Temmink 1974 Binding and cytochemical detection of cell bound concanavalin A. Exp. Cell Res., 86: 81-86. Cook, G. M. W., and R. W. Stoddart 1973 Surface Carbohydrates of t h e Eukaryotic Cell, New York, Academic Press, p. 79. Gaare, J. D., and J. Langman 1977 Fusion of nasal swellings in the mouse embryo: Surface coat and initial contact. Am. J. Anat., 150: 461-476.
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Greene, R. M., and D. M. Kochhar 1974 Surface coat on the epithelium of developing palatine shelves in the mouse as revealed by electron microscopy. J. Embryol. exp. Morph.. 31: 683-692. Greene, R. M., and R. M. P r a t t 1976 Developmental aspects of secondary palate formation. J. Embryol. exp. Morph., 36: 225-245. 1977 Inhibition by diazo-0x0-norleucine (DON) of rat palatal glycoprotein synthesis and epithelial cell adhesion in uitro. Exp. Cell Res., 105: 27-37. Greig, R. G., and M. N. Jones 1977 Mechanisms of intercellular adhesion. BioSystems, 9: 43-55. Lee, H. Y., J. B. Sheffield, R. G. Nagele and G. W. Kalmus 1976 The role of extracellular material in chick neurulation. I. Effects of concanavalin A. J. Exp. Morph., 198: 261-266. Lejour, M. 1970 Cleft lip induced in the rat. Cleft Palate J., 7: 169.186. Luft, J. H. 1976 The structure and properties of the cell surface coat. Int. Rev. Cytol., 45: 291-382. Moran, D., and R. W. Rice 1975 An ultrastructural examination of the role of cell membrane surface coat material during neurulation. J. Cell Biol., 64: 172-181. Oppenheimer, S. B. 1973 Utilization of L-glutamine in intercellular adhesion: Ascites tumor and embryonic cells. Exp. Cell Res., 77: 175-182. Pessac, B., and V. Defendi 1972 Cell aggregation: Role of acid mucopolysaccharides. Science, 175: 898.900. Pourtois, M. 1972 Morphogenesis of the primary and secondary palate. In: Developmental Aspects of Oral Biology. H. S. Slavkin and L. S. Bovetta, eds. Academic Press, New York, pp. 81-108. Pratt, R. M., and J . R. Hassell 1975 Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf. Dev. Biol., 45: 192.198. Roseman, S. 1974 Complex carbohydrates and intercellular adhesion. In: Biology and Chemistry of Eukaryotic Cell Surfaces. E. Y. C. Lee and E. E. Smith, eds. Academic Press, New York, pp. 317-354. Sadler, T. W. 1978 Distribution of surface coat material on fusing neural folds of mouse embryos during neurulation. Anat. Rec., 191: 345-350. Sharon, N., and H. Lis 1972 Lectins: Cell-agglutinating and sugar-specific proteins. Science, I 77: 949-959. Silver, M. H. 1978 Ultrastructure of neural fold fusion in chick embryos. Anat. Rec., 190: 541-542 (Abstract). Smuts, M. S. 1977 Concanavalin A binding to the epithelial surface of the mouse olfactory placode. Anat. Rec., 188: 29-38. Souchon, R. 1975 Surface coat of the palatal shelf epithelium during palatogenesis in mouse embryos. Anat. Embryol., 147: 133.142. Trader, D. G. 1968 Pathogenesis of cleft lip and its relation to embrvonic face shape in A/J and C57BL mice. Teratology, 1: 33-50.
w 0
4 Coronal l - F m section through the nasal folds and groove a t approximately the level of the ' in figure 4. Counts of silver grains, representing bound 3H-concanavalin A, were made in areas indicated by brackets (presumptive fusing surfaces) and between t h e arrows in the nasal groove (non-fusing surfaces). M, medial nasal fold; L, lateral nasal fold. X 10.
3 View of the roof of the primitive oral cavity of a twelfth day embryo (comma stage). The medial (M) and lateral (L) nasal folds have begun to fuse a t their posterior ends. Fusion proceeds in an anterior direction toward the top of the photograph.
2 Coronal l-Fm section through the nasal folds and groove a t approximately t h e level of the ' in figure 2. Counts of silver grains, representing bound 3HH-concanavalin A, were made in areas indicated by brackets (presumptive fusing surfaces) and between the arrows in the nasal groove (non-fusingsurfaces). M, medial nasal fold; L, lateral nasal fold. X 10.
1 View of the roof of the primitive oral cavity in an eleventh day embryo (oblong stage). The medial (M) and Lateral (L) nasal folds, which flank the nasal groove, have not yet contacted. R, Rathke's pocket. The arrow on the inset indicates the nasal groove of an eleventh day embryo viewed from t h e side.
EXPLANATION OF FIGURES
PLATE I
191
PLATE 2 E X P L A N A T I O N OF FIGLIKES
192
5
Electron micrograph of surfaces of pseudostratified columnar epithelial cells in t h e nasal groove of a n eleventh day embryo. Note numerous microvilli. Following incubation with CON A-HRP a n electron dense precipitate. representing cell surface coat material Iscm). I S present a t apical surfaces of cells. X 8.930.
6
Electron micrograph of surfaces of stratified squamous epithelial cells in t h e presumptive fusion r e o o n of a twelfth day embryo Note t h a t t h e surface is relatively smooth (free of projections) a s compared with t h e epithelium in figure 5 . scm. surface coat material. x 8,930.
7
Electron micrograph of surfaces of pseudostratified columnar epithelial cells in t h e nasal groove of a twelfth day embryo incubated with CON A plus a MG. S t a m i n g of surface coat material is elimited. c. cilium. X 8,930.
SURFACE COAT DISTRIBUTION ON NASAL FOLDS Dorothy Burk, T. W. Sadler and Jan Langman
PLATE 2
193
PLATE 3 EXPLANATIOK OF F I G U R E S
8
Autoradiograph of a coronal 1 - p m section through t h e lateral nasal fold a n d nasal groove of a n eleventh day embryo. Silver grains are restricted to apical surfaces of epithelial cells in t h e nasal groove; whereas, grains a r e located over t h e between epithelial cells in t h e presumptive fusion (transition, region. X 40.
9
Autoradiograph of a coronal 1 - p m section through t h e lateral nasal fold and nasal groove of a n eleventh day control ( = M G ) embryo a t a level similar t o t h a t shown in figure 8. X 40.
10 Autoradiograph of a coronal 1 - p m section through t h e nasal groove of a twelfth day embryo. Silver grains a r e present only a t apical surfaces of pseudostratified columnar epithelial cells. X 40. 11 Autoradiograph of a coronal 1 - p m section through t h e presumptive fusior region of t h e nasal folds of a twelfth day embryo. The folds a r e seen prior t o contact. Silver grains a r e present a t t h e apical surfaces of epithelial cells which will make contact. X 40.
SURFACE COAT DISTRIBUTION ON NASAL FOLDS Dorothy Burk. T W Sadler and J a n Langman
PLATE 3
195
E
Q)
0
C
V
0
3
c C v)
r
20
posterior
v)
20-
.
300
EXPLANATION OF FIGURES
E
Q,
0
C
U
3 0
c C
401
anterior
J
posterior
12th DAY
1 2 Comparison of mean number of silver grains (per 50 p m length) on aplcal surfaces of fusing and non-fusing nasal epithelium. Means represent pooled counts from groups of sections in anterior and posterior regions of t h e nasal folds in two stages of development.
anterior
30L1
401
fusing
non-fusing
11th DAY
PLATE 4
