Concanavalin A Binding to the Epithelial Surface of t h e Developing Mouse Olfactory Placode MARYBETH S . S M U T S ' Lahorntory of Developmental Biology and Anomalies, National Institute of Dental Research, Nntionctl Institutes of Health, Bethesdn, Maryland 2001 4
ABSTRACT Concanavalin A (Con A), a lectin binding to mannosyl and glucosy1 residues of glycoproteins and glycolipids, was used to study the appearance of carbohydrate-rich cell surface material o n the olfactory placode and nasal processes which contribute to formation of the primary palate. In vivo incorporation of 3H-thymidine was also used in a n attempt to correlate changes in labeling index with formation of the olfactory placode and nasal processes. The cell surface o f the early frontonasal epithelium binds Con A very little, if at all, but Con A binding was observed when the olfactory placode could be identified as a plate of cuboidal cells that exhibited a reduced labeling index. During the period of formation of the nasal processes, Con A binding was observed on the facial epithelium including the presumptive contact region. There was also a decline in the labeling index throughout primary palate formation. This study provides three criteria by which the olfactory placode can be identified: a morphological change of placode cells to a cuboidal shape, a synthesis or rearrangement of surface coat material that binds Con A, and a reduced labeling index.
The olfactory placodes, the most prominent feature of the early embryonic face (fig. la), contribute to a number of facial structures, primarily the nose and primary palate. Invagination of the placode participates in the formation of the lateral and medial nasal processes. Later stages of invagination and growth of the nasal processes sculpture the nasal pit so that its lower edges are in apposition (fig. lb). The lower edge of the medial nasal process contacts the lateral process. Mesenchymal penetration of this epithelial contact region consolidates the primary palate. A declining labeling index appears to be common for developing organs as they organize into rudiments; for example, feather and hair anlagen (Wessells, '65; Wessells and Roessner, '65), the lens (Modak et al., '68) and the chick thyroid (Smuts, '74). The olfactory rudiment is a thickening (placode) in the facial epithelium; therefore, a decline in its labeling index might be expected as the placode develops. Concanavalin A (Con A) is a lectin that specifically binds to mannosyl or glucosyl residues of glycoproteins and glycolipids ANAT. REC., 188: 29-38.
(Sharon and Lis, '72). It was selected to indicate changes in the surface coat during development of the olfactory placode. Prior to elevation of the secondary palatine shelves, an alteration occurs in the palatine surface coat as demonstrated by ruthenium red staining 48 hours before contact (Greene and Kochhar, '74) and by Con A binding (Pratt et al., '73). During neural tube closure, there is also an increase in surface associated material on the approaching neural folds (Moran and Rice, '75). Since a surface coat is conspicuous long before contact of the secondary palatine shelves and prior to contact of other structures about to undergo fusion, it is reasonable to postulate that there may be surface coat alterations prior to primary palate fusion. Although the formation and invagination of the olfactory placodes contribute to the development of a major facial region, there is little information about the mechanisms involved. This study was undertaken to Received May 7, ' 7 6 . Accepted Oct. 7, '76. Present address: Biology Department, The Catholic University of America, Washington, D.C., 20017. I
29
30
MARYBETH S . SMUTS
sected, and the embryos staged, The heads were fixed either in Carnoy's for eight minutes or 2 % glutaraldehyde in phosphate buffer (pH 7.2) for 20 minutes, dehydrated, embedded in paraffin or in Spurr's low MATERIALS AND METHODS viscositv embedding media and sectioned a t Pregnant C57B1/6J mice (The Jackson 5 pm or at 1 pm respectively. The sections Laboratory, Bar Harbor, Maine) of the were dipped in NTB-2, stored at 4 ° C for ninth, tenth, and eleventh day of gestation two weeks, and developed for autoradiog(plug day = d. 0) were killed by cervical raphy. The percentage of cells that had dislocation, the uteri removed and the em- incorporated tritiated thymidine was debryos dissected in Tyrode's solution. The termined by counting labeled and unlabeled embryos were staged according to the num- epithelial nuclei. A nucleus was considered ber of pairs of body somites or, once the to be labeled if it had four or more silver hind limb was present, the number of pairs grains over it. Every fifth section through of somites from the caudal edge of the hind the embryonic head was scored and at limb to the end of the tail. The heads were least 500 nuclei from each embryo were removed, fixed in 2.5% glutaraldehyde in counted. The labeling indices of the three 0.1 M cacodylate buffer (pH 7.2) for one embryos were averaged to obtain the lahour at 25°C. After fixation, the heads beling index for each embryonic stage and were rinsed in 0.1 M cacodylate buffer (pH the mean and standard error were cal6.9) for 20 minutes, then in several changes culated. of phosphate buffered saline (pH 6.9) for RESULTS 20 minutes, and finally divided into a conPre-placode morphology trol group and a n experimental group. In In the 14-somite mouse embryo (day 9), total, five embryos per stage were used. The experimental group went through an- the anterior neuropore is closed and the other saline wash while the control group frontal prominence is obvious (fig. la). was washed in 0.1 M a-methyl glucopyran- Cross-sections through the prospective oloside solution, a sugar that will compete factory placode area, reveal a squamous for Con A binding sites (Sharon and Lis, epithelial layer covering the frontonasal '72). The experimental heads were trans- surface. The squamous epithelium is very ferred to a plastic tube containing 5.0 pCi thin with prominent nuclei. At this stage 3H-Con A (New England Nuclear, sp. act. of facial development the squamous epi5.1 Ci/mmole) in 0.3 ml of saline buffer; thelium does not bind Con A (fig. 2) and the controls were added to a tube with 5.0 the labeling index of the frontonasal epithelium (fig. 3) was found to be 60.3 & pCi 3H-Con A in the presence of 0.1 M methyl glucopyranoside. After 30 minutes, 1.5%. all heads were washed in several changes Placode m o r p h o l o g y of saline buffer, postfixed for one hour in When the embryos have developed 20 1.25% glutaraldehyde and 0.05% osmium in 0.1 M cacodylate buffer (pH 7.2). They somites, the area of the olfactory placode were next rinsed, dehydrated and embedded can be recognized. Cells of the forming in Spurr's low viscosity embedding media placode become cuboidal with regions of (Ladd Research Industries, Inc.) and sec- apical cytoplasm. Concomitant with the tioned. Slides with 1-pm thick sections shape change, Con A binds predominately were dipped in Kodak NTB-2 emulsion, to the surface coat of the placode cells stored a t 4°C for two weeks and processed (fig. 4). There is little Con A binding to the for autoradiography. The developed sec- adjacent facial epithelium. The labeling tions were stained with 1% Toluidine blue. index of the placode area (fig. 5) is reduced Tritiated thymidine (New England Nu- to 41.7 -t 4.8% while the labeling index clear, sp. act. 20 Cilmmole) was injected of the adjoining epithelium remains at intraperitoneally a t 5 p C i per gram of 56.3 & 2.7%. body weight into pregnant C57Bl/6J mice. Early i n u a g i n a t w n After one hour. females were killed by The placode begins to invaginate at 26 cervical dislocation, the uteri were dis-
determine whether alterations in labeling index and surface coat properties might be used to characterize the early stages of primary palate formation.
Y
(Y-
CON A BINDING TO OLFACTORY PLACODE
31
of 3H-Con A containing a-methyl glucopyranoside, exhibited no Con A binding (fig. 7) showing that 3H-Con A was bound specifically. The invaginating placode exhibits a labeling index of 39.7 & 2 . 2 % .
Mandibular process
M O U S E EMBRYO. 14 Somites
10
Fig. l a Schematic drawing of a 14 pairs of body somites embryonic mouse head indicating the regions where the olfactory placodes will form. The line A indicates the plane of sectioning for figures 2 and 3.
ess (shoulder region!
M O U S E EMBRYO. 6 tail Somites
’
Ib
Fig. l b Schematic drawing of an embryonic mouse head at the stage of development of 6 pairs of tail somites just prior to closure of the primary palate. The regions labeled lateral process (shoulder region) and medial process (shoulder region) will fuse to form the primary palate. The line B indicates the section plane for figures 8 and 9.
somites. The invaginated sides are slightly curved inwards and appear as shoulders (figs. 6, 7). At the medial shoulder region of the placode, there is an abrupt boundary between the squamous facial epithelium and the pseudostratified-columnar placode cells. The lateral shoulder region, in contrast, does not end sharply but trails off into loose cuboidal cells (figs. 6, 7). At this stage, Con A binding occurs along the whole olfactory placode (fig. 6), as well as on the facial epithelium. Con A binds heavily to both lateral and apical surfaces of the cells. This binding to lateral cellular surfaces shows that Con A is able to penetrate between cells in the shoulder regions but not in the invaginated area. Control tissue, pre-washed in a-methyl glucopyranoside and exposed to a solution
Late invagination After hind limb formation, somite counts are taken from the caudal edge of the hind limb to the tip of the tail. These are referred to as tail somites. At 6-tail somites (day lo), the olfactory placode is still invaginating and the invaginated interior is considered the nasal pit or groove (fig. lb). The lateral and medial shoulders at the lower margins of the pit are coming into apposition. Con A binds to the entire olfactory area and facial epithelia. More lectin binds to the approaching borders of the lateral and medial nasal processes than to the base of the nasal pit (figs. 8, 9). Silver grains which indicate Con A binding were counted at the light microscopic level. Fields were counted in the shoulder regions and in the base of the nasal pit in representative sections. The number of apical silver grains in the shoulder regions was one and one-half times the number in the basal regions. At this time, the whole placode has decreased its DNA synthesis as indicated by an overall lower labeling index of 37.7 f 0 . 7 % . When the approaching contact regions of the lateral and medial nasal processes are counted separately, the lateral shoulder region epithelium labeled at 31.7 % 3.2% and the medial lower border labeled at 26.3 +- 5.4%. DISCUSSION
The acquisition of Con A binding can be used to identify the forming olfactory placode. Prior to placode formation, the epithelium covering the frontal prominence is squamous and does not bind Con A. When the embryo attains 20 pairs of body somites, however, the olfactory placode is recognizable as an area. of cuboidal cells which does bind the lectin. The onset of Con A binding suggests either that the placode is synthesizing surface carbohydrate-rich macromolecules or that surface rearrangements are occurring to expose lectin-binding sites. Initially, the presumptive olfactory placode region has a labeling index of 60.3 k 1 . 5 % . After placode formation, the area
32
MARYBETH S . SMUTS
of cuboidal cells has a labeling index of 41.6 -+ 4.8% which is significantly lower than the pre-placodal stage. The newly formed olfactory placode is thus composed of a population of cells, identifiable by their cuboidal appearance, by their reduced labeling index and by the change in their surface coat. A previous study on hamster nasal pit formation (Waterman and Meller, '73) describes the placode cells as smaller in apical surface area with more microvilli than the adjacent facial epithelium. In both of these studies, a gradual transition of these features occurs where the placode area ends and the facial epithelium begins; this is especially evident on the lateral border of the placode. Con A binds to the lateral and apical surfaces of cells in this border region but only to apical surfaces in the invaginated areas. This difference suggests that preparations for invagination or incorporation into the placode allow Con A to penetrate between these epithelial cells. Possible alterations of cellular junctional complexes that would permit this penetration should be investigated. Alterations in surface coat material and a decline in DNA synthesis during primary palate formation are analogous to changes occurring during secondary palate development. Ruthenium red staining of the secondary palate can be demonstrated as early as 48 hours prior to closure (Greene and Kochhar, '74). Although ruthenium red staining appeared on the surface of a large region of the palatal epithelium, the binding of Con A was more specific and confined to the presumptive adhesion region of the palatal epithelium (Pratt et al., '73). The epithelium at the medial edge of the secondary palate ceases DNA synthesis approximately 24 to 36 hours before fusion (Hudson and Shapiro, '73) and Con A binding also increased dramatically at this time (Pratt and Hassell, '75). The new surface coat material has been shown to be necessary for the adhesion of secondary palatine shelves in vitro (Greene and Pratt, '75). Unlike the secondary palate where only the medial-edge epithelium binds Con A and stops incorporating tritiated thymidine; all the epithelium of the primary palate region binds Con A. More Con A
binds to the shoulder regions of the lateral and medial nasal processes which will eventually contact and fuse than to the base of the nasal pit. This difference may be due either to surface alterations or to a greater surface area exposed to the lectin. The cells in the shoulder regions are oriented obliquely and so have more of their surface exposed. By contrast, the cells in the base of the nasal pit are vertically oriented to the external surface and have a very narrow apical surface exposed to the lectin. The cells of the shoulder regions may also have more surface projections, but this cannot be confirmed at the light microscopic level. At the stages studied, the epithelium of the contact region of the lateral and medial nasal processes continued to incorporate tritiated thymidine but the labeling index is lower than that of the rest of the placode. Carbohydrate-rich macromolecules are present on the fusion regions of the primary palate, but they have not yet been demonstrated to be necessary for contact and fusion of the nasal processes. ACKNOWLEDGMENTS
This investigation was supported by a National Institutes of Health Fellowship, 5F22DE02070-02, from the National Institute of Dental Research. LITERATURE CITED 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., 3 1 : 683-692. Greene, R. M., and R. M. Pratt 1975 Inhibition of palatal epithelial cell adhesion in nitro by diazo-0x0-norleucine. Teratology, I 1 ; 19A. Hudson, C., and B. Shapiro 1973 A radioautographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death. Archs. Oral Biol., 18: 77-84. Modak, S. P., G. Morris and T. Yamada 1968 DNA synthesis and mitotic activity during early development of chick lens. Devel. Biol., 14: 544561. 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. Pratt, R. M . , W. A. Gibson and J. R. Hassell 1973 Concanavalin A binding to the secondary palate of the embryonic rat. J. Dent. Res., 52: 111.
Pratt, R. M . , and J. R. Hassell 1975 Appearance and distribution of carbohydrate-rich macro-
CON A BINDING TO OLFACTORY PLACODE molecules on the epithelial surface of the developing rat palatal shelf. Devel. Biol., 45; 192-198. Sharon, N., and H . Lis 1972 Lectins: Cell-agglutinating and sugar-specific proteins. Science, 177: 949-959. Smuts, MB. S. 1974 Patterns of cellular proliferation during thyroid organogenesis. J. Cell Biol., 63: 323a. Waterman, R. E., and S. M . Meller 1973 Nasal
33
pit formation in the hamster: A transmission and scanning electron microscopic study. Devel. Biol., 34: 255-266. Wessells, N. 1965 Morphology and proliferation during early feather development. Devel. Biol., 12: 131-153. Wessels, N., and K. Roessner 1965 Nonproliferation and dermal condensations of mouse vibrissae and pelage hairs. Devel. Biol., 1 2 : 419433.
PLATE 1 EXPLANATION O F FIGURES
34
2
An autoradiograph of the pre-placode epithelium after exposure to [ W - C o n A. At 14 pairs of body somites, the epithelium of the presumptive olfactory placode is squamous (arrow). The pre-placode epithelium binds little if any [3H] -Con A. For section plane, see figure l a . X 400.
3
An autoradiograph depicting [3H] -thymidine incorporation into preplacode frontonasal epithelium at the stage of development of 14 pairs of body somites. This squamous epithelium has a labeling index of 60.3 % ; the arrow points to an epithelial cell that incorporated [3H] thymidine as indicated by the accumulation of silver grains over the nucleus. X 400.
4
Cross-section of a 20 pairs of body somites embryonic mouse head at the level of the olfactory placode. There is a heavy concentration of silver grains over the placode cells (arrow) which indicates that the cells are now binding [3Hj -Con A. x 400.
5
An autoradiograph depicting [3H] -thymidine incorporation by placode cells in a 20 pairs of body somites embryo. The boundary (” and arrow) between the lateral edge of the placode and the facial epithelia is made recognizable by the altered appearance of the placode cells; i.e., the basal position of their nuclei, their closer apposition and their cuboidal shape. The placode cells exhibit a reduced labeling index of 41.5% (Arrow indicates a labeled nucleus). The frontonasal epithelium still has a labeling index of 56.3 % . X 400.
CON A BINDING TO OLFACTORY PLACODE MaryBeth S. Smuts
PLATE 1
35
PLATE 2 EXPLANATION O F F I G U R E S
36
6
An autoradiograph of a section passing through the lateral process of a 2 6 pairs of body somites olfactory placode. [3H] -Con A binds to the surface of the placode cells. The lateral shoulder region of the placode is bracketed. X 400.
7
An autoradiograph of the lateral process, shoulder region i n brackets, of a 26 pairs of body somites olfactory placode. This control head was treated with buffer containing [3H] -Con A and a-methyl-glucopyranoside. In the presence of this competitive inhibitor, the tissue did not bind Con A. The placode cells are pseudostratified columnar with all mitotic figures at the apical surface (arrow). X 400.
8
Shoulder region of the medial process of the invaginated placode of the stage of 6 pairs of tail somites. [3H] -Con A binding was observed on the apical surfaces of cells of the shoulder region (arrow). For section plane, see figure l b . X 400.
9
Shoulder region of the lateral process of the invaginating olfactory placode at stage of development of 6 pairs of tail somites. [3H] -Con A binding to the apical cellular surface of the shoulder region i s evident (arrow). X 400.
CON A BINDING TO OLFACTORY PLACODE MaryBeth S . Stnuts
PLATE 2
37
ABSTRACT Concanavalin A (Con A), a lectin binding to mannosyl and glucosy1 residues of glycoproteins and glycolipids, was used to study the appearance of carbohydrate-rich cell surface material o n the olfactory placode and nasal processes which contribute to formation of the primary palate. In vivo incorporation of 3H-thymidine was also used in a n attempt to correlate changes in labeling index with formation of the olfactory placode and nasal processes. The cell surface o f the early frontonasal epithelium binds Con A very little, if at all, but Con A binding was observed when the olfactory placode could be identified as a plate of cuboidal cells that exhibited a reduced labeling index. During the period of formation of the nasal processes, Con A binding was observed on the facial epithelium including the presumptive contact region. There was also a decline in the labeling index throughout primary palate formation. This study provides three criteria by which the olfactory placode can be identified: a morphological change of placode cells to a cuboidal shape, a synthesis or rearrangement of surface coat material that binds Con A, and a reduced labeling index.
The olfactory placodes, the most prominent feature of the early embryonic face (fig. la), contribute to a number of facial structures, primarily the nose and primary palate. Invagination of the placode participates in the formation of the lateral and medial nasal processes. Later stages of invagination and growth of the nasal processes sculpture the nasal pit so that its lower edges are in apposition (fig. lb). The lower edge of the medial nasal process contacts the lateral process. Mesenchymal penetration of this epithelial contact region consolidates the primary palate. A declining labeling index appears to be common for developing organs as they organize into rudiments; for example, feather and hair anlagen (Wessells, '65; Wessells and Roessner, '65), the lens (Modak et al., '68) and the chick thyroid (Smuts, '74). The olfactory rudiment is a thickening (placode) in the facial epithelium; therefore, a decline in its labeling index might be expected as the placode develops. Concanavalin A (Con A) is a lectin that specifically binds to mannosyl or glucosyl residues of glycoproteins and glycolipids ANAT. REC., 188: 29-38.
(Sharon and Lis, '72). It was selected to indicate changes in the surface coat during development of the olfactory placode. Prior to elevation of the secondary palatine shelves, an alteration occurs in the palatine surface coat as demonstrated by ruthenium red staining 48 hours before contact (Greene and Kochhar, '74) and by Con A binding (Pratt et al., '73). During neural tube closure, there is also an increase in surface associated material on the approaching neural folds (Moran and Rice, '75). Since a surface coat is conspicuous long before contact of the secondary palatine shelves and prior to contact of other structures about to undergo fusion, it is reasonable to postulate that there may be surface coat alterations prior to primary palate fusion. Although the formation and invagination of the olfactory placodes contribute to the development of a major facial region, there is little information about the mechanisms involved. This study was undertaken to Received May 7, ' 7 6 . Accepted Oct. 7, '76. Present address: Biology Department, The Catholic University of America, Washington, D.C., 20017. I
29
30
MARYBETH S . SMUTS
sected, and the embryos staged, The heads were fixed either in Carnoy's for eight minutes or 2 % glutaraldehyde in phosphate buffer (pH 7.2) for 20 minutes, dehydrated, embedded in paraffin or in Spurr's low MATERIALS AND METHODS viscositv embedding media and sectioned a t Pregnant C57B1/6J mice (The Jackson 5 pm or at 1 pm respectively. The sections Laboratory, Bar Harbor, Maine) of the were dipped in NTB-2, stored at 4 ° C for ninth, tenth, and eleventh day of gestation two weeks, and developed for autoradiog(plug day = d. 0) were killed by cervical raphy. The percentage of cells that had dislocation, the uteri removed and the em- incorporated tritiated thymidine was debryos dissected in Tyrode's solution. The termined by counting labeled and unlabeled embryos were staged according to the num- epithelial nuclei. A nucleus was considered ber of pairs of body somites or, once the to be labeled if it had four or more silver hind limb was present, the number of pairs grains over it. Every fifth section through of somites from the caudal edge of the hind the embryonic head was scored and at limb to the end of the tail. The heads were least 500 nuclei from each embryo were removed, fixed in 2.5% glutaraldehyde in counted. The labeling indices of the three 0.1 M cacodylate buffer (pH 7.2) for one embryos were averaged to obtain the lahour at 25°C. After fixation, the heads beling index for each embryonic stage and were rinsed in 0.1 M cacodylate buffer (pH the mean and standard error were cal6.9) for 20 minutes, then in several changes culated. of phosphate buffered saline (pH 6.9) for RESULTS 20 minutes, and finally divided into a conPre-placode morphology trol group and a n experimental group. In In the 14-somite mouse embryo (day 9), total, five embryos per stage were used. The experimental group went through an- the anterior neuropore is closed and the other saline wash while the control group frontal prominence is obvious (fig. la). was washed in 0.1 M a-methyl glucopyran- Cross-sections through the prospective oloside solution, a sugar that will compete factory placode area, reveal a squamous for Con A binding sites (Sharon and Lis, epithelial layer covering the frontonasal '72). The experimental heads were trans- surface. The squamous epithelium is very ferred to a plastic tube containing 5.0 pCi thin with prominent nuclei. At this stage 3H-Con A (New England Nuclear, sp. act. of facial development the squamous epi5.1 Ci/mmole) in 0.3 ml of saline buffer; thelium does not bind Con A (fig. 2) and the controls were added to a tube with 5.0 the labeling index of the frontonasal epithelium (fig. 3) was found to be 60.3 & pCi 3H-Con A in the presence of 0.1 M methyl glucopyranoside. After 30 minutes, 1.5%. all heads were washed in several changes Placode m o r p h o l o g y of saline buffer, postfixed for one hour in When the embryos have developed 20 1.25% glutaraldehyde and 0.05% osmium in 0.1 M cacodylate buffer (pH 7.2). They somites, the area of the olfactory placode were next rinsed, dehydrated and embedded can be recognized. Cells of the forming in Spurr's low viscosity embedding media placode become cuboidal with regions of (Ladd Research Industries, Inc.) and sec- apical cytoplasm. Concomitant with the tioned. Slides with 1-pm thick sections shape change, Con A binds predominately were dipped in Kodak NTB-2 emulsion, to the surface coat of the placode cells stored a t 4°C for two weeks and processed (fig. 4). There is little Con A binding to the for autoradiography. The developed sec- adjacent facial epithelium. The labeling tions were stained with 1% Toluidine blue. index of the placode area (fig. 5) is reduced Tritiated thymidine (New England Nu- to 41.7 -t 4.8% while the labeling index clear, sp. act. 20 Cilmmole) was injected of the adjoining epithelium remains at intraperitoneally a t 5 p C i per gram of 56.3 & 2.7%. body weight into pregnant C57Bl/6J mice. Early i n u a g i n a t w n After one hour. females were killed by The placode begins to invaginate at 26 cervical dislocation, the uteri were dis-
determine whether alterations in labeling index and surface coat properties might be used to characterize the early stages of primary palate formation.
Y
(Y-
CON A BINDING TO OLFACTORY PLACODE
31
of 3H-Con A containing a-methyl glucopyranoside, exhibited no Con A binding (fig. 7) showing that 3H-Con A was bound specifically. The invaginating placode exhibits a labeling index of 39.7 & 2 . 2 % .
Mandibular process
M O U S E EMBRYO. 14 Somites
10
Fig. l a Schematic drawing of a 14 pairs of body somites embryonic mouse head indicating the regions where the olfactory placodes will form. The line A indicates the plane of sectioning for figures 2 and 3.
ess (shoulder region!
M O U S E EMBRYO. 6 tail Somites
’
Ib
Fig. l b Schematic drawing of an embryonic mouse head at the stage of development of 6 pairs of tail somites just prior to closure of the primary palate. The regions labeled lateral process (shoulder region) and medial process (shoulder region) will fuse to form the primary palate. The line B indicates the section plane for figures 8 and 9.
somites. The invaginated sides are slightly curved inwards and appear as shoulders (figs. 6, 7). At the medial shoulder region of the placode, there is an abrupt boundary between the squamous facial epithelium and the pseudostratified-columnar placode cells. The lateral shoulder region, in contrast, does not end sharply but trails off into loose cuboidal cells (figs. 6, 7). At this stage, Con A binding occurs along the whole olfactory placode (fig. 6), as well as on the facial epithelium. Con A binds heavily to both lateral and apical surfaces of the cells. This binding to lateral cellular surfaces shows that Con A is able to penetrate between cells in the shoulder regions but not in the invaginated area. Control tissue, pre-washed in a-methyl glucopyranoside and exposed to a solution
Late invagination After hind limb formation, somite counts are taken from the caudal edge of the hind limb to the tip of the tail. These are referred to as tail somites. At 6-tail somites (day lo), the olfactory placode is still invaginating and the invaginated interior is considered the nasal pit or groove (fig. lb). The lateral and medial shoulders at the lower margins of the pit are coming into apposition. Con A binds to the entire olfactory area and facial epithelia. More lectin binds to the approaching borders of the lateral and medial nasal processes than to the base of the nasal pit (figs. 8, 9). Silver grains which indicate Con A binding were counted at the light microscopic level. Fields were counted in the shoulder regions and in the base of the nasal pit in representative sections. The number of apical silver grains in the shoulder regions was one and one-half times the number in the basal regions. At this time, the whole placode has decreased its DNA synthesis as indicated by an overall lower labeling index of 37.7 f 0 . 7 % . When the approaching contact regions of the lateral and medial nasal processes are counted separately, the lateral shoulder region epithelium labeled at 31.7 % 3.2% and the medial lower border labeled at 26.3 +- 5.4%. DISCUSSION
The acquisition of Con A binding can be used to identify the forming olfactory placode. Prior to placode formation, the epithelium covering the frontal prominence is squamous and does not bind Con A. When the embryo attains 20 pairs of body somites, however, the olfactory placode is recognizable as an area. of cuboidal cells which does bind the lectin. The onset of Con A binding suggests either that the placode is synthesizing surface carbohydrate-rich macromolecules or that surface rearrangements are occurring to expose lectin-binding sites. Initially, the presumptive olfactory placode region has a labeling index of 60.3 k 1 . 5 % . After placode formation, the area
32
MARYBETH S . SMUTS
of cuboidal cells has a labeling index of 41.6 -+ 4.8% which is significantly lower than the pre-placodal stage. The newly formed olfactory placode is thus composed of a population of cells, identifiable by their cuboidal appearance, by their reduced labeling index and by the change in their surface coat. A previous study on hamster nasal pit formation (Waterman and Meller, '73) describes the placode cells as smaller in apical surface area with more microvilli than the adjacent facial epithelium. In both of these studies, a gradual transition of these features occurs where the placode area ends and the facial epithelium begins; this is especially evident on the lateral border of the placode. Con A binds to the lateral and apical surfaces of cells in this border region but only to apical surfaces in the invaginated areas. This difference suggests that preparations for invagination or incorporation into the placode allow Con A to penetrate between these epithelial cells. Possible alterations of cellular junctional complexes that would permit this penetration should be investigated. Alterations in surface coat material and a decline in DNA synthesis during primary palate formation are analogous to changes occurring during secondary palate development. Ruthenium red staining of the secondary palate can be demonstrated as early as 48 hours prior to closure (Greene and Kochhar, '74). Although ruthenium red staining appeared on the surface of a large region of the palatal epithelium, the binding of Con A was more specific and confined to the presumptive adhesion region of the palatal epithelium (Pratt et al., '73). The epithelium at the medial edge of the secondary palate ceases DNA synthesis approximately 24 to 36 hours before fusion (Hudson and Shapiro, '73) and Con A binding also increased dramatically at this time (Pratt and Hassell, '75). The new surface coat material has been shown to be necessary for the adhesion of secondary palatine shelves in vitro (Greene and Pratt, '75). Unlike the secondary palate where only the medial-edge epithelium binds Con A and stops incorporating tritiated thymidine; all the epithelium of the primary palate region binds Con A. More Con A
binds to the shoulder regions of the lateral and medial nasal processes which will eventually contact and fuse than to the base of the nasal pit. This difference may be due either to surface alterations or to a greater surface area exposed to the lectin. The cells in the shoulder regions are oriented obliquely and so have more of their surface exposed. By contrast, the cells in the base of the nasal pit are vertically oriented to the external surface and have a very narrow apical surface exposed to the lectin. The cells of the shoulder regions may also have more surface projections, but this cannot be confirmed at the light microscopic level. At the stages studied, the epithelium of the contact region of the lateral and medial nasal processes continued to incorporate tritiated thymidine but the labeling index is lower than that of the rest of the placode. Carbohydrate-rich macromolecules are present on the fusion regions of the primary palate, but they have not yet been demonstrated to be necessary for contact and fusion of the nasal processes. ACKNOWLEDGMENTS
This investigation was supported by a National Institutes of Health Fellowship, 5F22DE02070-02, from the National Institute of Dental Research. LITERATURE CITED 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., 3 1 : 683-692. Greene, R. M., and R. M. Pratt 1975 Inhibition of palatal epithelial cell adhesion in nitro by diazo-0x0-norleucine. Teratology, I 1 ; 19A. Hudson, C., and B. Shapiro 1973 A radioautographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death. Archs. Oral Biol., 18: 77-84. Modak, S. P., G. Morris and T. Yamada 1968 DNA synthesis and mitotic activity during early development of chick lens. Devel. Biol., 14: 544561. 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. Pratt, R. M . , W. A. Gibson and J. R. Hassell 1973 Concanavalin A binding to the secondary palate of the embryonic rat. J. Dent. Res., 52: 111.
Pratt, R. M . , and J. R. Hassell 1975 Appearance and distribution of carbohydrate-rich macro-
CON A BINDING TO OLFACTORY PLACODE molecules on the epithelial surface of the developing rat palatal shelf. Devel. Biol., 45; 192-198. Sharon, N., and H . Lis 1972 Lectins: Cell-agglutinating and sugar-specific proteins. Science, 177: 949-959. Smuts, MB. S. 1974 Patterns of cellular proliferation during thyroid organogenesis. J. Cell Biol., 63: 323a. Waterman, R. E., and S. M . Meller 1973 Nasal
33
pit formation in the hamster: A transmission and scanning electron microscopic study. Devel. Biol., 34: 255-266. Wessells, N. 1965 Morphology and proliferation during early feather development. Devel. Biol., 12: 131-153. Wessels, N., and K. Roessner 1965 Nonproliferation and dermal condensations of mouse vibrissae and pelage hairs. Devel. Biol., 1 2 : 419433.
PLATE 1 EXPLANATION O F FIGURES
34
2
An autoradiograph of the pre-placode epithelium after exposure to [ W - C o n A. At 14 pairs of body somites, the epithelium of the presumptive olfactory placode is squamous (arrow). The pre-placode epithelium binds little if any [3H] -Con A. For section plane, see figure l a . X 400.
3
An autoradiograph depicting [3H] -thymidine incorporation into preplacode frontonasal epithelium at the stage of development of 14 pairs of body somites. This squamous epithelium has a labeling index of 60.3 % ; the arrow points to an epithelial cell that incorporated [3H] thymidine as indicated by the accumulation of silver grains over the nucleus. X 400.
4
Cross-section of a 20 pairs of body somites embryonic mouse head at the level of the olfactory placode. There is a heavy concentration of silver grains over the placode cells (arrow) which indicates that the cells are now binding [3Hj -Con A. x 400.
5
An autoradiograph depicting [3H] -thymidine incorporation by placode cells in a 20 pairs of body somites embryo. The boundary (” and arrow) between the lateral edge of the placode and the facial epithelia is made recognizable by the altered appearance of the placode cells; i.e., the basal position of their nuclei, their closer apposition and their cuboidal shape. The placode cells exhibit a reduced labeling index of 41.5% (Arrow indicates a labeled nucleus). The frontonasal epithelium still has a labeling index of 56.3 % . X 400.
CON A BINDING TO OLFACTORY PLACODE MaryBeth S. Smuts
PLATE 1
35
PLATE 2 EXPLANATION O F F I G U R E S
36
6
An autoradiograph of a section passing through the lateral process of a 2 6 pairs of body somites olfactory placode. [3H] -Con A binds to the surface of the placode cells. The lateral shoulder region of the placode is bracketed. X 400.
7
An autoradiograph of the lateral process, shoulder region i n brackets, of a 26 pairs of body somites olfactory placode. This control head was treated with buffer containing [3H] -Con A and a-methyl-glucopyranoside. In the presence of this competitive inhibitor, the tissue did not bind Con A. The placode cells are pseudostratified columnar with all mitotic figures at the apical surface (arrow). X 400.
8
Shoulder region of the medial process of the invaginated placode of the stage of 6 pairs of tail somites. [3H] -Con A binding was observed on the apical surfaces of cells of the shoulder region (arrow). For section plane, see figure l b . X 400.
9
Shoulder region of the lateral process of the invaginating olfactory placode at stage of development of 6 pairs of tail somites. [3H] -Con A binding to the apical cellular surface of the shoulder region i s evident (arrow). X 400.
CON A BINDING TO OLFACTORY PLACODE MaryBeth S . Stnuts
PLATE 2
37
