ABSTRACT
R-cadherin is a newly identified member of the cadherin family of cell adhesion receptors. The expression of R-cadherin in early chicken embryos was studied using affinity-purified antibodies to this molecule, comparing it with that of N-cadherin. Inununoblot analysis of various organs of 10.5-day embryos showed that R-cadherin is most abundantly expressed in the retina and brain. Immunostaining of the cervical and thoracic regions of embryos revealed that R- and N-cadherin are expressed in all neural tissues. In the neural tube, R-cadherin appears at around stage 21, although N-cadherin expression begins at a much earlier stage. The distribution of R-cadherin in the neural tube differs from that of N-cadherin; for example, some regions of the tube express only R-cadherin, and other regions only N-cadherin. In the peripheral ganglia, these two cadherins are also expressed in different patterns which change during development. Some mesenchymal tissues including the notochord, the myotome, myotubes and perichondria also express these cadherins, again in different patterns. Thus, R- and N-cadherin are differentially expressed in all the tissues examined, and they may contribute to the spatial segregation of heterogeneous cells in a tissue.
Introduction
Cadherins are a family of Ca2+-dependent cell adhesion molecules that are crucial for compact intercellular connections, and each member of the family shows a distinct tissue distribution (Takeichi, 1988; 1990). These molecules interact with each other recognizing their own type; as a result, cells expressing identical cadherins preferentially adhere to each other. This property of the cadherins has been implicated in cell sorting mechanisms (Takeichi, 1990).
We have recently identified a novel cadherin by isolating cDNAs that hybridize with a nucleotide probe of the cytoplasmic domain of E-cadherin; the cDNAs were from libraries constructed with mRNA of chicken retina and brain (Inuzuka et al. 1991). This novel cadherin, shown to have a relative molecular mass of 124 × 103Mr in SDS –PAGE, is abundant in the differentiated neural retina and, therefore, it was termed retinal cadherin (R-cadherin). While R-cadherin is similar to other cadherins in the overall structure, it most resembles N-cadherin; 74% of the amino acids are conserved between these molecules, whereas only 49 % of the amino acids are conserved between R-cadherin and L-CAM, a member of the cadherin family expressed in chicken epithelia (Gallin et al. 1987).
Using cell lines transfected with cadherin cDNAs, it was found that R-cadherin can crossreact with N-cadherin, although both molecules prefer their own type in their binding interaction; thus, cells expressing these cadherins form chimeric aggregates when mixed, but they tend to segregate from each other within the aggregates (Inuzuka et al. 1991).
In the development of the neural retina, R- and N-cadherin are expressed in different spatiotemporal patterns (Inuzuka et al. 1991). N-cadherin is expressed from the beginning of the formation of the retina, while R-cadherin appears only after some degree of cellular differentiation has occurred. N-cadherin, however, finally disappears from most parts of the retina during development, whereas R-cadherin becomes a dominant cadherin in the matured neural retina. Also, these cadherins are expressed in different layers of the retina in different proportions. These results suggest that Rand N-cadherin may contribute to the segregation of heterotypic cells via their specific binding properties in retinal morphogenesis.
In the present study, we examined the expression of R-cadherin during the early development of chicken embryos, comparing it with N-cadherin expression. We found that the major tissues expressing R-cadherin are neural, and that R- and N-cadherin are expressed differentially in the neural tube as well as in the peripheral ganglia. R-cadherin is also expressed in some other tissues including developing muscles. We discuss the role of these cadherins in development, focusing on neural morphogenesis.
Materials and methods
Antibodies
Antiserum against R-cadherin was prepared by injecting a rabbit with a fusion protein of this molecule, and antibodies recognizing R-cadherin were affinity-purified from the antiserum using the fusion protein, as described (Inuzuka et al. 1991). For staining N-cadherin, rat monoclonal antibody NCD-2 (Hatta and Takeichi, 1986) was used. For staining neurofilaments, a mouse monoclonal antibody that was described previously (Hatta et al. 1987) was used.
Immunoblot analysis
Tissues were lyzed with a buffer containing 2 % SDS and after reducing with 5 % β-mercaptoethanol under boiling, the samples were subjected to SDS –polyacrylamide gel electrophoresis. The total amount of protein for each lane was adjusted to be equal. Proteins on the gels were then transferred to nitrocellulose sheets. The sheets were incubated with affinity-purified antibodies to R-cadherin, followed by incubation with 125I-labeled Protein A (Amersham) and then subjected to autoradiography.
Immunohistochemistry
Tissues of White Leghorn chicken embryos were fixed with 3.5% paraformaldehyde in Hepes-buffered Hanks’ solution (HBSS) for 1 –2 h at 4 °C. After subsequent washing in HBSS, they were incubated in a graduated series of sucrose solutions in HBSS (12 –18%) and frozen in Tissue-Tech (Miles Scientific) using liquid nitrogen. 10 μm thick cryostat sections were then made, mounted on slides coated with 1 % gelatin and 0.1% chromium potassium sulfate, and air dried. All slides were incubated in methanol at —20°C for 10 min, in 5 % skimmed milk in Tris-buffered saline (pH7.6) containing 1 mM CaCl2 (TBS-Ca) for 30min, and then with rabbit anti-R-cadherin or rat monoclonal anti-N-cadherin (NCD-2), diluted appropriately with TBS-CA, for 60 min at room temperature. The slides were then treated with second antibodies, biotinylated anti-rabbit IgG for R-cadherin staining, or FITC-labeled anti-rat IgG for N-cadherin staining, for 30min. For the R-cadherin staining, the samples were further incubated with Texas-red-labeled streptavidin for 30 min. For doublestaining for R- and N-cadherin, the samples were incubated in the mixtures of these antibodies at each step. In doublestaining for N-cadherin and neurofilaments, the former was detected with the same reagents as above, and the latter detected by the biotin –streptavidin system using biotinylated anti-mouse IgG at the second step in the above procedure. After the treatment with antibodies, the samples were washed with TBS-Ca, and mounted with 90% glycerol –10 % TBS-Ca containing 0.1 % paraphenylendiamine. They were examined with a Zeiss fluorescence microscope. The staging of the embryos was based on Hamburger and Hamilton (1951).
Results
Immunoblot analysis of tissue distribution of R-cadherin
To survey the tissue distribution of R-cadherin, various tissues of 10.5-day embryos were subjected to immunoblot analysis (Fig. 1). An intense 124 ×103 Mr band of R-cadherin was detected from the neural retina and the brain, a very faint band detected from the heart and skeletal muscle, and no positive bands detected from the stomach, liver, lung and kidney.
Immunoblot analysis for R-cadherin distribution in various organs of 10.5-day embryos. Arrow indicates the 124 × 103 Mr R-cadherin band. Br, whole brain; Re, neural retina; H, heart; Mu, skeletal muscle; St, stomach; Li, liver; Lu, lung; Ki, kidney.
The onset of R-cadherin expression
We immunostained sections of the embryos at developmental stages 16 (h 51 –56) to 34 (day 8.5) for R-cadherin, focusing on the cervical and thoracic levels of the body. For comparison, the same or adjacent sections were also stained for N-cadherin. In embryos younger than stage 16, we could not detect any positive staining for R-cadherin; the neural tube, somites and all other tissues examined were negative. However, when the myotome begins to be formed (around stage 17), this particular region of the somite becomes strongly positive (Fig. 2A) and, at similar stages, the notochord also begins to express R-cadherin (Fig. 2A). These R-cadherin-positive tissues also express N-cadherin (Fig. 2B), as reported previously (Hatta et al. 1987).
Immunofluorescent staining of the neural tube and surrounding tissues at different developmental stages. (A, B) Stage 20 (day 3 –3.5). (C, D) Stage 23 (day 4). (E, F) Stage 34 (day 8.5). Sections at the cervical level were doublestained for R-cadherin (A, C, E) and N-cadherin (B, D, F). Arrowhead in (C) indicates the motor column that is R-cadherin-positive. Small and large arrows in (C) and (F) indicate the region for the entry of sensory fibers. Small arrowheads in (E) and (F) represent some of the radial glia-like structures that are both R- and N-cadherin-positive. no, notochord; mt, myotome; nt, neural tube; sn, spinal nerve; drg, dorsal root ganglion; rp, roof plate; fp, floor plate; sg, sympathetic ganglion; vc, ventral commissure; dr, dorsal root; vr; ventral root. Magnification is identical for A –D, and for E and F. Scale bars, 100 μm.
Immunofluorescent staining of the neural tube and surrounding tissues at different developmental stages. (A, B) Stage 20 (day 3 –3.5). (C, D) Stage 23 (day 4). (E, F) Stage 34 (day 8.5). Sections at the cervical level were doublestained for R-cadherin (A, C, E) and N-cadherin (B, D, F). Arrowhead in (C) indicates the motor column that is R-cadherin-positive. Small and large arrows in (C) and (F) indicate the region for the entry of sensory fibers. Small arrowheads in (E) and (F) represent some of the radial glia-like structures that are both R- and N-cadherin-positive. no, notochord; mt, myotome; nt, neural tube; sn, spinal nerve; drg, dorsal root ganglion; rp, roof plate; fp, floor plate; sg, sympathetic ganglion; vc, ventral commissure; dr, dorsal root; vr; ventral root. Magnification is identical for A –D, and for E and F. Scale bars, 100 μm.
Expression in the neural tube
The neural tube does not express R-cadherin at stage 20 (day 3 –3.5) (Fig. 2A). At around stage 21 (day 3.5), the motor column and the associated ventral root, the lateral marginal zone and the ventricular zone acquire positive staining for R-cadherin (Fig. 2C), and this staining pattern persists up to stage 26 (day 5). A region in the marginal zone, that is adjacent to the dorsal root, exhibits bright R-cadherin staining, suggesting that sensory fibers entering from the dorsal root express this molecule.
The above pattern changes with the further differentiation of the neural tube, and a novel pattern of R-cadherin expression is established at around stages 30 to 34 (day 6.5 –8.5) (Fig. 2E). At these stages, the characteristic R-cadherin staining of the motor column and of the region adjacent to the dorsal root is lost. R-cadherin is now strongly expressed in bilateral streaks adjacent to the floor plate, and a similar staining pattern is observed in an area dorsal to the central canal. These R-cadherin-positive zones in the ventral and dorsal areas of the neural tube are connected to each other through the ventricular zone that is also R-cadherin-positive. R-cadherin is also expressed in other regions of the neural tube at these stages, including the mantle zone and the ventral and lateral white column, but it is not strongly expressed in the dorsal white column. Radial glia-like structures also express R-cadherin, especially at the ventral half of the tube.
These patterns of R-cadherin expression in the developing neural tube differ from those of N-cadherin as summarized below. (1) When R-cadherin begins to appear, N-cadherin is already expressed (Fig. 2B). (2) At the time when R-cadherin expression increases in the lateral motor column, N-cadherin expression in this region declines (Fig. 2D). (3) The ventral commissure stains intensely for N-cadherin but not so conspicuously for R-cadherin (Fig. 2C to F). (4) N-cadherin is expressed evenly in the regions dorsal and ventral to the central canal, rather than in bilateral streaks (Fig. 2D, F). (5) A region of the dorsal white column that is adjacent to the dorsal root strongly stains for N-cadherin at Stage 34 but this region is negative for R-cadherin at this stage (Fig. 2E,F), although the corresponding region is R-cadherin-positive at earlier stages, as mentioned above. (6) Radial glia-like structures coexpress R- and N-cadherin in the ventral and lateral white column, whereas they express mostly N-cadherin in the dorsal half of the neural tube (Fig. 2E,F).
Expression in peripheral ganglia
Dorsal root ganglia (DRG) and cranial ganglia begin to express R-cadherin at the same stages as the neural tube does (at around stage 21) (Figs 2C, 3A). In these ganglia, fibrous structures are stained, suggesting that sensory nerve fibers express R-cadherin. However, at later stages, this fibrous staining pattern disappears and the expression of this molecule tends to diminish (Figs 2E, 4A). Sensory fibers thus seem to express R-cadherin only transiently at early developmental stages.
Sympathetic ganglia also begin to express R-cadherin at around stage 21 (day 3.5) (data not shown). In contrast to the DRG, however, these ganglia acquire a higher expression of R-cadherin at later stages, when they stain much more strongly for this molecule than the DRG (Figs 2E, 4A).
Peripheral ganglia also express N-cadherin but its distribution patterns are different from those of R-cadherin. Such differences are most clearly seen in the trigeminal (Fig. 3) and sympathetic ganglia (Fig. 4A,B). In these ganglia, N-cadherin tends to be distributed evenly throughout the entire ganglia, but R-cadherin distribution is not homogenous and is restricted to certain regions. In the DRG at stage 34, N-cadherin is expressed in fibrous structures, but such a pattern is not observed for R-cadherin, as mentioned above.
Immunofluorescent staining of a trigeminal ganglion at stage 23. This section was double-stained for R-(A) and N-cadherin (B). Note that the staining pattern is different for R- and N-cadherin in the ganglion as well as in the hindbrain, tg, trigeminal ganglion; hi, hindbrain. Scale bar, 100 μm.
Immunofluorescent staining of a trigeminal ganglion at stage 23. This section was double-stained for R-(A) and N-cadherin (B). Note that the staining pattern is different for R- and N-cadherin in the ganglion as well as in the hindbrain, tg, trigeminal ganglion; hi, hindbrain. Scale bar, 100 μm.
Immunofluorescent staining of various ganglia at stage 34. (A, B) Complex of dorsal root (drg) and sympathetic (sg) ganglia, sectioned at the cervical level and double-stained for R-cadherin (A) and N-cadherin (B). Small arrows indicate the N-cadherin staining of fibrous structures. (C, D) Part of the intestine showing a ganglion of Remak (rem) and enteric ganglia (arrowheads). These are adjacent sections made at the lower thoracic level and stained for R-(C) and N-(D) cadherin. The staining of endodermal tissues in (C) and (D) shown by large arrows is due to the non-specific binding of the second or third antibodies, mt, myotome; vr, ventral root; rem, ganglion of Remak. Scale bar, 100 μm.
Immunofluorescent staining of various ganglia at stage 34. (A, B) Complex of dorsal root (drg) and sympathetic (sg) ganglia, sectioned at the cervical level and double-stained for R-cadherin (A) and N-cadherin (B). Small arrows indicate the N-cadherin staining of fibrous structures. (C, D) Part of the intestine showing a ganglion of Remak (rem) and enteric ganglia (arrowheads). These are adjacent sections made at the lower thoracic level and stained for R-(C) and N-(D) cadherin. The staining of endodermal tissues in (C) and (D) shown by large arrows is due to the non-specific binding of the second or third antibodies, mt, myotome; vr, ventral root; rem, ganglion of Remak. Scale bar, 100 μm.
Other ganglia including the nodose ganglia, the enteric ganglia, and the ganglia of Remak also express R- and N-cadherin (Fig. 4C,D).
Expression in the visceral arches
In the visceral arches, some groups of mesenchymal cells strongly express R-cadherin in a characteristic patchy pattern (Fig. 5A,B). In the mandibular arch, these cells form a U-shaped cluster. When this structure was stained for N-cadherin, not only the U-shaped region but also its core region was positive (Fig. 5C). We also found that the core region can be stained with HNK-1 antibodies but the U-shaped region cannot (data not shown), suggesting that the ceUs in the core region derive from the neural crest. To further characterize these structures, we double-stained them for N-cadherin and neurofilament, a cytoskeletal protein expressed in nerve cells. The core region stains for both these proteins (Fig. 6). Taken together, these results suggest that the U-shaped structure is a muscle precursor and the core contains a branch of the trigeminal nerve. Other R-cadherin-positive cell masses in the visceral arches are probably also muscle precursors.
Immunofluorescent staining of the visceral arches. (A) Phase-contrast-photomicrograph of a section of the visceral arches at stage 24. (B) R-cadherin staining on the same section as in A.===(C) N-cadherin staining on the same structure as shown by the arrow in B. (D and E) A hyoid arch from a distal region at stage 22 double-stained for R- and N-cadherin, respectively. Arrowhead indicates part of the ectoderm that is R-cadherin-positive. mx, maxillary arch; mn, mandibular arch; hy, hyoid arch. Scale bars, 200pm for A and B, 100 μm for C –E.
Immunofluorescent staining of the visceral arches. (A) Phase-contrast-photomicrograph of a section of the visceral arches at stage 24. (B) R-cadherin staining on the same section as in A.===(C) N-cadherin staining on the same structure as shown by the arrow in B. (D and E) A hyoid arch from a distal region at stage 22 double-stained for R- and N-cadherin, respectively. Arrowhead indicates part of the ectoderm that is R-cadherin-positive. mx, maxillary arch; mn, mandibular arch; hy, hyoid arch. Scale bars, 200pm for A and B, 100 μm for C –E.
Immunofluorescent staining of a trigeminal nerve at stage 24. (A) R-cadherin staining of a section of a trigeminal nerve at a proximal region of the maxillary and mandibular lobes. (B, C) A section adjacent to (A), double-stained for N-cadherin and neurofilaments, respectively. Note that neurofilaments are detected only in the regions that are N-cadherin-positive but R-cadherin-negative. ph, pharynx; mn, mandibular arch. Scale bar, 100 μm.
Immunofluorescent staining of a trigeminal nerve at stage 24. (A) R-cadherin staining of a section of a trigeminal nerve at a proximal region of the maxillary and mandibular lobes. (B, C) A section adjacent to (A), double-stained for N-cadherin and neurofilaments, respectively. Note that neurofilaments are detected only in the regions that are N-cadherin-positive but R-cadherin-negative. ph, pharynx; mn, mandibular arch. Scale bar, 100 μm.
In addition, a small region of the ectoderm that is adjacent to the tip of the R-cadherin-positive mesenchyme in the hyoid arch expresses R-cadherin (Fig. 5D). Such staining pattern was not observed for N-cadherin, but this cadherin shows other localized expression patterns in the ectoderm (Fig. 5E). Parts of other regions of the ectoderm also express R-cadherin, in particular where the ectoderm layers fuse to each other (Fig. 5B).
Expression in mesodermal tissues
The expression of R-cadherin in the myotome continues at the stages when this cell mass differentiates into myotubes. Early skeletal muscles thus express both R- and N-cadherin at least until stage 35 (day 9) (Fig. 7A –F), and this expression is localized to the boundaries between the muscle cells. The staining patterns of these two cadherins in a skeletal muscle are, however, not identical. The overall view of these patterns revealed some regional differences in the expression of these molecules; while most parts of a muscle express both R- and N-cadherin, some regions of the muscle stain more strongly either for R- or for N-cadherin (Fig. 7A –C). Closer examinations further revealed that individual muscle cells can express these two cadherins in different proportions (Fig. 7D –F). Some boundaries between muscle cells stain more strongly for R-cadherin than N-cadherin, while others show the opposite pattern. The same muscle cell can show both patterns with different neighboring cells.
Immunofluorescent staining of mesodermal tissues at stages 34 –35. (A –C) Part of an anterior muscle in the distal hind limb at stage 35. Note regional differences in R-cadherin (A) and N-cadherin (B) expression. (D –F) Higher magnification of R-cadherin (D) and N-cadherin (E) expression in the gastrocnemius muscle of distal hind limb at stage 35. Arrowheads indicate cell –cell boundaries that stain more strongly for R-cadherin, and arrows indicate those that stain more strongly for N-cadherin. (G, H) A proximal region of a forelimb at the ventral side at stage 34. Sections were double-stained for R-cadherin (A, D, G) and N-cadherin (B, E, H). (C) and (F) are Nomarski optics photomicrographs of the corresponding field, ca, cartilage; sm, skeletal muscle. Scale bars, 50 μm for (A –C), 20 μm for (D –F), and 200 μm for G, H.
Immunofluorescent staining of mesodermal tissues at stages 34 –35. (A –C) Part of an anterior muscle in the distal hind limb at stage 35. Note regional differences in R-cadherin (A) and N-cadherin (B) expression. (D –F) Higher magnification of R-cadherin (D) and N-cadherin (E) expression in the gastrocnemius muscle of distal hind limb at stage 35. Arrowheads indicate cell –cell boundaries that stain more strongly for R-cadherin, and arrows indicate those that stain more strongly for N-cadherin. (G, H) A proximal region of a forelimb at the ventral side at stage 34. Sections were double-stained for R-cadherin (A, D, G) and N-cadherin (B, E, H). (C) and (F) are Nomarski optics photomicrographs of the corresponding field, ca, cartilage; sm, skeletal muscle. Scale bars, 50 μm for (A –C), 20 μm for (D –F), and 200 μm for G, H.
The R-cadherin expression in skeletal muscle seems to diminish at later developmental stages since we could not detect this cadherin as an intense band if tissues of 10.5-day embryos were used in immunoblot analysis (see above); a similar down-regulation of expression was observed for N-cadherin during development (Hatta et al. 1987). The notochord continues to express R-cadherin strongly at least until day 8.5 (Fig. 2E). Some other tissues also stain for R-cadherin. These include the thyroid (data not shown) and some perichondrial regions of the cartilage (Fig. 7G,H).
Although these tissues co-express R- and N-cadherin, their distribution patterns are again not exactly the same. Endodermal organs, the lens of the eye, and differentiated cartilage did not give a positive signal for R-cadherin, at least at the developmental stages studied (day 3 –8.5).
Discussion
The present results demonstrate that the overall tissue distribution of R-cadherin is similar to that of N-cadherin. Both of them are expressed in neural tissues, the notochord, the myotome, early skeletal muscles and parts of the ectoderm. Such a pattern was not observed for the other cadherins so far identified. These findings, together with the molecular similarity of the two cadherins, suggest that R- and N-cadherin are more closely related to each other than to the other cadherins from an evolutionary point of view.
In spite of their apparent similarities in tissue distribution, the expression patterns of R- and N-cadherin were not identical. Firstly, N-cadherin is abundantly expressed in other tissues such as the connective tissues of endodermal organs and the lens (Hatta et al. 1987; Duband et al. 1988), while R-cadherin was not detected in these tissues. Secondly, the expression of R-cadherin occurs at later developmental stages than that of N-cadherin. For example, N-cadherin appears in the neural tube from the beginning of its formation, while R-cadherin appears only when some neuronal differentiation has taken place. A similar delay in R-cadherin expression was also observed during somite development. N-cadherin is expressed in this mesoderm-derived tissue even before somite morphogenesis begins (Hatta et al. 1987 ; Duband et al. 1988), but R-cadherin expression occurs only at later stages when the myotome is formed. Thirdly, the distribution of these molecules in a given tissue is not identical. This was seen not only in the tissues composed of heterogeneous cell types such as neural tissues, but also in those with relatively homogeneous cell types such as skeletal muscles.
We previously proposed that cadherin-mediated specific cell –cell adhesion may contribute to the segregation of cells in the development of the nervous system as well as of other tissues (Takeichi, 1988, 1990, 1991; Takeichi et al. 1991). In our recent work, we demonstrated that cells expressing R-cadherin can adhere to cells expressing N-cadherin but that the cells tend to form homotypic clusters within their chimeric aggregates in vitro (Inuzuka et al. 1991). The same mechanism could work for cell segregation in vivo, in the tissues expressing both of these cadherins.
From this point of view, the differential expression of R- and N-cadherin observed in many tissues is interesting. For example, in the early neural tube, R-cadherin is transiently expressed in the motor column and, concomitantly, N-cadherin expression in this region is suppressed. This pattern suggests that R-cadherin may function in placing early motor neurons together to establish the motor column, although other molecules might also be involved in such a process. In early trigeminal ganglia, R-cadherin expression is restricted to certain regions, while N-cadherin is distributed homogeneously. It is therefore again possible that R-cadherin may contribute to the selective grouping of particular sensory neurons. At later developmental stages, R- and N-cadherin are also differentially expressed in various regions of the spinal cord, including the ventral commissure, the floor plate and the dorsal white column, and also in the peripheral nervous system. Similar patterns of differential expression of these two cadherins were also found in the retina (Inuzuka et al. 1991). All these observations are consistent with the notion that cadherins may be involved in the segregation of heterogeneous cell types in the nervous system.
The present study also provides intriguing information on cadherin expression in skeletal muscle. This tissue expresses not only N-cadherin but also R-cadherin. Surprisingly, their distribution patterns in a muscle are different. This observation suggests that cadherin expression in myoblasts or myotubes in a muscle is variable and that these tissues express different amounts of R- or N-cadherin depending on their phenotype or location. Knudsen et al. (1990) suggested that N-cadherin may be involved in the fusion of myoblasts. If both R- and N-cadherin are involved in myoblast fusion, these molecules may control the fusion pattern of myoblasts; that is, the binding specificities of these cadherins may lead to the selective fusion between particular myoblasts. Such a mechanism could be important in muscle morphogenesis. During these observations, we did not find any correlation between the differential expression of R- and N-cadherin and slow versus fast muscle differentiation.
It should be noted that new members of the cadherin family have recently been identified, and many of them are expressed in the nervous system (Ranscht and Bronner-Fraser, 1991; Napolitano et al. 1991; Suzuki et al. 1991). Therefore, the actual pattern of cadherin expression in tissues is likely to be much more complex than we may currently assume. Such a complexity in the expression of adhesion molecules may be required for organizing a heterogeneous mass of cells into a highly ordered tissue architecture.
ACKNOWLEDGEMENTS
We thank H. Fujisawa for monoclonal antibodies to neuro filaments. This work was supported by research grants from the Ministry of Education, Science and Culture of Japan, the Special Coordination Fund of the Science and Technology Agency of the Japanese Government and the Naito Foundation. C.R. was a recipient of a Fellowship from the Japan Society for the Promotion of Sciences.