Early epochal maps of two different cell adhesion molecules - PNAS

Proc. Natl. Acad. Sci. USA Vol. 80, pp. 4384-4388, July 1983 Developmental Biology

Early epochal maps of two different cell adhesion molecules (embryogenesis/organogenesis/embryonic induction/molecular embryology)

G. M. EDELMAN*, W. J. GALLIN*, A. DELOUVIEt, B. A. CUNNINGHAM*,

AND J.-P. THIERYt

*The Rockefeller University, 1230 York Avenue, New York, New York 10021; and tInstitut d'Embryologie du Centre National de la Recherche Scientifique et du College de France, 49 bis Avenue de la Belle Cabrielle, 94130 Nogent/Marne, France

Contributed by Gerald M. Edelman, April 11, 1983 ABSTRACT N-CAM, the neural cell-adhesion molecule, has previously been found to be expressed during several epochs of development and function, first as an early marker in embryogenesis, later during organogenesis, and finally in adult life. LCAM, the liver cell-adhesion molecule, has now been localized in embryonic and adult tissues of the chicken by fluorescent antibody techniques. In the early embryonic epoch, L-CAM and NCAM appeared in epiblastic and hypoblastic tissues. L-CAM was distributed thereafter across all three germ layers. By the onset of neurulation, however, L-CAM disappeared in the region of the neural plate. and N-CAM increased in amount in that region. LCAM appeared strongly on all budding endodermal structures (liver, pancreas, lung, thyroid, parathyroid, thymus, and bursa of Fabricius) whereas N-CAM appeared most strongly in the neural plate, neural tube, and in cardiac mesoderm but was not found in endodermal derivatives. In placodes, both L-CAM and N-CAM were present until the formation of definitive neural structures, at which time L-CAM disappeared. In kidney precursors, the two CAMs followed a complex reciprocal pattern of appearance and disappearance. For the most part, however, the distributions of the two molecules did not overlap during organogenesis. Like NCAM, L-CAM persisted in a distinctive pattern of expression in adult tissues. During embryonic development, the two different CAMs were distributed on tissues derived from more than twothirds of the early embryonic surface. Interpretation of maps summarizing CAM distributions over a defined developmental epoch suggested a key role for both L-CAM and N-CAM in embryonic induction. Consistent with this interpretation and with the fact that the continuity of germ layers is lost when organ rudiments are formed, neither of the CAMs was limited in distribution to a single germ layer. The regions of the early epochal maps that lacked both L-CAM and N-CAM comprised some portions of the splanchnopleure and somatopleure. Certain adult tissues that derive from this lateral plate mesoderm such as smooth muscle also lacked L-CAM and N-CAM. Such observations suggest that at least one more CAM may exist in these and similarly derived tissues.

dynamic and sometimes transient. At later stages, N-CAM is deployed for formation of specific neural patterns (1, 2). During development, CAMs can undergo various forms of local cell surface modulation (3) either by chemical modification (4) or by changes in their prevalence on particular cells (1, 2). These modulation mechanisms, the widespread embryonic distribution of N-CAM (2), and the characterization (5) of a second major CAM from liver cells (L-CAM) prompted the conjecture (1) that only a small number of different CAMs will be found in the earliest epochs of embryogenesis. If this "small number" conjecture is correct, then these few molecules would be found distributed on a wide variety of different tissues during different developmental epochs. In the present study, we have localized L-CAM in the early embryogenetic epoch, in the organogenetic epoch, and in adult tissues of the chicken. The temporal and spatial distribution patterns of both L-CAM and N-CAM were compared with fate maps to yield maps of those cells that give rise to tissues expressing CAMs at successive developmental times. An analysis of these epochal maps supports the small-number conjecture mentioned above. The combined results suggest that CAMs are key molecules in the control of interacting systems of development and that they may play a specific role (1, 2) in embryonic induction. MATERIALS AND METHODS White Leghorn chicken embryos were staged according to (i) Vakaet (6) for the gastrulation period, (ii) number of somites, and (iii) Hamburger and Hamilton (7) for older stages. Embryos were fixed in 3.7% formaldehyde/phosphate-buffered saline (Pi/NaCI) for 1 hr at room temperature. After extensive washing with Pi/NaCl, embryos were infiltrated with increasing concentrations (12-18%) of sucrose in Pi/NaCl at 4°C. Embryos were wrapped in adult mouse abdominal muscle to facilitate handling and were mounted in OCT compound (LabTek Products, Naperville, IL) on frozen isopentane maintained in liquid nitrogen vapors; 8- to 10-,um sections made in a cryostat (Bright Instrument, Huntingdon, England) were attached to gelatin-coated slides according to Lohmann et al. (8). Immunofluorescence labeling was carried out with rabbit anti-LCAM or anti-N-CAM IgG (50 jig/ml) in Pi/NaCl containing bovine serum albumin at 5 mg/ml for 1 hr at room temperature; after washing with Pi/NaCl, sections were incubated for 30 min with a 1:150 dilution of fluorescein-conjugated sheep anti-rabbit IgG (Institut Pasteur, Paris). Slides were mounted in 90% glycerol/Pi/NaCl, pH 8.0/0.1% p-phenylenediamine to prevent bleaching (9). Sections were observed by fluorescence microscopy under epi-illumination with a Leitz Orthoplan (Leitz, Weitzlar, Federal Republic of Germany). Photo-

Cell adhesion plays major roles in the establishment and maintenance of tissues and organs during the earliest stages of embryogenesis as well as during adult life. The identification of cell-adhesion molecules (CAMs) from neural tissue (N-CAM) and liver (L-CAM) and knowledge of their structures and binding mechanisms have made it possible to suggest functions for CAMs in early embryogenesis, in organogenesis, and within mature tissues (1). During embryogenesis, N-CAM appears very early (2) and is enhanced in amount in regions concerned with primary induction (neural plate and tube, notochord, and somites). During secondary induction, it appears in neural crest cells, placodes, limb buds, cardiac mesoderm, and mesonephric primordia; the pattern of appearance in these structures is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: Pi/NaCl, phosphate-buffered saline; L-CAM, liver celladhesion molecule; N-CAM, neural cell-adhesion molecule.

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Developmental Biology: Edelman et al. graphs were taken with a Leitz Vario-Orthomat camera and TriX film (Kodak). Adult organs were snap frozen in an isopentane/dry ice bath and mounted in Lipshaw's M-1 medium; 10-,um sections were cut, attached to plain glass slides, air-dried, and fixed in 3.7% formaldehyde/Pi/NaCl for 15 min. Staining was as above, except that 1% goat serum was present in all solutions and rhodamine-conjugated goat-anti-rabbit Ig (Miles) was used as the

second antibody. RESULTS Early Expression of L-CAM and N-CAM in Embryogenesis. L-CAM and N-CAM were readily detected in the blastoderm by stage 2 of Vakaet (6). The upper layer and both the primitive endodermal cells and, the hypoblast cells that were released from the upper layer were stained by anti-N-CAM (Fig. 1A) and anti-L-CAM antibodies (Fig. 1B). These cells are released directly, without active migration through the primitive streak (10). Cells in the deep layer, which give rise to the extraembryonic endoderm, were more intensely labeled. At the -level of the primitive streak, both the upper layer cells and the cells that sank in the streak as a confluent mass contained LCAM. In contrast, migrating cells that were released from the streak no longer stained for L-CAM (Fig. 1C). Most of these cells undergo ingression and participate in the formation of the mesoderm, but some cells just caudal to the region of the presumptive Hensen's node were incorporated in the deep layer and progressively displaced the hypoblast (11); the definitive endoderm deriving from those cells expressed small amounts of L-CAM. Hensen's node stained intensely for L-CAM and N-CAM (2) in all layers and throughout its existence. As the primitive streak regressed (stage 6 of Vakaet), L-CAM disappeared rapidly from the medullary plate developing ahead of Hensen's node under the control of the presumptive notochord. In contrast, N-CAM staining increased rapidly at the level of the neural plate and was also found in the newly condensed lateral mesoderm (Fig. 1D). In time, the staining for L-CAM became restricted to the lateral ectoderm (Fig. 1E). The later history of the lateral ectoderm involved the formation of several placodes, which remained labeled with anti-L-CAM antibodies until they differentiated into a variety of tissues including cranial sensory ganglia and adenohypophysis (data not shown). N-CAM was also pres-

Proc. Natl. Acad. Sci. USA 80 (1983)

(2) at these early stages of placode development and remained in all the derivative neural structures after the disappearance of L-CAM. Appearance of L-CAM in Budding Endodermal Structures. Although the extraembryonic endoderm acquired L-CAM during its formation, the molecule was not easily detected in the cephalic region until the six-somite stage. The liver rudiment could be visualized with anti-L-CAM staining a few hours after it was determined (12) (Fig. 2A). Indeed, in every case in which gut appendages (lung, liver, pancreas, lymphoid organ) were established, a three-dimensional outgrowth of densely arranged cells staining strongly for L-CAM was found. Early stages in budding of the pharyngeal endoderm could be detected easily with fluorescent anti-L-CAM, and L-CAM appeared to be more abundant at sites of such intense morphogenetic processes in the pharynx. Interactions between ectoderm and endoderm that are known to lead to the formation of the branchial arches were reflected in L-CAM expression. At the site of transitory junctions between the ectoderm and the endoderm, for example, all the cells were uniformly stained; after staining by anti-L-CAM antibodies the boundary between these two tissues could not be distinguished (Fig. 2B). Local expansion of the endoderm was also found to be highly organized, with prominent anti-L-CAM staining in several tissues such as the lung (Fig. 2C); in all such cases of budding, the stained cells remained in close apposition. In the primary lymphoid organ rudiments, L-CAM was restricted to the endodermal component; neither the surrounding mesenchyme nor the hemopoietic cells that colonize the thymus and the bursa of Fabricius (13) acquired L-CAM at their surface (data not

ent

shown). On mesodermally derived tissues, only those elements comprising parts of the urogenital system showed L-CAM. The Wolffian duct was labeled during its earliest stage of development (15-somite stage). Primary condensates of nephric mesenchyme and the subsequent early tubules, which had been previously found to express N-CAM (2), lost N-CAM and developed further into more extensive tubules while expressing L-CAM at their surface (Fig. 2D). Thus, the sequence of expression of different CAMs in these tissues was Wolffian duct (LCAM), mesonephric rudiment (N-CAM), extension of mesonephric tubules (L-CAM).

_b=

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FIG. 1. Early appearance of L-CAM and N-CAM during the formation of three primary germ layers. (A) Full primitive streak stage [stage 7 of Vakaet (6)]: laterad to the streak, aggregates of cells released from the epiblast (ep) progressively replaced the cells of the endophyll (end). In addition to the epiblast, both the endophyll and the presumptive hypoblast (hyp) are stained with anti-N-CAM. (B) Similar stage, same region: cells from the same aggregates are also stained with anti-LCAM. (C) Stage 9: head-fold primitive streak (ps) level. In the upper level, the epiblast is labeled by anti-L-CAM antibodies; middle layer cells (ml) that have just been released from the upper layer are also stained. Migrating cells (arrows) are not stained. en, Definitive endoderm. (D) Ten-somite stage; neurulation and ectoderm formation: slightly below the last-formed somite, N-CAM was found in most tissues but the staining intensity was increased dramatically in the neural tube. nf, Neural fold; nt, neural tube; e, ectoderm; sm, somitic mesenchyme; en, endodern. (E) Same stage, same level: LACAM is found in all ectodermal cells. The last cells stained (arrow) are in the neural fold; note that the neural tube and the somitic mesenchyme (sm) donot stain with antiL-CAM and that the endoderm is weakly stained. (Bars = 30 Am.)

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FIG. 2. Appearance of L-CAM in morphogenesis of the liver, pharynx, and lung rudiments and in the ontogeny of the mesonephros. (A) Twenty-five-somite stage; level of the eighth somite: the liver rudiment (lv) appears as a rapidly expanding mass of aggregated cells budding from the open anterior intestine (ai). L-CAM is expressed in all of the presumptive liver cells. en, Endoderm; spm, splanchnopleural mesoderm. (B) Thirtyfive-somite stage; second branchial cleft: the expanding floor of the pharynx (ph) expresses much more L-CAM than the dorsal aspect of the pharynx. The arrow indicates that no discontinuity between ectoderm (e) and endoderm (en) can be discerned by anti-L-CAM staining. ba, Branchial arch. (C) Forty-somite stage; level of the ninth somite: the two lung rudiments (lg) are formed by outgrowing masses of endodermal cells (en) from the pharynx. All these cells remain in close contact while expanding to form a pair of lung rudiments. b-CAM is present on the surface of all the endodermal cells, particularly at the level of the evagination. (D) Stage 25 (Hamburger and Hamilton, ref. 7): proximal tubules that have formed early after the arrival of the Wolffian duct (wd) are now stained with anti-L-CAM antibodies while the newly formed distal tubules remain unstained. mt, Mesonephric tubules. (Bars = 40 ,um.)

Distribution of L-CAM in the Adult. L-CAM persisted in the organs of mature adults that arose from primordia that expressed L-CAM. As shown in Fig. 3A, the proliferative zone of the skin was intensely labeled. Adult hepatocytes also stained uniformly (Fig. 3B). In some regions of the digestive tract, the distribution of L-CAM on the endodermal epithelial cells was not homogeneous. In the pancreas, for example (Fig. 3C), the exocrine cells were stained at their apical-lateral surface whereas, in the proventricular glands (Fig. 3D), the staining was mostly concentrated at the basal surface. The distributions of N-CAM (2) and L-CAM during three developmental epochs-early embryonic, organogenetic, and adult-are summarized in Table 1. Like N-CAM, L-CAM lasts into the adult epoch and its expression in certain tissues derived from the ectoderm and endoderm is remarkably stable. Some mesodermal derivatives also contained L-CAM. An epochal map of the CAMs (Fig. 4A) based on the fate map described by Rudnick (14) was constructed, taking into account marking with carbon or iron particles (6, 15), grafts labeled by tritiated thymidine (16), experiments with chimeric chicken-quail blastoderm (11), and unpublished work of L. Vakaet (personal communication). This map revealed a well-delineated central area of blastoderm that will later express N-CAM. Given the staining intensities and the progressive disappearances of N-CAM in several tissues, we hypothesize that more quantitative studies will show a concentration gradient of N-CAM that increases from the lateral mesoderm to the neural plate (Fig. 4A). The central region expressing N-CAM was surrounded by a L-CAM region corresponding to the embryonic and extraembryonic ectoderm as well as the definitive endo-

derm. A particularly interesting feature of this map is the continuity of the endoderm with other L-CAM regions. The most caudal and unstained area corresponds to the earliest invaginating cells that participate in the formation of extraembryonic hemangioblastic cords. The only other region that remained unstained corresponded to that part of the splanchno- and somatopleural mesoderm that gives rise to smooth muscle and possibly blood elements. The sequential expression of the two CAMs over the three developmental epochs is schematized in Fig. 4B. While N-CAM and L-CAM both last into the adult epoch, the expression of N-CAM is particularly enhanced during primary induction. Secondary inductions, such as those related to the formation of sensory ganglia, also involve an enhancement of N-CAM; in contrast, other secondary inductions, such as the formation of kidney primordia, are more complex and demonstrate a reciprocal expression of both kinds of CAM. DISCUSSION The results of the present experiments can be combined with previous analyses (1, 2, 4) to yield a number of generalizations on the role of CAMs in various developmental epochs. Extension of these conclusions to a detailed interpretation of mechanisms must await more quantitative studies. Both L-CAM and N-CAM appear at a very early stage of development (Fig. 1 A and B). The distributions of the two CAMs overlap at this time but diverge sharply by the time of neurulation. An egregious exception to this rule is the simultaneous appearance and persistence of N-CAM and L-CAM in placodes; eventually, however, L-CAM disappears leaving N-CAM

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FIG. 3. (A) Adult skin from a foot pad shows stainmgfor b-CAM in the stratum germinativum (sg) but not in the dermis (d). se, Squamous epithelium. (B) Adult liver cells are stained on the cell surface with an apparent concentration of stain on the surfaces where two layers of cells adjoin. bv, Blood vessel. (C) Adult pancreas is stained on the apical-lateral surfaces of the acinar cells. is, Islet. (D) Glands of the adult proventriculus are stained on the basal surfaces of the glandular epithelium. (Bars = 30 gim.)

Developmental Biology:

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Proc. Natl. Acad. Sci. USA 80 (1983)

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Table 1. Distribution of b-CAM and N-CAM in three epochs 0- to 3-day embryo 5- to 13-day embryo Adult*

L-CAM Ectoderm Upper layer Epiblast Presumptive epidermis Placodes Mesoderm Wolffian duct

Endoderm Endophyll Hypoblast Gut primordium and buddings

Epidermis Extraembryonic

Skin: stratum

germinativum

ectoderm

Wolffian duct Ureter Most meso- and metanephric epithelium

Epithelium of Kidney

Epithelium of Oesophagus Proventriculus Gizzard Intestine Liver Pancreas Lung Thymus Bursa Thyroid Parathyroid Extraembryonic endoderm

Epithelium of Tongue Oesophagus

Oviduct

Proventriculus Gizzard Intestine Liver Pancreas

Lung Thymus Thyroid Parathyroid Bursa

N-CAM Ectoderm Upper layer Epiblast Neural plate Placodes Mesoderm Notochord Somites Dermomyotome Somato- and splanchnopleural mesoderm Heart Mesonephric

Nervous system

Nervous system

Striated muscle Adrenal cortex Gonad cortex Some mesonephric and metanephric epithelia Somato- and splanchnopleural elements Heart

primordium *

It is not yet known whether adult striated muscle contains N-CAM.

in the neural structures derived from placodes. It is not yet known whether the two CAMs appear on the same or different cells within the placodes but, in either case, it is clear that N-CAM and L-CAM do not bind to each other (1, 5). Their coexistence at different levels of modulation and binding strengths (1) might therefore serve as a segregating mechanism for groups of different cells within a tissue. The detection of L-CAM at stages comparable with the blastocyst in mammals, the calcium dependence of its binding function, and the similarities in the size of fragments released from the cell surface by trypsin (5) support the suggestion (1) that L-CAM and uvomorulin, a surface protein involved in compaction of mouse embryos (17), are identical or homologous molecules. But L-CAM also plays other roles in early embryogenesis, being present for example on the entire mass of Hensen's node and presumably on its constituent cells during their displacement caudally. While increases in staining for N-CAM accompany neuru-

B NP '.

A.t

7

T

K

t t

primary secondary Induction

I

E

0

A

FIG. 4. (A) CAM map showing actual and presumptive territories for L-CAM and N-CAM as well as territories not accounted for by either. The map collapses the CAM distributions at several times (B) into one diagram. IU, b-CAM distribution, a calcium dependent system; El, NCAM distribution, a calcium-independent system; vertical bar, primitive streak (PS); Ec, intraembryonic and extraembryonic ectoderm; En, endoderm; H, heart; Ha, hemangioblastic area; LP, lateral plate (splanchno-somatopleural mesoderm); N, nervous system; No, prechordal and chordo-mesoderm; S, somite; Sm, smooth muscle. The presumptive smooth muscle area is covered neither by the N-CAM nor the b-CAM distribution. U, urogenital system. Construction of the map was based on the classical map of Rudnick (14) and unpublished work of Vakaet, particularly in respect to the position of the definitive endoderm. In actuality, b-CAM and N-CAM are widely distributed at the prestreak stage. N-CAM increases later in derivatives of cells from those regions appropriately labeled in the map; note a progressive decrease of N-CAM from the neural region (N) to more distal regions. Note also that the b-CAM distribution crosses the border between the presumptive endodermal and mesodermal areas. (B) Epochal stages showing the sequential or alternative appearances of L-CAM and N-CAM in several structures: NP, neural plate; PI, placodes; K, kidney. The wide bar represents b-CAM while single lines represent embryonic N-CAM. Late expansion of the N-CAM lines represents the E -- A conversion (1, 4) which so far is not definitively proven for ganglia (dotted wide arrow). The bottom line represents epochs: E, early embryogenesis; 0, organogenesis; A, adult.

lation (1, 2), L-CAM dominates during budding and outgrowth of organ rudiments. During organogenesis, L-CAM appears and remains on all endodermally derived structures representing budding-the liver, pancreas, lung, thyroid, thymus, and bursa of Fabricius. Although quantitative studies remain to be carried out, it appears that L-CAM staining increases in intensity particularly in such regions of budding and in regions where fusions occur between two germ layers such as the areas of junction that form the branchial clefts. A fundamental question raised by these observations concerns control of the key differentiation events leading to expression of the different CAMs. Both the temporal relationships (Fig. 4B) of the enhanced appearances of L-CAM and NCAM and the map of their distribution in early embryogenesis (Fig. 4A) suggest the hypothesis that CAM expression is associated with inductive events. We stress that the role of these molecules has not been strictly defined by functional and bio-

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chemical experiments; nonetheless, the sequence of enhanced staining and disappearance is strikingly correlated with both primary and secondary inductions. In all cases, the appropriate CAM marker appeared prior to the corresponding morphogenetic event. Both L-CAM and N-CAM vary in their dynamics of appearance and distribution in early embryonic, organogenetic, and adult epochs. Neither molecule is restricted to derivatives of only one germ layer; instead, each appears to be distributed in relation to various potential functions of cell adhesion in each respective epoch. It is notable, however, that L-CAM is seen on extraembryonic tissues and that N-CAM is not present on any endodermal derivatives (Table 1). Thus, while the prediction (1) that L-CAM would be found on endodermal anlagen is confirmed, the distribution of this molecule is even more general than that of N-CAM. Together, the two CAMs are expressed on derivatives of more than two-thirds of the early embryonic surface (Fig. 4A). Only some of splanchnopleural and somatopleural derivatives fail to stain for these molecules; as shown in Fig. 2D, however, certain genitourinary derivatives of the splanchnopleure do stain in a dynamic fashion for one or the other CAM. Inasmuch as the two CAMs differ markedly in structure (5, 18, 19) and in their mechanisms of binding and modulation (1), the differential appearances described here must themselves reflect different regimes of binding affinities in cell-cell adhesion. Determination of the exact nature of these regimes and their relation to formation of different tissue structures will require more aggressive experiments and new assays. It is already known, however, that N-CAM undergoes local surface modulation by embryo to adult conversion, leading to a decrease in sialic acid residues (1, 4); so far, no chemical modulation event has been observed for L-CAM. Moreover, the N-CAM binding mechanism is calcium independent whereas L-CAM binding is calcium dependent (5, 20). Nonetheless, both N-CAM and LCAM show modulation by changes in cell surface prevalence. This form of modulation was seen previously for N-CAM on neural crest cells (2) and, in the present work, for L-CAM appearing at sites of budding and fusion as well as on early migrating cells. The polar staining observed in certain adult tissues such as the exocrine pancreas and glandular structures of the proventriculus (Fig. 3 C and D) raises the possibility that local cell surface modulation can also occur by means of polar redistribution of CAMs on the surface of individual cells. As shown in Table 1 and in the CAM map (Fig. 4A), there is a failure to map either CAM in some regions that derive from the somatopleure, such as smooth muscle. Intraembryonic endothelial and hemopoietic cells are also not accounted for by the two CAMs. This observation, and the consistent picture provided by the persistence into adult life of L-CAM and N-CAM (Table 1), raises the possibility that a third CAM may be found on certain adult structures deriving from the somatopleure and splanchnopleure. An obvious prediction is that antibodies to another CAM-present for example, on smooth muscle cellsmay fill the major gap in the CAM map. A minimal set of three "primitive" CAMs may have evolved to be expressed in the earliest epoch and also to be used in later ones. If this is so, then other "late" CAMs would not appear until the epoch of organogenesis. Alternatively, all CAMs may appear early but have grossly uneven distributions in the map. Moreover, certain morphogenetic events may require only coordination (1) between modulation of CAMs and of substrate adhesion molecules; if this is the case, there will be blank regions in the CAM map. In any event, it seems likely that the total number of different CAMs will not be large. With three different modulation mechanisms (1) and even a small number of different CAMs, each acting to alter or limit primary pro-

Proc. Natl. Acad. Sci. USA 80 (1983)

cesses other than adhesion (cell migration, division, differentiation, and death), it is clear that quite complex patterns could emerge during histogenesis. One of the challenging problems related to this emergence concerns the possible existence of local positional information or of fine structure within a CAM map. A broad gradient of N-CAM distribution does appear in early maps (Fig. 4A) but local information may in fact be expressed only as a difference in cell surface modulation (1, 3) within a certain radius and need not necessarily be expressed in the form of a gradient. Although CAMs carry out major functions in cell adhesion and movement, as well as in cell-cell recognition during the critical epochs of development, the details of these functions remain to be worked out. Studies of the regulation of genes for CAMs would obviously be particularly revealing in the analysis of the molecular basis of inductive events. The fact that homologous CAMs are found in a variety of different vertebrate species (21-23) opens up the possibility of new comparative studies of the different evolutionary contributions of each of the different primary (1) processes of development to morphogenetic events in different species. The known adhesion mechanisms of mapped CAMs could serve as a fundamental reference for such interspecies comparisons. Finally, the widespread but disparate tissue distributions of different CAMs in the adult (Table 1) suggest that these molecules may eventually be implicated in a number of generalized disease states. We would like to thank Professor L. Vakaet for critical discussions and for making available his unpublished results. This work was supported by National Institutes of Health Grants HD-16550, AI-11378, and AM-04256 and grants from the Centre National de la Recherche Scientifique ATP 82 (3701) and the Institut National de la Sante et de la Recherche Medicale CRL 82-4018. 1. Edelman, G. M. (1983) Science 219, 450-457. 2. Thiery, J.-P., Duband, J.-L., Rutishauser, U. & Edelman, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, 6737-6741. 3. Edelman, G. M. (1976) Science 192, 218-226. 4. Rothbard, J. B., Brackenbury, R., Cunningham, B. A. & Edelman, G. M. (1982)J. Biol. Chem. 257, 11064-11069. 5. Gallin, W., Edelman, G. M. & Cunningham, B. A. (1983) Proc. Nat. Acad. Sci. USA 80, 1038-1042. 6. Vakaet, L. (1962) J. Embryol. Exp. Morphol. 10, 38-57.

7. Hamburger, V. & Hamilton, H. L. (1951)J. Morphol. 88, 49-92. 8. Lohmann, S. M., Walter, U., Miller, P. E., Greengard, P. & De Camilli, P. (1981) Proc. Natl. Acad. Sci. USA 78, 653-657. 9. Johnson, G. D. & Nogueira-Araujo, G. M. C. (1981)J. Immunol. Methods 43, 349-350. 10. Duband, J.-L. & Thiery, J.-P. (1982) Dev. Biol. 94, 337-350. 11. Fontaine, J. & Le Douarin, N. M. (1977) J. Embryol. Exp. Morphol 41, 209-222. 12. Le Douarin, N. M. (1975) Med. Biol. 53, 427-455. 13. Le Douarin, N. M. (1978) in Differentiation of Normal and Neoplastic Hematopoietic Cells, eds. Clarkson, B., Marks, P. A. & Till, J. E. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 5-31. 14. Rudnick, D. (1948) Ann. N.Y. Acad. Sci. 49, 761-772. 15. Spratt, N. T. (1946)1. Exp. Zool. 103, 259-304. 16. Rosenquist, G. C. (1971) J. Embryol. Exp. Morphol. 25, 85-96. 17. Hyafil, F., Babinet, C. & Jacob, F. (1981) Cell 26, 447-454. 18. Hoffman, S., Sorkin, B. C., White, P. C., Brackenbury, R., Mailhammer, R., Rutishauser, U., Cunningham, B. A. & Edelman, G. M. (1982) J. Biol. Chem. 257, 7720-7729. 19. Cunningham, B. A., Hoffman, S., Rutishauser, U., Hemperly, J. J. & Edelman, G. M. (1983) Proc. Nat. Acad. Sci. USA 80, 31163120. 20. Brackenbury, R., Rutishauser, U. & Edelman, G. M. (1981) Proc. Natl. Acad. Sci. USA 78, 387-391. 21. Chuong, C.-M., McClain, D. A., Streit, P. & Edelman, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, 4234-4238. 22. Edelman, G. M. & Chuong, C.-M. (1982) Proc. Natl. Acad. Sci. USA 79, 7036-7040. 23. McClain, D. A. & Edelman, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, 6380-6384.