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

Early epochal maps of two different adhesion molecules (embryogenesis//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 dynamic and sometimes transient. At later stages, N-CAM is previously been found to be expressed during several epochs of deployed for formation of specific neural patterns (1, 2). development and function, first as an early marker in - During development, CAMs can undergo various forms of genesis, later during organogenesis, and finally in adult life. L- local cell surface modulation (3) either by chemical modifica- CAM, the cell-adhesion molecule, has now been localized in tion (4) or by changes in their prevalence on particular cells (1, embryonic and adult tissues of the chicken by fluorescent anti- 2). These modulation mechanisms, the widespread embryonic body techniques. In the early embryonic epoch, L-CAM and N- distribution of N-CAM (2), and the characterization (5) of a sec- CAM appeared in epiblastic and hypoblastic tissues. L-CAM was ond major CAM from liver cells (L-CAM) prompted the con- distributed thereafter across all three germ layers. By the onset CAMs will be of , however, L-CAM disappeared in the region of the jecture (1) that only a small number of different neural plate. and N-CAM increased in amount in that region. L- found in the earliest epochs of embryogenesis. If this "small CAM appeared strongly on all budding endodermal structures number" conjecture is correct, then these few molecules would (liver, , , , parathyroid, thymus, and bursa of be found distributed on a wide variety of different tissues dur- Fabricius) whereas N-CAM appeared most strongly in the neural ing different developmental epochs. plate, neural tube, and in cardiac but was not found In the present study, we have localized L-CAM in the early in endodermal derivatives. In placodes, both L-CAM and N-CAM embryogenetic epoch, in the organogenetic epoch, and in adult were present until the formation of definitive neural structures, tissues of the chicken. The temporal and spatial distribution at which time L-CAM disappeared. In precursors, the two patterns of both L-CAM and N-CAM were compared with fate CAMs followed a complex reciprocal pattern of appearance and maps to yield maps of those cells that give rise to tissues ex- disappearance. For the most part, however, the distributions of pressing CAMs at successive developmental times. An analysis the two molecules did not overlap during organogenesis. Like N- of these epochal maps supports the small-number conjecture CAM, L-CAM persisted in a distinctive pattern of expression in mentioned above. The combined results suggest that CAMs are adult tissues. During , the two different key molecules in the control of interacting systems of devel- CAMs were distributed on tissues derived from more than two- opment and that they may play a specific role (1, 2) in embry- thirds of the early embryonic surface. Interpretation of maps onic induction. summarizing CAM distributions over a defined developmental epoch suggested a key role for both L-CAM and N-CAM in em- bryonic induction. Consistent with this interpretation and with the MATERIALS AND METHODS fact that the continuity of germ layers is lost when rudi- to (i) ments are formed, neither of the CAMs was limited in distri- White Leghorn chicken were staged according bution to a single . The regions of the early epochal Vakaet (6) for the period, (ii) number of , maps that lacked both L-CAM and N-CAM comprised some por- and (iii) Hamburger and Hamilton (7) for older stages. Embryos tions of the splanchnopleure and somatopleure. Certain adult tis- were fixed in 3.7% formaldehyde/phosphate-buffered saline sues that derive from this such as smooth (Pi/NaCI) for 1 hr at room temperature. After extensive wash- muscle also lacked L-CAM and N-CAM. Such observations sug- ing with Pi/NaCl, embryos were infiltrated with increasing gest that at least one more CAM may exist in these and similarly concentrations (12-18%) of sucrose in Pi/NaCl at 4°C. derived tissues. Embryos were wrapped in adult mouse abdominal muscle to facilitate handling and were mounted in OCT compound (Lab- Cell adhesion plays major roles in the establishment and main- Tek Products, Naperville, IL) on frozen isopentane maintained tenance of tissues and organs during the earliest stages of em- in liquid nitrogen vapors; 8- to 10-,um sections made in a cryo- bryogenesis as well as during adult life. The identification of stat (Bright Instrument, Huntingdon, England) were attached cell-adhesion molecules (CAMs) from neural (N-CAM) to gelatin-coated slides according to Lohmann et al. (8). Im- and liver (L-CAM) and knowledge of their structures and bind- munofluorescence labeling was carried out with rabbit anti-L- ing mechanisms have made it possible to suggest functions for CAM or anti-N-CAM IgG (50 jig/ml) in Pi/NaCl containing CAMs in early embryogenesis, in organogenesis, and within bovine serum albumin at 5 mg/ml for 1 hr at room tempera- mature tissues (1). During embryogenesis, N-CAM appears very ture; after washing with Pi/NaCl, sections were incubated for early (2) and is enhanced in amount in regions concerned with 30 min with a 1:150 dilution of fluorescein-conjugated sheep primary induction (neural plate and tube, notochord, and so- anti-rabbit IgG (Institut Pasteur, Paris). Slides were mounted mites). During secondary induction, it appears in in 90% glycerol/Pi/NaCl, pH 8.0/0.1% p-phenylenediamine cells, placodes, limb buds, cardiac mesoderm, and mesoneph- to prevent bleaching (9). Sections were observed by fluores- ric primordia; the pattern of appearance in these structures is cence microscopy under epi-illumination with a Leitz Ortho- plan (Leitz, Weitzlar, Federal Republic of Germany). Photo- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: Pi/NaCl, phosphate-buffered saline; L-CAM, liver cell- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. adhesion molecule; N-CAM, neural cell-adhesion molecule. 4384 Downloaded by guest on October 1, 2021 Developmental Biology: Edelman et al. Proc. Natl. Acad. Sci. USA 80 (1983) 4385

graphs were taken with a Leitz Vario-Orthomat camera and Tri- ent (2) at these early stages of placode development and re- X film (Kodak). mained in all the derivative neural structures after the disap- Adult organs were snap frozen in an isopentane/dry ice bath pearance of L-CAM. and mounted in Lipshaw's M-1 medium; 10-,um sections were Appearance of L-CAM in Budding Endodermal Structures. cut, attached to plain glass slides, air-dried, and fixed in 3.7% Although the extraembryonic acquired L-CAM dur- formaldehyde/Pi/NaCl for 15 min. Staining was as above, ex- ing its formation, the molecule was not easily detected in the cept that 1% goat serum was present in all solutions and rho- cephalic region until the six- stage. The liver rudiment damine-conjugated goat-anti-rabbit Ig (Miles) was used as the could be visualized with anti-L-CAM staining a few hours after second antibody. it was determined (12) (Fig. 2A). Indeed, in every case in which RESULTS gut appendages (lung, liver, pancreas, lymphoid organ) were established, a three-dimensional outgrowth of densely ar- Early Expression of L-CAM and N-CAM in Embryogen- ranged cells staining strongly for L-CAM was found. Early stages esis. L-CAM and N-CAM were readily detected in the blas- in budding of the pharyngeal endoderm could be detected eas- toderm by stage 2 of Vakaet (6). The upper layer and both the ily with fluorescent anti-L-CAM, and L-CAM appeared to be primitive endodermal cells and, the cells that were more abundant at sites of such intense morphogenetic pro- released from the upper layer were stained by anti-N-CAM (Fig. cesses in the Interactions between en- 1A) and anti-L-CAM antibodies (Fig. 1B). These cells are re- pharynx. and leased directly, without active migration through the primitive doderm that are known to lead to the formation of the branchial streak (10). Cells in the deep layer, which give rise to the ex- arches were reflected in L-CAM expression. At the site of tran- traembryonic endoderm, were more intensely labeled. At the sitory junctions between the ectoderm and the endoderm, for -level of the , both the upper layer cells and the example, all the cells were uniformly stained; after staining by cells that sank in the streak as a confluent mass contained L- anti-L-CAM antibodies the boundary between these two tis- CAM. In contrast, migrating cells that were released from the sues could not be distinguished (Fig. 2B). streak no longer stained for L-CAM (Fig. 1C). Most of these Local expansion of the endoderm was also found to be highly cells undergo ingression and participate in the formation of the organized, with prominent anti-L-CAM staining in several tis- mesoderm, but some cells just caudal to the region of the pre- sues such as the lung (Fig. 2C); in all such cases of budding, sumptive Hensen's node were incorporated in the deep layer the stained cells remained in close apposition. In the primary and progressively displaced the hypoblast (11); the definitive lymphoid organ rudiments, L-CAM was restricted to the en- endoderm deriving from those cells expressed small amounts dodermal component; neither the surrounding of L-CAM. nor the hemopoietic cells that colonize the thymus and the bursa Hensen's node stained intensely for L-CAM and N-CAM (2) of Fabricius (13) acquired L-CAM at their surface (data not in all layers and throughout its existence. As the primitive streak shown). regressed (stage 6 of Vakaet), L-CAM disappeared rapidly from On mesodermally derived tissues, only those elements com- the medullary plate developing ahead of Hensen's node under prising parts of the urogenital system showed L-CAM. The the control of the presumptive notochord. In contrast, N-CAM Wolffian duct was labeled during its earliest stage of devel- staining increased rapidly at the level of the neural plate and opment (15-somite stage). Primary condensates of nephric mes- was also found in the newly condensed lateral mesoderm (Fig. enchyme and the subsequent early tubules, which had been 1D). In time, the staining for L-CAM became restricted to the previously found to express N-CAM (2), lost N-CAM and de- lateral ectoderm (Fig. 1E). The later history of the lateral ec- veloped further into more extensive tubules while expressing toderm involved the formation of several placodes, which re- L-CAM at their surface (Fig. 2D). Thus, the sequence of ex- mained labeled with anti-L-CAM antibodies until they differ- pression ofdifferent CAMs in these tissues was Wolffian duct (L- entiated into a variety of tissues including cranial sensory ganglia CAM), mesonephric rudiment (N-CAM), extension of meso- and adenohypophysis (data not shown). N-CAM was also pres- nephric tubules (L-CAM).

FIG. 1. Early appearance of L-CAM and N-CAM during the formation of three primary germ layers. (A) _b= Full primitive streak stage [stage 7 of Vakaet (6)]: lat- erad to the streak, aggregates of cells released from the (ep) progressively replaced the cells of the en- dophyll (end). In addition to the epiblast, both the en- dophyll and the presumptive hypoblast (hyp) are stained with anti-N-CAM. (B) Similar stage, same region: cells I from the same aggregates are also stained with anti-L- CAM. (C) Stage 9: head-fold primitive streak (ps) level. Inthe upper level, the epiblast is labeledby anti-L-CAM antibodies; middle layer cells (ml) that have just been released from the upper layer are also stained. Migrat- ing cells (arrows) are not stained. en, Definitive endo- derm. (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 mesen- chyme; 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 andthe somitic mesenchyme (sm) donotstain with anti- L-CAM and that the endoderm is weakly stained. (Bars = 30 Am.) Downloaded by guest on October 1, 2021 4386 Developmental Biology: Edelman et al. Proc. Nad Acad. Sci. USA 80 (1983) FIG. 2. Appearance of L-CAM in morphogenesis of the liver, pharynx, and lung rudiments and in the on- togeny ofthe mesonephros. (A) Twenty-five-somite stage; level of the eighth somite: the liver rudiment (lv) ap- pears 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, En- doderm; spm, splanchnopleural mesoderm. (B) Thirty- five-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 indi- cates that no discontinuity between ectoderm (e) and en- doderm (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, par- ticularly at the level of the evagination. (D) Stage 25 (Hamburger and Hamilton, ref. 7): proximal tubules that have formed early after the arrival ofthe 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 derm. A particularly interesting feature of this map is the con- the organs of mature adults that arose from primordia that ex- tinuity of the endoderm with other L-CAM regions. The most pressed L-CAM. As shown in Fig. 3A, the proliferative zone caudal and unstained area corresponds to the earliest invaginat- of the was intensely labeled. Adult hepatocytes also stained ing cells that participate in the formation of extraembryonic he- uniformly (Fig. 3B). In some regions of the digestive tract, the mangioblastic cords. The only other region that remained un- distribution of L-CAM on the endodermal epithelial cells was stained corresponded to that part of the splanchno- and not homogeneous. In the pancreas, for example (Fig. 3C), the somatopleural mesoderm that gives rise to and exocrine cells were stained at their apical-lateral surface whereas, possibly blood elements. in the proventricular (Fig. 3D), the staining was mostly The sequential expression of the two CAMs over the three concentrated at the basal surface. developmental epochs is schematized in Fig. 4B. While N-CAM The distributions of N-CAM (2) and L-CAM during three and L-CAM both last into the adult epoch, the expression of developmental epochs-early embryonic, organogenetic, and N-CAM is particularly enhanced during primary induction. adult-are summarized in Table 1. Like N-CAM, L-CAM lasts Secondary inductions, such as those related to the formation of into the adult epoch and its expression in certain tissues derived sensory ganglia, also involve an enhancement of N-CAM; in from the ectoderm and endoderm is remarkably stable. Some contrast, other secondary inductions, such as the formation of mesodermal derivatives also contained L-CAM. kidney primordia, are more complex and demonstrate a recip- An epochal map of the CAMs (Fig. 4A) based on the fate rocal expression of both kinds of CAM. map described by Rudnick (14) was constructed, taking into ac- count marking with carbon or iron particles (6, 15), grafts la- DISCUSSION beled by tritiated thymidine (16), experiments with chimeric The results of the present experiments can be combined with chicken-quail blastoderm (11), and unpublished work of L. previous analyses (1, 2, 4) to yield a number of generalizations Vakaet (personal communication). This map revealed a well-de- on the role of CAMs in various developmental epochs. Exten- lineated central area ofblastoderm thatwill later express N-CAM. sion of these conclusions to a detailed interpretation of mech- Given the staining intensities and the progressive disappear- anisms must await more quantitative studies. ances of N-CAM in several tissues, we hypothesize that more Both L-CAM and N-CAM appear at a very early stage of quantitative studies will show a concentration gradient of N-CAM development (Fig. 1 A and B). The distributions of the two CAMs that increases from the lateral mesoderm to the neural plate overlap at this time but diverge sharply by the time of neu- (Fig. 4A). The central region expressing N-CAM was sur- rulation. An egregious exception to this rule is the simultaneous rounded by a L-CAM region corresponding to the embryonic appearance and persistence of N-CAM and L-CAM in plac- and extraembryonic ectoderm as well as the definitive endo- odes; eventually, however, L-CAM disappears leaving N-CAM

tIt I FIG. 3. (A) Adult skin from a foot pad shows stain- a11 mgfor b-CAM in the stratum germinativum (sg) but not in the dermis (d). se, Squamous . (B) Adult liver cells are stained on the cell surface with an ap- parent concentration of stain on the surfaces where two layers of cells adjoin. bv, Blood vessel. (C) Adult pan- creas is stained on the apical-lateral surfaces of the aci- nar cells. is, Islet. (D) Glands ofthe adult proventriculus are stained on the basal surfaces of the glandular epi- thelium. (Bars = 30 gim.) Downloaded by guest on October 1, 2021 Developmental Biology: Edelman et aL Proc. Natl. Acad. Sci. USA 80 (1983) 4387 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 Skin: stratum Epiblast Extraembryonic germinativum Presumptive ectoderm epidermis Placodes Mesoderm Wolffian duct Wolffian duct Epithelium of Ureter Kidney Most meso- and Oviduct metanephric epithelium Endoderm Endophyll Epithelium of Epithelium of B Hypoblast Oesophagus Tongue Gut primordium Proventriculus Oesophagus NP and buddings Gizzard Proventriculus Intestine Gizzard '.- A.t 7 T Liver Intestine K Pancreas Liver t t Lung Pancreas primary secondary Induction Thymus Lung I Bursa Thymus E 0 A Thyroid Thyroid Parathyroid Parathyroid FIG. 4. (A) CAM map showing actual and presumptive territories Extraembryonic Bursa for L-CAM and N-CAM as well as territories not accounted for by either. endoderm The map collapses the CAM distributions at several times (B) into one diagram. IU, b-CAM distribution, a calcium dependent system; El, N- CAM distribution, a calcium-independent system; vertical bar, prim- N-CAM itive streak (PS); Ec, intraembryonic and extraembryonic ectoderm; En, Ectoderm endoderm; H, heart; Ha, hemangioblastic area; LP, lateral plate Upper layer Nervous system (splanchno-somatopleural mesoderm); N, nervous system; No, pre- Epiblast chordal and chordo-mesoderm; S, somite; Sm, smooth muscle. The pre- Neural plate sumptive smooth muscle area is covered neither by the N-CAM nor the Placodes b-CAM distribution. U, urogenital system. Construction of the map was Mesoderm based on the classical map of Rudnick (14) and unpublished work of Notochord Striated muscle Vakaet, particularly in respect to the position of the definitive endo- derm. In actuality, b-CAM and N-CAM are widely distributed at the Somites Adrenal cortex prestreak stage. N-CAM increases later in derivatives ofcells from those Dermomyotome Gonad cortex regions appropriately labeled in the map; note a progressive decrease Somato- and Some mesonephric and of N-CAM from the neural region (N) to more distal regions. Note also splanchno- metanephric epithelia that the b-CAM distribution crosses the border between the presump- pleural Somato- and splanchno- tive endodermal andmesodermal areas. (B) Epochal stages showingthe mesoderm pleural elements sequential or alternative appearances ofL-CAM and N-CAM in several Heart Heart structures: NP, neural plate; PI, placodes; K, kidney. The wide bar rep- Mesonephric resents b-CAM while single lines represent embryonic N-CAM. Late primordium 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). * It is not yet known whether adult striated muscle contains N-CAM. The bottom line represents epochs: E, early embryogenesis; 0, organ- ogenesis; A, adult. in the neural structures derived from placodes. It is not yet known whether the two CAMs appear on the same or different cells lation (1, 2), L-CAM dominates during budding and outgrowth within the placodes but, in either case, it is clear that N-CAM of organ rudiments. During organogenesis, L-CAM appears and L-CAM do not bind to each other (1, 5). Their coexistence and remains on all endodermally derived structures repre- at different levels of modulation and binding strengths (1) might senting budding-the liver, pancreas, lung, thyroid, thymus, therefore serve as a segregating mechanism for groups of dif- and bursa of Fabricius. Although quantitative studies remain ferent cells within a tissue. to be carried out, it appears that L-CAM staining increases in The detection of L-CAM at stages comparable with the blas- intensity particularly in such regions of budding and in regions tocyst in , the calcium dependence of its binding where fusions occur between two germ layers such as the areas function, and the similarities in the size of fragments released of junction that form the branchial clefts. from the cell surface by trypsin (5) support the suggestion (1) A fundamental question raised by these observations con- that L-CAM and uvomorulin, a surface protein involved in cerns control of the key differentiation events leading to compaction of mouse embryos (17), are identical or homologous expression of the different CAMs. Both the temporal relation- molecules. But L-CAM also plays other roles in early embryo- ships (Fig. 4B) of the enhanced appearances of L-CAM and N- genesis, being present for example on the entire mass of Hen- CAM and the map of their distribution in early embryogenesis sen's node and presumably on its constituent cells during their (Fig. 4A) suggest the hypothesis that CAM expression is as- displacement caudally. sociated with inductive events. We stress that the role of these While increases in staining for N-CAM accompany neuru- molecules has not been strictly defined by functional and bio- Downloaded by guest on October 1, 2021 4388 Developmental Biology: Edelman et al. Proc. Natl. Acad. Sci. USA 80 (1983) chemical experiments; nonetheless, the sequence of enhanced cesses other than adhesion (cell migration, division, differen- staining and disappearance is strikingly correlated with both tiation, and death), it is clear that quite complex patterns could primary and secondary inductions. In all cases, the appropriate emerge during histogenesis. One of the challenging problems CAM marker appeared prior to the corresponding morpho- related to this emergence concerns the possible existence of genetic event. local positional information or of fine structure within a CAM Both L-CAM and N-CAM vary in their dynamics of ap- map. A broad gradient of N-CAM distribution does appear in pearance and distribution in early embryonic, organogenetic, early maps (Fig. 4A) but local information may in fact be ex- and adult epochs. Neither molecule is restricted to derivatives pressed only as a difference in cell surface modulation (1, 3) of only one germ layer; instead, each appears to be distributed within a certain radius and need not necessarily be expressed in relation to various potential functions of cell adhesion in each in the form of a gradient. respective epoch. It is notable, however, that L-CAM is seen Although CAMs carry out major functions in cell adhesion on extraembryonic tissues and that N-CAM is not present on and movement, as well as in cell-cell recognition during the any endodermal derivatives (Table 1). Thus, while the predic- critical epochs of development, the details of these functions tion (1) that L-CAM would be found on endodermal anlagen is remain to be worked out. Studies of the regulation of genes for confirmed, the distribution of this molecule is even more gen- CAMs would obviously be particularly revealing in the analysis eral than that of N-CAM. Together, the two CAMs are ex- of the molecular basis of inductive events. The fact that ho- pressed on derivatives of more than two-thirds of the early em- mologous CAMs are found in a variety of different bryonic surface (Fig. 4A). Only some of splanchnopleural and species (21-23) opens up the possibility of new comparative somatopleural derivatives fail to stain for these molecules; as studies of the different evolutionary contributions of each of shown in Fig. 2D, however, certain genitourinary derivatives the different primary (1) processes of development to mor- of the splanchnopleure do stain in a dynamic fashion for one or phogenetic events in different species. The known adhesion the other CAM. mechanisms of mapped CAMs could serve as a fundamental Inasmuch as the two CAMs differ markedly in structure (5, reference for such interspecies comparisons. Finally, the wide- 18, 19) and in their mechanisms of binding and modulation (1), spread but disparate tissue distributions of different CAMs in the differential appearances described here must themselves the adult (Table 1) suggest that these molecules may eventually reflect different regimes of binding affinities in cell-cell adhe- be implicated in a number of generalized disease states. sion. Determination of the exact nature of these and regimes We would like to thank Professor L. Vakaet for critical discussions their relation to formation of different tissue structures will re- and for making available his unpublished results. This work was sup- quire more aggressive experiments and new assays. It is already ported by National Institutes of Health Grants HD-16550, AI-11378, known, however, that N-CAM undergoes local surface mod- and AM-04256 and grants from the Centre National de la Recherche ulation by embryo to adult conversion, leading to a decrease in Scientifique ATP 82 (3701) and the Institut National de la Sante et de sialic acid residues (1, 4); so far, no chemical modulation event la Recherche Medicale CRL 82-4018. has been observed for L-CAM. Moreover, the N-CAM binding whereas is 1. Edelman, G. M. (1983) Science 219, 450-457. mechanism is calcium independent L-CAM binding 2. Thiery, J.-P., Duband, J.-L., Rutishauser, U. & Edelman, G. M. calcium dependent (5, 20). Nonetheless, both N-CAM and L- (1982) Proc. Natl. Acad. Sci. USA 79, 6737-6741. CAM show modulation by changes in cell surface prevalence. 3. Edelman, G. M. (1976) Science 192, 218-226. This form of modulation was seen previously for N-CAM on 4. Rothbard, J. B., Brackenbury, R., Cunningham, B. A. & Edel- neural crest cells (2) and, in the present work, for L-CAM ap- man, G. M. (1982)J. Biol. Chem. 257, 11064-11069. pearing at sites of budding and fusion as well as on early mi- 5. Gallin, W., Edelman, G. M. & Cunningham, B. A. (1983) Proc. certain adult tis- Nat. Acad. Sci. USA 80, 1038-1042. grating cells. The polar staining observed in 6. Vakaet, L. (1962) J. Embryol. Exp. Morphol. 10, 38-57. sues such as the exocrine pancreas and glandular structures of 7. Hamburger, V. & Hamilton, H. L. (1951)J. Morphol. 88, 49-92. the proventriculus (Fig. 3 C and D) raises the possibility that 8. Lohmann, S. M., Walter, U., Miller, P. E., Greengard, P. & De local cell surface modulation can also occur by means of polar Camilli, P. (1981) Proc. Natl. Acad. Sci. USA 78, 653-657. redistribution of CAMs on the surface of individual cells. 9. Johnson, G. D. & Nogueira-Araujo, G. M. C. (1981)J. Immunol. As in Table 1 and in the CAM there Methods 43, 349-350. shown map (Fig. 4A), 10. Duband, J.-L. & Thiery, J.-P. (1982) Dev. Biol. 94, 337-350. is a failure to map either CAM in some regions that derive from 11. Fontaine, J. & Le Douarin, N. M. (1977) J. Embryol. Exp. Mor- the somatopleure, such as smooth muscle. Intraembryonic en- phol 41, 209-222. dothelial and hemopoietic cells are also not accounted for by the 12. Le Douarin, N. M. (1975) Med. Biol. 53, 427-455. two CAMs. This observation, and the consistent picture pro- 13. Le Douarin, N. M. (1978) in Differentiation of Normal and Neo- vided by the persistence into adult life of L-CAM and N-CAM plastic Hematopoietic Cells, eds. Clarkson, B., Marks, P. A. & Till, that a third CAM may be found J. E. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), (Table 1), raises the possibility pp. 5-31. on certain adult structures deriving from the somatopleure and 14. Rudnick, D. (1948) Ann. N.Y. Acad. Sci. 49, 761-772. splanchnopleure. An obvious prediction is that antibodies to 15. Spratt, N. T. (1946)1. Exp. Zool. 103, 259-304. another CAM-present for example, on smooth muscle cells- 16. Rosenquist, G. C. (1971) J. Embryol. Exp. Morphol. 25, 85-96. may fill the major gap in the CAM map. 17. Hyafil, F., Babinet, C. & Jacob, F. (1981) Cell 26, 447-454. A minimal set of three "primitive" CAMs may have evolved 18. Hoffman, S., Sorkin, B. C., White, P. C., Brackenbury, R., Mail- and also to be used in later hammer, R., Rutishauser, U., Cunningham, B. A. & Edelman, G. to be expressed in the earliest epoch M. (1982) J. Biol. Chem. 257, 7720-7729. ones. If this is so, then other "late" CAMs would not appear 19. Cunningham, B. A., Hoffman, S., Rutishauser, U., Hemperly, J. until the epoch of organogenesis. Alternatively, all CAMs may J. & Edelman, G. M. (1983) Proc. Nat. Acad. Sci. USA 80, 3116- appear early but have grossly uneven distributions in the map. 3120. Moreover, certain morphogenetic events may require only co- 20. Brackenbury, R., Rutishauser, U. & Edelman, G. M. (1981) Proc. Natl. Acad. Sci. USA 78, 387-391. ordination (1) between modulation of CAMs and of substrate D. P. & M. there will be blank re- 21. Chuong, C.-M., McClain, A., Streit, Edelman, G. adhesion molecules; if this is the case, (1982) Proc. Natl. Acad. Sci. USA 79, 4234-4238. gions in the CAM map. In any event, it seems likely that the 22. Edelman, G. M. & Chuong, C.-M. (1982) Proc. Natl. Acad. Sci. total number of different CAMs will not be large. With three USA 79, 7036-7040. different modulation mechanisms (1) and even a small number 23. McClain, D. A. & Edelman, G. M. (1982) Proc. Natl. Acad. Sci. of different CAMs, each acting to alter or limit primary pro- USA 79, 6380-6384. Downloaded by guest on October 1, 2021