REPORTS ture in vivo. So far, there is no evidence antibodies (Progen), affinity-purified rabbit anti- as follows. The staining of the lymphatic network in mouse collagen XVIII IgG (a gift from T. Pihlajaniemi), vivo was carried out as described (20). Briefly, that tumors can stimulate the growth of and rabbit anti-mouse laminin IgG (provided by E. 8-week-old mice were anesthetized and placed on a new lymphatic vessels (23), but further Lehtonen), by using the Vectastain ABC Elite kit heating pad to maintain a 37°C temperature. A 30- studies should establish the role of VEGF-C ( Vector Laboratories). Normal mouse or rabbit sera gauge needle, connected to a catheter filled with a were used as negative controls for the stainings. solution of FITC-dextran (2 M, 8 mg/ml in phos- in lymphangiomas and in tumor metastasis 27. In situ hybridization of sections was performed as phate-buffered solution), was injected intradermally via the lymphatic vasculature as well as in described [A. Kaipainen et al., J. Exp. Med. 178, into the tip of the tail. The solution was infused with a various other disorders involving the lym- 2077 (1993)]. The human VEGF-C antisense RNA constant hydrostatic pressure equivalent to a 50-cm phatic system and their treatment. probe was generated from linearized pCR™II plas- column of water (flow rate averaging roughly 0.01 mid (Invitrogen) containing an Eco RI fragment cor- ␮l/min) until the extent of network filling remained responding to nucleotides 628 through 1037 of hu- constant (approximately 2 hours). Flow rate and flu- REFERENCES AND NOTES man VEGF-C cDNA. The VEGFR-3 probe was de- orescence intensity were monitored continuously ______scribed earlier (18). The VEGFR-2 probe was an Eco throughout the experiment. The intravital fluores- 1. N. Ferrara and T. Davis-Smyth, Endocr. Rev.,in RI fragment covering base pairs 1958 through 2683 cence microscopy of blood vessels was as de- press. (GenBank accession number X59397, a gift from J. scribed (24). 2. B. Olofsson et al., Proc. Natl. Acad. Sci. U.S.A. 93, Rossant). 30. We thank E. Saksela for help with histological inter- 2576 (1996); S. Grimmond et al., Genome Res. 6, 28. For measurement of DNA synthesis, small (3 mm pretation; E. Lehtonen for help in electron micros- 124 (1996). by 3 mm) skin biopsies from four transgenic and copy; E. Hatva for critical reading of the manu- 3. V. Joukov et al., EMBO J. 15, 290 (1996); J. Lee et four control mice were incubated in Dulbecco’s script; E. Fuchs for the K14 expression cassette; al., Proc. Natl. Acad. Sci. U.S.A. 93, 1988 (1996). modification of Eagle’s medium with 10 ␮g/ml and E. Koivunen, M. Helantera¨ , T. Tainola, and E. 4. E. Kukk et al., Development 122, 3829 (1996). BrdU for 6 hours at 37°C, fixed in 70% ethanol for Rose for excellent technical assistance. Supported 5. D. Maglione, V. Guerriero, G. Viglietto, P. Delli-Bovi, 12 hours, and embedded in paraffin. After a 30-min through the Finnish Cancer Organizations, the M. G. Persico, Proc. Natl. Acad. Sci. U.S.A. 88, 9267 treatment with 0.1% pepsin in 0.1 M HCl at room Finnish Academy, the Sigrid Juselius Foundation, (1991). temperature to denature DNA, staining was carried the University of Helsinki, the State Technology 6. M. Klagsbrun and P. A. D’Amore, Cytokine Growth out as above with mouse monoclonal antibodies to Development Centre, and NIH. Factor Rev. 7, 259 (1996). BrdU (Amersham). 7. The human VEGF-C cDNA (GenBank accession 29. Fluorescence microlymphography was performed 22 January 1997; accepted 27 March 1997 number X94216) was blunt-end ligated to the Bam HI restriction site of the K14 expression cassette (8), and an Eco RI–Hind III fragment containing the K14 pro- moter, VEGF-C cDNA, and K14 polyadenylation sig- Geometric Control of Cell Life and Death nal was isolated and injected into fertilized oocytes of the FVB/NIH strain of mice. The injected zygotes Christopher S. Chen, Milan Mrksich, Sui Huang, were transplanted into oviducts of pseudopregnant C57BL/6 ϫ DBA/2J hybrid mice. We analyzed the George M. Whitesides, Donald E. Ingber* resulting founder mice for the presence of the trans- gene by polymerase chain reaction of tail DNA, with the primers: 5Ј-CATGTACGAACCGCCAG-3Ј and 5Ј- Human and bovine capillary endothelial cells were switched from growth to apoptosis AATGACCAGAGAGAGGCGAG-3Ј. The tail DNAs by using micropatterned substrates that contained extracellular matrix-coated adhesive were also subjected to endonuclease digestion, Southern blotting, and hybridization analysis using the islands of decreasing size to progressively restrict cell extension. Cell spreading also was transgene fragment as the probe. varied while maintaining the total cell-matrix contact area constant by changing the 8. R. Vassar, M. Rosenberg, S. Ross, A. Tyner, E. spacing between multiple focal adhesion-sized islands. Cell shape was found to govern Fuchs, Proc. Natl. Acad. Sci. U.S.A. 86, 1563 (1989). whether individual cells grow or die, regardless of the type of matrix protein or antibody 9. H. J. Leu and J. T. Lie, in Vascular Pathology (Chap- man & Hall, London, 1995), p. 509. to integrin used to mediate adhesion. Local geometric control of cell growth and viability 10. T. W. Fossum and M. W. Miller, J. Vet. Intern. Med. 6, may therefore represent a fundamental mechanism for developmental regulation within 283 (1992). the tissue microenvironment. 11. L. V. Leak, Microvasc. Res. 2, 361 (1970). 12. Y. Muragaki et al., Proc. Natl. Acad. Sci. U.S.A. 92, 8763 (1995); M. Rehn and T. Pihlajaniemi, ibid. 91, 4234 (1994). 13. M. Jeltsch et al., data not shown. he local differentials in cell growth and sons, adhesion-dependent control of apo- 14. M. Schmelz, R. Moll, C. Kuhn, W. W. Franke, Differ- T entiation 57, 97 (1994). viability that drive morphogenesis in com- ptosis has been assumed to be mediated by 15. K. Pajusola et al., Cancer Res. 52, 5738 (1992); F. plex tissues, such as branching capillary net- changes in integrin signaling. Analysis of Galland et al., Oncogene 8, 1233 (1993). works (1, 2), are controlled through modu- capillary regression in vivo, however, has 16. B. I. Terman et al., Biochem. Biophys. Res. Com- mun. 187, 1579 (1992); B. Millauer et al., Cell 72,83 lation of cell binding to extracellular matrix revealed that dying capillary cells remain in (1993). (ECM) (3–6). Local disruption of ECM by contact with ECM fragments, thus suggest- 17. J. Korhonen, A. Polvi, J. Partanen, K. Alitalo, Onco- pharmacologic or genetic means results in ing that the cell foreshortening caused by gene 9, 395 (1994). selective programmed cell death (apoptosis) ECM dissolution may be the signal that 18. A. Kaipainen et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3566 (1995). within adjacent cells (2, 6, 7). Soluble in- initiates the death program (2). This possi- 19. T. Yamaguchi, D. Dumont, R. Conion, M. Breitman, tegrin ␣V␤3 antagonists also induce apo- bility is supported by the finding that en- J. Rossant, Development 118, 489 (1993); D. Du- ptosis in cultured endothelial cells and pro- dothelial cells spread and grow on large mont et al., Dev. Dyn. 203, 80 (1995). 20. J. Folkman and Y. J. Shing, J. Biol. Chem. 267, mote capillary involution in vivo (8). Fur- (Ͼ100-␮m diameter) microcarrier beads 10931 (1992). thermore, death can be prevented by allow- (4), whereas they rapidly die when bound to 21. A. J. Leu, D. A. Berk, F. Yuan, R. K. Jain, Am. J. ing suspended cells to attach to immobilized small (4.5 ␮m) ECM-coated beads (10) Physiol. 267, 1507 (1994); M. A. Swartz, D. A. Berk, antibodies to integrins or by inhibiting ty- that cluster integrins and activate signaling R. K. Jain, ibid. 270, 324 (1996). 22. V. Joukov, unpublished data. rosine phosphatases (7, 9). For these rea- but do not support cell extension (11). 23. J. Folkman, N. Engl. J. Med. 334, 921 (1996). Understanding how this apoptotic 24. D. Fukumura et al., Cancer Res. 55, 4824 (1995). C. S. Chen, S. Huang, D. E. Ingber, Departments of switch is controlled in capillary cells has 25. For electron microscopy, tissue pieces from two Surgery and Pathology, Children’s Hospital–Harvard enormous clinical implications, because an transgenic skin biopsies were fixed in formaldehyde, - Medical School, Enders 1007, 300 Longwood Avenue, postfixed in 2% osmium tetroxide, and embedded in giogenesis is a prerequisite for tumor growth Boston, MA 02115, USA. LX 112. Sagittal ultrathin sections were studied with M. Mrksich and G. M. Whitesides, Department of Chem- (12). Thus, we set out to determine wheth- the Jeol1200EX electron microscope. istry, Harvard University, Cambridge, MA 02138, USA. er cell shape or integrin binding per se 26. For immunohistochemistry, we stained cryostat sec- tions of 5 to 10 ␮m from abdominal and back skin *To whom correspondence should be addressed. E-mail: governs life and death in these cells. We with anti-mouse desmoplakin I and II monoclonal [email protected] first measured apoptosis rates in suspended www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 276 ⅐ 30 MAY 1997 1425 human capillary endothelial cells attached 20 ␮m in diameter, cells spread until they switched on as cell and nuclear spreading to a range of different-sized beads coated took on the size and shape of the underlying were promoted (Fig. 2B). with fibronectin (FN). Most cells survived adhesive island (Fig. 1C). Significantly These results demonstrate that increased when spread on FN-coated planar dishes in more cells entered apoptosis when held in a cell spreading on a homogeneous, high- medium that contained saturating amounts round form on 20-␮m circles than when density coating of FN leads to cell survival of growth factors, whereas about 60% of spread on identically fabricated unpat- and growth. However, the total area of nonadherent cells entered the death pro- terned substrates (Fig. 1D). Furthermore, cell-ECM contact also increases under gram within 24 hours (Fig. 1, A and B). In the subtle decrease in cell and nuclear these conditions, so integrin binding, focal contrast, fewer than 10% of cells adherent spreading observed in cells on 10- compared adhesion formation, and accessibility to ma- to large (Ͼ25 ␮m) FN-coated beads under- with 20-␮m islands (Fig. 1C) was also ac- trix-bound growth factors (16) may all vary went programmed cell death. Unlike sus- companied by a statistically significant in- in parallel. To explore this mechanism pended cells, which remained small and crease in apoptosis (Fig. 1D). more fully, we evaluated apoptosis and spherical, these cells and their nuclei ap- Capillary cell spreading on ECM also peared to flatten as they extended around has been shown to modulate cell cycle pro- the beads (Fig. 1A). Importantly, as the gression (4, 11, 15). To determine the pre- bead diameter was decreased to 10 ␮m, cells cise spreading requirements for survival became more rounded, and the apoptotic rather than growth, we cultured cells syn- index increased to match that in nonadher- chronized in the quiescent phase of the cell ent cells (Fig. 1B). cycle on different-sized FN-coated adhesive The size of these spherical beads affects islands. The different-sized islands were not only the degree of cell and nuclear contained within a single substrate (Fig. spreading but also ECM curvature and bead 2A) to rule out the possibility that changes internalization. The 10-␮m beads appeared in cell behavior could be due to the release fully engulfed by cells within 4 hours, of paracrine growth modulators. When we whereas the 25-␮m beads were never fully plated cells on square-shaped islands coated internalized. To eliminate these complicat- with FN, square-shaped cells were produced ing factors, we used a microscale patterning that closely matched the size and shape of technique (13, 14) to fabricate planar ad- the adhesive island (Fig. 2A). Apoptosis hesive islands of defined size and shape, progressively declined when the island size separated by nonadhesive regions. When was increased from 75 to 3000 ␮m2, where- plated on circular FN-coated islands 10 or as DNA synthesis was concomitantly

Fig. 2. Effect of spreading on cell growth and Fig. 1. Effect of cell spreading on apoptosis. (A) Combined phase con- apoptosis. (A) Schematic diagram showing the trast–fluorescence micrographs of capillary endothelial cells cultured in initial pattern design containing different-sized suspension in the absence or presence of different-sized microbeads or square adhesive islands and Nomarski views of attached to a planar culture dish coated with FN for 24 hours (28). In the highly spread cell on the 25-␮m the final shapes of bovine adrenal capillary endo- bead, only the flattened 4Ј,6Ј-diamidino-2-phenylindole (DAPI)–stained nucleus is clearly visible. (B) thelial cells adherent to the fabricated substrate. Apoptosis in cells attached to different-sized beads, in suspension, or attached to a dish. The apoptotic Distances indicate lengths of the square’s sides. index was quantitated by measuring the percentage of cells exhibiting positive TUNEL staining (black (B) Apoptotic index (percentage of cells exhibiting bars) (Boehringer Mannheim), which detects DNA fragmentation; similar results were obtained by positive TUNEL staining) and DNA synthesis index analyzing changes in nuclear condensation and fragmentation in cells stained with DAPI at 24 hours (percentage of nuclei labeled with 5-bromode- (gray bars). Apoptotic indices were determined only within single cells bound to single beads. Error bars oxyuridine) after 24 hours, plotted as a function of indicate SEM. (C) Differential interference-contrast micrographs of cells plated on substrates micropat- the projected cell area. Data were obtained only terned with 10- or 20-␮m-diameter circles coated with FN (left), by a microcontact printing method (29) from islands that contained single adherent cells; or on a similarly coated unpatterned substrate (right). (D) Apoptotic index of cells attached to different- similar results were obtained with circular or sized adhesive islands coated with a constant density of FN for 24 hours; similar results were obtained square islands and with human or bovine endo- with human and bovine capillary endothelial cells (28). Bars same as in (B). thelial cells.

1426 SCIENCE ⅐ VOL. 276 ⅐ 30 MAY 1997 ⅐ www.sciencemag.org REPORTS growth in single cells spread across multiple, that preferentially use integrin ␤1 (type I signals elicited directly by integrin binding closely spaced adhesive islands of either 3 or collagen) or ␣V␤3 (vitronectin). Apoptosis and thereby modulate downstream signaling 5 ␮m in diameter to approximate the size of was greatly inhibited relative to the 60% (21). In fact, constitutive activation of the individual focal adhesions (Fig. 3, A and B). level observed in suspended cells when cells tyrosine kinase, pp125FAK, in the focal ad- Cell bodies spread across the intervening spread on unpatterned substrates regardless hesion complex can lead to shape- and ad- nonadhesive areas of the substrate, stretch- of the integrin ligand used (Fig. 4). How- hesion-independent cell survival and growth ing processes from one small adhesive island ever, survival was consistently greater in (22, 23). Alternatively, growth and viability to another. Immunofluorescence staining cells that adhered to intact ECM proteins. may be altered directly by mechanical stress- confirmed that adherent cells attached and When spreading was restricted by use of dependent changes in the organization or formed vinculin-containing focal adhesions 20-␮m circles, cells adherent to integrin ␤1 stiffness of the cytoskeleton and nucleus (18, only on the engineered islands (Fig. 3C). By ligands (FN, type I collagen, anti-␤1,or 19, 21, 24). For example, the increased flex- changing the spacing between adhesive is- anti-␤1 combined with anti-␣V␤3) exhibit- ibility of the cytoskeleton that has been ob- lands, we could increase cell spreading 10- ed much greater increases in apoptosis com- served in rounded cells (18) may permit fold without significantly altering the total pared with those on either intact vitronec- intracellular structural rearrangements that cell-ECM contact area (Fig. 3D). On these tin or on anti-␣V␤3 alone (Fig. 4). This was are lethal, including the characteristic struc- substrates, DNA synthesis scaled directly due to a change in sensitivity, rather than a tural degeneration of the cell and nucleus with projected cell area and not with cell- lack of response, because similar high levels that are hallmarks of apoptosis. The finding ECM contact area (Fig. 3D). Apoptosis was of apoptosis were induced when cell spread- that cell survival is more tightly coupled to similarly switched off by cell spreading, ing was further restricted by plating on 10- cell shape in cells adherent to ligands for even though the cell-ECM contact area ␮m circular islands. Thus, although geomet- integrin ␤1 compared with integrin ␣V␤3 is remained constant under these conditions ric switching between growth and apoptosis also consistent with the observation that ␤1 (Fig. 3D). Thus, cell shape per se appears to is a general phenomenon, different adhesion provides stronger ECM anchoring to resist be the critical determinant that switches receptors appear to be able to convey distinct cytoskeletal tension (18). From this perspec- cells between life and death and between death signals and thereby tune the cellular tive, adhesive substrates may prevent cell proliferation and quiescence. response to shape distortion. death and promote growth by resisting con- In vivo studies demonstrate that pro- The mechanism by which cells trans- tractile forces transmitted across integrins, grammed cell death and capillary regression duce changes in cell geometry into different thereby mechanically stabilizing the nucleo- can be induced by inhibiting integrin ␣V␤3 biochemical responses remains unclear. The cytoskeletal lattice. binding, whereas apoptosis can be prevent- specialized cytoskeletal structure, or focal During morphogenesis, growing, quies- ed in vitro by attachment of cells to immo- adhesion complex, that forms intracellu- cent, and dying cells often co-exist within bilized antibodies to integrin ␤1 (anti-␤1) larly at the site of integrin binding is a the same microenvironment (1, 2). In fact, (8, 9). Because cell binding to FN is medi- molecular bridge that mechanically couples the establishment of local differentials in ated by both ␤1 and ␤3 integrins, we chose integrins, and hence ECM, to the actin cell growth and viability drives pattern for- to explore their role in shape-dependent cytoskeleton (17–19). Because focal adhe- mation. Our results suggest that living cells control of apoptosis. We coated unpat- sions also orient much of the signal trans- can filter the same set of chemical inputs terned substrates and 20-␮m circular islands duction machinery of the cell (20), they (activation of integrin and growth factor with antibodies specific for ␤1 or ␣V␤3 in- may integrate mechanical signals associated receptor signaling) to produce different tegrins, FN, or physiological ECM ligands with changes in cell shape with chemical functional outputs (growth versus apo- ptosis) as a result of local mechanical defor- mation of the cell or nucleus. By sensing their degree of extension or compression, cells therefore may be able to monitor local changes in cell crowding or ECM compli-

Fig. 3. Cell-ECM contact area versus cell spreading as a regulator of cell fate. (A) Diagram of substrates used to vary cell shape independently of the cell-ECM contact area. Substrates were patterned with small, closely spaced circular islands (center) so that cell spreading could be promoted as in cells on larger, single round islands, but the ECM contact area would be low as in cells on the small islands. (B) Fig. 4. Role of different integrin ligands in cell Phase-contrast micrographs of cells spread on single 20- or 50-␮m-diameter circles or multiple 5-␮m shape–regulated apoptosis. Apoptotic indices circles patterned as shown in (A). (C) Immunofluorescence micrographs of cells on a micropatterned (percentage positive TUNEL staining) for cells cul- substrate stained for FN (top) and vinculin (bottom). White outline indicates cell borders; note circular tured for 24 hours on unpatterned substrates rings of vinculin staining, which coincide precisely with edges of the FN-coated adhesive islands. (D) (black bars) or on 20-␮m circles (gray bars) coat- Plots of projected cell area (black bars) and total ECM contact area (gray bars) per cell (top), growth index ed with FN, type I collagen (Col I), vitronectin ( VN),

(middle), and apoptotic index (bottom) when cells were cultured on single 20-␮m circles or on multiple anti-␤1, anti-␣V␤3, or antibodies to both integrin circles 5 or 3 ␮m in diameter separated by 40, 10, and 6 ␮m, respectively. ␤1 and integrin ␣V␤3 (29). www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 276 ⅐ 30 MAY 1997 1427 ance (for example, due to enhanced ECM 22. L. V. Owens et al., Cancer Res. 55, 2752 (1995); S. 29. We coated beads with FN (Collaborative Biomedical; 50 M. Frisch, K. Vuori, E. Ruoslahti, P. Y. Chan-Hui, J. ␮g/ml) using carbonate buffer (18). Patterned sub- remodeling or local application of cell ten- Cell Biol. 134, 793 (1996). strates containing islands coated with FN were fabricat- sion) and thereby couple changes in ECM 23. M. D. Schaller et al., Proc. Natl. Acad. Sci. U.S.A. 89, ed by a microcontact printing method (13, 14). Briefly, extension to expansion of cell mass within 5192 (1992); S. K. Hanks, M. B. Calalb, M. C. Harp- hexadecanethiol [HS(CH2)15CH3] was printed onto the local tissue microenvironment. Tissue er, S. K. Patel, ibid., p. 8487. gold-coated substrates with a flexible stamp containing 24. D. Stamenovic, J. J. Fredberg, N. Wang, J. P. Butler, a relief of the desired pattern. The substrate was im- involution may be promoted in other mi- D. E. Ingber, J. Theor. Biol. 181, 125 (1996); D. E. mersed immediately in 2 mM tri(ethylene glycol)-termi- croenvironments by inducing rapid break- Ingber, J. Cell Sci. 104, 613 (1993). nated alkanethiol [HS(CH2)11(OCH2CH2)3OH in etha- down of ECM and associated cell retrac- 25. D. E. Ingber, J. A. Madri, J. D. Jamieson, Proc. Natl. nol], which coated the remaining bare regions of gold. Acad. Sci. U.S.A. 78, 3901 (1981); D. E. Ingber and When these substrates were immersed in a solution of tion. During malignant transformation, pro- J. D. Jamieson, in Tumor Invasion and Metastasis,L. FN, vitronectin, or type I collagen (50 ␮g/ml in phos- gressive loss of shape-dependent regulation A. Liotta and I. R. Hart, Eds. (Nijhoff, The Hague, phate-buffered saline), the protein rapidly adsorbed only also may lead to cell survival in the absence Netherlands, 1982), pp. 335–357; D. E. Ingber and to the stamped regions. Antibody-coated substrates were prepared by first immersing surfaces in a solu- of ECM extension, unrestricted mass ex- J. D. Jamieson, in Gene Expression During Normal and Malignant Differentiation, L. C. Andersson, C. G. tion of goat antibody to mouse immunoglobulin G Fc pansion, and hence neoplastic disorganiza- Gahmberg, P. Ekblom, Eds. (Academic Press, Or- (50 ␮g/ml) and washed with 1% bovine serum albu- tion of tissue architecture (25–27). lando, FL, 1985), p. 13; D. E. Ingber, J. A. Madri, J. min in Dulbecco’s modified Eagle’s medium before D. Jamieson, Am. J. Pathol. 12, 248 (1985). immobilizing the mouse antibodies to integrin ␣V␤3 (1 ␮g/ml; LM609; Chemicon), ␤ (1 ␮g/ml; BD15; Bio- REFERENCES AND NOTES 26. I. MacPherson and L. Montagnier, Virology 23, 291 1 ______(1964); M. Stoker, C. O’Neill, S. Berryman, V. Wax- source), or a combination of the two (0.5 ␮g/ml man, Int. J. Cancer 3, 683 (1968). each). Cells cultured on substrates with no mouse 1. E. R. Clark and E. L. Clark, Am. J. Anat. 64, 251 27. S. C. Wittelsberger, K. Kleene, S. Penman, Cell 24, antibody or antibodies to intracellular proteins did not (1938). adhere under these conditions. 2. D. E. Ingber, J. A. Madri, J. Folkman, Endocrinology 859 (1981); R. W. Tucker, C. E. Butterfield, J. Folk- man, J. Supramol. Struct. Cell. Biochem. 15,29 30. This work was supported by grants from NIH 119, 1768 (1986). (HL57669, CA55833, GM30367), the Defense Ad- 3. D. E. Ingber and J. Folkman, Cell 58, 308 (1989); C. (1981); J. Folkman and H. P. Greenspan, Biochim. Biophys. Acta 417, 217 (1975). vanced Research Projects Agency, and the Office of D. Roskelley, A. Srebrow, M. J. Bissell, Curr. Opin. Naval Research; postdoctoral fellowships from the 28. Human pulmonary microvascular endothelial cells Cell Biol. 7, 736 (1995). American Cancer Society (M.M.) and the Swiss Na- (Clonetics) were cultured in EGM medium (Clonetics) 4. D. E. Ingber and J. M. Folkman, J. Cell Biol. 109, 317 tional Science Foundation (S.H.); and partial salary supplemented with 2% fetal calf serum, epidermal (1989). support (C.S.C.) from the Harvard–MIT Health Sci- growth factor (10 ng/ml), and fibroblast growth factor 5. M. S. Wicha, L. A. Liotta, B. K. Vonderhaar, W. R. ences Technology Program. Kidwell, Dev. Biol. 80, 253 (1980); C. J. Drake, D. A. (FGF) (5 ng/ml). Bovine adrenal capillary endothelial Cheresh, C. D. Little, J. Cell Sci. 108, 2655 (1995). cells were cultured in serum-free, chemically defined 6. C. J. Sympson et al., J. Cell Biol. 125, 681 (1994). medium supplemented with FGF (5 ng/ml) (4). 27 December 1996; accepted 7 April 1997 7. N. Boudreau, C. J. Sympson, Z. Werb, M. J. Bissell, Science 267, 891 (1995). 8. P. C. Brooks, R. A. Clark, D. A. Cheresh, ibid. 264, 569 (1994); P. C. Brooks et al., Cell 79, 1157 (1994); Lamina-Specific Connectivity in the Brain: P. C. Brooks et al., J. Clin. Invest. 96, 1815 (1995); S. Stromblad, J. C. Becker, M. Yebra, P. C. Brooks, D. Regulation by N-Cadherin, Neurotrophins, and A. Cheresh, ibid. 98, 426 (1996). 9. J. E. Meredith, B. Fazeli, M. A. Schwartz, Mol. Biol. Glycoconjugates Cell 4, 953 (1993); Z. Zhang, K. Vuori, J. C. Reed, E. Ruoslahti, Proc. Natl. Acad. Sci. U.S.A. 92, 6161 Akihiro Inoue and Joshua R. Sanes* (1995). 10. F. Re et al., J. Cell Biol. 127, 537 (1994). 11. H. P. McNamee, D. E. Ingber, M. A. Schwartz, ibid. In the vertebrate brain, neurons grouped in parallel laminae receive distinct sets of 121, 673 (1993); M. A. Schwartz, C. Lechene, D. E. Ingber, Proc. Natl. Acad. Sci. U.S.A. 88, 7849 synaptic inputs. In the avian optic tectum, arbors and synapses of most retinal axons are (1991); L. E. Dike and D. E. Ingber, J. Cell Sci. 109, confined to 3 of 15 laminae. The adhesion molecule N-cadherin and cell surface gly- 2855 (1996). coconjugates recognized by a plant lectin are selectively associated with these “reti- 12. J. Folkman, N. Engl. J. Med. 285, 1182 (1971); D. Hanahan and J. Folkman, Cell 86, 353 (1996); J. norecipient” laminae. The lectin and a monoclonal antibody to N-cadherin perturbed Folkman, K. Watson, D. Ingber, D. Hanahan, Nature laminar selectivity in distinct fashions. In contrast, neurotrophins increased the com- 339, 58 (1989); D. Ingber et al., ibid. 348, 555 (1990). plexity of retinal arbors without affecting their laminar distribution. Thus, cell surface 13. R. Singhvi et al., Science 264, 696 (1994). 14. K. L. Prime and G. M. Whitesides, ibid. 252, 1164 molecules and soluble trophic factors may collaborate to shape lamina-specific arbors (1991); A. Kumar, H. A. Biebuyck, G. M. Whitesides, in the brain, with the former predominantly affecting their position and the latter their size. Langmuir 10, 1498 (1994); M. Mrksich and G. M. Whitesides, Trends Biotech. 13, 228 (1995). 15. J. M. Folkman and A. Moscona, Nature 273, 345 (1978); D. E. Ingber, Proc. Natl. Acad. Sci. U.S.A. 87, 3579 (1990). Many parts of the vertebrate brain are of specific synaptic connectivity. 16. J. Folkman et al., Am. J. Pathol. 130, 393 (1988); T. organized into parallel laminae that bear To elucidate the mechanisms underlying Spivak-Kroizman et al., Cell 79, 1015 (1994); D. J. Falcone, T. A. McCaffrey, A. Haimovitz-Friedman, J. distinct neuronal subtypes and receive dis- lamina-specific retinotectal connectivity, we A. Vergilio, A. C. Nicholson, J. Biol. Chem. 268, tinct synaptic inputs (1–3). In the optic devised a coculture system in which a trans- 11951 (1993). tectum of the chick, for example, most verse tectal section was overlaid with a retinal 17. K. Burridge et al., Annu. Rev. Cell Biol. 4, 487 (1988); S. W. Craig and R. P. Johnson, Curr. Opin. Cell Biol. retinal axons terminate in just 3 of 15 strip, such that neurites from retinal ganglion 8, 74 (1996). laminae (Fig. 1A). Each individual axon cells had equal access to all tectal laminae 18. N. Wang, J. P. Butler, D. E. Ingber, Science 260, arborizes within a single lamina, even (Fig. 1B). Outgrowth and arborization on 1124 (1993); N. Wang and D. E. Ingber, Biophys. J. 66, 2181 (1994); Biochem. Cell Biol. 73, 327 (1995). though the dendrites of some postsynaptic these sections reproduced the lamina-selec- 19. A. Maniotis, C. S. Chen, D. E. Ingber, Proc. Natl. cells extend through many laminae (4–6). tive patterns observed in vivo, implicating Acad. Sci. U.S.A. 94, 849 (1997). The lamina-selective arborization of in- local tectal cues in axonal guidance (5). 20. E. A. Clarke and J. S. Brugge, Science 268, 233 coming axons may be a major determinant Moreover, lamina-selective growth persisted (1995); M. A. Schwartz, M. D. Schaller, M. H. Gins- berg, Annu. Rev. Cell Dev. Biol. 11, 549 (1995); D. E. when sections were chemically fixed before Ingber, Cell 75, 1249 (1993); G. E. Plopper, H. P. Department of Anatomy and Neurobiology, Washington retinal strips were added, which implied that McNamee, L. E. Dike, K. Bojanowski, D. E. Ingber, University School of Medicine, 660 South Euclid Avenue, some cues are associated with the cell surface Mol. Biol. Cell 6, 1349 (1995); S. Miyamoto, S. K. Box 8108, St. Louis, MO 63110, USA. Akiyama, K. M. Yamada, Science 267, 883 (1995). and are independent of activity (5). We later 21. D. E. Ingber, Annu. Rev. Phys. 59, 575 (1997). *To whom correspondence should be addressed. assessed the distribution of cell adhesion mol- 1428 SCIENCE ⅐ VOL. 276 ⅐ 30 MAY 1997 ⅐ www.sciencemag.org