18, cAMP production was measured In cells that were shaken in phosphate buffer for 4 hours, followed by 50-nM pulses of cAMP for an additional 3 hours (28). Cells were then collected, washed twice, and resus- pended at 5 x lo7 cells/ml. They were then stimulat- ed wlth 10 pM 2'-deoxy-CAMP, dithiothreitol was added to a final concentration of 5 mM, and 100-bl samples were removed at various tlmes, added to 100 p1 of 3.5% perchlorlc acld, and frozen. Total cAMP produced was determined with a cAMP RIA kit (Amersham). Before analysis, frozen samples were thawed and neutralized with 50% NaHCO,, the re- sulting lysates were centrifuged, and the superna- tants were assayed. Total cAMP production of cells developing on fllters was determined by scraping cells directly into 3.5% perchloric acld and process- ing the samples as described above.The cAMP stan- dard curves were determined by processing known concentrations of cAMP through the same sample preparation as the stimulated cells (perchlorlc acid treatment and so forth). Sensitivity to beef heart phos- phodiesterase(Sigma)was used to confirm the pres- ence of cAMP in samples. PDE treatment lowered cAMP concentrations in wild-type samples to those measured in acaA mutant strains. The amounts of "CAMP measured in extracts of acaA-null or acaA(PKA-C) cells were unaffected by the treatment with PDE. In these experiments,the detection limit of the assay was 0.1 pmol per 10' cells. 19. W. Roos, C. Sche~degger, G. Gerisch, Nature 266, 259 (1977); K. J. Tomchik and P. N. Devreotes, Science 212, 443 (1981); H. Levine, I. Aranson, L. Tsimring, T. V. Truong, Proc. Nati. Acad. Sci. U.S.A. 93, 6382 (1996). 20. E. Wallraff, D. L. Welker, K. L. Williams, G. Gerisch, J. Gen. Microbial. 130, 2103 (1984); G. Gerisch et ai., Cold Spring Harbor Symp. Quant Biol. 50, 813 (1 985). 21. K. B. Raper, J. Elisha Mitchell Sci. Soc. 56, 241 (1 940); J. G. Williams et ai., Cell 59, 1157 ( I989). 22. J. T. Bonner, Am. Nat. 86, 79 (1952); Proc. Nati. Acad. Sci. U.S.A. 45, 379 (1959); J. Sternfeld and C. N. David, Differentiation 20, 10 (1981); Dev. Biol. 93, 111 (1 982); C. J. Weijer, C. N. David, J. Sternfeld, Methods Cell Biol. 28, 449 (I987). 23. B. Wang and A. Kuspa, data not shown. 24. J. Franke, G. J. Podgorski, R. H. Kessln, Dev. Bioi. 124, 504 (1987); L. Wu, D. Hansen, J. Franke, R. H. Kessin, G. J. Podgorskl, ibid. 171, 149 (1995). 25,'To determine the extent of sporulation, we trans- ferred sori from individual fruiting bodies to 20 p1 of 20 mM potassium phosphate buffer (pH 6.2) con- taining 0.4% NP-40 nonionic detergent (Sigma)and triturated at room temperature. The number of re- fractile, ovoid spores was determined by phase-con- trast microscopy using a hemocytometer. Spores were counted for flve fruiting bodles produced by wild-type or acaA(PKA-C) cells. 26. The viablllty of the spores was determined after de- tergent treatment (25) by platlng 1500 vislble spores on 10 SM agar plates In association wlth Klebsielia aerogenes (27). The number of colonies formed was assumed to be representative of the number of via- ble spores In the origlnal sample. 27. M. Sussman, Methods Ceil Biol. 28, 9 (1987). 28. P. Devreotes, D. Fontana, P. Klein, J. Sherrlng, A. Theibert, ibid., p. 299. 29. The cell denslty dependence of development was measured by developing cells on phosphate-buff- ered agar at different cell densities (27). Cells were washed from growth medla, resuspended in phos- phate buffer, and diluted to various cell densities, and 0.1 ml of the suspension was spotted onto phosphate-buffered 1 % agar plates (Difco, Detroit, MI). A layer of cells within a 1.8-cm2 circle was formed after the buffer was absorbed by the agar. Total numbers of aggregates and fruiting bodies formed were scored on a dissecting microscope af- ter 14 hours and 36 hours of development, respec- tively. At the highest cell density tested, some of the aggregates formed by acaA(PKA-C) cells, which did not progress to normal fruiting bodies, formed termi- nal structures similar to those observed for wild-type cells overexpressing pkaC (7, 8) or pkaR-null mu- tants (6). The acaA mutant cells were never ob- served to produce aggregates at these cell densities (23). A similar cell density dependence for aggrega- tion of the acaA(PKA-C) cells was found on Millipore fllters (23). 30. M. L. Lacombe, G. J. Podgorski, J. Franke, R. H. Kessin, J. Biol. Chem. 261, 1681 1 (1 986). 31. K. L. Fosnaugh and W. F. Loomis, Nucleic Acids Res. 17, 9489 (1 989). 32. S. J. McRobbie, K. A. Jermyn, K. Duffy, K. Blight, J, G. Williams, Deveiopment 104, 275 (1988). 33. D. L. Richardson, C. B. Hong, W. F. Loomls, Dev. Biol. 144, 269 (1991). 34. We thank W. F, Loomis, D. Hereld, and S. Lu for many helpful discussions regarding this work; G. Souza, G. Shaulsky, and W. F. Loomis for sugges- tions that improved the manuscript; and R. Sucgang for help in preparing thefigures. Supported by grants from NIH and the State of Texas. A.K. is a Searle Scholar and an American Cancer Society Junior Faculty Research Fellow. 25 March 1997; accepted 30 May 1997 Specification of the Zebrafish Nervous System by Nonaxial Signals Katherine Woo* and Scott E. Fraser The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when graft- ed nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis. T h e developing vertebrate central nervous system is patterned by inductive interactions (1 ). The gastrula organizer (referred to as the "dorsal lip" in amphibians, "node" in am- niotes, and "shield" in fish) is thought to be the source of patterning information (2). Analyses using amphibian embryos have in- dicated temporally distinct signals within the organizer (3,4): A n activator signal from the anterior axial mesoderm defines the anterior neurectoderm. and a subseauent transformer signal from ;he chordagesoderm (noto- chord) repatterns nearby neural tissue into more posterior types. In mouse, chick, and fish embryos, the elimination of the organiz- er does not abolish anteroposterior (AP) pat- terning in the neurectoderm; hence, there is a source of pattern information in non-orga- nizer tissues (5. 6). . , , The neural fate map of zebrafish (7) shows patterning by 6 hours of development, when gastrulation has only advanced to the forma- tion of a thickened blastoderm margin (germ- ring) and an embryonic shield at its dorsal side. Forebrain progenitors are located far from the germring, spanning the dorsal mid- line (Fig. 1A). In contrast, hindbrain progen- itors lie close to the germring, lateral to the embryonic shield, with midbrain progenitors Dlvision of Biology and Biological Imaging Center, Beck- man Institute, California Institute of Technology, Pasade- na, CA 91 125, USA *Present address: UCSF/Gallo Center, Building 1, Room 101, San Francisco General Hospital, San Francisco, CA 941 10, USA. E-mail: kwooQitsa.ucsf.edu in between (Fig. 1A). The early regionaliza- tion of anterior (forebrain) and posterior (hindbrain) neural progenitors within the neurectoderm allowed us to investigate signals that may differentially pattern the neuraxis. We hypothesized that proximity to the germ- ring might specify more posterior neural fates. Indeed, labeled presumptive forebrain progen- itors (Fig. lB), transplanted (8) at shield stage to the position of the presumptive hindbrain, adopt the hindbrain fate (Fig. 1C). Moreover, presumptive hindbrain cells are not commit- ted to a specific fate at this stage (9). Thus, the signals that normally instruct or permit cells to adopt the hindbrain fate are still ac- tive in vivo at 6 hours. Because deletion of the shield disru~ts notochord but not hindbrain development (6), the signals responsible for hindbrain patterning probably do not come exclusive- lv from the shield. Germrine tissue mav be a source of such a posterioriz7ng signal. i'he shield contributes to axial mesoderm, noto- chord, and ventral neural tissues (10) (Fig. ID); the germring gives rise to somitic me- soderm, posterior mesoderm, and endoderm (Fig. 1, E and F) (1 1). To investigate pat- terning by nonaxial germring tissue, we transplanted sectors of the shield (0") and the germring at defined angular distances from the dorsal midline (45", 90°, 135", and 180") to the animal pole, a region fated to become forebrain (Fig. 1, A and B), of shield-stage zebrafish. If the germring were the source of a patterning signal, such grafts \ICE VOL. 277 11 JULY 1997 www.sciencemag.org