Economic Incentives for Rain Forest Conservation Across Scales

6. J. D. Axelrod, J. R. Miller, J. M. Shulman, R. T. Moon, N. Perrimon, Genes Dev. 12, 2610 (1998). 7. M. Boutros, N. Paricio, D. I. Strutt, M. Mlodzik, Cell 94, 109 (1998). 8. L. C. Sheldahl, M. Park, C. C. Malbon, R. T. Moon, Curr. Biol. 9, 695 (1999). 9. K. M. Bhat, Cell 95, 1027 (1998). 10. J. R. Kennerdell and R. W. Carthew, Cell 95, 1017 (1998). 11. H. Mueller, R. Samanta, E. Wieschaus, Development 126, 577 (1999). 12. P. Bhanot et al., Development 126, 4175 (1999). 13. C.-N. Chen and G. Struhl, Development 126, 5441 (1999). 14. K. M. Cadigan, M. P. Fish, E. J. Rulifson, R. Nusse, Cell 93, 767 (1998). 15. Fz1, Fz2, and the chimeric receptors were expressed with sevGal4 in the developing eye, in a specific subset of photoreceptor precursors (in R3 and R4 at the time of polarity determination). For analysis in imaginal discs, the enhancer trap line H123 was used as a marker for R4 and detected as described [M. Fanto and M. Mlodzik, Nature 397, 523 (1999)]. Eye imaginal discs and sections of adult eyes were prepared, photographed, and processed as described [D. I. Strutt, U. Weber, M. Mlodzik, Nature 387, 292 (1997)]. 16. J. Zhang and R. W. Carthew, Development 125, 3075 (1998). 17. A. H. Brand and N. Perrimon, Development 118, 401 (1993). 18. Several independent lines were generated for these and the chimeric constructs, and at least four were tested in the functional assays, all giving equivalent results. The transgenic strains showed comparable protein expression levels as determined by Western blotting with the inserted myc-epitope tag. 19. M. Boutros, J. Mihaly, M. Mlodzik, unpublished results. 20. Capped synthetic RNA of the different receptors was injected into both blastomeres of the two-cell embryo. At stage 9, animal cap ectoderm was explanted and cultured until sibling embryos reached stage 11. Reverse transcriptase–polymerase chain reaction (RT-PCR) was done as described [T. Bouwmeester, S.-H. Kim, Y. Sasai, B. Lu, E. De Robertis, Nature 382, 595 (1996)]. Protein levels were checked by Western blot analysis with anti-myc and anti-Fz1 antibodies. Dsh relocalization assays were done as described [J. D. Axelrod, J. R. Miller, J. M. Shulman, R. T. Moon, N. Perrimon, Genes Dev. 12, 2610 (1998)]. For Xnr-3 and Sia induction, 100 to 800 pg of RNA was injected; for Dsh relocalization, Fz receptors were injected at 100 to 200 pg of RNA together with 100 pg of Dsh-EGFP RNA. 21. M. Brannon and D. Kimelman, Dev. Biol. 180, 344 (1996). 22. F. Fagotto, K. Guger, B. M. Gumbiner, Development 124, 453 (1997). 23. K. Itoh and S. Sokol, Mech. Dev. 61, 113 (1997). 24. Both Fz1 and Fz2 recruited Dsh to the membrane at a concentration of 25 pg of injected RNA and were unable to do so at 12.5 pg, indicating that both receptors have the same threshold in this assay. 25. C. R. Vinson, S. Conover, P. N. Adler, Nature 338, 263 (1989). 26. P. Bhanot et al., Nature 382, 225 (1996). 27. We generated Fz1 and Fz2 chimeric constructs by assembling their CRD (Fz1, amino acids 1 to 166; Fz2, 1 to 219), seven-pass transmembrane region (Fz1, amino acids 167 to 557; Fz2, 220 to 617), and cytoplasmic tails (Fz1, amino acids 558 to 585; Fz2, 618 to 694). In brief, chimeric receptors consisted of 10 base pairs of the Fz1 59-untranslated region, the CRD domain (except for DCRD) linked to the transmembrane region (inserted by generating a Hind III site), and a myc tag in a nonconserved region. For COOH-terminal swaps, a Xho I site was created after the last transmembrane domain. We created chimeric receptors by assembling different fragments in pBS-SK and then shuttling them into Drosophila and Xenopus expression vectors. Constructs were confirmed by sequencing. For each construct, several independent transgenic Drosophila lines were generated and tested in the assays described. 28. The chimeric receptors were expressed under the control of the apGal4 driver in the dorsal wing and notum. The chimeras that showed mild dominantnegative behavior did not affect wg expression at the dorsal/ventral (D/V) margin, indicating that the observed effect is due to Wg read-out defects in the third larval instar. Planar polarity was analyzed in the wing cell hairs (Fig. 3) and by the orientation of the microchaetae on the notum. Effects on planar polarity in the notum were seen with Fz1 and Fz2-1, as is the case in the eye and wing. 29. To analyze the effects of the chimeric receptors in the legs, we used DllGal4 as a driver (other Gal4 drivers resulted either in all chimeras having a wild-type appearance irrespective of the Fz receptors expressed or were lethal). DllGal4.Fz1 gave no detectable planar polarity phenotype, whereas DllGal4.Fz2 was lethal. Fz2-1 and Fz2-2-1 were pupal lethal, showing loss of distal leg structures, a dominant-negative Wg-related effect. Fz1-2 and Fz1-1-2 showed a mild Wg GOF phenotype, as judged by loss of the dorsally derived claws (indicating a transformation to ventral Wg-induced structures) [W. J. Brook and S. M. Cohen, Science 273, 1373 (1996)]. 30. To determine the affinity of the different Fz chimeras for Wg, we expressed them with a dppGal4 driver perpendicular to the normal wg expression. All chimeras containing the Fz2 CRD behaved like Fz2 or Fz2CRD-GPI [K. M. Cadigan, M. P. Fish, E. J. Rulifson, R. Nusse, Cell 93, 767 (1998)], leading to Wg stabilization (Fig. 3). The inverse chimeric receptors with the Fz1 CRD had no effect on Wg stability. 31. The Fz1and Fz2-1–induced GOF planar polarity eye phenotype depends on the amount of Fz expressed in the R3 and R4 precursor cells. At high expression levels (as with Gal4-driven expression), the phenotype is the same in wild-type and in fz2/2 eye discs. At lower overexpression levels, the presence of the endogenous protein is important for the overall amounts to reach the threshold necessary for constitutive activation. 32. A small difference in Fz signaling levels between the neighboring R3 and R4 cells is critical to establish correct ommatidial polarity and leads reproducibly to correct polarity; the small difference is amplified by Delta/Notch signaling [M. Fanto and M. Mlodzik, Nature 397, 523 (1999); M. T. Cooper and S. J. Bray, Nature 397, 526 (1999); A. Tomlinson and G. Struhl, Development 126, 5725 (1999)]. The rescue experiments demonstrate that the CRD of Fz1 and a putative ligand are important to establish this difference. Fz2-1 cannot respond to the ligand and establish this difference. Nevertheless, for constitutive activation of downstream pathways the Fz1 CRD is not required. 33. A. Sato, T. Kojima, K. Ui-Tei, Y. Miyata, K. Saigo, Development 126, 4421 (1999). 34. Different signaling preferences of Fz1 and Fz2 are also corroborated by the observation that simultaneous overexpression of Wg and Fz1 in the wing disc neutralizes the GOF effects of each on either Wg signaling or the planar polarity pathway [ J. D. Axelrod, J. R. Miller, J. M. Shulman, R. T. Moon, N. Perrimon, Genes Dev. 12, 2610 (1998)]. 35. X. Lin and N. Perrimon, Nature 400, 281 (1999). 36. M. Tsuda et al., Nature 400, 276 (1999). 37. S. Romani, S. Campuzano, E. R. Macagno, J. Modolell, Genes Dev. 3, 997 (1989). 38. Wing imaginal discs were dissected from larvae and stained by standard methods. The discs were incubated with mouse antibody to Ac (1:200 dilution; gift of S. Carroll) or to Wg (gift of S. Cohen). Fluorescentlabeled secondary antibody mix (1:500 dilution). 39. We are grateful to P. Adler, K. Basler, S. Carroll, R. Carthew, S. Cohen, R. Nusse, and M. Strigini for fly strains and reagents. We thank members of the Bouwmeester and Mlodzik labs and M. Strigini and S. Cohen for discussions, and J. Curtiss for helpful comments on the manuscript. M.B. was supported by a predoctoral fellowship from the Boehringer Ingelheim Fonds, J.M. is a recipient of a long-term fellowship from the European Molecular Biology Organization.