letters to nature null mutation. Two different genotypes were used to mark mutant clones: yw tumours in Cdx2 mutant mice. Nature 396, 84–87 (1997). SA1 + - 24. Miller, D. J. & Miles, A. Homeobox genes and the zootype. Nature 365, 215–216 (1993). FLP122; FRT42D Dll /FRT42D Dp(y )44B or yw FLP122; FRT42D Dll 25. Sa´nchez, L. Casares, F., Gorfinkiel, N. & Guerrero, I. The genital disc of Drosophila melanogaster. II. SA1 − /FRT42D pwn. In the first genotype, Dll heat-shock-induced clones were Role of the genes hedgehog, decapentaplegic and wingless. Dev. Genes Evol. 207; 229–241 (1997). labelled with the cuticle marker y. As a possible transformation into hindgut 26. Murakami, R. et al. aproctous, a locus that is necessary for the development of the proctodeum in Drosophila embryos, encodes a homolog of the vertebrate Brachyury gene. Wilhelm Roux Arch. Dev. could not be scored with this system, in the second genotype the marker pwn Biol. 205, 89–96 (1995). labels the Dll+ clones twins of the DllSA1 clones. For Dll clones27 in the genital 27. Gorfinkiel, N., Morata, G. & Guerrero, I. The homeobox gene Distal-less induces ventral appendage SA1 development in Drosophila. Genes Dev. 11, 2259–2271 (1997). disc, larvae of genotype yw FLP122; FRT42D Dll /FRT42D arm–lacZ were 28. Diaz-Benjumea, F. & Cohen, S. M. Serrate signals through Notch to establish a wingless-dependent SA given 1-h heat shocks and Dll clones were marked by the loss of either Dll organizer at the dorsal-ventral compartment boundary of the Drosophila wing. Development 121, protein or lacZ activity. Partial loss of Dll function was studied in trans 4215–4225 (1995). 3 MP 8 combinations of the Dll , Dll and Dll–Gal4 alleles . We studied the require- Acknowledgements. We thank P. Fernandez, T. Kusch, C. Parras, R. Rivera, I. Rodriguez and G. Struhl for ments of the analia for hh, wg and dpp function using the hhts2, wgIL114, dppd12 antibodies, cDNAs and fly stocks; J. Casanova and E. Sanchez-Herrero for comments on the manuscript; S. Gonzalez for helping with the DNA injections; and R. Gonzalez and J. M. Gala´n for their technical help. d14 20 21 and dpp alleles as described . To activate the Hh pathway in the entire This work was supported by grants from the Direccio´n General de Investigacio´n Cientifica y Te´cnica and analia primordium we generated yw; cad–Gal4/UAS–ci; UAS–y flies, in which from the Human Frontier Science Program. An institutional grant from the Fundacion Ramon Areces to the Centro de Biologia Molecular is also acknowledged. E.M. is supported by a scholarship from the the Ci product is expressed in the whole of the analia primordium. The anal Comunidad Auto´noma de Madrid. plates of these flies are marked by the expression of the UAS–y gene, which Correspondence and requests for materials should be addressed to G.M. (e-mail: [email protected]). confers a dark colour. Ectopic cad expression. We used the Gal4/UAS method19.AUAS–cad gene was constructed by inserting the cad complementation DNA (a gift from R. Rivera-Pomar) in the pUAS vector as described8. The UAS–cad construct is function, as it can rescue the mutant phenotype of the cad–Gal4 insertions. The Notch signalling controls MS-248 (a gift from C. Parras) and ap–Gal48 driver lines gave the best results. Flies of genotype MS-248/UAS–cad and ap–Gal4/UAS–cad were mounted and pancreatic cell differentiation examined at the light microscope. A˚ sa Apelqvist*†, Hao Li*†, Lukas Sommer‡, Paul Beatus§, X-gal and antibody staining. X-gal staining of adult flies and imaginal discs David J. Andersonk, Tasuku Honjo¶, 8 was performed as described . For adults it is important that they had emerged Martin Hrabeˇ de Angelis#, Urban Lendahl§ shortly before fixing. Imaginal discs were immunostained using the normal & Helena Edlund* procedures for confocal microscopy. The specific antibodies used were rabbit * Department of Microbiology, University of Umea˚, S-901 87 Umea˚, Sweden anti-Byn (a gift from T. Kusch), rabbit anti-Cad (from G. Struhl), monoclonal ‡ Institute of Cell Biology, Swiss Federal Institute of Technology, anti-Dll (from S. Cohen and I. Duncan), rabbit anti-Eve (from M. Frusta) and ETH-Hoenggerberg HPM E38, CH-8093 Zu¨rich, Switzerland monoclonal anti-Abd-B (from S. Celniker and A. Macias). k Division of Biology 216-76, California Institute of Technology, 1201 E. California Blvd., Pasadena, California 91125, USA Received 17 May; accepted 28 June 1999. ¶ Department of Medical Chemistry, Kyoto University Faculty of Medicine, 1. Macdonald, P. & Struhl, B. 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