Cell, Vol. 37, 825-831, July 1984, Copyright 0 1984 by MIT 0092.8674/84/070825-07 $02.00/O Cloning and Transcriptional Analysis of the Segmentation Gene fushi farazu of Drosophila Atsushi Kuroiwa, Ernst Hafen,* and Walter J. Gehring Department of Cell Biology Biozentrum, University of Base1 Klingelbergstrasse 70 CH-4056 Basel, Switzerland Summary In the course of studying the Antennapedia (An@) locus, we found that one of the 3’ An@ exons has weak cross-homology to another gene affecting segmentation, fushi tarazu (ffz; meaning “not enough segments”), which is 30 kb to the left of An@. Homozygous ffz- embryos die before hatching and lack alternate body segments. The reduced number of segments results from the fusion of the anterior portion of one segment with the posterior portion of the next segment. The ftz gene encodes a single 1.9 kb poly(A)+ RNA expressed exclusively from the early blastoderm to gastrula stages of embryonic development. The structure of the ftz gene has been analyzed by Sl nuclease mapping and by restriction mapping of a cDNA clone. The ffz gene consists of two exons, and it is the 3’ exon that cross-hybridizes with the 3’ exon of Antp. The role of ftz in cell determination is discussed. Introduction The polarity and the spatial organization of the Drosophila embryo are under genetic control. The major body axes are laid down in the egg under the influence of the maternal genes and the genes of the oocyte prior to meiosis. Mutants affecting the major body axes, anterior-posterior and dorsoventral, have been isolated (see Ntisslein-Vol- hard, 1979) and shown to depend on the maternal geno- type. The phenotype of these mutants suggests that the egg contains determinants which specify the spatial coor- dinates within the egg. After fertilization, the egg first develops as a syncytium of synchronously dividing nuclei that subsequently migrate to the periphery of the egg, where they form a monolayer of cells-the blastoderm. Gynandromorph studies and nuclear transplantation experiments indicate that the cleav- age nuclei are equivalent and totipotent (Sturtevant, 1929; Zalokar, 1971; Okada et al., 1974; Illmensee, 1978). How- ever, when the cleavage nuclei reach the periphery and cellularization occurs, the blastoderm cells are determined (Chan and Gehring, 1971; Illmensee, 1976; Simcox and Sang, 1983). This indicates that there is an interaction between the cortical egg cytoplasm and the immigrating nuclei which leads to the determination of these nuclei and the cells they form. It is not known how precisely the spatial coordinates of the egg are defined or what the nature of l Present address: Department of Biochemistry, University of California, Berkeley. California 94720. these determinants of spatial organization is. Transplanta- tion of syncytial blastoderm nuclei with adhering cortical cytoplasm to ectopic sites in another embryo of the same age suggests that these nuclei have acquired at least an anterior versus posterior commitment (Kaufman, 1980). Cell lineage studies indicate that cell determination at the blastoderm stage is segment specific (Wieschaus and Gehring, 1976; Steiner, 1976; Lawrence and Morata, 1977), although segmentation becomes visible only later in development. Two classes of mutants are known to affect the spatial organization of the embryo into segments, the segmenta- tion mutants affecting segment number and polarity (Nijs- slein-Volhard and Wieschaus, 1980) and the homeotic mutants that transform one segment into another or parts of a segment into the corresponding parts of another segment (e.g. Lewis, 1978). One of the zygotic segmen- tation mutants is ftz, which deletes half the number of segments (Wakimoto and Kaufman, 1981; Ntisslein-Vol- hard et al., 1982). Homozygous ffz- embryos lack the posterior part of one segment and the anterior part of the next segment, and show a pairwise fusion of the remaining parts. These embryos are lethal at a late embryonic stage. In the course of our studies on the homeotic Anfp locus we discovered cross-homology between Antp and ftz, which allowed us to isolate the ffz gene. We describe the molecular cloning of this gene and the analysis of its transcripts. Results Isolation and Identification of the fushi tarazu (ftz) Gene We have reported the isolation of overlapping clones of chromosomal DNA from the 84B1,2 region including the Antp locus by chromosomal walking (Garber et al., 1983). In the course of this study, R. Garber found that one of the Antp cDNA clones weakly hybridizes to a DNA seg- ment located 30 kb to the left of Antp. When this cross- homologous fragment was used as a probe for Northern blotting of RNA from embryos, it hybridized strongly to a 1.9 kb poly(A)+ RNA whose size is different from that of the Antp transcripts. As shown in Figure 1, this region is deleted in the Df(3R)9A99 chromosome and is located to the right of the inversion Multiple sex comb, In(3R)Msc, breakpoint (Scott et al., 1983). Since Df(3R)9A99 leaves the Antp+ gene intact, the cross-homologous region is not included in the Antp gene itself. However, Df(3R)9A99 does not complement ftz and Sex comb reduced (Scr) (JOrgens et al., 1984), which are located just to the left of Anfp (Wakimoto and Kaufman, 1981; Hazelrigg and Kauf- man, 1983; Raff and Kaufman, 1983). Therefore, these genes are the best candidates for the 1.9 kb poly(A)+ RNA encoding gene. Scott et al. (1983) have mapped the ftz gene to the same chromosomal site on the basis of mutant lesions. Taking into account the spatial distribution of the transcripts of this gene (see accompanying paper, Hafen et al., 1984), we conclude that this gene corresponds to