Copyright 0 1985 by the Genetics Society of America APPARENT GENETIC COMPLEXITY GENERATED BY DEVELOPMENTAL THRESHOLDS: THE APTEROUS LOCUS IN DROSOPHILA MELANOGASTER MARY E. STEVENS' AND PETER J. BRYANT Developmental Biology Center and Department of Deuelopmental and Cell Biology, University of Cal$ornia, Imine, Calij-ornia 9271 7 Manuscript received June 15, 1984 Revised copy accepted February 14, 1985 ABSTRACT Mutations at the apterous (up) locus in Drosophila melanogaster give rise to three distinct phenotypes: aberrant wings, female sterility and precocious adult death. The wing phenotype includes five types of abnormality: blistering, de- ficiencies, duplications, high-order repetitions and transformation of structures. The mildest phenotype is seen with homozygous up"" animals which have either normal or slightly blistered wings. Most alleles produce, in the homozygote, a deficient wing in which part or all of the wing margin and wing blade is missing, but wing hinge and notum regions are normal. Animals hemizygous for each of 20 ap alleles, as well as ap*D/a$xnheterozygotes, show duplication of parts of the notum associated with complete wing deficiency. Animals het- erozygous for up' and the other tested ap alleles show repetitions of parts of the anterior wing margin, an engrailed-like transformation of posterior wing margin into anterior margin or both. Both up'" and upc show similar pheno- types in homozygotes and hemizygotes, yet both produce a less extreme phe- notype than that of the other hemizygotes, suggesting that neither mutation causes loss of the entire up' function. The 15 alleles that cause precocious death and female sterility occur in six complementation groups based on com- plementation for these phenotypes. This supports the previous conclusion that the effects of apterous mutations on the wing do not correlate with their effects on viability and fertility. We propose an explanation for the effects of apterous mutations on the wing in which quantitative reductions in the activity of gene product give rise to qualitatively different phenotypes because of different threshold requirements of the up+ function for critical events in wing disc development. HE complexity of complex genetic loci in higher organisms takes several T forms. Different mutations of such loci often give different phenotypes, and the mutant phenotypes are often pleiotropic. Furthermore, pairs of alleles often show partial or complete interallelic complementation, making definition of functional genetic units difficult. Consequently, there have been several interpretations of the structure and function of complex loci (for reviews, see CARLSON1959; JUDD 1976). One view is that such a locus may code for a single multifunctional protein with several domains, each of which is respon- Present address: Laboratory of Radiobiology, University of California, San Francisco, California 94 143. Genetics 110: 281-297 June, 1985. 282 M. E. STEVENS AND P. J. BRYANT sible for a particular function; for example, in Neurospora, the mom locus encodes a polypeptide with five enzyme activities (GAERTNERand COLE1977). Similarly, in Drosophila three enzyme activities controlled by the rudimentary locus appear to be associated with a single polypeptide (BROTHERSet al. 1978), and GRACE(1980) has proposed that the dumpy locus may also encode a multifunctional polypeptide. Complex loci have also been interpreted as con- sisting of clusters of closely linked genes which show genetic interaction; for example, bithorax (LEWIS 1978), Antennapedia (LEWISet al. 1980), decapen- taplegic (SPENCER, HOFFMANand GELBART1982) and Notch (PORTIN1975) of Drosophila have all been considered as multigene complexes. The apterous (up;2-55.2) function in Drosophila behaves as a complex locus in that different mutations give different phenotypes which are often pleio- tropic, and that complementation occurs between some pairs of alleles. The phenotypes caused by apterous mutations include several types of abnormal wings, female sterility, precocious adult death, abnormal gut morphology, per- sistence of larval fat body cells in the adult (BUTTERWORTHand KING 1965; BUTTERWORTH1972; WILSON 1980), leg deficiencies and duplications and oc- casional antenna-to-leg transformations (M. STEVENS,unpublished observa- tions). The female sterility is associated with nonvitellogenesis which is thought to be caused by juvenile hormone deficiency, since application of a juvenile hormone analog can stimulate vitellogenesis in up4 females (POSTLETHWAIT and WEISER1973). However, application of this compound before or during the temperature-sensitive period for adult death is not sufficient to prolong adult survival (WILSON 198 la). The relationship between juvenile hormone and the other up phenotypes has not been studied. By using a temperature-sensitive allele, WILSON (1981a) showed that the apterous locus has two distinct temperature-sensitive periods, one during the larval period for the wing phenotype and a second one during the pupal period for the precocious death and fertility phenotypes. The latter phenotypes are highly correlated with each other among genotypes (WILSON1980), but neither adult death nor female sterility shows correlation with the wing phenotype. These results indicate that the up gene product functions in different body parts at different developmental times and that mutations may preferentially interfere with one or another of its functions. The locus may, therefore, pro- vide an instructive example of gene activity under both temporal and spatial control. In this paper, we describe the phenotypes of several up mutants in detail, and by studying their behavior in homozygous, hemizygous and heter- ozygous conditions, we arrive at a new interpretation of the apparent genetic complexity of this locus. MATERIALS AND METHODS Stocks: Sixteen of the 24 ap alleles were kindly provided by T. WILSON;the other eight were obtained from Drosophila stock centers. We have classified the 24 alleles into six classes: in classes containing more than one allele, we refer to any allele within a particular class by using a super- script for that class. For example, ap” designates all of the class I1 alleles. WILSON(1980) noted that b pr ap*”’ homozygotes showed a much stronger phenotype than that described by LINDSLEY and GRELL(1968) for ap*’’;this observation and our genetic analysis indicate that ap”“ and ap*” COMPLEXITY OF THE APTEROUS LOCUS 283 are different alleles. Due to the low viability of most homozygotes, only the ap”’, up”’, apbUand a#‘ alleles were kept as homozygous stocks; 18 others were balanced over the SM5 chromosome (LINDSLEYand GRELL 1968). The two dominant alleles, ap’D and up-, were kept heterozygous with a chromosome carrying the homozygous lethal bw” mutation. A chromosome carrying M(2)S4, which is deficient for apterous, was used to produce animals hemizygous for various ap alleles. Except where noted otherwise, all stocks were maintained at 25” using a standard cornmeal, yeast, corn syrup and agar medium. Morphological phenotypes: Adult structures were prepared by removing the head, legs and ab- domen, heating the thorax and wings in 20% KOH for 5 min and mounting the parts between coverslips in either Euparal or Faure’s mounting medium. The wing disc derivatives were examined under a Zeiss microscope at X125 and X300 magnification and were scored using BRYANT’S(1975) fate map of the wing disc. Characterization of sterility and precocious death pheno*e: Ten pairs of animals from each geno- type were allowed to lay eggs for 24 hr at 25”. FI homozygotes or heterozygotes were scored for time of eclosion, and females were then tested for fertility and length of survival after eclosion by placing the animals into vials with several wild-type males and checking daily for dead animals and for the presence of Fa larvae. Heteroallelic combinations: All possible heteroallelic combinations of the 24 apterous alleles were made, using ten males and ten females in each cross. Reciprocal crosses were carried out, but since no significant differences were found between the results of the two crosses, the data for each combination were pooled. The parents were allowed to lay eggs for 6 days, transferred to fresh bottles for an additional 6 days and then discarded. Progeny from both sets of bottles were counted daily, with a minimum of 100 FI counted for each cross. The presence of the Cy marker on the SM5 chromosome (which carries a$+) enabled us to identify and use animals heterozygous for up+ as controls in the same culture vials. Some of the mutants that showed a clearly abnormal wing phenotype were nevertheless fertile and long-lived in both homozygous and hemizygous conditions. The normal phenotype seen in these hemizygotes indicates that one copy of such an allele is sufficient for a nonmutant phenotype; therefore, heterozygotes with these alleles were not used in the complementation analysis for sterility and precocious death. Heterozygotes between alleles that produce these mutant phenotypes when homozygous were checked daily for fertility and early death. The appearance of larvae in the vial indicated complementation for the sterility phenotype. In genotypes producing precocious death phenotypes, escapers occurred but with a frequency of 10% or less. We used the value of 20% of adults living for more than 5 days as our criterion for complementation of the precocious death phenotype. RESULTS The wing phenotypes of apterous: Several wing phenotypes other than the structural