Epidermal Growth Factor Receptor and Transforming Growth Factor-b Signaling Contributes to Variation for Wing Shape in Drosophila melanogaster

Wing development in Drosophila is a common model system for the dissection of genetic networks and their roles during development. In particular, the RTK and TGF-b regulatory networks appear to be involved with numerous aspects of wing development, including patterning, cell determination, growth, proliferation, and survival in the developing imaginal wing disc. However, little is known as to how subtle changes in the function of these genes may contribute to quantitative variation for wing shape, per se. In this study 50 insertional mutations, representing 43 loci in the RTK, Hedgehog, TGF-b pathways, and their genetically interacting factors were used to study the role of these networks on wing shape. To concurrently examine how genetic background modulates the effects of the mutation, each insertion was introgressed into two wild-type genetic backgrounds. Using geometric morphometric methods, it is shown that the majority of these mutations have profound effects on shape but not size of the wing when measured as heterozygotes. To examine the relationships between how each mutation affects wing shape hierarchical clustering was used. Unlike previous observations of environmental canalization, these mutations did not generally increase within-line variation relative to their wild-type counterparts. These results provide an entry point into the genetics of wing shape and are discussed within the framework of the dissection of complex phenotypes. IN quantitative and evolutionary genetics, the focus has primarily been on using QTL and linkage disequilibrium mapping to hunt for genes, but large-scale screens using mutagenesis have also been employed for traits such as bristle number and olfaction (Mackay et al. 1992; Anholt et al. 1996; Norga et al. 2003). These studies not only enrich the list of possible candidate genes harboring natural genetic variation, but also provide estimates for the mutational target size of these traits. Nonetheless it remains unclear if genes characterized in functional studies are good candidates for studies of natural variation. One facet that needs to be investigated is whether minor variation in gene function is sufficient to affect the expression of quantitative traits. In general, developmental processes such as patterning and determination have been addressed with classical Mendelian and molecular genetic approaches. However, a number of studies have demonstrated the utility of quantitative genetic methodologies for examining natural genetic variation for these developmental mechanisms (Gibson and Hogness 1996; Gibson and van Helden 1997; Polaczyk et al. 1998; Palsson and Gibson 2000; Atallah et al. 2004). We recently utilized association mapping to localize naturally occurring polymorphisms involved with variation for photoreceptor determination in Drosophila (Dworkin et al. 2003). Although evidence is still limited, these studies are consistent with genes of major effect harboring alleles that contribute to quantitative trait variation. With respect to the genetic dissection of development, the wing of Drosophila melanogaster is one of the best established model systems (Held 2002). During embryonic development, a set of 24 cells invaginate from the epithilium to form the wing disc rudiment (Cohen et al. 1991). During early larval development, broad patterning of the wing axes is established. In particular, the posterior region of the wing imaginal disc is patterned by the protein Engrailed (En) (GarciaBellido and Santamaria 1972; Lawrence and Morata 1976; Brower 1986). En activates the shortrange paracrine signaling ligand hedgehog ( 2–4 cell widths) at the boundary between the anterior and posterior territories (Hidalgo 1994; Tabata and Kornberg 1994; Sanicola et al. 1995). Hedgehog upregulates decapentaplegic, the canonical ligand of the TGF-b signaling pathway (Zecca et al. 1995). While dpp RNA is present only in an 5-cell-wide region just anterior to the anterior– posterior (A–P) boundary, the Dpp secreted protein elicits long-range effects throughout the future wing blade Corresponding author: Department of Genetics, North Carolina State University, 3632 Gardner Hall, Raleigh, NC 27695-7614. E-mail: [email protected] Genetics 173: 1417–1431 ( July 2006) (at least 35 cell diameters from its source), regulating a number of downstream target genes that specify domains along the A–P axis (Podos and Ferguson 1999; Held 2002). These future wing territories are further subdivided into vein and intervein fates by the regulation of epidermal growth factor receptor signaling. During early pupal development, the TGF-b pathway is reutilized in the maintenance of vein–intervein fates (Held 2002; De Celis 2003; Crozatier et al. 2004). The above description is a gross simplification of the process and of the role of these genes. For instance, members of both the TGF-b and receptor tyrosine kinase (RTK) signaling pathways have been implicated in other developmental processes in the wing such as cell growth and survival (Martin et al. 2004). In addition, there is evidence for ‘‘cross-talk’’ between pathways with respect to vein–intervein determination (Crozatier et al. 2002; Yan et al. 2004; Sotillos and De Celis 2005), and they appear to interact as networks, rather than linear pathways. While there is a wealth of information with respect to the role of these genes during the development of the wing, little is known about the genetic modification of wing shape. That is, it is unclear how the wing takes on its final adult dimensions. Geometric morphometric methods, a recent development in the analysis of shape (Bookstein 1991; Zelditch et al. 2004), allow for sensitive discrimination between groups or treatments (Klingenberg 2002;Houle et al. 2003). Thesemethods have primarily been used to characterize naturally occurring variation, with little attempt to understand the developmental basis for shape differences. With respect to wing shape in D. melanogaster, a number of studies have demonstratedmoderate to high heritability for the phenotype (Weber 1990; Birdsall et al. 2000; Zimmerman et al. 2000; Palsson and Gibson 2004; Mezey et al. 2005), and there is little evidence for constraints on the evolution of shape (Mezey and Houle 2005). Consistent with a large mutational target size for wing shape, 20% of novel P-element insertion lines demonstrated replicable phenotypic effects on shape (Weber et al. 2005). In addition, it is clear that there is considerable segregating genetic variation in natural populations for wing shape (Weber 1990; Weber et al. 1999; Birdsall et al. 2000; Zimmerman et al. 2000; Mezey et al. 2005). Concerning the contribution of individual genes on wing shape, deficiency complementation mapping has been used to investigate the role of candidate gene function on shape (Palsson and Gibson 2000; Mezey et al. 2005). In addition, a series of studies have demonstrated how a putative regulatory polymorphism in the Egfr gene is associated with natural variation for wing shape (Palsson and Gibson 2004; Dworkin et al. 2005; Palsson et al. 2005). Unfortunately, none of this work was performed in controlled genetic backgrounds to investigate the individual effects ofmutations in these genes. One exception is the study byWeber et al. (2005), which demonstrated that 11 of 50 random P-element insertion lines had a significant effect in an isogenic background on the basis of at least one of four univariate measures of wing allometry. Plasmid rescue of these insertions suggests that putative genes were involved in a variety of developmental and physiological processes. Notably, themethodused to examine shape for this study likely underestimated phenotypic variation in the wing. In this study we investigate the potential role of genes in the EGF, TGF-b, and Hedgehog signaling pathways with respect to wing shape in Drosophila. Fifty P-element insertional mutations in genes from these pathways were introgressed into each of two standard lab wild-type strains. Wing shape was then measured on heterozygotes for each mutation and compared to their respective wild-type congenics. With this experimental framework, we addressed several questions: (1) Given that genes in the TGF-b and EGF/RTK signaling pathways are involved with various aspects of wing development, what role might they and their interacting factors play in wing shape?, (2)What are the effects of themutations when measured in a heterozygous state?, (3) How important is genetic background when estimating the effects of the mutations on shape?, (4) Do the effects of the mutations on shape make sense on the basis of their known developmental roles?, and (5) Domutations within genes from the same pathway tend to have ‘‘related’’ effects on shape when compared with mutations in genes from different pathways? We demonstrate that the mutations in most of the genes under study show a significant effect on shape relative to their wild-type counterparts when measured in a heterozygous state. However, it is clear that genetic background plays an important role in describing shape both as marginal and as epistatic effects. Furthermore we demonstrate that while some of themutations clearly affect shape in a similar manner with respect to their known function, the effects of the mutations on shape do not cluster on the basis of pathways, consistent with extensive cross-talk. These results are discussed within the framework of the role of TGF-b and RTK signaling on wing shape and their potential as candidate genes that harbor segregating variation for shape. MATERIALS AND METHODS Stocks: Insertional mutations were selected from the Bloomington Stock Center (Table 1). Many of the insertions were considered ‘‘within’’ genes if they were within 5 kb of the ORF of that gene or showed a failure to complement with other knownmutations in those genes (Table 1). Regardless of the original source of the insertion, each transposon used was marked with a mini-white (P{w}), as this facilitated the backcross procedure. All insertions were introgressed into two wild-type lab strains, Samarkand (Sam) and Oregon-R (Ore), both marked withwhite (w), resulting in white-eyed flies. Introgressions were 1418 I. Dworkin and G. Gibson