Tubby-RFP Balancers for Developmental Analysis: FM7c 2xTb-RFP, CyO 2xTb-RFP and TM3 2xTb-RFP

An extremely useful tool available to the Drosophila geneticist is the “balancer” chromosome. This marked chromosome allows recessive lethal mutants to be maintained as true-breeding stocks thanks to the very efficient suppression of recombination between homologues. Moreover, since the balancer chromosome can be easily identified through a visible, dominant trait, segregation of the recessive lethal chromosome can be readily followed in genetic crosses [Greenspan, 1997]. The origin of balancers goes back to the beginnings of Drosophila genetics. In 1918, Hermann Muller determined that a lethal mutation could be kept as a stock when another lethal mutation was located on the corresponding homologous chromosome. Muller referred to these as ‘balanced lethals’ [Muller, 1918]. Shortly after, Alfred Sturtevant proposed that inversions present on one of the homologous chromosomes suppressed recombination between the pair [Sturtevant, 1921]. Inversions in combination with lethals allowed balancing of larger chromosomal regions and created the foundation for the balancers in use today. Today’s autosomal balancers are complex chromosomes, containing 1) multiple, overlapping inversions that effectively suppress recombination across much of the chromosome, 2) a lethal, recessive mutation that prevents survival of homozygous balancer flies, and 3) at least one visible, dominant marker that unequivocally identifies the balancer in the adult fly. X chromosome balancers are similarly constructed except for the lack of recessive lethal loci. However, most include a recessive female-sterile locus, thereby restricting fertility to the heterozygous females while ensuring the viability of the hemizygous males. As mentioned above, all balancers carry a visible dominant adult marker, such as the 3rd chromosome traits stubble bristles (Sb) and serrated wings (Ser) found on TM3 balancers, or the curly wings (Cy) associated with all 2nd chromosome balancers, including CyO or the SM# series. Only the 3rd chromosome balancers TM6B and TM6C also carry a dominant visible larval/pupal marker, Tb1, in addition to the dominant adult bristles markers Humeral (Hu) and Sb, respectively. The Tubby phenotype due to the Tb1 mutant allele consist of a short and stout larva, and eventually pupa, clearly distinguishable from the slender body shape of the wt particularly at the L3 and pupal stages [Crymer 1980, 1984]. This larval marker makes the TM6B and TM6C balancers more versatile than any other and a much appreciated tool of the developmental geneticist. First identified by Charlotte Auerbach in 1943, the Tb1 phenotype can always be distinguished from Tb+ in both homozygous and heterozygous larvae (L2, L3) or pupae [Craymer, 1980]. The phenotype is visible to the naked eye and thus allows one to quickly detect the segregation of mutant alleles balanced over TM6B or TM6C at the larval and pupal stages, thus facilitating the analysis of developmental mutants. Another particularly effective use of this feature is in large-scale screens for the identification of recessive lethal alleles on the 3rd chromosome. Scoring of the Tb marker at the pupal stage avoids the need to carefully score adult progeny, greatly improving the efficiency of such screens. Moreover, thanks to its normal fertility and viability, the use of the Tb marker does not adversely affect any crosses [Guan et al., 2006]. Over time, transgenic embryonic and larval markers have been added to several 1st, 2nd, and 3rd chromosome balancers for the study of developmental loci. GFP-expressing transgenes allow visual scoring of progeny at these early stages through the use of fluorescence microscopy [Reichhart and Ferrandon, 1998; Casso et al., 1999]. Nonetheless, the simplicity of the Tb1 marker, which can be detected visually without use of a stereomicroscope, remains unbeaten. Recently, the Tb locus has been identified as a member of the Twdl gene family. Twdl type proteins are involved in cuticle formation and are characterized by 2 conserved motifs found in all members of this insect protein family (Guan et al., 2006). By sequence analysis, Guan and colleagues (2006) identified a 23 amino acid deletion within one of the evolutionarily conserved domains of TwdlA to be the cause of the dominant Tb1 phenotype. A similar deletion in TwdlD, TwdlD1, could also induce the short body type (Guan et al., 2006). The mutant phenotype was dose dependent and best visible at 1:1, or greater, ratios of mutant to wt alleles (Guan et al., 2006). A quantitative description of this trait is provided by the axial ratio (AR), defined as the ratio of the larval/pupal body length to the body width. This value has been shown to possess a constant mean that is characteristic of a fly strain and independent of the overall size of a pupa [Letsou et al., 1991]. Tb+ stocks have a pupal AR of ~3.0, whereas the AR of Tb1 pupae is around 2.0. We report the construction of new Tb-RFP balancers for the X, II and III chromosomes of Drosophila. We first generated two types of ‘Tubby’ P-element constructs: one with two copies of the Tb1-RFP fusion and another with one copy of Tb1-RFP and one of TwdlD1-RFP (Guan et al., 2006) (Fig. 1A). Two Tb-inducing alleles were used in order to ensure at least a 1:1 ratio of mutant to wt alleles. Both constructs were then transformed into w1118 creating several Tb-looking fly lines. Transgenic lines containing the 2xTb1-RFP transgene displayed a more severe Tb phenotype than TwdlD1-RFP + Tb1-RFP lines and were used as donors for transposition of the P-elements onto balancers for the X (FM7c, Bar), the 2nd (CyO, Cy) and 3rd (TM3, Sb) chromosomes (Fig. 1B-D). Figure 1 Constructs and genetic crosses The Tb phenotype of the resulting new balancers is easily scored at the L2, L3 and pupal stages, and the presence of RFP extends this range to include late embryos, L1 larvae and adults (Fig. 2). In addition, since expression of the Tb1-RFP fusion protein is associated nearly exclusively with the cuticle, its presence does not interfere with the staining of internal tissues (e.g. larval imaginal discs) permitting triple-staining of balancer siblings as controls. Figure 2 Tubby and RFP detection in embryos, larvae, pupae and adults As expected for a 1:1 ratio of Tb to Tb+ loci, the mean AR of pupae from the 2xTb1-RFP balancers is ~2.0, significantly lower than the ~3.0 AR of w1118 pupae (Fig. 2E, 2F) or of the original balancer lines (see Fig. 2 legend). Tb1-RFP expression begins in late embryogenesis during larval cuticle deposition, and the fusion protein can be detected with a fluorescence stereomicroscope at this time (Fig. 2A′). In larvae, Tb1-RFP is present throughout the cuticle and is easily visible during movement when the cuticle is compressed permitting detection in live larvae even at the L1 stage (Fig. 2B′, 2C′, 2D′). Adult flies also fluorescence. In the abdominal region, two lines of fluorescent dots are clearly visible through the dorsal cuticle (Fig. 2G) and diffuse fluorescence is present on the ventral side (Fig. 2H). Whereas detection of the RFP will be most informative at the embryonic and L1 stages, it may also be useful in adult flies particularly if the adult visible trait associated with the balancer (Bar, Cy or Sb) is masked by, or similar it is to, phenotypes associated with other chromosomes present in the stock (e.g., the dominant Bar eye associated with FM7 cannot be scored in an genetic background that causes an adult eyeless phenotype). In summary, the presence of the 2xTb1-RFP transgene on these balancers (FM7c, Bar 2xTb-RFP; CyO, Cy 2xTb-RFP; TM3, Sb 2xTb-RFP) greatly facilitates the analysis of lethals and other developmental mutants in L2/L3 larvae and pupae, but also extends this range from late embryos all the way to adults. At the time of this submission, the Gatti group reported the construction of Tb-tagged FM7a and CyO-Sbw balancers (Lattao et al., 2011). Our balancers differ in that they carry 2 copies of Tb1 and additionally the RFP-fusion marker. Moreover, FM7a and FM7c differ significantly in that FM7a is homozygous fertile whereas FM7c is sterile and better suited for the maintenance of X-linked lethals and weaker X chromosome. Thus, these new balancers significantly expand the options of available Tb balancers. The FM7c, Bar 2xTb-RFP, CyO, Cy 2xTb-RFP, and TM3, Sb 2xTb-RFP balancer stocks listed in Fig. 2I have been deposited at the Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu).