“A fly appeared”: sable, a classic Drosophila mutation, maps to Yippee, a gene affecting body color, wings, and bristles

Abstract

Insect body color is an easily assessed and visually engaging trait that is informative on a broad range of topics including speciation, biomaterial science, and ecdysis. Mutants of the fruit fly Drosophila melanogaster have been an integral part of body color research for more than a century. As a result of this long tenure, backlogs of body color mutations have remained unmapped to their genes, all while their strains have been dutifully maintained, used for recombination mapping, and part of genetics education. Stemming from a lesson plan in our undergraduate genetics class, we have mapped sable1, a dark body mutation originally described by Morgan and Bridges, to Yippee, a gene encoding a predicted member of the E3 ubiquitin ligase complex. Deficiency/duplication mapping, genetic rescue, DNA and cDNA sequencing, RT-qPCR, and two new CRISPR alleles indicated that sable1 is a hypomorphic Yippee mutation due to a mdg4 element insertion in the Yippee 5’-UTR. Further analysis revealed additional Yippee mutant phenotypes including curved wings, ectopic/missing bristles, delayed development, and failed adult emergence. RNAi of Yippee in the ectoderm phenocopied sable body color and most other Yippee phenotypes. Although Yippee remains functionally uncharacterized, the results presented here suggest possible connections between melanin biosynthesis, copper homeostasis, and Notch/Delta signaling; in addition, they provide insight into past studies of sable cell nonautonomy and of the genetic modifier suppressor of sable.

Demonstration ofin vivoengineered tandem duplications of varying sizes using CRISPR and recombinases inDrosophila melanogaster

“…Designs were conducted using Geneious Prime (Biomatters, Inc.). Assembly of the w-FRT construct, CRISPaintL, was detailed in (Dean et al 2022). The FRT-w construct, CRI-SPaintR, was built similarly but using a different Level 1 vector:…”

“…Briefly, homologyarm PCR products were assembled with a vector fragment and a marker-FRT insert fragment. The same vector, H-arm-CFP, from (Dean et al 2022) was used; this vector is marked with 3xP3-CFP to detect improper insertions. For the marker-FRT inserts, variants of that paper's FRT-w-FRT insert plasmid were designed.…”

“…Our initial approach to insert marker-FRT sites applied the CRI-SPaint strategy (Schmid-Burgk et al 2016;Bosch et al 2020;Dean et al 2022). In this approach, the CRISPR/Cas9-induced DNA break in the injected embryo is repaired by nonhomologous end joining.…”

“…We next attempted to insert marker-constructs into the same sites using the homology-directed-repair (HDR) strategy for CRISPR (Gratz et al 2014), which has worked effectively for us in the past (Loehlin et al 2022;Dean et al 2022). Marker constructs were assembled for each site: either a w + FRT or FRT w + insert flanked with ~500bp homology arms that match the sequence flanking the double-strand-break.…”

“…Guide RNA plasmids were built using the KLD procedure into vector pU6-3-chiRNA (Gratz et al 2014;Dean et al 2022) with primers listed in File S1. Guide sequences are: MX2 CT-GAATAATAAGTGGTTGT, MX5 CGAAACCGCTACTCTGGCT, MX6 TAGATGTGCTTAATTATGA, MX10 TTAGCCAGCCAA-GATTTAT.…”