Drosophila melanogaster is a well-established genetic model system that has been an excellent source for gene discovery, in particular utilizing developing tissue(s) such as the eye (Morgan 1911) . All manners of genetic screening have been conducted in Drosophila, ranging from developmental screens to behavioral screens (St Johnston 2002) . A Flp/FRT EMS screen was conducted to identify developmental cell growth and cell division regulators on chromosome 2R. This screen identified mutations that disrupt cell growth, cell cycle, and organ development (Kagey et al. 2012). Additionally, the screen was conducted in a genetic background in which cell death is homozygous blocked (via the Dark ⁸² allele present on the starting chromosome) (Akdemir et al. 2006). Here, students from the Fly-CURE consortium worked within undergraduate laboratory courses to characterize and map one mutant from this screen, mutant M.3.2 (Merkle et al. 2023).

Mosaic eyes were generated by mating the control (starting chromosome) ;FRT42D, Dark ⁸² /CyO and ;FRT42D, Dark ⁸² , M.3.2/CyO to virgin females of ;FRT42D, Ey-Flp (BDSC). The Dark ⁸² allele was generated with a mw+ containing transposon, so the Dark ⁸² containing mutant tissue is pigmented in both eyes. Straight-winged F1 progeny were scored for their mosaic eye phenotype. As previously reported, the Dark ⁸² mosaic eye results in a slight over representation of red tissue to white tissue (60-70% pigmented tissue, and Figure 1A ) (Akdemir et al. 2006; Kagey et al. 2012). Contrastingly, the Dark ⁸² , M.3.2 mosaic eye results in a deformed mosaic eye that has an expansion of cuticle, additional hair in the eye, and an overall smaller eye size than the control ( Figure 1A compared with 1B). The expansion of the cuticle coupled with the overall smaller adult eye size, suggests that there may be a non-autonomous aspect of the M.3.2 mutation.

The genetic mapping of the location of mutant M.3.2 was done by three independent groups of researchers within undergraduate genetics laboratory courses at Ohio Northern University, Morehouse College, and the University of Detroit Mercy as part of the Fly-CURE consortium (Merkle et al. 2023). Genetic mapping was accomplished by setting up complementation tests between the Dark ⁸² , M.3.2 mutant stocks and the 2R deficiency kit (BDSC) (Cook et al. 2012). Virgin females from the ;FRT42D, Dark ^82, M.3.2 / CyO stock were mated in series to male flies from the 87 deficiency stocks that comprise the Bloomington Stock Center 2R Deficiency kit (and are distal to the FRT42D location). Triplicate data from the three groups of mapping students confirmed that M.3.2 failed to complement Df(2R) Exel6284 and Df (2R) ED2354. Although these two deficiencies do not overlap ( overlap Chr2R:14,574,557 and14,574,750) there are two genes that span this gap, leaving a region that fails to complement of only two predicted genes ( Figure 1B, yellow box, and Table 1).

Further genetic mapping found that the M.3.2 mutation failed to complement two independent alleles of the gene tout-velu (ttv ^00681b and ttv ^k11904 ) , suggesting that M.3.2 is a novel allele of tout-velu, ttv ^M.3.2 . Once we had identified the causative locus for the M.3.2 mutation, we utilized WGS of heterozygous flies to look for the specific heterozygous mutations within the ttv locus that are unique to M.3.2 and not present in any other 2R mutant we have sequenced Bieser et al. (in preparation). From this we identified a point mutation in the ttv locus that introduces a stop codon at the 515 amino acid (Trp515*) ( Figure 1C ). TTV is a 772 amino acid protein and having a truncation at amino acid 515 suggests that the M.3.2 mutation in ttv is a truncation mutation and a novel null allele of ttv ( Figure 1C ). Given the extensive genetic and molecular data presented here we can reasonably conclude that M.3.2 is a lethal (and novel) allele of ttv and there are likely no additional lethal mutations (other than Dark ⁸² ) on the chromosome. However, a future rescue experiment would be necessary to demonstrate the phenotype observed is based solely on the ttv and Dark mutations.

Tout-velu is a homologue of the human gene, EXT1 , which is a tumor suppressor needed for the Hedgehog protein diffusion (Kim et al. 2001). In flies, Hedgehog signaling is essential for proper development of many different tissues, including the processing of the morphogenetic furrow in the developing eye (Heberlein et al. 1993). Given that previous studies have found a non-autonomous role for TTV in promoting Hh signaling, and the non-autonomous mosaic phenotypes we observed, we provide additional data supporting a non-autonomous role of TTV function (Bellaiche et al. 1998). The Hedgehog protein is a morphogen essential for embryonic development, cell growth, cell specialization, and the patterning or shaping of the body, as TTV ensures receiving cells can interpret Hh signaling. Thus, it is likely that the ttv ^M.3.2 mutant clones disrupted the MF progression in the developing eye both autonomously and non-autonomously. Additionally, loss of TTV results in cells that are unable to interpret Hh signaling from the MF, resulting in loss of Hh signaling for homozygous mutant cells, which would lead to arrested development of ommatidia (Ma and Moses 1995) .

Lastly, the inclusion of the Dark ⁸² allele allowed for the discovery of this ttv allele as a conditional regulator of cell growth and cell division in this Flp/FRT system. Previous studies disrupting Hh signaling in the developing eye through mutations in Ptc , Cos2 , or Smo have resulted in both autonomous and non-autonomous apoptosis (Christiansen et al. 2012; Kagey et al. 2012; Moore et al. 2022). Given that ttv mutations disrupt Hh signaling, it is reasonable to suggest that ttv ^-/- clones would undergo apoptosis, and thus may have been missed in the initial round of Flp/FRT growth mutants (Moberg et al. 2001; Tapon et al. 2002; Harvey et al. 2003). This would define ttv ^M.3.2 as a conditional negative regulator of cell growth and development, only identified in a genetic background of blocked apoptosis (Gilbert et al. 2011; Kagey et al. 2012).