Description

The lethal allele E7.25d.7 was isolated in a previously described FLP/FRT screen for conditional growth suppressors on chromosome 3L (Gilbert et al., 2011). Briefly, male w¹¹¹⁸;; Df(3L)H99,FRT80B/TM6B flies were fed 25mM EMS and mated en mass to eyFLP;;ubi-GFP, FRT80B virgin females to screen a total of ~20,000 chromosome arms. Although most alleles from the screen were selected based on overgrowth of mutant white tissue relative to red wild-type twin spots, E7.25d.7 retained a mostly 50:50 white:red ratio, and apparent cell shape and bristle defects (Fig. 1A). To test whether our E725d.7 allele could synergize with Df(3L)H99 to override organ size controls, we crossed H99,E7.25d.7,FRT80B/TM6B to eyFLP;; RpL14¹,FRT80B/TM6B, a Minute allele (Saeboe-Larssen et al., 1997), thus allowing us to create entire heads that were mutant for E725d.7 and lacked developmental cell death. Compared to Df(3L)H99,FRT80B/FRT80B,RpL14¹ control heads, H99,E7.25d.7,FRT80B/FRT80B,RpL14¹ heads were larger and malformed, contained increased numbers of thick bristles, evidence of necrosis, and tumor-like morphological abnormalities (Fig. 1B). These phenotypes had different levels of expressivity, with females consistently exhibiting the most severe phenotypes.

In order to identify the gene, Df(3L)H99 was recombined off of the mutant chromosome, which was then checked for maintenance of lethality. E7.25d.7,FRT80B/TM6B female flies were then crossed in single vials to males from each of the 77 stocks of the Bloomington 3L Deficiency Kit (Cook et al., 2012). E7.25d.7 failed to complement Df(3L)Aprt-32 (3L: 1,668,654 … 2,587,456; Cook, 2016), but complemented Df(3L)BSC181 (3L: 1,688,724 … 1,841,694; BDSC, 2008) and Df(3L)ED4287(3L: 1,795,442 … 2,551,761; DrosDel Project, 2007) (Table 1). We obtained loss-of-function alleles for two candidates, daughter of sevenless (dos^P115) and mishappen (msn¹⁰²). msn¹⁰² failed to complement E7.25d.7. In a separate experiment E7.25d.7 failed to complement an independent allele of msn (msn¹⁷²) (Table 2).

msn encodes a ste-20-like Ser/Thr kinase of the MAPK4 family, and alleles were originally identified in two independent screens for genes required for eye development (Xu and Rubin, 1993), and those regulated by the photoreceptor transcription factor Glass (Treisman et al., 1997). The phenotypic abnormalities in the eye of the original msn alleles included cell shape and orientation changes in photoreceptors. It was also reported that msn transcripts are expressed in cells undergoing cell migration and shape changes, and ultimately it was hypothesized that Msn plays a role in cell signaling during cytoskeletal rearrangement. Msn has recently shown to act, along with other MAPK4-family kinases, to phosphorylate the Hippo pathway kinase Warts (Wts) to restrict Yorkie (Yki) activity (Meng et al., 2015; Li et al., 2015). Importantly, Msn activity is partially redundant to both Wts and other MAPK4 family members in restricting Yki activity, which could explain the conditional nature of the allele.