Genetic mapping of EgfrL.3.1 in Drosophila melanogaster

Figure 1.

A. Mosaic (FRT42D Dark⁸²), and Dark⁸² Egfr^L.3.1 (FRT42D Dark⁸²Egfr^L.3.1) eyes. In both eyes the homozygous mutant tissue is pigmented (w^+mC). B. Region of chromosome 2R that failed to complement L.3.1 by deficiency mapping (2R:21,497,290..21,806,350). Arrow denotes location of Egfr gene. B is adapted from flybase.org (Gramates et al., 2017).

Description

An EMS screen was conducted utilizing the Flp/FRT system to identify mutations that caused an array of phenotypic alterations in the size of the eye including the ratio of mutant to wild type tissue (red over white) or the developmental patterning of the mosaic eye. This screen was done in the genetic background of blocked apoptosis in the homozygous mutant cells to identify conditional regulators of cell growth and eye development (Kagey et al., 2012). The block in apoptosis in the mosaic mutant tissue was achieved by using a FRT42D Dark⁸² chromosome as a starting point for the EMS mutagenesis (Akdemir et al., 2006). The Dark⁸² allele was generated by an imprecise excision of the P{lacW}Ark^CD4, this allele retains the w^+mC (Akdemir et al., 2006) One of the mutants identified was L.3.1 which generated a small rough eye mosaic phenotype, with a smaller percentage of pigmented tissue than the FRT42D, Dark⁸² control (Figure 1A). The Dark⁸² mosaic eye is approximately 60% pigmented tissue, while the Dark⁸² L.3.1 mosaic eye was smaller overall and approximately 50% mutant tissue (w^+mC). The ‘rough eye’ phenotype indicates a disruption in the ommatidial organization. In both images the pigmented (w^+mC) tissue is homozygous mutant and the unpigmented tissue is homozygous wild type.

The genetic mapping of the location of mutant L.3.1 was done by two independent groups of undergraduate researchers at the University of Detroit Mercy and University of Evansville in undergraduate genetics laboratory courses as part of the Fly-CURE consortium (Bieser et al., 2018). Complementation mapping was conducted independently and the results confirmed between groups. Virgin females from the FRT42D L.3.1 Dark⁸²/CyO stock were mated in series to male flies from the 87 deficiency stocks that comprise the Bloomington Stock Center 2R Deficiency Kit (only stocks distal to the FRT42D site were used for mapping) (Cook et al., 2012). Mutant L.3.1 failed to complement Deficiency stock Df(2R)BSC664 (2R:21,341,647..21,872,028), while complementing the flanking overlapping deficiencies Df(2R)BSC821 and Df(2R)BSC597. This left a region of failure to complement of 2R:21,497,290..21,806,350, which is pictured above in Figure 1 B. Lethal alleles of candidate genes within this region were mated independently to L.3.1 to test for complementation. L.3.1 failed to complement an apparent loss of function allele Egfr^k05115 (Dworkin et. al. 2006), indicating that L.3.1 is likely a novel Egfr allele, Egfr^L.3.1.

Reagents

FRT42D Dark⁸²/CyO (Akdemir et al., 2006)

FRT42D Dark⁸² Egfr^L.3.1/CyO (this manuscript)

Ey>Flp; FRT42D (BDSC 5616)

y¹ w^67c23; P{w^+mC=lacW}Egfr^k05115/CyO (BDSC 10385)

Bloomington Drosophila Stock Center 2R Deficiency Kit (Cook et al., 2012):

w¹¹¹⁸; Df(2R)BSC664/SM6a

w¹¹¹⁸; Df(2R)BSC821, P+PBac{ w^+mC =XP3.RB5}BSC821/SM6a

w¹¹¹]; Df(2R)BSC597/SM6a