The use of genetic screens in Drosophila melanogaster has been a powerful tool for identifying genes associated with a variety of different biological phenomena, including cell growth and cell cycle regulation  . As a result of evolutionary conservation, information gleaned from the study of Drosophila can often be applied to improve both our understanding of the genetic causes of human diseases and our approaches to combating such diseases   . One such gene example is hippo (hpo), a gene involved in regulation of apoptosis and cell division in Drosophila, which has two human homologs, Mst 1 and Mst 2. Mutations in hpo have been shown to inhibit apoptosis and promote cell division; tissue overgrowth from hpo mutations in Drosophila has been found to be conserved in humans, with Mst 1/2 mutations being linked to human cancer  . In addition to hpo, additional other genes first identified in Drosophila have been found to play a role in human cancer development   -  .
To study mutations that otherwise would be homozygous lethal to the entire organism, the Flp/FRT system is utilized to restrict the homozygous mutant tissue to unessential organs or tissues, thus allowing the survival of the heterozygous organism into adulthood  . The expression of flippase (Flp) is under regulation of the eyeless promoter (Ey>Flp), which restricts the expression of Flp and results in a restriction of mitotic recombination in developing eye tissue. The FRT site used here is located on chromosome 2R and when Flp is expressed, recombination is facilitated between the FRT sites of the homologous chromosomes. Using ethyl methanesulfonate (EMS) to induce mutagenesis along with the Flp/FRT system directed to the Drosophila eye allows for a screen in which the ratio of homozygous mutant tissue to homozygous wild type tissue can be analyzed. Previous Flp/FRT screens of this design relied on a single EMS mutation to look for regulators of cellular pathways involved in governing tissue size, cell division, cell growth, and apoptosis. These screens resulted in the discovery of a variety of genes involved in different developmental and cellular processes     -  . In the present study, we utilized a “two-hit” Flp/FRT screen on chromosome 2R. We began the screen with the ;FRT42D, ark82 chromosome, which would block the canonical apoptotic pathway in homozygous mutant tissue, and then induced the mutations with EMS  . This screen, along with several others, has identified a conditional class of regulators of cell growth and cell division that are dependent on a block in apoptosis for phenotypic changes  -  . One of the mutants identified from this ark82 based screen was named Cruella (Cru), as a result of the abnormal black coloration of homozygous mutant tissue. The mutant Cru is the focus of the present study. It maps genetically to the gene capping protein α (cpa) and represents a novel allele of this gene, cpacru.
2.1. Phenotypic Characterization of Cruella through Mosaic Eye and Wing Crosses
The experiments performed stem from a conditional EMS mutagenesis screen of chro- mosome 2R for abnormalities in growth and development  . To study these mutations, the Ey > Flp/FRT system was employed. For the screen, male ;FRT42D, ark82 flies were fed EMS. Then, F1 mutants were mated to Ey > Flp ;FRT42D virgin females, and F2 progeny were screened for eyes that depicted an increase in tissue size or a change in the ratio of mutant to wild type tissue. It should be noted this in this screen, the mutant tissue is pigmented due to the mw + gene associated with the ark82 allele  .
The Cru, ark82 mutant eye was characterized by imaging F1 progeny from the cross of virgin females Ey > Flp ;FRT42D mated to males ;FRT42D, Cru, ark82. Similarly, mosaic clones were created in the wing by mating male Cru, ark82 mutants to virgin females UBX > Flp ;FRT42D. To analyze the phenotype of an eye entirely comprised of Cru, ark82 homozygous tissue, mutant males were mated to virgin females with a cell lethal mutation associated with the FRT chromosome Ey > Flp ;FRT42D, M(2)/CyO. To determine whether a block in apoptosis was necessary for the Cru phenotype, the wild type ark allele was recombined onto the ;FRT42D, Cru chromosome. This recombinant male was then mated to virgin females of the Ey > Flp ;FRT42D, ubi GFP. In this final experiment, the wild type tissue was pigmented with the mw+ gene, and the ;FRT42D, Cru chromosome was unpigmented. All mosaic eyes were imaged under ethanol, and mosaic wings were imaged under vegetable oil. Images were taken with Am Scope 5.0 MP digital microscope camera.
2.2. Genetic Mapping
To determine the locations of the mutant phenotype in Cru, we performed a series of complementation tests using Bloomington Stock Center Deficiency Kits for chromosome 2R. Crosses were set up between male ;FRT42D, Cru, ark82/CyO and virgin female deficiency stocks: Df2(R)/CyO to test for complementation. The F1 progeny were scored for complementation for each cross, and a region of non-complementation was identified. Complementation was determined by the presence of both curly and straight wing flies in the F1 generation. Non-complementation was identified when all F1 progeny had curly wings. Overlapping deficiencies were used to further narrow the region of chromosome 2R from the original failure to compliment genomic region. Finally, mutant alleles of candidate genes were mated to the Cru mutant to identify which gene the mutation is located. In particular, Cru was mated to the alleles cpa 107E and cpa 69E of the gene cpa  .
3.1. Phenotypic Characterization of Cruella
We initially identified Cru from the conditional screen due to the striking nature of the homozygous mutant tissue. As shown in Figure 1(B), ;FRT42D, Cru, ark82 resulted in a mosaic eye phenotype in which mutant tissue appeared black, as opposed to the red coloration typically seen in homozygous mutant tissue of mosaic eyes, compare tissue pigmentation of Figure 1(B) to that of Figure 1(A). Despite the change in pigmentation, the size of the eye and the relative ratio of mutant to wild type tissue appeared similar to the control ;FRT42D, ark82 mosaic (Figure 1(A)).
The entire eye was made homozygous by flipping the ;FRT42D, Cru, ark82 chromo-
Figure 1. Cru mosaic eye depicts a homozygous mutant tissue that is black in nature. A) control mosaic ;FRT42D, ark82 B) FRT42D, ark82, cpacru, mutant tissue is pigmented C) FRT42D, ark82, cpacru/FRT42D, M (2), mutant tissue is pigmented D) FRT42D, cpacru wild type tissue is pigmented. Eyes were imaged at 40 X magnification under 70% ethanol.
some over a cell lethal mutation on 2R (;FRT42D, M (2)). The result of this experiment was a complete pupal lethality, suggesting that Cru plays a necessary role in head and eye development. The dissected pharate pupae from this cross have underdeveloped eyes, and the eyes are almost entirely comprised of darkly pigmented tissue in a similar manner to the mosaic eye (Figure 1(C)).
To determine whether the Cru phenotype was dependent on a blockage of apoptosis and to demonstrate that the alteration in pigmentation is not related to the ark82 allele, we recombined a wild type ark allele onto the ;FRT42D, Cru chromosome. As can be seen in Figure 1(D), the mutant tissue (note that the wild type tissue now has the mw + gene) is still black in appearance, suggesting that the ark82 allele is not associated with the phenotype that alters the appearance of the pigment. Without blocked apoptosis, however, the relative amount of mutant tissue is decreased, suggesting that Cru homozygous mutant tissue may result in apoptosis, compare ratio of black pigmented tissue in Figure 1(B) to Figure 1(D).
To determine whether the Cru phenotype was exclusive to the eye or was more ubiquitous in the body, we created mosaic clones in the developing wing disc through the use of the UBX driver. Several notable phenotypic changes were observed in the Cru, ark82 mosaic wing including disruption in wing vein patterning compare Figure 2(B) to Figure 2(A). In addition to the alterations in wing vein patterning, the adult flies demonstrated a “wings held out” phenotype (Figure 2(C)) a commonly seen characteristic of alterations in wing development  .
3.2. Genetic Mapping of Cruella
To map the location of the Cru mutation, crosses were setup between males ;FRT42D, Cru, ark82 and virgin females of the deficiency stocks representing the overlapping deficiency stocks from the Bloomington Deficiency Kit for chromosome 2R. Complementation mapping identified that the lethal mutation of Cru resides on 2R between bases 21,056,798 and 21,088,247. A previously characterized gene in this region, capping protein α (cpa), was selected as a candidate. Matings between ;Cru, ark82 and two previously identified cpa alleles, cpa 107E and cpa 69E, resulted in a failure to complement
Figure 2. Cru mosaic wings result in vein alterations and a “wings held out” phenotype in adult drosophila. A) control mosaic adult wing, B) FRT42D, ark82, cpacru mosaic wing, C) FRT42D, ark82, cpacru adult with mosaic wings. All animals and wings are imaged from female flies. Wings were imaged at 70 X magnification under vegetable oil.
with Cru, suggesting that Cru is a novel allele of cpa  . Therefore, Cru will be referred to as cpacru from this point forward.
In the present study, we present the mapping and characterization of a novel allele of cpa, cpacru. The cpa protein functions as the α subunit of an actin capping protein. An α/β heterodimer is necessary for proper functionality. Previous evidence has shown that both the α and β subunits are necessary for a functional CP and that both subunits play a role in the regulation of one another  . A mutation in either subunit can therefore lead to an inability to perform F-actin capping, resulting in an accumulation of actin   . The phenotype resulting from cpacru homozygous tissue causes a loss of F-ac- tin capping ability in the eye tissue of Drosophila, resulting in improper actin polymerization. Such accumulation of actin filaments has led to what appears to be the discoloration of customarily red mutant eye tissue, causing it to appear black in Drosophila with cpacru alleles. Our finding is in agreement with the results of a study by Delalle et al., in which a similar alteration in pigmentation was observed  . Although the mosaic eye of the ;cpacru, ark82 has a similar ratio of mutant to wild type tissue as the ;ark82 mosaic eye, there is an essential role for cpa in eye development. When the ;cpacru, ark82 mosaic eyes were flipped over a cell lethal mutation, the result was pupal lethality, suggesting a necessary role of cpa in eye development. Previous D. melanogaster studies in retinal CP mutants have suggested that F-actin accumulating resulted in alteration of the regulation of actin-binding activities throughout the cell. This, in turn, could inhibit signaling events due to its effects on cell movement and polarity  . It is likely that the ;cpacru mutant heads are defective in developmental signaling. Future experimentation could look into the necessary role that cpa plays in regulating eye development in Drosophila.
In addition to phenotypic alterations in the mosaic eye ;cpacru, ark82 also demonstrated mosaic wing phenotypes. The accumulation of F-actin, resulting from the cpacru cells, led to the flies’ inability to form proper wing veins. Other studies have also observed wing alterations in cpa and cpb mutant cells in both wing imaginal discs and adult wings   . Furthermore, cpa or cpb mutations have also been found to lead to alterations in bristles, in which transheterozygous mutants displayed significantly shorter bristles   . This, in turn, caused disorganization and abnormal bundling of the actin cytoskeleton, resulting in the abnormal bristle phenotype shown in these studies    .
Many of the genes involved in the cytoskeletal pathways are evolutionarily conserved from Drosophila to humans. The relationship between cpa and cpb is well conserved in humans through the gene Cap Z, a subunit of the alpha-beta heterodimer, which functions in F-actin regulation and polymerization   . Because of the high levels of conservation between cpa and its human homolog Cap Z, the results of the present study have various implications for study of human diseases and disorders such as cell growth and nuerodegeneration  . Cap Z also plays a gatekeeper role in inhibiting Yap/Taz oncogenes in human cells, similarly to the way cpa and cpb inhibit Yki    . Mutations in Cap Z subsequently up-regulate Yap/Taz activity, which has been associated with human cancer development  . This type of evolutionary conservation in basic protein function, as well as in the associated cell signaling, makes Drosophila a powerful model for an array of human systems.
The authors wish to acknowledge S. Kelly and M. Andrzejak for comments on initial drafts, J. Roche and T. Hibbard of the University of Detroit Mercy Honors program, E. Kagey for providing editorial guidance, and F. Janody for providing CPA stocks. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.
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