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A Thesis Entitled A Visual Screen for Centrosome Mutants in Drosophila melanogaster by Sarah E. Hynek Submitted to the Graduate Faculty as partial fulfillments of the requirements for the Master of Science Degree in Biology Dr. Tomer Avidor-Reiss, Committee Chair Dr. Deborah Chadee, Committee Member Dr. Scott Leisner, Committee Member Dr. John Plenefisch, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2015 Copyright 2015, Sarah Elizabeth Hynek This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author An Abstract of A Visual Screen for Centrosome Mutants in Drosophila melanogaster by Sarah E. Hynek Submitted to the Graduate Faculty as partial fulfillment of the requirements for a Master of Science Degree in Biology University of Toledo May 2015 Centrosomes are highly conserved organelles that are composed of two microtubule-based centrioles surrounded by an amorphous protein cloud of pericentriolar material (PCM), which is able to nucleate astral microtubules. They serve as microtubule organizing centers during cell division and are important for fertilization. During fertilization, upon fusion with the ovum, the sperm contributes modified centrioles and a haploid set of genetic material. These modified centrioles recruit maternal PCM proteins and then form the microtubule sperm aster. These microtubules extend to find the female pronucleus and facilitate its movement towards, and eventual fusion with, the male pronucleus. This process creates a complete genome, and allows the first zygotic cell division to take place. During spermatogenesis, the centrioles undergo a variety of changes such as elongation, duplication, and separation, all of which precede centrosome reduction. This is the process which creates modified centrioles in the mature sperm to be contributed to the oocyte. iii During this phenomenon, the centrosome loses its astral microtubule nucleating function, PCM, and many centriolar proteins. By using a forward genetic approach, we have created a random mutagenesis screen for centriolar mutants in the testes, with the overall goal of finding those with centrosome reduction defects. We have chosen ethyl methylsulfonate (EMS) as our mutagen. Using Ana1-GFP or Asl-GFP, which label the centrioles and PCM respectively, we dissect and visualize Drosophila testes using fluorescence microscopy. In total we have examined 1436 mutants, finding many defects including those in testes morphology, centriole length, spermatid nucleus morphology, and one mutant of particular interest with Asl-GFP labeling in the mature sperm, indicating a defect in centrosome reduction. iv Acknowledgements This thesis would not be possible without the unyielding support of my family, friends, fiancé, advisor, and other dedicated faculty here at UT. No amount of thank you-s could express how grateful I am for every positive thought, motivational pep talk, and helpful discussion along the way. v Contents Abstract iii Acknowledgements v Contents vi List of Tables ix List of Figures x List of Abbreviations 1 xii Introduction 1 1.1 The Centrosome is Important for Fertility . . . . . . . . . . . . . . . . . 1 1.2 Centrosome Reduction Creates Modified Centrioles in the Sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3 Male Infertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.4 Spermatogenesis in Drosophila melanogaster . . . . . . . . . . . . . . 5 1.5 Drosophila melanogaster as a model organism . . . . . . . . . . . . . . 7 vi 2 3 Methods and Materials 10 2.1 Fly Stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2 EMS Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.3 Virgin Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.4 Crossing Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.5 Lethality Scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.6 Heat Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.7 Dissections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.8 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.9 Complementation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.10 Motility Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.11 Photon Counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.12 Penetrance Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 A Visual Screen for Centrosome Mutants 16 3.1 Screen Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 3.2 Testes Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.3 Nuclear Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . 19 vii 3.4 Developmental Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.5 Mutant UT737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.6 Mutant UT1446 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.7 Zuker Collection Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.7.1 Mutant z2984 has Asl-GFP in the Spermatozoa . . . . . . 23 3.7.2 Mutant z2984 is not a Plk4 Mutant . . . . . . . . . . . . . . . . 24 3.7.3 Mutant z2984 is a Complex Genetic Interaction . . . . . . 25 4 Discussion and Future Directions References 28 31 viii List of Tables Table 1 Numerical analysis of the screen . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 2 Phenotypic groups of mutants identified . . . . . . . . . . . . . . . . . . 18 ix List of Figures Figure 1 The Centrosome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Figure 2 Sperm Aster Formation in the Zygote . . . . . . . . . . . . . . . . . . . . . 3 Figure 3 Model of Centrosome Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Figure 4 Spermatogenesis in Drosophila melanogaster . . . . . . . . . . . . . . 6 Figure 5 Model of Forward Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Figure 6 Crossing Scheme for the Screen . . . . . . . . . . . . . . . . . . . . . . . . . 12 Figure 7 Testes Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Figure 8 Sperm Nucleus Morphology Defects . . . . . . . . . . . . . . . . . . . . . 19 Figure 9 Testes Developmental Defects . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Figure 10 Mutant UT737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Figure 11 Mutant UT1446 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Figure 12 Mutant z2984 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Figure 13 Mutant z2984 Penetrance Quantification . . . . . . . . . . . . . . . . . 24 x Figure 14 Mutant z2984 Photon Counting Quantification . . . . . . . . . . . . 24 Figure 15 Schematic of a Complementation Test . . . . . . . . . . . . . . . . . . . 25 Figure 16 Genetic Interaction Quantification . . . . . . . . . . . . . . . . . . . . . . 27 xi List of Abbreviations Asl . . . . . . . . . . . . . . . . . . Asterless CyO . . . . . . . . . . . . . . . . . Wings (Marker Mutation/Balancer) Dr . . . . . . . . . . . . . . . . . . Drop (Eye Marker Mutation) EMS . . . . . . . . . . . . . . . . . Ethyl Methanesulfonate Hu . . . . . . . . . . . . . . . . . . Humeral (Bristle Marker Mutation) MKRS . . . . . . . . . . . . . . . Balancer PCL . . . . . . . . . . . . . . . . . Proximal Centriole-Like PCM . . . . . . . . . . . . . . . . . Pericentriolar Material Tb . . . . . . . . . . . . . . . . . . . Tubby (Pupa Marker Mutation) TM6B . . . . . . . . . . . . . . . . Balancer xii Chapter One Introduction 1.1 The Centrosome is Important for Fertility The centrosome is a large organelle in the cell that is conserved throughout studied animal species, with the known exception of the flatworms, planarians (Azimzadeh et al., 2012). A pair of microtubule-based centrioles form the foundation of a typical animal centrosome, however in higher plants, these are absent (Brown and Lemmon, 2011). These centrioles recruit and are surrounded by an amorphous matrix of proteins called the pericentriolar material, or PCM. From the PCM, the centrosome can nucleate astral microtubules (Azimzadeh and Bornens, 2007) (Figure 1). The centrosome functions as the major microtubule organizing center of a cell during cell division, nucleates cilia, and plays a critical role in successful fertilization (Nigg and Raff, 2009). The centrosome plays a key role in fertilization by contributing its centrioles to the zygote (Sutovsky and Schatten, 2000). During oocyte formation in animals (oogenesis), the centrioles are eliminated, and PCM proteins become abundant in the cytoplasm (Schatten, 1994). Conversely, during sperm 1 formation in animals (spermatogenesis), the centrosomal components are eliminated leaving only the centrioles (Manandhar et al., 2005). In all nonrodent animals, when a sperm fuses with an oocyte, its genetic material (pronucleus) and centrioles, , are contributed to the zygote. The centrioles are then able to recruit the PCM proteins present in the zygotic cytoplasm and nucleate astral microtubules (Figure 2) (Callaini and Riparbelli, 1996), referred to as the sperm aster (Sathananthan et al., 1997; Terada et al., 2010). The astral microtubules extend throughout the zygote to locate the female pronucleus, which is then able to move along the astral microtubules towards the male pronucleus. After fusion of the pronuclei, each centriole duplicates forming two centrosomes, which allows the first zygotic cell division to take place (Blachon et al., 2014). Figure 1 – The centrosome consists of three components: two centrioles (green), an amorphous pericentriolar material cloud (red), and astral microtubules (red lines). 2 Figure 2 – The modified centrioles (green) from the sperm recruit PCM proteins (red) and nucleate astral microtubules (white) to locate the female pronucleus (pink) and help it move to fuse with the male pronucleus (blue). ♀ ♂ 1.2 Centrosome Reduction Creates Modified Centrioles in the Sperm During animal spermatogenesis, early spermatids have a typical centrosome with a pair of centrioles surrounded by PCM that can nucleate astral microtubules (Manandhar and Schatten, 2000). As spermatids mature (spermiogenesis), the centrosome loses many of its characteristics via a process called centrosome reduction. First, the centrosome loses its microtubule nucleating function; second, the PCM no longer surrounds the centrioles; and finally, the centrioles degenerate, however, this degeneration varies between different species (Figure 3) (Manandhar et al., 2000; Manandhar et al., 1998). For example, mice have completely degenerated centrioles (Manandhar et al., 1998), monkeys and humans have one intact and one degenerated centriole (Manandhar and Schatten, 2000), and Drosophila have one partially degenerated centriole and a partially degenerated secondary centriolar structure, the PCL (Blachon et al., 2009). 3 This knowledge comes from very few studies, and importantly, the mechanism and function behind centrosome reduction remains unknown. 1 2 3 Figure 3 – Centrosome reduction takes place in three steps: 1. Loss of astral microtubules 2. Loss of PCM 3. Loss of centriolar proteins. 1.3 Male Infertility Approximately 10 – 15% of couples in the United States are infertile (Practice Committee of American Society for Reproductive Medicine in collaboration with Society for Reproductive and Infertility, 2008) and it is estimated that in one-third of these cases, the male is the sole contributor to the problem. Despite recent advances in the diagnosis of infertility factors, in the male nearly 50% of all infertility cases have no known cause (Jose-Miller et al., 2007). Assisted reproductive technology (ART), such as in vitro fertilization and intracytoplasmic sperm injection (ICSI), is making it possible for those with male factor infertility to conceive. However, defects in the events after sperm entry into the oocyte cannot be overcome with this method (Terada, 2007). It is thought that in some of these cases, the centrosome may have defects that are still causing the infertility (Kovacic and Vlaisavljevic, 2000; 4 Rawe et al., 2000). We hypothesize that one such defect may be in centrosome reduction. 1.4 Spermatogenesis in Drosophila melanogaster Spermatogenesis is a highly complex process that must be tightly controlled to produce correct, functional male gametes. In Drosophila, spermatogenesis begins at the most apical end of the testes, the tip. Here we find a group of seven to nine stem cells referred to as the hub (de Cuevas and Matunis, 2011). Each stem cell asymmetrically divides to form one daughter cell, or spermatagonium, and one stem cell, keeping the hub replenished at all times (Kiger et al., 2001). The spermatagonium undergoes four mitotic divisions with incomplete cytokinesis to give a syncytium of sixteen spermatocytes. During this time, the cells increase to roughly thirty times their original size, and each spermatocyte has two pairs of centrioles which have elongated dramatically, and are perpendicular to one another (Gottardo et al., 2013). These cells are surrounded on either side by two cyst cells, ensuring that each mass of sixteen cells travel and develop together throughout spermatogenesis (Figure 4). After mitosis, the syncytium of spermatocytes, referred to as a bundle, completes meiosis one and meiosis two. These two cell divisions create a total of sixty-four haploid cells, each with only one centriole, called the giant centriole (Fuller, 1993). This group of cells has a round nucleus, and the giant 5 centriole docks to the nucleus while forming the cilium, which serves as the tail (flagellum) of the sperm cell (Figure 4). As the nucleus begins to change shape from round to leaf (Fabian and Brill, 2012), the formation of a unique atypical centriolar structure called the Proximal Centriole Like (PCL) occurs (Blachon et al., 2009), which serves as the second centriolar structure in the zygote (Blachon et al., 2014). The nucleus continues to change from leaf to canoe and then to needle, and in parallel both the major and minor mitochondria change from round to rod shaped (Fabian and Brill, 2012). During these changes, centrosome reduction takes place resulting in modified centrioles in the mature sperm (spermatozoa) which are finally stored in the seminal vesicle (Figure 4). Figure 4 – Spermatogenesis in the testes occurs in chronological order, and begins with stem cells and spermatogonia at the tip (red) and continues to spermatocytes (blue), then onto spermatids where the nucleus and mitochondria change shape (orange), and finally to a mature sperm stored in the seminal vesicle with reduced centriole and PCL. (purple). 6 1.5 Drosophila melanogaster as a model organism The fruit fly, Drosophila melanogaster, has been used in basic biological studies for over 100 years, serving as an excellent model for many cellular processes and human diseases (Roote and Prokop, 2013). Drosophila has a short generation time of ten days, and can be easily genetically manipulated with an array of tools and techniques. Using Drosophila to understand an observable phenotype is advantageous due to its ease of use in forward genetic screens. The forward genetic approach utilizes the observance of a particular phenotype followed by random mutagenesis until alteration or elimination of the phenotype is identified via screening. Forward genetics has five steps: 1) random mutagenesis, 2) identify a phenotype of interest, 3) identify the gene responsible, 4) study the function of the protein, and 5) study the ortholog in other species (Roote and Prokop, 2013) (Figure 5). 7 Figure 5 - We utilize the 5 step forward genetic approach in order to study centrosome reduction in the fruit fly, Drosophila melanogaster. Ethyl methanesulfonate (EMS), radiation, and transposons are common ways to induce random mutations (St Johnston, 2002). For the purposes of this screen, we have chosen to use EMS for three reasons: 1 – EMS generates single point mutations, and rarely, small deletions (Sega, 1984), often affecting protein domains, which will generate large numbers of alleles unlike other techniques that affect the whole protein. 2 – Unlike radiation and transposons, which have biases towards certain types of genes, EMS has the potential to mutate all genes. 3 – Identifying the specific mutation is simple with techniques such as whole genome sequencing. The original goal of our EMS screen was to identify mutants that do not undergo normal centrosome reduction. 8 To do this, we utilized a visual approach, where we dissected the testes of flies whose centrioles were labeled with Ana1-GFP, a centriolar protein. After dissection, the testes were stained with hoescht dye to see the nucleus, and observed under a fluorescence microscope. The phenotype we wanted to identify was flies containing spermatozoa in the seminal vesicle that still retained Ana1-GFP labeling, indicating a defect in centrosome reduction had taken place. Finding this phenotype would represent the first mutant in centrosome reduction, allowing for both mechanistic and functional insights to be gained. 9 Chapter Two Methods and Materials 2.1 Fly Stocks All flies were cultured on standard media at 25°C (Roote and Prokop, 2013). The HS-Hid-Dr and FRT82 stocks were obtained from the Bloomington Stock Center (7758 and 2035, respectively). The Asl reduction mutant was obtained from Dr. Barbara Wakimoto (Wakimoto et al., 2004), who screened through flies originally from Dr. Charles Zuker (Koundakjian et al., 2004). Transgenic constructs of Asl-GFP and Ana1-GFP were made in the Avidor-Reiss lab and sent to BestGene to create fly lines expressing the transgenic protein under its endogenous promoter. 2.2 EMS treatment Males 24 – 48 hours old were collected and starved, but not dehydrated, for approximately 18 hours in a chamber with damp filter paper. After approximately 18 hours, the males were moved to a chamber with filter paper dampened with a 25 mM EMS solution for 8 – 10 hours. The EMS solution was composed of 1% sucrose and green food coloring. Flies were then scored for green abdomens indicating that the EMS solution was ingested. Flies 10 without green abdomens were disposed of and those with green abdomens were moved to a fresh food vial overnight to recover before being crossed (adapted from (Koundakjian et al., 2004). 2.3 Virgin Collection Females were collected in a vial every 12 hours and considered virgin if no larva were produced after 3 – 4 days. Those that had a meconium were separated and labeled as “M+” and were able to be used right away. 2.4 Crossing Scheme For the first of three crosses, EMS treated males with an isogenized third chromosome that contained an FRT site (Bloomington stock #2035) were crossed en masse to virgin females containing the transgene on the second chromosome, Ana1-GFP, which labels the centrioles. These females also have the apoptotic gene Hid coupled with a heat inducible Hid promoter on their third chromosome; this is phenotypically marked with the dominant marker mutation Drop (Dr). For the second cross, single males were selected from the first cross’s progeny that had Ana1-GFP over the balancer CyO, which has the dominant phenotype of curly wings on the second chromosome. The males had a mutated third chromosome over the balancer TM6B. Each individual male was then crossed to 3-4 virgin females as stated above. Virgin collection was avoided by using a heat shock. Male and female progeny of heat shocked vials 11 were scored for successful heat shock by the absence of flies with the eye phenotype Dr and then moved to a new vial (cross #3) to mate. After 10 days, vials were scored for lethality (Figure 6). Figure 6 – Crossing scheme used to generate mutant lines that have mutations on the third chromosome. 2.5 Lethality Scoring Flies in cross 3 contain the balancer TM6B on the third chromosome with the dominant phenotypic markers Tubby (Tb) for pupae and Humeral (Hu) for adults. Absence of these marker phenotypes (denoted by a +) indicates fly is homozygous for any mutations on the third chromosome. The absence of Tb+ pupae in a vial indicated an embryo or larva lethal phenotype when the EMS induced mutation was homozygous. Tb+ pupae that did not develop enough to determine the sex were considered pupa lethal. Pupae that developed to determine the sex but did not have flies exit out of their pupae were considered pharate adult. Finally, Tb+ pupae with flies that exitd but did not yield live Humeral plus (Hu+) flies were deemed adult lethal. Vials with Tb+ pupae and Hu+ flies were considered to have viable mutations. 12 2.6 Heat Shock The heat shock was performed two times during cross two (refer to Figure 6). Parent flies were moved to new vials 24 hours after the creation of cross two, and the empty vials containing food and embryos were placed in a 37°C water bath for one hour to activate the heat inducible Hid promoter to kill the embryos. After another 24 hours, parents flies were disposed of, and the empty vials containing food and embryos were again placed in a 37°C water bath for one hour. This resulted in two copies of each cross two fly line. After 10 - 12 days, progeny flies were scored for Dr to confirm the success of the heat shock. If Dr flies were found, the flies and their vial were discarded because they were undesired progeny of cross two. 2.7 Dissections All dissections were performed on very late pupae or adults 1 – 3 days old as described in (Basiri et al., 2013) with the following changes: the testes were cut into 2 pieces for higher quality staining, and incubated in the stain (1:1000 µg DAPI or Hoescht) for 10 minutes. 2.8 Microscopy Screening of the mutant testes was done with a Leica SP5 fluorescent microscope using a 100x objective. Centriolar mutations were imaged with a Leica SP8 confocal microscope using a 63x objective. Confocal images were 13 taken as Z-stacks and formatted with maximum projection for image generation purposes. 2.9 Complementation Tests Complementation tests were performed if a mutant phenocopied a known mutation on the third chromosome. Heterozygote flies of the known gene and the phenocopying gene were crossed, and the pupae with one copy of the known mutation and one copy of the unknown mutation were dissected. Testes that maintained the mutant phenotype were considered to not complement, and those that exhibited a wild type phenotype were considered to complement. Mutants that did not complement were suggested to be new alleles of known genes. Mutants that complemented were suggested to represent a new gene. 2.10 Motility Assays Sperm motility was determined using males 24 – 48 hours old. Their testes were dissected and placed in 3µL of 0.5% NaCl and crushed with a glass coverslip. Slides were analyzed using a Leica SP5 microscope and phase contrast light with a 100x objective for spermatozoa with motile tails. 2.11 Photon Counting Testes were dissected as stated above in “Dissections,” with the following changes: the seminal vesicle was punctured before staining to release the 14 spermatozoa, and slides were not fixed. We used three different stages of sperm development to determine photon levels of Asl-GFP in the centrosome: round spermatid (onion stage), late spermatid, and spermatozoan. Five sperm at each stage from five different testes (25 measurements total) were collected using the Leica SP8 Confocal Microscope. The Asl-GFP images were taken as Z-stacks using the counting mode and then analyzed for number of photons at maximum projection. Statistics were generated using a two-tailed Student’s T test. 2.12 Asl-GFP in the Spermatozoa Quantification Testes were dissected as stated above in “Dissections,” with the following change: the seminal vesicle was punctured before fixing and staining. Testes from 5 different flies were analyzed, and 20 sperm from each were counted as either having or not having Asl-GFP in their spermatozoa. Statistics were generated using a two-tailed Student’s T test. 15 Chapter Three A Visual Screen for Centrosome Mutants 3.1 Screen Statistics In total, 7466 mutant lines have been attempted, and of those 2361 survived. The surviving lines were scored for lethality, and 887 lines were embryo or larva lethal, 38 were pupa lethal, and 199 were pharate adult/adult lethal. Mutations that were embryo, larva, or pupa lethal were discarded, and those that were pharate adult/adult lethal were retained because they still produced pupae that were screened. Pharate adults develop fully but do not exit from the pupa. In total, 1124 lines out of the established 2361 lines were lethal, a 48% lethality rate. Therefore, a total of 1436 lines were screened (Table 1). We targeted the third chromosome in this screen due to the availability of genetic tools in our lab. The third chromosome contains around 6000 genes with 4300 of them representing genes estimated to be essential (Koundakjian et al., 2004). By assuming a Poisson distribution (Pollock and Larkin, 2004), we calculated that we have induced 2.86 mutations per chromosome, resulting in approximately 4107 mutations visually screened for centrosome 16 and testes defects. Poisson distribution takes advantage of knowing the number of total genes on the targeted chromosome, and of those, how many are essential. By comparing the lethality rate of the EMS-induced mutations with the number of predicted essential genes, we are able to estimate the number of mutations per chromosome the EMS has caused. Lines Attempted 7466 Total Surviving Lines 2361 Early Lethal (No Tb+ pupa) 887 Pupa Lethal (unable to tell male/female) 38 Pharate Adult/Adult Lethal (No Hu+ flies) 199 Total Lethal 1124 Viable 1237 Lines Able to be Screened (Viable + Pharate Adult) 1436 Screened 1436 Mutations per chromosome 2.86 (48% lethality) Total mutation studied in the screen 2.86 x 1436 = 4107 Total Centriole Mutants 34 (2%) Table 1 – Numerical analysis of the screen to date, including lines created, lethal phenotypes scored, induced mutations based on Poisson Distribution, and total centriolar mutants found. 17 Phenotype Number of Mutants No spermatozoa in seminal vesicle 358 Testes Deformity 54 Developmental Defects 21 Nuclei Defects 16 Long Giant Centriole 29 No PCL 1 Short Giant Centriole 3 Centrosome Reduction 0 3.2 Testes Morphology Mutants We have identified 54 mutants with testes deformities (Table 2). These deformities represent a range of phenotypes. Many of the mutants have a large bulge in the tip of the testes, leaving the base very small and skinny (Figure 7). Other variations of a testes deformities include small testes and round, ball-shaped testes (Not shown). 18 Figure 7 – Mutant 20 has a massive bulb testes tip and mutant 5402 has a less severe defect with a large tip. Testes tips denoted with an arrow. 3.3 Nuclear Morphology Mutants Mutants we identified that have defects in their nuclear morphology exhibit a large range of phenotypes. In the screen, we identified 16 mutants with some sort of nuclear defect. Of these, 8 had curved nuclei, and 10 showed a developmental arrest (Table 2). The most common is an arrest between leaf and canoe stage which gives an amorphous shape that never progresses to the needle like shape of a normal mature sperm. In these mutants, the nucleus often elongates but is unable to morphologically change shape. We have also found mutants that showed a curved (Figure 8) or abnormal nucleus shape as a spermatozoa, and mutants arrest at the round spermatid stage, referred to in the mouse and human system as globozoospermia. Figure 8 – Mutant with spermatozoa nuclei (blue) that are curved (right) instead of needle shaped (left). 19 3.4 Developmental Mutants We have found a subset of mutations that affect the location of various stages in spermatogenesis, 21 in total exhibit this phenotype (Table 2). Normally in the testes, certain developmental stages are confined to specific areas of the testes, for example, stem cells and spermatocytes are always found at the tip of the testes. Along the outer edge of the testes before spiraling begins is where meiotic cells can be found (Figure 9). In mutants with developmental defects, we see spermatocytes and meiotic cells present very late, where we normally find bundles of maturing sperm that are canoe to needle shaped. We have also identified a mutant that appears to have too many stem cells in its hub (not shown). Figure 9 – Control testes (left) with spermatocytes in their proper position. Defective testes (mutant 850; right) with spermatocytes much later towards the base of the testes where typically more mature sperm are located. 20 3.5 Mutant UT737 Mutant UT737 exhibits a short giant centriole (Figure 10), and this phenocopies a known mutation in Bld10, a centriolar protein. We performed a complementation test between mutant UT737 and a Bld10 mutant, and found that the phenotype failed to complement, suggesting that this mutation is an allele of Bld10. Interestingly, we find that this mutant has motile sperm, but the known Bld10 mutant does not have motile sperm. This suggests that it may be a mutation in a different protein domain or a more mild mutation than the known allele. Flies with one copy of UT737 and one copy of the known Bld10 allele do not have motile sperm, but do exhibit a short giant centriole. Figure 10 – Mutant UT737 (right) has a short giant centriole compared to control (left). Centriole and PCL labeled by Ana1-GFP, nucleus stained with DAPI (blue). 3.6 Mutant UT1446 Ana1-GFP labels both the giant centriole and the PCL in the spermatids (Blachon et al., 2009). Mutant UT1446 is devoid of Ana1-GFP labeling of the 21 PCL in the spermatids (Figure 11). This mutant phenocopies a mutation in the know centriolar protein Poc1. A complementation test between the Poc1 allele k245 and mutant UT1446 failed to complement, suggesting that this mutant is an allele of Poc1. Flies with one copy of the k245 allele and one copy of the UT1446 allele show a normal giant centriole length, yet still does not have Ana1-GFP labeling of the PCL. Sequencing of the mutant confirmed that indeed, it is a mutation in Poc1. There are 3 other known alleles of Poc1, each exhibiting a PCL without Ana1 labeling and having a short giant centriole. Mutant UT1446 has the PCL phenotype, but has a normal length giant centriole, suggesting that its mutation is PCL specific. Figure 11 – Ana1-GFP labels the giant centriole and PCL in wild type spermatids (left), however in the UT1446 mutant (Poc1, right) there is no Ana1-GFP labeling of the PCL. 3.7 Zuker Collection Mutants A small collection of male sterile mutants originally from the Zuker Collection (Koundakjian et al., 2004) is available in our lab. Mutations in these flies were induced by EMS treatment. The flies have Asl-GFP, a PCM protein, instead of Ana1-GFP. In parallel to the Ana1-GFP screen, we also screened these Asl-GFP mutants. Surprisingly, we found that mutant z2984 22 as a heterozygote has Asl-GFP labeling in the spermatozoa (Figure 12), indicating a dominant mutation in which centrosome reduction did not go to completion. Figure 12 – Spermatozoa from control (left) and mutant z2884 (right) flies. Mutant z2984 retains Asl-GFP labeling of the centrosome (arrow), while the control does not, indicating a defect in centrosome reduction. 3.7.1 Mutant z2984 has Asl-GFP in the Spermatozoa In heterozygotes with the z2984 mutation, 89%(±8) of their mature spermatozoa have Asl-GFP labeling compared to 0% (±0) in control flies (n=5 testes, p<0.0001) (Figure 13). Photon counting quantification of this phenotype reveals that the mutant has a statistically significant increase in the total amount of Asl present in the spermatozoa compared to the control (Figure 14), confirming that this mutant does indeed have a defect in centrosome reduction of Asl. 23 Figure 13 – Analysis of spermatozoa in both wildtype and z2984 for Asl-GFP labeling. The z2984 mutant has a significant increase in the amount of spermatozoa with Asl-GFP labeling, n = 5, p<0.001 Percent Spermatozoa with Asl-GFP labeling *** 100 80 60 % Szoa with Asl-GFP 40 20 0 Wild type z2984 Photon Counting Asl-GFP 100,000 10,000 1,000 * 100 z2984 10 control 1 Figure 14 – Photon counting at round, intermediate, and mature stage sperm shows a significant increase in the amount of Asl-GFP in the mature sperm, n = 5, p < 0.001. Graph shown in a logarithmic scale. 3.7.2 Mutant z2984 is not a Plk4 Mutant Mutant z2984 phenocopies a known, dominant mutation in Plk4, which does not undergo complete reduction of Asl in the spermatozoa. Plk4 is on the third chromosome, and mutant z2984 came from an EMS screen for the third chromosome. A complementation test was performed with Plk4 and z2984 heterozygotes, with both mutations over the balancer, TM6B (Figure 15). This balancer has two dominant markers, Tb for pupae and Hu for flies, 24 allowing for those with plk4 and z2984 to be identified as Tb+ pupae or Hu+ adults. Because both mutations are dominant, the presence of Asl-GFP in the mature spermatozoa could not be used as a criteria for complementation, therefore the plk4 homozygote phenotype of uncoordination and meiotic defects was used. Upon analysis of flies containing plk4 and z2984, we found that they were coordinated, and had no meiotic defects in the testes, suggesting that mutation z2984 was not a Plk4 mutation. Figure 15 – Schematic representation of a complementation test between plk4 (blue) and z2984 (green). 3.8.3 Mutant z2984 is a Complex Genetic Interaction Because the z2984 mutation is a dominant mutation, it is not guaranteed that it resides on the third chromosome. We next needed to map the mutation to a chromosome using genetic crosses. Using a z2984 mutant male crossed to a wild type female, we negatively controlled for the X chromosome, and 25 positively controlled for the 2nd and 3rd chromosome as fathers do not pass their X chromosome onto their sons. Our results showed that males with a wild type X chromosome and wild type 2nd and 3rd chromosomes over the balancer CyO (2nd) or TM6B (3rd) had Asl-GFP in 0% of their spermatozoa (n = 5 testes). Males with a wild type X chromosome and the z2984 mutation on the 3rd chromosome over the balancer TM6B had Asl-GFP labeling in about 2% of their mature sperm (n = 5 testes), significantly less than the original phenotype of 89%. Lastly, we found that males with a wild type X chromosome and z2984 mutation on the 3rd chromosome over the balancer MKRS had Asl-GFP labeling in 0% of their spermatozoa (n = 5 testes). Statistical analysis of the percent of spermatozoa with Asl-GFP labeling shows no significant difference between the three groups. Together these data suggest that the mutation is located on the X chromosome. Using z2984 mutant females, we positively controlled for the X chromosome. Our results show unexpectedly that progeny males with a predicted mutated X chromosome and a wild type 3rd chromosome over the balancer TM6B have Asl-GFP labeling in only 1% of their mature sperm on average (n = 5 testes). Male progeny that had a predicted mutated X chromosome and the z2984 mutation on the 3rd chromosome over the balancer TM6B showed Asl-GFP in the spermatozoa in 80% of their mature sperm (n = 5 testes). Even more surprising was that when males had a predicted mutated X and the z2984 mutation over the balancer MKRS we saw Asl-GFP labeling in 0% of the 26 spermatozoa (n = 5 testes) (Figure 16). Statistical analysis of these data show a significant difference between the percent spermatozoa with Asl-GFP in mutant x, z2984 on the third, and the balancer TM6B compared to all other male progeny. These data suggest a complex genetic interaction between a mutation on the X chromosome, the z2984 mutation, and the balancer TM6B. Due to the complex nature of this interaction, we have chosen not to pursue further studies on it. However, determining the genes responsible would be possible with whole genome sequencing. In order to do this, we would need to obtain the original fly line that the mutant was created from and compare its sequence to mutant z2984. z2984 Males x Wild type Females Wild type Males x z2984 Females *** Figure 16 – Percent of spermatozoa with Asl-GFP labeling in z2984 males crossed with wild type females (left) as positive control for the 2 nd and 3rd chromosomes and negative control for the X chromosome. Percent of spermatozoa with Asl-GFP labeling in z2984 females crossed with wild type males (right) as positive control for the X chromosome and negative control for the 2nd and 3rd chromosomes. N = 5 testes, p<0.0001 for x*; 3*/TM6B compared to all other progeny scored. 27 Chapter Four Discussion and Future Directions Even though we did not succeed in our original goal to find a centrosome reduction mutant in the centriolar protein Ana1, the screen was still successful in many ways. Here we have shown that the visual approach to screening in the Drosophila testes is an effective way to find mutations in both the centrosome and in the many areas of spermatogenesis. Previously, centrosome mutations were found using a behavioral screen that looked for uncoordinated flies. Overall in this screen, we were able to identify 34 centrosome mutations, about 2% of the total mutants found. This represents one of the largest collections of centrosome mutations, which can be further investigated by our lab and by other labs in the field. We are currently following up with 2 mutants from this screen, the Bld10 (UT737) mutant and the Poc1 (UT1446) mutation. UT737 is being sequenced to find its specific mutation. We are interested in this mutant because it has motile sperm unlike any other Bld10 mutants. The UT1446 mutant is currently being examined by multiple members of the lab, both biochemically 28 and genetically. A paper is in progress regarding this mutant and its impact on male fertility. The third chromosome of Drosophila contains roughly 6000 genes (Koundakjian et al., 2004). In our EMS screen, we generated an estimated 4107 mutations, potentially covering 68% of the genes on the third chromosome. This leaves nearly 2000 genes that were potentially not hit based on our estimations. We also did not find more than one allele for any known gene that we could score. This screen was done over 2 years, so in order to generate enough mutations to potentially mutate every gene on the third chromosome once, the screen would need to continue for at least 1 more year. Instead of extending the EMS screen, we have chosen to pursue an RNAi screen in the lab. The RNAi screen will be targeting all known kinases and phosphatases in the Drosophila genome due to our lab’s recent finding that the kinase, Plk4, regulates the centrosome reduction of the PCM protein Asl. RNAi lines for all of the 251 kinases and 86 phosphatases are available in the Vienna Drosophila RNAi Center and the Transgenic RNAi Center (Dietzl et al., 2007). The advantage of doing an RNAi-based screen is that the gene causing the phenotype is already known, and that the RNAi can be specifically expressed in the germline cells, minimizing its impact on fly viability. 29 Our hypothesis regarding centrosome reduction was that it is essential for male fertility, and recent work in the lab supports this. The plk4 mutant in our lab has a defect in the centrosome reduction of the PCM protein Asl. When Asl is labeled with GFP in this mutant, fluorescence is still observed in the mature sperm, just like the z2984 mutant (Figure 12) Embryos that are fathered by sperm with this defect have an arrest or a delay in development, resulting in a decrease in fertility by about 20%. We would anticipate that having multiple mutations that affect the reduction of various centrosomal proteins would lead to a greater reduction in the fertility of these sperm. One important future goal of the screen is to create a resource of these mutations for other labs that have an interest in any of the phenotypes we identified. For example, a group in Pennsylvania we met at the recent Drosophila conference was very interested in our testes deformity mutants that have large bulb tips. The Drosophila community greatly benefits from mutant collections of EMS screens that are made available to the public. 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