Regulation of Egg Activation in Drosophila

1 Regulation of Egg Activation in Drosophila Alex MacLellan Dr. Andrew Swan Lab Department of Biomedical Sciences University of Windsor 2 Abstract Egg activation in Drosophila describes a series of events that signify the transition of mature oocyte to embryo. This includes completion of meiosis, destabilization of maternally deposited mRNA transcripts, and cortical microtubule reorganization. This project investigates the poorly understood upstream regulation of transcript destabilization and cortical microtubule reorganization to better understand eukaryotic embryogenesis. Many of the events of egg activation are regulated by the Anaphase Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase which facilitates the metaphase to anaphase transition in mitosis and meiosis. Transcript destabilization and cortical microtubule reorganization are both presumed to be APC/C dependent; however, this is still an area of ongoing investigation. The APC/C, activated by Cyclin-dependent kinase 1 (Cdk1) mediated phosphorylation and binding of Cort and Fzy subunits, ubiquitinates substrates which are subsequently degraded by the proteasome. We propose a model based on previous work for transcript destabilization that involves Cdk1-Cyclin B3 activating APC/CCort/Fzy, which subsequently degrades Cyclin B3, causing a drop in Cdk1 activity. This drop in Cdk1 activity allows an enzyme known as the PAN GU kinase complex to transition to an active, unphosphorylated state. Active PAN GU kinase can then phosphorylate well characterized downstream targets which mediate transcript destabilization. Cortical microtubule reorganization, although likely APC/C dependent, remains poorly understood. This project employs mutant alleles and RNAi knockdown to investigate Cyclin B3, Cort, Fzy, and APC/C roles, assessing transcript destabilization using qRT-PCR and cortical microtubule reorganization via immunostaining. Our data suggests that Fzy is required for transcript destabilization. Other components are still in the process of investigation. 3 Acknowledgements First and foremost, an enormous amount of appreciation goes to Rajni. The amount of time you spent training me, explaining concepts, helping me whenever I needed it, all while being incredibly kind and patient, is truly admirable. I’ve been truly honored to work alongside you on your project nearly every day for the last 8 months. I wish nothing but the absolute best for you in all aspects of your life and I hope you keep me updated on this project. Dr. Swan, you gave me the opportunity to join your lab back in January 2023 as an undergraduate volunteer who’s main responsibility was washing vials. Who knew that would pave the way for me to work on this unforgettable project. I cannot thank you enough for the experience this has been, along with your helpfulness and kindness along the way. A thank you to Fatima, who went with me on this journey as doing an undergraduate thesis together. You were always helpful and wish you the best with your future endeavors. A thank you to the other members in this lab: Paria, Emma, Aleks, and Hoda. Without your efforts, none of this would be possible. Lastly, thank you to all the fruit flies (even though they caused me lots of problems at times) which allowed me to gain so much skill and experience, ultimately creating an unforgettable part of my life. 4 Table of Contents Abstract……………………………………………………………………………………………………2 1. Introduction ………………………………………..……………………………………….……..5 1.1 Cell Division ……………………………………………………………………………….…5 1.2 Cyclin Dependent Kinases ……………………………………………………………………5 1.3 Cyclins ………………………………………………………………………………………..6 1.4 The APC/C, Fzy, and Cort ……………………………………………………………………7 1.5 Egg Activation ………………………………………………………………………………..9 1.6 Objectives …………………………………………………………………………………...12 2. Materials and Methods …………………………………………………………………………..14 2.1. Gal4-UAS System ……………………………………………………………………..…....14 2.2. Crosses ………………………………………………………………………………...……14 2.3. Sample Preparation for Western Blot ……………………………………………………….16 2.4. Western Blot Protocol ………………………………………………………………………16 2.5. Bacterial Transformation and Plasmid Isolation ……………………………………………17 2.6. Restriction Digest …………………………………………………………………………...18 2.7. Embryo Collection ………………………………………………………………………….18 2.8. RNA Extraction ……………………………………………………………………………..18 2.9. cDNA Synthesis …………………………………………………………………………….19 2.10. qRT-PCR ………………………………………………………………………………...19 2.11. Methanol Fixation of Embryos ………………………………………………………….19 2.12. Immunostaining of Methanol Fixed Embryos …………………………………………..20 2.13. Formaldehyde Fixation of Embryos …………………………………………………….20 2.14. Immunostaining of Formaldehyde Fixed Embryos ……………………………………..21 3. Results …………………………………………………………………………………………...22 3.1. Fzy Knockdown ……………………………...……………………………………………..22 3.2. APC/C Knockdown ………………………………………………………………………...23 3.3. Non-Degradable Cyclin B3 …………………………………………………………………24 3.4. cort;cycB3 Double Mutant ………………………………………………………………….27 3.5. Microtubule Reorganization of cort Mutant to Determine Best Technique ………………..27 4. Discussion 4.1. Fzy Knockdown …………………………………………………………………………….29 4.2. APC/C Knockdown ………………………………………………………………………...29 4.3. Non-Degradable Cyclin B3 …………………………………………………………………30 4.4. cort;cycB3 Double Mutant ………………………………………………………………….32 4.5. Determination of Best Technique for Microtubule Staining ………………………………..33 4.6. Conclusions and Additional Future Steps …………………………………………………..33 Statement of Contribution ……………………………………………………………………………….35 References………………………………………………………………………………………………..36 5 Chapter 1 Introduction 1.1 Cell Division Meiosis and mitosis are the two types of cellular divisions fundamental to all multicellular eukaryotes. The mechanisms governing mitosis and meiosis are under tight regulation, although meiotic regulation is less understood. Mitosis consists of a single round of cellular division that produces two genetically identical diploid daughter cells, whereas meiosis involves two successive divisions yielding four haploid gametes of different genetic composition. The first meiotic division involves separation of homologous chromosomes, whereas the second meiotic division involves separation of sister chromatids, analogous to mitosis. Mitosis is how complex multicellular organisms with differentiated cells arise from a single zygote to form complex organisms and is also largely involved in repair of damaged tissues. Meiosis is a mechanism that allows for sexual reproduction in eukaryotes, allowing for genetic diversity via recombination of maternal and paternal genes, creating genetically unique offspring. 1.2 Cyclin-dependent Kinases All phases of the cell cycle, including cellular divisions, are intricately regulated by various cyclin-dependent kinases (Cdks). Cdks are serine/threonine kinases that rely on binding of cyclins to expose their active site (Ding et al., 2020). Cdks can bind to different cyclins, leading to different substrate specificity and ensuring coordinated progression throughout the cell cycle (Minshull et al., 1990). Cdk1 is a well conserved enzyme across eukaryotes that is necessary for mitotic and meiotic cellular divisions, hence also referred to as M-Cdk. Cyclin A, Cyclin B, and Cyclin B3 all interact with Cdk1. In addition to cyclin binding, two other criteria are necessary for Cdk1 to be fully activated: Cdc25 phosphatase mediated dephosphorylation of inhibitory phosphorylated sites on Cdk1 and 6 phosphorylation on Cdk1’s T-loop mediated by Cdk7. (Gavet & Pines, 2010; Merrick et al., 2008; Sur & Agrawal, 2016). The activity of Cdk1 activity orchestrates precise progression through cellular divisions. In both mitosis and meiosis, Cdk1 phosphorylates nuclear lamins and nuclear pore complexes to trigger nuclear envelope breakdown (NEB). NEB is a crucial initiating step of cellular division which allows cytosolic proteins to interact with nuclear contents and vice versa. Additionally, Cdk1 phosphorylates the condensin complex required for chromosome condensation in prophase (Abe et al., 2011). Cdk1 also phosphorylates a crucial cell cycle regulator, the anaphase promoting complex/cyclosome (APC/C). The APC/C is discussed in detail in later paragraphs. 1.3 Cyclins Cyclins comprise a protein family known for their interaction with various Cdks. Structurally, they share a defined feature known as the cyclin box, a domain consisting of approximately 100 amino acid residues which form five alpha-helix secondary structures. (Malumbres, 2014). Cyclins were first discovered by Evans et al., 1983 due to there oscillating expression levels in dividing cells of sea urchin eggs. Cyclin A, Cyclin B, and Cyclin B3 are all implicated with mitosis and appear to have partially redundant roles (Furuno et al., 1999; Swan & Schüpbach, 2005). In Drosophila, mutations to cycA are lethal whereas cycB mutants, which are sterile, and cycB3, which are female sterile, are viable (Reber, 2006; Bourouh & Swan, 2018; Jacobs et al., 1998). In contrast, both cycA and cycB mutants are lethal in vertebrates. cycB and cycB3 double mutants are lethal in Drosophila, implicating an overlapping role in mitosis (Bourouh & Swan, 2018; Jacobs et al., 1998). All three mitotic cyclins get degraded during Drosophila mitosis, with Cyclin A usually being first, followed by Cyclin B and lastly Cyclin B3 (Swan 7 & Schüpbach, 2005). Cyclin A in complex to Cdk1 is directly involved in phosphorylation of nuclear lamins associated with NEB, Cyclin B also is involved in NEB when localized to the nucleus, along with other functions (Stiffler et al., 1999; Onischenko et al., 2005). Cyclin B3 is involved in chromosome condensation in prophase, mitotic progression, and anaphase onset via APC/C activation (Jacobs et al., 1998; Yuan & O’Farrell, 2015; Garrido et al., 2020). The role of each cyclin in Drosophila meiosis appears to be more complex and again seemingly redundant in certain aspects. All three cyclins contribute to NEB, although cyclin A has the greatest contribution (Bourouh et al., 2016). Cyclin A is required early in meiosis I for proper homolog segregation (Bourouh et al., 2016). Cyclin B is necessary for the metaphase I arrest in mature oocytes, the timely advancement through the meiosis II, and proper spindle organization (Bourouh et al., 2016). Cyclin B3 in Drosophila meiosis is poorly understood, but Cyclin B3 activates the APC/C in meiosis (Garrido et al., 2020). Cyclin B3 is required for anaphase progression in meiosis I and II (Bourouh et al., 2016). In cycB3 mutant females, defects in meiosis are seen only after metaphase I (Jacobs et al., 1998). 1.4 The APC/C, Fzy, and Cort The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit protein with E3 ubiquitin ligase enzymatic activity which facilitates the metaphase to anaphase transition in both mitosis and meiosis, although its role in meiosis is less characterized. Structurally, the APC/C is comprised of 13 subunits with a molecular weight of over 1 MDa, making it an incredibly large protein complex (McLean et al., 2011). The APC/C ubiquitinates substrates on lysine residues (Van Voorhis & Morgan, 2014). Following ubiquitination of substrates, they are recognized by the 26S proteasome for destruction where they are cleaved into peptide fragments (McLean et al., 2011). 8 With respect to the mitotic cell cycle, the APC/C becomes active during the metaphase to anaphase transition. The spindle assembly checkpoint (SAC) is a complex of proteins and prevents premature activation of the APC/C, hence preventing improper or premature sister chromatid separation (McLean et al., 2011). One of the proteins of the SAC, Mad2, sequesters Cdc20 until proper mitotic spindle attachment has been achieved for all chromosomes in a cell. Cdc20 is one of two activating subunits of the APC/C, with the other one being Cdh1. APC/CCdc20 is active predominantly in anaphase, where APC/CCdh1 regulates Cdk activity after completion of anaphase. In addition to its activating subunit, the APC/C requires phosphorylation via Cdk1 to be active. The active APC/C has two relevant targets, Securin and M-phase cyclins. The APC/C recognizes a destruction box (D-box) motif on both cyclins and Securin substrates (Kraft et al., 2005). Prior to APC/C activation, Securin is bound to the enzyme Seperase; this interaction inhibits Seperase (Luo & Tong, 2020). Securin ubiquitination mediated by the APC/CCdc20 and subsequent destruction allows for freeing of Seperase (Guo et al., 2015). Seperase cleaves Scc1, which is a part of the cohesin protein complex holding sister chromatids together (Uhlmann, 2001). The cleavage of Scc1 permits the sister chromatid separation characteristic of anaphase (Uhlmann, 2001). The other target of APC/CCdc20 is the mitotic cyclins. This ubiquitination of the cyclins results in a decline of Cdk1 activity. Simply put, cyclins activate the machinery that causes its own destruction. This occurs in anaphase allowing migration of sister chromatids to their respective poles of the cell. Interestingly, in Drosophila meiosis, there is no evidence suggesting the SAC for APC/C function is required for meiosis (Batiha & Swan, 2012). SAC mutants are viable and fertile. In Drosophila, the Cdc20 homologue is Fizzy (Fzy). Cortex (Cort) is another germline specific Cdc20 homologue in Drosophila females. Cort and Fzy have both distinct and redundant functions in meiosis. Both APC/CCort and APC/CFzy are implicated in meiotic cyclin A destruction, however APC/CCort has 9 much greater affinity (Vardy et al., 2009). APC/CCort ubiquitinates cyclin B associated with the spindle midzone, whereas APC/CFzy targets cyclin B localized to spindle microtubules in Drosophila meiosis (Swan and Schüpbach, 2007). cort mutants arrest in metaphase II, however the earliest requirement for Cort is chromosome segregation in anaphase I (Page & T.L. Orr-Weaver, 1996). In contrast, fzy mutants arrest in anaphase II, however once again the earliest requirement of Fzy is in anaphase I (Swan and Schüpbach, 2007). Swan and Schüpbach, 2007 found that double mutants of cort and fzy arrest in metaphase II, furthermore suggesting distinct yet overlapping roles. 1.5 Egg Activation Egg activation in Drosophila encompasses a series of events that signify the transition of mature oocyte to embryo. This includes completion of meiosis from their metaphase I arrest, microtubule reorganization around the cortex, degradation of certain maternally deposited messenger RNA (mRNA) transcripts, and increased protein translation in the absence of transcription (Theurkauf & Hawley, 1992; Page & T.L. Orr-Weaver, 1996; Hara et al., 2017; Tadros et al., 2003). Interestingly, fertilization is not a requirement for egg activation, and it is activation, not fertilization that alleviates meiotic arrest (Page & T.L. Orr-Weaver, 1996; Mahowald et al., 1983). In Drosophila, egg activation typically occurs when the oocyte reaches the oviducts (Theurkauf & Hawley, 1992). Destabilization of maternally deposited mRNA transcripts is a developmental process well conserved across the animal kingdom. This process occurs in the 3–5-hour time interval following eggs being laid. Transcripts are stable in the 0-2 hour time interval. During early embryogenesis, protein synthesis is dependent on maternally deposited transcripts rather than zygotic transcription to be translated into proteins (Tesarik, 2022). Maternal transcript deposition and destabilization has been observed in insects, mice, humans, and many other organisms (Preuss et al., 2012; Tora & Vincent, 2021; Tesarik, 2022). Egg activation alone is both necessary and sufficient to cause transcript 16 APC knockdown: ♀𝑚𝑎𝑡67 𝑚𝑎𝑡67;+ + 𝑥 ♂+ +;𝑈𝐴𝑆 𝐴𝑃𝐶3𝑅𝑁𝐴𝑖 𝑈𝐴𝑆 𝐴𝑃𝐶3𝑅𝑁𝐴𝑖 → ♀𝒎𝒂𝒕𝟔𝟕 +;𝑼𝑨𝑺 𝑨𝑷𝑪𝟑𝑹𝑵𝑨𝒊 + Balanced 𝑐𝑦𝑐𝐵3𝑑 stock: ♂𝑈𝐴𝑆 𝑣𝑒𝑛𝑢𝑠𝑐𝑦𝑐𝐴 𝑐𝑦𝑜 ;𝑈𝐴𝑆 𝐺𝐹𝑃𝑐𝑦𝑐𝐵3𝑑 𝑇𝑚6(10.20) 𝑥 ♀ 𝐶𝑦𝑂 𝑠;𝑇𝑚6 𝑃𝑟 (9.04) → 𝑪𝒚𝑶 𝒔;𝑼𝑨𝑺 𝑮𝑭𝑷𝒄𝒚𝒄𝑩𝟑𝒅 𝑻𝒎𝟔 Expression of 𝑐𝑦𝑐𝐵3𝑑 from 10.20 stock: ♀𝑚𝑎𝑡67 𝑚𝑎𝑡67;+ + 𝑥 ♂𝐶𝑦𝑂 𝑠;𝑈𝐴𝑆 𝐺𝐹𝑃𝑐𝑦𝑐𝐵3𝑑 𝑇𝑚6 → ♀𝒎𝒂𝒕𝟔𝟕 𝑪𝒚𝑶/𝒔 ;𝑼𝑨𝑺 𝑮𝑭𝑷𝒄𝒚𝒄𝑩𝟑𝒅 + Expression of 𝑐𝑦𝑐𝐵3𝑑 from 10.17 stock: ♀𝑚𝑎𝑡67 𝑚𝑎𝑡67;+ + 𝑥 ♂𝐶𝑦𝑂 𝑠;𝑝𝑖𝑚𝑑𝑘1,𝑈𝐴𝑆 𝐺𝐹𝑃𝑐𝑦𝑐𝐵3𝑑 𝑇𝑚6(10.17) → ♀𝒎𝒂𝒕𝟔𝟕 𝑪𝒚𝑶/𝒔 ;𝒑𝒊𝒎𝒅𝒌𝟏,𝑼𝑨𝑺 𝑮𝑭𝑷𝒄𝒚𝒄𝑩𝟑𝒅 + 2.3 Sample Preparation for Western Blot Ovaries were dissected in 1X PBS. Following dissection, they were transferred to Eppendorf tube, flash froze in liquid nitrogen then ground in 20 µL of 2X SB per pair of ovaries, followed by boiling of the sample at 65 ºC for 5 minutes. They were then stored at -20 ºC until ready to use. 2.4 Western Blot Protocol 10 µL of each sample was loaded into a 7.5% polyacrylamide gel and ran at 120 V. The transfer to a nitrocellulose membrane using 350 mA current for 1 hour. The nitrocellulose membrane was stained in Ponceau Red for 5 minutes. Blocking was done using 5% milk solution in TBST for 1 hour. The membrane was then sealed in a hybridization bag with 1/1000 Rabbit anti-GFP IgG primary antibody 17 (Torrey Pines Biolabs, Lot: 081211) in TBST and left to incubate overnight. Following TBST washing, the CyTM5 AffiniPureTM Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch Laboratories Inc.) was added at 1/10000 for 1 hour. The blot was then washed in TBST and imaged using SuperSignal West Pico Plus Chemiluminescent Substrate and imaged using BIORAD ChemiDec Imaging System. 2.5 Bacterial Transformation and Plasmid Isolation 0.05 mL of NEB 5-alpha Competent E. coli cells (New England BioLabs) were transformed with pUAS-GFPcycB3dSDM plasmid according to NEB 5-alpha Competent E. coli High Efficiency transformation protocol, with the exception of using 2TY media rather than SOC. 1 mL was added to a LB-amp selection plates and spread using a plate spreader. Using a sterilized plate spreader, the initial spread-out aliquot was spread to a second LB-amp plate. Both plates were incubated overnight at 37 ºC, then wrapped in parafilm and stored ~48 hours at 4 ºC. Using a pipette tip, four individual colonies were selected, two from each the diluted and non-diluted plate. The pipette tip was then added to an autoclaved glass vial containing 2 mL of 2TY media and 4 µL of filter sterilized 100mg/mL ampicillin solution and incubated overnight at 37 ºC in a 220 rpm shaker. The following day, the plasmid was isolated using Qiagen QIAprep Spin Miniprep kit. The plasmid concentration following isolation was measured using the Thermo Scientific NanoDrop OneC Microvolume UV-Vis Spectrophotometer. The plasmid concentration of the chosen colony was 385.4 ng/µL and the 260/280 value was 1.90. (The 260/280 value is a ratio of absorbance of the sample at 260 nm light to absorbance at 280 nm light, indicative of nucleic acid purity). 18 2.6 Restriction Digest The restriction digest contained 0.3 µL each of EcoRI and EcoRV restriction endonucleases, 1 µL of buffer, and variable amounts of DNA plasmid and nuclease free water for a reaction volume of 10 µL with 1 µg DNA. Reaction was carried out at 37 degrees for 1 hr. 2 µL DNA loading dye was then added into each of the restriction digest tubes. Each sample was run on a 1% agarose gel (containing 5 µL of ethidium bromide) at 120 V until the dye front showed sufficient migration. The gel was then imaged using the BIORAD ChemiDoc MP imaging system. 2.7 Embryo Collections Flies were placed in egg laying chamber on apple juice agar plates with dried yeast solution. The egg laying chamber was at 25ºC, with the exception of APC knockdown which was kept at 22ºC. The plates were changed no longer than 48 hours apart. For embryo collection, the plates were swapped with a fresh new pre-lay plate. Following 1 hour with the pre-lay plate, the pre-lay was removed and a new plate was added for a 2-hour time interval. Following the 2-hour interval, embryos were collected immediately for 0–2-hour samples, or the plate was placed at 25ºC for an additional 3 hours for 3–5hour samples. Embryos were then rinsed with bleach and poured into an embryo basket, rinsed with water, then rinsed with embryo wash, rinsed again with water, and then placed in embryo wash. Embryos were transferred to Eppendorf tube and embryo wash was removed by pipette. The samples were either flash frozen with liquid nitrogen and stored at -80ºC or fixed in methanol or formaldehyde (described below). 2.8 RNA Extraction RNA extraction and purification was carried out using the Qiagen RNeasy Mini Kit. Although, there was large amount of variability, roughly 10-20 µL of embryos was used for each RNA extraction. 19 RNA concentration was measured using the Thermo Scientific NanoDrop OneC Microvolume UV-Vis Spectrophotometer. The results of each extraction used for cDNA synthesis is listed below: Sample RNA concentration (ng/µL) 260/280 yw 0-2 26.5 2.07 yw 3-5 63.9 2.16 Fzy 0-2 (1st replicate) 147.2 2.17 Fzy 3-5 (1st replicate) 76.5 2.36 Fzy 0-2 (2nd replicate) 69.5 2.13 Fzy 3-5 (2nd replicate) 21.3 1.94 2.9 cDNA Synthesis The Thermo Scientific RevertAid First Strand cDNA Synthesis kit was used to make cDNA from purified RNA. Each cDNA reaction was adjusted that each reaction would have between 0.20 and 0.25 µg of RNA. The reaction took place at 42 ºC for 1 hour, followed by termination at 70 ºC for 5 minutes. The cDNA was then stored at -80 ºC. 2.10 qRT-PCR Two master mixes were made with SYBR Green reagent, one for control gene (rpa1) and one for target gene (nanos). Each master mix consisted of 80 µL of SYBR Green, 6.4 µL of 5 uM forward and reverse primers for rpa1 or nanos. cDNA of each sample was diluted to 1/5 in nuclease-free water. The reactions were carried out in 384 well plate so that each reaction received 5.6 µL of SYBR + primer master mix, and 4.6 µL of diluted cDNA of each sample, or 4 µL of nuclease-free water as a negative control. Each reaction was performed in triplicate. The reaction was performed in the Thermo Fischer Viia 7 Real-Time PCR System. 2.11 Methanol Fixation of Embryos 600 µL of heptane was added to Eppendorf tube containing the embryos, followed immediately by 600 µL of methanol and then shook vigorously by hand for ~10-15 seconds. Heptane and most 20 methanol were removed. Another 600 µL of methanol was added, mixed, removed, then repeated one more time. The embryos were stored at -20ºC in ~600 µL of methanol. 2.12 Immunostaining of Methanol Fixed Embryos Following methanol fixation, embryos were gradually rehydrated and then blocked in a 1% BSA solution in PBST for ~1 hour at room temperature. The embryos were then incubated overnight at 4 ºC followed by 1 hour at room temperature in PBST containing Thermo Fisher Scientific Invitrogen alpha Tubulin Monoclonal Antibody (YL1/2, Lot: XB3238702A) at 1/1000, RNase A (1/250) and 1% BSA. After incubation, embryos were rinsed and washed in fresh 1X PBST. The CyTM3 AffiniPureTM Goat Anti-Rat IgG (H+L) secondary antibody (Jackson ImmunoResearch Laboratories Inc.) was then added (1/1000) with OliGreen (1/500) in 1% BSA and PBST. Rinsing and washing in PBST followed overnight at incubation 4 ºC. The embryos were then incubated with methanol for 5 minutes, then isopropanol for 10 minutes. Isopropanol was removed and 60 µL of tetrahydronaphthalene was added to embryos that were subsequently mounted on glass slides and sealed with nail polish. The slides were stored at 4 ºC until imaging. 2.13 Formaldehyde Fixation of Embryos A fixative solution was prepared using 600 µL heptane, 540 µL PBST, 60 µL of 37% formaldehyde, and 1 µL EGTA (ethylene glycol-bis-N,N,N’,N’-tetraactetic acid). This solution was mixed and added to embryos where it was fixed on a nutator for 10 minutes, then removed. Another 600 µL of heptane was added, followed immediately by 600 µL of methanol and then shook vigorously by hand for ~60 seconds. Heptane and most methanol were removed, along with embryos at the interface. Another 600 µL of methanol was added, mixed, removed, then repeated one more time. The embryos were stored at -20ºC in ~600 µL of methanol. 21 2.14 Immunostaining of Formaldehyde Fixed Embryos Immunostaining was done analogously to staining of methanol embryos described in Section 2.12, with the exception of using Anti-histone, H1+core proteins mouse monoclonal, clone F152.C25.WJJ (Millipore Sigma, Lot: 2465294) primary antibody at 1/1000 in place of Oligreen and RNAse A. 22 Chapter 3 Results 3.1 Transcript Destabilization in Fzy Knockdown Destabilization of maternally deposited mRNA transcripts is a process of activation that occurs during the 3–5-hour time interval following egg laying. One of the well-known mRNA transcripts that undergo this destabilization is nanos, which is used in these experiments as the experimental transcript (Tadros et al., 2003). rpa1 is a mRNA transcript which remains at relatively constant levels in embryos, thus is used as the control transcript (Tadros et al., 2003). To test our proposed model and replicate previous findings, nanos mRNA levels in 0-2 hour and 3-5 hour time interval were analysed relative to rpa1 mRNA using qRT-PCR. yw is a wildtype control. Our model suggests that Fzy is required for transcript destabilization. Fzy is an activator of the APC/C required to lower Cdk1 activity hypothesized to be necessary for transcript destabilization, such that PNG kinase becomes active. Results of the qRT-PCR of the fzy knockdown are seen below in Figure 2. Each qRT-PCR reaction was preformed in triplicate. The data suggests that transcript destabilization is Fzy dependant, since nanos transcripts are stable in the 3-5 hour time interval. This result nicely agrees with preliminary data by Boruouh, 2018, and our proposed model such that Fzy is necessary for transcript destabilization. Interestingly, nanos transcript levels are noticeably increased in the 3-5 hour time interval; the significance of this is discussed in detail in the discussion section. The large standard deviation in the three triplicates for the wildtype control 0-2 hour embryos is noted, and is something that needs to be redone in the future. The data analysis from the qRT-PCR results was preformed by Rajni. 23 Figure 2. mRNA levels in fzy knockdown examined using qRT-PCR. Relative nanos mRNA levels relative to wildtype control (yw). The fzy knockdown sample shows stable transcript levels in the 3-5 hour time interval compared to yw embryos aged 3-5 hours, which supports that Fzy is required for transcript destabilization. Data is from a single experiment, each qRT-PCR reaction was formed in triplicate. In addition to transcript destabilization, fzy embryos have been fixed in methanol and immunostained to look at cortical microtubule reorganization. We are waiting for access to the confocal microscope to view these embryos. Wildtype control staining is also something that needs to be done in order to make a comparison. 3.2 APC/C Knockdown Preliminary data from Boruouh, 2018 found that when a subunit of the APC/C is knocked down using RNA interference, transcript destabilization and cortical microtubule reorganization do not occur, -6.000 -4.000 -2.000 0.000 2.000 4.000 6.000 8.000 yw 0-2 yw 3-5 fzy0-2 fzy3-5 nanos mRNA RQ Levels Sample Relative mRNA Transcript Levels in Fzy Knockdown 24 suggesting that both processes are APC/C dependant. Regarding our model for transcript destabilization, we propose that APC/C mediated ubiquitination of Cyclin B3 is necessary for inactivation of Cdk1. This drop in Cdk1 activity would allow for PNG kinase to transition to an active state. When APC/C is knocked down, ubiquitination of Cyclin B3 would not occur, therefore Cdk1 would remain active. Cdk1 activity will cause PNG kinase to remain phosphorylated and inactive, therefore unable to phosphorylate the downstream target required for transcript destabilization. Unfortunately, repeated fertility was observed in the flies that should have had a subunit of the APC/C knocked down via UASAPC3RNAi germline expression. The observed fertility can be interpreted as that there is no RNA interference occurring. The cross is currently being repeated one final time on a small scale to test if the stock is useful. Testing another stock (𝑈𝐴𝑆 𝐺𝐹𝑃 𝐶𝑦𝑂 ;𝑈𝐴𝑆 𝐴𝑃𝐶3𝑅𝑁𝐴𝑖 𝑇𝑚6) to see if sterility is observed is also something being done currently. 3.3 Non-Degradable Cyclin B3 Expressing a non-degradable Cyclin B3 in the female germline was initially a goal of this research. This non degradable Cyclin B3, Cyclin B3-d, was previously designed in our lab using site directed mutagenesis. We predicted that since Cyclin B3 levels will stay elevated, Cdk1-Cyclin B3 activity will remain high, thus PNG kinase will remain inhibited, such that transcript destabilization will not occur. Germline expression of Cyclin B3-d should result in sterility; their eggs should not hatch into larvae. However, repeated trials of two different stocks continuously yielded fertile females. Both a western blot and genomic prep analysis was done using the 10.20 (𝑈𝐴𝑆 𝑣𝑒𝑛𝑢𝑠𝑐𝑦𝑐𝐴 𝑐𝑦𝑜 ;𝑈𝐴𝑆 𝐺𝐹𝑃𝑐𝑦𝑐𝐵3𝑑 𝑇𝑚6) stock to test for the presence of the transgene. Both showed no evidence of CyclinB3-d, however lacked the necessary positive controls to make a definitive conclusion. Despite a definitive result, the 10.17 25 (𝐶𝑦𝑂 𝑠;𝑝𝑖𝑚𝑑𝑘1,𝑈𝐴𝑆 𝐺𝐹𝑃𝑐𝑦𝑐𝐵3𝑑 𝑇𝑚6) stock was tested instead. Figure 3 shows the western blot performed on dissected ovarian tissue of flies that were supposed to be expressing the transgene from the 10.17 stock. The results showed no reason to believe the transgene was being expressed. Figure 3. Western blot to confirm absence of GFP-Cyclin B3-d. Ponceau red stain of nitrocellulose membrane (left), chemiluminescence of western blot (middle), and ladder used for western blot (right). The expected band size of GFP-Cyclin B3-d from the 10.17 stock is ~95 kDa, which was concluded to be absent. Given that both 10.20 and 10.17 were presumed to be not useful, the next step was to transform competent E. coli cells with the pUAS-GFPcycB3dSDM plasmid such that the plasmid could be purified and sent away for microinjection. Following transformation and purification of the plasmid, a restriction digest using EcoRI and EcoRV restriction endonucleases to confirm the identity of the isolated genetic material. The plasmid should yield DNA fragments of 7008 bp, 2888 bp, and 2450 bp. The results of the restriction digest are seen in Figure 4. Colonies 1,2, and 4 all showed anticipated band size. Colony 2 was chosen to be sent away for microinjection. 32 destabilization and cortical microtubule reorganization in embryos expressing a non-degradable form of Cyclin B3 can be experimentally determined in future investigations. 4.4 cort;cycB3 Double Mutant The cort;cycB3 double mutant is something that is crucial to test our proposed model of transcript destabilization. We propose that APC/C activation is necessary to degrade Cyclin B3. This leads to a drop in Cdk1 activity so that the PNG kinase can transition to an active state. Cort is a germline specific activator of the APC/C in female meiosis. cort mutants fail to undergo transcript destabilization (Page & T.L. Orr-Weaver, 1996; Lieberfarb et al., 1996; Boruouh, 2018). Transcript destabilization does occur in cycB3 mutants which can be explained by low Cdk1 activity allowing PNG kinase to remain active, although this is still considered preliminary (Boruouh, 2018). Therefore, we predict that the low Cdk1 activity in the double mutant will allow PNG kinase to remain unphosphorylated, despite the APC/C being inactive due to the cort mutant. If transcript destabilization still occurs in the cort;cycB3 double mutant, it can be considered strong evidence that our proposed model is correct. Generating necessary balanced stocks to ultimately achieve the double mutant is a process that extended the duration of nearly this entire project, but was achieved successfully. The offspring of the final cross are currently being screened for the correct genotype, and cort;cycB3 double mutant females are being collected. Embryo collection followed by either microtubule staining or RNA extraction, cDNA synthesis, and qRT-PCR to examine transcript destabilization is something that will be done in the near future. 33 4.5 Determination of Best Technique for Microtubule Staining Investigating the success of formaldehyde fixing compared to methanol fixation for looking at cortical microtubule reorganization is something that was supposed to be done. Ideally, the two techniques would have been used to stain yw and cort mutant embryos to compare which technique produced the best results. cort mutants fail to undergo cortical microtubule reorganization, hence would have served as a positive control (Page & T.L. Orr-Weaver, 1996). Unfortunately, due to loss of samples when preparing slides and using contaminated PBST solution, we were unable to look at the success of cortical microtubule staining following formaldehyde fixation. cort mutant embryos fixed in methanol are shown in Figure 5 but are essentially meaningless with such a small sample number and nothing to compare them to. Therefore, determination of the best technique is something that still needs to be done. 4.6 Conclusions and Additional Future Steps The result of transcript levels to investigate transcript destabilization in the fzy knockdown embryos is very promising and validates preliminary data (Boruouh, 2018). Further repeats of this experiment are needed to make a definitive conclusion that Fzy is required for transcript destabilization following egg activation. Additionally, investigating why nanos mRNA is increased in 3-5-hour fzy knockdown embryos compared to 0-2 hour embryos is something that should be explored in the future. The APC/C knockdown is still a work in progress, but hopefully we can ultimately validate preliminary findings of transcript destabilization and cortical microtubule reorganization soon. The non-degradable Cyclin B3 and cort;cycB3 double mutant experiments has yet to be preformed, yet promising steps have been made through the duration of this project. Investigating the role of other mitotic/meiotic cyclins, namely Cyclin A and Cyclin B, in transcript destabilization and cortical microtubule reorganization is also something that should be done later on. 34 Observing the phosphorylation states of PNG kinase in the various mutants and knockdowns is also something that should be done in future experiments to further validate our model. Active PNG kinase is ultimately required for transcript destabilization (Hara et al., 2017). The phosphorylation state of GNU, one of the subunits of the PNG kinase complex, determines whether the enzyme is active or inactive. Phosphorylated and unphosphorylated GNU migrates differently in a western blot, therefore we can use antibodies against GNU to observe phosphorylation states for all previously discussed mutants and RNAi knockdowns. For example, in the fzy knockdown we predict that since the APC/C will not be activated by Fzy, Cyclin B3 will not be ubiquitinated hence Cdk1 activity will remain high. High Cdk1 activity will lead to GNU being phosphorylated and thus inhibited. The phosphorylation state of GNU would be determined using western blotting. Ultimately, determining the phosphorylation states of PNG kinase in each discussed genotype would provide further support to our proposed model. Overall, this project made good progress in better understanding two key events of activation: transcript destabilization and cortical microtubule reorganization. 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