The properties of ovaries of Drosophila melanogaster has turned it into a resource for studying effects of genetic dysregulation upon both germline and somatic cells and tissues (Kirilly & Xie, 2007). Within egg chambers of developmental stages between 7 and 10, one cytoophidium is normally seen in follicle cells, whereas a nurse cell holds multiple micro-cytoophidia in addition to a macro-cytoophidium. The latter usually spans the diameter of the nurse cell nucleus. Nonetheless, the consistency in size and length presented by cytoophidia in ovaries allowed inferences to be made of perturbations to this ‘normal’, in the case where they are seen under systemic overexpression of a particular miRNA. In order to efficiently utilize these tissues for screening, emergent F1 flies were transferred into a fresh vial smeared with wet yeast to encourage oogenesis. After two days, ovaries were quickly dissected out of flies of selective phenotype and processed for wither immunohistochemistry or RNA extraction purposes.
Immunoflurosecence and viewing
Antibody-based histochemistry against CTPsyn ensued. Adjustments to the protocol was only made in terms of the types of primary and secondary antibodies applied to a tube of ovaries for different permutations of staining. These changes will be denoted clearly on each confocal imagery presented within this thesis. Along with anti-CTPsyn, antibodies were employed for their efficacy in highlighting important structural components of the egg chambers, and thus enhancing visualization. Hu-li-tai-shao or HTS is a spectrin-binding protein; in egg chambers, spectrin is disseminated around the outer cytoplasmic boundaries of individual follicle cells, and anti-HTS is thus used to distinguish these cells from one another. Rhodamine Phalloidin conjugates with high-affinity to the ubiquitously expressed structural protein F-actin. For drosophilid ovaries, this property of the counterstain explicitly highlights the cell-to-cell barriers between neighbouring nurse cells.Phenotyping and counting
Once cytoophidia-altering candidates were identified, the subsequent phenotyping phase in the context of this screening entailed these following characteristics: (a) number of ovary pairs displaying altered cytoophidia for the purpose of calculating penetrance, (b) comparison of length and thickness of cytoophidia in ovaries of control females to those of candidate-miRNA overexpressing females, (c) increment or decrement in micro-cytoophidia numbers within follicle and nurse cells, and (d) changes in shape and/or size of individual follicle cells surrounding egg chambers. All quantifications were subjected to simple statistical analyses through Student T-test. Significance was set for a p-value of
Preservation and extraction of RNA from ovarian cells
Ovaries from a minimum of fifteen females were collected for a single RNA extract. All individuals were aged on wet yeast for two days prior to dissection in filter-sterilized PBST. A time limit was set for each dissection round; no tissues were left in PBST for more than ten minutes before being processed. In this case, 100µl of TRIZol (Ambion, Invitrogen, Life Technologies) was added once most PBST was removed from the tube. Ovaries were re-suspended well in the reagent followed by flash freezing at -80°C. Tissues were left in TRIZol for no longer than three days before RNA extraction. RNA was kept in -20°C and subjected to minimal freezing and thawing cycles. Quantification and integrity assessment of extracts in 1% gel was typically followed immediately by conversion of RNA into cDNA. Once diluted by a factor of 5, cDNA was either utilized straightaway in qPCR, or stored in -20°C until required.
Establishing a reference phenotype
Previous studies have either utilized y w or wild-typed flies as controls in gauging the ‘normal’ lengths, shapes and sizes of cytoophidia within D. melanogaster ovaries (Aughey, Grice, & Liu, 2016; Aughey et al., 2014; Strochlic et al., 2014). Initially, we had followed suit, and Oregon-R, a common wild-type strain, was meticulously characterized to be referred to as our standards for the duration of this project.
However, the expression of GAL4 under the influence of certain drivers are known to produce unexpected phenotypes (Hara et al., 2017; Kramer & Staveley, 2003). Upon further scrutiny, we did in fact observe differential micro and macro-cytoophidia formation patterns in driver-GAL4 lines. These phenotypes were retained in F1 populations even after they were mated to Oregon-R, a cross which should not have borne any phenotypical consequences. As ectopic expression of GAL4 alone appeared consequential in determining cytoophidia traits, we had to therewith readjust the reference or control phenotypes used as a standard of measurement for future crosses. These induced characteristics of individual GAL4-drivers would be applied to recalibrate outcomes in miRNA-overexpressing lines.