Forward and Reverse genetics approach to understanding Border Cell Migration & Permeabilisation of Drosophila melanogaster embryos

Sarkar, Arunabha (2012) Forward and Reverse genetics approach to understanding Border Cell Migration & Permeabilisation of Drosophila melanogaster embryos. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (07MS28 BS-MS Dissertation) Arunabha_Sarkar_-_Dissertation_Thesis_-_IISER_Kolkata_-_Forward_and_Reverse_genetics_approach_to_understanding_Border_Cel~1.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Border Cell Migration Directed cell migration is characteristic of various biological processes like embryonic development, immune response, wound healing and cancer metastasis. Single cell migration has been mostly studied in fibroblasts, keratinocytes and Dictyostelium model system but the mechanism of coordinated and directed cell migration as in cancer metastasis, etc is far from conspicuous. Border Cell migration in Drosophila ova has emerged over the past two decades as a prime model system to study coordinated group cell migration. At stage 9 of ova development (out of a total of 14 stages), a clique of 6-8 cells from the follicle delaminate and undergo directed invasive migration through the nurse cells to the anterior oocyte boundary before dorsal migration. As the X-chromosome has not been screened for loci involved in border cell migration, we employed forward genetics to generated X-chromosome EMS mutants. These were screened for potential border cell migration regulatory factors during Drosophila oogenesis. One of the screened lines named ‘B 2-11’ exhibited a partial migration defect phenotype. Border cells with the migration cues (from oocyte) undergo dynamic changes in their cytoskeleton network and migrate invasively through the 15 compact & interconnected nurse cells to the posterior end to the oocyte of the egg chamber. So far it is only the growth factors from the oocyte and cell adhesion intermediates between nurse & border cells that are known to have a paracrine affect on migrating border cells. Since the timely migration of these cells is critical for fertilisation, we hypothesised that depletion of any nurse cell specific factor critical for border cell migration will also result in sterile eggs. So, RNAi silencing constructs of a subset of such genes were ordered and screened for such migration delay/defect. We found three genes α-catenin, lin19 and MED11 exhibiting border cell migration delay/defect as well as novel egg chamber phenotypes. Embryo Permeabilisation Drosophila melanogaster has emerged as one of the most versatile model systems in biological studies utilised in studies of development, population, genetics, cellular locomotion, reproduction, etc. Yet even after a century since its first usage for genetics, its innumerable genetic strains and variants are still maintained by intensive and expensive culturing and sub-culturing method. With increasing popularity of this model system in diverse research fields, there is an ever increasing demand for an efficient and effective protocol for cold storage of the flies for extended period of time. Attempts at cryo-preservation of Drosophila melanogaster have achieved very limited success over trails since the past two decades. The major challenge lies in the embryo permeabilisation step whilst maintaining viability for absorption of cryopreservants like glycerol, urea, saccharides, etc. Moreover, the few successful protocols are too cumbersome for everyday use by a typical Drosophila melanogaster model system based labs. I have explored for possibilities of a simpler yet efficient protocol for permeabilisation of Drosophila melanogaster whilst maintaining viability designed for easy use and management of various genotypes for use extensively by any such labs for the purpose of intended future Cryopreservation.

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