Understanding Collective Cell Movement by Employing the Model of Border Cell Migration During Drosophila oogenesis

Sharma, Aditi (2019) Understanding Collective Cell Movement by Employing the Model of Border Cell Migration During Drosophila oogenesis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

The development of an individual includes several processes such as cell division, proliferation and migration, which gives rise to an adult structure. Of all these essential phenomena, cell migration is one of the important processes, aberration of which may lead to several diseased conditions including orofacial clefting and tumor cell metastasis. Cell migration could be either single or collective. A large amount of literature addresses the mechanism through which single cell migrates, in several developmental contexts. However, a clear understanding of how cells employ collective mode of motility still remains elusive. Quite a few genetic models of group cell movement have been described by a number of authors, of which, border cell migration, during Drosophila oogenesis has emerged as an excellent, genetically tractable system to study such kind of movement. Therefore, I have employed this model to decipher the role of Insulin signaling and Mucin91C, one of the fly mucins, during collective cell movement. The first part of the thesis addresses how canonical Insulin signaling (InS) modulates border cell migration. This has been demonstrated by the knockdown and knockout studies of the Insulin-like Receptor, InR, that hampered migration and resulted in incomplete migration of border cell cohorts. Subsequently, suppression of migration defects observed on knockdown of InR by over-expressing the downstream effectors of Insulin cascade including Phosphoinositide-3-kinase (PI3K) and Protein kinase B (Akt) suggests that InS functions canonically to modulate cell migration. This is also substantiated by the Chico (Insulin Receptor Substrate), loss of function studies that exhibited strong migration defects. Further, the removal of a copy of Forkhead box Class O protein (FoxO), which antagonises InS, also suppressed the migration defects caused by depletion of InR. The second part of the thesis gives an important molecular insight as to how Insulin signaling regulates the movement of border cells. Besides defective migration, the time-lapse studies of InR depleted clusters suggest that inefficient detachment of the border cell cluster from the anterior follicular epithelium, could be one of the factors that would impede border cell movement. Given the well documented role of Partitioning defective, PAR-1 gene, in the regulation of detachment of border cell cluster and the fact, that InR mimicked the PAR-1 phenotype in terms of detachment and protrusion dynamics, I found that InR genetically interacts with PAR-1. This is supported when I observed proportionate increase in the number of un-detached clusters on simultaneous depletion of InR and PAR-1 function compared to individual controls. Moreover, the levels of PAR-1 also seemed to be affected in clusters depleted of InR. PAR-1 function is known to stabilize Myosin filaments through the inhibition of Myosin binding subunit, Mbs. Mbs activity leads to the inactivation of Spaghetti squash, Sqh, which is the regulatory subunit of Non-muscle Myosin-II. As Mysoin-II function is required to facilitate border cell detachment, supplementation of a copy of phospho-mimetic form of Sqh rescued the migration defects imparted by InR depletion. Correspondingly, the migration defects were also suppressed on depletion of Mbs function too. Taken together, InR has been shown to function canonically through PAR-1 to modulate Sqh activity, which is critical to aid in border cell detachment and forward movement. The third part of the thesis delineates the role of Muc91C in the regulation of border cell migration. This is supported by the observation of defective migration and altered nurse cell morphology, in Muc91C knock-down border cell clusters. The defect was suppressed by over-expressing Muc91C alone in Muc91 depleted clusters. The clusters depleted of Muc91C function exhibited altered protrusion and F-actin dynamics. The actin dynamics in border cells is mainly influenced by the guidance cue, PDGF and VEGF-related factor, PVF, secreted from the oocyte. Consistent with my expectation I observed a genetic interaction between PDGF and VEGF-related receptor, PVR and Muc91C. Moreover, PVR is known to function through Pak3 to regulate F-actin in migrating border cells and interestingly, an elevation in the migration defects was observed when Muc91C function was depleted in Pak3 heterozygous background. Correspondingly, Muc91C was also found to genetically interact with Rhea, a fly homolog of Talin, that exacerbated the migration defects. Altogether, the results in this section suggests that Muc91C functions along with guidance cues to modulate actin dynamics in the migrating border cluster. Overall, my thesis outlines how Insulin and Mucin91c regulate border cell movement. Given that Insulin and Mucins have implications in disease biology, findings from this study will have long term therapeutic implications.

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