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A genetic approach to identify Hox regulatory microRNAs during Drosophila development

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posted on 2023-06-09, 01:12 authored by Wan Liu
The Hox genes encode a family of transcriptional regulators that activate distinct developmental programs along the anterior-posterior (AP) axis of animals. Recent observations in Drosophila demonstrate that at least two miRNAs can repress Hox gene expression during development suggesting that miRNA-based regulation might be a general mechanism of Hox gene regulation. Here explore this possibility by applying a comprehensive genetic approach to identify miRNAs able to repress Hox gene expression during development. Given that the reduction of Drosophila Hox gene Ultrabithorax (Ubx) expression leads to easily tractable homeotic transformations in haltere, I use Ubx to test the repressive effects of dozens of miRNAs in an overexpression screen. Scoring over 10,000 halteres showed that out of 106 miRNAs tested, ~28% produced Ubx mutant phenotypes suggesting that miRNA-dependent Hox regulation might be a pervasive mechanism controlling Hox gene function during development. I classify phenotypes into four major categories: Ubx mutant effects (Class I and II) and others (Class III and IV). Through the combination of RNA-Seq data and TaqMan RT-PCR approaches, I confirm that there is no correlation between the phenotypic strength and miRNA expression level indicating that haltere phenotypes emerge from miRNA qualitative roles. Furthermore, using protein expression analysis and Ubx 3’ UTR fluorescent reporters, I confirmed that at least nine miRNAs affect Ubx protein expression and that six of these directly target Ubx 3’ UTR in vivo. Lastly, I explore the nature and effects of miRNA regulation of Ubx at the cellular level in the Drosophila embryonic CNS and find that miR-252 is sufficient and necessary to repress Ubx expression in specific neural lineages. Our work thus contributes to the understanding of miRNA-mediated Hox gene regulation and, more generally, to the study of miRNA-target interactions within the physiological context of metazoan development.

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File Version

  • Published version

Pages

201.0

Department affiliated with

  • Biology and Environmental Science Theses

Qualification level

  • doctoral

Qualification name

  • phd

Language

  • eng

Institution

University of Sussex

Full text available

  • Yes

Legacy Posted Date

2016-05-25

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