Title

Novel Newt Proteins Regulate Evolutionarily Conserved Wingless Signaling Pathway to Rescue Drosophila Eye Mutants

Date of Award

1-1-2019

Degree Name

Ph.D. in Biology

Department

Department of Biology

Advisor/Chair

Advisor: Amit Singh

Abstract

Pathways involved during regeneration/restoration of missing body parts are conserved throughout animal kingdom, but only few animals like newts have a remarkable ability to repeatedly regenerate/restore missing organs/tissues throughout its lifespan. It remains to be determined if such an exceptional ability of the newt is attributed to strategic regulation of evolutionarily conserved pathways by genes that newt may have evolved. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, our group identified a novel family of 5 protein members expressed in adult tissues during regeneration in Notophthalmus viridescens (Red-spotted newt). The presence of a putative signal peptide suggests that all these proteins are secretory in nature. Here we employed iterative threading assembly refinement (I-TASSER) server to generate a three-dimensional structures of these novel newt proteins and predicted their function. The data suggest that these proteins could act as ion transporters, and be involved in redox reactions. To investigate downstream events (signaling pathways) that can be modulated by newt genes, and due to absence of transgenic approaches in N. Viridescens, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster in which these genes were misexpressed using GAL4/UAS binary system. Expression of more than 2700 transcripts were compared between these 5 newly identified newt genes. Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. Wingless (Wg) / Wnt was one of the important evolutionarily conserved pathways that was reported to be differentially regulated. To validate the RNA Seq. data, expression of six highly regulated genes (Pka-C1, hsp70Bb, PGRP-SB2, CG12224, Syp, Unc-115b ) were verified using real time Quantitative Polymerase Chain Reaction (RT-qPCR). Wg signaling pathway is one of the evolutionarily conserved pathway that has been reported to modulate biological processes like regeneration, growth, development etc. in newts as well as in Drosophila. In order to visualize the phenotype transformative effect of regulating Wg signaling pathway we misexpressed newt genes in Lobe2 (L2)-mutant background (L2/+; ey>C4). L2, a dominant-negative allele of Lobe (L), causes upregulation in Wg resulting in loss of ventral eye in the L2/+ background in 100% of Drosophila (flies). We determined that misexpression of these 5 unique genes from newt (Notophthalmus viridescens) in Drosophila downregulated Wg in L2-mutant background, resulting in partial rescue of missing photoreceptor cells in the ventral compartment of the developing Drosophila eye. Perturbing positive and negative regulators of Wg signaling pathway confirmed that newt genes regulate Wg signaling pathway to transform L2-mutant phenotype. We further tracked that growing photoreceptor cells showed cell Non-Autonomous trend in growth but did not breach the eye field i.e. we did not notice any change in eye fate. To receive a whole picture of the mechanism behind the rescue of the mutant phenotype, we calculated mitotic index, and did cell death assays. Comparatively, we found that misexpressing newt genes under L2-mutant background showed robust increase in cell proliferation, and significant downregulation in cell death at the ventral margins. Our current results demonstrate the possibility of using unique genes from newt to correct pattern defect mutations in the developing eye by promoting growth, and survival of missing photoreceptor cells in the Drosophila eye field. In the future such gene therapeutic technique of using unique genes from animals with high regeneration potential (e.g. newts) (as gene or protein/drug) can have strong implications in the field of regenerative medicine.

Keywords

Biology, Bioinformatics, Biomedical Engineering, Biophysics, Cellular Biology, Developmental Biology, Regeneration, Rescue, Development, Evolutionary, Wingless, Eye, Mutant, Lobe2, RNA sequencing, Ab initio, Gene therapy

Rights Statement

Copyright 2019, author

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