Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Neural Engineering

Neural Engineering - Poster Session E

Poster Z15 - RNAi Analysis of Patterning Factors in the Drosophila Thoracic Peripheral Nervous System

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Understanding the mechanisms behind biological patterning is crucial for understanding how organisms' biological structures develop. This study investigates the cell-cell signaling pathways responsible for patterning the peripheral nervous system (PNS) in the dorsal thorax of the fruit fly, Drosophila melanogaster. Neural precursor cells in the PNS utilize cytonemes, long and thin signaling filopodia, to interact and influence cell fate decisions, apoptosis, and cell division timing in neighboring cells. We implement an RNAi screen based on previously published RNAseq data to analyze and identify new genes contributing to cytoneme-mediated interactions during PNS patterning. The first stage of our screening targeted approximately 120 genes, using quantitative live microscopy to detect defects in bristle patterning. We quantify changes in the arrangement and density of bristle precursors. Through this screening, we identified 21 genes whose knockdown significantly altered bristle precursor density or cell fate switching rates. To further understand the implications of these genetic variations, we examined their effects on bristle precursor cell morphology and adult fly behavior in the second stage of our screen.

Materials and

Methods:  Screen stage one: To identify genes essential for the organization of the PNS we analyzed pupae and focused specifically on the dorsal thoracic epithelium. This developmental stage allowed us to examine how gene knockdowns affect final bristle density. For live imaging, non-Tubby pupae expressing UAS-RNAi under the neuralized-GAL4 driver (expressed in bristle precursors only), aged 24 hours after pupariation, were secured on double-sided tape. The pupal casing was carefully removed from the thorax and a coverslip was placed over the exposed pupae to ensure clear imaging of the array of bristles formed. As shown in Figure 1, bristle cell density was assessed by counting the number of bristles within a region of interest with a minimum of three pupae per RNAi knockdown condition.
Screen stage two: To analyze cytoneme morphology, we observed shape changes in bristle precursor cells resulting from gene knockdown using time-lapse imaging of cells co-expressing RNAi and a GFP actin reporter, quantifying their morphology and behavior. At least two pupae per genotype were imaged. In Figure 2, bristle precursor cells expressing a GFP reporter were imaged to identify cytonemes. The number of cytonemes produced in a 15-minute window was quantified.
Additionally, a cleaning behavior assay was conducted by dusting 48-hour-old adult RNAi-expressing fruit flies with 100 mg of talc powder. After excess talc powder was removed, the flies were allotted one hour of recovery time before performing the assay. A minimum of 10 2-day-old adults per assay, with at least two biological replicates per genotype, are assessed.

Results, Conclusions, and Discussions: Thus far, we have identified 21 genes whose knockdown led to significant alterations in bristle patterning. These alterations included changes in overall bristle pattern compared to controls. The significant changes in the bristle pattern suggest the 21 genes have essential roles in the organization of the PNS. Phenotype classifications indicated genes with fewer or more error pairs compared to control, showing decreased or increased bristle cell density. With the characterization of each gene, we aim to uncover their specific roles in PNS development.
The grooming assay showed that some flies with patterning defects displayed impaired cleaning behavior. Figure 4 shows defective flies with an inability to clean properly highlighting the critical role of the organization of the PNS and proper bristle function. Wild-type bristle function is essential for stimulating cleaning behavior in response to dirt, dust, and other debris.
The sensory organ precursor (SOP) and epithelial cells (EC) ratio compares mRNA expression. The SOP/EC ratio is derived from data from Buffin and Gho (PLoS One 2010). By analyzing this ratio, there are transcriptional differences that drive sensory organ development. Each gene is associated with molecular functions and biological processes. Highly expressed biological processes include development, transport/localization, and an unknown factor. Similarly, molecular functions included transporters and another unknown factor. The presence of these unknown factors suggests there may be uncharacterized genes involved in sensory organ development.
Our genetic screen has identified new genes essential for bristle precursor cell communication. Functions like forming bristle patterns, sensory function, cytonemes, cell death, and switching rely on effective cell communication. When knocking down these genes an organism's ability to develop and maintain proper sensory function leads to defects. The discovery of these 21 genes, which had not previously been linked to bristle pattern formation, provides new opportunities for research into the underlying effects of cell communication. Our objective is to determine how each of these genes contributes to the communication between brittle precursor cells during tissue patterning.

Acknowledgements (Optional):  1. Cohen et al 2010 Dev Cell. 2. Koto et al 2011 Curr Biol. 3. Lacoste et al 2022 eLife. 4. Buffin & Gho 2010 PLoS One. 5. Gramates et al 2022 Genetics
This work is supported by NIH R03NS130395 to GH. CN is supported by R25EB033080.
Thank you to former lab members: Jacob Inyang, Caitlin Minardo, Mia Greco, Rylee Marin, Delaney Fatcheric, and Sara Custer.