Many transcription factors contribute to C. elegans growth and fat storage

Abstract

Reverse genetic screens by RNA interference (RNAi) in model organisms such as the nematode Caenorhabditis elegans have provided numerous insights into gene function, thereby connecting genotype to phenotype. However, genes that contribute only subtly are often missed because relatively large numbers of measurements and reliable quantification are required to overcome experimental and biological noise that may mask subtle phenotypic effects. Here, we address this challenge by focusing on two phenotypes in C. elegans: growth and fat storage. We carried out comprehensive RNAi knockdown of transcription factors (TFs), as these are known important regulators of biological processes during development and the maintenance of homeostasis. Microscopy images of TF knockdown animals stained with Oil Red O (ORO) were captured, and body size (proxy for growth) and ORO staining intensity (proxy for fat storage) were precisely quantified using a newly developed imaging tool we named IPPOME (Image Processing for Precise and Objective MEasurement). We found that a surprisingly large proportion of TFs contribute to growth and fat storage, but that most TFs have only subtle, yet significant effects. This study provides a blueprint for studies of other genes and phenotypes in C. elegans.

Nuclear receptor NHR-49 promotes peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency inC. elegans

“…We then queried footprintDB for transcription factors that were predicted to bind these motifs (Sebastian and Contreras-Moreira, 2014), which yielded 42 transcription factors. An additional 10 transcription factors that were reported to regulate fat storage were considered (Arda et al, 2010;Mori et al, 2017;Pathare et al, 2012). Next, we curated a cherry-picked RNAi library of these 52 transcription factors in order to test if their depletion would modulate alh-4(-) phenotypes.…”

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans

“…We then queried footprintDB for transcription factors that were predicted to bind these motifs [51], which yielded 42 transcription factors. An additional 10 transcription factors that were reported to regulate fat storage were considered [32,52,53]. Next, we curated a cherry-picked RNAi library of these 52 transcription factors in order to test if their depletion would modulate alh-4(-) phenotypes.…”

Workflow for Segmentation of Caenorhabditis elegans from Fluorescence Images for the Quantitation of Lipids

“…Label-free imaging techniques such as spectroscopic coherent Raman [12] and coherent anti-Stokes Raman scattering imaging [13,14] are important tools for C. elegans lipid-storage studies, but require expensive equipment. The most commonly used technique is histochemical staining, e.g., with Oil Red O [15,16] or the solvatochromatic dye Nile red [17][18][19]. There are controversial opinions on which stains and methodologies are best suited for lipid content quantification [4,12,15].…”

“…There are several well-established procedures for the segmentation of C. elegans on brightfield images. The online tool IPPOME accurately segments images of worms on agar pads by noise-reduction and the Chan-Vese algorithm [16]. The wormachine is a MATLAB-based image analysis tool which incorporates worm segmentation from agar brightfield images by auto-thresholding.…”