Abstract
The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.
Highlights
Basic metabolic principles exhibit a remarkable degree of evolutionary conservation
Worm Histopathology – Usefulness of Classical Stains Worm anatomy is often imaged in the sagittal plane of the semitransparent body of C. elegans obtained by differential interference contrast microscopy (DIC, Figure 1A) and, more rarely, by transmission electron microscopy (TEM)
We started with conventional hematoxylin and eosin (H&E) staining, as this is the most widely used medical staining technique that gives a good overview of general morphology, and can be used as a reference for other dyes
Summary
Basic metabolic principles exhibit a remarkable degree of evolutionary conservation. While many key regulators of metabolism have been identified through biochemical and molecular approaches using mammalian model systems, invertebrate genetic model systems including the nematode C. elegans have widely accelerated the discovery of genes essential for the maintenance of an organism’s metabolic homeostasis. A number of genes involved in the regulation of lipid synthesis and storage, mitochondrial function and insulin signaling have been identified using C. elegans as a model system. Many of these genes are described as important modifiers of lifespan in C. elegans, probably by regulating metabolic shifts during reproduction and aging [1]. Various methods have been developed to assess metabolic changes in worms [2,3,4,5] They tend to be performed in a bulk manner where whole worm homogenates are analyzed, precluding analyses and understanding of metabolic changes in specific tissues. Even highly significant changes in a specific tissue or organ are often under-represented or masked in such bulk analyses, highlighting the need for refinement of existing methods
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