The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Apical extracellular matrices (aECMs) are associated with all epithelia and form a protective layer against biotic and abiotic threats in the environment. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. Several protease inhibitors are implicated in molting, but their functions remain poorly understood. Here we characterize mlt-11, an unusual protease inhibitor with 10 conserved Kunitz domains. MLT-11 oscillates and is localized in the cuticle and in lysosomes in larvae and in the embryonic sheath starting at the 3-fold embryo stage. mlt-11 (RNAi) produced a developmental delay, motility defects, failed apolysis, and a defective cuticle barrier. mlt-11 null and C-terminal Kunitz domain deletion mutants are embryonic lethal while N-terminal deletions cause a rolling phenotype indicative of cuticle structure abnormalities. mlt-11 activity is primarily necessary in seam and hypodermal cells and accordingly mlt-11 (RNAi) causes defects in localization of the collagens ROL-6 and BLI-1 over the cuticle. mlt-11 (RNAi) molting phenotypes can be suppressed by genetically inhibiting endocytosis. Our model is that MLT-11 is acting in the aECM to coordinate remodeling and timely ecdysis.

Nematode molting is a remarkable process where the animals must essentially build a new epidermis underneath the old skin and then rapidly shed the old skin. The study of molting provides a gateway into the developmental program of many core cellular and physiological processes, such as oscillatory gene expression, coordinated intracellular trafficking, steroid hormone signaling, developmental timing, and extracellular remodeling. The nuclear hormone receptor NHR-23/NR1F1 is an important regulator of molting. Imaging and western blot time-courses revealed oscillatory NHR-23::GFP expression in the epithelium that closely followed the reported mRNA expression. Timed depletion experiments using the auxin-inducible degron system revealed that NHR-23/NR1F1 depletion early in a given larval stage caused animals to arrest with only weak molting defects, whereas later depletion resulted in highly penetrant severe molting and morphological defects. This larval arrest was independent of insulin signaling. Despite the weakly penetrant molting defects following early NHR-23/NR1F1 depletion, the epidermal barrier was defective suggesting that NHR-23/NR1F1 is necessary for establishing or maintaining this barrier. NHR-23/NR1F1 coordinates the expression of factors involved in molting, lipid transport/metabolism, and remodeling of the apical extracellular matrix. We propose that NHR-23/NR1F1 is a regulator in a recently discovered large-scale gene oscillatory network coordinating rhythmic skin regeneration.Summary StatementThis work provides insight into how a recently discovered large-scale gene expression oscillator promotes rhythmic skin regeneration in C. elegans.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23/NR1F1 is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism, and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells following NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic disease. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a C. elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation was critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1) we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.