Biology subjects

Levenson, M. T.

Publications and source records attributed to Levenson, M. T..

6 recordsLinked to original sources

bioRxiv2025

MLT-11 is necessary for C. elegans embryogenesis and conserved sequences play distinct roles in cuticle structure

Apical extracellular matrices (aECMs) are associated with many epithelia and many form a protective layer against biotic and abiotic threats in the environment. Despite their importance, we lack a deep understanding of their structure and dynamics in development and disease. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. Here, we show that the poorly characterized putative protease inhibitor gene, mlt-11, is directly regulated by the NHR-23 transcription factor. We identify key cis-regulatory elements required for robust mlt-11 expression. An internal MLT-11::mNeonGreen translational fusion transiently localized to the aECM in the cuticle and embryo. MLT-11::mNeonGreen was also detected in lining openings to the exterior (vulva, rectum, mouth). mlt-11 is necessary to pattern all layers of the adult cuticle, and reduction of MLT-11 levels disrupted the barrier function of the cuticle. Deletion of conserved Kunitz protease inhibitor domains or intervening sequences produced a range of defects including either left or right rollers, and small separations of the cuticle along the length of the animal (microblisters). MLT-11 is processed into at least two fragments and internal and C-terminal mNeonGreen knock-ins display distinct localization patterns. Predicted mlt-11 null mutations caused fully penetrant embryonic lethality and elongation defects. Together, this work suggests that MLT-11 localizes similarly to pre-cuticle components and conserved sequences play distinct roles in promoting proper assembly of the aECM.

developmental biology↗

bioRxiv2024

MLT-11 is a transient apical extracellular matrix component required for cuticle patterning and function

C. elegans molting offers a powerful entry point to understanding developmentally programmed apical extracellular matrix remodeling. However, the gene regulatory network controlling this process remains poorly understood. Focusing on targets of NHR-23, a key transcription factor that drives molting, we confirmed the Kunitz family protease inhibitor gene mlt-11 as an NHR-23 target. Through reporter assays, we identified NHR-23-binding sites that are necessary and sufficient for epithelial expression. We generated a translational fusion and demonstrated that MLT-11 is localized to the cuticle and lined openings to the exterior (vulva, rectum, mouth). We created a set of strains expressing varied levels of MLT-11 by deleting endogenous cis-regulatory element sequences. Combined deletion of two cis-regulatory elements caused developmental delay, motility defects, and failure of the cuticle barrier. Inactivation of mlt-11 by RNAi produced even more pronounced defects. mlt-11 is necessary to pattern every layer of the adult cuticle, suggesting a broad patterning role prior to the formation of the mature cuticle. Together these studies provide an entry point into understanding how individual cis-regulatory elements function to coordinate expression of oscillating genes involved in molting and how MLT-11 ensures proper cuticle assembly.

developmental biology↗

bioRxiv2022

Single-nucleus resolution mapping of the adult C. elegans and its application to elucidate inter- and trans-generational response to alcohol.

Single-cell RNA transcriptomic platforms have significantly contributed to our understanding of tissue heterogeneity as well as of developmental and cellular differentiation trajectories. They also provide an opportunity to map an organisms response to environmental cues with high resolution and unbiasedly identify the cell types that are the most transcriptionally sensitive to exposures. Here, we applied single nucleus RNA-seq experimental and computational approaches to C. elegans to establish the transcriptome of the adult nematode and comprehensively characterize the transcriptional impact of ethanol as a model environmental exposure on the entire organism at cell type-resolution over several generations. Clustering, tissue and phenotype enrichment, and gene ontology analyses identified 31 clusters representing a diverse number of adult cell types, including those from syncytial and multi-nucleated tissues which are difficult to assess by single cell RNA-seq, such as the mitotic and meiotic germline, hypodermal cells, and the intestine. We applied this method to identify the impact of inter- and trans-generational exposure to two human-relevant doses of alcohol. Cell type proportions were not significantly altered by ethanol. However, Euclidean distance analysis identified several germline, striated muscle, and neuronal clusters as being major transcriptional targets of ethanol at both the F1 and F3 generations although the relative order of clusters changed between generations. The impact on germline clusters was further confirmed by phenotypic enrichment analysis as well as functional validation, namely a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq of the adult C. elegans represents a powerful approach for the detailed examination of an adult organisms response to environmental cues.

genetics↗

bioRxiv2022

Experimental considerations for study of C. elegans lysosomal proteins

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.

molecular biology↗

bioRxiv2022

The conserved, secreted protease inhibitor MLT-11 is necessary for C. elegans molting and embryogenesis

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.

developmental biology↗

bioRxiv2021

Conditional depletion reveals temporal requirements for the oscillating transcription factor NHR-23/NR1F1 in C. elegans larval progression

SUMMARY STATEMENTThis work shows how a C. elegans transcription factor controls remodeling of the apical extracellular matrix during development and in which tissues it acts. ABSTRACTNematode 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.

molecular biology↗