Mercado, S. H. | Search bioRxiv

bioRxiv2025

Multiscale patterning of a model apical extracellular matrix revealed by systematic endogenous protein tagging

Barrier epithelia are shielded from the external environment by their apical extracellular matrices (aECMs). The molecular complexity of aECMs has challenged understanding of their organization in vivo. To define the molecular architecture of a model aECM we generated a toolkit of 102 fluorescently tagged aECM components using gene editing in C. elegans, focusing on proteins secreted by the epidermis to form the collagen-rich cuticle. We developed efficient pipelines for modular protein tagging and rapid fluorophore swapping. Most tagged collagens were functional and exhibited exquisitely specific patterning across stages, cell types, and matrix substructures. We define multiple reference markers for key substructures including the little-understood cortical layer, as well as the helical crossed fiber arrays that function as a hydrostatic skeleton to maintain organismal shape. We further tagged >30 members of key aECM protein classes including proteases, protease inhibitors, and lipid transporters. Our standardized markers will allow dissection of the mechanistic basis of aECM spatiotemporal patterning in vivo. HighlightsO_LIFirst large-scale protein tagging resource for the apical extracellular matrix C_LIO_LIOptimization of CRISPR methods for protein tagging including color swaps C_LIO_LITagged proteins are functional and exhibit a high degree of stage-, cell- and compartment specificity C_LIO_LIReference localization patterns for multiple aECM compartments and markers for newly defined compartments C_LI

developmental biology↗

bioRxiv2024

Sensory integration of food availability and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the ASJ chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrate population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of non-committed dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, a CaM-kinase pathway, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution. Summary/Significance StatementAnimals must respond appropriately to multiple sensory stimuli to make informed decisions. It remains unclear how the nervous system is able to integrate different sensory cues and propagate that information towards making decisions over longer timescales. We use the nematode Caenorhabditis elegans to investigate how sensory integration occurs during the decision to exit diapause, a stress-resistant developmentally arrested state triggered by multiple sensory inputs including food availability and population density. We show how expression of an insulin-like peptide critical to dauer exit reflects the sensory integration process and decision commitment, and we dissect the regulation of this insulin-like peptides expression. Our study explicitly analyzes the relationship between neuronal activity and neuropeptide expression during a complex decision with diverse sensory inputs.

neuroscience↗