Roles and regulation of the Kunitz domain protein MLT-11 during C. elegans cuticle synthesis and molting

Apical extracellular matrices (aECMs) are associated with many epithelia and often 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. Caenorhabditis 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 also lined openings to the exterior (vulva, rectum, and 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 roller phenotypes and small separations of the cuticle along the length of the animal (microblisters). MLT-11 is processed into at least 2 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 precuticle components, and conserved sequences play distinct roles in promoting proper assembly of the aECM.