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.