The mucosal barrier in the gut, lungs and other vital tubular organs consist primarily of mucins and protects the underlying cells against pathogens, dehydration and physical or chemical injury. Mucins are large, high molecular weight glycoproteins that contain a central polymorphic domain composed of tandem repeats, rich in amino acids serine, threonine and proline. These highly O-glycosylated proteins form densely arrayed structures that provide mutivalency and high stoichiometric power. In Drosophila, twenty-three mucins and mucin-related proteins that are expressed dynamically during different stages of the life cycle were identified. To understand the role of Drosophila mucins during development, we used the transgenic RNA interference (RNAi) system to systematically knockdown mucins. Ubiquitous knockdown of mucins expressed in the gut resulted in larval and pupal lethality, indicating that they are essential and suggesting their requirement for protective barrier formation in these tissues. Interestingly, tissue-specific knockdown of one mucin (Muc26B), which is expressed in the proventriculus, showed 1st instar larva lethality along with a dramatic up-regulation of genes encoding antimicrobial peptides. Additionally, loss of Muc26B resulted in activation of conserved signaling pathways. Immunostaining for MUC26B showed that it is expressed in the specialized secretory cells of the proventriculus called the PR cells, which are responsible for the synthesis and secretion of the peritrophic membrane (PM). Indeed, MUC26B staining could be seen in this protective membrane in wild type flies. We therefore hypothesize that MUC26B serves as a crucial component of the PM and we are currently investigating the cellular processes and signaling cascades activated in response to the loss of MUC26B. We are generating transgenic flies that endogenously express Muc26Blacking mucin-domains and associated chitin-binding domains to investigate the role of MUC26B in PM formation and barrier function. These transgenic flies will aid in elucidating changes in PM structure and function upon modulation of different MUC26B domains. In addition, we will investigate the role of MUC26B glycosylation in the packaging, secretion and formation of the PM using correlative light and electron microscopy (CLEM). These studies will ultimately advance our understanding of mucins and their role in the protection of epithelial cell surfaces.