Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates. Here, we show that the bicistronic gene würmchen is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Würmchen1 and Würmchen2, which are both necessary for normal epithelial tissue development. Würmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Würmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development.