Timed Knickkopf function is essential for wing cuticle formation in Drosophila melanogaster

The insect cuticle is an extracellular matrix that consists of the polysaccharide chitin, proteins, lipids and organic molecules that are arranged in distinct horizontal layers. In Drosophila melanogaster, these layers are not formed sequentially, but, at least partially, at the same time. Timing of the underlying molecular mechanisms is conceivably crucial for cuticle formation. To study this issue, we determined the time period during which the function of Knickkopf (Knk), a key factor of chitin organization, is required for wing cuticle differentiation in D. melanogaster. Although knk is expressed throughout metamorphosis, we demonstrate that its expression 30 h prior and 48 h after pupariation is essential for correct wing cuticle formation. In other words, expression beyond this period is futile. Importantly, manipulation of Knk expression during this time causes wing bending suggesting an effect of Knk amounts on the physical properties of the wing cuticle. Manipulation of Knk expression also interferes with the structure and function of the cuticle surface. First, we show that the shape of surface nano-structures depends on the expression levels of knk. Second, we find that cuticle impermeability is compromised in wings with reduced knk expression. In summary, despite the extended supply of Knk during metamorphosis, controlled amounts of Knk are important for correct wing cuticle differentiation and function in a concise period of time.