Abstract
The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control.
Highlights
The Obstructor-A (Obst-A) protein[17] that binds chitin-fibrils to form a scaffold for localizing chitin deacetylases Serpentine (Serp) and Vermiform (Verm), to mature physical and chemical properties of the chitin-matrix[16,18,19]
Investigating the molecular network in Cht[2] and idgf[6] RNA interference (RNAi) induced mutants, which showed strongest lethality and most severe cuticle defects, we propose that certain Chts are involved in organizing the chitin-matrix scaffold, which is essential for further enzyme-mediated extracellular matrix (ECM) maturation and cuticle thickness
The Idgf proteins contain one N-terminal Signal-Peptide for protein secretion and one glyco-18 domain (Supplementary Fig. S1a). It was shown by an RNAi injection screen in Tribolium castaneum that Cht[5], Cht[10] and idgf[4] genes are involved in the molting process[34]
Summary
The Obstructor-A (Obst-A) protein[17] that binds chitin-fibrils to form a scaffold for localizing chitin deacetylases Serpentine (Serp) and Vermiform (Verm), to mature physical and chemical properties of the chitin-matrix[16,18,19]. At the apical cell surface they need to be organized into a tight and compact ECM architecture This process of chitin-ECM assembly and replacement is repeated several times due to cuticle molting and upon wounding and may involve a strict genetic control. By individually knocking down genes in the cuticle secreting tissues, we show that a large number of the Chts and idgfs are involved in cuticle molting during larval as well as in pupal stages. This is supported by gene specific spatial-temporal expression profiles and by developmental lethality profiles upon gene knockdowns. In contrast to Chts, the non-enzymatic Idgfs play an essential role in the protection of the newly synthesized cuticle matrix against degradation in order to stabilize and expand the ECM size in the larvae
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