ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila

Abstract

Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects.

The localization of chitin synthase mediates the patterned deposition of chitin in developing Drosophila bristles

“…worth noting that previously we detected several patches of extracellular chitin fibers on the apical 611 surface of 29 hr awp pupal wings (Sobala et al, 2015). This is 11 hrs prior to when we first detect chitin 612 in hairs (Adler et al, 2013) and 3 hours prior to the start of hair outgrowth (Wong and Adler, 1993).…”

“…We did not 198 see any evidence for the secretion of Kkv::NG in these experiments. As an internal positive control, the 199 larvae also expressed a secreted ChtVis-tdTomato chitin reporter (in red) (Sobala et al, 2015). As 200 expected, Cht-Vis could be detected in the extracellular space between the disc epithelium and 201 peripodial membrane cells.…”

“…As noted in the introduction the relationship between the patterned deposition of chitin and the 271 localization of CS in insects is not clear. To investigate this we carried out experiments where we 272 localized both Kkv::NG and our chitin reporter (Cht-Vis) in bristles in living pupae (Sobala et al, 2015). 273…”

Short distance non-autonomy and intercellular transfer of chitin synthase in Drosophila

“…As morphogenesis continues the delayed hairs catch up to the 129 earlier forming ones. To determine if the deposition of cuticle followed a similar time course I used a 130 chitin reporter and examined wing cuticle deposition by in vivo imaging (Sobala et al, 2015). At all times 131 examined (42, 44 and 46 hr awp (after white prepupae)) neighboring cells showed similar levels of the 132 reporter ( Fig 2C).…”

A Tandem Green–Red Heterodimeric Fluorescent Protein with High FRET Efficiency

“…Likewise, a dimeric version of the Heteractis crispa red FP (HcRed) and an Entacmaea quadricolor derived red FP dimer tdKatushka2, were made into tandem dimers. Notably, tdTomato is one of the brightest and most widely used red FPs for in vivo imaging applications …”