Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss

Abstract

Organ renewal is governed by the dynamics of cell division, differentiation and loss. To study these dynamics in real time, we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem-cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12–16 hr and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. By applying a pipeline for movie processing and analysis, we uncover new and intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of adult organ renewal.

Myt1 kinase promotes mitotic cell cycle exit inDrosophilaintestinal progenitor cells

“…Since myt1 mutant ECs fail to display the mature EC marker Pdm1, our results suggest that Myt1-mediated G2 phase arrest is a critical stage for EBs to exit the mitotic cell cycle and receive differentiation cues. Consistent with this idea, it was recently demonstrated that EB exit from the mitotic cell cycle occurs prior to EC fate commitment (Martin et al, 2018). Moreover, Notch-dependent cell fate decisions are also determined during G2 phase arrest in Drosophila sensory organ precursors Hunter et al, 2016).…”

“…Having shown that loss of Myt1 perturbs EB quiescence, we next assessed EB cell fate using an esg ts -driven reporter line that differentially labels ISCs and EBs (Martin et al, 2018).…”

Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila

“…esgGal4 (112304) was obtained from the Kyoto Drosophila Genetic Resource Center. The following stocks were gifts: mexGal4 (Carl Thummel), breathlessGal4 , UAS-his2b::CFP (Yoshihiro Inoue), ubi-his2avD::YFP (Pavel Tomancak), and GBE-Su(H)-GFP:nls (Joaquin de Navascues), UAS-CD8-GFP, hs-flp 12 ; tubGal4; FRT82, tubGal80 and FRT82 (David Bilder). The ‘fate sensor’ line ( esgGal4, UAS-his2b::CFP, GBE-Su(H)-GFP:nls; ubi-his2av::mRFP ) was generated in a previous publication 12 .…”

“…Some newer approaches preserve animal viability but cannot visualize individual cells 6–8 . Other recent advances enable high-resolution imaging but require opening the abdominal cuticle, which leads to death of the fly 9–12 .…”

“…As in vertebrate intestine, stem cells in the fly midgut continuously divide to replenish terminally differentiated epithelial cells that form the intestinal barrier 13 . Although current live imaging methods capture single divisions 12 , their timescales are not sufficient to track multiple divisions of the same stem cell or to monitor differentiation of cellular progeny.…”