Raeppli: a whole-tissue labeling tool for live imaging of Drosophila development

Abstract

Observation of how cells divide, grow, migrate and form different parts of a developing organism is crucial for understanding developmental programs. Here, we describe a multicolor imaging tool named Raeppli (after the colorful confetti used at the carnival in Basel). Raeppli allows whole-tissue labeling such that the descendants of the majority of cells in a single organ are labeled and can be followed simultaneously relative to one another. We tested the use of Raeppli in the Drosophila melanogaster wing imaginal disc. Induction of Raeppli during larval stages irreversibly labels >90% of the cells with one of four spectrally separable, bright fluorescent proteins with low bias of selection. To understand the global growth characteristics of imaginal discs better, we induced Raeppli at various stages of development, imaged multiple fixed discs at the end of their larval development and estimated the size of their pouch primordium at those developmental stages. We also imaged the same wing disc through the larval cuticle at different stages of its development; the clones marked by Raeppli provide landmarks that can be correlated between multiple time points. Finally, we used Raeppli for continuous live imaging of prepupal eversion of the wing disc.

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

“…The vhhGFP4 coding fragment (Saerens et al, 2005) was inserted at the C-terminal end of Nrv1::TagBFP. A Drosophila Kozak sequence (CAAA) was added and subsequently vhhGFP4::Nrv1::TagBFP was inserted into the multiple cloning site (MCS) of the pUASTLOTattB vector(Kanca et al, 2014). …”

“…In addition, the 2316 base pair minimal apical localization sequence of Bazooka (obtained from Krahn et al, 2010) was attached C-terminally to mCherry. A Drosophila Kozak sequence (CAAA) was added when inserting vhhGFP4::T48-Baz::mCherry into the MCS of the pUASTLOTattB vector (Kanca et al, 2014). …”

A cellular process that includes asymmetric cytokinesis remodels the dorsal tracheal branches in Drosophila larvae

“…In order to gain an overview of tracheal remodeling at the cellular level, we made use of Raeppli, a novel technique for cell labeling based on the differential expression of four spectrally separable fluorescent proteins (Kanca et al, 2014). We used two copies of Raeppli for a stronger signal and a broader color palette, under the control of the Tub-Gal80 ts ;UAS-Flp,Act-Gal4 induction system (Kanca et al, 2014).…”

“…We used two copies of Raeppli for a stronger signal and a broader color palette, under the control of the Tub-Gal80 ts ;UAS-Flp,Act-Gal4 induction system (Kanca et al, 2014). The animals were first reared at 18°C until L2 stage and were then transferred to the non-permissive temperature of 29°C, unlocking Raeppli and irreversibly forcing a stochastic color selection for each copy of the Raeppli construct.…”

Oxygenation and adenosine deaminase support growth and proliferation of ex vivo cultured Drosophila wing imaginal discs

“…These snapshots have been used to reconstruct the temporal progression of events and to deduce cause-effect relationships, but methods to follow the development of individual discs over time are lacking. One such method recently reported is to repeatedly image a wing disc through the larval cuticle at different points of development (Kanca et al, 2014;Nienhaus et al, 2012). Novel approaches would become possible, however, if wing imaginal discs could be cultured ex vivo for live imaging.…”