MultiColor FlpOut – Stochastic Combinatorial Cell Labeling

Nern et al (2015) describe a new method for labeling cells called MultiColor FlipOut (MCFO) and its application to the study of the neuroanatomy of the optic lobe of Drosophila melanogaster.

Brainbow is an example of a stochastic combinatorial cell labeling method using different fluorescent proteins to produce a range of colors.

There are several methods for combinatorial stochastic labeling of cells – most notably Brainbow and its derivatives.

Nern et al (2015) discuss the limitations of existing methods including, in particular, the absence of precise control over the density of labeled cells.

The density of labeled cells using MCFO can be controlled in a number of ways allowing users to tune the system for their needs.

Cover image expansion

Drosophila visual system Image credit JCB 2010; 189 (5)

MCFO involves the expression of multiple, independently epitope tagged versions of an inactive, membrane form of GFP called ‘spaghetti monster GFP’ (smGFP). More about smGFP can be found in Yiswanathan et al (2015), but it is enough to know that this protein is essentially just an epitope scaffold. Each epitope tag allows for independent imaging using indirect immunofluorescence with different fluorophore-labeled secondary antibodies. Different combinations of tagged smGFP expression result in different combination of fluorphores. Which epitope tagged smGFP is expressed is controlled in two ways. First, transcription is regulated by a UAS-containing promoter and an appropriate Gal4 driver. Second, translatable transcripts are produced only after an insert (a stop-tag) in the middle of the smGFP coding region is ‘flipped out’ (Flp/FRT system or something similar). Controlling the frequency of excision of the stop-tag determines how dense the marked cells will be in the tissue of interest.

Nern et al describe two ways in which they regulated the frequency and timing of stop-tag excision. First, by using a recombinase under the regulatory control of a heat shock promoter. Short heat-shocks administered after cell division in the optic lobe was complete resulted in a low density of labeled cells. Longer heat shocks at the same time resulted in higher densities of labeled cells. If heat shocks were given while mitosis was occurring in the optic lobe, marked clones arose. The second means by which the density of marked cells was controlled was by using different alleles of Flp recombinase with different activities. In this case, the recombinase was expressed ubiquitously. Some Flp recombinase alleles had low activities and resulting in low labeling densities and others, with more activity resulted in higher densities of labeled cells.

Nern et al were interested in certain neuroanatomical aspects of the optic lobe and the majority of their paper describes those observations and their significance.

As for MCFO and its applicability to other animals, the system could be widely useful so long as on has appropriate driver lines and the ability to create the necessary genotypes.

Nern, A., Pfeiffer, B. D., and G. M. Rubin (2015) Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system PNAS 2015 112 (22) E2967E2976; published ahead of print May 11, 2015, doi:10.1073/pnas.1506763112

Facebooktwittergoogle_plusredditpinterestlinkedinmailFacebooktwittergoogle_plusredditpinterestlinkedinmail

Post a Comment

Your email address will not be published. Required fields are marked *