by Isabel D’Alessandro and Rachel Wilson

MultiColor FlpOut #

MultiColor FlpOut (MCFO) is a method for the combinatorial stochastic labeling of cells. You can read more about MCFO in the original paper (Nern et al. 2015), and in this document.  The protocol for MCFO immunostaining is described in this document. 

Split-Gal4 #

The Drosophila split-GAL4 system, first described in Luan et al., 2006, allows for restricted expression of regulatory targets only in cells where the two components of the split-GAL4 activator are co-expressed. The GAL4 DNA-binding domain fused to the Zip- protein-pairing domain can be expressed in one pattern, and a transcriptional activation domain fused to the Zip+ protein-pairing domain can be expressed in another pattern. The UAS reporter construct will be expressed in the intersection of the two patterns. We now have thousands of stocks that carry either 1) a construct that expresses a DNA-binding domain protein derived from either GAL4 or lexA or 2) a construct that expresses a transcriptional activation domain derived from either GAL4, VP16 or p65. Read more about the split-Gal4 system in Dionne et al. 2018, and Tirian and Dickson, 2017. 

Tagging cells by neurotransmitter or receptor#

Lacin et al. 2019 generated and validated split-Gal4 hemidrivers to target glutamatergic, cholinergic, and GABAergic cells. Kondo et al. (2020) generated T2A-GAL4 knockins targeting neurotransmitter receptor genes.

DenMark #

DeMark is a genetic tool to mark pre-and postsynaptic compartments of neurons in the Drosophila brain. It makes use of an mcherry fluorescent protein fused with the mammalian intercellular adhesion molecule ICAM5, which selectively marks the somatodendritic compartment, and the presynaptic marker synaptotagmin tagged with GFP to mark axons. The specific form of the genetic tool we use contains repo(glia)-Gal80, and UAS-DenMark and UAS-syt-eGFP combined. Read more about DenMark in the original paper (Nicolai et al. 2010). The protocol for DenMark immunostaining is described in this document. These days, DenMark is not used as often as it was previously, because we can get polarity information from connectome data (e.g., the segregation index of Schneider-Mizell 2016).

TransTango #

transTango is a tool which uses an exogenous signaling mechanism(human glucagon and its receptor) to allow transsynaptic marking of presynaptic and postsynaptic partners. In the starter population of neurons, a ligand is attached to neurexin1(presynaptic cell adhesion protein) domains, tethering it to the presynaptic membrane.Exogeneous receptors are expressed at the surface of cells throughout the nervous system. In cells that are postsynaptic to the starter neurons, the ligand activates the receptors, which triggers proteolytic (TEV) release of the transcriptional activator (QF) from the plasma membrane and the expression of a target reporter gene (mtdtomato) in the postsynaptic cell. Read more about transTango in the original paper (Talay et al. 2017). The protocol for transTango immunostaining is described in this document. These days, DenMark is not used as often as it was previously, because we can get polarity information from connectome data.