Optogenetic Control of Gene Expression in <i>Drosophila</i>

<div><p>To study the molecular mechanism of complex biological systems, it is important to be able to artificially manipulate gene expression in desired target sites with high precision. Based on the light dependent binding of cryptochrome 2 and a cryptochrome interacting bHLH protein, we developed a split lexA transcriptional activation system for use in <i>Drosophila</i> that allows regulation of gene expression <i>in vivo</i> using blue light or two-photon excitation. We show that this system offers high spatiotemporal resolution by inducing gene expression in tissues at various developmental stages. In combination with two-photon excitation, gene expression can be manipulated at precise sites in embryos, potentially offering an important tool with which to examine developmental processes.</p></div

Kinetics of the light inducible transcription system driven by <i>elav</i>^<i>c155</i>-Gal4 with LexAOP2-Gal80 feedback control.

<p>(A) Effect of light intensity on GFP expression levels in embryos and adults. The average GFP intensity of the nervous system was measured in embryos after 4 hours of blue light illumination (n>10, mean±s.d.). The total GFP intensity of the whole brain was measured in the adults because of an uneven GFP distribution among the various brain regions. Adult flies were illuminated with continuous blue light for ~16 hours (n>5, mean±s.d.). (B) Effect of exposure time on GFP expression levels in embryos and adults. The GFP expression levels in the nervous system were measured after illuminating embryos or adults with blue light (474nm, ~2.5mWcm^-2) for the specified periods of time (n>7, mean±s.d.). (C) Selective spatial induction of gene expression using two-photon excitation. The region of interest is highlighted with the red line (left image) and exposed to two photon excitation (860nm, 80MHz, 200fs pulse width) for 4 hours. The Z-stack projection of the GFP expression pattern in the nervous system was imaged immediately afterwards (right image). A 3D-reconstruction of the exposed region is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138181#pone.0138181.s006" target="_blank">S1 Movie</a> (Scale bar = 100μm).</p

Live imaging of GCaMP3.0 fluorescence in <i>fruitless</i> neurons.

<p>(A) Males expressing the light–inducible LexA system (<i>fru</i>-Gal4 UAS-CIBN::LexA-mcherry-p65::CRY2 LexAOP-GCaMP3.0) were exposed to blue light (474nm, ~2.5mWcm^-2) for 24 hours. Clear increase in GCaMP3.0 fluorescence was observed after the administration of 10mM carbamylcholine. (B) Time-lapse changes in GCaMP3.0 fluorescence in the mcAL neuronal cluster region (highlighted in dotted circle). Each line represented a response curve from an individual brain (n = 6). The arrow indicates when the perfusion of carbamylcholine (CCh) started (~20 sec). (C) Males expressing the light–inducible LexA system were kept in darkness before the assay. Little changes in GCaMP3.0 fluorescence was observed after the administration of 10mM carbamylcholine. (D) Time-lapse changes in GCaMP3.0 fluorescence in the mcAL cluster (highlighted in dotted circle). Each line represents a response curve from an individual brain (n = 4).</p

<i>In vivo</i> use of the light-inducible gene expression system.

<p>(A) Schematic representation of the transgenic construct carrying the total light inducible LexA transcription system. T2A ribosomal skipping signals were added to the C-terminus of CIBN::LexA and mcherry to maintain stoichiometry of the required proteins. (B, C) Induction of GFP expression using the light switchable system in embryos (B) and adults (C). GMR18G07-Gal4 and orco-Gal4 were used to drive the transcription system in embryos and in adults respectively. GFP expression was seen in neuroblast cells in embryos or antennal lobe neurons in adults after blue light illumination (embryo: 4 hours; 474nm, ~1.1mWcm^-2; adult ~16 hours, 474nm, ~2.5mWcm^-2). (D) Addition of a Gal80 feedback mechanism minimizes basal expression without illumination. <i>elav</i>^<i>c155</i>-Gal4 were used to drive the transcription system in adults. Flies were illuminated with blue light for ~16 hours or kept in constant darkness. The incorporation of LexAOP2-Gal80 dramatically lowered the basal GFP expression in control without blue light illumination. This construct also greatly reduced the expression of the transcription system marked by mcherry. The reduction in mcherry expression was observed in samples with the LexAOP2-Gal80 feedback mechanism even if the exposure time was 6 times longer than that of samples without the LexAOP2-Gal80 (Scale bar = 100μm).</p

A light inducible transcription system.

<p>(A) Schematic representation of the light inducible LexA transcription system. Upon blue light (474nm, ~2.5mWcm^-2) illumination, CRY2 undergoes a conformational change that allows it to bind to CIBN to form a functional LexA transcription activator. (B) Light induced GFP expression in S2 cells. S2 cells were transfected with the two constructs that should generate a functional transcription factor upon blue light illumination (pActPL-CIBN-LexA and pActPL-p65-CRY2) and a LexAOP-GFP reporter construct. Cells were exposed to blue light for 1 hour or kept in the dark. (Scale bar = 100μm) (C) Comparison of the efficacy of the VP16 and p65 transcription activation domains. The average number of GFP positive cells per field of view (n = 10, mean±s.e.m. t-test p<0.0001).</p

Display of courtship behavior in males expressing TrpA1 in <i>fruitless</i> neurons using the light-inducible transcription system.

<p>Males expressing the light-inducible LexA system (<i>fru</i>-Gal4 UAS-CIBN::LexA-mcherry-p65::CRY2 LexAOP-TrpA1) displayed significantly more unilateral wing extensions at 30°C, a typical courtship behavioral pattern, after blue light exposure (474nm, ~2.5mWcm^-2) for 24 hours (p<0.001, Fisher’s exact test, n = 17 and 13 for light and dark respectively). The photo shows a typical wing extension behavior displayed by males with prior exposure to blue light.</p