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. Author manuscript; available in PMC: 2020 May 8.
Published in final edited form as: Neuron. 2019 Mar 12;102(3):574–586.e5. doi: 10.1016/j.neuron.2019.02.015

Figure 2. A genetically-encoded RAR-reporter shows increased RAR-signaling in-vivo.

Figure 2.

(A) Schematic representation of the reporter construct. Red fluorescent protein (RFP) is under control of the cytomegalovirus promoter (CMV) resulting in constitutive expression. Green fluorescent protein (GFP) is under the control of a triple repeat of the Retinoic Acid Response Element (RARE) upstream to a weak SV40 promoter, resulting in RA-regulated expression.

(B) Left: fluorescence images of HEK293 cells expressing the RAR reporter. Cells were lipofectamine-transfected and 48 hrs later, either vehicle alone (0.1% DMSO in PBS) or with 1 μM all-trans retinoic-acid (ATRA) were added to the culture medium for an additional 48 hrs. Images show strong RFP expression after either vehicle or ATRA treatment, but a significant increase in GFP expression only following ATRA treatment. Right: quantification of RFP and GFP fluorescence, normalized to mean RFP fluorescence values in vehicle-treated cells. Values are shown as mean ± SEM, 3 separate experiments were conducted in duplicate wells of HEK cells grown in serum-free media. n.s.: non-significant, ***p<0.001,2-tailed t-test. See also Figure S2.

(C,D) The RAR reporter shows RA signaling in the ganglion cell layer (GCL) from WT and degenerated mouse retina (rd1, C), and WT and degenerated rat retina (s334ter,D). RA-dependent signaling was quantified in individual cells by measuring GFP fluorescence in RFP-expressing cells (mean gray value in arbitrary units, ‘a.u.’). Histograms show the distribution of GFP fluorescence in RFP-positive cells. Data for individual cell values were pooled from 8–10 retinas in 4–5 animals per strain. Each retina was divided into 3–4 pieces, and 4–6 fields of view were imaged and analyzed for each piece. Images correspond to 2D projections of Z-stacks from a spinning disk confocal microscope. Blue vertical line indicates the median value of GFP fluorescence for each distribution. Animals were injected intravitreally at P30–45 with AAV2-RAR-reporter and analyzed ~60–90 days later. See also Figure S3. For full datasets, see Table S1.