pgen.1009613.g006.tif (2.11 MB)
Download file

Novel motif combinations yield a series of progressively restricted expression patterns.

Download (2.11 MB)
posted on 2021-06-23, 20:15 authored by Clara Poupault, Diane Choi, Khanh Lam-Kamath, Deepshe Dewett, Ansa Razzaq, Joseph Bunker, Alexis Perry, Irene Cho, Jens Rister

(A) Schematic comparison of wild type Rh3 promoter and hybrids of the distal Rh3 promoter combined with the proximal core promoters of Rh4, Rh5, or Rh6. Note in the proximal region the shared K50 motif within the Rh6 RCSI and the ATTCII motif at the same position within the Rh5 RCSI, as well as the same spacing of the Rh5 RCSI and the TATA box. The dotted vertical line indicates the break/fusion point of the hybrids. (B)—(E) Wild type vs. hybrid promoters driving GFP reporter expression (green) in the R7 layer. Rh3 (blue) labels pR7s and Rh4 (red) labels yR7s. Bar graphs show GFP co-expression in the Rh3 or Rh4 subset, respectively. (B’)—(E’) Wild type reporter vs. hybrid promoter driving GFP reporter expression (green) in the R8 layer. Rh5 (blue) labels pR8s and Rh6 (red) labels yR8s. Bar graphs show GFP co-expression in the Rh5, Rh6, or R1-R6 subset. Green numbers indicate the mean percentage of GFP co-expressing photoreceptors, error bar represents standard error of the mean. (B) and (B’) The wild type Rh3 promoter drives subtype-specific GFP expression in the pR7/Rh3 subset. Green staining in (B’) is from the axons of pR7s that traverse the R8 layer. N = 18 retinas and n = 1,188 R7s for (B); N = 11 retinas, n = 958 R8s and 5,748 R1-R6 PRs for (B’). (C) and (C’) The Rh3-Rh6 hybrid drives photoreceptor class-restricted GFP expression in all ‘inner’ photoreceptors, i.e. both the R7 and R8 photoreceptor types (pR7s, yR7s, pR8s, and yR8s). N = 10 retinas and n = 1,307 R7s for (C); N = 12 retinas, n = 872 R8s and 5,232 R1-R6 PRs for (C’). (D) and (D’) The Rh3-Rh4 hybrid drives photoreceptor type-restricted GFP expression in both R7 subtypes (pR7s and yR7s). GFP signal in (D’) is from traversing R7 axons. N = 12 retinas and n = 1,569 R7s for (D); N = 11 retinas, n = 708 R8s and 4,248 R1-R6 PRs for (D’). (E) and (E’) The Rh3-Rh5 hybrid drives photoreceptor subtype-specific GFP expression in pR7s. GFP signal in (D’) is from pR7 axons. N = 11 retinas and n = 1,133 R7s for (E); N = 9 retinas, n = 967 R8s and 5,802 R1-R6 PRs for (E’). Scale bars, 10 μm.