Analysis of opsin expression intensity across the mouse retina.

(A, E, I) Whole mounted C57BL/6 mouse retina stained for M-opsin (green) and S-opsin (blue). (B, F, J) Heatmap displaying the log relative density of pixels that have opsin signal identified in a 25 mm2 region. (A) M-opsin signal. (B) Heatmap of total M-opsin density bins. (C) Graph of the relative density of pixels that are expressing M-opsin summed horizontally (D—V). (D) Graph of the relative density of pixels that are expressing M-opsin summed vertically (T—N). (E) S-opsin signal. (F) Heatmap of total S-opsin density bins. (G) Graph of the relative density of pixels that are expressing S-opsin summed horizontally (D—V). (H) Graph of the relative density of pixels that are expressing S-opsin summed vertically (T—N). (I) M-opsin and S-opsin (co-expression) signal. (J) Heatmap of co-expressing opsin density bins. (K) Graph of the relative density of pixels that are co-expressing S- and M-opsin summed horizontally (D—V). (L) Graph of the relative density of pixels that are co-expressing S- and M-opsin summed vertically (T—N). T = Temporal, N = Nasal, D = Dorsal, V = Ventral.</p

Simulated cone mosaic produced by the quantitative model.

Simulated cone photoreceptor mosaic generated by the quantitative model displaying expression of S-opsin (blue), and M-opsin (green). A dorsal to ventral region is shown. (A1, B1, C1, D1) Complete simulated D-V strip. (A2, B2, C2, D2) Zoom in the dorsal region. (A3, B3, C3, D3) Zoom in the central region. (A3, B3, C3, D3) Zoom in the ventral region. (A1-4) S-opsin only cones. (B1-4) S-opsin expression in CEC cones. (C1-4) M-opsin expression in CEC cones. (D1-4) S- and M-opsin expression in CEC cones (E1-4) All cones including S-opsin only and CEC cones.</p

Modeling binary and graded cone cell fate patterning in the mouse retina - Fig 6

Model for cone cell fate specification (A) A naïve cell (grey) makes a binary decision between S-opsin only (blue) or co-expressing competent (CEC) cone fate (green, cyan or blue). The CEC cone expresses graded levels of M- and S-opsin dependendent on the D-V concentration of thyroid hormone. (B1-4) T3 (Thyroid hormone), Thrβ2* (active Thrβ2 binding T3), FD (fate determinate function), U (undifferentiated cell), S (S-only cone), C (Co-expressing cone), H (Hill function), ϕ (degradation constant of opsin proteins). (B1) Binding of T3 to Thrβ2 activates Thrβ2 (Thrβ2*) (B2) Thrβ2 controls the binary decision between S-opsin only/FD(S) or CEC/FD(C) cone fate (B3) Thrβ2* promotes M-opsin expression (B4) Thrβ2* inhibits S-opsin expression, whereas inactive Thrβ2 promotes S-opsin expression.</p

S- and M-opsin intensities in cones.

(A—B) Clustering analysis of cone populations. Cluster one = dark blue; cluster two = maroon. (C-H) Cones are ranked according to the intensity of S- and M-opsin expression levels. Intensity values are represented in arbitrary units. Each point is colored according to the log10[Probability] of expression levels. A line is drawn on the graph to show the separation between the two discrete populations of S-opsin only and CEC cone populations. (C) All cones in the regions imaged. (D) Cones in the dorsal 500–750 mm. (E) Cones in the dorsal 1500–1750 mm. (F) Cones in the central 2500–2750 mm. (G) Cones in the ventral 3500–3750 mm. (H) Cones in the ventral 4500–4750 mm.</p

ThrB2Δ mouse Intensity Plots.

Relative intensity of S-opsin cone cells (X-axis) displayed as a function of dorsal to ventral position. Each point is colored according to the log10[Probability] of expression levels. (A) Control Retina, as seen in Fig 5F. (B) Thrβ2Δ retina.</p

Intensity of M- and S-opsins in cones.

Relative intensity of M- or S-opsin in a cone population (X-axis) is displayed as a function of dorsal to ventral position. Each point is colored according to the log10[Probability] of expression levels. (A-E) Relative intensity of M-opsin expression (F-J) Relative intensity of S-opsin expression (A, F) All M-opsin expressing cells. (B, G) All S-opsin expressing cells. For (B), arrow heads mark two distinct groups of cells in the dorsal region. (C, H) CEC cones co-expressing both S- and M-opsins. (D, I) M-opsin only expressing CEC cones. (E, J) S-opsin only expressing CEC cones.</p

Spatial distribution of M- and S-opsins in cone cells.

Relative density of a cone population summed horizontally across the image and displayed in the dorsal to ventral position. Dotted line represents midpoint of transition zone. (A) All M-opsin expressing cells. (B) All S-opsin expressing cells. (C) M-opsin only expressing cells. (D) S-opsin only expressing cells. (E) Co-expressing cells.</p

D-V cone pattering in simulated and experimental data.

Cones are ranked according to the intensity of S- and M-opsin expression levels. Intensity values are represented in arbitrary units. Each point is colored according to the log10[Probability] of expression levels. (A-F) Experimental data, as seen in Fig 4C–4H. (G-L) Simulated data. (A, G) All cone cells (B, H) Cones in the dorsal 500–750 mm. (C, I) Cones in the dorsal 1500–1750 mm. (D, J) Cones in the central 2500–2750 mm. (E, K) Cones in the ventral 3500–3750 mm. (F, L) Cones in the ventral 4500–4750 mm.</p

Correlation between CEC fate and S-opsin transitions.

(A) The fraction of CEC cells at the point where the S-opsin transition is at its midpoint. Data are shown for both experimental (red) and modeled (cyan) retinas. (B) The slope of the CEC transition at the S-opsin midpoint, for both experimental (red) and modeled (cyan) retinas. Note: the distributions of only 5 of the 6 retinas are included here, as one of the images had major disruptions at the transition zone due to disecting and mounting.</p

Identification of cone subtypes.

(A-S) Retina stained with antibodies against M-opsin (green) and S-opsin (blue) (A) High-resolution region spanning the dorsal to ventral retina. (B-G) A region of the dorsal retina. (F-M) A region of the central retina. (N-S) A region of the ventral retina. (B, H, N) Blue and green channels. (C, I, O) Green channel only. (D, J, P) Blue channel only. (E, K, Q) White outline indicates identified M-opsin expressing cells. (F, L, R) White outline indicates identified S-opsin expressing cells. (G, M, S) White outline indicates identified co-expressing cells.</p