Research Article

Thyroid hormone signaling specifies cone subtypes in human retinal organoids

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Science  12 Oct 2018:
Vol. 362, Issue 6411, eaau6348
DOI: 10.1126/science.aau6348
  • Temporally regulated TH signaling specifies cone subtypes.

    (A) Embryonic stem cell–derived human retinal organoids [wild type (WT)] generate S and L/M cones. Blue, S-opsin; green, L/M-opsin. (B) Organoids that lack thyroid hormone receptor β (Thrβ KO) generate all S cones. (C) Early activation of TH signaling (WT + T3) specifies nearly all L/M cones. (D) TH-degrading enzymes (such as DIO3) expressed early in development lower TH and promote S fate, whereas TH-activating regulators (such as DIO2) expressed later promote L/M fate.

  • Fig. 1 S and L/M cone generation in human retinal organoids.

    (A) Decision between S and L/M cone subtype fate. (B and C) S-opsin (blue) and L/M-opsin (green). (B) Human adult retina age 53. (C) iPSC-derived organoid, day 200 of differentiation. (D to K) Bright-field images of organoids derived from iPSCs. (D) Undifferentiated iPSCs. (E) Day 1, aggregation. (F) Day 4, formation of neuronal vesicles. (G) Day 8, differentiation of retinal vesicles. (H) Day 12, manual isolation of retinal organoid. (I) Day 43, arrow indicates developing retinal tissue, and arrowhead indicates developing retinal pigment epithelium. (J) Day 199, arrow indicates outer segments. (K) Day 330, arrow indicates outer segments.

  • Fig. 2 Human cone subtype specification is recapitulated in organoids.

    (A to K) S-opsin (blue) and L/M-opsin (green) were examined in human iPSC-derived organoids [(A), (C) to (E), and (G) to (M)] and human retinas [(B), (D), (F), and (H)]. [(A) to (C) and (E) to (G)] Arrows indicate outer segments, solid arrowheads indicate inner segments, and open arrowheads indicate nuclei. [(A) and (E)] CRX (a general marker of photoreceptors) is expressed in S cones and L/M cones. [(B) to (D)] S cones display short outer segments and thin inner segments in both human retinas and organoids. [(F) to (H)] L/M cones display long outer segments and wide inner segments in both human retinas and organoids. [(D) and (H)] Quantification of outer segment lengths and inner segment widths (adult retina, L/M, n = 13 cones, S, n = 10 cones; organoid, L/M, n = 35 cones, S, n = 42 cones). [(I) to (N)] S cones are generated before L/M cones in organoids. (L) Ratio of S:L/M cones during organoid development. (M) Density of S and L/M cones during organoid development. (N) S-opsin expression precedes L/M-opsin expression in human iPSC-derived organoids. CRX expression starts before opsin expression. TPM, transcripts per kilobase million.

  • Fig. 3 Thyroid hormone signaling is necessary and sufficient for the temporal switch between S and L/M fate specification.

    (A to K) S-opsin (blue) and L/M-opsin (green) were examined in human ESC-derived organoids. (A) Wild-type (WT). (B) Thrβ2 early termination mutant (Thrβ2 KO). (C) Quantification of (A) and (B) (WT, n = 3 organoids; Thrβ2 KO, n = 3 organoids). (D) WT. (E) Thrβ KO. (F) WT treated with 20 nM T3 (WT + T3). (G) Thrβ KO treated with 20 nM T3 (Thrβ KO + T3). (H) Quantification of (D) to (G) (WT, n = 9 organoids; Thrβ KO, n = 3 organoids; WT + T3, n = 6 organoids; Thrβ KO + T3, n = 3 organoids. Tukey’s multiple comparisons test: WT versus Thrβ KO, P < 0.0001; WT versus WT + T3, P < 0.01; WT + T3 versus Thrβ KO + T3, P < 0.0001). (I) Length of outer segments. WT, L/M n = 66 cells; WT, S n = 66 cells; Thrβ KO, n = 50 cells (Tukey’s multiple comparisons test, WT L/M versus WT S, P < 0.0001; WT L/M versus Thrβ KO, P < 0.0001; WT S versus Thrβ KO, not significantly different). (J) Width of inner segments. WT, L/M n = 78 cells; WT, S n = 78 cells; Thrβ KO, n = 118 cells (Tukey’s multiple comparisons test, WT L/M versus WT S, P < 0.0001; WT L/M versus Thrβ KO, P < 0.0001; WT S versus Thrβ KO, not significantly different). (K) T3 acts through Thrβ to increase total cone number. Quantification of density of S and L/M cones; WT, n = 6 organoids; Thrβ KO, n = 3 organoids; WT + T3, n = 3 organoids; Thrβ KO + T3, n = 3 organoids (Tukey’s multiple comparisons test between total cone numbers, WT versus Thrβ KO, not significantly different; WT versus WT + T3, P < 0.01; WT + T3 versus Thrβ KO + T3, P < 0.0001).

  • Fig. 4 Dynamic expression of thyroid hormone signaling regulators during development.

    (A to C) Heat maps of log(TPM + 1) values for genes with (A) changing expression, (B) consistent expression, and (C) no expression. Numbers at the bottom of heat maps indicate organoid age in days. (D) Model of the temporal mechanism of cone subtype specification in humans. For simplicity, only the roles of DIO3 and DIO2 are illustrated. In step 1, expression of DIO3 degrades T3 and T4, leading to S cone specification. In step 2, expression of DIO2 converts T4 to T3 to signal Thrβ to repress S and induce L/M cone fate.

Supplementary Materials

  • Thyroid hormone signaling specifies cone subtypes in human retinal organoids

    Kiara C. Eldred, Sarah E. Hadyniak, Katarzyna A. Hussey, Boris Brenerman, Ping-Wu Zhang, Xitiz Chamling, Valentin M. Sluch, Derek S. Welsbie, Samer Hattar, James Taylor, Karl Wahlin, Donald J. Zack, Robert J. Johnston Jr

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
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    Table S1
    List of identified dominant bacterial phylotypes from soils across the globe. This list contains information on the taxonomic identity of each phylotype, the ecological cluster it was assigned to, and the most closely related reference genome, cultivated strain and isolate.

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