Transcriptional control of cone photoreceptor diversity by a thyroid hormone receptor

Abstract

Cone photoreceptor diversity allows detection of wavelength information in light, the first step in color (chromatic) vision. In most mammals, cones express opsin photopigments for sensitivity to medium/long (M, "green") or short (S, "blue") wavelengths and are differentially arrayed over the retina. Cones appear early in retinal neurogenesis but little is understood of the subsequent control of diversity of these postmitotic neurons, because cone populations are sparse and, apart from opsins, poorly defined. It is also a challenge to distinguish potentially subtle differences between cell subtypes within a lineage. Therefore, we derived a Cre driver to isolate individual M and S opsin-enriched cones, which are distributed in counter-gradients over the mouse retina. Fine resolution transcriptome analyses identified expression gradients for groups of genes. The postnatal emergence of gradients indicated divergent differentiation of cone precursors during maturation. Using genetic tagging, we demonstrated a role for thyroid hormone receptor β2 (TRβ2) in control of gradient genes, many of which are enriched for TRβ2 binding sites and TRβ2-regulated open chromatin. Deletion of TRβ2 resulted in poorly distinguished cones regardless of retinal location. We suggest that TRβ2 controls a bipotential transcriptional state to promote cone diversity and the chromatic potential of the species.

Keywords: THRB; color vision; cone photoreceptor; retina; thyroid hormone receptor.

Fig. 1.

Cone transcriptome diversity. (A) Cones labeled with Thrb^b2Cre and Rosa26^Ai6 reporter, costained with cone markers (TRβ2, Arr3). DIC, differential interference contrast view. (B) Thrb^b2Cre knockin. Cre replaces the TRβ2-specific exon, leaving the rest of the gene and cone enhancer intact. (C) Diagram of opsin gradients beside a retinal flatmount. Boxes, zones from which cones were isolated. (D) Volcano plot identifying gradient genes with superior or inferior expression bias (P < 0.01; Student t test). RNA-seq data for 6,469 genes, cutoff 20 cpm; 53 superior, 50 inferior cones; 2-mo-old mice. (E) Gene ontology categories for 157 gradient genes. (F) Distribution plot of 103 cones showing opsin counter-gradients. Abbreviations: INBL, inner neuroblastic layer; INL, inner nuclear layer; IS/OS, inner/outer segments; ONBL, outer neuroblastic layer; ONL, outer nuclear layer; RPE, pigmented epithelium.

Fig. 2.

Gradient emergence during cone maturation. (A) Distribution (tSNE) plot of 200 cones; RNA-seq; 21–30 cells/group. Initially, cones cluster together then diverge by location after ~P8 (interpretative diagram, Right) (B) Heatmap of gradient emergence for representative genes (as sup/inf expression ratios). (C) Selected gradient genes corroborated by qPCR. Levels are relative to the highest expression (assigned as “1”) among the four groups at both ages; three pools of ≥3 mice; *P < 0.05, Student’s t test. (D) Distribution plots (152 cones) showing that all cones express Opn1sw before Opn1mw is induced and counter-gradients appear. Pde6h and Gnat2, control genes lacking gradients.

Fig. 3.

Disrupted gene expression in TRβ2-KO cones. (A) tSNE plots showing dispersed clusters of superior and inferior cones in control mice and mixed clusters in TRβ2-KO mice. RNA-seq, adults, 48–53 cones/group. (B) Box plots of normalized expression averaged for gradient gene groups, showing loss of gradients in KO cones. Z score for each gene = (each cone value−mean of both genotypes)/SD. P values, calculated by Student’s t test (and in panel E). (C) Heatmap of superior/inferior gene expression ratios. Superior bias, yellow; inferior bias, blue. Reference gradient genes noted. (D) TRβ2-KO disrupts expression of a high proportion of gradient genes (~40%; 63/157) relative to other genes (6.3%; 398/6,312). (E) Box plots for nongradient, TRβ2-dependent genes.

Fig. 4.

Chromatin binding and remodeling by TRβ2. (A) HAB-tagged endogenous TRβ2 is biotinylated by BirA, then affinity purified (AP) from mice with Thrb^HAB (HAB) and Rosa26^BirA (BirA) alleles. (B) TRβ2-HAB peaks relative to TSS determined by ChAP-seq (Left) and association with TRβ2-regulated ATAC peaks determined by differential analysis of control and TRβ2-KO datasets; cutoff >1.5-fold, FDR q < 0.05 (Right). (C) Top consensus motifs at TRβ2 binding and TRβ2-regulated chromatin sites, identified by Homer analysis. (D) Association of TRβ2-HAB binding sites with gradient genes; heatmap as in Fig. 3C (157 gradient genes, 85 [54%] of which have a binding site within 50 kb). (E) TRβ2-HAB binding and TRβ2-regulated open chromatin (ATAC) associated with gradient genes, total cone-expressed genes (6,469) or total genes in genome (25,672). (F) Inka2 gradient gene illustrating TRβ2 binding (HAB) and TRβ2-regulated open chromatin (ATAC) sites (red arrows; >2.5-fold difference, both ATAC peaks). BirA, Rosa26^BirA/BirA control for ChAP-seq. IGV reads scale, numbers on Left.

Fig. 5.

TRβ2-mediated gradient formation. (A) Dot plot of representative gradient genes with diminished gradients in KO cones; RNA-seq, 48–53 cones/group. TRβ2-independent gradient examples, below. (B) In situ hybridization of example genes. Sagittal cryosections; outer nuclear layer (ONL) of wild-type and TRβ2-KO mice at P28. (Scale bar applies to all panels.) Arrowheads, cone nuclei location. (C) Opsin gradient genes with TRβ2-regulated open chromatin (ATAC) sites (control vs. KO; FDR q < 0.05; fold changes: Opn1mw >60, Opn1sw = 1.8, Ccdc136 = 1.4). Red arrows; sites with TRβ2 binding; red outline arrow, site lacking detectable TRβ2 binding; IGV reads scale, numbers on Left. (D) TRβ2-dependent gene expression. Single cones ordered from lowest to highest expression; +, mean; **P < 0.01 control vs. KO (Student’s t test).

Fig. 6.

TRβ2 and cone diversity. A simplified model in which TRβ2 controls gene expression gradients underlying cone diversity in mice. Gradient genes are also influenced by spatial location on the superior–inferior plane of the retina. Gradient emergence postnatally occurs during a maturation phase for many organs (including eye opening) that is prompted by rising levels of thyroid hormone (L-triodothyronine [T3], active form; thyroxine [T4], precursor of T3).