The Atoh7 remote enhancer provides transcriptional robustness during retinal ganglion cell development

Abstract

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SE^del/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

Keywords: glaucoma; human genetic disorders; optic disc area; optic nerve; shadow enhancer.

Fig. 1.

Targeted deletion of the murine Atoh7 SE. (A) Human and mouse Atoh7 locus maps. (Top) VISTA plots (114) show conserved noncoding elements within SEs (CNE1-3) and PEs (Di, Pr). Human NCRNA and mouse SE deletions (brackets) and transcription start sites (blue arrows) are indicated. Sequence identity (50 to 100%) is plotted for a 50-bp moving window. DNA segments are identified as intergenic (red), repetitive (green), coding (blue), or UTR (yellow). Primers used to amplify SE deletion (Δ) junction and WT endpoints are indicated below the mouse VISTA plot. (Bottom) Multiz species alignment, adapted from the University of California Santa Cruz (UCSC) browser (build mm10). (B) Agarose gel showing diagnostic PCRs from +/+, SE/+, and SE/SE genomic DNA. (C) Sanger chromatograms of PCR products spanning the SEΔ junction and WT endpoints. Deleted nucleotides (yellow highlight), Cas9 cut sites (arrowheads), and protospacer adjacent motifs (PAMs) are indicated. (D) Schema of Atoh7 alleles. (E) Adult eyes from Atoh7 genotypic series. The optic nerves are thin in SE/KO eyes (with 56.6 ± 10.6% of +/+ cross-sectional area, SI Appendix, Table S4) and absent in KO/KO eyes. (F) Histology of adult eyes (hematoxylin/eosin) with similar retinal morphology. X. tropicalis, Xenopus tropicalis; nt, nucleotides. (Scale bars: Top, 500 µm; and Middle and Bottom, 100 µm.)

Fig. 2.

The SE regulates Atoh7 mRNA abundance. (A) Triplex RT-PCR strategy to quantify Atoh7 mRNAs in +/HA and SE/HA retinas. The FAM (*)-labeled 5′ primer (black arrowhead) recognizes both alleles; unlabeled 3′ primers are specific for UT (black/blue) or 3xHA knock-in (red) alleles. (B) Capillary electrophoresis profiles of PCR products from +/HA and SE/HA cDNA, and HA/+ genomic DNA. The average ratio of UT (244 nt) and HA (273 nt) cDNA peak areas, at the right-hand corner of each trace, reflects the relative abundance of allelic transcripts. The gDNA trace (UT/HA product ratio = 1.8 ± 0.11, n = 3) was included as a PCR efficiency control, with a 1.0 fixed molar template ratio. Atoh7 gene and cDNA are colinear with no introns. SS, 250-nt ROX size standard. (C) UT/HA cDNA peak area ratios are plotted at four ages, normalized to +/HA cDNA controls. The ratio of Atoh7 mRNAs transcribed from SEΔ versus WT alleles is 0.16 ± 0.02 at E11.5, 0.16 ± 0.009 at E14.5, 0.22 ± 0.007 at E16.5, and 0.20 ± 0.015 at P0.5. (D) Temporal profile of Atoh7 mRNA extrapolated from qPCR (8) and triplex RT-PCR data. The SE deletion uniformly reduces Atoh7 expression at all time points. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.

Abundance of Atoh7⁺ and Isl1/2⁺ cells decreases across the Atoh7 genotypic series. (A–F) At E14.5, Atoh7⁺ cells (magenta) reside within the neuroblast layer (nb) layer while Isl1/2⁺ cells (green) are located in the nb and ganglion cell layer (gcl). KO/KO retinas lack Atoh7 immunoreactivity. (G) Atoh7⁺ and Isl1/2⁺ cell counts per 10,000 µm². Each mutant genotype has fewer Atoh7⁺ cells than WT (+/+) and most have fewer Isl1/2⁺ cells. (H) In bivariate plots, the number of Atoh7⁺ and Isl1/2⁺ cells is directly related to Atoh7 mRNA abundance (for Atoh7⁺ cells, linear regression P < 0.01, r² = 0.94, df = 4). Atoh7 mRNA values were calculated from triplex RT-PCR data as 1.00 (+/+), 0.58 (SE/+), 0.50 (KO/+), 0.16 (SE/SE), 0.08 (SE/KO), and 0.00 (KO/KO). (I) The numbers of Atoh7⁺ and Isl1/2⁺ cells are strongly correlated across the genotypic series. *P < 0.05, **P < 0.01 and ****P < 0.0001. (Scale bar: 100 µm.)

Fig. 4.

Atoh7 declines faster in terminal S-phase in SE deletion mutants. (A–E) Distribution of Atoh7⁺ and EdU⁺ cells in E14.5 retinas. Double-positive cells, which have initiated Atoh7 expression during terminal S-phase (90-min EdU pulse), are present in all genotypes (Inset). (F) A greater fraction of Atoh7⁺ cells are EdU⁺ in SE/SE and SE/KO retinas compared to +/+. *P < 0.05. (Scale bar: 50 µm.)

Fig. 5.

Fewer RGCs in adult SE mutants. (A) P30 retinal flatmounts immunostained with Rbpms antisera. SE/KO retinas have noticeably fewer Rbpms⁺ RGCs while KO/KO retinas have essentially none. Insets show 4×-magnified views. (B) Automated cell count data showing fewer total Rbpms⁺ RGCs in SE/SE, SE/KO, and KO/KO retinas than in +/+ retinas. (C) Plot showing how adult RGC counts vary with size of the RGC-competent progenitor pool, indicated by the planimetric density of E14.5 Atoh7⁺ cells. The curve rises steeply but flattens sharply for Atoh7⁺ cell densities >30% of WT. ***P < 0.001 and ****P < 0.0001. (Scale bar: 500 µm.)

Fig. 6.

Secondary effects associated with graded loss of Atoh7 expression. (A) Pax2⁺ cells (magenta) delimit the ONH in an E14.5 mutant series. The intraretinal ONH domain (yellow outline in +/+ retina) does not include the stalk. (B) Comparison of Pax2⁺ ONH cells per meridional section across Atoh7 genotypes. The abundance of Pax2⁺ APCs is correlated with the number of Atoh7⁺ progenitors. SE/KO retinas have significantly fewer Pax2⁺ cells than +/+ retinas. (C) P30 retinal flatmounts showing surface vasculature (Ib4 lectin) and RGC axons (Tubb3). Fewer major blood vessels emanate from the ONH in SE/SE and SE/KO retinas compared to WT (number ± SD, n = 3). Axon fasciculation is abnormal in SE/SE and SE/KO retinas, with large aggregates in the central retina (yellow arrowheads) adjacent to the ONH. ***P < 0.001 and ****P < 0.0001. (Scale bar: 100 µm.)

Fig. 7.

Open chromatin signatures surrounding Atoh7. (A) (A) ATAC-seq profiles of +/+ and SE/SE mutant E14.5 retinas across the Atoh7 locus. The total number of aligned reads per nucleotide are shown as bigWig tracks in the UCSC mm10 genome browser, with 250 reads indicated (red line). Each plot reflects pooled data from three embryo replicates (six retinas, >69 million reads). Open chromatin peaks are apparent over CNE2, CNE3, Di, Pr, a small nonconserved element (*) and the Atoh7gene body. No reads were mapped to CNE2 or CNE3 in SE/SE chromatin; otherwise, the ATAC-seq profiles are similar. The open chromatin status and accessibility of PE does not depend on SE. (B) Proposed mechanism for Atoh7 regulation by SE and PE during retinal histogenesis. SE (CNE2/3 elements) and PE (distal element) loop independently with the Atoh7 promoter to activate transcription and are functionally redundant. Each modular enhancer is sufficient to control spatiotemporal expression, but they act additively to increase activity.