Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish

Abstract

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.

A Stage-Specific OTX2 Regulatory Network and Maturation-Associated Gene Programs Are Inherent Barriers to RPE Neural Competency

“…Differentially accessible peak summits in the developing RPE were found to be highly enriched for the TAATCC homeobox motif, which is recognized by the DNA-binding homeodomain of a number of homeobox factors that regulate eye development, including OTX2, CRX, and PITX1 ( Figure 6H ). Moreover, we observed a high overrepresentation of motifs resembling the TEA motif recognized by TEAD1, TEAD3, and TEAD4, which is notable given the known roles for TEAD transcription factors in governing RPE specification and function ( Miesfeld et al, 2015 ; Buono et al, 2021 ). Moreover, the NR motifs recognized by NR2F1 and NR2F2 were highly overrepresented in the observed DARs, which are known to regulate RPE vs neural retina identity throughout the differentiating optic vesicle ( Tang et al, 2010 ).…”

“…Although MITF and VSX2 are two of the earliest indicators of optic vesicle regionalization, they are part of an intricate gene regulatory network that is not fully defined. A recent study leveraged gene expression and chromatin accessibility profiling to resolve intrinsic transcription factor networks that delineate early RPE and neural retina cell differentiation ( Buono et al, 2021 ). This study uncovered a conserved, sequential transcription factor program that specifies the RPE and activates functional attributes, exemplified by the TEAD-mediated activation of RPE desmosome function.…”

Trap-TRAP, a Versatile Tool for Tissue-Specific Translatomics in Zebrafish

“…Finally, mRNA abundance was calculated in Reads Per Kilo base per Million mapped reads (RPKM) allowing the comparison among different samples. For the comparative expression analysis between whole-embryo (GEO accession number: GSM4521281) and vsx2:TRAP transcripts, we examined RPKM values for a list of transcription factor (TF) encoding genes that have been shown as enriched at the neural retina domain vs the RPE in cell populations isolated by FACS (Buono et al, 2021). To reduce noise, we included in our analysis only those genes with a RPKM value above 5 (89 TFs).…”

“…To further examine the eGFP-rpl10a reported capacity to efficiently pull-down tissue-specific transcripts in zebrafish (Tryon et al, 2013), we fused this cassette to the neural-retina specific promoter vsx2.2 (Nicolas-Perez et al, 2016;Buono et al, 2021). The resulting construct Tol2_vsx2.2:TRAP was microinjected into one-cell embryos to generate the corresponding zebrafish line Tg[vsx2.2:TRAP] (Figures 1D,E).…”

“…Then, polysomes from 22 hpf Tg[vsx2.2:TRAP] embryos were harvested using a standard TRAP protocol, and captured RNA (120 ng) was deep sequenced (see Methods). To assess the retrieval efficiency of specific transcripts in that sample, we focused on the expression of a retina-enriched cluster of TFs (cluster 1), identified in our group in vsx2.2-positive retinal cells sorted by FACS at stage 23 hpf (Buono et al, 2021). We compared TFs relative mRNA abundance (RPKM) between affinity purified RNAs from vsx2.2:TRAP embryos at 22 hpf (TRAP-seq), and control RNA-seq samples obtained from whole embryos also at 22 hpf (GSM4521281).…”