The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6

Abstract

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

Signaling and Gene Regulatory Networks in Mammalian Lens Development

“…In recent years, lens studies have greatly benefited from the implementation of a full repertoire of genome-wide and proteomic studies, including RNA-seq, ChIP-seq, and mass spectrometry, and a growing number of genes available for mouse genetic studies that include double and triple knockouts [52, 91, 96, 100, 144, 145, 239, 240]. Use of the CRISPR-Cas9 system allows an effective dissection of regulatory mechanisms at the DNA level including complex enhancers [100].…”

“…Use of the CRISPR-Cas9 system allows an effective dissection of regulatory mechanisms at the DNA level including complex enhancers [100]. A major gap exists in our understanding of the posttranscriptional, translational, and posttranslational regulatory mechanisms functioning in the lens [241–243], as well as numerous cross-talks occuring between individual signal transduction pathways [221, 244], nuclear organization [245, 246], and how noncoding RNAs regulate nuclear organization and gene expression [247].…”

“…Regulation of Pax6 expression in lens and other cell types, including retinal progenitor cells, pancreas cells, and radial glia cells, is an important subject of current investigations [100, 101]. Earlier studies have shown that the mouse Pax6 locus resides within a 420 kb region of chromosome 2 [102] and contains a landscape of interdigitated distal enhancers, many of them being highly conserved throughout vertebrate evolution [103].…”

“…Two distal enhancers of the Pax6 locus were identified through the evolutionarily conserved non-coding regions [ectodermal enhancer, EE, 106, 107], and through the analysis of DNaseI hypersensitivity and human DNA translocation within the 3’-region of PAX6 locus called SIMO [SIMO, 108, 109]. Genomic deletion of EE partially disrupted Pax6 expression [110], prompting a systematic analysis of both 5’-EE and 3’-SIMO (Figure 3A), including their compound effects [100]. The current data support the idea that EE and SIMO function as shadow enhancers and employ several common factors, including Meis1/2 and Pax6 [100, Figure 4B]; nevertheless, whether these enhancers also bind SRTFs downstream of BMP and FGF signaling and trigger Pax6 expression remain unknown (Figure 3B).…”

“…Genomic deletion of EE partially disrupted Pax6 expression [110], prompting a systematic analysis of both 5’-EE and 3’-SIMO (Figure 3A), including their compound effects [100]. The current data support the idea that EE and SIMO function as shadow enhancers and employ several common factors, including Meis1/2 and Pax6 [100, Figure 4B]; nevertheless, whether these enhancers also bind SRTFs downstream of BMP and FGF signaling and trigger Pax6 expression remain unknown (Figure 3B). …”