Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation

Abstract

The eye field transcription factor, Six6, is essential for both the early (specification and proliferative growth) phase of eye formation, as well as for normal retinal progenitor cell differentiation. While genomic regions driving six6 optic cup expression have been described, the sequences controlling eye field and optic vesicle expression are unknown. Two evolutionary conserved regions 5′ and a third 3′ to the six6 coding region were identified, and together they faithfully replicate the endogenous X. laevis six6 expression pattern. Transgenic lines were generated and used to determine the onset and expression patterns controlled by the regulatory regions. The conserved 3′ region was necessary and sufficient for eye field and optic vesicle expression. In contrast, the two conserved enhancer regions located 5′ of the coding sequence were required together for normal optic cup and mature retinal expression. Gain-of-function experiments indicate endogenous six6 and GFP expression in F1 transgenic embryos are similarly regulated in response to candidate trans-acting factors. Importantly, CRISPR/CAS9-mediated deletion of the 3′ eye field/optic vesicle enhancer in X. laevis, resulted in a reduction in optic vesicle size. These results identify the cis-acting regions, demonstrate the modular nature of the elements controlling early versus late retinal expression, and identify potential regulators of six6 expression during the early stages of eye formation.

Genomic Signatures of Fine-Scale Local Adaptation in Atlantic Salmon Suggest Involvement of Sexual Maturation, Energy Homeostasis, Behaviour, and Immune Defence-Related Genes

“…Barson et al (2015) showed that a second conserved enhancer, necessary for six6 expression in the mouse forebrain (Lee et al, 2012) maps close to candidate SNP ss1868315338. Additional putative enhancers, with poorly characterized functions, are also located within our candidate region in other species (Conte et al, 2010; Ledford et al, 2017; Lee et al, 2012).…”

“…Barson et al (2015) did not find any nonsynonymous mutations in the six6 coding region, and results indicate that the selective target could be a regulatory element for this gene. Studies in various species have identified multiple 5’ evolutionarily conserved enhancer elements which regulate six6 expression in different tissues and at different development stages (Conte et al, 2010; Ledford et al, 2017; Lee et al, 2012). Six6 regulation also involves at least one 3’ enhancer (Conte et al, 2010; Ledford et al, 2017), and may also be mediated by local chromatin structure (Gómez-Marín et al, 2015).…”

“…Studies in various species have identified multiple 5’ evolutionarily conserved enhancer elements which regulate six6 expression in different tissues and at different development stages (Conte et al, 2010; Ledford et al, 2017; Lee et al, 2012). Six6 regulation also involves at least one 3’ enhancer (Conte et al, 2010; Ledford et al, 2017), and may also be mediated by local chromatin structure (Gómez-Marín et al, 2015). We found that one conserved 5’ enhancer, responsible for six6 expression in the maturing medaka retina (Conte et al, 2010) maps between two candidate SNPs (ss1868043109 and ss1868793364).…”

Genomic signatures of fine‐scale local selection in Atlantic salmon suggest involvement of sexual maturation, energy homeostasis and immune defence‐related genes

“…Many putative six6 enhancer elements are located within our candidate region in other species. Several have directly been shown to regulate six6 expression in different tissues and at different developmental stages, while others remain poorly characterized (Conte et al., ; Ledford et al., ; Lee, Rizzoti et al., ).…”

Aberrant activity of NKL homeobox gene NKX3-2 in a T-ALL subset

“…Physiological expression of SIX6 has been described in eye development [ 24 ]. In this context SIX6 is regulated via defined enhancers and TFs neither of which are known to involve NKX3-1 or NKX3-2 [ 44 , 45 ]. During eye-development SIX6 sets the balance between proliferation and differentiation in the retina and suppresses specific non-lineage genes [ 33 , 46 ].…”