Abstract
Purpose :
During development of the visual system, a handful of transcription factors are essential for specification of the eye field and later for differentiation of specific neuronal subtypes. Zebrafish six7 (sine oculis homeobox 7) in conjunction with six3b is required for forebrain patterning (Inbal et al., 2007), and independently regulates photoreceptor progenitor mitosis and green-sensitive cone survival (Sotolongo-Lopez et al., 2016). We propose a tractable system in zebrafish to identify putative cis-regulatory elements that control the spatial and temporal expression pattern of six7.
Methods :
Putative regulatory elements were identified from published data (Sotolongo-Lopez et al., 2016) as well as publicly available ChIP-seq and genome conservation data sets. Combinations of CRISPR/Cas9 gRNA/Cas9 complexes flanking these putative regulatory elements were injected into one cell stage zebrafish embryo. six7 and six3b null alleles were generated using CRISPR/Cas9 gene targeting of coding sequence. Forebrain development and photoreceptor cell number were analyzed via morphology and immunolabeling.
Results :
Targeting of the six7 coding sequence yielded germ line mutations that are predicted to result in a frameshift mutation and premature stop codon upstream of the majority of the six domain and the entirety of the homeobox domain. The mutant larvae phenocopy previously described six7 nulls (Sotolongo-Lopez et al., 2016); forebrain patterning is normal, but the retina shows loss of green cones and an 8 fold increase in rods (p<0.001,t-test). Homozygous adults were viable. Targeting of the six3b gene on six7 null background yielded a much stronger phenotype than achieved by morpholino knockdown with the complete loss of the eyes and diminished volume in the developing forebrain. Targeting of a putative enhancer 40kb upstream of the six7 transcription start site previously identified in a hypomorphic allele of six7 yielded indels and two deletions of ~400bp spanning 2 ChIP-seq peaks. However, these lesions did not alter forebrain patterning, and photoreceptor development appears normal.
Conclusions :
The ability to use gene targeting to show that that six7 and six3b are redundant during eye field specification demonstrates the potential of sensitized-lines for the rapid analysis of gene function. These will be used to probe the function of putative regulatory elements during eye development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.