Abstract
Purpose :
Sox11 and Sox4 are members of the SoxC family of HMG-box containing transcriptional regulators. Previously, we demonstrated by morpholino-mediated gene knockdown that Sox4 and Sox11 regulate ocular morphogenesis and photoreceptor development through inhibition of Hh signaling. To investigate the long-term consequences of loss of SoxC activity, we are generating targeted mutations in each SoxC co-orthologue using CRISPR/Cas genome editing. Here we describe the phenotypes observed in maternal zygotic (MZ) mutants for sox4a.
Methods :
Zebrafish embryos were obtained from natural crosses and staged as previously described. Single strand guide RNA (sgRNA) targeting the sox4a locus and Cas9 mRNA were microinjected into zebrafish embryos at the single-cell stage. High resolution melt curve analysis (HRMA) was performed to screen for mutations in sox4a. For mRNA rescue experiments, WT sox4a mRNA and TdTomato mRNA were made by in-vitro transcription and injected into mutant embryos at the 1-cell stage. Gene expression in control and mutant zebrafish embryos was analyzed by quantitative RT-PCR and by whole mount in situ hybridization (WISH). Cell death was analyzed by acridine orange (AO) staining. Retinal cell type differentiation was analyzed by immunohistochemistry and using fluorescent reporter transgenic lines.
Results :
We recovered a sox4a mutant allele that results in a frame-shift and premature stop codon upstream of the Sox4a transactivation domain. Approximately 50% of sox4a maternal zygotic (MZ) mutant eggs were either unfertilized or did not survive past 24 hours post fertilization (hpf). Of the surviving sox4a MZ mutants, defects such as heart edema, tail curvature, increased cell death, and microphthalmia were observed in 30-50% of embryos, and a small number of embryos in this group also displayed coloboma. The remaining sox4a MZ mutant embryos displayed reduced eye size at 5 days post fertilization (dpf) and reduced numbers of rod photoreceptors at 5 dpf and 2 weeks post fertilization (wpf). Sox4a MZ mutant embryos demonstrated an increase in expression of the Hh signaling pathway genes ihhb and ptc2, and a reduction in expression of bmp4.
Conclusions :
These data support our previous results, and indicate that Sox4 is required in early development for proper ocular morphogenesis, rod photoreceptor differentiation, and to maintain the appropriate levels of Hh and Bmp signaling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.