Investigating the functional role of SMOC-1 in zebrafish
Sexton, David James
True anophthalmia is the most severe congenital eye malformation. With absence of the eye, optic nerve, chiasm and optic tracts. Identifying the genes that cause genetic true anophthalmia should improve our understanding of the critical processes required for development of the eye. Recessive loss-of-function mutations in SMOC1 have been identified as the cause of Ophthalmo-acromelic syndrome (OAS), a multisystem disorder which has true anophthalmia as a prominent feature with characteristic limb and facial malformations. In order to establish the function of SMOC1 in development I used the zebrafish as a model organism to support a link between SMOC-1 and BMP signalling. As a first step I characterised the genomic structure of zebrafish smoc1 gene. I was able to correct an error in the zebrafish genome (Zv8) that annotated zsmoc1 as two fragmented and rearranged orthologous loci. However, using RTPCR I could show that there is in fact a single intact zsmoc1 transcript. In addition, I was able to identify an un-annotated 5’ coding exon using 5' RACE which showed that the full open reading frame includes a signalling peptide. RT-PCR was also used to identify several novel zsmoc1 splice isoforms. To explore the link between zsmoc1 and bmp signalling I used injection of antisense morpholino oligonucleotide and capped mRNA to examine the effects of loss-of-function and overexpression respectively of smoc1 and genes functioning in the bmp signalling pathway. The resulting embryos were analysed using morphometric analysis (Kishimoto scale), a quantitative assay of dorsalisation/ventralisation and live imaging of reporter transgenic fish. I developed a quantitative RT-PCR assay for expression of dorsal (otx2 and runx3) and ventral (eve1 and gata2) marker genes. I established a reliable system for live imaging of zebrafish development between 8 hpf and 24 hpf. By combining this system with fluorescent transgenic reporters marking the eye field (rx3:gfp reporter) and BMP-signaling (BRE:gfp reporter) I was able to accurately quantitate the effect of smoc1 depletion on eye size and SMAD1/5/8 signalling in the eye. These results support the predictions from the Drosophila homologue Pent that zsmoc1 functions as an antagonist of bmp signalling. Finally, I describe my attempt to produce a zebrafish model for OAS using genome editing technology. I designed, produced and validated transcription activator like effectors nucleases (TALENs) targeted to the zsmoc1 open reading frame using the Voytas Goldengate method. I designed and optimised a novel strategy to demonstrate targeted cutting activity for in vitro validation. Following injections of the in vitro validated TALEN into zebrafish embryos I used Ion Torrent sequencing to assess the in vivo activity of the engineered TALEN pairs. Unfortunately these TALENs were not able to cut the targeted locus in vivo.
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