Development of novel molecular tools for the characterisation of zebrafish renin
Item statusRestricted Access
Embargo end date30/11/2021
The renin angiotensin system (RAS) is highly conserved across vertebrates. However, until recently it has not been extensively explored in zebrafish, an important model organism suitable for developmental studies, high-resolution in vivo imaging, and genetic and chemical screens. In common with mammals, the adult zebrafish kidney contains specialised renin-expressing mural cells that contain granules indicative of the processing and regulated secretion of active renin. Due to the translucent nature of zebrafish larvae, the functional pronephros is easily accessible for real-time imaging and in vivo studies of the RAS. The primary aim of this project was to develop novel tools to help elucidate the function of renin and the role of the RAS in zebrafish. I designed a zebrafish renin-luciferase reporter transgene using a previously published promoter sequence to investigate the transcriptional regulation of renin in real-time. The expression vector was injected into fertilised WIK zebrafish eggs and successful integration of the plasmid into the zebrafish genome was demonstrated. An in vivo assay was designed to select for highest luciferase expressers, but bioluminescent imaging revealed that the high signal stemmed from the yolk sac of zebrafish. Yolk sac expression appeared to originate from episomal transcription of the injected plasmid and, in the F0 fish, masked potential bona fide expression from the renin-expressing cells of the pronephros. A new transgene was generated using two reporters, driven by the same promoter sequence and the two reporters were separated by a ’2A’ sequence which codes for a self-cleaving peptide. This zebrafish line was generated to permit selection of tg(ren:LUC-2A-mCherry) fish for the strongest mCherry signal originating from renin expressing cells. However, no expression was observed at the anterior mesenteric artery (AMA). Further investigation revealed that when generating novel transgenic zebrafish, the reporter proteins are transiently expressed in the yolk sac of F0 founder fish and the ectopic expression can influence studies relying on quantitative reporters. Current studies largely rely on the transcriptional activity of RAS components and are restricted to timepoint-specific observations. The lack of antibodies prevents the measurement of RAS proteins in zebrafish. The amino acid sequence for zebrafish angiotensinogen is known and by comparison to mammalian sequences, I identified the amino acid sequences for angiotensin (Ang) I and II. The production of zebrafish Ang I and II peptides by solid phase peptide synthesis provided standards for the development of assays for zebrafish angiotensins. I used these assays to demonstrate that Captopril, which in mammals prevents the conversion of Ang I to Ang II by inhibiting the Angiotensin Converting Enzyme (ACE), is active in zebrafish, and that its administration leads to a dramatic decrease in Ang II. In parallel I designed and synthesised a fluorescent resonant energy transfer (FRET) probe to enable zebrafish renin activity to be measured. Using various zebrafish models I was able to demonstrate dynamic changes in renin activity and measure these using the first renin zebrafish FRET probe. I characterised a renin knockout zebrafish, generated by CrispRCas9 gene editing. Knockouts were identified by DNA sequencing which identified an 8bp deletion in exon 2 of the renin gene, causing a frameshift mutation and early termination of renin translation. Renin knockout fish proved to be viable and were screened for a phenotype using the high throughput automated screening system (VAST) which permits the precise imaging of a large number of live fish. Imaging revealed delayed development of the swim bladder and reduced fish length compared to age-matched controls. This was indicative of an overall developmental delay. The knockout fish were intercrossed with existing renal reporter lines to investigate phenotypes at a cellular level. Ren−/− tg(wt1b:EGFP) fish showed a delay in glomerular fusion of the pronephric kidney and a cross of the ren−/− with tg(ren:RFP-LifeAct) fish indicated a dramatic increase in renin-expressing cell along the renal mesonephric vasculature. Moreover, morphological examination revealed vacuolation of proximal tubules in the mesonephric kidney. The intercrossing of the ren−/− zebrafish to the tg(ren:RFP-LifeAct) and tg(acta2:EGFP) fluorescent reporters lead to the optimisation of a FAC sorting protocol and resulted in the first zebrafish ren- and acta2-expressing cells to be cultured and imaged using high-resolution microscopy imaging. In summary, my work has led to the first successful measurement of zebrafish AngI and AngII in mesonephric zebrafish tissue to demonstrate the effectiveness of Captopril in zebrafish as well as the first development of a zebrafish renin FRET probe to allow the measurement and quantification of renin activity in zebrafish, enabling more accurate studies of the zebrafish RAS. Lastly, successful phenotypic characterisation of the ren−/− fish will further our understanding of the role of renin and the RAS in zebrafish.