An intestinal cell type in zebrafish is the nexus for the SARS-CoV-2 receptor and the Renin-Angiotensin-Aldosterone System that contributes to COVID-19 comorbidities

Abstract

People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with the coronavirus SARS-CoV-2. These COVID-19 comorbidities are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure or dehydration via the peptide Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, thus counteracting its chronic effects. Ace2 is also the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we don’t yet understand how. To exploit zebrafish (Danio rerio) as a disease model to understand mechanisms regulating the RAAS and its relationship to COVID-19 comorbidities, we must first identify zebrafish orthologs and co-orthologs of human RAAS genes, and second, understand where and when these genes are expressed in specific cells in zebrafish development. To achieve these goals, we conducted genomic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have an ortholog in zebrafish and some have two or more co-orthologs. Results further identified a specific intestinal cell type in zebrafish larvae as the site of expression for key RAAS components, including Ace, Ace2, the coronavirus co-receptor Slc6a19, and the Angiotensin-related peptide cleaving enzymes Anpep and Enpep. Results also identified specific vascular cell subtypes as expressing Ang II receptors, apelin, and apelin receptor genes. These results identify specific genes and cell types to exploit zebrafish as a disease model for understanding the mechanisms leading to COVID-19 comorbidities.SUMMARY STATEMENTGenomic analyses identify zebrafish orthologs of the Renin-Angiotensin-Aldosterone System that contribute to COVID-19 comorbidities and single-cell transcriptomics show that they act in a specialized intestinal cell type.

A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2

“…Examination of ACE2 amino acid motifs in the region involved in binding SARS-CoV-2 S protein revealed zebrafish ACE2 has 50%/64% sequence similarity with the corresponding human ACE2 region compared to 71%/78% in macaques ACE2 or 57%/71% in ferret ACE2 (Table S2) (Liu et al, 2020). Combined, these data agree with previous reports and reveal that key amino acids involved in the interactions between human ACE2 and SARS-CoV-2 S protein are not conserved in zebrafish (Fu et al, 2020; Postlethwait et al, 2020).…”

“…Interestingly, we did not observe any changes in ace2 expression after 3h immersion with SARS-CoV-2 S RBD protein. Recently, enterocytes were found to be the main cell type expressing Ace2 in 5 dpf-old zebrafish larvae (Postlethwait et al, 2020); and therefore it is possible that the down-regulation in ace2 expression observed in our experiments was the result of enterocyte responses to SARS-CoV-2. However, an olfactory epithelial cell cluster was not identified in this dataset, probably because these cells constitute too small a fraction of the cells of an entire larva.…”

“…qPCR was performed using SSOadvanced supermix (Biorad, 1725270) and primers listed in Table S3 (Supplemental Methods). Gene expression changes were quantified using the Pfaffl method (Pfaffl, 2001).…”

“…Zebrafish offer many advantages over other animal models due to their high reproductive ability, rapid development, low maintenance costs, and small transparent bodies. Importantly, zebrafish olfactory, immune, and cardiovascular physiology share a significant degree of conservation with humans (Postlethwait et al, 2020; Saraiva et al). Genetically, more than 80% of disease related genes have a zebrafish orthologue (Howe et al, 2013).…”

Exploring zebrafish larvae as a COVID-19 model: probable SARS-COV-2 replication in the swim bladder

“…Despite these in silico predictions, host susceptibility requires experimental validations, especially given that many other receptors and co-receptors for SARS-CoV-2 have been identified (Zamorano Cuervo & Grandvaux, 2020). In zebrafish larvae, based on single cell transcriptomics, ace2 is strongly expressed in a subtype of enterocytes (Postlethwait et al, 2020); the gut is also the organ with strongest ace2 expression in humans.…”