key: cord-325610-n3zb36am authors: Postlethwait, John H.; Farnsworth, Dylan R.; Miller, Adam C. title: 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 date: 2020-09-02 journal: bioRxiv DOI: 10.1101/2020.09.01.278366 sha: doc_id: 325610 cord_uid: n3zb36am 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 STATEMENT Genomic 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. orthologs and previously unrecognized co-orthologs of important components; and second, that a specific 113 under-characterized cell type expresses many RAAS components and is hence a focal cell type for the RAAS 114 that merits further exploration. An apparently similar cell type in humans allows SARS-CoV-2 infection, the 115 production of infectious virus, and likely some COVID-19 pathologies (Stanifer et al., 2020) . These studies 116 support zebrafish as a model for investigating the relationship of the RAAS to COVID-19 pathologies. To identify RAAS components in the zebrafish genome and to detect potential COVID-19-related cellular 119 systems, we combined our ongoing efforts in defining the relationships of zebrafish and human genomes 120 (Braasch et Ang I sequences vary more among ray-finned than lobe-finned vertebrates (Fig. 2) . Because coelacanth 179 and several basally diverging ray-finned vertebrates (sturgeon, elephant fish) have the sequence 180 NRVYVHPFNL, this is likely the ancestral Ang I sequence for all bony vertebrates. Several ray-finned 181 vertebrates have isoleucine at position 5, representing independent mutations that happen to match placental 182 mammals. Position 9 is highly variable in ray fins as in lobe fins. Zebrafish Ang I is NRVYVHPFNL, differing 183 from the human form at positions 1, 5, and 9. The Angiotensin system was likely already active in stem vertebrates because chondrichthys (cartilaginous 185 fish) and even agnathans (jawless vertebrates) possess Angiotensinogen genes (Takei et al., 1993) . At 186 position 1, some cartilaginous fish have arginine like most ray-finned vertebrates, but others have asparagine 187 or tryptophan, and position 9 is variable. The ancestral Ang I sequence in jawed vertebrates was likely the 188 same as in ancestral bony fishes (NRVYVHPFNL). Angiotensinogen genes in jawless vertebrates appear to encode an Angiotensin that shares the amino-190 terminal four residues with mammals but varies in the carboxy-terminal six residues (Wong and Takei, 2011). At position 1, lampreys have either aspartic acid or glutamic acid, but the conserved isoleucine or leucine at 192 position 5 is replaced with methionine followed by glutamine replacing the otherwise invariant histidine. Lamprey Ang II alters cardiovascular dynamics in live lampreys but teleost Ang II (NRVYVHPF) did not 194 (Wong and Takei, 2011) Angiotensin II (Ang II) forms when Ace cleaves two C-terminal amino acid residues from Ang I (Fig. S1 .3). Ang II contributes to hypertension, an important COVID-19 comorbidity, and likely promotes the inflammation 207 that leads to poor disease outcomes. Human and zebrafish Ang II differ only at the first and fifth residues 208 (DRVTIHPF vs. NRVTVHPF, Fig. 2) . Importantly, the fish and human peptides act about equally in Zebrafish has a single ortholog of human AGTR2 with conserved syntenies (Fig. 3G) Conserved syntenies suggest a mechanism for this situation. In human, MAS1 lies in the gene sequence: . Importantly, recent analyses showed that SLC6A20 is at 320 the peak of a genome wide association study for poor outcomes from COVID-19 (Ellinghaus et al., 2020). These findings raise the novel hypothesis that SLC6A20 variants contribute to variation in COVID-19 322 outcomes due to differences in expression or protein function related to interactions with ACE2, the SARS- CoV-2 receptor. 324 Adam17 (Fig. S1 .13) is a metalloendopeptidase that cleaves the membrane isoform of Ace2 to make the 325 soluble protein sAce2 (Lambert et al., 2005) . Zebrafish has two copies of adam17 in double conserved 326 synteny with human ADAM17 (Fig. 6A) , showing that they are co-orthologs of human ADAM17 from the TGD. The zebrafish Atlas showed expression of adam17a (ENSDARG00000043213) stronger in embryonic 328 intestinal epithelium than in larval ace2-expressing c152 cells (Fig. 6B ). In addition, several cells in the 329 vascular endothelium expressed adam17a (Fig. 6C) . The TGD duplicate adam17b (ENSDARG00000093093) 330 was expressed in the Atlas in a few widely dispersed individual cells. Because expression of adam17 331 paralogs was not detected in c152, zebrafish may not have soluble Ace2; alternatively, a different enzyme in 332 zebrafish might cleave Ace2 to make the soluble form or expression levels may be too low for detection. Analysis of the zebrafish aplnra and aplnrb genes in the same tree (ENSGT01000000214406) showed that 385 most teleost clades have orthologs of each gene, that the bonytongues, which branch deep in the teleosts, 386 root each clade; furthermore, spotted gar and reedfish serve as pre-TGD outgroups, as expected from 387 historical species relationships (Fig. 7B) . The tree showed that the sister group of the ray-finned 388 aplnra+alpnrb clade is a lobe-finned vertebrate clade rooted on coelacanth and amphibia as expected and 389 includes 'reptiles' and birds (Fig. 7B ). This clade, which we tentatively here call the Aplnrl clade, contains only 390 monotremes and marsupials among mammals, indicating that this gene was lost in eutherian mammals 391 (Zhang et al., 2018) . Conserved syntenies showed that chicken (Gallus callus) chromosome Gga15, which 392 contains Aplnrl (ENSGALG00000047717), has orthologs on both Dre8 and Dre10, the sites of aplnra and 393 aplnrb, respectively, as well as Dre5 (Fig. 7D) . These conserved syntenies independently verify that: 1) Aplnrl 394 was present in the last common ancestor of human and zebrafish; 2) Aplnrl was lost from eutherians after 395 they diverged from marsupials; and 3) the TGD produced aplnra and aplnrb paraogs. These analyses support the hypothesis that the last common ancestor of zebrafish and human had at least 397 two Aplnr-related genes; one became APLNR in human and 'aplnr2' in teleosts and the other was lost in 398 eutherians but retained in other tetrapods, and subsequently duplicated in the TGD, becoming 'aplnra' and 399 'aplnrb' in zebrafish. Renaming aplnr2 (ENSDARG00000004447) to aplnr, aplna (ENSDARG00000002172) 400 to aplnrla, and aplnrb (ENSDARG00000036670) to aplnrlb would better connect zebrafish to human biology. Atlas cells expressing aplnr (ENSDARG00000004447) occupied midline fates, including prominently the 402 floorplate (c176), hypochord (c218), and scleroderm (c33), as well as an embryonic intestinal epithelium cell 403 type (c101). Expression of alpnr was detected in the spleen and heart of zebrafish adults by qPCR (Zhang et conserves genes encoding Ang II receptors Agtr1 and Agtr2. Zebrafish also has orthologs encoding Slc6a19, 435 which binds Ace2 and Adam17, the enzyme that creates the soluble form of Ace2. Furthermore, zebrafish 436 has an ortholog encoding Tmprss2, which activates coronavirus spike protein for binding to ACE2 and 437 bringing the virus into human cells. Zebrafish also has the ligand and receptors for the Apelin signaling 438 system. Zebrafish has a single ortholog of most RAAS genes but has duplicates of some. Many zebrafish duplicates 440 of human RAAS genes derive from the teleost genome duplication event, including 1) agtr1a and agtr1b, 2) 441 anpepa and anpepb, 3) slc6a19a and slc6a19b, and 4) aplnrla and aplnrlb. Other duplicated RAAS genes 442 appeared by tandem duplication, including 1) anpep and anpepl, 2) anpepla.1 and anpepla.2; and 3) 443 slc6a19a.1 and slc6a19a.2. The significance of these discoveries is that the actions of both zebrafish co-444 orthologs must be considered when translating zebrafish science to human biology. The RAAS/Apelin system also provides an example of 'ohnologs gone missing', in which one ohnolog Zebrafish express COVID-19-related RAAS genes in tissues similar to human. Results showed that, like humans and other mammals, zebrafish liver cells express Angiotensinogen. In the 473 Atlas, 5dpf zebrafish larvae had three types of hepatocytes, two of which expressed agt, adding cellular 474 precision to expression studies in adult zebrafish (Cheng et al., 2006) . Induction of Agt expression in 475 mammals relies on cortisol and inflammation (Brasier and Li, 1996 Ace, which cleaves Ang I to Ang II, was surprisingly shown by our scRNA-seq analysis to be co-expressed Significantly, SLC6A20 is at the peak of the strongest of two genome wide association study loci for 509 undesirable COVID-19 outcomes (Ellinghaus et al., 2020) . We suggest the hypothesis that genetic variants 510 near SLC6A20 affect the severity of COVID-19 symptoms due to variations in interactions with ACE2. Angiotensin peptides are related to COVID-19 comorbidities because they bind to Agtr1 and Agtr2 receptors 512 on vascular cells to help regulate vasoconstriction, and on the adrenal to stimulate secretion of aldosterone, 513 leading to salt and water retention related to the comorbidity of obesity-related kidney damage (Fyhrquist and 514 Saijonmaa, 2008). As in humans, zebrafish scRNA-seq analysis showed that agtr1b is expressed in 515 endothelial cells and confirmed that agtr2 is also expressed in endothelial cells (Wong et al., 2009 ). The conserved expression of zebrafish RAAS-related genes supports the contention that RAAS regulation The zebrafish RAAS is pharmacologically similar to that of mammals. The Ace inhibitor lisinopril blocks the 540 effects of Ang I on sodium uptake in zebrafish, as predicted if Ace were required to convert Ang I to Ang II 541 (Kumai et al., 2014) . Zebrafish cultured from 24hpf to 96dpf in water containing the Ace inhibitor captopril do not differ in survival from controls and neither do fish in water with 5% of the normal salt concentration (Rider (E), anpepa (F), and dpp4 (G). 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