A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio)

Abstract

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

Coevolution between cannabinoid receptors and endocannabinoid ligands

“…This requirement made taxon sampling a challenge. PTPN22, for example, may be limited to amniotes -no PTPN22 ortholog was identified in the Tr genome, or in the zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis), and medaka (Oryzias latipes) genomes, as previously reported (McPartland et al, 2007). Species sampling limited to amniotes, however, did not provide adequate divergence for CB1, DAGLα, and MAGL phylograms, whose sequences are highly conserved (all branch length correlations were significant, data not shown).…”

“…A NAAA ortholog from Bos taurus, another laurasiatherian, was substituted for the missing Cf ortholog so the root of the Euarchontoglires branch could be determined, but the branch leading to Bos taurus was excluded in analyses. FAAH2 orthologs did not occur in several mammalian genomes (McPartland et al, 2007), so no FAAH2 phylogram was constructed. Sequence accession numbers are provided in Supplementary online Table 2 The use of cladistic approaches made it possible to investigate ancestral branches; we calculated branch lengths from five ancestral sequences, estimated from internal nodes in gene trees: primate (ancestor of Hs and Mamu), murid (ancestor of Rn and Mm), Euarchontoglires (ancestor of primate and murid), Mammalia (ancestor of Euarchontoglires and Cf), and amniote (ancestor of Mammalia and Gg).…”

“…The paralogs CNR1 and CNR2 have been attributed to a whole genome duplication (WGD) event that occurred in the ancestor of vertebrates; a second WGD occurred in the ancestor of teleost fishes (Yamaguchi et al, 1996). However, at least 50% of paralogs have been secondarily lost, which explains why duplicated genes are not a universal phenomenon, and why all teleost fishes are not tetraploids (McPartland et al, 2007).…”

A Putative ‘Pre-Nervous’ Endocannabinoid System in Early Echinoderm Development

“…In this context, it is worth noting evidence for cannabinoid receptors in vertebrates in addition to CB1 and CB2, such as the orphan receptor GPR55 [McPartland et al, 2007a;McPartland et al, 2007b], which has been identified as a novel cannabinoid receptor with ligand-binding and signaling properties partly similar to and partly unique from CB1 and CB2 [Pertwee, 2007;Ryberg et al, 2007;Lauckner et al, 2008]. Interestingly, AM-251, an analog of the CB1-selective antagonist SR141716 [Palmer et al, 2002], is a potent agonist for human GPR55 [Ryberg et al, 2005;Ryberg et al, 2007] and functions as a CB receptor agonist, mimicking the inhibitory effects of AEA and THC on fertility of sperm in the sea urchin S. intermedius [Berdyshev, 1999].…”

Nuclear receptor research in zebrafish

“…They regulate gene expression by binding to PPAR response elements (PPREs) as a heterodimer with RXR. In silico analysis showed that the zebrafish genome contains five genes encoding PPARs: duplicate PPARα and PPARβ genes and a single gene for PPARγ (Bertrand et al 2007, McPartland et al 2007, Den Broeder et al 2015. Studies on PPREs in the promoter regions of zebrafish fatty acid-binding protein (fabp) genes showed that different PPAR isoforms activate specific target genes (Laprairie et al 2016).…”