Tek/Tie2 is not required for cardiovascular development in zebrafish

Angiopoietin/TIE signalling plays a major role in blood and lymphatic vessel development.  In mouse, Tek/Tie2 mutants die prenatally due to a severely underdeveloped cardiovascular system.  In contrast, in zebrafish, previous studies have reported that while embryos injected with tek morpholinos (MOs) exhibit severe vascular defects, tek mutants display no obvious vascular malformations.  To further investigate the function of zebrafish Tek, we generated a panel of loss-of-function tek mutants, including RNA-less alleles, an allele lacking the MO-binding site, an in-frame deletion allele, and a premature termination codon-containing allele.  Our data show that all these mutants survive to adulthood with no obvious cardiovascular defects.  MO injections into tek mutants lacking the MO-binding site or the entire tek locus cause similar vascular defects as those observed in MO-injected +/+ siblings, indicating off-target effects of the MOs.  Surprisingly, comprehensive phylogenetic profiling and synteny analyses reveal that Tek was lost in the largest teleost clade, suggesting a lineage-specific shift in the function of TEK during vertebrate evolution.  Altogether, these data show that Tek is dispensable for zebrafish development, and probably dispensable in most teleost species.

Methods
Evolutionary analysis of TEK, TIE1 and Angiopoietins

We used HaMStR-OneSeq v1.8.0 (Ebersberger et al., 2014) (https://github.com/BIONF/HaMStR) to determine the presence-absence pattern of orthologs to human TEK (UniprotID: Q02763), TIE1 (UniprotID: P35590), Angiopoietin-1 (UniprotID: Q15389) and Angiopoietin-2 (UniprotID: O15123) across 255 species represented in the RefSeq partition of non-mammalian vertebrate genomes available at NCBI Genome (Februrary 2020) (https://ftp.ncbi.nlm.nih.gov/genomes/refseq/), complemented with the Quest For Orthologs reference proteomes (https://www.ebi.ac.uk/reference_proteomes/), and with the sea urchin (Strongylocentrotus purpuratus) proteome from NCBI Genome.  The full list of taxa is provided in Table S3.  Pairwise protein feature architecture similarities (Koestler et al., 2010) between the human seed proteins and their respective orthologs were assessed and scored with feature architecture similarity (https://github.com/BIONF/FAS).  Phylogenetic profiles together with the protein feature architecture were visualized and analysed with PhyloProfile (Tran et al., 2018).  Representative orthologs from 5 Sarcopterygians, 76 Actinopterygians, 5 Chondrichthyans, and additionally from 2 chordate outgroup species were aligned with Muscle v3.8.31 (Edgar, 2004), and alignment columns with more than 50% gaps were excluded prior to phylogeny reconstruction using a custom Perl script.  The processed alignment served as an input for a maximum likelihood tree reconstruction with RAxML v.8.2.11 (Stamatakis, 2014) choosing the option to automatically select the optimal model of sequence evolution (option PROTGAMMAAUTO).  Branch support was assessed with 100 non-parametric bootstrap replicates using the rapid bootstrapping algorithm implemented into RAxML (Stamatakis et al., 2008).  Tree display and editing were performed with iTOL (Letunic and Bork, 2019).