Evolutionary and developmental analysis reveals KANK genes were co-opted for vertebrate vascular development

Abstract

Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.

Figure 1. Extended majority-rule consensus tree for the Bayesian phylogenetic analysis of KANK proteins.

Numbers at each node indicate posterior probability (pp) values based on twenty million runs. Branch lengths are proportional to means of the pp densities for their expected replacements per site. The ML phylogenetic tree (Fig. S1.) was generally in agreement with the BP phylogeny: all the invertebrates have one KANK, while there are four KANKs in vertebrates (KANK1-KANK4). There are extra Kank1 and Kank4 proteins in teleosts. The tunicate (urochordate) and lancelet (cephalochordate) form the closest outgroups of vertebrates. The tree was rooted with Tricoplax and Hydra.

Figure 2. KANK1 gene is located in an evolutionary conserved synteny in vertebrates.

Seven representative vertebrate species were analyzed. The illustration of the genes and their sizes are not proportional to the length of the bars. KANK1 is highlighted in red, and the synteny is highlighted in blue dashed-line boxes. The zebrafish kank1a is within the evolutionary conserved synteny. The zebrafish kank1b is distinct from the rest as it is only linked with smarca4. Hs, human; Mm, mouse; Gg, chicken; Dr, zebrafish; Lo, Spotted gar; Cm, elephant shark. Chr, chromosome. LG2, linkage group 2.

Figure 3. Gene expression patterns of kank1a and kank1b in zebrafish embryos.

Whole-mount in situ hybridization of zebrafish embryos at stages 15 hpf (a,b,h and i), 24 hpf (c–e,j–l) and 48 hpf (f,g,m,n). Anterior is to the left in all the whole-mount images, and dorsal is to the top in all transverse sections. (a,b). Lateral and dorsal view of the expression of kank1a at 15 hpf. (c,f). Lateral view of kank1a expression at 24 hpf and 48 hpf, respectively. (h,i). Lateral and dorsal view of the expression of kank1b at 15 hpf. (j,m). Lateral view of kank1b expression at 24 hpf and 48 hpf, respectively. The dashed lines indicate the positions of sections. The letters below the dashed lines correspond to the panels. b, brain; cf, caudal fin bud; di, diencephalon; e, eye; hb, hindbrain; hv, head blood vessels; kv, Kupffer’s vesicle; lpm, lateral plate mesoderm; mb, midbrain; n, notochord; nt, neural tube; op, optic vesicle; ov, otic vesicle; pa, pharyngeal arch; pf, pectoral fin.

Figure 4. Kank2, kank3 and kank4 gene expression during zebrafish development.

Whole mount in situ hybridization of zebrafish embryos at stages 15 hpf (a,b,j,k,s,t), 18 hpf (v), 24 hpf (c–e,l,m,u) and 48 hpf (f–i,n–q,w–z’). Anterior is to the left in all whole-mount images, and dorsal is to the top in all transverse sections. (a–i). Gene expression of kank2. (j–r). Gene expression of kank3. (s–z’). Gene expression of kank4. The dashed lines indicate the positions of section. The letters below the dashed lines correspond to the panels. The inset within panel (u) is the transverse section of the dashed line above. The white arrow points to the kank4 expression on the vascular vessels on the surface of the yolk. af, anal fin bud; cf, caudal fin bud; cv, caudal vein; da, dorsal aorta; df, dorsal fin bud; dlv, dorsal longitudinal vein; e, eye; hb, hindbrain; hv, head vessels; llp, lateral line premordia; psm, presomatic mesoderm; lpm, lateral plate mesoderm; mb, midbrain; nt, neural tube; ov, otic vesicle; pa, pharyngeal arch; pcv, posterior cardinal vein; pf, pectoral fin bud; so, somite.

Figure 5. The molecular evolution of KANK genes and the origin of vertebrate blood vessels.

The phylogenetic relationships of bilateria and vertebrata are based on the recent phylogenomic analyses and our past collagen gene phylogenetic analyses. The branch lengths are not proportional to the time of diversification. The two vertical dark blue bars on the tree indicate the WGD events (R1 and R2) at the origin of the vertebrates. The other light blue vertical bar represents the teleost specific WGD. The green bar on the bird lineage indicates the loss of KANK2. The process of KANK gene duplication and diversification through WGD is illustrated by the brown bars. The co-option of the vertebrate four KANK genes’ expression coincides with the vertebrate vascular vessels.