Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development

Abstract

Nitric oxide (NO) is an ancestral key signalling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission and immunity. Although our knowledge of NO synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore nos gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that nos expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes' roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

Keywords: gene duplication and loss; genome duplication; nos; phylogenomics; synteny; vertebrate evolution.

Figure 1.

Evolution of the Nos gene family. (a) Phylogenetic analysis of Nos proteins in chordates. The tree topology was inferred by Bayesian inference and maximum-likelihood methods, with the exact topology obtained from the former shown here (see the electronic supplementary material, figure S7 for the maximum-likelihood tree). Numbers at nodes represent posterior probability values (left) and maximum-likelihood bootstrap support for 1000 replicates (right). (b,c), Evolutionary scenarios indicating the loss of Nos2 event in Neoteleostei (b) and Nos3 in Clupeocephala (c) as grey lines. Nos3 in Elopomorpha is absent, although parsimony suggests it was present in stem elopomorphs, and it is indicated with a dashed line. TGD stands for teleost-specific genome duplication. (Online version in colour.)

Figure 2.

Conserved microsynteny of nos2 and nos3. (a) The nos2 paralogues derived from different duplication modalities: carp-specific genome duplication (Cs4R) (nos2ba and nos2bb in the goldfish and blind barbel); salmonid-specific genome duplication (Ss4R) (nos2α and nos2β in the Atlantic salmon and rainbow trout); tandem gene duplication occurred independently in five lineages (nos2.1 and nos2.2 in the northern pike, Atlantic herring, electric eel, Mexican tetra and channel catfish). An additional nos2 duplicate (nos2a) is present in cyprinids (zebrafish, goldfish, and blind barbel) (see the electronic supplementary material, figure S2). (b) A conserved synteny map of genomic regions around the nos3 gene locus highlights the loss in Clupeocephala (including zebrafish and medaka), and in Osteoglossomorpha (arowana). Consecutive genes are represented as arrows and are colour coded according to their orthology and ohnology. The direction of arrows indicates gene transcription orientation. // indicates long-distance on the chromosome (>600 kb), * indicates scaffold 72 of the freshwater elephantfish genome [15]. (Online version in colour.)

Figure 3.

Spotted gar nos3 localization during development. Expression of nos3 is localized in the pharyngeal area in 4 dpf (a,b) and 6 dpf (c,d) embryos, in pharyngeal arches in 7 dpf larvae (e–g) schematized in (h), in developing gills in 11 dpf late larvae (i–l), and in gill lamellae in 14 dpf juveniles (m–p). Coronal (n) and transversal section (o) planes are indicated with a red dashed line in (m). ey, eye; gi, gill; dv, dorsal view; lv, lateral view. Scale bar is 1 mm in (a,c,e,i,m); 100 µm in (b,d,l,n,o,p). (Online version in colour.)

Figure 4.

Expression of nos genes in developing gills of sturgeon, bichir, and shark embryos. The expression of nos2 in the gills of sterlet sturgeon Acipenser ruthenus (14 mm stage, a–c) and bichir Polypterus senegalus (stage 31, d,e); nos1 in the shark Scyliorhinus torazame (stage 27, g–i). Higher magnification views of the gill structure of (a,d,g) are shown in (b,e,h), respectively. The arrowheads indicate sectioning plane (a,d,g): transversal sections (c,f, 50 µm) and frontal section (I, 10 µm). gi, gill; yo, yolk; pf, pectoral fin. Scale bar in (a,d,g) is 0.5 mm. (Online version in colour.)

Figure 5.

Expression patterns of nosA and nosB in larvae of the arctic lamprey. At stages 24–25 the nosA is expressed in the brain, mouth, upper and lower lip, dorsal midline epidermis, and cloaca (a). At stage 28, nosA expression is restricted to the mouth (b). The nosB is exclusively expressed in the cheek process, consisting of upper and lower lips (c,d), and faint expression in the dorsal midline epidermis (c). br, brain; cl, cloaca; dme, dorsal midline epidermis; gp, gill pouches; mo, mouth; ll, lower lip; ul, upper lip; lv, lateral view; vv, ventral view. Scale bar in (a) is 0.5 mm. (Online version in colour.)

Figure 6.

Nos evolution in light of recent gene findings in vertebrates. The proposed evolution of nos genes in gnathostomes (a) supposes an ancestral loss of a predicted fourth nos gene, based on the linkage of human Nos and Hox clusters (b). Loss of nos3 occurred in stem Clupeocephala and loss of nos2 in stem Neoteleostei (a). Species-specific nos2 duplications occurred in some Otomorpha, including Cyprinidae and Characidae families. (Online version in colour.)