Mechanism of subfunctionalization

Hatching of euteleostean fishes

Hatching enzymes are one of the proteases that digest the egg envelope at the time of hatching. Species of lower Actinopterygii possess a single type of hatching enzyme, and then, gene duplication occurred in the ancestor of Teleostei. The species of Teleostei possess two types of hatching enzyme genes, called HCE and LCE. The egg envelope is composed of ZP proteins (ZPB and ZPC), which possess the ZP domain. The HCE cleaves the N-terminal region of ZPB and ZPC to swell the envelope, and then, the LCE cleaves two sites, the middle of the ZP domain of ZPB and the N-terminal of ZPC to solubilize the swollen envelope (Fig. 1). The former cleavage site is called "mid-ZPd site" and the latter is called "N-ZPd site".

Hatching of sticklebacks

We found that subfunctionalization of the hatching enzyme gene occurred during the evolution of sticklebacks. Although both three-spined sticklebacks (genus Gasterosteus) and nine-spined sticklebacks (genus Pungitius) belong to the same family Gasterosteidae, they differed in the egg envelope digestion manner (Fig. 2). Three-spined sticklebacks have both HCE and LCE, and hatch by dissolving the egg envelope as same as other euteleostean fishes do. The LCE gene can be found as a single copy gene in the genome of most euteleostean fishes. On the other hand, nine-spined sticklebacks underwent gene duplication of LCE, and they possess two types (α-type and β-type) of the LCE genes in their genome. After the gene duplication, subfunctionalization occurred, and the two types of LCE cleave different sites of the egg envelope protein. The α-type LCE cleaves only the "N-ZPd" site, and β-type LCE cleaves only the "mid-ZPd" site. Therefore, in order to dissolve the egg envelope, both α-type and β-type LCEs are necessary for nine-spined sticklebacks.

Fig. 2：Comparison of egg envelope digestion manner of three-spined stickleback and nine-spined stickleback.

Evolution of stickleback hatching enzyme genes

The above results indicate that the LCE gene duplication occurred during the evolution of the family Gasterosteidae, and then, subfunctionalization occurred. Therefore, in order to clarify the timing of gene duplication and functional differentiation, we further analyzed hatching enzyme genes of other species in the family Gasterosteidae (genera Culaea, Apeltes, and Spinachia). Furthermore, we synthesized ancestral hatching enzymes. As the results, we conclude that gene duplication occurred in the common ancestor of genera Pungitius and Culaea, and then, subfunctionalization occurred in the genus Pungitius.

Fig. 3： Evolution of hatching enzyme genes in the family Gasterosteidae.

Original Paper

Mari Kawaguchi, Hiroshi Takahashi, Yusuke Takehana, Kiyoshi Naruse, Mutsumi Nishida, and Shigeki Yasumasu, 2013. Sub-functionalization of duplicated Genes in the evolution of nine-spined stickleback hatching enzyme. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 320: 140-150.
DOI: 10.1002/jez.b.22490