EVOLUTION AND EXPRESSION OF THE METAL RESPONSE IN THE ASIAN LANCELET

BACKGROUND: Lancelets (Branchiostoma spp.) occupy a basal position in chordate evolution and their shallow, coastal habitats are highly susceptible to the effects of ocean warming and pollution. In response to trace metal exposure, metal-activated transcription factor-1 (MTF-1) induces the expression of metallothionein (Mth) in both vertebrates and invertebrates. We investigated (a) the evolution of this protein in three Branchiostoma species and (b) its role in the metal response of the Asian lancelet, B. belcheri. METHODS: (a) i.The domain architectures of MTF-1 orthologs were compared among an invertebrate (sea urchin), vertebrate (zebrafish) and three Branchiostoma species (belcheri, lanceolatum, floridae). ii. MTF-1 and MTF-2 orthologs from 10 species were aligned and Bayesian inference was used to construct a phylogenetic tree with high bootstrap confidence. iii. Sequence conservation and phylogeny were established among Mth proteins in Asian and Florida lancelets and four reference species. iv. The fine structure of the Mth promoter was compared among Asian and Florida lancelets, oysters and zebrafish. (b) Lancelets were collected in Pak Lap Wan Bay near Hong Kong and maintained for 72 h in seawater alone or in seawater supplemented with 100 ppb cadmium (Cd), nickel (Ni) or chromium (Cr). Total RNA purified from each animal was analyzed by QPCR for the abundance of Mth and MTF-1 transcripts relative to the expression of actin mRNA. RESULTS: (a) The genomes of B. belcheri, B. lanceolatum and B. floridae all encode an MTF-1 that has six C₂H₂-type zinc fingers, the same domain architecture that is found in vertebrates. Bayesian phylogeny confirms the basal position of cephalochordates in the chordate lineage and indicates that MTF-1 in the Asian lancelet diverged earlier from ancestral lancelet gene than did MTF-1 in the European and Florida species. (b) Relative to actin, mRNA levels for Mth were increased by exposure to Cd (35-fold) and Ni (24-fold) (p < 0.05). Expression of MTF-1 itself was also increased by Cd (75-fold) and Ni (22-fold) (p < 0.005). The order of activity of these metals, Cd > Ni > Cr = Control, correlates with the predicted ability of their hydrated ions to compete for a zinc binding site (p < 0.01). DISCUSSION: The Asian lancelet genome encodes a highly conserved MTF-1, and low concentrations of Cd and Ni lead to significantly elevated levels of Mth mRNA. We also observed a surprising increase in the expression of the transcription factor MTF-1 itself. A similar increase in the expression of MTF-1 has previously been observed in zebrafish cells, though at much higher levels of exposure to metals (Cheuk et al 2008). The MTF-1 promoters in both the Asian lancelet and zebrafish appear to lack a conserved metal response element (MRE), so the upregulation by metals of MTF-1 may reflect an increase in mRNA stability, rather than an increase in de novo transcription.