Endogenous Green Fluorescent Protein (GFP) in Amphioxus DIMITRI D. DEHEYN 1, *, KAORU KUBOKAWA 2 , JAMES K. MCCARTHY 1 , AKIO MURAKAMI 3 , MAGALI PORRACHIA 1 , GREG W. ROUSE 1 , AND NICHOLAS D. HOLLAND 1 1 Marine Biology Research Division, Scripps Institution of Oceanography (UCSD), La Jolla, California 92093-0202; 2 Research Institute, University of Tokyo, Nakano, Tokyo, 164-8639, Japan; 3 Research Center for Inland Seas, Kobe University, Awaji, Hyogo, 656-2401, Japan Green fluorescent proteins (GFPs) are well known for their intensive use in cellular and molecular biology in applications that take advantage of the GFPs self-folding and built-in fluorophore characteristics as biomarker. Oc- currence and function of GFPs in nature is less known. For a long time GFPs were described only from some cnidar- ians, and it is only recently that they were also found in copepod crustaceans. Here we describe the occurrence of a GFP from three species of amphioxus, namely Branchio- stoma floridae, B. lanceolatum, and B. belcheri (Chordata: Cephalochordata). This is the first time an endogenous GFP has been found in any representative of the deuterostome branch of the Animal Kingdom. We have isolated and char- acterized a gene (AmphiGFP) from B. floridae that encodes a GFP protein related to those of cnidarians and copepods in both its amino acid sequence and its predicted higher order structure (an 11-stranded -barrel enclosing a flu- orophore). Bayesian and maximum parsimony phylogenetic analyses demonstrate that the AmphiGFP protein is mark- edly more closely related to copepod than to cnidarian GFPs. In adults of all three amphioxus species, the green fluorescence is strikingly concentrated anteriorly. The an- terior end is the only body part exposed to light in these shallow-water dwellers, suggesting possible photoreceptive or photoprotective functions for the endogenous GFP. Green fluorescent proteins (GFPs) are familiar to most biologists as invaluable tools for cellular and molecular biology (1). However, in spite of the considerable effort spent on developing GFPs for laboratory reagents, much remains to be learned about the taxonomic distribution and biological function of these proteins in nature. To date, GFPs have been found in only two major groups in the metazoan tree: specifically, in a number of cnidarians, rel- atively near the base of the tree, and in a few copepod crustaceans, relatively derived within the protostome branch (2, 3). The cnidarian GFPs are often associated with biolu- minescence, but those found so far in copepods are not. We now report that the limited taxonomic distribution of ani- mals with endogenous GFPs may be partially due to inad- equate sampling efforts, because we have found such mol- ecules in the cephalochordate amphioxus. About 10 years ago, we began to suspect that endogenous GFPs are present in amphioxus, because the eggs and embryos emit a uniform green fluorescence when illuminated with UV light (this phenomenon is illustrated in reference 4). The present note reports on the isolation and molecular characterization of indubitable GFPs from three amphioxus species (none of which are bioluminescent). This is the first demonstration of the presence of these distinctive molecules in any deutero- stome. In addition, the tissue distribution of amphioxus fluorescence, interestingly localized at the anterior end of the adult body, gives insights into possible functions of the endogenous GFPs (discussed below). The present note concerns three amphioxus species: Branchiostoma floridae Hubbs, 1922 (the Florida am- phioxus, collected in Tampa, Florida), Branchiostoma lan- ceolatum (Pallas, 1774) (the European amphioxus, collected in Banyuls-sur-Mer, France), and Branchiostoma belcheri Gray, 1847 (the Asian amphioxus, collected in Enshu-nada Sea, Japan). For adults of these three species, the fluores- cence spectra, stimulated by incident UV (380 nm), had Received 23 January 2007; accepted 8 May 2007. * To whom correspondence should be addressed. E-mail: ddeheyn@ ucsd.edu Reference: Biol. Bull. 213: 95–100. (October 2007) © 2007 Marine Biological Laboratory 95