Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates-a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

The Drosophila retinal determination gene network occurs in animals generally as a Pax-Six-Eyes absent-Dachshund network (PSEDN). For amphioxus, we describe the complete network of nine PSEDN genes, four of which-AmphiSix1/2, AmphiSix4/5, AmphSix3/6, and AmphiEya-are characterized here for the first time. For amphioxus, in vitro interactions among the genes and proteins of the network resemble those of other animals, except for the absence of Dach-Eya binding. Amphioxus PSEDN genes are expressed in highly stage- and tissue-specific patterns (sometimes conspicuously correlated with the local intensity of cell proliferation) in the gastrular organizer, notochord, somites, anterior central nervous system, peripheral nervous system, pharyngeal endoderm, and the likely homolog of the vertebrate adenohypophysis. In this last tissue, the anterior region expresses all three amphioxus Six genes and is a zone of active cell proliferation, while the posterior region expresses only AmphiPax6 and is non-proliferative. In summary, the topologies of animal PSEDNs, although considerably more variable than originally proposed, are conserved enough to be recognizable among species and among developing tissues; this conservation may reflect indispensable involvement of PSEDNs during the critically important early phases of embryology (e.g. in the control of mitosis, apoptosis, and cell/tissue motility).

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model.

Members of the subphylum Cephalochordata, which include the genus Branchiostoma (i.e. amphioxus), represent the closest living invertebrate relatives of the vertebrates. To date, developmental studies have been carried out on three amphioxus species (the European Branchiostoma lanceolatum, the East Asian B. belcheri, and Floridian-Caribbean B. floridae). In most instances, adult animals have been collected from the field during their ripe season and allowed (or stimulated) to spawn in the laboratory. In any given year, dates of laboratory pawning have been limited by two factors. First, natural populations of these three most studied species of amphioxus are ripe, at most, for only a couple of months each year and, second, even when apparently ripe, animals spawn only at unpredictable intervals of every several days. This limited supply of living material hinders the development of amphioxus as a model system because this limitation makes it more difficult to work out protocols for new laboratory techniques. Therefore we are developing laboratory methods for increasing the number of amphioxus spawning dates per year. The present study found that a Mediterranean population of B. lanceolatum living near the Franco-Spanish border spawned naturally at the end of May and again at the end of June in 2003. Re-feeding experiments in the laboratory demonstrated that the gonads emptied at the end of May refilled with gametes by the end of June. We also found that animals with large gonads (both, obtained from the field and kept and fed at the laboratory during several weeks) could be induced to spawn in the laboratory out of phase with the field population if they were temperature shocked (spawning occurred 36 hours after a sustained increase in water temperature from 19 degrees C to 25 degrees C).

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