The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Abstract

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.

Figure 1.

Juvenile amphioxus (Branchiostoma floridae). This specimen has not yet developed gonads. Anterior to left. (C) Buccal cirri surrounding mouth; (G) pharyngeal gill slits; (A) atrial cavity; (D) digestive diverticulum. Scale bar, 0.5 mm.

Figure 2.

The amphioxus genome contains 15 Hox genes. (A) Genomic organization of the 470-kb amphioxus Hox gene cluster and linked Evx gene pair; beneath the horizontal line are shown the two Hox15 cosmid clones analyzed. Black vertical lines represent small sequence gaps; asterisks indicate genes with an intron within the homeobox; arrows denote transcriptional orientation. FISH to amphioxus interphase nucleus (B) and metaphase chromosomes (C–E). (Red signal) Cosmid MPMGc117I2469 (AmphiHox15); (green signal) cosmid MPMGc117L0856 (AmphiHox12 and AmphiHox13). Methods as described in Castro and Holland (2002).

Figure 3.

Homeobox gene loss in chordates. Homeobox gene loss has been extensive during the evolution of the Olfactores (vertebrates plus tunicates), yet in amphioxus, no homeobox gene loss has occurred since the last common ancestor of the chordates (LCAC). (Blue) ANTP class; (red) PRD class; (green) other classes. Gene losses along the tunicate branch occurred before the last common ancestor of Ciona and Oikopleura. Photo of Goodrich reproduced by permission of Department of Zoology, University of Oxford.

Figure 4.

Predicted amphioxus endocrine pathways in which a red box identifies homologs found in the amphioxus genome. (A) Reproductive axis in which missing components include classical pituitary hormones (LH, luteinizing hormone; FSH, follicle stimulating hormone; Prl, prolactin) and their receptors. GPR54: glycoprotein receptor 54 (kisspeptin receptor); GnRH: gonadotropin-releasing hormone; E: estrogen; ER: estrogen receptor; SR: steroid receptor. (B) Thyroid metabolism showing that the thyroglobulin gene could not be identified in the amphioxus genome (hence, crossed out). CTHBP: cytosolic thyroid hormone binding protein; SIS: sodium iodide symporter; Tg: thyroglobulin; T2: diiodothyronine; T3: triiodothyronine; T4: thyroxine; TR: thyroid hormone receptor; RXR: retinoid X receptor; TRE: thyroid hormone response element. (C) Retinoid metabolism in which CRABP and AKR genes could not be identified in the amphioxus genome (hence, crossed out). ADH: alcohol dehydrogenase; AKR: aldo-keto reductase; ARAT: acylCoA:retinol acyltransferase; BCOX: beta,beta-carotene-15,15'-oxygenase; CRABP: cellular retinoic acid binding protein; CRBP: cellular retinol binding protein; CYP26: cytochrome P450–26; LRAT: lecithin:retinol acyltransferase; RALDH: retinaldehyde dehydrogenase; RDH: retinol dehydrogenase; REH: retinyl ester hydrolase; RPE65: retinal pigment epithelium-specific protein 65 kDa; RAR: retinoic acid receptor; RXR: retinoid X receptor; RARE: retinoic acid response element.

Figure 5.

Expansion of innate immunity genes in amphioxus (right) compared with the human system (left). Novel domain architectures and (predicted) varied receptor conformations found in amphioxus proteins are expected to increase the specificity of innate immunity in amphioxus. We propose that some TLR-like proteins are localized in the endosome, as also seen in mammals. Domains: TIR (Toll/interleukin-1 receptor) domain, NACHT (NAIP, CIITA, HET-E and TP1), LRR (leucine-rich repeat), CARD (caspase recruitment domain), PYRIN (N-terminal domain of protein pyrin), DD (death domain), DED (death effector domain), SAM (sterile alpha motif) domain, BIR (Baculovirus Inhibitor of apoptosis Repeat).

Figure 6.

Amphioxus-human conserved noncoding sequences function as enhancers. (A–G) LacZ reporter gene expression driven by the conserved noncoding element (CNE) near amphioxus ZNF503/703 in mouse (A–C) and amphioxus (D–G). (H–L) Expression driven by the orthologous CNE near human ZNF503 in mouse (H–J) and amphioxus (K,L). (M) Expression of the endogenous amphioxus ZNF503/703 gene in the anterior central nervous system (n), somites (s), pharynx (p), and weakly in the notochord. (N–T) Expression of the orthologous CNE near ZNF703 in mouse (N–P) and amphioxus embryos (Q–T). B, I, and O show dorsal views of the mouse hindbrain; all other views are lateral, with anterior to the left. Mouse embryos shown at 9.5 d post conception. The mouse ZNF503 construct directs expression to the forebrain, midbrain, hindbrain, eye, branchial arches. Blue arrowheads show expression in the eye directed by the two human elements, but not the amphioxus element. The red arrowhead shows expression in a lateral domain that is directed by the amphioxus element, but not the human elements. Purple arrowheads indicate sites of expression observed for all three CNEs. The amphioxus ZNF503/703 CNE construct expresses at a high level in the amphioxus notochord (n) and somites(s) and at a lower level in the ectoderm (e) and central nervous system (nc). The embryo in D has an abnormal head, presumably due to injection trauma. Ectopic expression in necrotic cells in the gut lumen as in G, L, and S is common in amphioxus embryos injected with reporter constructs. Twenty-four-hour embryos (D,E,Q–T), 30 h larvae (K–M), 36 h larvae (F,G).