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First published online 24 August 2005
doi: 10.1242/dev.01973

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The evolutionary history of placodes: a molecular genetic investigation of the larvacean urochordate Oikopleura dioica

Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

View larger version (124K): [in a new window]   Fig. 1. Olfactory- and pituitary-like organs in Oikopleura. (A) Schematized adult morphology, lateral right view. (B,C) Lateral right (B) and dorsal (C) views of adult anterior trunk showing arrangement of the anterior brain (br), paired rostral nerves (rn), ciliary funnel (cf). (C) Cell bodies (green dots) line the rostral nerve. Rows of clustered lip receptor (lr) sensory cilia protrude from the lower lip. (D) Right lateral view of a juvenile, showing the rostral nerve in cross section, the ventral organ (vo) and cilia (ci). Thickened epithelia in the mouth are made up of rows of ciliated sensory cells in the lower lip and stomodeal roof (pink dots). (E) Developing hatchling (slightly ventral, right lateral view) with thick, immature walls of the funnel connected to the pharynx roof, the rostral nerve thickening (rn), the primordium of the ventral organ (vo) and the sensory bristle cilium (sb) of one of two upper lip cells. (F) Tailshift stage hatchling (slightly dorsal, right lateral view) showing the ciliary funnel in cross-section near its base in the pharynx roof and the right rostral nerve in longitudinal section. Insets in E and F schematize the funnel (blue) and rostral nerves (green). bg, buccal gland; br, anterior brain; cf, ciliary funnel; cg, caudal ganglion; ci, sensory cilia; e, endostyle; go, gonad; gu, gut; li, lips; lr, lip receptors; mo, mouth; mu, muscle; n2 and n3, second and third paired nerves; nc, trunk nerve cord; no, notochord; ph, pharynx; rn, rostral nerve; sb, sensory bristle; sc, spinal cord; sl, statolith; sv, sensory vesicle; tf, tail fin; vo, ventral organ. Scale bars: 10 µm.

View larger version (110K): [in a new window]   Fig. 2. Ventral organ development proceeds with placode-like cell behavior. Cells (red asterisk) that will ensheath the rostral nerve originate from a focal thickening in the rostral ectoderm at the anterior margin of the CNS in hatchling larvaceans (left lateral view). They delaminate over several hours and associate with a bundle of axons from the ventral organ and brain. Ciliated epithelial cells of the ventral organ also form from an ectodermal thickening (blue asterisk) where they infold into a slit-shaped pit and protrude cilia into the environment. br, brain; ci, sensory cilia; e, endostyle; mo, mouth; ph, pharynx. Scale bar: 20 µm.

View larger version (34K): [in a new window]   Fig. 3. Oikopleura orthologs of vertebrate placode-marking genes. (A) Gene structures for five larvacean genes (red, Eya domain; black, homeodomain; blue, Six domain; green, intron unique to larvacean Six3/6 duplicates). (B) Topologies of neighbor-joining gene phylogenies of aligned protein sequences are consistent with assignment of Oikopleura genes to Eya, Pitx, Six1/2 and Six3/6 orthologies.

View larger version (114K): [in a new window]   Fig. 4. Orthologs of ANR-marking genes are expressed anterior to the CNS. All panels are left lateral views, all insets are frontal. (A) In incipient-tailbud stage, the stomodeal primordium expresses six3/6a. (B) The anterior pharynx primordium and the tail muscle precursors express pitx at incipient-tailbud stage. (C) Schematic overlay of pitx and six3/6a in which the anterior pitx domain lies just interior to the ectodermal six3/6a expression. (D) In mid-tailbud stage the six3/6a ectodermal expression (blue arrowhead) changes shape (see inset). (E) Ectodermal cells begin to express pitx by mid-tailbud stage in weak (purple arrowhead) and strong (purple double arrowheads) domains. (F) Double in situ reveals that only the ventral rank of ectodermal cells weakly expressing pitx overlaps six3/6a expression, while the dorsal pitx-expressing cells do not. (G) Schematic summarizing overlapping (checkered) and non-overlapping pitx and six3/6a domains. (H) A horizontal row of ectodermal cells expresses eya in mid-tailbud embryos. (I) Schematic shows inferred overlapping (checkered) and non-overlapping eya and pitx domains. ec, ectoderm; en, endoderm; gi, endodermal gill pouch primordium; ph, pharynx primordium. Black arrowhead indicates separation between trunk and tail. Scale bars: 20 µm.

View larger version (126K): [in a new window]   Fig. 5. The developing ventral organ expresses placode-marking gene orthologs. In early hatchlings, a ring of ectodermal cells concentric with the mouth primordium expresses eya (A) and six1/2 (B). (B) Expression of six1/2 in the ventral ectoderm reveals bilaterally paired domains (red asterisk) where the ventral organ develops (insets dorsal view, and ventral superficial view). The epidermis of most the trunk also diffusely expresses six1/2, but this expression is excluded from cells just posterior to the rostral ring. (C) A ring of ectoderm around the mouth also expresses pitx in animals just hatched (inset, superficial ventral view). Cells of the developing mouth itself and the pharynx also express pitx. (D) In mid-hatchling stages, pitx expression continues in the ring, and expands in the anterior pharynx endoderm. Other expression domains include the dorsal brain, the migrating buccal glands, the left side of the stomach, and epidermis of rostral tail fin and trunk/tail junction. Red asterisks in C,D indicate ventral organ primordium. (E,F) In late hatchlings, cells along the rostral nerve trunks express pitx (E, dorsal view; F, left lateral view). A cell (bc) bulging from the left side of rostral-most brain expresses pitx, as do the cells of the rostral pharynx. (G) In late hatchlings, the cells lining the rostral nerves also express six3/6a. The anterior brain expresses six3/6a, as does the pharyngeal roof (see Fig. 4), and the ascending esophageal wall. bg, buccal gland; bc, brain cell; br, brain; cf, ciliary funnel; e, endostyle; ep, epidermal expression; gi, gill pouches; li, lip; ph, pharynx; rn, rostral nerve; st, stomach. Scale bars: 20 µm.

View larger version (100K): [in a new window]   Fig. 6. The ciliary funnel expresses orthologs of placode-marking genes. Left lateral (A) and dorsal (C) views of early hatchlings shows that the roof of the pharynx expresses six3/6a. six3/6a is also expressed in the anterior brain, rostral towards the sensory vesicle. Left lateral (B) and dorsal (D) views of the same stage as in A,C show that six3/6b is expressed in a more limited domain in the right side of the pharynx roof in just three cells. When the ciliary funnel can be easily seen as a projection to the right of the brain in late hatchlings, six3/6a (E) and six3/6b (F) are both expressed in the funnel walls. In dorsal (G) and lateral (H) views of late hatchlings, pitx expression can be seen in both the dorsal and ventral walls of the pharynx (ph), as well as in the ventral-most part of the funnel wall where it meets the pharynx. br, brain expression; cf, ciliary funnel; ep, epidermal expression; lb, bristle-bearing lip cell; mo, mouth; ph, pharynx. Scale bars: 10 µm.

View larger version (122K): [in a new window]   Fig. 7. Paired ciliated sensory organs of the lips and posterior trunk express Eya and Six genes. (A) Bilaterally paired patches of eya expression in a tailbud stage embryo at the border between the posterior trunk and tail where the Langerhans receptors develop. (B) Optical section at dashed plane shown in A. (C) Unilateral, right side incipient tailbud expression of six3/6a precedes later bilateral expression. (D) eya expression continues in paired patches into early hatchling stages (ventral view). (E) six3/6a expression becomes bilateral in the Langerhans primordia by early hatchling stages. (F) A living, late hatchling with fully formed Langerhans receptors (la) bulging from the posterior trunk near the tail. (G) Scanning electron micrograph (SEM) of one Langerhans receptor. (H) six1/2 expression in a late hatchling narrows to the lips (li) where rows of ciliated receptors form. (I) SEM of an adult mouth showing the rows of sensory cilia (inset) emerging from a groove made up of epithelial cells that nearly cover the innervated receptor cells of the lower lip and inner stomodeum roof. a, anterior; br, brain; cg, caudal ganglion; en, endoderm; l, left; la, Langerhans receptor; li, lips; no, notochord; p, posterior; r, right; st, stomach; vo, ventral organ. Red asterisks in A-E indicate Langerhans primordium. Scale bars: 20 µm in A-F,H; 5 µm in G,I.

View larger version (32K): [in a new window]   Fig. 8. Comparison of larvacean and vertebrate placodes. An Oikopleura early hatchling and a representative vertebrate embryo (24-hour zebrafish) are schematized in dorsal view to show the correspondence in topography between the larvacean ventral organ (VO) and vertebrate olfactory (OL) placodes (green), and between the ciliary funnel (CF) and adenohypophyseal (AD) placodes (yellow). The larvacean Langerhans (LA) and vertebrate otic (OT) placodes (blue) may also be orthologous, or they could have evolved independently from a common mechanosensory placode precursor. The larvacean ciliated circumoral mechanosensory organ (CO, orange), however, might be homologous to ascidian and cephalochordate oral mechanosensory systems, but might have no counterpart in modern vertebrates. FB, forebrain; HB, hindbrain; no, notochord; sc, spinal cord.