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First published online 16 June 2004
doi: 10.1242/dev.01201

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Amphioxus and ascidian Dmbx homeobox genes give clues to the vertebrate origins of midbrain development

¹ Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
² Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

View larger version (30K): [in a new window]   Fig. 1. Comparisons of homeodomains from Dmbx family genes. Sequences of vertebrate and hydra proteins from GenBank: Homo sapiens (Accession Number AB037699), Mus musculus (AF499446), Gallus gallus (AF461038), Danio rerio (AF398526), Takifugu rubripes (AY071923) and Hydra vulgaris (AF126249). Sequences of AmphiDmbx (Branchiostoma floridae) and CiDmbx (Ciona intestinalis) cDNA sequences have been deposited in GenBank (Accession Numbers AY588475 and AY588476, respectively).

View larger version (17K): [in a new window]   Fig. 2. (A) Unrooted phylogenetic tree of paired-like homeobox genes from the PRD class, constructed from the homeodomain only. Bf, Branchiostoma floridae (amphioxus); Ce, Caenorhabditis elegans; Ci, Ciona intestinalis (ascidian); Dm, Drosophila melanogaster; Hv, Hydra vulgaris; Mm, Mus musculus; Sm, Schistosoma mansoni. Q50 and K50 denote the amino acid of homeodomain position 50. (B) Molecular phylogenetic tree of Dmbx family proteins. This tree is constructed from the entire amino acid sequences (after removal of unalignable regions) and is rooted by H. vulgaris manacle protein. Both trees are constructed by the neighbour-joining method. Branch lengths are proportional to evolutionary distance corrected for multiple substitutions. Scale bar: 0.1 underlying amino acid substitutions per site. Figures on branches indicate robustness of each node, estimated from 1000 bootstrap replicates.

View larger version (22K): [in a new window]   Fig. 3. Exon-intron genomic structure of Dmbx genes. Coding sequence exons are shown as rectangles; homeodomains are indicated by grey shadows, OAR domains by dotted boxes and the conserved domains similar to the GEH/eh-1 domain by black boxes. The numbers at the corner of rectangles indicate the nucleotide position at the exon-intron boundary. AmphiDmbx genomic sequences are available in GenBank (Accession Numbers AY588477 and AY588478). Genomic sequences of other species are from GenBank (human, NT 004852; mouse, NT 039264; Takifugu CAAB01001099) and the JGI website (ascidian). Each genomic structure was deduced from comparison of genomic sequence with cDNA sequence in accordance with the GU-AG splice site rule.

View larger version (66K): [in a new window]   Fig. 4. Spatial and temporal expression of AmphiDmbx deduced by whole-mount in situ hybridisation. Anterior of whole mounts towards the right; cross-sections (counterstained pink) viewed from the posterior end of animal. (A) Lateral view of the embryo at the late gastrula stage (9 hours). AmphiDmbx expression is detected at the anterior mesendoderm. (B,C) Lateral views of the embryo at the neurula stage (13 hours, 20 hours). AmphiDmbx expression is seen in dorsal anterior mesendoderm. (D) Lateral view of the embryo at the mid neurula stage (24 hours). The anterior mesendoderm expression extends into Hatschek's diverticula. (E) Lateral view of the embryo at the late neurula stage (36 hours). Expression is detected in anterior endoderm, including Hatschek's diverticula, and also in the most rostral tip of the notochord. (F) Cross-section through f in E, showing expression in the anterior endoderm. (G) Cross-section through g in E, showing expression in anterior endoderm and notochord, but not neural tube. (H) Lateral view of 60-hour swimming larva. AmphiDmbx is expressed in anterior endoderm including part of the club shaped gland, endostyle and pharyngeal endoderm. Expression is also observed in a few dorsal cells of the preoral ciliated pit. No expression is detected in the neural tube at any stage of development examined. a, anterior; d, dorsal; v, ventral; r, right; l, left; csg, club shaped gland; Hd, Hatschek's diverticula; n, notochord; nt, neural tube; pcp, preoral ciliated pit; pe, pharyngeal endoderm; s1, the first somite.

View larger version (55K): [in a new window]   Fig. 5. Spatial and temporal expression of CiDmbx deduced by whole-mount in situ hybridisation. Anterior towards the right in all specimens except A. (A) Lateral view of 32-cell embryo; animal pole towards the right. (B) Dorsal view of a neurula stage embryo. No CiDmbx expression is observed at these stages. (C) Lateral view of mid tail-bud stage embryo. CiDmbx expression is detected exclusively in the neural tube. (D) Dorsal view of the same developmental stage, showing that expression is in one bilateral pair of cells. (E) Comparison of expression between CiDmbx (blue stain, arrowhead) and Ci-Pax2/5/8-A (red stain, arrow) at mid tail-bud stage (lateral view). CiDmbx is expressed in cells immediately posterior to cells expressing Ci-Pax2/5/8-A, with no gap and no overlap. (F) Comparison of Ci-Fgf8/17/18 (blue stain) and CiDmbx (red stain) at mid tail-bud stage (lateral view) showing co-localised expression (arrowhead). Additional expression of Ci-Fgf8/17/18 is detected in the trunk-lateral cells and at the tip of the tail after prolonged staining. (G) Comparison of CiHox3 (blue stain) and CiDmbx (red stain) at later mid tail-bud stage embryo (lateral view), showing co-localised expression (arrowhead). Dorsal (H) and lateral (I) views of swimming tadpole larva. CiDmbx expression is observed in the visceral ganglion, just behind the sensory vesicle. o, otolith and ocellar pigment spots (expression negative); pb, polar bodies (expression negative); vg, visceral ganglion (expressing CiDmbx).

View larger version (24K): [in a new window]   Fig. 6. Comparison of expression domains between chordate neural tubes and proposed homology of brain regions. An underlying tripartite organisation is depicted by brackets, expression of transcription factors by shading and the organiser activity of FGF8/17/18 proteins by arrows (not proven in ascidia). In all three chordate taxa, the most anterior of the three regions is marked by expression of Otx. This region is divided into two regions in vertebrates: an anterior region without Dmbx expression (forebrain) and a posterior region with Dmbx expression (midbrain). Amphioxus and ascidia do not possess the Dmbx-expressing domain (midbrain) anterior to Pax2/5/8 expression (MHB). In vertebrates and ascidians, expression of Pax2/5/8 family genes fills the gap between the Otx-expressing region and the posterior region expressing Hox genes; this is not seen in amphioxus. Dmbx has a later expression domain in the hindbrain of vertebrates (shown by longitudinal stripes), coincident with Hox gene expression (up to rhombomere 2); co-localisation of Hox (CiHox3) and Dmbx also occurs in the ascidian anterior visceral ganglion.

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