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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01119

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Investigating the origins of triploblasty: `mesodermal' gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa)

¹ Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813, USA
² Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA

View larger version (28K): [in a new window]   Fig. 1. Evolutionary relationships and germ layer composition of Cnidaria and Bilateria. (A) The topology of the tree summarizes the results of recent molecular phylogenetic analyses (Odorico and Miller, 1997; Schuchert, 1993). The phylum Cnidaria is an outgroup of the Bilateria, perhaps the sister group. The class Anthozoa is the sister of the Medusozoa (Odorico and Miller, 1997; Schuchert, 1993). Anthozoans exhibit only the polyp stage, while most medusozoans exhibit both polyp and medusa life stages. The Medusozoa comprises the members of the classes Hydrozoa, Cubozoa and Scyphozoa. Multiple independent scyphozoan lineages are depicted because a recent analysis suggests that the Scyphozoa may be paraphyletic (Collins, 2002). (B) The germ layer composition of representative taxa is indicated using diagrams of the cross-sectional anatomy. The anthozoan polyp is sectioned through the pharynx. Notice that the lumen of the pharynx is lined with ectoderm, while the outer surface of the pharynx is lined with endoderm. The hydrozoan polyp lacks a pharynx. Both animals exhibit two epithelial layers (endoderm and ectoderm). The hydrozoan medusa exhibits a third epithelial layer, the entocodon, that surrounds a coelom-like internal cavity, the subumbrellar cavity (Boero et al., 1998). Mesoderm lines the coelomic cavity of both protostomes and deuterostomes.

View larger version (99K): [in a new window]   Fig. 2. Nematostella development. In Nematostella, the sexes are separate and adults generate either eggs or sperm. The cleavage program is chaotic with no two embryos developing identically. (A) A hollow blastula is formed. Yolk fragments break off from the apical regions of cells and remain trapped in the blastocoel. (B) Gastrulation proceeds by the invagination of the future oral end of the embryo (*). Cells entering the blastocoel from the prospective oral pole will give rise to endoderm (en) surrounded by outer ectoderm (ec). (C) The planula `larva' is initially shaped like a teardrop and swims with its apical sensory tuft (at) directed forward. The endoderm (en) forms a solid ball of cells at the planula stage. (D) The coelenteron (gut cavity) has formed by the hollowing out of the solid planula endoderm (ct). Tentacle buds (tn) begin to form around the oral opening (*) in the swimming larva prior to settlement. Note that the pharynx (ph) extends into the coelenteron. (E) The polyp forms 7 days after fertilization. When it first settles onto the substrate, it has four tentacles and eight endodermal mesenteries. Endoderm lines the hollow tentacles. The thickened pharyngeal wall connects the mouth/anus to the coelenteron.

View larger version (76K): [in a new window]   Fig. 3. Nv-forkhead. Phylogenetic analysis and gene expression. (A) Alignment of 81 amino acids from the conserved forkhead domain (top), 35 amino acids from the transcription activation domain II (bottom left) and 16 amino acids from the transcription activation domain III (bottom right) (Pani et al., 1992). (B) Neighbor-joining tree produced from the 81 amino acids of the forkhead domain. Branch lengths are proportional to the number of substitutions per residue. Numbers at nodes indicate bootstrap support (percentage of 2000 bootstrap replicates in which the given clade was recovered). Some branch lengths were truncated by 50% for presentation purposes (indicated by x2). Sequences included in the analysis are: bin (biniou), Drosophila, residues 312-391; budhead, Hydra vulgaris, residues 82-159; CG32006-PA (hypothetical protein), Drosophila, residues 147-220; fkh (fork head), Drosophila, residues 211-288; FOXA2 (forkhead box A2), Homo, residues 160-237; FOXB1, Homo, residues 14-91; FOXC1, Homo, residues 79-157; FOXD1, Homo, residues 126-203; FOXE1, Homo, residues 54-131; FOXM1, Homo, residues 237-312; slp2 (sloppy paired 2), Drosophila, residues 182-258; fox (fork head box protein), Nematostella, residues 40-117. (C) Surface view of a late blastula showing localized expression at the future site of the blastopore. (D) Lateral view of the same stage. (E) The first cells that enter the blastocoel express Nv-forkhead. (F-J) Only cells of the pharynx (pha) and pharyngeal mesenteries (mes) express this gene. Asterisks indicate the site of the blastopore, the future mouth.

View larger version (81K): [in a new window]   Fig. 4. Nv-GATA. Phylogenetic analysis and expression. (A) Alignment of 80 amino acids from various GATA transcription factors spanning a central C2C2 zinc finger motif. (B) Phylogeny of GATA sequences. The tree was constructed and labeled as in Fig. 3. Sequences included in the analysis are: CG10278-PA (hypothetical protein), Drosophila, residues 514-595; GATA, Nematostella, residues 248-321; GATA-1 (GATA binding protein 1), Homo, residues 235-316; GATA-2, Homo, residues 326-406; GATA-3, Homo, residues 294-373; GATA-4, Homo, residues 193-273; GATA-5, Homo, residues 221-300; GATA-6, Homo, residues 274-356; grain, Drosophila, residues 292-378; MGC2306 (hypothetical protein), Homo, residues 326-407; pannier, Drosophila, residues 148-224; serpent, Drosophila, residues 451-535; TRPS1, Homo, residues 873-959. (C-H) Expression of Nv-GATA. (C,D) Scattered cells express Nv-GATA at the blastula stage. Nv-GATA-expressing cells appear to move into the blastocoel (arrows). (E,F) At gastrulae stages expression is confined to endodermal cells. In the late planula (G) and early polyp (F) stages, ectodermal cells at the base of the developing tentacles also express Nv-GATA. Asterisks indicate the site of the mouth. end, endoderm; ect, ectoderm; tn, tentacles.

View larger version (39K): [in a new window]   Fig. 5. Nv-mef2. Phylogenetic analysis and gene expression. (A) Alignment of 68 amino acids from the conserved MADS domain of various mef2 genes plus blistered and serum response factor (SRF). (B) Phylogeny of mef2 sequences. The tree was constructed and labeled as in Fig. 3. Phylogenetic analysis was based on the amino acid sequence of the MADS domain. Sequences included in the analysis are: blistered, Drosophila, residues 166-233; mef2 (myocyte enhancing factor 2), Drosophila, residues 2-69; mef2, Nematostella, residues 2-69; mef2, Podocoryne, residues 2-69; MEF2A (myocyte enhancer factor 2A), Homo, residues 2-69; MEF2B, Homo, residues 2-69; MEF2C, Homo, residues 2-69; MEF2D, Homo, residues 2-69; SRF (serum response factor), Homo, residues 142-209. (C-G) Expression of Nv-mef2. (C) Expression begins in individual cells distributed broadly around the circumference of the blastula. (D,E) Throughout the development of the planula, expression remains confined to epidermal cells. (F,G) As development continues, Nv-mef2 expression comes to be preferentially expressed at the oral pole (arrows), particularly along the surface of the developing tentacle buds (tb) and tentacles (tn).

View larger version (48K): [in a new window]   Fig. 6. Nv-muscle LIM. Phylogenetic analysis and gene expression. (A) Alignment of 62 amino acid residues from the conserved LIM domain. Conserved residues are indicated in red (Conserved Domain Database at NCBI: pfam00412.8, LIM). (B) Phylogeny of LIM sequences based on neighbor-joining analysis. The tree was constructed and labeled as in Fig. 3. Sequences included in the analysis are: CG4656-PA (hypothetical protein), Drosophila, residues 4-63; CG30174-PA (hypothetical protein), Drosophila, residues 93-152; CG30179-PA (hypothetical protein), Drosophila, residues 104-163; CRIP1 (cysteine-rich protein 1), Homo, residues 4-63; CSRP1 (cysteine and glycine-rich protein 1), Homo, residues 10-68; CSRP2 (smooth muscle LIM protein), Homo, residues 10-68; CSRP3 (cysteine and glycine-rich protein 3, cardiac LIM protein), Homo, residues 10-68; FHL1 (four and a half LIM domains 1), Homo, residues 101-160; limpet, Drosophila, residues 220-277; M-LIM (muscle LIM protein), Nematostella, residues 4-62; Mlp60A (muscle LIM protein at 60A), Drosophila, residues 11-69; Mlp84B (muscle LIM protein at 84B), Drosophila, residues 12-70. The tree is drawn as though rooted between the Limpet/CRIP clade and the muscle LIM/CSRP clade. (C-H) Expression of Nv-muscle LIM. (C,D) Expression begins at mid-late planula stages in the precursors of the eight endodermal mesenteries (arrows). (E) Mid level optical section of a late planula, indicating expression in the gastrodermal lining of the coelenteron. The endodermal mesenteries are not in the plane of focus. Note that the pharynx and outer epidermis do not express Nv-muscle LIM. (F-H) In early polyp stages, the highest expression is in the endodermal lining (arrows) of the developing tentacles (tn). (G,H) Lateral views. In F, the mouth is bent towards the viewer to reveal the tip of the developing tentacle (arrow). The asterisk indicates the site of the mouth.

View larger version (76K): [in a new window]   Fig. 7. Nv-snailA and NvsnailB. Phylogenetic analysis and gene expression. (A) Alignment of 105 amino acids containing repeated C2H2 zinc-finger motifs. (B) Phylogeny of snail sequences. The tree was constructed and labeled as in Fig. 3. Sequences included in the analysis are: escargot, Drosophila, residues 344-446; Kruppel, Drosophila, residues 250-354; Kruppel-like protein (zinc finger protein 443), Homo, residues 449-477 plus 505-580; scratch, Drosophila, 498-600; scratch1, Homo, 222-324; snail, Drosophila, residues 280-383; snail, Podocoryne, residues 226-238; snail 1, Homo, residues 154-257; snail 2, Homo, residues 159-261; snail A, Nematostella, residues 156-258; snail B, Nematostella, residues 154-256. The tree is drawn as though rooted using the Kruppel clade as an outgroup. (C-J) Expression of Nv-snailA through development. (C) Nv-snailA transcript is not detectable at early cleavage stages. (D) Expression becomes visible in the late blastula at the site of the blastopore. (E) The embryo initiates gastrulation at the site of Nv-snailA expression. (F) The first cells that invaginate into the blastocoel express Nv-snailA. (G) Expression continues throughout the process of gastrulation as cells move in to the blastocoel. (H-J) All of the cells of the gastrodermis, but none of the cells of the epidermis express Nv-snailA up through the polyp stages. The asterisk indicates the site of the mouth. The expression of Nv-snailB is indistinguishable from Nv-snailA. Arrows in G,H,J indicate endoderm.

View larger version (33K): [in a new window]   Fig. 8. Nv-twist. Phylogenetic analysis and gene expression. (A) Alignment of 56 amino acids from twist, atonal and nautilus proteins. (B) Phylogeny of twist sequences. The tree was constructed and labeled as in Fig. 3. Sequences included in the analysis are: ato (atonal), Drosophila, residues 256-311; ATOH1 (atonal homolog), Homo, residues 160-215; nautilus, Drosophila, residues 162-216; twi (twist), Drosophila, residues 363-417; twist, Nematostella, residues 37-92; twist, Podocoryne, residues 52-106; TWIST1, Homo, residues 109-163. (C-G) Developmental expression of Nv-twist. (C) Expression begins after gastrulation has started in a ring of endodermal cells encircling the presumptive mouth. (D) Expression continues in endodermal cells surrounding the future mouth. (E) Low magnification image of early planula stage embryos seen from the oral pole showing the ring of expression surrounding the future mouth. (F,G) As development proceeds, expression remains confined to a circum-oral ring, in the endodermal epithelium of the pharynx facing (arrow 1) and external gastrodermal wall under the tentacles (arrow 2).