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Origins of Bilateral Symmetry: Hox and Dpp Expression in a Sea Anemone

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Science  28 May 2004:
Vol. 304, Issue 5675, pp. 1335-1337
DOI: 10.1126/science.1091946

Abstract

Over 99% of modern animals are members of the evolutionary lineage Bilateria. The evolutionary success of Bilateria is credited partly to the origin of bilateral symmetry. Although animals of the phylum Cnidaria are not within the Bilateria, some representatives, such as the sea anemone Nematostella vectensis, exhibit bilateral symmetry. We show that Nematostella uses homologous genes to achieve bilateral symmetry: Multiple Hox genes are expressed in a staggered fashion along its primary body axis, and the transforming growth factor–β gene decapentaplegic (dpp) is expressed in an asymmetric fashion about its secondary body axis. These data suggest that bilateral symmetry arose before the evolutionary split of Cnidaria and Bilateria.

The Bilateria is an evolutionary lineage that encompasses more than 1.5 million modern-day animal species, including such diverse forms as humans, fruit flies, and soil nematodes (1). Bilateral symmetry has been implicated as a “key innovation” of the Bilateria, associated with an evolutionary transition from stationary or drifting planktonic animals to active burrowers and swimmers. In the Bilateria, bilateral symmetry is achieved by the orthogonal intersection of the anterior-posterior (A-P) axis and the dorsal-ventral (D-V) axis. The conserved deployment of homologous developmental regulatory genes argues for the underlying homology of both the A-P axis and the D-V axis (fig. S1). Hox genes play a conserved role in patterning the A-P axis (2), and decapentaplegic (dpp) plays a conserved role in patterning the D-V axis (3, 4).

Because bilateral symmetry was already present in the ancestral bilaterian over 500 million years ago, modern-day bilaterians cannot shed much light on the origin of bilateral symmetry. Outgroup taxa, animals that do not fall within the Bilateria, may reveal key steps in the evolution bilateral symmetry. One important outgroup to the Bilateria is the phylum Cnidaria (sea anemones, corals, hydras, and jellyfishes). Modern cnidarians resemble the earliest known fossil animals (5), and they may be “representative of a grade of late Precambrian organization from which bilaterians evolved” (6). Current textbooks characterize cnidarians as radially symmetrical, like simple cylinders (79). However, it has long been recognized that many cnidarians exhibit bilateral symmetry (10). For example, the sea anemone Nematostella vectensis possesses two orthogonal body axes (Fig. 1). The primary body axis, the oral-aboral axis, runs from the mouth to the foot (11). The secondary body axis, the directive axis, traverses the pharynx at a right angle to the primary body axis (10).

Fig. 1.

Anatomy of Nematostella. The body consists of an outer ectodermal epithelium and an inner endodermal epithelium, separated by an intervening layer of largely acellular connective tissue known as mesoglea. The mouth connects to the gut via the pharynx, a bilayered invagination of the body wall at the oral pole. (A and B) Longitudinal section through the oral-aboral axis. (A) Planula larva. The primary body axis of the planula is the apical-blastoporal axis. The animal swims in the direction of the apical tuft (arrow head). The blastopore becomes the mouth of the adult polyp. (B) Adult polyp. The primary body axis is known as the oral-aboral axis. The epithelium lining the lumen of the pharynx (ph) is ectodermal in origin. The oral extremity of the body axis occurs where the pharynx empties into the gut cavity. bw, body wall. (C) Cross section through the pharyngeal region. The pharynx is attached to the outer body wall via eight endodermal mesenteries. Each mesentery bears a retractor muscle on one face. The only plane of mirror symmetry passes through the directive axis.

The co-occurrence of bilateral symmetry in cnidarians and bilaterians suggests two possibilities: homology or convergence. If bilateral symmetry is homologous in Cnidaria and Bilateria, then homologous molecular mechanisms are expected to pattern their body axes. Gene expression data from Nematostella support the hypothesis of homology. During Nematostella development, five Hox genes are expressed in staggered domains along the primary body axis, and decapentaplegic (dpp) is expressed asymmetrically about the secondary body axis, a pattern of gene expression reminiscent of bilaterians.

Five Hox genes were recovered from Nematostella. Several previous studies have reported the recovery of anterior and posterior Hox genes from various cnidarians (1218). Phylogenetic analyses performed here are consistent with these earlier studies. According to neighbor-joining and maximum-likelihood analyses of homeodomain sequences (Materials and Methods), three genes recovered from Nematostella appear related to anterior Hox genes (anthox6, anthox7, and anthox8), whereas two appear related to posterior Hox genes (anthox1 and anthox1a) (1214). Additional evidence consistent with the identification of these genes as Hox genes includes genomic linkage data (19), the presence of Hox-specific hexapeptide sequences upstream of the homeodomain in anthox7 and anthox8, and the simultaneous recovery of numerous other members of the extended Hox family from Nematostella including Gsx, Evx, Mox, and Gbx (20). Two cnidarian-specific gene duplications appear to have produced two pairs of sister genes, anthox1-anthox1a and anthox7-anthox8, suggesting that the common ancestor of Cnidaria and Bilateria likely possessed three distinct Hox genes [Supporting Online Material (SOM) Text].

Hox gene expression was assayed by in situ hybridization throughout the blastula stage, during gastrulation, in the ciliated swimming larvae (planulae), and in newly settled polyps that are displaying their first four tentacles. Hox expression was observed in all regions along the primary body axis and in both body layers, endoderm and ectoderm (Fig. 1). Anthox1 is first expressed in early cleavage stages at the aboral pole. In the blastula, anthox1 is expressed over a wide area of the aboral blastoderm (Fig. 2A). After gastrulation in the two-layered planula larva, the expression of anthox1 is restricted to a small circular patch of ectodermal cells at the extreme aboral pole (Fig. 2, B and C).

Fig. 2.

Developmental expression of Hox genes. All images are seen from the lateral aspect, except for (B), which is an aboral view. The apical or aboral pole is toward the left. The asterisks indicates the blastoporal pole, the site of the future mouth. Expression of anthox1 in (A) early blastula stage and (B and C) planula larva stage. Expression of anthox1a in (D and E) gastrula and (F) late larval stages. Expression of anthox7 in (G and H) gastrula and (I) late larval stages. Expression of anthox8 in (J and K) gastrula and (L) late larval stages. Expression of anthox6 in (M) early larval stage and (N) juvenile polyp. bc, blastocoel; ec, ectoderm; ecph, pharyngeal (ph) ectoderm; en, endoderm; enph, pharyngeal (ph) endoderm; enbw, body wall (bw) endoderm; mes, mesentery; and tn, tentacles.

Three genes are expressed primarily in the body wall endoderm: anthox1a, anthox7, and anthox8. Each of these genes is first expressed in the endoderm near the blastopore during the gastrula stage (Fig. 2, D, G, and J). Throughout larval development, expression remains restricted to the endoderm, primarily along the outer body wall. Where the outer body wall joins the pharynx, the expression of anthox1a and anthox8 extends into the endoderm of the pharynx (arrowheads in Fig. 2, F and L).

In addition to being restricted along the oral-aboral axis, the expression of anthox1a, anthox7, and anthox8 is also restricted along the directive axis (Fig. 2, E, H, and K). Anthox1a is expressed in a thin strip of body wall endoderm at one end of the directive axis, flanked on either side by expression of both anthox7 and anthox8. The anthox8 expression overlaps with the anthox1a expression.

Anthox6 is expressed solely in the outer, endodermal layer of the pharynx (Fig. 2, M and N). Anthox6 expression first becomes apparent in endoderm near the blastoporal pole in the gastrula (Fig. 2M), and its expression remains fairly constant through the early and late larval stages and in the juvenile polyp (Fig. 2N).

Hox expression in Nematostella, consisting of staggered domains that collectively span nearly the entire oral-aboral axis, is reminiscent of Hox expression in bilaterian animals (Fig. 3). The interpretation of axial expression in Nematostella is complicated by the fact that the pharynx is an inversion of the body wall at the oral end of the animal (10). The region of the pharynx that protrudes most deeply into the coelenteron may be regarded as axially equivalent to the oral tip of cnidarians that lack a pharynx, such as Hydra (Fig. 1B). Therefore, the expression of anthox6 deep in the pharynx (Fig. 2N) is closer to the oral extremity than the expression of anthox1a or anthox8 at the junction of the pharynx with the outer body wall (Fig. 2, F and L). Data from the hydrozoan jellyfish Podocoryne are consistent with the conclusion that Hox genes are involved in patterning the primary body axis of cnidarian larvae, though the axial expression boundaries of specific homologs at the larval stage do not appear to be evolutionarily conserved between Podocoryne and Nematostella (17, 21).

Fig. 3.

Summary of Hox and TGFβ gene expression. (A) Provisional homology of Nematostella Hox genes based on phylogenetic analysis of homeodomains (Materials and Methods). Vertebrate Hox paralogs are numbered from 1 to 13. Arthropod Hox paralogs are named with Drosophila gene terminology (lab, labial; pb, proboscipedia; zen, zerknullt; Dfd, Deformed; scr, sex combs reduced; ftz, fushi tarazu; Antp, Antennapedia; Ubx, Ultrabithorax; abd-A, abdominalA; and AbdB, AbdominalB). (B) Gene expression along the oral-aboral and directive axes. The germ layer composition of Nematostella is shown in longitudinal section. To simplify the depiction of the primary body axis, the pharynx has been drawn as though everted. The mesenteries are not shown. Collectively, the five Hox expression domains span practically the entire oral-aboral axis. Anthox1a, anthox7, and anthox8 are restricted to one side of the directive axis. Likewise, both TGFβ genes, dpp and GDF5-like, exhibit asymmetric expression about the directive axis. Only the asymmetric aspects of their expression are shown. Dpp is expressed in the pharyngeal ectoderm on the side of the directive axis opposite the sector expressing anthox1a, anthox7, and anthox8. GDF5-like is expressed in the endoderm on the same side as anthox1a, anthox7, and anthox8.

We next assayed dpp expression because dpp is implicated in dorsal-ventral patterning in bilaterians. We recovered a single dpp ortholog from Nematostella (Materials and Methods). Expression of dpp commences at gastrulation, initially skewed to one side of the blastopore (Fig. 4, A and B) but later expanding to surround the blastopore (Fig. 4C), as it does in the coral Acropora (22). In the planula, the entire gastrodermis expresses dpp, including the eight mesenteries, the pharyngeal endoderm, and the body wall endoderm (Fig. 4D). Ectodermal expression occurs in the area of the apical organ at the aboral end of the planula, and in a thin strip of ectodermal tissue where the pharynx meets the mouth (Fig. 4, D and E). The expression in the pharynx is highly asymmetrical relative to the directive axis (Fig. 4, D and E). This pharyngeal staining is transient (lasting 48 to 72 hours) and disappears before the polyp stage (Fig. 4F). The pharyngeal expression of dpp occurs on the opposite side of the directive axis from the expression of anthox1a, anthox7, and anthox8 (SOM Text and fig. S7). In addition to dpp, we recovered another member of the transforming growth factor–β (TGFβ) family from Nematostella, a GDF-5-like gene, which also displays asymmetric expression, although at the opposite pole of the directive axis (SOM Text).

Fig. 4.

Developmental expression of Nv-dpp.(A) Early gastrula in lateral view. (B) Early gastrula in blastoporal view. There is asymmetric expression along the blastopore. (C) Late gastrula in oblique view. (D) Lateral view of planula larva. ao, apical organ. (E) Optical section through the oral-aboral axis at the level of the pharynx of a planula larva. There is asymmetric expression in the pharyngeal ectoderm (ecph). mes, mesentery. (F). Lateral view of a later stage planula with tentacle buds (tn). Strong expression is seen throughout the gastrodermis. bc, blastocoel; asterisks, blastopore.

Symmetry is an unresolved issue in metazoan evolution [reviewed in (23)]. Were the earliest metazoans radially symmetrical, or were they bilaterally symmetrical? The phylum Cnidaria is central to the debate because it is a closely related outgroup to the Bilateria and because major cnidarian lineages differ with respect to symmetry. Radial symmetry predominates in the class Hydrozoa (hydras and hydromedusae), whereas bilateral symmetry predominates in the class Anthozoa (sea anemones, corals, etc.). Most theories suggest that the cnidarian-bilaterian ancestor was a radially symmetrical animal [e.g., (11, 2429)]. According to this view, extant hydrozoans retain the ancestral radial symmetry. Competing theories propose that the cnidarian-bilaterian ancestor was a bilaterally symmetrical animal [e.g., (3033)]. According to this view, the Hydrozoa evolved radial symmetry secondarily, perhaps in conjunction with the evolution of the pelagic medusa phase of their life history.

The data summarized here suggest that bilateral symmetry evolved before the split between Cnidaria and Bilateria. Both taxa exhibit bilateral symmetry. Both taxa exhibit staggered Hox expression domains along the primary body axis and asymmetric dpp expression along the secondary body axis. Homology is the most parsimonious explanation for the shared possession of these morphological and molecular traits. If we invoke homoplasy as an explanation, we must presume that one or both of these complex axial patterning systems evolved convergently in two independent evolutionary lineages.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1091946/DC1

Materials and Methods

SOM Text

Figs. S1 to S7

References and Notes

References and Notes

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