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Homeoboxes in sea anemones (Cnidaria; Anthozoa): a PCR-based survey of Nematostella vectensis and Metridium senile.

Introduction

Homeobox genes encode a family of transcription factors that are characterized by the presence of a DNA-binding domain consisting of 60 amino acids and known as the homeodomain. Homeobox genes have been found in all metazoan phyla that have been surveyed, as well as in plants and fungi (reviewed in Burglin, 1994). Recent reports of homeobox genes from basal metazoans, such as sponges and cnidarians, are revising our understanding of the evolution of this multigene family (Murtha et al., 1991; Schierwater et al., 1991; Miles and Miller, 1992; Naito et al., 1993; Kruse et al., 1994, Seimiya et al., 1994; Degnan et al., 1995; Kuhn et al., 1996). Research on homeobox genes has stimulated an ongoing synthesis between the fields of evolutionary biology and developmental genetics because of the possibility that homeobox genes have played an evolutionary role in the diversification of metazoan body plans.

We identified homeobox-containing genes in two species of sea anemone, Metridium senile and Nematostella vectensis. Sea anemones belong to the Cnidaria, a prylum of tentacle-bearing, radially symmetric animals that possess nematocysts (Hyman, 1940). Substantial phylogenetic evidence places the Cnidaria near the base of the Eumetazoa, possibly as the sister group to the Bilateria. The Cnidaria share with bilaterian animals several derived characters that distinguish eumetazoans from sponges, including the possession of epithelio-muscle cells and nerve cells. However, the cnidarian body plan lacks several derived features of the Bilateria: organ-level organization, a well-differentiated mesoderm, a coelom. Furthermore, cnidarians display radial or biradial symmetry instead of bilateral symmetry. The relative simplicity of the cnidarian body plan suggests that cnidarians may possess a network of developmental regulatory genes that is relatively simple and retains more of the primitive characteristics of early eumetazoan animals than do networks found among the bilaterians. Therefore, study of cnidarians may provide unique insights into the evolution of developmental regulatory genes, such as homeobox genes. In any event, the Cnidaria are an outgroup to the Bilateria, so regardless of the degree of simplicity in their developmental regulatory networks, they will provide an important phylogenetic perspective on the evolution of development in the Metazoa.

Among extant Cnidaria, the Anthozoa (sea anemones and corals) are thought to be the sister group to a clade comprising the Cubozoa, Scyphozoa, and Hydrozoa, and it has been argued that the anthozoan body plan most resembles that of the ancestral cnidarians (GrassholT, 1984; Schick, 1991; Bridge et al., 1992; Odorico and Miller, 1997). Furthermore, the Anthozoa have a simpler life cycle than other Cnidaria, lacking the presumably derived medusoid stage that characterizes the Scyphozoa and the Hydrozoa. We have chosen to study homeobox genes in the Anthozoa that we might better understand the ancestral function of these genes in the Cnidaria and in the Eumetazoa.

Two techniques have been used to rapidly survey metazoan genomes for the presence of homeoboxes: (1) PCR (polymerase chain reaction) amplification of genomic DNA with degenerate primers corresponding to conserved regions of the homeodomain, and (2) library screening with a conserved oligonucleotide probe. More than 20 homeoboxes have been isolated from 8 cnidarian species by using these techniques (Murtha et al., 1991; Schierwater et al., 1991; Miles and Miller, 1992; Schummer et al., 1992; Naito et al., 1993; Shenk et al., 1993; Aerne et al., 1995). As many as five distinct homeoboxes have been recovered from individual hydrozoans, Chlorohydra viridissima (Schummer et al., 1992) and Eleutheria dichotoma (Kuhn et al., 1996). In a recent classification scheme (Naito et al., 1993) cnidarian homeoboxes are sorted into eight mutually paralogous classes (Table I), but 18 of 20 of the cnidarian genes are from members of the class Hydrozoa. Little is known of homeobox genes in other classes, including the corals and anemones of the class Anthozoa. We have used PCR to survey the genome of two anthozoans for the presence of homeobox genes.

Materials and Methods

DNA extraction

Live anemones were obtained from William Zamer at Lake Forest College (Lake Forest, IL; Metridium) and Kevin Uhlinger at Bodega Bay Marine Laboratory (Bodega Bay, CA; Nematostella). All animals were starved for 2 weeks and rinsed several times in sterile artificial seawater prior to DNA extraction. DNA extraction was performed with proteinase K and RNase digestion followed by several phenol/chloroform extractions (Sambrook et al., 1989).

PCR amplification

Degenerate primers corresponding to highly conserved regions in helices one and three of the homeodomain were used to amplify an 82-bp fragment from genomic DNA (nucleotides 60 through 141 of the homeobox, not including the primers). The upstream primer (5[prime]-GARYTIGARAARGARTT-3[prime]) corresponded to the amino acid sequence ELEKEF, and the downstream primer (5[prime]-CKNCKRTTYTGRAACCA-3[prime]) corresponded to the reverse complement for the amino acid sequence WFQNRR. The 5[prime] ends of the primers were phosphorylated to facilitate cloning PCR products. Both primers were synthesized commercially (Operon Technologies, Alameda, CA).

PCR was performed in 50-[[micro]liter] reactions containing 50 mM Tris (pH 8.3), 10 mM KCl, 3.5 mM Mg[Cl.sub.2], 200 [[micro]molar]dNTPs, 0.6 Units Taq polymerase, 12.5 pmol of each primer, and 150 ng genomic DNA. Thermal cycling was performed in a MiniCycler (MJ Research, Watertown, MA) under the following parameters: 5 cycles (45-s each) of denaturation at 94 [degrees] C, 45 s of primer annealing at 37 [degrees] C, and 45 s of primer extension at 72 [degrees] C, followed by 35 cycles in which the annealing temperature was raised to 42 [degrees] C. Eight separate 50-[[micro]liter] PCR reactions were performed for each species, and these were pooled prior to cloning to minimize the effects of stochastic variation known as PCR drift (Wagner et al., 1994) Twenty-five microliters of the pooled reaction products were visualized on a 2% agarose gel stained with ethidium bromide, and the expected amplification product (114 bp) was observed. Control reactions lacking one primer of a primer pair failed to produce the expected amplification product. Control reactions lacking template produced no visible amplification products.

Cloning and sequencing

PCR products were blunted prior to cloning by using T4 DNA Polymerase (Costa and Weiner, 1994). The entire reaction was then run out on a 0.8% SeaPlaque GTG agarose gel (FMC Bioproducts, Rockland, ME) in a modified TAE buffer (0.04M Tris-acetate, 0.2 mM EDTA). The appropriate band was excised from the gel, melted, and added directly to a ligation reaction. PCR products were cloned into pUC 18 plasmid that had been cut with Smal and treated with phosphatase (Kalvakolanu and Livingston, 1991). Following alkaline denaturation, double-stranded sequencing was performed on recombinant clones; the Sequenase v2.0 kit (USB, Cleveland, OH) was used with 35S-labeled dATP and the M13 forward primer. Alternatively, automated sequencing was performed with dye-labeled dideoxynucleotides and [TABULAR DATA FOR TABLE I OMITTED] an ABI 373A sequencer. Homeobox-containing clones were sequenced in the reverse direction with the M13 reverse primer. Of the 44 clones sequenced for Metridium, 32 (73%) contained recognizable homeobox sequences. Of the 47 clones sequenced for Nematostella, 35 (74%) contained recognizable romeobox sequences.

Results and Discussion

Homeoboxes identified by the PCR-based survey

Our PCR-based survey identified 12 distinct homeoboxes in two species of sea anemone: 7 genes from Metridium senile and 5 from Nematostella vectensis [ILLUSTRATION FOR FIGURE 1 OMITTED]. Consensus sequences for each of these 12 anemone homeobox gene fragments were submitted to GenBank (accession numbers U42726-U42737). To our knowledge, the 7 homeoboxes identified in Metridium are the most recovered from any single species of cnidarian. The recovery of so many distinct genes may be attributable to the initial pooling of 8 separate PCR reactions (Wagner et al., 1994). The 12 anemone homeoboxes appear to represent 9 paralogous homeobox genes referred to as anthox1, anthox1a, anthox2, anthox4, anthox5, anth-hbxA, anth-hbxB, anth-hbxC, and anth-eve. The names were chosen to reflect putative homology between anemone homeoboxes and those from other cnidarians (see legend to Fig. 1). The PCR surveys identified overlapping but different sets of homeoboxes in Metridium and Nematostella. Anthox1, anth-eve, and anth-hbxA were recovered from both Metridium and Nematostella. Anthox2, anthox4, anthox5, and anth-hbxB were recovered only in Metridium. Anthox1a and anth-hbxC were recovered only in Nematostella.

Comparison of homeoboxes from anemones and other cnidarians

We wanted to know if the homeoboxes we isolated are orthologous to previously identified homeoboxes from cnidarians (Table I; Naito et al., 1993), or if they represent new classes of cnidarian homeoboxes. We used phylogenetic methods to evaluate the evolutionary relationships between representatives of the 8 putative cnidarian classes and the 12 homeoboxes recovered from Metridium and Nemalostella (Saitou and Nei, 1987; Fig. 2). A neighbor-joining tree (Saitou and Nei, 1987) was constructed using the program Phylip (version 3.5, Felsenstein, 1989; [ILLUSTRATION FOR FIGURE 2 OMITTED]), and a parsimony tree was constructed using the program PAUP (Phylogenetic Analysis Using Parsimony, version 3.1; Swofford, 1991; [ILLUSTRATION FOR FIGURE 3 OMITTED]).

On the gene phylogenies [ILLUSTRATION FOR FIGURE 2 AND 3 OMITTED], Class I, Class II, and Class IV, as defined by Naito et al. (1993; Table I) are monophyletic. Among the anemone homeodomains, anthoxl of Metridium seems to be orthologous to anthoxl of Nematostella. Anth-eve of Metridium and Nematostella also appear orthologous, as do anth-hbxA of Metridium and Nematostella. The phylogenies also permit a tentative assignment of some of the anemone romeodomains to specific cnidarian classes. Anth-eve appears orthologous to eveCoral (Class VII). Anthox1 and anthox1a cluster with members of cnidarian Class I that are believed to be related to the Antennapedia-like, or Hox class, homeoboxes (Naito et al., 1993). Anthox5 appears orthologous to cnox5 (Class V). Other associations appear more tenuous.

Identity of anemone homeoboxes assessed by searching the Genbank database

The sequences of anemone homeodomains inferred from the gene fragments recovered in this study were compared to sequences in GenBank with the BLASTx network search algorithm (Basic Local Alignment Search Tool; Altschul et al., 1990). The top five matches recovered in the BLAST searches are aligned to their anemone homeodomain counterparts in Figure 4. For each of the unique anemone homeodomain fragments, the search was limited to the 50 most similar genes in the GenBank database. In every case, all 50 of the genes identified were homeobox genes. At the amino acid level, anemone homeodomains are 59%-85% identical to the most similar homeobox genes in the GenBank database. The BLAST searches reveal that five of the anemone sequences are most similar to homeodomains of the Hox class: these are anthox1, anthox1a, anthox2, anthox4, and anthox5. The anth-eve homeodomains are most similar to even-skipped homologs from other species, particularly the even-skipped homolog of the staghorn coral (Miles and Miller, 1992). The anth-hbxA romeodomains from both Metridium and Nematostella appear most similar to the flatworm homeodomain EgHbx2 (Oliver et al., 1992). EgHbx2 has been referred to as an NK type homeobox, but this assignment is not supported by a recent homeobox gene tree (Burglin, 1994). The anth-hbxA homeodomains also resemble the engrailed homeodomain of Drosophila. Anth-hbxB of Metridium is most similar to empty-spiracles homologs from the mouse (EMX1 and EMX2). Anth-hbxC from Nematostella exhibits similarity to a diverse group of genes, including Hox A8 from Branchiostoma and msx2 from mouse, two members of the msh-class of homeodomains.

Phylogenetic relationships of homeoboxes from sea anemones and higher metazoans

Phylogenetic analyses of cnidarians and bilaterians were used to further assess the identity of the anemone homeoboxes (Saitou and Nei, 1987). In addition to the 12 anemone homeodomains and 13 sequences from other cnidarians, our analysis included 20 sequences from bilaterian animals, each representing a different class of homeodomains (Burglin, 1994). A neighborjoining tree [ILLUSTRATION FOR FIGURE 5 OMITTED] supports the finding that some of the anemone homeoboxes belong to previously proposed cnidarian classes (Naito et al., 1993; see previous section Comparison of homeoboxes from anemones and other cnidarians). Anthox1 allies with members of Class I (cnox1.Hm and SAox3), anthox2 with members of Class II (cnox2.Cv and SAox2), anthox4 with members of Class IV (cnox4.Hm and cnox1.Cv), anthox5 with Class V (cnox5.Hm), and anth-eve with Class VII (eveC). All of these findings, with the exception of the precise relationships of anthox4, are supported by a parsimony analysis [ILLUSTRATION FOR FIGURE 6 OMITTED].

The phylogenetic analyses suggest that certain anemone homeoboxes represent classes previously unidentified in the Cnidaria [ILLUSTRATION FOR FIGURE 5 AND 6 OMITTED]. Anthox1a from Nematostella appears as the sister group to the Class I genes and may represent a new class of cnidarian homeoboxes (Class Ia). Anth-hbxA, found in both Metridium and Nematostella, appears most closely related to the homeobox from the human gene TCL-3, a non-cluster member of the Hexapeptide superclass (Biirglin, 1994). As suggested by the BLASTx search results [ILLUSTRATION FOR FIGURE 3 OMITTED], Anth-hbxB from Metridium appears most closely related to the Drosophila empty-spiracles homeobox, a gene known to be involved in head development in both Drosophila and vertebrates (Walldorfand Gehring, 1992; Simeone et al., 1992). Finally, Anth-hbxC appears most closely related to the C. elegans homeobox ceh-9. The PCR surveys of sea anemones failed to recover representatives of cnidarian classes 3, 4, 6, or 8. The BLAST searches and phylogenetic analyses suggest to us an expanded working classification of homeobox genes in the Cnidaria (Naito et al., 1993) to encompass the new data from sea anemones (Table II).

The Hox class in basal metazoans

In distantly related taxa, such as vertebrates, insects, and nematodes, the genes of the Hox class are located in evolutionarily conserved genomic clusters. Furthermore, individual Hox genes are expressed along the anterior-posterior axis of the developing embryo in the same relative order as their position in the Hox cluster: genes located towards the 3[prime] end of the cluster are expressed more anteriorly than genes located towards the 5[prime] end of the cluster. This evidence suggests that a linked cluster of Hox genes is a shared, derived metazoan character that is involved in patterning the anterior-posterior axis (Slack et al., 1993; Miller and Miles, 1993). However, very little is known about the composition of the Hox cluster in basal metazoans such as the Cnidaria, or even whether such a cluster exists.

As our knowledge of Hox genes in basal metazoans increases, we should be able to establish the antiquity of the Hox cluster and of individual Hox genes, and to make inferences about the primitive function of Hox genes. Among the relevant questions are (1) How many Hox genes are found in the Cnidaria? and (2) What are the relationships of cnidarian Hox genes to Hox genes from bilaterian animals? Phylogenetic analyses of vertebrate and insect Hox genes suggest a very early trichotomy in the evolution of the Hox class - giving rise to an Antennapedia/deformed precursor, a labial/proboscipedia precursor, and an AbdominalB precursor (Schubert et al., 1993). Previous studies revealed similarities between individual cnidarian homeoboxes and homeoboxes from the Drosophila genes labial, proboscipedia, Deformed, and Antennapedia (Murtha et al., 1991; Schierwater et al., 1991; Schummer et al., 1992; Naito el al., 1993). These homeobox genes have been referred to as anterior and central members of the Drosophila HOM/Hox cluster because their anterior borders of expression lie in the anterior half of the Drosophila embryo (Bartels el al., 1993). A homolog of the posterior Hox genes (AbdominalB-like) has not been recognized in the Cnidaria.

With few exceptions (e.g., Naito el al., 1993), the identities of cnidarian Hox genes have been considered in isolation: Hox class genes cloned from a single species are compared to Hox sequences from Drosophila and vertebrates without reference to Hox genes from other cnidarians or to non-Hox homeoboxes; these comparisons have been made using pairwise alignments (e.g., Schummer et al., 1992; Miles and Miller, 1992; Miller and Miles, 1993) or, more rarely, phylogenetic techniques (Aerne et al., 1995; Kuhn et al., 1996). In this study we have attempted a more systematic approach: we used phylogenetic techniques to simultaneously assess the relationships between all the reported cnidarian homeoboxes and a broad representation of homeoboxes from bilaterian animals. As in several previous studies, our phylogenetic analyses suggest that certain cnidarian Hox genes are closely related to anterior and central members of the Hox cluster in higher metazoans (Murtha et al., 1991; Schierwater et al., 1991; Schummer et al., 1992; Naito et al., 1993). The members of cnidarian classes II, IV, V, and VI appear to be more closely related to the labial and Antennapedia homeoboxes of Drosophila than to the AbdominalB homeobox or any non-Hox homeobox. Our analyses indicate that cnidarians may possess homologs to posterior members of the complex as well as to the more anterior genes. The phylogenetic analyses [ILLUSTRATION FOR FIGURE 5 AND 6 OMITTED] suggest that members of the cnidarian homeobox classes I and Ia are most closely related to the Drosophila Abdominal-B homeobox, the most posterior member of the Drosophila Hox cluster. Therefore, distinct anterior/central (Antennapedia/labial-like) and posterior (AbdominalB-like) Hox gene lineages may have predated the split between the Cnidaria and the Bilateria (Schubert el al., 1993). Additional data on cnidarian Hox genes, such as their genomic organization and axial expression, may serve to corroborate or contradict this hypothesis.

A recent paper by Kuhn et al. (1996) suggested a very different scenario for the origin of the HOM/Hox genes. Hox genes from triploblasts (Drosophila and humans) were compared with Hox genes from a cnidarian (the hydrozoan Eleutheria dichotoma) by using parsimony and distance methods. In the phylogenetic analyses, the resulting topologies can be rooted such that genes from triploblasts form a monophyletic group to the exclusion of all cnidarian Hox-type genes, suggesting that the common ancestor of triploblasts and cnidarians possessed only a single Hox-type gene. In other words, Kuhn et al. (1996) find no evidence of a Hox cluster in the common ancestor of diploblasts and triploblasts, whereas the analyses presented here are consistent with the existence of an ancestral cluster.

The difference in the two phylogenetic conclusions derives from the choice of phylogenetic characters - the analyses reported here were based on the inferred amino acid residues within the homeodomain; those of Kuhn et al. (1996) were based on the entire nucleotide sequence of the homeobox, including rapidly evolving, silent nucleotide sites within codons. But the relevant evolutionary time scale is very long: both cnidarian and bilaterian fossils are known from Cambrian sediments laid down more than 500 million years ago (e.g., Sepkoski, 1981), so more than I billion years of independent evolution separate the last common ancestor of modern-day triploblasts and cnidarians. Therefore, rapidly evolving sites are likely to be saturated with phylogenetic noise. Furthermore, if there are differences in codon bias between taxa, the inclusion of silent sites in the analysis may falsely indicate phylogenetic affinity between nonhomologous gene sequences derived from closely related taxa. When the phylogenetic analysis of Kuhn et al., (1996) is repeated with the inferred amino acid substitutions as phylogenetic characters, the result is consistent with the existence of an ancestral cluster [ILLUSTRATION FOR FIGURE 7 OMITTED].

Confidence in inferring orthology of homeodomain sequences

In trying to infer the evolutionary relationships of homeobox genes, we are confronted by historical limitations. Our inferences are based on a small region of sequence that is highly conserved across very distantly related phyla. Sequence conservation outside the homeodomain is generally poor, so sequence alignment is difficult. The phylogenetic utility of sequences outside the homeodomain may therefore be limited. Considering this historical limitation, our inferences about the identity of several of the cnidarian homeodomains appear surprisingly robust. Several partitions on the neighbor-joining tree that suggest homology between cnidarian and bilaterian homeodomains are supported by more than 50% of bootstrap trials (for example, cnidarian even-skipped homologs + Drosophila eve: bootstrap proportion [greater than or equal to] 75%). Results from computer simulations and experimental bacteriophage phylogenies suggest that bootstrap proportions over 50% can indicate a much higher than 50% probability that the corresponding branch is correct (Hillis and Bull, 1993; Felsenstein and Kishino, 1993). The results presented here agree with an emerging pattern: newly discovered homeoboxes can often be assigned to previously described classes with reasonable confidence, though the interrelationships between different classes of homeobox genes are difficult to reconstruct (e.g., Finnerty el al., 1996). This pattern suggests that many of the homeobox classes are extremely ancient, and that the sequences of these genes have been very strongly conserved.

Acknowledgments

We are extremely grateful to William Zamer at Lake Forest College and Kevin Uhlinger at the Bodega Bay Marine Laboratory for supplying live sea anemones. This work was supported by NSF grant #9315653 to M.Q.M. and NIH grant HD07136 to J.R.F.

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Title Annotation:Polymerase Chain Reaction
Author:Finnerty, John R.; Martindale, Mark Q.
Publication:The Biological Bulletin
Date:Aug 1, 1997
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