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Hox) Genes
MOLECULAR PHYLOGENETICS AND EVOLUTION
Vol. 6, No. 1, August, pp. 30–38, 1996 ARTICLE NO. 0055
Homeobox Genes in the Cnidarian Eleutheria dichotoma: Evolutionary Implications for the Origin of Antennapedia-Class (HOM/Hox) Genes KERSTIN KUHN,* BRUNO STREIT,*
AND
BERND SCHIERWATER*,†
*Department of Ecology and Evolution, J.W. Goethe-Universita¨t, Postfach 111932, Siesmayerstr. 70, D-60054 Frankfurt am Main Germany; and †Yale University, Department of Biology, 165 Prospect Street, New Haven, Connecticut 06511 Received June 5, 1995; revised October 24, 1995
1991; Salser and Kenyon, 1994) and most arthropods (Beeman et al., 1989; Averof and Akam, 1993) possess a single homeobox gene cluster. The homeobox cluster (HOM-C) in Drosophila is subdivided into two complexes, the Antennapedia (Ant-C) (Kaufmann et al., 1980, 1990) and Bithorax complexes (BX-C) (Lewis, 1978; Duncan, 1987). This split probably occurred late in insect evolution (Akam, 1989; Kenyon, 1994). The HOM-C contains the Antennapedia (Antp)-class genes: eight homeotic genes, which specify pattern formation along the anteroposterior body axis (for reviews see McGinnis and Krumlauf, 1992; Lawrence and Morata, 1994), plus three non-homeotic genes (zen1, zen2, ftz). Vertebrates possess four homeobox gene clusters (HoxA to Hox-D) located on four different chromosomes, each containing between 9 and 13 Antp-class homeobox genes (for review see Krumlauf, 1992, 1994). The four clusters most likely arose through gene cluster duplication events, followed by the loss of some homeobox genes (Garcia-Ferna`ndez and Holland, 1994). Among nematodes, arthropods, and vertebrates, the linear organization of homeobox genes along the chromosome correlates with their expression patterns along the anterior–posterior axis of the body (Duboule and Dolle´, 1989; Graham et al., 1989; Bu¨rglin et al., 1991; Stuart et al., 1991). The nearer the genes are located to the 3′ end of the chromosome, the more anterior is their expression in the embryo. Based on sequence similarities and conservation of genomic organization, a common ancestral homeobox gene cluster for vertebrates and arthropods as well as for arthropods and nematodes has been proposed (Fig. 1; Holland, 1992; Averof and Akam, 1993; Bu¨rglin and Ruvkun, 1993; Kappen and Ruddle, 1993; Schubert et al., 1993; Garcia-Ferna`ndez and Holland, 1994). Phylogenetic analyses of vertebrate and arthropod homeobox genes suggested the presence of a single gene cluster containing three Antp-class homeobox genes early in metazoan evolution (before the separation of diploblasts and triploblasts). This hypothetical ancestral cluster should contain homeobox genes corresponding to the differen-
In order to test current hypotheses on the early evolution of Antp-class (HOM/Hox) genes in metazoan animals, we surveyed the diploblastic hydrozoan Eleutheria dichotoma for the presence of these genes. By means of PCR with different sets of degenerate primers, three new homeobox sequences were identified, in addition to two previously found fragments. Following RACE amplification of the 3′ and 5′ flanking regions, we classified all five genes, Cnox-1 to Cnox-5, as belonging to the Antp-class. Homeodomain identity to the most similar HOM/Hox genes from Drosophila and human ranges from 58 to 72%. Each of the Cnox genes contains diagnostic residues of more than one Antp-class family known from triploblastic animals. These data are at odds with a previously proposed ancestral HOM/Hox cluster composition, with respect to both the number and types of Antp-class homeobox genes found. 1996 Academic Press, Inc.
INTRODUCTION The study of homeobox containing genes, which serve as transcription factors (for review and refs. see Scott et al., 1989; Gehring, 1993; Gehring et al., 1994), has generated a stimulating synthesis among the fields of molecular genetics, development, and evolution (e.g., Spradling, 1993; Quiring et al., 1994; Tautz, 1994; Schierwater, 1995). Homeobox genes, which are most likely ubiquitous among metazoans, were first discovered in Drosophila melanogaster and have subsequently been detected in most major phyla, including sponges, cnidarians, platyhelminthes, nematodes, annelids, arthropods, echinoderms, and chordates (e.g., McGinnis et al., 1984; Scott and Weiner, 1984; Dolecki et al., 1988; Bu¨rglin et al., 1989; Garcia-Ferna`ndez et al., 1991; Murtha et al., 1991; Schierwater et al., 1991; Oliver et al., 1992; Schummer et al., 1992; Dick and Buss, 1994; Seimiya et al., 1994). In the species from which homeobox genes have been mapped, they are arranged in clusters. Nematodes (Kenyon and Wang, 1055-7903/96 $18.00 Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tiation in head, trunk, and tail (3′, central- and 5′ precursor gene) (Averof and Akam, 1993; Schubert et al., 1993). Subsequent gene duplication events are believed to have extended the cluster in higher metazoa. Homeobox sequences in diploblastic metazoans are available from cnidarians (reported homeobox genes from sponges do not belong to the Antp-class, Seimiya et al., 1994). However, in contrast to the above scenario, Antp-class homeobox genes in cnidarians seemed to resemble the more anteriorly expressed genes at the 3′ end of the HOM cluster (lab-, pb-, and Dfd-like) (Murtha et al., 1991; Schierwater et al., 1991; Schummer et al., 1992). Therefore, Murtha et al. (1991) suggested that duplications of homeobox genes forming the cluster in higher metazoa have tended to occur in one direction: toward the 5′ end of the complex. Since Schummer et al. (1992) report that only two of five isolated homeobox genes from Chlorohydra were closely related to the Antp-class, and because of similar evidence from homeobox genes found in flatworms, it has been assumed that primitive organisms probably possess only a few Antp-class homeobox genes (Oliver et al., 1992). In order to shed some light on the early evolution of Antp-class homeobox genes, we performed an extended survey of Antp-class genes in a diploblastic cnidarian. We report the full-length homeodomain sequences of five Antp-class genes from the hydrozoan Eleutheria dichotoma. These findings are not consistent with a hypothetical ancestral head–trunk–tail gene model, which has been suggested for diploblasts. MATERIALS AND METHODS Amplification and Cloning of Homeobox Fragments E. dichotoma medusae were collected in Southern France (Banyuls-sur-Mer) and have been maintained in culture for several years (Schierwater, 1989). Total RNA was isolated from whole tissue of medusae and polyps, using a total RNA-Isolation kit (Promega). First-strand cDNA was synthesized with an oligo (dT)adapter–primer (Frohmann et al., 1988), M-MLV reverse transcriptase (Superscript, Gibco, BRL), and total RNA as template, following manufacturer’s protocol. Two different sets of degenerate primers, designed to recognize conserved regions in HOM/Hox genes, were used to PCR-amplify homeobox gene fragments from cDNA. The primers HoxA/HoxB (Murtha et al., 1991) amplify a 77-bp fragment from positions 62 to 138 of homeobox genes, whereas the primer set HoxE/ HoxF (Pendleton et al., 1993) amplifies an 82-bp fragment spanning positions 63 to 144. We used cDNA instead of genomic DNA templates for the amplification, since homeoboxes may be interrupted by introns (e.g., lab, pb, and Abd-B). PCR reactions were performed in 12.5-µl reaction volumes, containing 10 mM Tris–HCl,
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pH 7.5, 50 mM KCl, 2 mM MgCl2 , 0.1% gelatin, 0.1 mM each dNTP, 2.5 pmol each primer (HoxA/HoxB or HoxE/HoxF), 0.3 U Taq polymerase (BoehringerMannheim), and 5 ng cDNA. The cycling conditions for the PCR reactions were as follows: 92°C 2 min denaturation, followed by 30 cycles: 92°C (20 s), 45°C (20 s), and 72°C (30 s; ramp 1′06″) (Perkin-Elmer/Cetus Thermocycler 9600). PCR products were verified by gel electrophoresis on 2% agarose gels. All products with the expected size of 166 and 146 bp (homeobox fragments plus primer lengths), respectively, were cut out of the gel, and cloned into the pGEM-T Vector (Promega), according to manufacturer’s instructions. DNA minipreparations of single clones were performed as described in Sambrook et al. (1989). The isolated plasmids were checked for the presence of inserts by digesting with restriction endonucleases SacI and ApaI and separated on 2% agarose gels. Sequencing Both strands of the inserts were sequenced by means of cycle sequencing, using digoxigenin-labeled standard sequencing primers (Dig-Taq-sequencing kit, Boehringer-Mannheim). Sequencing reactions were performed in 8-µl reaction volume in the Perkin-Elmer/ Cetus Thermocycler 9600 programmed as follows: 95°C 1 min denaturation, followed by 25 cycles: 93°C (25 s), 43°C (15 s), and 72°C (15 s) using the fastest available temperature transitions. 5′RACE and 3′RACE (Rapid Amplification of cDNA Ends) Using sequence information from the homeobox fragments, 5′RACE and 3′RACE (Frohmann et al., 1988) were performed to amplify the cDNA ends of the homeobox genes in order to obtain full-length sequences. Four gene-specific primers were designed for each homeobox gene. For the 5′RACE procedure, the firststrand cDNA was purified with GLASSMAX Spin Cartridges (Gibco, BRL) according to manufacturer’s instructions, prior to the tailing reaction with cytosine (Frohmann et al., 1988). The oligo(dC)-tailed cDNA was used as template for the PCR reaction. Nested PCR was performed using the gene-specific outer (RACE-1) and inner (RACE-2) primers and Frohmann’s Anchor Primer and Universal Amplification Primer. For the 3′RACE, the first-strand cDNA was used without modification in the PCR reaction. The gene-specific outer and inner primers were used in combination with Frohmann’s 3′RACE primers, both corresponding to sequences of the oligo(dT)adapter–primer, which was used in the reverse transcription reaction. The PCR reaction conditions for RACE were identical to those described above. Primary RACE reactions were carried out as follows: 90°C 2 min denaturation, followed by 35 cycles: 92°C (20 s), 50°C (20 s), and 72°C (30 s; ramp 1′06″) followed by 72°C for 2 min. Secondary RACE re-
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actions were performed using 0.1 µl of the RACE-1 reactions as template. The PCR program was identical to RACE-1, except that the annealing temperature was increased to 52°C and 40 instead of 30 cycles were performed. Digoxigenin Labeling of DNA Sequenced plasmids, each containing a specific homeobox gene fragment, were used as templates to PCRamplify specific Cnox- gene fragments for the Dig-labeling reactions. We used the primer set, HoxE/HoxF, and identical PCR conditions as used for the amplification of homeobox fragments (see above). One hundred nanograms of the PCR products (146 bp) was directly labeled with digoxigenin–dUTP, using the random primed DNA labeling kit (Boehringer-Mannheim), and used as probes in Southern analyses. Southern Analyses Typically, multiple bands resulted from the second RACE reactions. Consequently, the RACE-2 products were separated on 1.4% agarose gels and transferred onto a charged nylon membrane (Boehringer-Mannheim) via alkaline blotting for subsequent Southern hybridization (Sambrook et al., 1989). The blots were treated according to the manufacturer’s instructions (DIG Nucleic Acid Detection kit, Boehringer-Mannheim). Hybridization was carried out overnight at 42°C in a buffer containing 50% formamide, 53 SSC, 2% blocking reagent (Boehringer-Mannheim), 0.1% (w/v) N-lauroylsarcosine, and 40 ng/ml Dig-labeled DNA. The blots were washed twice with 23 SSC (0.1% SDS)
at RT for 5 min and then twice with 0.53 SSC (0.1% SDS) at 68°C for 15 min. The hybridized probes were detected by enzyme immunoassay according to the manufacturer’s protocol (DIG Nucleic Acid Detection kit, Boehringer-Mannheim). Positive 5′ and 3′RACE-2 fragments were cloned and sequenced as described above. RESULTS Identification of Three New Homeobox Genes Using two different sets of degenerate oligonucleotides, we PCR-amplified different homeobox fragments from E. dichotoma medusae and polyp first-strand cDNA. Sequencing a total of 22 clones, we identified three new homeobox genes, Cnox-3, Cnox-4, and Cnox5, as well as the two previously reported genes, Cnox1 and Cnox-2 (Schierwater et al., 1991). All genes were amplified using the primer set HoxE/HoxF, and only Cnox-2 was amplified with both primer sets (HoxA/ HoxB and HoxE/HoxF). 5′RACE and 3′RACE revealed the full-length sequences for all five homeoboxes (Fig. 2; Schierwater et al., in preparation) needed for the identification of certain residues that are diagnostic for the different Antp-class gene families (Table 1). Antp-Class Genes Comparison of the 60 amino acid residues of E. dichotoma homeodomains with Drosophila and vertebrate homeodomains revealed that the four invariant positions in the third helix, as well as the eight highly
TABLE 1 E. dichotoma Homeodomains Aligned with Selected HOM/Hox-Type Genes of Drosophila and Human
Note. Asterisks below the Antp reference sequence indicate the eight highly conserved positions among homeodomains (homeodomain positions according to Gehring et al., 1990). Open circles denote amino acids at the four invariant positions. Eleutheria homeodomains aligned with selected homeodomain sequences from Drosophila and human (Hox: for further sequences see Duboule, 1994). Dashes indicate identity with the Drosophila Antp reference sequence. Amino acid sequences are taken from the UEMBL40 GenBank (October 1994).
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TABLE 2 Comparison of E. dichotoma Homeodomains with other HOM/Hox Genes
lab pb zen1 zen2 Dfd Scr ftz Antp Ubx abd-A Abd-B Hox-B3 Hox-D3 Hox-A4 Hox-C4 Hox-D4 Hox-B5 Hox-B6 Hox-B7
Cnox-1 (%)
Cnox-2 (%)
Cnox-3 (%)
Cnox-4 (%)
Cnox-5 (%)
52 53 53 57 63 62 65 65 63 62 55 65 63 60 62 62 63 62 65
62 67 67 67 70 68 65 67 60 63 53 68 70 72 68 70 70 65 67
52 53 55 60 60 63 58 60 57 58 55 57 58 60 57 58 58 58 60
50 50 52 52 57 55 55 55 55 55 50 53 52 55 58 57 57 55 55
67 57 58 57 65 65 58 65 62 65 52 65 67 60 63 62 62 67 67
Note. Comparison of Cnox-1 to Cnox-5 homeodomains from E. dichotoma with HOM-C homeodomains of Drosophila and the most similar human homeodomains. The percent identities are based on the comparison of the 60 amino acids of the homeodomains. Numbers in bold indicate the most similar sequences. Amino acid sequences are taken from the UEMBL40 85 GenBank (October 1994).
conserved and homeodomain diagnostic positions (Scott et al., 1989), are present in all five homeodomains from E. dichotoma (Table 1), except for the substitution of the Ile45 residue by Val45 in Cnox-2 and Cnox-3 and the Lys57 residue by Arg57 in Cnox-4. For further classification of the five homeodomains a SwissProt 30 (release October 1994) data base search was performed, searching for proteins with 50% or more sequence similarity allowing no gaps. All most similar homeodomains found belonged to the Antpclass, indicating that the five homeodomains of E. dichotoma likewise belong to this class. As shown in Table 2, each Cnox homeodomain of E. dichotoma shows about equal similarity to Antp-class homeodomains from different families. For example, Cnox-1 shows 65% similarity with ftz, Antp-, Hox-B3, and Hox-B7, and Cnox-5 is 67% identical to lab, Hox-D3, Hox-B6, as well as to Hox-B7. Since a high number of closely related homeodomains was found, we looked for diagnostic amino acid residues which are conserved within distinct families of Antp-class genes, but which vary between families (Scott et al., 1989). Representatives of distinct families are listed in Table 1. Surprisingly, we could find conserved residues for diverse homeobox families at
various positions in each of the five Cnox genes, and no unequivocal one-to-one correspondents in Drosophila or vertebrates were identified. For example, Cnox-5 has the archetypal lab phenylalanine residue at position 8, and alanine at position 29, but also the Abd-B typical lysine at positions 3 and 4 (see Table 1). Outside the homeoboxes, we searched for the short conserved hexapeptide or pentapeptide sequence which is commonly located 15 bp upstream of the homeobox in Antp-class homeobox genes (excluding Abd-B). These motifs were not found in any deduced protein sequence of Eleutheria homeobox genes. Since the hexapeptide does not exist in Abd-B-like homeoboxes, we looked for conserved residues specific for this gene. The typical GC rich region at the 5′ end of Abd-B-like genes, encoding a high number of proline, glycine, and alanine residues, as well as the Trp–Met duplet, does not exist in any of the five homeobox genes. However, the conserved tryptophan residue at position 27 upstream from the homeobox, which has not been found at this position in any homeobox other than Abd-B-like genes (Izpisu´a-Belmonte et al., 1991), is present in Cnox-1 and Cnox-3. Thus, none of the five homeobox genes of E. dichotoma can be grouped to any distinct family of Antp-class homeobox genes; rather, all of them represent intermediate forms. These findings are supported by both cluster and parsimony analyses (see Figs. 3 and 4). In contrast to some previously published trees, which were based on partial sequence information of homeoboxes, the trees built upon the full-length homeobox nucleotide sequences (180 bp) do not suggest homologous counterparts for any Cnox gene to Drosophila or human Antp-class genes. Other Cnidarian Homeodomains The homeobox genes in E. dichotoma are only slightly related to each other, but as shown in Table 3, Cnox-1, Cnox-2, and Cnox-5 correspond to homeobox genes from other cnidarian species. Since Cnox-3 and Cnox-4 seem to be different, we placed them into separate groups (see Table 3). DISCUSSION Both the number and types of Antp-class homeobox genes found in the cnidarian E. dichotoma confound existing views on the ancestral state of this gene class at the time of the origin of triploblastic animals from diploblastic ancestors. The findings suggest that conclusions drawn solely from information on triploblastic organisms may result in an oversimplified picture and that the evolution of Antp-class homeobox genes may have been more complex than expected. The large number of Antp-class genes found in Eleutheria was unexpected (cf. Oliver et al., 1992; Miller and Miles, 1993) and does not easily fit to an ancestral head, trunk, and tail model, which has been
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TABLE 3 Alignment of E. dichotoma Homeodomains with Homeodomains from Other Cnidarian Species
Note. Deduced amino acid sequences of homeobox genes in E. dichotoma compared with other cnidarian homeodomains. Dashes indicate amino acid residues identical with the Antp gene of Drosophila. Cnox-1 from E. dichotoma shows highest similarity with Cnox3 from Hydra (80%), Cnox-2 is 100% identical to SAox2 and 98% identical to Cnox2 from Chlorohydra, H. vulgaris, and H. magnipapillata, and Cnox-5 shows 84% similarity to SAox1. The most similar homeobox gene to Cnox-3 (62%) as well as to Cnox-4 (58%) is Cnox5 of H. magnipapillata. References: Sarsia sp. (Murtha et al., 1991), Hydractinia symbiolongicarpus (Schierwater et al., 1991), Chlorohydra viridissima (Schummer et al., 1992), H. magnipapillata (Naito et al., 1993), H. vulgaris (Schenk et al., 1993).
proposed for diploblasts (Fig. 1; Averof and Akam, 1993; Schubert et al., 1993). Additional, and more important, evidence against an ancestral three-gene-cluster model in cnidarians derives from the characterization of the Cnox genes. Phylogenetic reconstructions from Drosophila and vertebrate HOM/Hox genes suggested a three-gene-cluster (head, trunk, and tail) which evolved before the separation of diploblasts and triploblasts more than 600 million years ago (Schubert et al., 1993). Under this hypothesis one should expect to find genes in E. dichotoma to be lab-like (3′ or head), Antp-like (central or trunk: Dfd, Scr, Antp, Ubx, and abd-A), and Abd-B-like (5′ or tail). Our results do not readily conform to this expectation. The Eleutheria Cnox genes are not closely related to any distinct homeobox gene family; rather, they simultaneously share features with different families. Thus, for none of the Cnox genes a homologue to Drosophila or vertebrate homeobox genes can be defined. The latter is further supported by tree inferring analyses if homeobox sequences are used. Based on the fact that the characterized homeobox genes of E. dichotoma do not belong to any distinct homeobox gene family, our data fail to support the hypothesis that cnidarian homeobox genes are more related to the genes at the 3′ end than to genes
at the 5′ end of the insect and vertebrate homeobox clusters (Murtha et al., 1991). Our findings generally agree with a relationship analysis of 337 homeodomains using a distance matrix approach by Kappen et al. (1993). At least 30 distinguishable families were generated, with cnidarian homeobox genes divided into several separate families. The authors assumed that the majority of clearly definable homeobox families were established prior to the emergence of the vertebrate, arthropod, and even nematode lineages. Furthermore, they point out that once established, a distinct homeobox family should be strongly conserved in its sequence (cf. Bu¨rglin, 1994). Naito et al. (1993) proposed eight homeobox families in cnidarians, three of them consisting of non-homeotic genes (even-skipped-like, msh-like, and BarH2-like). Although this analysis was based on only 25 amino acid residues for most homeodomains, together with our findings it seems obvious that distinct homeobox families do exist for cnidarians which differ from the HOM/ Hox families known for triploblasts. For example, similarities between 96 and 100% are found in one family, which consists of six Cnox-2 homeodomains from different hydrozoans (Hydra vulgaris, Hydra magnipapillata, E. dichotoma, Hydractinia symbiolongicarpus,
CNIDARIAN HOMEOBOX GENES
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FIG. 1. Suggested evolution of homeobox gene clusters. The proposed homeobox gene cluster of the arthropod/vertebrate ancestor is shown with six precursor genes (Holland, 1990, 1992; Bu¨rglin and Ruvkum, 1993; Garcia-Ferna`ndez and Holland, 1994). The evolution of Scr (Drosophila) and the paralog group 4 (human) precursor gene is controversial (Averof and Akam, 1993; Schubert et al., 1993; Kappen and Ruddle, 1993). The last common ancestral homeobox cluster of arthropods and nematodes is taken from Bu¨rglin and Ruvkun (1993). Precursor genes of triploblasts and diploblasts are described in Schubert et al. (1993). The arrangement of the precursor genes shown is hypothetical. Abbreviations are as follows: lab (labial), pb ( proboscipedia), zen1 (zerknu¨llt), zen2, Dfd (deformed ), Scr (Sex combs reduced), ftz (fushi tarazu), Antp (Antennapedia), Ubx (Ultrabithorax), abd-A (abdominal-A), Abd-B (Abdominal-B).
Chlorohydra viridissima, and Sarsia sp.) (see Table 3). Nevertheless, homeobox genes belonging to this family have previously been classified as homologous to three different families ( pb, Dfd, and Gsh-1/Gsh-2) (Murtha et al., 1991; Schummer et al., 1992; Bu¨rglin, 1994). The homeobox genes of E. dichotoma represent intermediate sequences of the distinct Antp-families of Drosophila and vertebrates. These findings call for more elaborate explanations concerning the types of homeobox genes present in a hypothetical ancestor of cnidarians and triploblasts. Before doing so, two main objections, however, should be considered: (I) Since cnidarians diverged more than 600 million years ago, one may argue against a high degree of sequence conservation between cnidarian and Drosophila homeobox genes. On the other hand, conserved regions diagnostic for distinct homeobox families, as well as the complex organization of the cluster, have been maintained for over 500 million years in nematodes, Drosophila, and humans. High sequence conservation was also found between flatworms and Drosophila Antp-class genes (Oliver et al., 1992). Furthermore, it must be noted that cnidarian homeobox genes which do not belong to the Antp-class, like msh from C. viridissima (Schummer et al., 1992) and eveC from Acropora formosa (Miller and Miles, 1993), do have homologous counterparts in Dro-
sophila. (II) It could be argued that Eleutheria is a very derived organism, not representing the typical picture for hydrozoa in general. However, this argument is not compelling, because other hydrozoan species exhibit homologous sequences with the same peculiarities. In addition, other available DNA sequence data do not support a particularly derived position for Eleutheria within the hydrozoa (Bridge, 1994). More likely explanations may be based on the assumption that the proposed head–trunk–tail precursor genes do not exist in the hydrozoa. At present, it seems more likely that the cnidarian lineages diverged prior to the fixation of the Antp-families present in nematodes, arthropods, and vertebrates and that a proposed ancestral three-gene Antp-class cluster may be novel to triploblasts. A common ancestor of hydrozoans and triploblasts may have possessed a single Antp-class precursor gene. Independent duplications of this precursor gene could have led to different distinct Antp-families in both lineages. At this point, information on the chromosomal arrangement of Cnox genes, i.e., whether these Antp-class genes are arranged in clusters or not, would not resolve the questions. We would need more comparative information on Antp-class genes in other diploblastic animals, including the anthozoan cnidarians (so far only one Antp-
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FIG. 3. UPGMA-dendrogram of E. dichotoma, Drosophila, and human homeobox nucleotide sequences (180 bp) of the 17 homeotic genes in Table 2. The tree was created with the software Treecon (Van de Peer and De Wachter, 1993), using the distance metric of Kimura (1980).
FIG. 2. Nucleotide and derived amino acid sequences of three new Antp-class homeobox genes from E. dichotoma (Cnox-3 to Cnox5). The homeodomains are underlined.
FIG. 4. Parsimony analysis (PAUP, Swofford, 1991) of unweighted nucleotide sequences of homeoboxes from E. dichotoma, Drosophila, and human (see Fig. 3). The tree is a 50% majority-rule consensus of eight equally parsimonious trees, found by heuristic search using random addition (100 replicates) and subtree-pruning – regrafting branch-swapping. Tree length is 693 steps, consistency index is 0.397, and retention index 0.423.
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class homeobox gene has been detected outside the hydrozoa (Miller and Miles, 1993)), before the evolution of HOM/Hox genes in early metazoan evolution can be adduced.
of the homeodomain and its functional implications. Trends Genet. 6: 323–329. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G., and Wu¨thrich, K. (1994). Homeodomain-DNA recognition. Cell 78: 211–223.
ACKNOWLEDGMENTS
Graham, A., Papalopulu, N., and Krumlauf, R. (1989). The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57: 367–378. Holland, P. W. H. (1990). Homeobox genes and segmentation: Cooption, co-evolution, and convergences. Dev. Biol. 1: 135–145.
We acknowledge collaborative research with Leo Buss, comments and critical discussions from Cort Anderson, Leo Buss, Rob DeSalle, Matt Dick, Klaus Schwenk, and Thomas Sta¨dler, and financial support from DFG (277 Schi-3-1), NATO (CRG-91004), NSF (IBN9119437), BMFT (Helmholtz-Programm), and BMFT support to B. Streit.
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Holland, P. W. H. (1992). Homeobox genes in vertebrate evolution. BioEssays 14: 267–272. Izpisu´a-Belmonte, J.-C., Falkenstein, H., Dolle´, P., Renucci, A., and Duboule, D. (1991). Murine genes related to Drosophila Abd-B homeotic gene are sequentially expressed during development of the posterior part of the body. EMBO J. 10: 2279–2289. Kappen, C., and Ruddle, F. H. (1993). Evolution of a regulatory gene family: HOM/HOX genes. Curr. Opin. Genet. Dev. 3: 931–938. Kappen, C., Schughart, K., and Ruddle, F. H. (1993). Early evolutionary origin of major homeodomain sequence classes. Genomics 18: 54–70. Kaufmann, T. C., Lewis, R., and Wakimoto, B. (1980). Cytogenetic analysis of chromosome 3 in Drosophila melanogaster. The homeotic gene complex in polytene chromosomal interval 84A, B. Genetics 94: 115–133. Kaufmann, T. C., Seeger, M., and Olsen, G. (1990). Molecular and genetic organization of the Antennapedia gene complex of Drosophila melanogaster. Adv. Genet. 27: 309–362. Kenyon, C. (1994). If birds can fly, why can’t we? Homeotic genes and evolution. Cell 78: 175–180. Kenyon, C., and Wang. B. (1991). A cluster of Antennapedia-class homeobox genes in a nonsegmented animal. Science 253: 516–517. Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111–120. Krumlauf, R. (1992). Evolution of the vertebrate HOX homeobox genes. BioEssays 14: 245–251. Krumlauf, R. (1994). HOX genes in vertebrate development. Cell 78: 191–201. Lawrence, P. A., and Morata, G. (1994). Homeobox genes: Their function in segmentation and pattern formation of Drosophila. Cell 78: 181–189. Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276: 565–570. McGinnis, W., Levine, M., Hafen, E., Kuroiwa, A., and Gehring, W. J. (1984). A conserved DNA sequence in homeotic genes of the Drosophila Antennapedia and Bithorax complexes. Nature 308: 428– 433. McGinnis, W., and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68: 283–302. Miller, D. J., and Miles, A. (1993). Homeobox and the zootype. Nature 365: 215–216. Murtha, M. T., Leckman, J. F., and Ruddle, F. H. (1991). Detection of homeobox genes in development and evolution. Proc. Natl. Acad. Sci. USA 88: 10711–10715. Naito, M., Ishiguro, H., Fujisawa, T., and Kurosawa, Y. (1993). Presence of eight distinct homeobox-containing genes in cnidarians. FEBS Lett. 333: 271–274. Oliver, G., Vispo, M., Mailhos, A., Martinez, C., Sosa-Pineda, B., Fielitz, W., and Ehrlich, R. (1992). Homeboxes in flatworms. Gene 121: 337–342. Pendleton, J. W., Nagai, B. N., Murtha, M. T., and Ruddle, F. H.
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(1993). Expansion of the Hox gene family and the evolution of chordates. Proc. Natl. Acad. Sci. USA 90: 6300–6304. Quiring, R., Walldorf, U., Kloter, U., and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the small eye gene in mice and Aniridia in humans. Science 265: 785–789. Salser, S. J., and Kenyon, C. (1994). Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations. Trends Genet. 10: 159– 164. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schenk, M. A., Bode, H. R., and Steele, R. E. (1993). Expression of Cnox2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation. Development 117: 657–667. Schierwater, B. (1989). Allometric changes during growth and reproduction in Eleutheria dichotoma (Hydrozoa, Athecata) and the problem of estimating body size in a microscopic animal. J. Morphol. 200: 255–267. Schierwater, B. (1995). Developmental genes as a potential tool to population ecology? Experientia 51: 539–544. Schierwater, B., Murtha, M. T., Dick, M. H., Ruddle, F. H., and Buss, L. W. (1991). Homeoboxes in Cnidarians. J. Exp. Zool. 260: 413– 416. Schubert, F. R., Nieselt-Struwe, K., and Gruss, P. (1993). The Antennapedia-type homeobox genes have evolved from three precursors separated early in metazoan evolution. Proc. Natl. Acad. Sci. USA 90: 143–147. Schummer, M., Scheurlen, I., Schaller, C., and Galliot, B. (1992).
HOM/HOX homeobox genes are present in hydra (Chlorohydra viridissima) and are differentially expressed during regeneration. EMBO J. 11: 1815–1823. Scott, M. P., and Weiner, A. J. (1984). Structural relationships among genes that control development: sequence homology between the Antennapedia, Ultrabithorax and fushi tarazu loci of Drosophila. Proc. Natl. Acad. Sci. USA 81: 4115–4119. Scott, M. P., Tamkun, J. W., and Hartzell, G. W. (1989). The structure and function of the homeodomain. Biochim. Biophys. Acta 989: 25– 38. Seimiya, M., Ishiguro, K., Miura, K., and Watanabe, Y. (1994). Homeobox-containing genes in the most primitive metazoa, the sponges. Eur. J. Biochem. 221: 219–225. Spradling, A. C. (1993). ‘‘Evolutionary Conservation of Developmental Mechanisms,’’ Wiley–Liss, New York. Stuart, J., Brown, S., Beeman, R., and Denell, R. (1991). A deficiency of the homeotic complex of the beetle Tribolium. Nature 350: 72– 74. Swofford, D. L. (1991). PAUP: Phylogenetic Analysis Using Parsimony, version 3.1. Illinois Natural History Survey, Champaign. Tautz, D. (1994). Evolutionary analysis of genes involved in early embryonic pattern formation in Drosophila. In ‘‘Molecular Ecology and Evolution: Approaches and Applications’’ (B. Schierwater, B. Streit, G. P. Wagner, and R. DeSalle, Eds.), pp. 525–536, Birkha¨user, Basel/Boston. Van de Peer, Y., and De Wachter, R. (1993). Treecon: A software package for the construction and drawing of evolutionary trees. Comput. Appl. Biosci. 9: 177–182.
Vol. 6, No. 1, August, pp. 30–38, 1996 ARTICLE NO. 0055
Homeobox Genes in the Cnidarian Eleutheria dichotoma: Evolutionary Implications for the Origin of Antennapedia-Class (HOM/Hox) Genes KERSTIN KUHN,* BRUNO STREIT,*
AND
BERND SCHIERWATER*,†
*Department of Ecology and Evolution, J.W. Goethe-Universita¨t, Postfach 111932, Siesmayerstr. 70, D-60054 Frankfurt am Main Germany; and †Yale University, Department of Biology, 165 Prospect Street, New Haven, Connecticut 06511 Received June 5, 1995; revised October 24, 1995
1991; Salser and Kenyon, 1994) and most arthropods (Beeman et al., 1989; Averof and Akam, 1993) possess a single homeobox gene cluster. The homeobox cluster (HOM-C) in Drosophila is subdivided into two complexes, the Antennapedia (Ant-C) (Kaufmann et al., 1980, 1990) and Bithorax complexes (BX-C) (Lewis, 1978; Duncan, 1987). This split probably occurred late in insect evolution (Akam, 1989; Kenyon, 1994). The HOM-C contains the Antennapedia (Antp)-class genes: eight homeotic genes, which specify pattern formation along the anteroposterior body axis (for reviews see McGinnis and Krumlauf, 1992; Lawrence and Morata, 1994), plus three non-homeotic genes (zen1, zen2, ftz). Vertebrates possess four homeobox gene clusters (HoxA to Hox-D) located on four different chromosomes, each containing between 9 and 13 Antp-class homeobox genes (for review see Krumlauf, 1992, 1994). The four clusters most likely arose through gene cluster duplication events, followed by the loss of some homeobox genes (Garcia-Ferna`ndez and Holland, 1994). Among nematodes, arthropods, and vertebrates, the linear organization of homeobox genes along the chromosome correlates with their expression patterns along the anterior–posterior axis of the body (Duboule and Dolle´, 1989; Graham et al., 1989; Bu¨rglin et al., 1991; Stuart et al., 1991). The nearer the genes are located to the 3′ end of the chromosome, the more anterior is their expression in the embryo. Based on sequence similarities and conservation of genomic organization, a common ancestral homeobox gene cluster for vertebrates and arthropods as well as for arthropods and nematodes has been proposed (Fig. 1; Holland, 1992; Averof and Akam, 1993; Bu¨rglin and Ruvkun, 1993; Kappen and Ruddle, 1993; Schubert et al., 1993; Garcia-Ferna`ndez and Holland, 1994). Phylogenetic analyses of vertebrate and arthropod homeobox genes suggested the presence of a single gene cluster containing three Antp-class homeobox genes early in metazoan evolution (before the separation of diploblasts and triploblasts). This hypothetical ancestral cluster should contain homeobox genes corresponding to the differen-
In order to test current hypotheses on the early evolution of Antp-class (HOM/Hox) genes in metazoan animals, we surveyed the diploblastic hydrozoan Eleutheria dichotoma for the presence of these genes. By means of PCR with different sets of degenerate primers, three new homeobox sequences were identified, in addition to two previously found fragments. Following RACE amplification of the 3′ and 5′ flanking regions, we classified all five genes, Cnox-1 to Cnox-5, as belonging to the Antp-class. Homeodomain identity to the most similar HOM/Hox genes from Drosophila and human ranges from 58 to 72%. Each of the Cnox genes contains diagnostic residues of more than one Antp-class family known from triploblastic animals. These data are at odds with a previously proposed ancestral HOM/Hox cluster composition, with respect to both the number and types of Antp-class homeobox genes found. 1996 Academic Press, Inc.
INTRODUCTION The study of homeobox containing genes, which serve as transcription factors (for review and refs. see Scott et al., 1989; Gehring, 1993; Gehring et al., 1994), has generated a stimulating synthesis among the fields of molecular genetics, development, and evolution (e.g., Spradling, 1993; Quiring et al., 1994; Tautz, 1994; Schierwater, 1995). Homeobox genes, which are most likely ubiquitous among metazoans, were first discovered in Drosophila melanogaster and have subsequently been detected in most major phyla, including sponges, cnidarians, platyhelminthes, nematodes, annelids, arthropods, echinoderms, and chordates (e.g., McGinnis et al., 1984; Scott and Weiner, 1984; Dolecki et al., 1988; Bu¨rglin et al., 1989; Garcia-Ferna`ndez et al., 1991; Murtha et al., 1991; Schierwater et al., 1991; Oliver et al., 1992; Schummer et al., 1992; Dick and Buss, 1994; Seimiya et al., 1994). In the species from which homeobox genes have been mapped, they are arranged in clusters. Nematodes (Kenyon and Wang, 1055-7903/96 $18.00 Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tiation in head, trunk, and tail (3′, central- and 5′ precursor gene) (Averof and Akam, 1993; Schubert et al., 1993). Subsequent gene duplication events are believed to have extended the cluster in higher metazoa. Homeobox sequences in diploblastic metazoans are available from cnidarians (reported homeobox genes from sponges do not belong to the Antp-class, Seimiya et al., 1994). However, in contrast to the above scenario, Antp-class homeobox genes in cnidarians seemed to resemble the more anteriorly expressed genes at the 3′ end of the HOM cluster (lab-, pb-, and Dfd-like) (Murtha et al., 1991; Schierwater et al., 1991; Schummer et al., 1992). Therefore, Murtha et al. (1991) suggested that duplications of homeobox genes forming the cluster in higher metazoa have tended to occur in one direction: toward the 5′ end of the complex. Since Schummer et al. (1992) report that only two of five isolated homeobox genes from Chlorohydra were closely related to the Antp-class, and because of similar evidence from homeobox genes found in flatworms, it has been assumed that primitive organisms probably possess only a few Antp-class homeobox genes (Oliver et al., 1992). In order to shed some light on the early evolution of Antp-class homeobox genes, we performed an extended survey of Antp-class genes in a diploblastic cnidarian. We report the full-length homeodomain sequences of five Antp-class genes from the hydrozoan Eleutheria dichotoma. These findings are not consistent with a hypothetical ancestral head–trunk–tail gene model, which has been suggested for diploblasts. MATERIALS AND METHODS Amplification and Cloning of Homeobox Fragments E. dichotoma medusae were collected in Southern France (Banyuls-sur-Mer) and have been maintained in culture for several years (Schierwater, 1989). Total RNA was isolated from whole tissue of medusae and polyps, using a total RNA-Isolation kit (Promega). First-strand cDNA was synthesized with an oligo (dT)adapter–primer (Frohmann et al., 1988), M-MLV reverse transcriptase (Superscript, Gibco, BRL), and total RNA as template, following manufacturer’s protocol. Two different sets of degenerate primers, designed to recognize conserved regions in HOM/Hox genes, were used to PCR-amplify homeobox gene fragments from cDNA. The primers HoxA/HoxB (Murtha et al., 1991) amplify a 77-bp fragment from positions 62 to 138 of homeobox genes, whereas the primer set HoxE/ HoxF (Pendleton et al., 1993) amplifies an 82-bp fragment spanning positions 63 to 144. We used cDNA instead of genomic DNA templates for the amplification, since homeoboxes may be interrupted by introns (e.g., lab, pb, and Abd-B). PCR reactions were performed in 12.5-µl reaction volumes, containing 10 mM Tris–HCl,
31
pH 7.5, 50 mM KCl, 2 mM MgCl2 , 0.1% gelatin, 0.1 mM each dNTP, 2.5 pmol each primer (HoxA/HoxB or HoxE/HoxF), 0.3 U Taq polymerase (BoehringerMannheim), and 5 ng cDNA. The cycling conditions for the PCR reactions were as follows: 92°C 2 min denaturation, followed by 30 cycles: 92°C (20 s), 45°C (20 s), and 72°C (30 s; ramp 1′06″) (Perkin-Elmer/Cetus Thermocycler 9600). PCR products were verified by gel electrophoresis on 2% agarose gels. All products with the expected size of 166 and 146 bp (homeobox fragments plus primer lengths), respectively, were cut out of the gel, and cloned into the pGEM-T Vector (Promega), according to manufacturer’s instructions. DNA minipreparations of single clones were performed as described in Sambrook et al. (1989). The isolated plasmids were checked for the presence of inserts by digesting with restriction endonucleases SacI and ApaI and separated on 2% agarose gels. Sequencing Both strands of the inserts were sequenced by means of cycle sequencing, using digoxigenin-labeled standard sequencing primers (Dig-Taq-sequencing kit, Boehringer-Mannheim). Sequencing reactions were performed in 8-µl reaction volume in the Perkin-Elmer/ Cetus Thermocycler 9600 programmed as follows: 95°C 1 min denaturation, followed by 25 cycles: 93°C (25 s), 43°C (15 s), and 72°C (15 s) using the fastest available temperature transitions. 5′RACE and 3′RACE (Rapid Amplification of cDNA Ends) Using sequence information from the homeobox fragments, 5′RACE and 3′RACE (Frohmann et al., 1988) were performed to amplify the cDNA ends of the homeobox genes in order to obtain full-length sequences. Four gene-specific primers were designed for each homeobox gene. For the 5′RACE procedure, the firststrand cDNA was purified with GLASSMAX Spin Cartridges (Gibco, BRL) according to manufacturer’s instructions, prior to the tailing reaction with cytosine (Frohmann et al., 1988). The oligo(dC)-tailed cDNA was used as template for the PCR reaction. Nested PCR was performed using the gene-specific outer (RACE-1) and inner (RACE-2) primers and Frohmann’s Anchor Primer and Universal Amplification Primer. For the 3′RACE, the first-strand cDNA was used without modification in the PCR reaction. The gene-specific outer and inner primers were used in combination with Frohmann’s 3′RACE primers, both corresponding to sequences of the oligo(dT)adapter–primer, which was used in the reverse transcription reaction. The PCR reaction conditions for RACE were identical to those described above. Primary RACE reactions were carried out as follows: 90°C 2 min denaturation, followed by 35 cycles: 92°C (20 s), 50°C (20 s), and 72°C (30 s; ramp 1′06″) followed by 72°C for 2 min. Secondary RACE re-
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actions were performed using 0.1 µl of the RACE-1 reactions as template. The PCR program was identical to RACE-1, except that the annealing temperature was increased to 52°C and 40 instead of 30 cycles were performed. Digoxigenin Labeling of DNA Sequenced plasmids, each containing a specific homeobox gene fragment, were used as templates to PCRamplify specific Cnox- gene fragments for the Dig-labeling reactions. We used the primer set, HoxE/HoxF, and identical PCR conditions as used for the amplification of homeobox fragments (see above). One hundred nanograms of the PCR products (146 bp) was directly labeled with digoxigenin–dUTP, using the random primed DNA labeling kit (Boehringer-Mannheim), and used as probes in Southern analyses. Southern Analyses Typically, multiple bands resulted from the second RACE reactions. Consequently, the RACE-2 products were separated on 1.4% agarose gels and transferred onto a charged nylon membrane (Boehringer-Mannheim) via alkaline blotting for subsequent Southern hybridization (Sambrook et al., 1989). The blots were treated according to the manufacturer’s instructions (DIG Nucleic Acid Detection kit, Boehringer-Mannheim). Hybridization was carried out overnight at 42°C in a buffer containing 50% formamide, 53 SSC, 2% blocking reagent (Boehringer-Mannheim), 0.1% (w/v) N-lauroylsarcosine, and 40 ng/ml Dig-labeled DNA. The blots were washed twice with 23 SSC (0.1% SDS)
at RT for 5 min and then twice with 0.53 SSC (0.1% SDS) at 68°C for 15 min. The hybridized probes were detected by enzyme immunoassay according to the manufacturer’s protocol (DIG Nucleic Acid Detection kit, Boehringer-Mannheim). Positive 5′ and 3′RACE-2 fragments were cloned and sequenced as described above. RESULTS Identification of Three New Homeobox Genes Using two different sets of degenerate oligonucleotides, we PCR-amplified different homeobox fragments from E. dichotoma medusae and polyp first-strand cDNA. Sequencing a total of 22 clones, we identified three new homeobox genes, Cnox-3, Cnox-4, and Cnox5, as well as the two previously reported genes, Cnox1 and Cnox-2 (Schierwater et al., 1991). All genes were amplified using the primer set HoxE/HoxF, and only Cnox-2 was amplified with both primer sets (HoxA/ HoxB and HoxE/HoxF). 5′RACE and 3′RACE revealed the full-length sequences for all five homeoboxes (Fig. 2; Schierwater et al., in preparation) needed for the identification of certain residues that are diagnostic for the different Antp-class gene families (Table 1). Antp-Class Genes Comparison of the 60 amino acid residues of E. dichotoma homeodomains with Drosophila and vertebrate homeodomains revealed that the four invariant positions in the third helix, as well as the eight highly
TABLE 1 E. dichotoma Homeodomains Aligned with Selected HOM/Hox-Type Genes of Drosophila and Human
Note. Asterisks below the Antp reference sequence indicate the eight highly conserved positions among homeodomains (homeodomain positions according to Gehring et al., 1990). Open circles denote amino acids at the four invariant positions. Eleutheria homeodomains aligned with selected homeodomain sequences from Drosophila and human (Hox: for further sequences see Duboule, 1994). Dashes indicate identity with the Drosophila Antp reference sequence. Amino acid sequences are taken from the UEMBL40 GenBank (October 1994).
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TABLE 2 Comparison of E. dichotoma Homeodomains with other HOM/Hox Genes
lab pb zen1 zen2 Dfd Scr ftz Antp Ubx abd-A Abd-B Hox-B3 Hox-D3 Hox-A4 Hox-C4 Hox-D4 Hox-B5 Hox-B6 Hox-B7
Cnox-1 (%)
Cnox-2 (%)
Cnox-3 (%)
Cnox-4 (%)
Cnox-5 (%)
52 53 53 57 63 62 65 65 63 62 55 65 63 60 62 62 63 62 65
62 67 67 67 70 68 65 67 60 63 53 68 70 72 68 70 70 65 67
52 53 55 60 60 63 58 60 57 58 55 57 58 60 57 58 58 58 60
50 50 52 52 57 55 55 55 55 55 50 53 52 55 58 57 57 55 55
67 57 58 57 65 65 58 65 62 65 52 65 67 60 63 62 62 67 67
Note. Comparison of Cnox-1 to Cnox-5 homeodomains from E. dichotoma with HOM-C homeodomains of Drosophila and the most similar human homeodomains. The percent identities are based on the comparison of the 60 amino acids of the homeodomains. Numbers in bold indicate the most similar sequences. Amino acid sequences are taken from the UEMBL40 85 GenBank (October 1994).
conserved and homeodomain diagnostic positions (Scott et al., 1989), are present in all five homeodomains from E. dichotoma (Table 1), except for the substitution of the Ile45 residue by Val45 in Cnox-2 and Cnox-3 and the Lys57 residue by Arg57 in Cnox-4. For further classification of the five homeodomains a SwissProt 30 (release October 1994) data base search was performed, searching for proteins with 50% or more sequence similarity allowing no gaps. All most similar homeodomains found belonged to the Antpclass, indicating that the five homeodomains of E. dichotoma likewise belong to this class. As shown in Table 2, each Cnox homeodomain of E. dichotoma shows about equal similarity to Antp-class homeodomains from different families. For example, Cnox-1 shows 65% similarity with ftz, Antp-, Hox-B3, and Hox-B7, and Cnox-5 is 67% identical to lab, Hox-D3, Hox-B6, as well as to Hox-B7. Since a high number of closely related homeodomains was found, we looked for diagnostic amino acid residues which are conserved within distinct families of Antp-class genes, but which vary between families (Scott et al., 1989). Representatives of distinct families are listed in Table 1. Surprisingly, we could find conserved residues for diverse homeobox families at
various positions in each of the five Cnox genes, and no unequivocal one-to-one correspondents in Drosophila or vertebrates were identified. For example, Cnox-5 has the archetypal lab phenylalanine residue at position 8, and alanine at position 29, but also the Abd-B typical lysine at positions 3 and 4 (see Table 1). Outside the homeoboxes, we searched for the short conserved hexapeptide or pentapeptide sequence which is commonly located 15 bp upstream of the homeobox in Antp-class homeobox genes (excluding Abd-B). These motifs were not found in any deduced protein sequence of Eleutheria homeobox genes. Since the hexapeptide does not exist in Abd-B-like homeoboxes, we looked for conserved residues specific for this gene. The typical GC rich region at the 5′ end of Abd-B-like genes, encoding a high number of proline, glycine, and alanine residues, as well as the Trp–Met duplet, does not exist in any of the five homeobox genes. However, the conserved tryptophan residue at position 27 upstream from the homeobox, which has not been found at this position in any homeobox other than Abd-B-like genes (Izpisu´a-Belmonte et al., 1991), is present in Cnox-1 and Cnox-3. Thus, none of the five homeobox genes of E. dichotoma can be grouped to any distinct family of Antp-class homeobox genes; rather, all of them represent intermediate forms. These findings are supported by both cluster and parsimony analyses (see Figs. 3 and 4). In contrast to some previously published trees, which were based on partial sequence information of homeoboxes, the trees built upon the full-length homeobox nucleotide sequences (180 bp) do not suggest homologous counterparts for any Cnox gene to Drosophila or human Antp-class genes. Other Cnidarian Homeodomains The homeobox genes in E. dichotoma are only slightly related to each other, but as shown in Table 3, Cnox-1, Cnox-2, and Cnox-5 correspond to homeobox genes from other cnidarian species. Since Cnox-3 and Cnox-4 seem to be different, we placed them into separate groups (see Table 3). DISCUSSION Both the number and types of Antp-class homeobox genes found in the cnidarian E. dichotoma confound existing views on the ancestral state of this gene class at the time of the origin of triploblastic animals from diploblastic ancestors. The findings suggest that conclusions drawn solely from information on triploblastic organisms may result in an oversimplified picture and that the evolution of Antp-class homeobox genes may have been more complex than expected. The large number of Antp-class genes found in Eleutheria was unexpected (cf. Oliver et al., 1992; Miller and Miles, 1993) and does not easily fit to an ancestral head, trunk, and tail model, which has been
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TABLE 3 Alignment of E. dichotoma Homeodomains with Homeodomains from Other Cnidarian Species
Note. Deduced amino acid sequences of homeobox genes in E. dichotoma compared with other cnidarian homeodomains. Dashes indicate amino acid residues identical with the Antp gene of Drosophila. Cnox-1 from E. dichotoma shows highest similarity with Cnox3 from Hydra (80%), Cnox-2 is 100% identical to SAox2 and 98% identical to Cnox2 from Chlorohydra, H. vulgaris, and H. magnipapillata, and Cnox-5 shows 84% similarity to SAox1. The most similar homeobox gene to Cnox-3 (62%) as well as to Cnox-4 (58%) is Cnox5 of H. magnipapillata. References: Sarsia sp. (Murtha et al., 1991), Hydractinia symbiolongicarpus (Schierwater et al., 1991), Chlorohydra viridissima (Schummer et al., 1992), H. magnipapillata (Naito et al., 1993), H. vulgaris (Schenk et al., 1993).
proposed for diploblasts (Fig. 1; Averof and Akam, 1993; Schubert et al., 1993). Additional, and more important, evidence against an ancestral three-gene-cluster model in cnidarians derives from the characterization of the Cnox genes. Phylogenetic reconstructions from Drosophila and vertebrate HOM/Hox genes suggested a three-gene-cluster (head, trunk, and tail) which evolved before the separation of diploblasts and triploblasts more than 600 million years ago (Schubert et al., 1993). Under this hypothesis one should expect to find genes in E. dichotoma to be lab-like (3′ or head), Antp-like (central or trunk: Dfd, Scr, Antp, Ubx, and abd-A), and Abd-B-like (5′ or tail). Our results do not readily conform to this expectation. The Eleutheria Cnox genes are not closely related to any distinct homeobox gene family; rather, they simultaneously share features with different families. Thus, for none of the Cnox genes a homologue to Drosophila or vertebrate homeobox genes can be defined. The latter is further supported by tree inferring analyses if homeobox sequences are used. Based on the fact that the characterized homeobox genes of E. dichotoma do not belong to any distinct homeobox gene family, our data fail to support the hypothesis that cnidarian homeobox genes are more related to the genes at the 3′ end than to genes
at the 5′ end of the insect and vertebrate homeobox clusters (Murtha et al., 1991). Our findings generally agree with a relationship analysis of 337 homeodomains using a distance matrix approach by Kappen et al. (1993). At least 30 distinguishable families were generated, with cnidarian homeobox genes divided into several separate families. The authors assumed that the majority of clearly definable homeobox families were established prior to the emergence of the vertebrate, arthropod, and even nematode lineages. Furthermore, they point out that once established, a distinct homeobox family should be strongly conserved in its sequence (cf. Bu¨rglin, 1994). Naito et al. (1993) proposed eight homeobox families in cnidarians, three of them consisting of non-homeotic genes (even-skipped-like, msh-like, and BarH2-like). Although this analysis was based on only 25 amino acid residues for most homeodomains, together with our findings it seems obvious that distinct homeobox families do exist for cnidarians which differ from the HOM/ Hox families known for triploblasts. For example, similarities between 96 and 100% are found in one family, which consists of six Cnox-2 homeodomains from different hydrozoans (Hydra vulgaris, Hydra magnipapillata, E. dichotoma, Hydractinia symbiolongicarpus,
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FIG. 1. Suggested evolution of homeobox gene clusters. The proposed homeobox gene cluster of the arthropod/vertebrate ancestor is shown with six precursor genes (Holland, 1990, 1992; Bu¨rglin and Ruvkum, 1993; Garcia-Ferna`ndez and Holland, 1994). The evolution of Scr (Drosophila) and the paralog group 4 (human) precursor gene is controversial (Averof and Akam, 1993; Schubert et al., 1993; Kappen and Ruddle, 1993). The last common ancestral homeobox cluster of arthropods and nematodes is taken from Bu¨rglin and Ruvkun (1993). Precursor genes of triploblasts and diploblasts are described in Schubert et al. (1993). The arrangement of the precursor genes shown is hypothetical. Abbreviations are as follows: lab (labial), pb ( proboscipedia), zen1 (zerknu¨llt), zen2, Dfd (deformed ), Scr (Sex combs reduced), ftz (fushi tarazu), Antp (Antennapedia), Ubx (Ultrabithorax), abd-A (abdominal-A), Abd-B (Abdominal-B).
Chlorohydra viridissima, and Sarsia sp.) (see Table 3). Nevertheless, homeobox genes belonging to this family have previously been classified as homologous to three different families ( pb, Dfd, and Gsh-1/Gsh-2) (Murtha et al., 1991; Schummer et al., 1992; Bu¨rglin, 1994). The homeobox genes of E. dichotoma represent intermediate sequences of the distinct Antp-families of Drosophila and vertebrates. These findings call for more elaborate explanations concerning the types of homeobox genes present in a hypothetical ancestor of cnidarians and triploblasts. Before doing so, two main objections, however, should be considered: (I) Since cnidarians diverged more than 600 million years ago, one may argue against a high degree of sequence conservation between cnidarian and Drosophila homeobox genes. On the other hand, conserved regions diagnostic for distinct homeobox families, as well as the complex organization of the cluster, have been maintained for over 500 million years in nematodes, Drosophila, and humans. High sequence conservation was also found between flatworms and Drosophila Antp-class genes (Oliver et al., 1992). Furthermore, it must be noted that cnidarian homeobox genes which do not belong to the Antp-class, like msh from C. viridissima (Schummer et al., 1992) and eveC from Acropora formosa (Miller and Miles, 1993), do have homologous counterparts in Dro-
sophila. (II) It could be argued that Eleutheria is a very derived organism, not representing the typical picture for hydrozoa in general. However, this argument is not compelling, because other hydrozoan species exhibit homologous sequences with the same peculiarities. In addition, other available DNA sequence data do not support a particularly derived position for Eleutheria within the hydrozoa (Bridge, 1994). More likely explanations may be based on the assumption that the proposed head–trunk–tail precursor genes do not exist in the hydrozoa. At present, it seems more likely that the cnidarian lineages diverged prior to the fixation of the Antp-families present in nematodes, arthropods, and vertebrates and that a proposed ancestral three-gene Antp-class cluster may be novel to triploblasts. A common ancestor of hydrozoans and triploblasts may have possessed a single Antp-class precursor gene. Independent duplications of this precursor gene could have led to different distinct Antp-families in both lineages. At this point, information on the chromosomal arrangement of Cnox genes, i.e., whether these Antp-class genes are arranged in clusters or not, would not resolve the questions. We would need more comparative information on Antp-class genes in other diploblastic animals, including the anthozoan cnidarians (so far only one Antp-
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FIG. 3. UPGMA-dendrogram of E. dichotoma, Drosophila, and human homeobox nucleotide sequences (180 bp) of the 17 homeotic genes in Table 2. The tree was created with the software Treecon (Van de Peer and De Wachter, 1993), using the distance metric of Kimura (1980).
FIG. 2. Nucleotide and derived amino acid sequences of three new Antp-class homeobox genes from E. dichotoma (Cnox-3 to Cnox5). The homeodomains are underlined.
FIG. 4. Parsimony analysis (PAUP, Swofford, 1991) of unweighted nucleotide sequences of homeoboxes from E. dichotoma, Drosophila, and human (see Fig. 3). The tree is a 50% majority-rule consensus of eight equally parsimonious trees, found by heuristic search using random addition (100 replicates) and subtree-pruning – regrafting branch-swapping. Tree length is 693 steps, consistency index is 0.397, and retention index 0.423.
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class homeobox gene has been detected outside the hydrozoa (Miller and Miles, 1993)), before the evolution of HOM/Hox genes in early metazoan evolution can be adduced.
of the homeodomain and its functional implications. Trends Genet. 6: 323–329. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G., and Wu¨thrich, K. (1994). Homeodomain-DNA recognition. Cell 78: 211–223.
ACKNOWLEDGMENTS
Graham, A., Papalopulu, N., and Krumlauf, R. (1989). The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57: 367–378. Holland, P. W. H. (1990). Homeobox genes and segmentation: Cooption, co-evolution, and convergences. Dev. Biol. 1: 135–145.
We acknowledge collaborative research with Leo Buss, comments and critical discussions from Cort Anderson, Leo Buss, Rob DeSalle, Matt Dick, Klaus Schwenk, and Thomas Sta¨dler, and financial support from DFG (277 Schi-3-1), NATO (CRG-91004), NSF (IBN9119437), BMFT (Helmholtz-Programm), and BMFT support to B. Streit.
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