Hemichordates occupy a critical phylogenetic position among deuterostomes because they exhibit echinoderm-like larval morphology and chordate-like adult morphology. Analyses of the expression and function of hemichordate developmental genes will therefore provide insight into the evolution of deuterostome body plans. The distal-less/dlx gene encodes a homeodomain transcription factor and plays roles in the development of appendages and the brain in a variety of animals. Here we have characterized a distal-less gene (Pf-dlx) of the hemichordate Ptychodera flava. During embryogenesis, Pf-dlx is expressed in the whole aboral ectoderm of the blastula and gastrula. Later, its expression appears in several cells in the boundary region between the oral and aboral ectoderm. The tornaria larvae express Pf-dlx in some specific cells of the ciliary band. The results are discussed in terms of an ancestral function of the distal-less/dlx gene in the formation of the nervous system.
INTRODUCTION
Our present knowledge of the molecular mechanisms of evolutionary developmental processes has mainly been obtained from investigations using model animals such as flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans), fish (Danio rerio), frogs (Xenopus laevis) and mice (Mus musucurus). From comparative studies of these animals, a number of conserved regulatory systems have been found to play roles in putatively comparable developmental processes among such model organisms. For example, the HOMC/Hox complex has been shown to play a central role in the antero-posterior axis formation in a variety of animals (Miller and Miles, 1993).
Interestingly, such correspondences are seen even in the formation of structures which are not necessarily thought to involve plegiomorphy. For example, the formation of the Drosophila ‘wing’ and the chick ‘wing’ utilizes some shared developmental regulatory systems (Laufer et al., 1997), although these structures are thought of as a good instance of evolutional convergence (see Hall, 1998). Such similarity of gene expression pattern between vertebrates and insects appears to be surprising for several reasons. One reason is that these two phyla are phylogenetically distant from each other. Another reason is that modes of their development may not have persisted well after evolving from their ancestral condition, but rather they have been altered, since most invertebrate taxa display different modes of development than vertebrate taxa, particularly concerning the initial process of cell specification (reviewed by Davidson, 1991). Therefore, in order to understand the evolution of animal body plans, we need to accumulate much more basic molecular information on the development of animals, including small phyla of marine invertebrates, because they display a major part of the biodiversity of metazoa.
Hemichordates, acorn worms, occupy a unique phylogenetic location because they exhibit echinoderm-like larval morphology as well as chordate-like adult morphology (e.g., Brusca and Brusca, 1990). Early embryogenesis of acorn worms is a definitive example of indirect development. Recently several developmental regulatory genes from the acorn worm Ptychodera flava have been characterized, and their expression patterns during embryogenesis have been investigated (Tagawa et al., 1998b, 2000; Peterson et al., 1999; Taguchi et al., 2000; Harada et al., 2000). It is obvious that better understanding of the ontogeny of hemichordates will fill a gap in our understanding of the two quite dissimilar developmental modes of echinoderms and chordates and help us to determine the evolutionary scenario of the developmental processes of all deuterostomes.
The distal-less (dll)/dlx gene encodes a homeodomain transcription factor (Duboule, 1994). The distal-less gene of D. melanogaster is required for the development of both appendages and the central nervous system (CNS) (Cohen, 1990). Localization of the distal-less gene product in developing appendages has been reported in various taxa (Panganiban et al., 1997; Lowe and Wray, 1997). Vertebrates have multiple copies of dlx class genes as a result of tandem and clustered gene duplications (Stock et al., 1996). These dlx genes are expressed in a variety of sites, including limb buds and forebrain (e.g., Price et al., 1991; Dollé et al., 1992). A single copy of the amphioxus distal-less gene is also expressed in the forebrain (Holland et al., 1996). Thus, the distal-less/dlx genes share roles in the formation of the appendages and the anterior nervous system.
In the present study, we characterized the distal-less/dlx gene of the acorn worm P. flava. We report here the dynamics of its expression pattern during early embryogenesis, which are suggestive about its ancestral function.
MATERIALS AND METHODS
Embryos
Mature adult Ptychodera flava were collected in December at a sand bar in Kaneohe Bay, Oahu, Hawaii. Natural spawning was induced as described by Tagawa et al. (1998a). Fertilized eggs were allowed to develop to the desired developmental stages at room temperature in the Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii.
Molecular cloning
PCR amplification was carried out using a P. flava gastrula cDNA library (Tagawa et al., 1998b) as template DNA. The primer sequences were as follows: DLX-F: 5′-MGNAARCCNMGNACNATHTA-3′ and DLX-R: 5′-CKRTTYTGRAACCADATYTT-3′ (where D = not C, H = not G, K = G or T, M = A or C, N = any, R = A or G, and Y = C or T). We screened the cDNA library by probing with the PCR fragments obtained. The probes were labelled with [32P]dCTP. Several positive cDNA clones were isolated. We selected a clone which contained the longest insert and a poly A tail. Then the phage cDNA clone was converted into plasmids. Both strands of the cDNA clone were sequenced using an automated DNA sequencer (ABI PRISM, Perkin Elmer).
Sequence comparisons and molecular phylogenetic analysis
The putative amino acid sequence of Pf-Dlx was deduced from its nucleotide sequence. The amino acid sequence was aligned with sequences of related homeobox gene products. The relationship was analyzed molecular phylogenetically by means of the neighbor-joining method using PHYLIP ver. 3.5 (Felsenstein, 1993).
Whole-mount in situ hybridization
Whole-mount specimens were hybridized in situ basically as described by Tagawa et al. (1998b). Embryos were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M MOPS, pH 7.5 on ice overnight. Probes for Pf-dlx were synthesized following the instructions supplied with the kit (Boehringer Mannheim DIG RNA Labelling kit) and used at 0.5 μg/ml in hybridization buffer. The hybridization signal was detected using NBT and BCIP following the supplier's instructions.
Double in situ hybridization was preformed as described in our previous report (Shoguchi et al., 2000). RNA probes for Pf-dlx and Pf-otx were synthesized, and specimens were simultaneously hybridized with a mixture of the two kinds of riboprobes labelled with digoxigenin and fluorescein, respectively. After washing, coloring reactions were made first to produce a brown Pf-otx signal and then to produce a red Pf-dlx signal.
RESULTS AND DISCUSSION
Isolation and characterization of a hemichordate distal-less gene
The longest cDNA clone we isolated possessed a single open reading frame, which predicted a polypeptide of 320 amino acids. The full-length nucleotide and predicted amino acid sequences of the cDNA clone are available in the database under DDBJ/EMBL/GenBank Accession Number, AB028221. We named the gene Pf-dlx (P. flava dlx).
Fig. 1. shows a comparison of the amino acid sequences of the homeodomain of Pf-Dlx with those of other distal-less/dlx class gene products. Based on a comparison of the amino-acid sequences, we constructed a molecular phylogenetic tree of the related homeobox gene products by using mouse Nkx2-3 and Msx-3 gene products as outgroups. As shown in Fig. 2, the clade of Dlx proteins, including Pf-dlx, was supported with a 100% bootstrap value. Thus, we concluded that Pf-dlx is a hemichordate ortholog of the distal-less/dlx gene. The vertebrate dlx family is divided into two major subgroups as a consequence of tandem gene duplication, and each subgroup contains several subclass members (Fig. 2; Stock et al., 1996). However, Pf-dlx does not show any affinity to a specific subclass, in contrast to amphioxus AmphiDll and distal-less of Drosophila.
Fig. 1
Alignment of amino acid sequences of the homeobox region of Pf-Dlx with those of other Dlx orthologs. Asterisks indicate amino acids identical among all members, while dots indicate similar amino acids.
Fig. 2
Molecular phylogenetic analysis of relationships of distal-less/dlx subclass and related-class homeobox gene products using the homeodomain amino acid sequences. Branch lengths are proportional to evolutionary distance corrected for multiple substitutions with the scale denoting 0.1 amino acid substitutions per site. The numbers indicate the relative robustness of each node as assessed by bootstrap analysis (100 replications).
Pf-dlx is expressed in the aboral ectoderm of the blastula and gastrula and in cells of the oral/aboral boundary of the tornaria larva
The expression of Pf-dlx was detected as early as the blastula stage. It was expressed in half of the ectodermal region of the blastula and early gastrula (Fig. 3A, B). The Pf-dlx-positive half corresponds to what will become the aboral side, and in situ hybridization signal was stronger in the aboral side than in the oral/aboral boundary side. This expression pattern suggests that the ectoderm of the acorn worm blastula can be subdivided into oral and aboral halves, as in the case of the ectoderm of the sea urchin blastula (reviewed by Davidson et al., 1998). Since the tornaria larva of acorn worms and the larva of echinoderms show remarkable morphological similarity to each other, it will be interesting to examine whether the ectoderm of acorn worm embryos is specified by mechanisms similar to those for the ectoderm of echinoderm embryos (discussed in Davidson et al., 1998).
Fig. 3
Spatial expression of Pf-dlx. (A, B) An early gastrula (18 hr of development), vegetal pole view (A) and lateral view (B). ae, aboral ectoderm; ar, archenteron; bp, blastopore; oe, oral ectoderm. (C) A late gastrula (24 hr of development). The intensity of the signal in the aboral ectoderm decreases, while a distinct signal is detected in the oral/aboral boundary (arrowhead). (D–F) Simultaneous detection of Pf-dlx (red signals, arrowheads) and Pf-otx expression (brown signals, arrows) in early tornaria larvae (3 days of development), viewed from the aboral side (D), animal pole (E) and lateral side (F), respectively. D and E show the same larva. hp, hydropore. (G) An enlargement of the left lateral ectoderm region of a tornaria larva shown in the insert. Red signals of Pf-dlx mRNA are detected in several cells at the oral/aboral boundary, which partially overlap the region with brown signals of Pf-otx expression (white asterisks).
The expression of Pf-dlx in the aboral ectoderm decreased during gastrulation, except in cells on the edge of the expression domain, or else expression appeared in this region de novo. At the late gastrula stage, cells with strong Pf-dlx expression were observed in the oral/aboral boundary (Fig. 3C). Figure 3D–G shows Pf-dlx expression in the tornaria larva. Judging from specimens viewed from the animal pole, the aboral ectodermal expression continued at this stage (Fig. 3E), although it became weaker. Pf-dlx signals were evident in cells located discontinuously in the line of the oral/aboral boundary (Fig. 3D, F). This boundary may correspond to the aboral part of the post-oral ciliary band, which contains cells of the nervous system, by analogy to the sea urchin embryo (Nakajima, 1986; Cameron et al., 1993). Figure 3D shows an aboral view of the early tornaria; Pf-dlx-positive cells are buried in the epidermis, and distribution of cells with Pf-dlx expression in the larva is not left-and-right symmetrical (Fig. 3D). This pattern of Pf-dlx expression persisted in the 6-day-old larva, the last stage we observed.
The distal-less/dlx gene is expressed in the appendages of animals in many phyla (e.g., Panganiban et al., 1997). However, it is thought that the distal-less/dlx gene is older than the putative outgrowth-bearing ancestor. In addition, the genes are expressed in the CNS and peripheral nervous system, including parts of the brain involved in optic function (Price et al., 1991; Kaphingst and Kunes, 1994). The distal-less/dlx gene is also expressed in the CNS of nematode embryos (Panganiban et al., 1997). Therefore, it has been suggested that the ancestral function of the gene is associated with the CNS formation. Although the function of Pf-dlx in the ciliary band remains to be elucidated, the pattern of its expression suggests its role in the nervous system formation. We recently found that the hemichordate otx gene (Pf-otx) is expressed along the ciliary band (Harada et al., 2000). To determine whether the expression domains of Pf-dlx and Pf-otx are overlapping, larvae were double-hybridized with two probes, one for Pf-dlx and the other for Pf-otx, and the relative locations of the regions showing expression of these genes were monitored simultaneously. As shown in Fig. 3G, cells with Pf-dlx expression were located within the region of cells with Pf-otx expression. However, it was not determined whether individual cells express both Pf-dlx and Pf-otx simultaneously. These molecular probes can be used as tools for examining further a putative relationship between the ciliary bands of the tornaria larva and the CNS of chordates, an issue which has long been debated by numerous researchers (Garstang, 1928; Crowther and Whittaker, 1992; Nielsen, 1999).
Acknowledgments
Y. H. was supported by a postdoctoral fellowship from the JSPS with Research Grant #8910. This research was also supported in part by a Grant-in-Aid from the Monbusho, Japan, to N. S.
