Hox and paraHox genes from the anthozoan Parazoanthus parasiticus

https://doi.org/10.1016/S1055-7903(03)00062-9Get rights and content

Abstract

We surveyed the genome of the Caribbean zoanthid Parazoanthus parasiticus for Hox and paraHox genes, and examined gene expression patterns for sequences we uncovered. Two Hox genes and three paraHox genes were identified in our surveys. The Hox genes belong to anterior and posterior classes. In phylogenetic analyses, the anterior Hox sequence formed an anthozoan-specific cluster that appears to be a second class of cnidarian anterior Hox gene. The presence of an anterior Gsx-like paraHox gene supports the hypothesis that duplication of a protoHox gene family preceded the divergence of the Cnidaria and bilaterians. The presence of two Mox class paraHox genes in P. parasiticus deserves further attention. Expression analysis using RT-PCR, indicated that one Mox gene and the anterior paraHox gene are not expressed in adult tissue, whereas the other three sequences are expressed in both dividing and unitary polyps. Dividing polyps showed slightly lower Ppox1 (i.e., Mox) expression levels. Our data add to the number of published anthozoan sequences, and provide additional detail concerning the evolutionary significance of cnidarian Hox and paraHox genes.

Introduction

Hox and paraHox genes comprise the Antenapedia class of homeotic genes, and encode a class of DNA-binding transcription factors that are highly conserved among and within animal lineages (Akam, 1989; Bürglin, 1994; Bürglin et al., 1989; Davidson et al., 1995; de Rosa et al., 1999; Gehring, 1987; Kenyon, 1994; Mito and Endo, 1997; Murtha et al., 1991; Schierwater et al., 1991). These gene families play a crucial role in axial patterning and segmental identity in all metazoan animals (Aerne et al., 1995; Arenas-Mena et al., 1998; Dolecki et al., 1986; Garcia-Fernandez et al., 1991; Holland and Hogan, 1986; Kenyon and Wang, 1991; Knoll and Carroll, 1999; Krumlauf, 1994; Laughon and Scott, 1984; McGinnis et al., 1984; Oliver et al., 1992; Schummer et al., 1992; Shenk et al., 1993a, Shenk et al., 1993b). One major feature of these genes is that they are arranged in clusters, and the linear organization of the homeobox genes correlates with their expression patterns along the anterior–posterior axis of the developing animal (Akam, 1989; Graham et al., 1989; Kenyon and Wang, 1991; Krumlauf, 1994; Pendelton et al., 1993; Peterson et al., 2000; Valentine et al., 1996). There is evidence that the paraHox and Hox clusters are evolutionary sisters (Brooke et al., 1998), which has important implications for our understanding of the current distribution of these genes in the animal kingdom (Brooke et al., 1998; Finnerty, 2001; Finnerty and Martindale, 1997; Finnerty and Martindale, 1999; Kourakis and Martindale, 2000).

Among bilaterians, we currently recognize four classes of Hox gene families (anterior, group 3, central and posterior) and three classes of paraHox gene families (Gsx, Xlox, and Cdx) (see Gauchat et al., 2000). While anterior and posterior Hox and paraHox representatives have been identified in the diploblasts, the cnidarians appear to lack the middle Hox (group 3 and central) and paraHox (Xlox) genes (e.g., Finnerty, 2001). Currently, the early evolutionary history of the Hox and paraHox gene clusters is unresolved, but several hypotheses have been proposed. The leading hypothesis posits a protoHox gene cluster consisting of at least three genes that existed before the Hox/paraHox split (Brooke et al., 1998; Ferrier and Holland, 2001; Finnerty, 2001; Finnerty and Martindale, 1998, Finnerty and Martindale, 1999; Gauchat et al., 2000; Kuhn et al., 1996, Kuhn et al., 1999; Masuda-Nakagawa et al., 2000). There are several lines of support for this hypothesis including the fact that in all phylogenetic analyses to date, the paraHox genes cluster within established Hox gene families (e.g., Kourakis and Martindale, 2000). Under this scenario, metazoans lost the central paraHox gene while cnidarians lost the central genes in both Hox and paraHox gene clusters. Kourakis and Martindale (2000) recently proposed a protoHox cluster, predating the Hox/paraHox split, that contained all four Hox families.

With additional homeobox gene surveys of different classes and species of cnidarians, the Hox/paraHox gene families continue to grow, and it has been suggested that additional work with cnidarians is likely to broaden our understanding of the origin and evolution of these genes, as well as the structure and function of ancestral protoHox genes (Ferrier and Holland, 2001; Finnerty, 2001; Gauchet et al., 2000). For example, it is possible (albeit remotely) that the “middle” Hox/paraHox genes have been missed in the numerous surveys that have been conducted to date (e.g., Kourakis and Martindale, 2000). Additional surveys and analysis of genome organization (i.e., linkage analysis) of basal animal groups should elucidate the early evolution of these developmentally important gene families.

In the present study we adopted the former approach, and surveyed a previously unexamined anthozoan species (Parazoanthus parasiticus) for Hox and paraHox genes. Our goal was to obtain novel gene sequences, and expand the number of anthozoans surveyed to date. P. parasiticus was chosen because it is common throughout the Caribbean, it is conspicuous due to its epibiotic growth habit (Hill, 1998), and can be collected in various stages of asexual reproduction. In addition to the Hox/paraHox sequences obtained, we present data on the expression of these genes in P. parasiticus tissues in the process of dividing (i.e., asexual reproduction) compared to unitary polyps. Our data lend support to the hypothesis that cnidarians diverged from the metazoan lineage after the duplication of the protoHox cluster. Larger scale sequencing efforts have produced useful phylogenetic hypotheses concerning the molecular evolution of cnidarian Hox and paraHox genes, and our data add detail to these gene trees.

Section snippets

Isolation and sequencing of DNA

Parazoanthus parasiticus polyps were collected from the surface of the sponge Callyspongia vaginalis in the Florida Keys in the summer of 1999. Tissue samples of both dividing and unitary P. parasiticus polyps were stored at −80 °C. Polyps showing any evidence of fission were classified as “dividing.” DNA was isolated from P. parasiticus using Qiagen’s DNeasy kit following the manufacturer’s protocol. Two sets of degenerate primers were used to survey the genome for Hox genes. The first primer

Sequence and phylogenetic analyses

We isolated five homeodomain sequences from P. parasiticus corresponding to four distinct paraHox or Hox gene families (Fig. 1). The results of the phylogenetic analysis are shown in Fig. 2. The neighbor-joining tree indicated that Ppox1 and Ppox2 grouped within the Mox/Cnox5 paraHox family. The Ppox3 sequence grouped within the Gsh/Cnox2 paraHox gene family. Ppox4, an anterior Hox sequence, was found in the AntHox-Cnox1 clade, and Ppox5, a member of the posterior Hox (Cnox3) family, was

Phylogenetic analysis and evolutionary implications

Our survey of the P. parasiticus genome increases the number of currently identified anthozoan Hox and paraHox genes to just over 15 (Finnerty, 2001). At least three interesting observations can be made when our P. parasiticus sequences are compared to other anthozoan Hox/paraHox genes. First, Ppox4 shows a strong affinity for two of the Nematostella sequences (Fig. 2). Thus, there appear to be two distinct anterior Hox groupings in the Cnidaria, as has been suggested previously (Finnerty, 1998

Acknowledgements

We would like to thank Tom Wilcox for his assistance during the collection of P. parasiticus. This research was made possible by a grant from the National Science Foundation (IBN) as well as by a Fairfield University Fellows Scholar Research Stipend.

References (46)

  • T. Mito et al.

    A PCR survey of Hox genes in the sea star, Asterina minor

    Mol. Phylogenet. Evol.

    (1997)
  • G. Oliver et al.

    Homeoboxes in flatworms

    Gene

    (1992)
  • M. Shenk et al.

    Expression of Cnox-2, a HOM/HOX gene, is suppressed during head formation in hydra

    Dev. Biol.

    (1993)
  • J. Valentine et al.

    Developmental evolution of metazoan body plans: the fossil evidence

    Dev. Biol.

    (1996)
  • C. Arenas-Mena et al.

    Expression of the Hox gene complex in the indirect development of a sea urchin

    Proc. Natl. Acad. Sci. USA

    (1998)
  • N.M. Brooke et al.

    The ParaHox gene cluster is an evalutionary sister of the Hox gene cluster

    Nature

    (1998)
  • T. Bürglin
  • T. Bürglin et al.

    Caenorhabditis elegans has scores of homeobox-containing genes

    Nature

    (1989)
  • E.H. Davidson et al.

    Origin of bilaterian body plans: evolution of developmental regulatory mechanisms

    Science

    (1995)
  • R. de Rosa et al.

    Hox genes in brachiopods and priapulids and protostome evolution

    Nature

    (1999)
  • G. Dolecki et al.

    Stage-specific expression of a homeobox-containing gene in the non-segmented sea urchin embryo

    EMBO J.

    (1986)
  • D. Ferrier et al.

    Ancient origin of the Hox gene cluster

    Nature

    (2001)
  • J.R. Finnerty

    Cnidarians reveal intermediate stages in the evolution of Hox clusters and axial complexity

    Am. Zool.

    (2001)
  • View full text