Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
A conserved cluster of three PRD-class homeobox genes (homeobrain, rx and orthopedia) in the Cnidaria and Protostomia.
Mazza ME
,
Pang K
,
Reitzel AM
,
Martindale MQ
,
Finnerty JR
.
???displayArticle.abstract???
BACKGROUND: Homeobox genes are a superclass of transcription factors with diverse developmental regulatory functions, which are found in plants, fungi and animals. In animals, several Antennapedia (ANTP)-class homeobox genes reside in extremely ancient gene clusters (for example, the Hox, ParaHox, and NKL clusters) and the evolution of these clusters has been implicated in the morphological diversification of animal bodyplans. By contrast, similarly ancient gene clusters have not been reported among the other classes of homeobox genes (that is, the LIM, POU, PRD and SIX classes).
RESULTS: Using a combination of in silico queries and phylogenetic analyses, we found that a cluster of three PRD-class homeobox genes (Homeobrain (hbn), Rax (rx) and Orthopedia (otp)) is present in cnidarians, insects and mollusks (a partial cluster comprising hbn and rx is present in the placozoan Trichoplax adhaerens). We failed to identify this 'HRO' cluster in deuterostomes; in fact, the Homeobrain gene appears to be missing from the chordate genomes we examined, although it is present in hemichordates and echinoderms. To illuminate the ancestral organization and function of this ancient cluster, we mapped the constituent genes against the assembled genome of a model cnidarian, the sea anemone Nematostella vectensis, and characterized their spatiotemporal expression using in situ hybridization. In N. vectensis, these genes reside in a span of 33 kb with the same gene order as previously reported in insects. Comparisons of genomic sequences and expressed sequence tags revealed the presence of alternative transcripts of Nv-otp and two highly unusual protein-coding polymorphisms in the terminal helix of the Nv-rx homeodomain. A population genetic survey revealed the Rx polymorphisms to be widespread in natural populations. During larval development, all three genes are expressed in the ectoderm, in non-overlapping territories along the oral-aboral axis, with distinct temporal expression.
CONCLUSION: We report the first evidence for a PRD-class homeobox cluster that appears to have been conserved since the time of the cnidarian-bilaterian ancestor, and possibly even earlier, given the presence of a partial cluster in the placozoan Trichoplax. Very similar clusters comprising these three genes exist in Nematostella and diverse protostomes. Interestingly, in chordates, one member of the ancestral cluster (homeobrain) has apparently been lost, and there is no linkage between rx and orthopedia in any of the vertebrates. In Nematostella, the spatial expression of these three genes along the body column is not colinear with their physical order in the cluster but the temporal expression is, therefore, using the terminology that has been applied to the Hox cluster genes, the HRO cluster would appear to exhibit temporal but not spatial colinearity. It remains to be seen whether the mechanisms responsible for the evolutionary conservation of the HRO cluster are the same mechanisms responsible for cohesion of the Hox cluster and other ANTP-class homeobox clusters that have been widely conserved throughout animal evolution.
Figure 1. Alignment of octapeptide regions, homeodomains and OAR domains from representative hbn, otp and rx genes of cnidarians, protostomes and deuterostomes. Homeodomain sequences are aligned against the Drosophila antennapedia homeodomain. Nematostella, human and fruit fly representatives were taken from Ryan et al. [32]. Homeodomains from additional taxa were identified by similarity searches (tBLASTn and BLASTp) conducted through the BLAST interface at National Center for Biotechnology Information (see Methods) and at respective genome assemblies available through the Joint Genome Institute. Abbreviations for taxa are as follows: (cnidarians: Acropora millepora (Acm), Hydra magnipapillata (Hym), Nematostella vectensis (Nev); placozoan: Trichoplax adhaerens (Tra); protostome bilaterians: Aedes aegypti (Aea), Anopheles gambiae (Ang), Apis mellifera (Apm), Caenorhabditis elegans (Cae), Capitella teleta (Cap), Lottia gigantea (Log), Tribolium castaneum (Trc); deuterostome bilaterians: Branchiostoma floridae (Brf), Ciona intestinalis (Cii), Danio rerio (Dar), Gallus gallus (Gag), Homo sapiens (Hos), Mus musculus (Mum), Saccoglossus kowalevskii (Sak), Strongylocentrotus purpuratus (Stp), Xenopus laevis (Xel)). The octapeptide [72,73] and/or the OAR domain [51,75] are indicated when identified. Two different versions of the Rx homeodomain of Nematostella are shown, and the polymorphic residues are highlighted.
Figure 2. Neighbor-joining phylogeny of PRD class genes based on homeodomain sequences. Pairwise distances between homeodomains were calculated using the PAM matrix in the computer program protdist in the Phylip package (Phylogenetic Inference Package, V.3.61). Abbreviations for taxa are as in Figure 1. Numbers above nodes indicate the percentage of 1000 bootstrap replicates in which the given node was recovered in a neighbor-joining analysis. The tree is rooted using Hox1 and distalless homeodomains from the lancelet and the fruit fly.
Figure 3. Organization of the homeobrain-rx-orthopedia cluster of Nematostella. Homeobrain orthopedia and rx ESTs map to a ~35 kb region within a single scaffold 1,036,593 nucleotides long in the Joint Genome Institute's assembly of the Nematostella genome (scaffold 062, Joint Genome Institute Nematostella vectensis genome portal, V.1.0). Arrows above the cluster indicate the transcriptional orientation of each gene. The nucleotide length of each intron, exon and intergenic region is indicated below the cluster.
Figure 4. Phylogenetic distribution of the Otp-Rx-Hbn cluster. Several sequenced metazoan genomes were searched for evidence of linkage between orthopedia, rx and homeobrain. The genomic organization of the fruit fly homeobrain related gene was previously reported identified by Walldorf et al. [31]. Predicted linkage of these three genes was determined for insects (mosquito, honeybee, flour beetle), chordates (vertebrates, amphioxus, ascidian), sea urchin, two lophotrochozoans (limpet, polychaete annelid), sea anemone and placozoan by querying the available genome sequences using the annotated/predicted gene sequences for each gene (this study). Solid lines between genes represent linkage on a single chromosome, scaffold or contig. Approximate distance (kb between genes) is noted above each line, and the relative transcriptional orientation of each gene is indicated by the direction of the arrow (the arrow points from 5' to 3'). Question marks indicate uncertain linkage. Genes confirmed to be on different chromosomes or genomic scaffolds are represented by squares with no interconnecting lines.
Figure 5. Expression of NvOtp. In situ hybridization using a 1556 bp probe of otp. In all panels, the asterisk denotes the blastopore or oral end. (a) Expression is not detected in the egg, (b) during early cleavage or (c,d) during gastrulation. (e,f) Expression is detected in the planula stage in the ectoderm around the pharynx (ph) and in single ectodermal cells (arrows). (g) Oral view of the same planula from (f). (h,i) Similar expression in later stages as tentacles (t) begin to grow. (j,k) Polyp stages showing high expression in an oral ectodermal ring.
Figure 6. Expression of NvRx. In situ hybridization using a 1419 bp probe of rx. The asterisks denote the blastopore or oral end. (a,b) No expression is detected in the egg or early cleavage stages. (c,d) Mid gastrula stages showing expression around the aboral ectoderm. (e) Late gastrula stage showing a band of expression towards the aboral pole. (f) The same embryo in (e) as viewed from the aboral pole, showing a lack of expression where the future apical tuft (at) will form. (g-j) Planula stages, with (i) showing a surface view of the patchy expression. (k) Polyp stage, showing a close-up view of expression in the ectoderm of the tentacles (t).
Figure 7. Expression of NvHbn. In situ hybridization using a 749 bp probe of hbn. The asterisks mark the blastopore or oral end. (a,b) Egg and early cleavage stages showing a lack of expression. (c) Blastula stage embryo showing expression everywhere except in the blastopore. (d) Mid gastrula showing lack of expression in the invaginating endoderm (en). (e,f) Lateral and oral views of a late gastrula, stage embryo. (g) Planula stage, showing strong expression around the future tentacles (arrow) and in the putative neurons (n) of the mid body. (h,i) Early polyp stage showing expression around the base of the tentacles (t). (j,k) Older polyp stages, where expression is confined to the base of the tentacles.
Acampora,
The role of Otx and Otp genes in brain development.
2000, Pubmed
Acampora,
The role of Otx and Otp genes in brain development.
2000,
Pubmed
Acampora,
Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene.
1999,
Pubmed
Arnone,
Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity.
2006,
Pubmed
,
Echinobase
Bailey,
Regulation of vertebrate eye development by Rx genes.
2004,
Pubmed
Balavoine,
Hox clusters and bilaterian phylogeny.
2002,
Pubmed
Barad,
A novel, tissue-specific, Drosophila homeobox gene.
1988,
Pubmed
Brouwer,
The OAR/aristaless domain of the homeodomain protein Cart1 has an attenuating role in vivo.
2003,
Pubmed
Burke,
A genomic view of the sea urchin nervous system.
2006,
Pubmed
,
Echinobase
Burri,
Conservation of the paired domain in metazoans and its structure in three isolated human genes.
1989,
Pubmed
,
Echinobase
Casarosa,
Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development.
1997,
Pubmed
Cavalieri,
Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells.
2007,
Pubmed
,
Echinobase
Cavalieri,
Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos.
2003,
Pubmed
,
Echinobase
Chesterman,
Comparative analysis of Prx1 and Prx2 expression in mice provides evidence for incomplete compensation.
2002,
Pubmed
Chourrout,
Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements.
2006,
Pubmed
Chuang,
Expression of three Rx homeobox genes in embryonic and adult zebrafish.
1999,
Pubmed
Chuang,
Zebrafish genes rx1 and rx2 help define the region of forebrain that gives rise to retina.
2001,
Pubmed
Coulier,
MetaHox gene clusters.
2000,
Pubmed
D'Aniello,
The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function.
2006,
Pubmed
Davis,
Drosophila retinal homeobox (drx) is not required for establishment of the visual system, but is required for brain and clypeus development.
2003,
Pubmed
Del Giacco,
Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons.
2006,
Pubmed
Di Bernardo,
Homeobox genes and sea urchin development.
2000,
Pubmed
,
Echinobase
Di Bernardo,
Spatially restricted expression of PlOtp, a Paracentrotus lividus orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos.
1999,
Pubmed
,
Echinobase
Duboule,
The rise and fall of Hox gene clusters.
2007,
Pubmed
Edgar,
MUSCLE: multiple sequence alignment with high accuracy and high throughput.
2004,
Pubmed
Eggert,
Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development.
1998,
Pubmed
Finnerty,
Early evolution of a homeobox gene: the parahox gene Gsx in the Cnidaria and the Bilateria.
2003,
Pubmed
Fröbius,
Capitella sp. I homeobrain-like, the first lophotrochozoan member of a novel paired-like homeobox gene family.
2006,
Pubmed
Furukawa,
rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina.
1997,
Pubmed
Galliot,
Evolution of homeobox genes: Q50 Paired-like genes founded the Paired class.
1999,
Pubmed
Garcia-Fernà ndez,
The genesis and evolution of homeobox gene clusters.
2005,
Pubmed
Granadino,
EVG, the remnants of a primordial bilaterian's synteny of functionally unrelated genes.
2003,
Pubmed
Hughes,
Evolution of the integrin alpha and beta protein families.
2001,
Pubmed
Hui,
Features of the ancestral bilaterian inferred from Platynereis dumerilii ParaHox genes.
2009,
Pubmed
Jiang,
Identification and comparative analysis of the protocadherin cluster in a reptile, the green anole lizard.
2009,
Pubmed
Kern,
A novel murine homeobox gene isolated by a tissue specific PCR cloning strategy.
1992,
Pubmed
Kerner,
Evolutionary history of the iroquois/Irx genes in metazoans.
2009,
Pubmed
Kurosawa,
Organization and structure of hox gene loci in medaka genome and comparison with those of pufferfish and zebrafish genomes.
2006,
Pubmed
Larroux,
The NK homeobox gene cluster predates the origin of Hox genes.
2007,
Pubmed
Lemons,
Genomic evolution of Hox gene clusters.
2006,
Pubmed
Lewis,
A gene complex controlling segmentation in Drosophila.
1978,
Pubmed
Lin,
Identification, chromosomal assignment, and expression analysis of the human homeodomain-containing gene Orthopedia (OTP).
1999,
Pubmed
,
Echinobase
Loosli,
Medaka eyeless is the key factor linking retinal determination and eye growth.
2001,
Pubmed
Lopreato,
Evolution and divergence of sodium channel genes in vertebrates.
2001,
Pubmed
Lowe,
Anteroposterior patterning in hemichordates and the origins of the chordate nervous system.
2003,
Pubmed
Maclean,
Rhox: a new homeobox gene cluster.
2005,
Pubmed
Mann,
Why are Hox genes clustered?
1997,
Pubmed
Marlow,
Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian.
2009,
Pubmed
Martinez,
Genomics of the HOX gene cluster.
2002,
Pubmed
Mathers,
Regulation of eye formation by the Rx and pax6 homeobox genes.
2000,
Pubmed
Mazet,
An ancient Fox gene cluster in bilaterian animals.
2006,
Pubmed
Mazza,
Genomic organization, gene structure, and developmental expression of three clustered otx genes in the sea anemone Nematostella vectensis.
2007,
Pubmed
Mazza,
A conserved cluster of three PRD-class homeobox genes (homeobrain, rx and orthopedia) in the Cnidaria and Protostomia.
2010,
Pubmed
Miura,
Expression of a novel aristaless related homeobox gene 'Arx' in the vertebrate telencephalon, diencephalon and floor plate.
1997,
Pubmed
Nederbragt,
Novel and conserved roles for orthodenticle/ otx and orthopedia/ otp orthologs in the gastropod mollusc Patella vulgata.
2002,
Pubmed
Norris,
The identification of Prx1 transcription regulatory domains provides a mechanism for unequal compensation by the Prx1 and Prx2 loci.
2001,
Pubmed
Norris,
Identification of domains mediating transcription activation, repression, and inhibition in the paired-related homeobox protein, Prx2 (S8).
2001,
Pubmed
Norris,
Human PRRX1 and PRRX2 genes: cloning, expression, genomic localization, and exclusion as disease genes for Nager syndrome.
2000,
Pubmed
Ohuchi,
Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development.
1999,
Pubmed
Patel,
Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals.
2008,
Pubmed
Pollard,
Evidence for 14 homeobox gene clusters in human genome ancestry.
2000,
Pubmed
Putnam,
Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization.
2007,
Pubmed
Ryan,
Pre-bilaterian origins of the Hox cluster and the Hox code: evidence from the sea anemone, Nematostella vectensis.
2007,
Pubmed
Ryan,
The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis.
2006,
Pubmed
Schneitz,
Molecular genetics of aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila.
1993,
Pubmed
Seo,
Hox cluster disintegration with persistent anteroposterior order of expression in Oikopleura dioica.
2004,
Pubmed
Shibuya,
Comparative genomics of the keratin-associated protein (KAP) gene clusters in human, chimpanzee, and baboon.
2004,
Pubmed
Shimeld,
Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster.
2010,
Pubmed
Simeone,
Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila.
1994,
Pubmed
Smith,
A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo.
1996,
Pubmed
Spitz,
Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes.
2005,
Pubmed
Stock,
The Dlx gene complement of the leopard shark, Triakis semifasciata, resembles that of mammals: implications for genomic and morphological evolution of jawed vertebrates.
2005,
Pubmed
Sullivan,
Conserved and novel Wnt clusters in the basal eumetazoan Nematostella vectensis.
2007,
Pubmed
Sullivan,
StellaBase: the Nematostella vectensis Genomics Database.
2006,
Pubmed
Technau,
Maintenance of ancestral complexity and non-metazoan genes in two basal cnidarians.
2005,
Pubmed
Tiong,
Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity.
1987,
Pubmed
Umesono,
A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain.
1997,
Pubmed
Umesono,
Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes.
1999,
Pubmed
Vogel,
Chromosomal clustering of a human transcriptome reveals regulatory background.
2005,
Pubmed
Walldorf,
Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain.
2000,
Pubmed
Wang,
The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus.
2000,
Pubmed
Wotton,
Conservation of gene linkage in dispersed vertebrate NK homeobox clusters.
2009,
Pubmed
Zhou,
Evolution of OTP-independent larval skeleton patterning in the direct-developing sea urchin, Heliocidaris erythrogramma.
2003,
Pubmed
,
Echinobase