Expression of the pair-rule gene homologs runt, Pax3/7, even-skipped-1 and even-skipped-2 during larval and juvenile development of the polychaete annelid Capitella teleta does not support a role in segmentation
© Seaver et al; licensee BioMed Central Ltd. 2012
Received: 28 November 2011
Accepted: 18 April 2012
Published: 18 April 2012
Annelids and arthropods each possess a segmented body. Whether this similarity represents an evolutionary convergence or inheritance from a common segmented ancestor is the subject of ongoing investigation.
To investigate whether annelids and arthropods share molecular components that control segmentation, we isolated orthologs of the Drosophila melanogaster pair-rule genes, runt, paired (Pax3/7) and eve, from the polychaete annelid Capitella teleta and used whole mount in situ hybridization to characterize their expression patterns.
When segments first appear, expression of the single C. teleta runt ortholog is only detected in the brain. Later, Ct-runt is expressed in the ventral nerve cord, foregut and hindgut. Analysis of Pax genes in the C. teleta genome reveals the presence of a single Pax3/7 ortholog. Ct-Pax3/7 is initially detected in the mid-body prior to segmentation, but is restricted to two longitudinal bands in the ventral ectoderm. Each of the two C. teleta eve orthologs has a unique and complex expression pattern, although there is partial overlap in several tissues. Prior to and during segment formation, Ct-eve1 and Ct-eve2 are both expressed in the bilaterial pair of mesoteloblasts, while Ct-eve1 is expressed in the descendant mesodermal band cells. At later stages, Ct-eve2 is expressed in the central and peripheral nervous system, and in mesoderm along the dorsal midline. In late stage larvae and adults, Ct-eve1 and Ct-eve2 are expressed in the posterior growth zone.
C. teleta eve, Pax3/7 and runt homologs all have distinct expression patterns and share expression domains with homologs from other bilaterians. None of the pair-rule orthologs examined in C. teleta exhibit segmental or pair-rule stripes of expression in the ectoderm or mesoderm, consistent with an independent origin of segmentation between annelids and arthropods.
KeywordsCapitella Eve Pax3/7 polychaete Runt Segmentation
The evolution of segmentation in bilaterian animals is an ongoing area of investigation and debate (reviewed in [1–4]). Three major animal clades contain segmented representatives, the Annelida, the Arthropoda and the Chordata. According to molecular phylogenies [5, 6], each of these clades is more closely related to animals that lack a segmented body than to each other. Thus, the question remains whether segmentation arose independently in distinct lineages, or whether most extant clades lost the segmented body plan that was present in a common segmented ancestor. One approach to address this question is to compare the molecular mechanisms controlling segment generation across taxa, with the assumption that shared molecular mechanisms reflect a common evolutionary history.
Segment formation is best understood in Drosophila melanogaster, in which a detailed understanding of the genetic control of segmentation has served as a framework for comparative studies in other arthropods. Briefly, a segmentation gene cascade sequentially divides the embryo into smaller units through the hierarchical action of the gap, pair-rule and segment polarity genes (reviewed in ). The pair-rule genes are the first to be expressed in a periodic pattern in D. melanogaster, and include the genes paired (Pax3/7), even-skipped (eve) and runt. These genes are expressed with a two-segment periodicity in ectodermal stripes along the anterior-posterior axis. Mutants of pair-rule genes lack alternating segmental structures such as denticle bands in the larval cuticle . In contrast, mutants in segment polarity genes exhibit defects in the pattern of every segment.
The mode of segmentation in D. melanogaster is derived compared to other arthropods (reviewed in ). In D. melanogaster, all segments form at virtually the same time (long germ band development) and much of the patterning critical for segment formation occurs prior to cellularization. In most other arthropods, segments form in a temporal progression from anterior to posterior and the earliest segments form before the tissue for additional segments is present (short germ band development). Although many insects also develop via an early syncytial stage, this is a derived feature within arthropods, and many arthropods form all or most segments within a cellular environment.
The expression patterns of pair-rule and segment polarity gene orthologs have been examined across arthropods, including in insects, and also in chelicerates, myriapods and crustaceans. In general, pair-rule genes show more variability in their expression patterns than do the highly conserved segment polarity genes, and there are examples lacking periodic expression patterns even within insects . In several arthropods, pair-rule gene orthologs are expressed in stripes in every segment; in some cases the onset of expression is clearly prior to segment generation. This pattern is more consistent with a segment polarity role than a pair-rule function. For example, in the centipede Lithobius atkinsoni and the spider Cupiennius salei, Pax3/7 is expressed in a portion of every segment. In the spider mite Tetranychus urticae and the millipede Glomeris marginata, runt has a segmental pattern. Likewise, eve is expressed with a segmental periodicity in L. atkinsoni, C. salei and in the insect Oncopeltus fasciatus. In D. melanogaster it is notable that several pair-rule genes, including even-skipped, runt and paired, have an initial pair-rule expression pattern of seven alternating stripes that later matures into a segmental 14-stripe pattern [18–20]. These data suggest that the pair-rule orthologs have a general, and likely ancestral, function in arthropod segment formation. The extent to which pair-rule patterning is conserved across arthropods, and whether pair-rule patterning was utilized primarily in holometabolous insects (flies, bees, beetles and moths) or ancestrally at the base of arthropods, remains an open question [7, 11, 18]. Regardless, the pair-rule genes are useful markers for inter-taxonomic comparisons to reconstruct the evolution of segmentation.
The mechanistic understanding of the molecular control of segment formation in annelids is poor compared to that in chordates and arthropods. Efforts to identify genes involved in annelid segmentation have largely utilized a candidate gene approach. Examining the expression of genes involved in arthropod segmentation and vertebrate somitogenesis and identification of shared components of a common genetic program for segment formation would support a shared evolutionary origin of segmentation. Expression patterns of segment polarity gene orthologs have been examined in a number of annelid species; the most well-characterized is the segment polarity gene engrailed. In contrast to the highly conserved expression pattern of en across arthropods [15, 21–23], en expression patterns among annelids exhibit substantial variability. Although en is expressed in ectodermal stripes in the polychaete annelid Platynereis dumerilii, this pattern is not apparent in any other annelid examined, including in the polychaetes Chaetopterus sp. , Hydroides elegans and Capitella teleta Blake, Grassle & Eckelbarger, 2009  (previously known as Capitella sp. I) and in the leech Helobdella triserialis. Thus, there is currently a discrepancy in en expression patterns among annelids and the P. dumerilii pattern may represent a convergence, rather than a common origin, with the arthropod pattern. Alternatively, if the P. dumerilii and arthropod patterns represent a conservation, there has been extensive divergence in en expression among annelids.
Previously, we investigated the evolution of bilaterian segmentation by characterizing the expression of orthologs of the segment polarity genes, en, wg and hh and of the pair-rule genes hairy and odd-paired in C. teleta. Here, we report the expression of the pair-rule gene orthologs runt, paired (also called pax group III or Pax3/7) and two eve genes in C. teleta immediately prior to and during larval segment formation. Additionally, we characterize Pax3/7 and eve expression during adult segment formation. C. teleta eve, Pax3/7 and runt genes lack segmental or pair-rule stripes of expression in both the ectoderm and mesoderm, even though Ct-Pax3/7, Ct-eve1 and Ct-eve2 expression is initiated prior to overt segmentation. Each ortholog examined has a distinct expression pattern and exhibits expression domains conserved with those found in other bilaterians.
Cloning and sequencing of C. teleta runt, Pax3/7, and eve orthologs
Fragments corresponding to conserved regions of runt, Pax3/7 and eve orthologs were isolated from C. teleta by degenerate polymerase chain reaction (PCR) using a cDNA template prepared from mixed embryonic and larval stages. To recover a fragment of runt, two rounds of amplification were performed in a semi-nested PCR reaction using the following primers from published sequences : runfw-1: 5'- RCNRYNATGAARAAYCARGTNGC -3' and runbw: 5'-CKNGGYTCNCKNGGNCCRTC-3' followed by runfw-2: 5'-MRNTTYAAYGAYYTNMGNTTYGTNGG -3' and runbw. To isolate a fragment of Pax3/7 from mixed stage cDNA, a semi-nested approach was utilized with the following published primers  representing the conserved paired domain and paired-like homeodomain: Prbyfout: 5'- GGNGGNGTNTTYATHAAYGG -3' and Prbyr: 5'-RTTNSWRAACCANACYTG -3' followed by Prbyfin: 5'-MARATHGTNGARATGGC -3' and Prbyr in a second round of amplification. A 226 base pair (bp) fragment of eve was recovered from a genomic DNA template using the following degenerate primers: ab-evefw: 5'-MGTTAYMGTACIGCITTYAC-3' and eve-bw1: 5'-CKYTGNCKYTTRTCYTTCAT-3' . Additional sequences for Ct-eve, Ct-Pax3/7 and Ct-runt were obtained from a mixed stage embryonic and larval stage template using gene-specific primers with the Smart rapid amplification of cDNA ends (RACE) amplification kit (Clontech, Mountain View, CA, USA). Resulting fragments were cloned into the pGEM-T Easy vector (Promega, Fitchburg, WI, USA) and sequenced by the University of Hawaii sequencing facility (Honolulu, HI, USA) or by Macrogen Inc. (Seoul, South Korea). The resulting products were: a 702 bp 5' RACE fragment and a 1269 bp 3' RACE fragment for Ct-Pax3/7, a 644 bp 5' RACE fragment and a 945 bp 3' RACE fragment for Ct-runt. For eve, a 460 bp 5' RACE fragment was recovered by PCR using gene-specific primers. Sequencing of multiple cDNA clones from 5' RACE reactions revealed the presence of a five amino acid insertion in some clones. Two distinct 3' RACE fragments were recovered: a 583 bp and a 734 bp fragment. Gene-specific primers designed to each of the 5' and 3' eve sequence variants were used to amplify and characterize features of individual cDNAs. Subsequent to the isolation of Ct-runt, Ct-Pax3/7, Ct-eve1 and Ct-eve2 by degenerate PCR, sequences became available for the C. teleta genome project (Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA ). Cloned sequences were consistent with predicted gene models from the genome. The genome was searched for additional runt, Pax3/7 and eve paralogs; none were detected. Linkage analysis of Ct-eve1 and Ct-eve2 genes was performed through searches of the C. teleta genome using nucleotide sequences obtained from degenerate and RACE PCR clones. Accession numbers for Ct-runt, Ct-eve1, Ct-eve2 and Ct-Pax3/7 are listed in Additional file 1: Table S1.
Sequence alignments and phylogenetic analyses
Sequences isolated by degenerate PCR and RACE were assigned putative orthologies based on BLASTX searches of the GenBank database from the National Center for Biotechnology Information. In addition, tBLASTn searches of the C. teleta genome (Joint Genome Institute (JGI), Walnut Creek, CA, USA) were conducted to find all homologs of eve, runt and the paired homeobox (pax) family. Two putative orthologs were found for eve, one for runt and six for pax family members. Amino acid sequences from diverse animal taxa were downloaded from the protein database in GenBank. Alignments of conserved domains were generated with ClustalX, using default parameters in MacVector 11.1.1 (MacVector, Inc., Cary, NC, USA). The domains analyzed were the homeodomain and 10 amino acids flanking the 5'- and 3'-ends of the homeodomain for Eve, the runt domain for Runt and the 127 amino acid paired domain for the Pax family. Alignments were edited by hand to correct obvious alignment errors. Nexus alignments are available upon request. Sequences, abbreviations and accession numbers used are included in Additional file 1: Table S1.
ProtTest v2.4  was run for each alignment to determine the appropriate model of protein evolution. The Jones model was recommended and used for the Eve and the Pax family, and the RtRev model for Runt. Both Bayesian and maximum likelihood methods were used for each gene family. Bayesian analysis was conducted using MrBayes v3.1.2 , with the model determined by ProtTest. For the Runt and Pax alignments, three million generations were run, sampled every 100 generations, with four independent runs and four chains. Ten million generations were run for the Eve alignment. Once convergence was reached, majority rule consensus trees were generated with burnin values of 25,000 (Eve), 9,700 (Runt) and 5,400 (Pax). Maximum likelihood analyses were performed with RAxML v7.0.0  using the same models as for Bayesian analysis with 1,000 bootstrap replicates. Trees were visualized with FigTree v1.3.1  and drawn using Adobe Illustrator version CS4.
Whole mount in situ hybridization
Whole mount in situ hybridization was performed according to previously published protocols . Digoxigenin-labeled riboprobes (dig-11-UTP; Roche Diagnostics, Indianapolis, IN, USA) were transcribed with the MEGAscript High Yield Transcription Kit (Ambion, Austin, TX, USA) and diluted to working concentrations of 3 ng/μL for Ct-runt, 0.25 ng/μL for Ct-Pax3/7, 0.1 ng/μL for Ct-eve1 and 0.2 ng/μL for Ct-eve2. Probe lengths were as follows: 1041 bp for Ct-runt, 1270 bp for Ct-Pax3/7, 1379 bp for Ct-eve1 and 1224 bp for Ct-eve2. Following termination of the development reaction, specimens were equilibrated and stored in 80% glycerol, 20% 5× phosphate buffered saline (PBS). The detailed protocol is available upon request.
Visualization of mid-body mesoderm
Animals were fixed and labeled according to previously described conditions (fixation #4, ). Specimens were exposed to a 1:500 dilution of mouse anti-histone antibody (F152, C25.WJJ; Millipore, Billerica, MA, USA) and a 1:400 dilution of goat anti-mouse rhodamine (Invitrogen, Carlsbad, CA, USA). After antibody labeling, animals were incubated in 1:100 BODIPY FL phallacidin (Invitrogen) in PBS, 0.1% Triton X-100 (PBT) for two hours at room temperature followed by several PBT washes over 45 minutes. Animals were mounted in Slow Fade Gold (Invitrogen) and analyzed.
Riboprobe-labeled specimens were analyzed using differential interference contrast (DIC) optics on an Axioskop 2 compound microscope (Zeiss, Munich, Germany). Digital images were captured with either a stem-mounted 4.0 megapixel Nikon Coolpix 4500 (Nikon, Inc., Melville, NY, USA) or SpotFlex digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Confocal laser scanning microscopy was performed using an Axioplan 2 LSM510 microscope (Zeiss). Three-dimensional reconstructions were generated from confocal images with ImageJ (National Institutes of Health, Bethesda, MD, USA). Figures and diagrams were constructed using Photoshop CS4 and Illustrator CS4 (Adobe Systems Inc., New York, NY, USA). Some panels in figures seven and eight (see Figure legends) are composites of multiple DIC focal planes that were merged using Helicon Focus (Helicon Soft Ltd., Kharkov, Ukraine).
Cloning and phylogenetic analyses of C. teleta runt, eve and Pax3/7 genes
The C. teleta runt ortholog we recovered lacks the characteristic motif VWRPY at the C-terminal end, which is necessary for Groucho-mediated transcriptional repression activity . To confirm that the missing motif was not due to a cloning artifact, 3' RACE PCR was repeated with a second primer set and an independently generated cDNA pool. The resulting fragment was identical in sequence to the original Ct-runt 3' RACE product. In addition, we identified a single runt sequence from searches of the C. teleta genome , which has the same amino acid sequence as the sequence isolated by PCR and lacks the C-terminal Groucho-binding domain. Searches of the Lottia gigantea genome  reveal that its predicted runt homolog also lacks the VWRPY C-terminal motif (Protein ID 119594).
A 226 bp fragment of eve was initially identified by degenerate PCR from a C. teleta genomic DNA template. Subsequent RACE PCR recovered a 5' fragment and two distinct 3' fragments. From sequence searches of the C. teleta genome, two distinct eve genes were identified, which we designate Ct-eve1 and Ct-eve2. The Ct-eve1 and eve2 predicted ORFs are 325 and 330 amino acids, respectively. The Ct-Eve1 and Ct-Eve2 predicted ORFs are largely identical within the predicted ORF; the exceptions are the two amino acids immediately 5' of the stop codon (KS for Ct-eve1 and PK for Ct-eve2), and a small five amino acid insertion present only in Ct-Eve2. This insertion immediately follows amino acid residue 85 and is located 17 amino acids 5' of the homeodomain. The homeodomain in both Eve sequences contains conserved amino acids characteristic of eve gene sequences (Figure 1C). Bayesian and maximum likelihood analyses of the homeodomain confirm the identity of Ct-eve1 and Ct-eve2 as eve gene orthologs within the homeodomain superfamily (Additional file 3: Figure S2). Ct-Eve1 and Ct-Eve2 cluster together with Eve proteins from other animal taxa and separately from the homeodomain-containing Gbx and Engrailed outgroups. There is 100% Bayesian posterior probability and 90% bootstrap support for the Eve node. Within the predicted ORFs, there are only seven nucleotide differences between Ct-eve1 and Ct-eve2, aside from sequence differences due to the five amino acid insertion in Ct-Eve2 and the two amino acids immediately 5' of the stop codon. In contrast, the two sequences diverge in sequence and length in the 3' UTR (Ct-eve1, 167 bp and Ct-eve2, 349 bp).
C. teleta development
The segmented ectoderm and mesoderm have distinct embryonic origins in C. teleta. All segmental ectoderm is generated from the blastomere 2d, which also generates nonsegmental posterior ectoderm. The segmented mid-body ectoderm segregates from nonsegmental ectoderm within the first few divisions of 2d, such that 2d11 generates segmented ectoderm and PGZ, but not the telotroch or the posterior unsegmented pygidium . Most of the mid-body mesoderm arises from the two cells 3c and 3d, whose descendants form the right and left mesodermal bands, respectively (Figure 4). Initially, a pair of large posterior mesodermal cells is visible beneath the ectoderm (Figure 4A,D). Subsequently, two mesodermal bands become visible as a row of subsurface cells in the lateral part of the body (Figure 4B,C,E,F). Over time, the bands increase in cell number and expand circumferentially (Figure 4G-L). By stage 5, when the first segments are visible, longitudinal and circular muscle fibers extend from the mesodermal bands (Figure 4G). Extension of a single circular muscle fiber per segment is one of the earliest signs of segmentation in the mesoderm and follows soon after ectodermal segmentation. These circular muscle fibers are positioned within the intersegmental grooves (Figure 4I) .
Expression of eve genes
Ct-eve2 has distinct expression domains from Ct-eve1, although there are clear overlapping areas of expression. When the blastopore is present (Stage 3b), Ct-eve2 is detected in the mesodermal precursor cells (Figure 8A) and on the posterior face of the blastopore at the ventral midline (Figure 8B). Later during Stage 3, there is continued expression in the mesodermal precursor cells, and novel expression in the presumptive hindgut (Figure 8C) and in a ring of ectodermal cells near the posterior end of the embryo (Figure 8D). At Stages 4 and 5, after the telotroch has formed, it is apparent that these posterior ectodermal cells are immediately anterior to the telotroch in the position of the presumptive PGZ (not shown). Ct-eve2 expression in the mesodermal precursor cells and hindgut persists (Figure 8E,G). At Stage 5, Ct-eve2 also appears in several additional ectodermal domains, including weak expression in the foregut and mid-body ectoderm (between the prototroch and telotroch). The most prominent expression is in the forming ventral nerve cord (Figure 8F), which persists as the left and right sides fuse at the ventral midline at Stage 6 (Figure 8H). In the mid-body, there is expression in both the ectoderm and mesoderm at Stage 6. The mesodermal expression is particularly pronounced in the PGZ and in the mesodermal precursor cells (Figure 8I). In the ectoderm, small clusters of cells express Ct-eve2 more strongly relative to surrounding cells (Figure 8H arrowheads), which become refined to three columns of discrete, segmentally iterated cell clusters on each side of the animal by Stage 7 (Figure 8 J,K,L). The three columns are in the following positions: between the ventral nerve cord and the neuropodial chaetae, between the neuro- and notopodial chaetae, and dorsal to the notopodial chaetae (Figure 8J). The most dorsal column is the first to express Ct-eve2. These three columns of Ct-eve2-expressing cell clusters span the ectoderm epithelia and have a very similar expression pattern to elav and synaptotagmin (Meyer and Seaver, unpublished data), conserved markers of neuronal differentiation and of differentiated neurons, respectively. These columns of cell clusters are likely peripheral sensory structures. In addition, at Stages 6 and 7, there is ectodermal expression in a single line of cells across the dorsal surface of some larvae in the PGZ (Figure 8L). This expression domain is not visible at later stages. By Stage 8, there is only weak expression in the columns of ectodermal cell clusters (not shown) and they are undetectable by Stage 9. Expression persists in the hindgut and PGZ into Stage 8 (Figure 8M,O,P), and at this stage it is clear from the subsurface position of the hindgut expression that Ct-eve2 is localized to the rectum. A new expression domain appears at Stage 8 in mesodermal cells along the dorsal midline in the posterior mid-body (Figure 8M,N,O). These Ct-eve2-expressing cells can be seen extending from the dorsal midline along the anterior edge of segments (Figure 8N). It is unknown to what structures these cells contribute. At Stage 9, expression is limited to a small domain in the rectum (Figure 8Q) and weak expression in the PGZ (Figure 8Q) and dorsal midline (not shown). In juveniles, Ct-eve2 is expressed in the PGZ, hindgut and weakly in posterior ganglia (Figure 9B,D).
In summary, Ct-eve1 and Ct-eve2 have unique spatio-temporal expression patterns, although they are both expressed in mesodermal precursor cells, foregut and hindgut, and the PGZ. Prior to the generation of the mesodermal bands, Ct-eve1 and Ct-eve2 are similarly expressed in the mesodermal precursor cells (compare Figures 7A and 8A). Once the mesodermal bands appear, however, Ct-eve1 is expressed in both mesodermal precursor cells and their descendant bands, whereas Ct-eve2 is restricted to the mesodermal precursor cells and is not expressed in their progeny. The temporal dynamics of hindgut expression between the two genes is distinct. Ct-eve2 is continuously expressed in the hindgut from Stage 3 through metamorphosis into juvenile stages (Figures 8 and 9). In contrast, Ct-eve1 expression is transient in the hindgut, is initially detected a day later than for Ct-eve2 at Stage 4, and is not detectable in late stage larvae (compare Figures 7L and 8Q). Another difference between Ct-eve1 and Ct-eve2 expression is in the mid-body, where Ct-eve2 is largely expressed in ectodermal derivatives such as the ventral nerve cord and peripheral sensory structures. This differs dramatically from Ct-eve1, which has broad expression throughout the mid-body mesoderm. The unique expression patterns for Ct-eve1 and Ct-eve2 indicates a divergence of regulatory elements between these two genes.
Runt, Pax3/7 and eve genes in C. teleta
The C. teleta genome has clear orthologs of the D. melanogaster pair-rule segmentation genes runt, paired (Pax3/7) and eve. Two eve genes are present in the C. teleta genome and, based on amino acid sequence similarity and genomic linkage, they appear to be either the result of a recent duplication or concerted evolution, possibly due to gene conversion event(s) [55, 56]. Gene conversion has previously been proposed to occur between the two Dlx genes in the C. teleta genome . Both Ct-Pax3/7 and Ct-runt lack a Groucho-binding domain, which is conserved in orthologs of these genes from other species. However, the Pax3/7A ortholog in the leech Helobdella sp.  and the sea squirt Ciona intestinalis, and the D. melanogaster paired gene  also lack this motif, as does the predicted limpet Lottia gigantea runt homolog. The C. teleta genome contains a groucho gene (scaffold_692 1858), and other C. teleta genes contain the groucho repression motif, including the hes genes . These features confirm that the groucho repression motif is present and likely utilized in the context of transcriptional co-repression in C. teleta. Although we do not know the functional consequences of the lack of a groucho domain on the C. teleta runt and paired homologs, the lack of a groucho domain in D. melanogaster paired suggests that this domain is not necessary for a segmentation function.
Relationship of Ct-runt, Ct-Pax3/7, Ct-eve1 and Ct-eve2 expression to segment formation
If annelids and arthropods shared a common segmented ancestor, the expectation is that pair-rule gene orthologs would be expressed in transverse stripes with a consistent relationship to segment boundaries in C. teleta, with either a pair-rule or segmental periodicity. However, none of the genes we examined, Ct-runt, Ct-Pax3/7, Ct-eve1 or Ct-eve2, have a spatio-temporal expression pattern consistent with a possible role in segment formation. When the first larval segments are forming, Ct-runt expression is limited to a subset of cells in the brain and there is no expression in the mid-body where new segments form. Although Ct-Pax3/7 is expressed in the mid-body prior to overt segment formation in larvae, its spatial pattern does not prefigure segments, either in a pair-rule or segmental pattern. Both Ct-eve genes are expressed in the mid-body as larval segments begin to form. At these stages, the Ct-eve1 transcript is ubiquitously expressed in the mid-body mesoderm and lacks spatially restricted positional expression expected for a role segment boundary formation. Ct-eve2 expression has more restricted expression in the mid-body, limited to mesodermal precursor cells and the ventral nerve cord, and also lacks pair-rule or segmental stripes of expression in the ectoderm or mesoderm. At late larval stages and during juvenile growth when segments form from the PGZ, all four genes are expressed in the PGZ. However, none of them have patterns that prefigure segments, such as circumferential banded expression or expression limited to either the anterior or posterior region of the growth zone. Ct-Pax3/7 and Ct-runt have restricted expression around the circumference of the PGZ, in a small ventrolateral domain and ventrolateral domain of the ectoderm, respectively. Both Ct-eve genes have broad posterior expression domains that include the PGZ and nascent segments in late larval stages and juveniles, but do not resolve into more restricted patterns. In some animals that form segments sequentially, such as in vertebrates, centipedes and spiders, segment formation is preceded by waves of dynamic gene expression [61–63]. Of the genes examined here, we did not observe dynamic expression in the PGZ, since we did not detect multiple patterns in the PGZ for a gene in similarly staged larvae.
We previously examined expression of orthologs of the pair-rule genes odd-paired and hairy in C. teleta[29, 30]. During early stages of segment formation, Ct-zic, the C. teleta ortholog of odd-paired, is expressed along the lateral edge of the ventral neural ectoderm, and in segmentally iterated domains of mesoderm expression in the mid-body soon after segments form. However, there are no stripes of expression in either the ectoderm or mesoderm. One of the hairy orthologs, CapI-hes1, is expressed in the mesoderm of presumptive larval segments in a segmentally iterated pattern, although it is very distinct from arthropod patterns of hairy expression [7, 64, 65]. Of the pair-rule orthologs for which we have examined the expression pattern in C. teleta, CapI-hes1 is the most promising candidate to function in segmentation . Ct-Pax3/7, Ct-runt, Ct-eve1, Ct-eve2, CapI-hes2 and Ct-zic all have very distinct expression patterns, all of which indicate that these genes are not likely to be involved in segmentation. Perhaps a subset of these genes are post-transcriptionally regulated and play a role in establishing segmental boundaries; however, functional studies will be necessary to obtain a definitive confirmation of the role of these pair-rule homologs in C. teleta.
Comparison of runt, Pax3/7 and eve expression among species
Expression of the pair-rule genes Pax3/7 and eve have been characterized in a few other annelids, but expression patterns for runt have not been reported for any other lophotrochozoan. Runt orthologs appear to have a conserved role in D. melanogaster and in Danio rerio, chick  and mammalian neural development (reviewed in ), and expression of Ct-runt in the brain and ventral nerve cord of C. teleta extends this conservation. For Pax3/7 orthologs, expression has been examined in two other annelids. Expression of Pax3/7 in P. dumerilii is somewhat similar to the pattern in C. teleta. Both Pdu-pax3/7 and Ct-Pax3/7 are expressed in bilaterally symmetric longitudinal bands in the ventral trunk ectoderm, adjacent to the ventral nerve cord. In P. dumerilii, each band of expression is much broader than in C. teleta, possibly reflecting differences in the size of the neurogenic field at these stages. Helobdella sp. Austin has two Pax3/7 orthologs. The expression pattern has been reported for Hro-Pax3/7A, and it is expressed in the mesodermal bands and later in mesodermal derivatives . This is in contrast to the longitudinal bands of Pax3/7 expression in the ventral ectoderm in C. teleta and P. dumerilii. A better understanding of the extent to which these genes have conserved expression domains in annelids will emerge as data from additional species are reported. Outside of annelids, Pax3/7 orthologs function in neural specification in the ventral nerve cord of D. melanogaster and the vertebrate neural tube. However, their expression patterns are quite distinct in that there are transverse bands of expression in D. melanogaster and longitudinal bands of expression in vertebrates [71, 72].
Comparison of eve expression patterns across bilaterian animals reveals several common expression domains. In annelids, eve is expressed in the growth zone during posterior growth of C. teleta and H. robusta, and in the PGZ during regeneration of P. dumerilii[73, 74]. However, in P. dumerilii, expression in the PGZ is ectodermal, whereas in H. robusta and C. teleta it is both ectodermal and mesodermal. Furthermore, there is prominent expression in the mesodermal bands of C. teleta and H. robusta, but not in P. dumerilii. Additionally, eve is expressed in the posterior gut of both C. teleta and P. dumerilii. Outside of annelids, posterior expression of eve has been reported in Danio rerio, Xenopus laevis, B. floridae, nematodes , and in a range of arthropods [10, 15, 17, 78–80]. Eve also appears to have an important and widespread role in neural specification or neurogenesis in bilaterians, including in insects (D. melanogaster[48, 81], Schistocerca americana), Caenorhabditis elegans, B. floridae, X. laevis and Mus musculus. In annelids, eve is expressed in the developing ventral nerve cord of C. teleta, P. dumerilii and H. robusta, and a role in neural development appears to be the most conserved function of eve. Although less well characterized, eve has also been implicated in mesodermal patterning in Danio rerio and D. melanogaster. The expression of eve in pericardial progenitors along the dorsal midline in D. melanogaster and in mesodermal cells along the dorsal midline of C. teleta may indicate a shared role in protostome cardiovascular development. In summary, eve genes show conserved roles across bilaterians in multiple domains, indicating conservation of several developmental roles, and the C. teleta eve gene expression patterns appear to show conservation with several of these functions. This broad conservation for eve is in contrast with its function in arthropod segmentation, which appears to be more of a clade-specific role.
To the best of our knowledge, no orthologs of D. melanogaster pair-rule genes show striped patterns of ectodermal expression in annelids, either in a segmental or pair-rule-like pattern. Vertebrate orthologs of runt, Pax3/7 and eve do not have a role in somitogenesis. Furthermore, a pair-rule-like expression pattern has not been reported for any gene in annelids. In the polychaete P. dumerilii, a number of genes are reported to be expressed in segmental ectodermal stripes during larval segment formation, adult segment generation and/or during regeneration. These include members of the NK gene family, specifically NK4, Lbx and Msx orthologs , the wnt genes Wnt1, Wnt4, Wnt5, Wnt10, Wnt11, Wnt16[24, 87], hh and en. However, with the exception of en, hh, Wnt1 and Wnt5, these genes do not function in either arthropod or vertebrate segmentation, and thus are unlikely to be present in an ancestral bilaterian segmentation cascade. Furthermore, there are no functional data implicating any of these genes in the initial formation of segmental boundaries in annelids. Thus, there are likely fundamental differences in how segments form in annelids and arthropods, with no evidence of pair-rule patterning as part of the annelid segmentation program.
We thank Alexa Bely for the forward eve primer, Maja Makagon for cloning the initial fragment of eve from C. teleta, Lori Kaneshige for technical assistance with cloning and in situ hybridization, Danielle de Jong for help with genomic analysis of the eve genes, and Greg Davis and Nipam Patel for the Prby degenerate primer sequences prior to publication. We are grateful to members of Kewalo Marine Laboratory for discussions and comments on the preparation of this manuscript. The use of the PBRC cluster for RAxML maximum likelihood analyses comes from The University of Hawaii RCMI Program (G12RR003061). This work was supported by the National Science Foundation (IOB00-94925 and EAR-0342392 to ECS).
- Blair SS: Segmentation in animals. Curr Biol. 2008, 18: R991-995. 10.1016/j.cub.2008.08.029.View ArticlePubMed
- Chipman A: Parallel evolution of segmentation by co-option of ancestral gene regulatory networks. Bioessays. 2010, 32: 60-70. 10.1002/bies.200900130.View ArticlePubMed
- Peel A, Akam M: Evolution of segmentation: rolling back the clock. Curr Biol. 2003, 13: R708-710. 10.1016/j.cub.2003.08.045.View ArticlePubMed
- Seaver EC: Segmentation: mono- or polyphyletic. Int J Dev Biol. 2003, 47: 583-595.PubMed
- Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P, Baguñà J, Bailly X, Jondelius U, Wiens M, Müller WE, Seaver E, Wheeler WC, Martindale MQ, Giribet G, Dunn CW: Rooting the bilaterian tree with scalable phylogenomic and supercomputing tools. Proc Biol Sci. 2009, 276: 4261-4270. 10.1098/rspb.2009.0896.PubMed CentralView ArticlePubMed
- Pick KS, Philippe H, Schreiber F, Erpenbeck D, Jackson DJ, Wrede P, Wiens M, Alie A, Morgenstern B, Manuel M, Worheide G: Improved phylogenomic taxon sampling noticeably affects nonbilaterian relationships. Mol Biol Evol. 2010, 27: 1983-1987. 10.1093/molbev/msq089.PubMed CentralView ArticlePubMed
- Damen WGM: Evolutionary conservation and divergence of the segmentation process in arthropods. Dev Dyn. 2007, 236: 1379-1391. 10.1002/dvdy.21157.View ArticlePubMed
- Nüsslein-Volhard C, Wieschaus E: Mutations affecting segment number and polarity in Drosophila. Nature. 1980, 287: 795-801. 10.1038/287795a0.View ArticlePubMed
- Davis GK, Patel NH: Short, long, and beyond: molecular and embryological approaches to insect segmentation. Annu Rev Entomol. 2002, 47: 669-699. 10.1146/annurev.ento.47.091201.145251.View ArticlePubMed
- Patel NH, Ball EE, Goodman CS: The changing role of even-skipped during the evolution of insect pattern formation. Nature. 1992, 357: 339-342. 10.1038/357339a0.View ArticlePubMed
- Davis GK, D'Alessio JA, Patel NH: Pax3/7 genes reveal conservation and divergence in the arthropod segmentation hierarchy. Dev Biol. 2005, 285: 169-184. 10.1016/j.ydbio.2005.06.014.View ArticlePubMed
- Schoppmeier M, Damen WG: Expression of Pax group III genes suggests a single-segmental periodicity for opisthosomal segment patterning in the spider Cupiennius salei. Evol Dev. 2005, 7: 160-169. 10.1111/j.1525-142X.2005.05018.x.View ArticlePubMed
- Dearden P, Donly C, Grbic M: Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae. Development. 2002, 129: 5461-5472. 10.1242/dev.00099.View ArticlePubMed
- Janssen R, Budd GE, Prpic N-M, Damen WG: Expression of myriapod pair rule gene orthologs. EvoDevo. 2011, 2 (1): 5-10.1186/2041-9139-2-5.PubMed CentralView ArticlePubMed
- Hughes CL, Kaufman TC: Exploring myriapod segmentation: the expression patterns of even-skipped, engrailed, and wingless in a centipede. Dev Biol. 2002, 247: 47-61. 10.1006/dbio.2002.0683.View ArticlePubMed
- Damen WGM, Janssen R, Prpic N-M: Pair rule gene orthologues in spider segmentation. Evol Dev. 2005, 7: 618-628. 10.1111/j.1525-142X.2005.05065.x.View ArticlePubMed
- Liu PZ, Kaufman TC: Even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect. Development. 2005, 132: 2081-2092. 10.1242/dev.01807.View ArticlePubMed
- Davis GK, Patel NH: Playing by pair-rules?. Bioessays. 2003, 25: 425-429. 10.1002/bies.10278.View ArticlePubMed
- Gutjahr T, Frei E, Noll M: Complex regulation of early paired expression: initial activation by gap genes and pattern modulation by pair-rule genes. Development. 1993, 117: 609-623.PubMed
- Ingham PW, Baker NE, Martinez-Arias A: Regulation of segment polarity genes in the Drosophila blastoderm by fushi tarazu and even skipped. Nature. 1988, 331: 73-75. 10.1038/331073a0.View ArticlePubMed
- Damen WG: Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis. Development. 2002, 129: 1239-1250.PubMed
- Patel NH, Kornberg TB, Goodman CS: Expression of engrailed during segmentation in grasshopper and crayfish. Development. 1989, 107: 201-212.PubMed
- Scholtz G, Patel NH, Dohle W: Serially homologous engrailed stripes are generated via different cell lineages in the germ band of amphipod crustaceans (Malacostraca, Peracarida). Intl J Dev Biol. 1994, 38: 471-478.
- Prud'homme B, de Rosa R, Arendt D, Julien JF, Pajaziti R, Dorresteijn A, Adoutte A, Wittbrodt J, Balavoine G: Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation. Curr Biol. 2003, 13: 1876-1881. 10.1016/j.cub.2003.10.006.View ArticlePubMed
- Seaver EC, Paulson DA, Irvine SQ, Martindale MQ: The spatial and temporal expression of Ch-en, the engrailed gene in the polychaete Chaetopterus, does not support a role in body axis segmentation. Dev Biol. 2001, 236: 195-209. 10.1006/dbio.2001.0309.View ArticlePubMed
- Seaver EC, Kaneshige LM: Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans. Dev Biol. 2006, 289: 179-194. 10.1016/j.ydbio.2005.10.025.View ArticlePubMed
- Blake JA, Grassle JP, Eckelbarger KJ: Capitella teleta, a new species designation for the opportunistic and experimental Capitella sp. I, with a review of the literature for confirmed records. Zoosymposia. 2009, 2: 25-53.
- Lans D, Wedeen CJ, Weisblat DA: Cell lineage analysis of the expression of an engrailed homolog in leech embryos. Development. 1993, 117: 857-871.PubMed
- Thamm K, Seaver EC: Notch signaling during larval and juvenile development in the polychaete annelid Capitella sp. I Dev Biol. 2008, 320: 304-318.View ArticlePubMed
- Layden MJ, Meyer NP, Pang K, Seaver EC, Martindale MQ: Expression and phylogenetic analysis of the zic gene family in the evolution and development of metazoans. EvoDevo. 2010, 1 (1): 12-10.1186/2041-9139-1-12.PubMed CentralView ArticlePubMed
- Grassle J, Grassle JF: Sibling species in the marine pollution indicator Capitella (polychaeta). Science. 1976, 192: 567-569. 10.1126/science.1257794.View ArticlePubMed
- Seaver EC, Thamm K, Hill SD: Growth patterns during segmentation in the two polychaete annelids, Capitella sp. I and Hydroides elegans: comparisons at distinct life history stages. Evol Dev. 2005, 7: 312-326. 10.1111/j.1525-142X.2005.05037.x.View ArticlePubMed
- Damen WGM, Weller M, Tautz D: Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod. PNAS. 2000, 97: 4515-4519. 10.1073/pnas.97.9.4515.PubMed CentralView ArticlePubMed
- Davis GK, Jaramillo CA, Patel NH: Pax group III genes and the evolution of insect pair-rule patterning. Development. 2001, 128: 3445-3458.PubMed
- Capitella teleta v1.0. [http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html]
- Abascal F, Zardoya R, Posada D: ProtTest: selection of best-fit models of protein evolution. Bioinformatics. 2005, 21: 2104-2105. 10.1093/bioinformatics/bti263.View ArticlePubMed
- Huelsenbeck JP, Ronquist F: Bayesian analysis of molecular evolution using MrBayes. Statistical methods in molecular evolution. Edited by: Nielsen R. 2005, New York: Springer, 504-
- Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006, 22: 2688-2690. 10.1093/bioinformatics/btl446.View ArticlePubMed
- FigTree v1.3.1. [http://tree.bio.ed.ac.uk/software/figtree/]
- Meyer NP, Seaver EC: Neurogenesis in an annelid: characterization of neural progenitors in the polychaete Capitella sp. I Dev Biol. 2009, 335: 237-252.View ArticlePubMed
- Aronson BD, Fisher AL, Blechman K, Caudy M, Gergen JP: Groucho-dependent and -independent repression activities of Runt domain proteins. Mol Cell Biol. 1997, 17: 5581-5587.PubMed CentralView ArticlePubMed
- Lottia gigantea v1.0. [http://genome.jgi-psf.org/Lotgi1/Lotgi1.info.html]
- Balczarek KA, Lai ZC, Kumar S: Evolution of functional diversification of the paired box (Pax) DNA-binding domains. Mol Biol Evol. 1997, 14: 829-842. 10.1093/oxfordjournals.molbev.a025824.View ArticlePubMed
- Matus DQ, Pang K, Daly M, Martindale MQ: Expression of Pax gene family members in the anthozoan cnidarian, Nematostella vectensis. Evol Dev. 2007, 9: 25-38. 10.1111/j.1525-142X.2006.00135.x.View ArticlePubMed
- Schmerer M, Savage RM, Shankland M: Paxβ: a novel family of lophotrochozoan Pax genes. Evol Dev. 2009, 11: 689-696. 10.1111/j.1525-142X.2009.00376.x.View ArticlePubMed
- Ferrier DE, Minguillon C, Cebrian C, Garcia-Fernandez J: Amphioxus Evx genes: implications for the evolution of the midbrain-hindbrain boundary and the chordate tailbud. Dev Biol. 2001, 237: 270-281. 10.1006/dbio.2001.0375.View ArticlePubMed
- Brown SJ, Parrish JK, Beeman RW, Denell RE: Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum. Mech Dev. 1997, 61: 165-173. 10.1016/S0925-4773(96)00642-9.View ArticlePubMed
- Frasch M, Hoey T, Rushlow C, Doyle H, Levine M: Characterization and localization of the even-skipped protein of Drosophila. EMBO J. 1987, 6: 749-759.PubMed CentralPubMed
- Fröbius AC, Seaver EC: Genomic organization and expression demonstrate spatial and temporal Hox gene colinearity in the lophotrochozoan Capitella sp. I. PLoS ONE. 2008, 3: e4004-10.1371/journal.pone.0004004.PubMed CentralView ArticlePubMed
- Hui J, McDougall C, Monteiro A, Holland P, Arendt D, Balavoine G, Ferrier D: Extensive chordate and annelid macrosynteny reveals ancestral homeobox gene organization. Mol Biol Evol. 2011, 29: 7-165.
- Holland LZ, Albalat R, Azumi K, Benito-Gutierrez E, Blow MJ, Bronner-Fraser M, Brunet F, Butts T, Candiani S, Dishaw LJ, Ferrier DE, Garcia-Fernàndez J, Gibson-Brown JJ, Gissi C, Godzik A, Hallböök F, Hirose D, Hosomichi K, Ikuta T, Inoko H, Kasahara M, Kasamatsu J, Kawashima T, Kimura A, Kobayashi M, Kozmik Z, Kubokawa K, Laudet V, Litman GW, McHardy AC, Meulemans D, Nonaka M, Olinski RP, Pancer Z, Pennacchio LA, Pestarino M, Rast JP, Rigoutsos I, Robinson-Rechavi M, Roch G, Saiga H, Sasakura Y, Satake M, Satou Y, Schubert M, Sherwood N, Shiina T, Takatori N, Tello J, Vopalensky P, Wada S, Xu A, Ye Y, Yoshida K, Yoshizaki F, Yu JK, Zhang Q, Zmasek CM, de Jong PJ, Osoegawa K, Putnam NH, Rokhsar DS, Satoh N, Holland PW: The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 2008, 18: 1100-1111. 10.1101/gr.073676.107.PubMed CentralView ArticlePubMed
- Minguillon C, Garcia-Fernandez J: Genesis and evolution of the evx and Mox genes and the extended Hox and ParaHox gene clusters. Genome Biol. 2003, 4: R12-10.1186/gb-2003-4-2-r12.PubMed CentralView ArticlePubMed
- Ryan JF, Mazza ME, Pang K, Matus DQ, Baxevanis AD, Martindale MQ: Pre-bilaterian origins of the hox cluster and the hox code: evidence from the sea anemone. Nematostella vectensis. PLoS ONE. 2007, 2: e153-10.1371/journal.pone.0000153.PubMed CentralView ArticlePubMed
- Meyer NP, Boyle MJ, Martindale MQ, Seaver EC: A comprehensive fate map by intracellular injection of identified blastomeres in the marine polychaete Capitella teleta. EvoDevo. 2010, 1: 8-10.1186/2041-9139-1-8.PubMed CentralView ArticlePubMed
- Slightom JL, Blechl AE, Smithies O: Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell. 1980, 21: 627-638. 10.1016/0092-8674(80)90426-2.View ArticlePubMed
- Smithies O, Powers PA: Gene conversions and their relation to homologous chromosome pairing. Philos Trans R Soc Lond B Biol Sci. 1986, 312: 291-302. 10.1098/rstb.1986.0008.View ArticlePubMed
- McDougall C, Korchagina N, Tobin JL, Ferrier DE: Annelid Distal-less/Dlx duplications reveal varied post-duplication fates. BMC Evol Biol. 2011, 11: 241-10.1186/1471-2148-11-241.PubMed CentralView ArticlePubMed
- Woodruff JB, Mitchell BJ, Shankland M: Hau-Pax3/7A is an early marker of mesoderm involved in segmental morphogenesis, nephridial development, and body cavity formation. Dev Biol. 2007, 306: 824-837. 10.1016/j.ydbio.2007.03.002.View ArticlePubMed
- Wang W, Zhong J, Wang YQ: Comparative genomic analysis reveals the evolutionary conservation of Pax gene family. Genes Genet Syst. 2010, 85: 193-206. 10.1266/ggs.85.193.View ArticlePubMed
- Frigerio G, Burri M, Bopp D, Baumgartner S, Noll M: Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell. 1986, 47: 735-746. 10.1016/0092-8674(86)90516-7.View ArticlePubMed
- Chipman AD, Akam M: The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues. Dev Biol. 2008, 319: 160-169. 10.1016/j.ydbio.2008.02.038.View ArticlePubMed
- Dequeant ML, Pourquie O: Segmental patterning of the vertebrate embryonic axis. Nat Rev Genet. 2008, 9: 370-382.View ArticlePubMed
- Stollewerk A, Schoppmeier M, Damen WG: Involvement of Notch and Delta genes in spider segmentation. Nature. 2003, 423: 863-865. 10.1038/nature01682.View ArticlePubMed
- Carroll SB, Laughon A, Thalley BS: Expression, function, and regulation of the hairy segmentation protein in the Drosophila embryo. Genes Dev. 1988, 2: 883-890. 10.1101/gad.2.7.883.View ArticlePubMed
- Sommer RJ, Tautz D: Involvement of an orthologue of the Drosophila pair rule gene hairy in segment formation of the short germ band embryo of Tribolium (Coleoptera). Nature. 1993, 361: 448-450. 10.1038/361448a0.View ArticlePubMed
- Dormand E-L, Brand AH: Runt determines cell fates in the Drosophila embryonic CNS. Development. 1998, 125: 1659-1667.PubMed
- Kalev-Zylinska M, Horsfield J, Flores M, Postlethwait J, Vitas M, Baas A, Crosier P, Crosier K: Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis. Development. 2002, 129: 2015-2030.PubMed
- Chen A, de Nooij J, Jessell T: Graded activity of transcription factor Runx3 specifies the laminar termination pattern of sensory axons in the developing spinal cord. Neuron. 2006, 49: 395-408. 10.1016/j.neuron.2005.12.028.View ArticlePubMed
- Zagami CJ, Zusso M, Stifani S: Runx transcription factors: lineage-specific regulators of neuronal precursor cell proliferation and post-mitotic neuron subtype development. J Cell Biochem. 2009, 107: 1063-1072. 10.1002/jcb.22221.View ArticlePubMed
- Denes AS, Jekely G, Steinmetz PR, Raible F, Snyman H, Prud'homme B, Ferrier DE, Balavoine G, Arendt D: Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in Bilateria. Cell. 2007, 129: 277-288. 10.1016/j.cell.2007.02.040.View ArticlePubMed
- Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Gruss P: Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 1991, 10: 1135-1147.PubMed CentralPubMed
- Jostes B, Walther C, Gruss P: The murine paired box gene, Pax-7, is expressed specifically during the development of the nervous and muscular system. Mech Dev. 1991, 33: 27-38.View Article
- de Rosa R, Prud'homme B, Balavoine G: Caudal and even-skipped in the annelid Platynereis dumerilii and ancestry of posterior growth. Evol Dev. 2005, 7: 574-587. 10.1111/j.1525-142X.2005.05061.x.View ArticlePubMed
- Song MH, Huang FZ, Chang GY, Weisblat DA: Expression and function of an even-skipped homolog in the leech Helobdella robusta. Development. 2002, 129: 3681-3692.PubMed
- Sordino P, Duboule D, Kondo T: Zebrafish Hoxa and Evx-2 genes: cloning, developmental expression and implications for the functional evolution of posterior Hox genes. Mech Dev. 1996, 59: 165-175. 10.1016/0925-4773(96)00587-4.View ArticlePubMed
- Ruiz i Altaba A, Melton DA: nvolvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis. Cell. 1989, 57: 317-326. 10.1016/0092-8674(89)90969-0.View ArticlePubMed
- Ahringer J: Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7. Genes Dev. 1996, 10:
- Brown SJ, Parrish JK, Beeman RW, Denell RE: Molecular characterization and embryonic expression of the even-skipped orthologue of Tribolium casaneum. Mech Dev. 1997, 61: 165-173. 10.1016/S0925-4773(96)00642-9.View ArticlePubMed
- Copf T, Rabet N, Celniker SE, Averof M: Posterior patterning genes and the identification of a unique body region in the brine shrimp Artemia franciscana. Development. 2003, 130: 5915-5927. 10.1242/dev.00835.View ArticlePubMed
- Patel NH, Condron BG, Zinn K: Pair-rule expression patterns of even-skipped are found in both short-and long-germ beetles. Nature. 1994, 367: 429-434. 10.1038/367429a0.View ArticlePubMed
- Doe C, Smouse D, Goodman CS: Control of neuronal fate by the Drosophila segmentation gene even-skipped. Nature. 1988, 333: 376-378. 10.1038/333376a0.View ArticlePubMed
- Esmaeili B, Ross JM, Neades C, Miller DM, Ahringer J: The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory. Development. 2002, 129: 853-862.PubMed
- Ruiz i Altaba A: Neural expression of the Xenopus homeobox gene Xhox3: evidence for a patterning neural signal that spreads through the ectoderm. Development. 1990, 108: 595-604.PubMed
- Bastian H, Gruss P: A murine even-skipped homologue, Evx 1, is expressed during early embryogenesis and neurogenesis in a biphasic manner. EMBO J. 1990, 9: 1839-1852.PubMed CentralPubMed
- Seebald JL, Szeto DP: Zebrafish eve1 regulates the lateral and ventral fates of mesodermal progenitor cells at the onset of gastrulation. Dev Biol. 2011, 349: 78-89. 10.1016/j.ydbio.2010.10.005.View ArticlePubMed
- Saudemont A, Dray N, Hudry B, Le Gouar M, Vervoort M, Balavoine G: Complementary striped expression patterns of NK homeobox genes during segment formation in the annelid Platynereis. Dev Biol. 2008, 317: 430-443. 10.1016/j.ydbio.2008.02.013.View ArticlePubMed
- Janssen R, Le Gouar M, Pechmann M, Poulin F, Bolognesi R, Schwager EE, Hopfen C, Colbourne JK, Budd GE, Brown SJ, Prpic NM, Kosiol C, Vervoort M, Damen WG, Balavoine G, McGregor AP: Conservation, loss, and redeployment of Wnt ligands in protostomes: implications for understanding the evolution of segment formation. BMC Evolutionary Biology. 2010, 10: 374-10.1186/1471-2148-10-374.PubMed CentralView ArticlePubMed
- Dray N, Tessmar-Raible K, Le Gouar M, Vibert L, Christodoulou F, Shipany K, Guillou A, Zantke J, Snyman H, Behague J, Vervoort M, Arendt D, Balavoine G: Hedgehog signaling regulates segment formation in the annelid Platynereis. Science. 2010, 329: 339-342. 10.1126/science.1188913.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.