Capitella sp. I homeobrain-like, the first lophotrochozoan member of a novel paired-like homeobox gene family

Gene Expression Patterns 6 (2006) 985–991 www.elsevier.com/locate/modgep

Capitella sp. I homeobrain-like, the Wrst lophotrochozoan member of a novel paired-like homeobox gene family Andreas C. Fröbius 1, Elaine C. Seaver ¤ Kewalo Marine Lab, PaciWc BioScience Research Center, University of Hawaii, 41 Ahui St., Honolulu, HI 96813, USA Received 2 February 2006; received in revised form 24 March 2006; accepted 10 April 2006 Available online 9 June 2006

Abstract The paired-like class of homeobox genes contains numerous distinct families, many of which have been implicated in a variety of developmental functions. We report the isolation and expression of a gene with high similarity to Drosophila melanogaster homeobrain from the polychaete annelid Capitella sp. I. The homeobrain-like (hbnl) gene is a paired-like gene that contains a conserved homeodomain, octapeptide region, alanine stretches, and an OAR domain. Gene orthology analyses of the homeodomain from CapI-hbnl places this gene in a new family of paired-like homeodomain genes that includes D. melanogaster homeobrain (hbn) and representatives from all major bilaterian clades as well as a cnidarian gene. CapI-hbnl expression is largely restricted to subsets of cells in the brain and eyes during larval development in Capitella sp. I. The earliest expression of CapI-hbnl is in small discrete cell clusters in the cerebral ganglia. This expression persists through late larval developmental stages whereas expression is absent in postmetamorphic juveniles. Outside the brain, expression is present on the ventral side of the larva in two small cell clusters, at the brain/pharyngeal border and in the anterior-most segment. CapI-hbnl shares features of brain expression with hbn, although in contrast to hbn, which is expressed along the length of the ventral nerve cord, CapI-hbnl has a restricted anterior expression pattern. CapI-hbnl represents an important neural marker for characterization of the annelid nervous system. © 2006 Elsevier B.V. All rights reserved. Keywords: Homeobrain; Paired-like; Polychaete; Brain development; Eye; Hbnl; Homeobrain-like; Capitella sp. I

1. Results and discussion Homeodomain transcription factors are structurally and often functionally conserved across wide phylogenetic distances and play a variety of major roles during metazoan development. These regulatory genes form a large and diverse group, and have been categorized into distinct classes according to diVerences in sequence within the homeodomain as well as the presence or absence of additional conserved domains outside the homeodomain. The paired

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Corresponding author. Tel.: +1 808 539 7324; fax: +1 808 599 4817. E-mail addresses: [email protected] (A.C. Fröbius), [email protected] (E.C. Seaver). 1 Present address: Institut für Phytopathologie und Angewandte Zoologie, Justus-Liebig-Universität Gießen, Heinrich-BuV-Ring 26-32, 35392 Gießen, Germany. 1567-133X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.04.002

class of homeodomain genes have an additional conserved 130 amino acid DNA-binding domain upstream of the homeodomain, called the ‘paired’ domain (Bopp et al., 1986). The ‘paired-like’ class shows sequence similarities in its homeodomain to the homeodomain of the paired class, but it lacks the paired domain (Duboule, 1994). Most of the reported paired-like genes have been isolated from Drosophila, Caenorhabditis, and vertebrate model organisms within the last decade. Many paired-like genes are expressed in the nervous system, among them several families that are characteristically expressed during brain and eye morphogenesis, such as the Rx (retinal homeobox), Arx (aristaless-related homeobox), and Vsx (visual system homeobox) families. Rx genes are expressed in the forebrain and developing retina of vertebrates (Casarosa et al., 1997; Furukawa et al., 1997; Mathers et al., 1997), and in Drosophila, Rx is involved in the development of a central

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brain structure and clypeus precursors, although expression in eye primordia or larval eye-antennal imaginal discs is absent (Eggert et al., 1998). Chx/Vsx genes encode transcription factors involved in formation and maintenance of ocular tissues in vertebrates (Ohtoshi et al., 2001). In mouse and zebraWsh, Arx expression suggests a role in the maintenance of speciWc neuronal subtypes in the forebrain and axon guidance in the Xoor plate (Miura et al., 1997), and al is expressed in the brain of developing Xy embryos (Schneitz et al., 1993). Paired-like homeodomain genes have been less well-characterized in lophotrochozoans. Expression patterns for orthologues of the goosecoid (gsc) family in a mollusc and a polychaete (Arendt et al., 2001; Lartillot et al., 2002), and of otp in a mollusc (Nederbragt et al., 2002) show conservation with orthologous family members from other bilaterian clades. Here, we report the isolation and characterization of a paired-like homeodomain gene from a third paired-like family in the polychaete annelid Capitella sp. I. Its developmental expression proWle is highly restricted; during brain development, it is limited to a subset of anterior structures, mostly nervous system elements in the head. Furthermore, gene orthology analysis places it within a family distinct from other previously described paired-like homeobox gene families. 1.1. IdentiWcation of CapI-hbnl Using degenerate PCR to isolate homeobox genes, a 129 bp DNA fragment of a novel paired-like homeodomain gene was ampliWed from a mixed embryonic and larval stage Capitella sp. I cDNA library. From the 87 bp unique sequence, gene-speciWc primers were used in a RACE reaction to identify additional sequence using the cDNA library as a template. Sequence (1.5 kb) was recovered and conceptually spliced together, which we name homeobrain-like (CapI-hbnl), reXecting its high similarity to Drosophila homeobrain (hbn). The CapI-hbnl cDNA contains a complete 1068 bp ORF and codes for a predicted protein of 356 amino acids. It is Xanked by a 44 bp 5⬘ UTR and a 331 bp 3⬘UTR, which is terminated by a poly(A) tract. Two putative polyadenylation sites (AATAAT and AATAAA) are located immediately upstream of the poly(A)-tail. The deduced CapI-hbnl protein sequence contains an N-terminal octapeptide/GEH domain, a motif which functions as a transcriptional repressor (Smith and Jaynes, 1996), a homeodomain, and a C-terminal OAR transcriptional activation domain (named after the Wrst initials of otp, aristaless, and rax, where it was Wrst found (Simeone et al., 1994; Brouwer et al., 2003)) (Fig. 1A). 1.2. CapI-hbnl is a member of a novel paired-like family Gene orthology analysis of the CapI-hbnl homeodomain by Bayesian phylogenetic inference and neighbor joining reveals that CapI-hbnl is a member of the paired-like class of homeodomain genes (Fig. 2). Within the paired-like class, there are a number of distinct families present in

diverse taxa including Rx, aristaless (alx, arx), Vsx, orthopedia (otp) (see review by Galliot et al., 1999), goosecoid (Gsc) (Cho et al., 1991; Blum et al., 1992), and Pitx-genes (Gage et al., 1999), which, together with the paired genes, comprise the PRX superclass of homeodomain genes (Duboule, 1994). The CapI-hbnl gene does not cluster within one of these subgroups. However, CapI-hbnl clusters with Drosophila melanogaster hbn (Walldorf et al., 2000), in a wellsupported node that also contains several other paired-like homeodomain genes (Fig. 2). These gene sequences are from the following species: the insect Anopheles gambiae, the urchin Strongylocentrotus purpuratus, the hemichordate Sarcoglossus kowalevskii, and the cnidarian Hydra vulgaris. Thus, we have identiWed a novel paired-like homeodomain family, which we call homeobrain-like (hbnl), one that includes D. melanogaster Hbn and CapI-hbnl. Our analyses place the hbnl homeodomain family closest to the pairedlike Rx genes, although this association is not strongly supported. The presence of two glutamines, at position 15 and 24 of the homeodomain, distinguish the hbnl family from other paired-like and pax homeodomain members (Fig. 1D). Although glutamines at these two positions are widely found among diverse classes of homeodomain genes, they have been lost from other paired-like homeodomain families, occurring only in the vertebrate Prop genes. The hbnl family of paired-like homeodomain genes was likely present in the bilaterian plus cnidarian ancestor, since we have identiWed members representing all three major bilaterian clades as well as a Hydra sequence (cnidarian). We were unable to identify a vertebrate homeobrain orthologue by database searches of whole genomes of several vertebrates (see Section 2), consistent with a loss of this paired-like homeodomain family in the vertebrate lineage. One feature of paired-like homeodomain genes is the presence of conserved domains in addition to the homeodomain. The predicted ORF of CapI-hbnl contains an OAR domain and an octapeptide domain (Fig. 1A), and all three domains in the CapI-hbnl sequence show sequence conservation with other taxa, illustrated by domain alignments from paired-like class representatives from other species (Fig. 1B–D). It is notable that although the presence/ absence of the homeodomain, OAR, and octapeptide domains is generally conserved within families of pairedlike homeodomain genes (Table 1), the presence of particular domains among the genes that cluster with Dme-hbn in the hbnl family varies among species. For example, the CapI-hbnl and the S. purpuratus sequences have all three domains, and Dme-hbn and the H. vulgaris sequences contain the homeodomain and octapeptide, but not the OAR domain (Table 1). It will be interesting to determine whether these domain diVerences among members of the hbnl family correlate with distinct functions. 1.3. Expression of CapI-hbnl The expression of CapI-hbnl was examined by wholemount in situ hybridization. No signal was detected during

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Fig. 1. Schematic of the Capitella sp. I-hbnl gene product and comparisons with conserved domains from paired-like homeodomain genes from other species. (A) Structure of the CapI-hbnl predicted open reading frame (ORF). The CapI-hbnl transcript contains a predicted 1068 bp ORF, coding for 356 amino acid residues. CapI-hbnl features an amino-terminal octapeptide, a paired-like homeodomain, two polyalanine stretches and a carboxy-terminal OAR domain. Amino acid alignments of the CapI-hbnl octapeptide (B), homeodomain (D), and OAR domain (C) with related domains of other pairedclass homeodomain genes. Residues identical with corresponding residues of CapI-hbnl are marked “.”, missing residues are marked “-”. See Fig. 2 legend for species abbreviations and Section 2 for sequence accession numbers.

embryonic and early larval stages. The majority of CapIhbnl expression is restricted to a subpopulation of cell clusters in the developing brain. Brain development in Capitella sp. I larvae has previously been described (Bhup and Marsden, 1982). The cerebral ganglia initially appear as two lateral epidermal thickenings in the head (Stage 4), which expand through localized cell divisions and eventually become internalized as bilaterally symmetric structures anterior to the mouth connected by a cerebral commissure. Cerebral ganglia development can be followed morphologically from very early stages using a nuclear stain such as Hoechst, since the brain primordia appear as areas of higher nuclear density relative to surrounding tissue (e.g. Seaver et al., 2005; Fröbius and Seaver, 2006) and in unstained specimens as areas that are clear of yolk in the anterior region of the head (Fröbius and Seaver, unpublished observations). CapI-hbnl expression is initiated at Stage 5 (staging system after Seaver et al., 2005) during later phases of cerebral ganglia formation. Expression can Wrst be detected in a big medial cluster (Fig. 3B) and two small lateral clusters of approximately 2–4 cells/cluster on each side of the brain (Fig. 3C). During

Stage 6, the two big bilateral patches expressing CapI-hbnl are replaced by a number of smaller clusters of expression (some of which contain only two cells). In addition, a new expression domain appears in the lateral epithelium adjacent to these smaller clusters (Fig. 3D and E), reXecting initial expression of CapI-hbnl in the epithelio–optic nerves (Bhup and Marsden, 1982). Expression is also detected in a few nerve cells connecting the epithelio–optic nerve and the medial expression domain (Fig. 3D). Discrete expression in a few cells of the cerebral commissure also becomes visible (Fig. 3D). Outside the brain, expression of CapIhbnl is now detected in two small domains located between the posterior boundary of the cerebral ganglia and the anterior wall of the larval pharynx (Stage 6) (Fig. 3D and E). At Stage 7, CapI-hbnl expression becomes more pronounced in the epithelio–optic nerves and more medial cell clusters in the brain (Fig. 3F and G). In the lateral epithelium, expression now clearly coincides with the position of the eyes (compare Fig. 3G with Fig. 3H). Between Stages 6 and 8, the medial brain cells expressing CapI-hbnl move closer together towards the midline (compare Fig. 3D and F). At late Stage 7/Stage 8, two small clusters

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100

77 88

95

99

100 65 HsaVsx1 100 HsaChx10 100 MmuChx10 95 DreVsx2 100 SpuAl 78 100 LvaAlx1 Alx 79 MmuAlx3 96 GgaAlx4 73 DreRx3 PduRx XlaN HsaRx Rx 100 GgaRAX 100 DreRx2 DreRx1 DmeRx 72 HvuAlx 83 SpuN 77

PvuGsc DmeGsc PduGsc MmuGsc MmuPax6

95

CapIHbn-l

85 91

100 99

SkoN DmeHbn AgaN

Gsc

MmuPax4

Pax 4/6

Vsx/Chx10

Hbn-l

PvuOtp MmuOtp Otp 100 DreOtp 100 DmeOtp MmuPax3 100 Pax 3/7 MmuPax7 100 HsaProp1 100 Prop1 MmuProp1 100 BbePitx 100 Pitx DmePitx 100 95 90

54 60

100 100 99

91 DmeAl 100 AmeAlx DreArx TniN MMuArx XlaAlx SpuAlx DmeDll CinDll 58 DmeAbdB 54 CapIXlox 55 CapIGsx

CapICdx

0.1 Fig. 2. CapI-hbnl is a member of a novel family of paired-like homeodomain genes, called ‘homeobrain-like’ (hbnl), which includes D. melanogaster hbn and sequences from diverse taxa. Consensus tree shows the relationship of CapI-hbnl with other paired-like class homeodomain sequences. The Dll-genes, Xlox, Gsx, Cdx, and AbdB belong to other homeodomain gene classes and have been used to root the tree. The tree shown is a Bayesian interference consensus tree with posterior probabilities >50% marked above the nodes. Values below nodes are bootstrap support values from neighbor joining analysis. Species abbreviations: Aga, Anopheles gambiae; Ame, Apis mellifera; Bbe, Branchiostoma belcheri; CapI, Capitella sp. I; Cin, Ciona intestinalis; Dme, Drosophila melanogaster; Dre, Danio rerio; Gga, Gallus gallus; Hsa, Homo sapiens; Hvu, Hydra vulgaris; Lva, Lytechinus variegatus; Mmu, Mus musculus; Pdu, Platynereis dumerulii; Pvu, Patella vulgata; Sko, Sarcoglossus kowalevskii; Spu, Strongylocentrotus purpuratus; Tni, Tetraodon nigroviridis; Xla, Xenopus laevis. See Section 2 for sequence accession numbers.

of CapI-hbnl-expressing cells appear in the anterior-most segment lateral to the mouth (Fig. 3J). Expression levels in all domains decrease by late larval stages (Stage 8) and

appear to be expressed in fewer cells/cluster (Fig. 3I and J). CapI-hbnl expression in post-metamorphic juveniles was not detected.

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Table 1 Distribution of domains in CapI-hbnl and other paired-class homeodomain genes

CapI-hbnl SpuN SkoN HvuAlx DmeHbn Rx-genes Chx10-genes Bilaterian Alx-genes HsaVsx HsaVsx2 DmeOtp Gsc-genes

Octapeptide

Homeodomain

Pos15 Hom

Pos50 Hom

OAR

F F ? F F F — — F — — F

F F F F F F F F F F F F

Q Q Q Q Q E E E E E E Varying

Q Q Q Q Q Q Q Q Q Q Q K

F F F — — F F F — F F —

Paired-like homeodomain gene families have distinct combinations of octapeptide and OAR domains. Within the ‘hbnl’ family, there is variation among species in the presence and/or absence of particular domains. Horizontal lines indicate absence of motif, check indicates presence. Hom, homeodomain; OAR, OAR domain; Pos15 Hom, and Pos50 Hom, aa at position 15 and 50 of the homeodomain, respectively.

1.4. Comparison of expression patterns among hbnl family members The temporal and spatial expression of CapI-hbnl is consistent with a role in diVerentiation of the larval eye and brain, possibly conferring identity to distinct subsets of cells. The exact identity of these neural cell clusters is unknown since detailed knowledge of the Wne structure and molecular development of the capitellid brain is currently limited. Drosophila hbn and CapI-hbnl expression patterns share a number of features, although the two genes also have distinct expression characteristics. Both are expressed in the developing brain, initially as large patches, which resolve into smaller expression domains as the pattern matures. While CapI-hbnl transcripts are detected in larval eyes and the optic nerves, expression of Drosophila hbn in the visual system has not been reported. Furthermore, hbn is expressed in the ventral nerve cord and in the midgut/hindgut boundary, expression domains absent in Capitella sp. I. In general, CapI-hbnl has more restricted expression than hbn. Less is known about expression of the other members of the hbnl family. The expression of HyAlx, the aristaless-related gene isolated from Hydra, is conWned to a band of ectodermal epithelial cells at the base of the tentacles in polyps (Smith et al., 2000), expression consistent with a role in tentacle patterning. Expression of HyAlx in nerve cells is not observed. To our knowledge, expression patterns of the genes from S. purpuratus, S. kowalevskii, and A. gambiae are not yet reported. The eye and brain expression of CapI-hbnl shows similar expression to other families of paired-like homeodomain genes, particularly the Rx, Arx, and Vsx families, and this conservation may reXect an ancestral role of paired-like homeodomain genes. 2. Experimental procedures 2.1. Capitella sp. I culture Capitella sp. I was reared in Wnger-bowls containing mud and Wltered seawater (FSW) as described previously (Seaver et al., 2005). Embryos of Stages 2–4 were Wrst permeabilized by treatment with 0.5 M sucrose/ 0.125 M sodium citrate for 3 min., older stages were relaxed in 0.19 mol/l

MgCl2 in FSW for 15 min prior to Wxation in 3.7% formaldehyde in FSW at 4 °C overnight. All developmental stages were washed, dehydrated, and stored in methanol at ¡20 °C.

2.2. Isolation of a paired-like gene Eighty-seven base pairs of a gene fragment with identity to Drosophila hbn were isolated from Capitella sp. I by degenerate PCR. Primers targeted against conserved regions of the homeodomain (AQLVELEKE (5⬘-GCB CAR YTN GTH GAR YTV GAR AAR G-3⬘) and WFQNRR (5⬘-CKN CKR TTY TGR AAC CA-3⬘)), were used under conditions favoring the ampliWcation of small fragments (2£ PCR buVer, 4.5 mM MgSO4) using MasterTaq-Polymerase and Taqmaster (Eppendorf). An ampliWed cDNA library generated from total RNA isolated from Capitella sp. I embryos and larvae (Stages 1–8), was used as a template, following excision from the lambda ZAPII vector. 5⬘- and 3⬘-regions of the transcript were obtained by direct PCR from the excised cDNA library using a nested PCR-strategy with gene-speciWc primers and pBluescript-speciWc primers. For ampliWcation of the 5⬘-region, ccPrd3out (5⬘-ACC TGG ACC CTG GCC T-3⬘) was combined with T3 (5⬘-AAT TAA CCC TCA CTA AAG GG-3⬘) followed by ccPrd3in (5⬘-GGT CCA GCC TCA GCG CCA ACT CCT C-3⬘), and T3RAC (5⬘-CTG GAG CTC CAC CGC GGT GGC-3⬘). Isolation of the 3⬘-region involved ccPrd5out (5⬘-GAG AAG ACA CAG TAC CCA GAT G-3⬘) and M13E (5⬘-GTA AAA CGA CGG CCA GTG-3⬘), followed by a second round using ccPrd5in (5⬘-GGA GGA GTT GGC GCT GAG GCT GGA C-3⬘) and T7RAC2 (5⬘-GAC GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG C-3⬘). The Wrst ampliWcation was performed with standard conditions (94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 50 °C for 40 s, and 72 °C for 2 min and an additional elongation-cycle of 72 °C for 5 min). The product from the Wrst round was diluted 1/100 and used as a template in a touchdown-PCR involving 5 cycles of (94 °C for 30 s, 72 °C for 2.5 min), followed by 5 cycles of (94 °C for 30 s, 70 °C for 2.5 min) and 20 cycles of (94 °C for 30 s, 68 °C for 40 s, 72 °C for 2 min). PCR products were puriWed utilizing the MinElute-Kit (Qiagen) and cloned into the pGEM-TEasy plasmid vector (Promega). Clones were sequenced by Macrogen, Seoul, Korea with an ABI3730 XL automatic DNA sequencer.

2.3. Whole mount in situ hybridization A 0.6 kb digoxygenin-labeled CapI-hbnl anti-sense riboprobe corresponding to a region immediately 5⬘ of the homeodomain was synthesized by in vitro transcription using the T7-Megascript-kit (Ambion). Whole mount in situ hybridization was carried out as described previously (Seaver and Kaneshige, 2006) on Stage 4–8 Capitella sp. I larvae. The Wnal probe concentration was 2 ng/l. Specimens were cleared, mounted in 80% glycerol, and viewed by DIC optics on a Zeiss Axioskop II plus. Digital images were taken with a Nikon Coolpix 4500.

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Fig. 3. Spatial expression of CapI-hbnl during larval development of Capitella sp. I shown by whole-mount in situ hybridization. For each stage, both a dorsal mid-focal plane view (B, D, F, and I) and a lateral view (C, E, G, H, and J) are shown. In all panels anterior is to the left and for all lateral views, ventral is down. (A) Schematic of the Capitella sp. I larval body plan and position of the CNS (shown in grey). Ventral view is shown on the left and lateral view is on the right. (B and C) Stage 5 larva. (D and E) Stage 6. (F, G, and H) Stage 7. (I and J) Stage 8. Note that in panel (F), the mid-body has non-speciWc staining in the chaetal sacs (reiterated spots). (H) Anterior region of an unstained Stage 7 larva showing the position of the eye (red spot), lateral view. Big grey arrows indicate the earliest expression of CapI-hbnl in large medial clusters near the posterior wall of the forming brain. Small grey arrows indicate CapI-hbnl in small clusters near the posterior wall of the forming brain. White arrowheads show expression in the position of the eyes, white arrows indicate nerve cells connecting the medial expression domain and the epithelio–optic nerve. Black arrowheads show two clusters of CapI-hbnl expressing cells located between the brain and oesophagus. The black arrow shows CapI-hbnl expression in the commissure between the two brain halves. The grey arrowhead points towards late onset of CapI-hbnl expression in a cell cluster located posterior to the mouth and lateral to the pharynx. The asterisk marks the position of the mouth; p, prototroch; t, telotroch.

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2.4. Sequence and phylogenetic analysis Sequence similarities to paired-like genes were ascertained for CapI-hbnl by BLASTX searches of the GenBank database from NCBI. An amino acid (aa) alignment of the homeodomain was generated with sequences identiWed from BLASTX searches and other representatives of the paired-like homeodomain class, including a range of distinct family members. Searches for hbnl and hbn orthologues from additional species were performed by directly searching the genome and/or EST databases from the following species: Homo sapiens, Xenopus laevis, Mus musculus, Bos taurus, and Danio rerio. Alignments were generated with the MacVector software (Accelyrs, v. 7.2.3) using default alignment parameters (CLUSTALW) (Matrix, BLOSUM Series; GapOpen, 10; GapExt, 0.05; GapDist, 8) and then examined and hand edited for any obvious alignment errors. Bayesian phylogenetic analysis was performed with MrBayes V3.1.1 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) using the ‘wag’ amino acid model with two independent runs of 1,000,000 generations and four chains each. A majority rule consensus tree was generated from 9500 trees representing 950,000 stable generations. Neighbor Joining analysis was performed in PAUP*4.0b10 (SwoVord, 2002) with 1000 replicates for bootstrap analysis. Nexus alignments available upon request. Accession #s for sequences included in the analyses are as follows: CapI-hbnl, (DQ333220); AgaN (EAA11051); AmeAlx (XP_624630); BbePitx (AAF03901); CapICad (DQ102389); CapIGsx (DQ132894); CapIXlox (DQ102390); CinDll (CAC08327); DmeAbd (CAB57859); DmeAl (AAA28840); DmeDll (AAB24059); DmeGsc (CAA64699); DmeHbn (CAC06429); DmeOtp (NP_995909); DmePitx (018400); DmeRx (CAA11241); DreArx (O42115); DreOtp (AAD42021); DreRx1 (AAH60890); DreRx2 (NP_571301); DreRx3 (NP_571302); DreVsx2 (NP_571537); GgaRAX (NP_989435); GgaAlx4 (AAC61772); HsaChx10 (P58304); HsaPitx1 (AAH03685); HsaVsx1 (NP_055403); HvuAlx (AAG03082); HsaProp1 (AAC77453); HsaRx (AAD23438); LvaAlx1 (AAP34699); MmuAlx3 (NP_031467); MmuAlx4 (NP_031468) MmuArx, (O35085); MmuChx10 (AAH58806); MmuGsc (NP_034481); MmuOtp (NP_035151); MmuPax3 (AAH48699); MmuPax4 (NP_035168); MmuPax6 (CAC80518); MmuPax7 (AAG16663); MmuProp1, (Q5NCG9); TniN, (CAF97990); AgaN (EAA11051); PduGsc (CAC19336); PduRx (AAU20320); PvuGsc (CAD45551); PvuOtp (AAM33145); SpuAlx (Q26657); SpuAlx3 (AAP34698); SpuN (XP_781057); XlaAlx (AAS91656); XlaN (unnamed from Xenbase; http://www.xenbase.org/); SkoN is an unpublished sequence provided by Chris Lowe.

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