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Genomic Organisation, Embryonic Expression and Biochemical Interactions of the Zebrafish Junctional Adhesion Molecule Family of Receptors

Genomic Organisation, Embryonic Expression and Biochemical Interactions of the Zebrafish Junctional Adhesion Molecule Family of Receptors

  • Gareth T. Powell, 
  • Gavin J. Wright
PLOS
x

Abstract

The mammalian JAM family is composed of three cell surface receptors. Interactions between the proteins have well-characterised roles in inflammation and tight junction formation, but little is known about their function in early development. Recently, we identified a role for jamb and jamc in zebrafish myocyte fusion. Genome duplication in the teleost lineage raised the possibility that additional JAM family paralogues may also function in muscle development. To address this, we searched the zebrafish genome to identify potential paralogues and confirmed their homology, bringing the total number of zebrafish jam family members to six. We then compared the physical binding properties of each paralogue by surface plasmon resonance and determined the gene expression patterns of all zebrafish jam genes at different stages of development. Our results suggest a significant sub-functionalisation of JAM-B and JAM-C orthologues with respect to binding strength (but not specificity) and gene expression. The paralogous genes, jamb2 and jamc2, were not detected in the somites or myotome of wild-type embryos. We conclude that it is unlikely that the paralogues have a function in primary myogenesis.

Introduction

The JAM (Junctional adhesion molecule) family is a small, deuterostome lineage-restricted subgroup of the immunoglobulin superfamily [1]. There are three JAM genes in the human genome, each encoding a type I cell surface receptor with two immunoglobulin domains, a single transmembrane domain and a short cytoplasmic region ending in a C-terminal PDZ domain-binding motif [2]. The members of the family are named JAM-A, JAM-B and JAM-C (official symbols: F11R, JAM2, JAM3 respectively; for clarity and consistency with recent literature we use the systematic, unofficial nomenclature, as suggested previously [3]).

To date, the main focus of research into mammalian JAM proteins is their role in inflammation [4]. Heterophilic and homophilic interactions between JAM family proteins are important for their function: JAM-A interacts homophilically [5][8] and with JAM-C [9], JAM-B interacts with itself [10] and JAM-C [11][14] and JAM-C interacts with itself and JAM-A [9], [12], [13]. To date, no interaction between JAM-B and JAM-A has been demonstrated. Interactions between JAM proteins and integrins, expressed by vascular endothelia and leukocytes, are thought to play a key role in controlling migration of leukocytes into and away from sites of inflammation [5], [9], [11][13], [15], [16]. Many other roles have been proposed for each member of the family, including: angiogenesis [17][20], cancer [9], [19], [21][23], spermatid development and motility [24][26], and the maintenance of the myelin sheath formed by Schwann cells [27]. These diverse processes are most likely unified by an important function of the JAM proteins – the formation and maintenance of adhesion and tight junctions between cells [12], [13], [28][35].

Although the JAMs are known to be expressed during embryogenesis, no developmental function has been described in the various mouse models to date [20], [25], [26], [36][40]. Our recent work on cell surface protein interactions identified a function for zebrafish orthologues of JAM-B and JAM-C (named Jamb and Jamc, respectively) in myogenesis [41]. Using cellular transplantation assays, we were able to determine that the heterophilic interaction of Jamb and Jamc between muscle precursor cells (myocytes), in trans, was essential for cellular fusion and the formation of normal, syncytial muscle fibres. Loss-of-function of either protein resulted in a potent block in fusion, resulting in an overabundance of mononuclear muscle fibres. The zebrafish orthologue of JAM-A has also been shown to play a role in the development of the lateral line, though the exact function of the protein remains to be elucidated [42].

In Drosophila, the cell surface receptors Kirre [43] and Sns [44], and their paralogues Rst [45] and Hbs [46], play a critical role in the development of the larval body wall musculature. The interaction between Kirre and Sns [47] is thought to be the critical step in initiating myocyte fusion between sub-types of precursor cells – founder cells and fusion competent myoblasts (FCMs); the mutually exclusive expression of these proteins in the different sub-types of myocytes is a key regulatory feature [48], [49]. Kirre is expressed by founder cells [43], and Sns is expressed by FCMs [44], so that only founder cell-FCM fusion events can occur. However, loss-of-function of Kirre or Sns alone doesn’t cause a complete loss of fusion. The paralogues of these receptors, Rst and Hbs, play partially redundant functions as they have overlapping expression patterns and are capable of interacting with each other, and Kirre or Sns, to initiate fusion [45][47]. The zebrafish genome contains a high number of paralogous genes because of an ancient duplication of the genome in the teleost lineage [50], [51]. The possibility that jamb and jamc paralogues may also participate in myocyte fusion in zebrafish was of particular interest to us because such a discovery might aid in further elucidating the molecular biology of myogenesis. To address this question, we sought to establish if the JAM family had been duplicated in zebrafish and if so, compare the embryonic expression and physical binding properties of the paralogues.

Results

Cloning and Homology of Zebrafish Jam Family Members

Four zebrafish JAM family orthologues, F11r, Jam2a, Jam2b and Jam3b (hereafter referred to as Jama, Jamb, Jamb2 and Jamc respectively; see Materials and Methods for official nomenclature and database entries), were included in recent AVEXIS screens for cell surface protein interactions that are important for zebrafish development [52][54]. The interaction between Jamb and Jamc was identified [52] and later found to be essential for myocyte fusion during muscle development [41]. To identify paralogues of jama and jamc that may have arisen because of genome duplication in the teleost lineage [50], [51], we searched the zebrafish genome using the protein sequence of the extracellular domains of Jama and Jamc, reasoning that the immunoglobulin superfamily (IgSF) domains are likely to be the most conserved amongst JAM family members. We identified and cloned the full length sequences of potential candidates by 3′ RACE or RT-PCR (Figure 1). For clarity, hereafter we refer to these candidates as jama2 (official symbol si:ch211-89p1.1) and jamc2 (official symbol jam3a).

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Figure 1. Cloning of putative jama2 and jamc2 paralogues by 3′ RACE or RT-PCR.

Agarose gel electrophoresis results of amplification of full-length jama2 by 3′ RACE (A) or jamc2 by RT-PCR (B) from cDNA prepared from total RNA extracts of wild-type 24 h. p. f. zebrafish embryos. (A) Lanes: - 3′ RACE negative control; 1–4 igsf11 positive control: 3′ RACE –1 5′ primer, 2 nested 5′ primer; nested PCR –3 negative control, 4 nested 5′ primer; 5–8 jama2∶3′ RACE –5 5′ primer, 6 nested 5′ primer; nested PCR –7 negative control, 8 nested 5′ primer. (B) Lanes: - negative control; + ef1α positive control; 1 jamb positive control; 2 & 4 jamc2 primers designed to amplify predicted full-length ORF; 3 & 5 jamc2 primers designed to amplify IgSF domains. M, DNA size markers with size of selected bands indicated in kbp (left).

https://doi.org/10.1371/journal.pone.0040810.g001

To assess the homology of both genes to zebrafish jama, jamc, and the mammalian orthologues, we took four complementary approaches. Alignments of the ectodomains of zebrafish and mammalian JAM family proteins showed conservation of key features (Figure S1) such as the putative binding motif in the distal IgSF domain (R-V/I/L-E; [7], [8]), or in the case of JAM-B and JAM-C orthologues, the non-canonical disulphide bond between A and G β-strands in the proximal IgSF domain. In addition, five of the zebrafish paralogues are predicted to have a transmembrane domain and a conserved C-terminal type II PDZ-domain binding motif; in Jama2 the cytoplasmic region has been replaced with a single domain predicted to encode a coiled-coil motif (Figure 2).

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Figure 2. Sequence and gene structure analysis confirms homology to JAM family.

Phylogenetic tree (left) drawn from protein sequence alignments of the ectodomains of mammalian (human and mouse, red lines) and putative zebrafish (blue lines) JAM family orthologues and related cell surface proteins ESAM, GPA33, and CXADR, which serve as an outgroup. Schematic of intron-exon structure of human and zebrafish JAM family genes and human ESAM, GPA33, and CXADR (right; drawn to scale). Regions encoding a signal peptide (SP), distal IgSF domain (IgSF d1), proximal IgSF domain (IgSF d2), transmembrane domain (TM) and cytoplasmic domain (CYT) are separated by vertical lines and annotated accordingly. The final coding exon of jama2 (*) is predicted to encode a coiled-coil motif, but not a transmembrane domain.

https://doi.org/10.1371/journal.pone.0040810.g002

We constructed a phylogeny from alignments of the protein sequences of the IgSF domains of human, mouse and zebrafish JAM family proteins, using other mammalian two-IgSF-domain-containing cell surface proteins as an outgroup (ESAM, GPA33 and CXADR; Figure 2). Jama2 and Jamc2 were shown to be most closely related to JAM-A and JAM-C orthologues, respectively. We compared the intron-exon structure of human and zebrafish JAM family genes and those of the human genes ESAM, GPA33 and CXADR (Figure 2 and Figure S1). The zebrafish JAM family orthologues were found to have a more similar gene structure to the human JAM genes than that of ESAM, GPA33 and CXADR – particularly the unusual division of the distal IgSF domain into three exons, but also in the short cytoplasmic domains composed of multiple exons (with the obvious exception of jama2).

Finally, we compared the genomic loci of JAM family orthologues in human, mouse, chick (where possible; no JAM-A orthologue could be identified in the chick genome) and zebrafish (Figure S2). The conservation of presence, orientation and relative position of other genes at these loci in all four genomes demonstrated an evolutionary relationship between the JAM family orthologues.

All four analyses supported the conclusion that the putative paralogues, jama2 and jamc2 were indeed orthologues of JAM-A and JAM-C, respectively. Having established the presence of six zebrafish jam family genes, we sought to assess if the paralogues shared biochemical properties and gene expression patterns.

Biochemical Properties of Zebrafish Jam Family Ectodomains

To assess the biochemical properties of each zebrafish JAM family cell surface receptor, we cloned the IgSF-domain-encoding regions of each gene and expressed them as tagged, monomeric proteins using a mammalian expression system [52]. We performed a systematic all-against-all screen using surface plasmon resonance (SPR) to identify and quantify any interactions. Because homophilic interactions within the analyte would lead to an underestimation of analyte activity and consequently an underestimation of the equilibrium binding constant, we chose to measure dissociation rate constants (kd) as they are independent of analyte activity, assuming first-order kinetics. Previously, we observed a 1∶1 stoichiometry of binding between Jamb and Jamc [41], so we expected that all Jam family interactions we detected would also have first-order dissociation kinetics. These measurements allowed us to compare the relative binding strengths of each interaction between family members. Each analyte was purified and then resolved by gel filtration chromatography immediately before use to ensure the removal of aggregated proteins that can influence the measurement of kinetic parameters. Each ectodomain eluted as a monodisperse peak from the gel filtration column at similar retention volumes.

We identified 27 interactions amongst family members, within the 36 that we tested (Table S1), with a good fit of the dissociation phase data to a first-order decay curve, again suggesting a 1∶1 binding stoichiometry and validating our assumption of first-order kinetics (Figure 3A). Of these, 16 appeared to have a t½ ≤0.1 s, below the frequency of detection of the instrument used (10 Hz), and could not be reliably quantified. All heterophilic interactions were identified in both orientations of analyte and ligand, with the exception of the weak interaction between Jama and Jama2.

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Figure 3. Zebrafish Jam paralogues have similar interaction specificity, but differing binding strengths.

(A) Graph showing examples of SPR dissociation phase data from interactions between zebrafish Jam family ectodomains. Coloured lines represent mean of three experiments for a given interaction; error bars represent standard deviation; dashed lines indicate the theoretical first-order decay curve for each interaction shown. (B) Scale bar indicating the average half-life of each heterophilic (above the scale bar) and homophilic (below) interaction that could be quantified (t½ ≥0.1 s at 28°C). (C) Diagram of interactions (arrows) between zebrafish Jam family ectodomains, coloured according to half-life. The zebrafish JAM-B and JAM-C orthologues show similar specificity of binding, but with differing strengths. Double-headed arrows represent heterophilic interactions tested in both orientations of analyte and ligand; curly arrows represent homophilic interactions. No quantifiable interactions were detected for Jama2 (grey).

https://doi.org/10.1371/journal.pone.0040810.g003

The interactions identified between the zebrafish orthologues closely match the interactions between their mammalian counterparts: JAM-B interacts homophilically [10] and heterophilically with JAM-C [11], [13], [14] and JAM-C also interacts with itself and with JAM-A [9], [13]. We have observed a novel class of interaction: weak heterophilic interactions between the JAM-B orthologues and JAM-A orthologues. Both JAM-A orthologues and both JAM-B orthologues showed the same specificity of binding for other Jam family ectodomains, but only one JAM-C orthologue, Jamc2, was found to interact with both JAM-A orthologues. These results suggest similarity of heterophilic binding specificity between the paralogues. Interestingly, no homophilic interaction was identified for either Jama or Jama2, but they were found to bind each other. In contrast, homophilic interactions of Jamc and Jamc2 were observed, but not a heterophilic interaction between them.

There was a wide range of dissociation constants and half-lives observed amongst the 11 interactions that could be quantified: ∼0.11–7.00 s (average t½ for both orientations of ligand and analyte; Figure 3). The relative strengths of interactions amongst the paralogues were found to be quite different; for example, Jamb bound Jamc with t½ ∼1.63 s, but bound Jamc2 with a much greater t½ ∼7.00 s. Similarly, the homophilic interactions of Jamc and Jamc2 were different: 0.67 s and 0.11 s, respectively. Taken together, these results suggest sub-functionalisation of binding affinity amongst the paralogues, but not binding specificity, as each ectodomain contains the same conserved putative binding motif [7], [8].

Gene Expression Patterns of Zebrafish Jam Family during Development

To assess any similarity of spatio-temporal gene expression patterns between zebrafish jam family paralogues during development, we performed a systematic wholemount in situ hybridisation screen using antisense riboprobes transcribed from the extracellular IgSF-encoding regions of each paralogue. We observed expression in wild-type zebrafish embryos at different stages of development (Figure 4, Table S2): shield (∼6 h. p. f.; data not shown), 10–13 somites (∼14–15½ h. p. f.; [41]), 21 somites (∼19½ h. p. f.; data not shown), 24 h. p. f. [41] and 48 h. p. f. [41].

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Figure 4. Expression patterns of zebrafish jam family genes suggest different transcriptional regulation of paralogues.

Wholemount in situ hybridisation of jama (top row), jama2 (second row), jamb (third row), jamb2 (fourth row), jamc (fifth row) and jamc2 (bottom row) to wild-type embryos at three different stages of development: approximately 10–13 somites (left column), 24 h. p. f. (middle column) and 48 h. p. f. (right column). JAM-B and JAM-C orthologues show little overlap in expression during embryogenesis, suggesting different regulation of the paralogues.

https://doi.org/10.1371/journal.pone.0040810.g004

There was very little similarity between the expression patterns of the JAM-B orthologues until 48 h. p. f., where both paralogues were expressed in the pectoral fin; there may also be some overlap in expression in the branchial/mandibular arches and craniofacial mesoderm at this stage. The expression patterns of the JAM-C orthologues were also quite different; jamc2 was predominantly expressed in the neural tube and forebrain, while jamc was strongly expressed in the somites and myotomes until 24 h. p. f., and thereafter in the brain. However, jamc also appeared to be expressed ubiquitously at low levels, a result that was replicated with a second riboprobe targeted to the 3′ UTR (data not shown). In contrast, both JAM-A orthologues are expressed in similar tissues at each stage tested, primarily the otic vesicle, pronephric ducts and lateral line primordium. The similarity in expression may be due to shared regulatory elements in the genome, as both genes are in close proximity on chromosome 5 (Figure S2). However, it may also be due to cross-hybridisation, as the IgSF-domain encoding region of jama and jama2 is highly conserved –82% nucleotide identity by alignment (data not shown). To distinguish between these possibilities, we used antisense riboprobes targeted to the divergent C-terminal domains and 3′ UTRs of either gene. The riboprobe templates were amplified from wild-type cDNA, indicating that both genes are expressed (data not shown). The expression pattern of jama was replicated with the C-terminal/3′ UTR probe, but jama2 expression was not observed (data not shown).

These results show that the transcriptional regulation is different between the paralogues, with the exception of jama and jama2. We conclude that it is unlikely for jamb2 and jamc2 to play a role in muscle development, at least during primary myogenesis.

Discussion

The duplication of the JAM family in the teleost lineage provides a unique opportunity to assess the functions and properties of these genes during vertebrate development. In this study, we identified two additional members of the zebrafish jam family, bringing the total number of zebrafish jam genes to six. We confirmed their homology – and that of the previously identified genes – to the mammalian orthologues by comparing gene structure, genomic loci and protein features. We then compared the zebrafish paralogues with respect to physical binding properties and gene expression patterns. We observed aspects of sub-functionalisation and innovation within the expanded zebrafish jam family.

The JAM-A orthologue identified in this study, jama2, presents a clear example of innovation. The C-terminal transmembrane domain and cytoplasmic region of jama have been replaced with a single exon in jama2. This exon is predicted to contain a coiled-coil motif, suggesting that jama2 is expressed as a secreted multimer. Both paralogues may have similar transcriptional regulation, possibly because of their close proximity in the genome, but this may also be a result of cross-hybridisation – there is a very high degree of identity between the IgSF domains of jama and jama2, greater than any other pair of zebrafish paralogues and similar to the degree of conservation between human and mouse JAMs. We attempted to discriminate between regulation and cross-hybridisation, but were unable to confirm the spatio-temporal expression of jama2 by in situ hybridisation using a riboprobe targeted to the divergent 3′ end of the transcript. To date, we have been unable to identify a jama2 homologue in other fish species (stickleback, fugu, medaka and tetraodon) using BLAST searches, suggesting that jama2 may have arisen from a segmental duplication event unique to zebrafish. In mammals, JAMs have been found to play a role in cell polarity [26], [29], [30] and JAM-A has been identified as a component of tight junctions in slit diaphragms – a specialised junction essential for glomerular filtration [55]. The expression of jama in the polarised epithelia of the pronephric ducts suggests this may be a function conserved in zebrafish. What function the potentially multimeric, secreted Jama2 protein may have is difficult to evaluate. Soluble forms of JAM-A have been found to increase in inflammation models [56], and soluble forms of JAM-C protein have been linked to angiogenesis [57]. Whether these ectodomains alone have a biological function or are merely a consequence of other processes remains to be ascertained.

Sub-functionalisation within the zebrafish jam family is evident when comparing the gene expression patterns and relative binding strengths of the JAM-B and JAM-C orthologues. Protein interaction experiments showed that there is similar specificity of binding amongst the paralogues, but with wide-ranging half-lives: from ∼0.26 s (Jamb2–Jamc) to ∼7 s (Jamb–Jamc2). It is difficult to extrapolate any biological functions for the identified interactions based solely upon relative binding strengths without knowledge of the site of expression of the proteins [58]. Our systematic gene expression analysis suggested that the interaction between JAM-A orthologues and JAM-B orthologues may have a function in the development of the otic vesicle, and interactions between Jamb and either JAM-C orthologue may be relevant in the brain. Of course, homophilic interactions, such as those identified for both JAM-B and JAM-C orthologues, may be biologically relevant in any tissue in which they are expressed. There is little similarity in the timing and distribution of gene expression between the paralogues, suggesting differing transcriptional regulation; this may be because the paralogues are separated onto different chromosomes and are influenced by local transcriptional enhancers.

The results of the protein interaction screen support the conclusion that conservation of the putative binding motif between paralogues retained specificity, but divergence in the binding surface affected relative binding strength. For example, both JAM-C orthologues are capable of binding JAM-B orthologues and interacting homophilically, but are not able to bind each other. In other words, the Jamc paralogues have retained the ability to bind JAM-B orthologues, indicating conservation of the binding site, but lost the ability to bind each other, indicating a divergence in the binding surface. We speculate that heterophilic interactions, in trans, might require a different binding surface or binding site to that of homophilic interactions. Our current understanding of the mechanism of interaction between JAM proteins is based upon the structure of homomeric JAM-A dimers, arranged in cis in a crystal lattice [7], [8]. Each zebrafish Jam ectodomain contains the conserved putative binding motif but interacts with other family members with different relative strengths. This suggests that changes in the context of the motif between paralogues, the binding surface, are responsible for the heterogeneity of affinity, or indeed, compatibility.

Our main aim was to determine if the zebrafish paralogues of jamb and jamc might also have a role in early muscle development, as is the case of the Drosophila receptor paralogues kirre/rst [43], [44] and sns/hbs [45], [46]. We know from our previous study that the interaction between Jamb and Jamc, measured at t½ ∼1.63 s, is physiologically relevant [41]. It remains to be determined if the weaker interactions between the zebrafish paralogues play any role in development. The gene expression patterns of each of the JAM-B and JAM-C orthologues show that only jamb and jamc are detectable in the somitic muscle precursors. This makes it unlikely that jamb2 and jamc2 have a role in primary myogenesis. However, it remains possible that either gene may be involved in later muscle development, such as that of the craniofacial or pectoral fin musculature.

Materials and Methods

Nomenclature and Accession Numbers

We refer to the zebrafish orthologues of JAM-A, JAM-B and JAM-C as jama, jama2, jamb, jamb2, jamc and jamc2, respectively, for the sake of clarity and consistency with other recent literature concerning the JAM family [3]. The official symbols and accession/reference numbers are as follows: jama (official symbol f11r) – Entrez Gene: 323696; jama2 (official symbol si:ch211-89p1.1) – Entrez Gene: 100005566; jamb (official symbol jam2a) – Entrez Gene: 100005261; jamb2 (official symbol jam2b) – Entrez Gene: 100005301; jamc (official symbol jam3b) – Entrez Gene: 569217; jamc2 (official symbol jam3a) – Entrez Gene: 797651.

3′ RACE and RT-PCR

Zebrafish embryonic RNA was extracted from approximately 30–50 wild-type 24 h. p. f. embryos fixed in methanol, using the Nucleospin RNA II kit (Macherey-Nagal). First strand cDNA was then transcribed from RNA using the SMART RACE kit (Clontech) for jama2, or for jamc2, using a T20VN oligomer and Superscript III reverse transcriptase (Invitrogen). Negative control synthesis reactions, without reverse transcriptase, were prepared in parallel.

Gene-specific nested 5′ primers and SMART RACE universal primers were used to amplify jama2 by touchdown PCR with Advantage II Polymerase Mix (Clontech). The PCR products were analysed by agarose gel electrophoresis and purified using the QIAquick Gel Extraction kit (QIAGEN). The purified jama2 3′ RACE product was cloned into pCR4-TOPO (Invitrogen) and sequenced using the ABI PRISM big dye terminator cycle sequencing ready reaction kit according to the manufacturer’s instructions, in an ABI 3730×1 automatic sequencer.

Different combinations of jamc2-specific primers were used in touchdown PCR reactions using 24 h. p. f. wild-type embryo cDNA, and the products were analysed by agarose gel electrophoresis. The full-length product was purified, cloned and sequenced as described above.

Sequence Alignments and Synteny

Amino acid sequences for mouse and human JAM-A, JAM-B, JAM-C, ESAM, CXADR and GPA33 were retrieved from the NCBI database (www.ncbi.nlm.nih.gov). Alignments of nucleotide and amino acid sequences were performed using ClustalW (www.ebi.ac.uk/ClustalW). A neighbour-joining tree was drawn from this alignment using the Poisson Distribution model in MEGA (v3.1; [59]) using 500 bootstrap replicates. Signal peptide cleavage sites and transmembrane domains were predicted from amino acid sequences using SignalP (v3.0; [60]) and TMHMM (v2.0; [61]).

Ensembl (www.ensembl.org) annotations of all genes within a 500 kbp window centered on a JAM family member were manually compared between human, mouse, chick (where possible) and zebrafish genomes for their relative position, strand orientation, exon-intron structure and orthology.

Protein Production, Purification, and Surface Plasmon Resonance

The extracellular IgSF domains of all zebrafish Jam family members were expressed as a soluble fusion protein with rat Cd4 domains 3 and 4 and either a 6-histidine (Cd4d3+4–6H) or an enzymatically biotinylatable peptide (Cd4d3+4-bio) C-terminal tag. These were purified and used in SPR experiments, essentially as previously described [52]. Dissociation rate constants (kd) were calculated by averaging the dissociation phase of three different concentrations of purified Jam protein ectodomain and fitting to a simple first-order decay curve. Fits to the data were good, suggesting a 1∶1 stoichiometry of binding. Half lives (t½) were calculated by t½ = ln 2/kd.

Zebrafish Husbandry and Embryo Culture

Zebrafish were maintained according to standard fish husbandry conditions and UK Home Office and local ethical regulations and guidelines. Embryos were raised at 28°C in egg water (0.18 g/l sea salt, 2 mg/l methylene blue). Embryos were staged accordingly to morphology, as previously described [62].

Wholemount in situ Hybridisation

Wholemount in situ hybridisations using digoxygenin-labelled antisense riboprobes were performed using standard protocols [63]. Riboprobe templates were generated from plasmids containing the extracellular IgSF domains of each zebrafish jam family member: jama, jama2, jamb, jamb2, jamc or jamc2. Riboprobe templates of the 3′ ends of jama and jama2 were amplified by RT-PCR, using total RNA extracted from wild-type 24 h. p. f. embryos with TRIzol (Invitrogen), as per manufacturer’s instructions.

Image Acquisition and Processing

Wholemount in situ hybridisation images were obtained using a Zeiss Imager M1 microscope, Zeiss AxioCam Hrc camera, and Zeiss AxioVision software. Entire images were adjusted for contrast, brightness, dynamic range, and resampled to a standardised resolution (300 d. p. i.) using Adobe Photoshop CS2.

Supporting Information

Figure S1.

Conserved protein features of JAM family ectodomains. Protein sequence alignments of the extracellular IgSF domains of human, mouse and zebrafish JAM family. Predicted signal peptides are shown in bold text, consensus N-linked glycosylation sites are highlighted in grey, disulphide bridge cysteines in red, alternating black/blue text colour indicates alternate exons and red text indicates codon/exon boundary overlap. Also marked are the putative dimerisation motif highlighted in green and the linker region, VLV residues, underlined and italicised text.

https://doi.org/10.1371/journal.pone.0040810.s001

(DOC)

Figure S2.

Conservation of presence, order and orientation of genes between mammalian, avian and zebrafish JAM family loci confirms orthology. Schematic showing the arrangement and relative position of annotated genes (boxes) within 0.5 Mbp of sequence from human (Hs), mouse (Mm), chicken (Gg) and zebrafish (Dr) genomes, centred on JAM family orthologues (dark blue boxes): JAM-A (A), JAM-B (B) and JAM-C (C). Genes conserved between mammalian, avian and zebrafish JAM family loci are represented in red, except for JAM-A, where no chicken homologue could be identified. Genes conserved only between mammalian and avian loci are shown in light blue. Genes indicated in white are unique to each loci. Black boxes (B) indicate mett121c paralogues shared between zebrafish JAM-B loci but not present in avian or mammalian loci. Grey box (B) indicates a gene within an intron of the conserved gene mrpl39 that is not present in avian or mammalian JAM-B loci. Asterisks represent conserved genes present in other loci outside of the 0.5 Mbp window. Drawn to scale.

https://doi.org/10.1371/journal.pone.0040810.s002

(TIF)

Table S1.

Dissociation rate constants and half-lives of interactions amongst zebrafish Jam family proteins. Dissociation rate constant and calculated half-life is presented for each positive interaction observed. Interactions that were too weak to quantify are given the nominal value ≥6.9, equivalent to a half-life of 0.1 seconds. Interactions that could be quantified are presented as a mean ± S. D. (n = 3) and are highlighted in bold; kd is presented as a mean ± S. D. (n = 3), t½ is calculated using the mean kd value.

https://doi.org/10.1371/journal.pone.0040810.s003

(DOC)

Table S2.

Annotation of zebrafish jam family gene expression patterns during development. ‘-’ indicates no expression observed; bold indicates major sites of gene expression at each stage.

https://doi.org/10.1371/journal.pone.0040810.s004

(DOC)

Acknowledgments

We thank Cécile Crosnier for comments on the manuscript.

Author Contributions

Conceived and designed the experiments: GTP GJW. Performed the experiments: GTP. Analyzed the data: GTP. Wrote the paper: GTP GJW.

References

  1. 1. Ruan J, Li H, Chen Z, Coghlan A, Coin LJM, et al. (2008) TreeFam: 2008 update. Nucleic Acids Res 36: D735–D740.J. RuanH. LiZ. ChenA. CoghlanLJM Coin2008TreeFam: 2008 update.Nucleic Acids Res36D735D740
  2. 2. Ebnet K, Suzuki A, Ohno S, Vestweber D (2004) Junctional adhesion molecules (JAMs): more molecules with dual functions? J Cell Sci 117: 19–29.K. EbnetA. SuzukiS. OhnoD. Vestweber2004Junctional adhesion molecules (JAMs): more molecules with dual functions?J Cell Sci1171929
  3. 3. Mandell KJ, Parkos CA (2005) The JAM family of proteins. Adv Drug Delivery Rev 57: 857–867.KJ MandellCA Parkos2005The JAM family of proteins.Adv Drug Delivery Rev57857867
  4. 4. Weber C, Fraemohs L, Dejana E (2007) The role of junctional adhesion molecules in vascular inflammation. Nat Rev Immunol 7: 467–477.C. WeberL. FraemohsE. Dejana2007The role of junctional adhesion molecules in vascular inflammation.Nat Rev Immunol7467477
  5. 5. Bazzoni G, Martinez-Estrada OM, Mueller F, Nelboeck P, Schmid G, et al. (2000) Homophilic interaction of Junctional Adhesion Molecule. J Biol Chem 275: 30970–30976.G. BazzoniOM Martinez-EstradaF. MuellerP. NelboeckG. Schmid2000Homophilic interaction of Junctional Adhesion Molecule.J Biol Chem2753097030976
  6. 6. Liang TW, DeMarco RA, Mrsny RJ, Gurney A, Gray A, et al. (2000) Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation. Am J Physiol Cell Physiol 279: C1733–1743.TW LiangRA DeMarcoRJ MrsnyA. GurneyA. Gray2000Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation.Am J Physiol Cell Physiol279C17331743
  7. 7. Kostrewa D, Brockhaus M, D'Arcy A, Dale G, Nelboeck P, et al. (2001) X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif. EMBO J 20: 4391–4398.D. KostrewaM. BrockhausA. D'ArcyG. DaleP. Nelboeck2001X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif.EMBO J2043914398
  8. 8. Prota AE, Campbell JA, Schelling P, Forrest JC, Watson MJ, et al. (2003) Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc Natl Acad Sci U S A 100: 5366–5371.AE ProtaJA CampbellP. SchellingJC ForrestMJ Watson2003Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding.Proc Natl Acad Sci U S A10053665371
  9. 9. Santoso S, Orlova VV, Song K, Sachs UJ, Andrei-Selmer CL, et al. (2005) The homophilic binding of Junctional Adhesion Molecule-C mediates tumor cell-endothelial cell interactions. J Biol Chem 280: 36326–36333.S. SantosoVV OrlovaK. SongUJ SachsCL Andrei-Selmer2005The homophilic binding of Junctional Adhesion Molecule-C mediates tumor cell-endothelial cell interactions.J Biol Chem2803632636333
  10. 10. Cunningham SA, Arrate MP, Rodriguez JM, Bjercke RJ, Vanderslice P, et al. (2000) A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions. J Biol Chem 275: 34750–34756.SA CunninghamMP ArrateJM RodriguezRJ BjerckeP. Vanderslice2000A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions.J Biol Chem2753475034756
  11. 11. Cunningham SA, Rodriguez JM, Arrate MP, Tran TM, Brock TA (2002) JAM2 interacts with alpha 4beta 1. Facilitation by JAM3. J Biol Chem 277: 27589–27592.SA CunninghamJM RodriguezMP ArrateTM TranTA Brock2002JAM2 interacts with alpha 4beta 1. Facilitation by JAM3.J Biol Chem2772758927592
  12. 12. Lamagna C, Meda P, Mandicourt G, Brown J, Gilbert RJC, et al. (2005) Dual interaction of JAM-C with JAM-B and alphaMbeta2 integrin: function in junctional complexes and leukocyte adhesion. Mol Biol Cell 16: 4992–5003.C. LamagnaP. MedaG. MandicourtJ. BrownRJC Gilbert2005Dual interaction of JAM-C with JAM-B and alphaMbeta2 integrin: function in junctional complexes and leukocyte adhesion.Mol Biol Cell1649925003
  13. 13. Arrate MP, Rodriguez JM, Tran TM, Brock TA, Cunningham SA (2001) Cloning of human Junctional Adhesion Molecule 3 (JAM3) and its identification as the JAM2 counter-receptor. J Biol Chem 276: 45826–45832.MP ArrateJM RodriguezTM TranTA BrockSA Cunningham2001Cloning of human Junctional Adhesion Molecule 3 (JAM3) and its identification as the JAM2 counter-receptor.J Biol Chem2764582645832
  14. 14. Liang TW, Chiu HH, Gurney A, Sidle A, Tumas DB, et al. (2002) Vascular Endothelial-Junctional Adhesion Molecule (VE-JAM)/JAM 2 interacts with T, NK, and dendritic cells through JAM 3. J Immunol 168: 1618–1626.TW LiangHH ChiuA. GurneyA. SidleDB Tumas2002Vascular Endothelial-Junctional Adhesion Molecule (VE-JAM)/JAM 2 interacts with T, NK, and dendritic cells through JAM 3.J Immunol16816181626
  15. 15. Ostermann G, Weber KSC, Zernecke A, Schroder A, Weber C (2002) JAM-1 is a ligand of the beta2 integrin LFA-1 involved in transendothelial migration of leukocytes. Nature Immunol 3: 151–158.G. OstermannKSC WeberA. ZerneckeA. SchroderC. Weber2002JAM-1 is a ligand of the beta2 integrin LFA-1 involved in transendothelial migration of leukocytes.Nature Immunol3151158
  16. 16. Santoso S, Sachs UJ, Kroll H, Linder M, Ruf A, et al. (2002) The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1. J Exp Med 196: 679–691.S. SantosoUJ SachsH. KrollM. LinderA. Ruf2002The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1.J Exp Med196679691
  17. 17. Naik MU, Mousa SA, Parkos CA, Naik UP (2003) Signaling through JAM-1 and alphavbeta3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and alphavbeta3 complex. Blood 102: 2108–2114.MU NaikSA MousaCA ParkosUP Naik2003Signaling through JAM-1 and alphavbeta3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and alphavbeta3 complex.Blood10221082114
  18. 18. Naik MU, Vuppalanchi D, Naik UP (2003) Essential role of Junctional Adhesion Molecule-1 in basic fibroblast growth factor-induced endothelial cell migration. Arterioscler Thromb Vasc Biol 23: 2165–2171.MU NaikD. VuppalanchiUP Naik2003Essential role of Junctional Adhesion Molecule-1 in basic fibroblast growth factor-induced endothelial cell migration.Arterioscler Thromb Vasc Biol2321652171
  19. 19. Lamagna C, Hodivala-Dilke KM, Imhof BA, Aurrand-Lions M (2005) Antibody against Junctional Adhesion Molecule-C inhibits angiogenesis and tumor growth. Cancer Res 65: 5703–5710.C. LamagnaKM Hodivala-DilkeBA ImhofM. Aurrand-Lions2005Antibody against Junctional Adhesion Molecule-C inhibits angiogenesis and tumor growth.Cancer Res6557035710
  20. 20. Cooke VG, Naik MU, Naik UP (2006) Fibroblast growth factor-2 failed to induce angiogenesis in Junctional Adhesion Molecule-A-deficient mice. Arterioscler Thromb Vasc Biol 26: 2005–2011.VG CookeMU NaikUP Naik2006Fibroblast growth factor-2 failed to induce angiogenesis in Junctional Adhesion Molecule-A-deficient mice.Arterioscler Thromb Vasc Biol2620052011
  21. 21. Murakami M, Francavilla C, Torselli I, Corada M, Maddaluno L, et al. (2010) Inactivation of Junctional Adhesion Molecule-A enhances antitumoral immune response by promoting dendritic cell and T lymphocyte infiltration. Cancer Res 70: 1759–1765.M. MurakamiC. FrancavillaI. TorselliM. CoradaL. Maddaluno2010Inactivation of Junctional Adhesion Molecule-A enhances antitumoral immune response by promoting dendritic cell and T lymphocyte infiltration.Cancer Res7017591765
  22. 22. McSherry EA, McGee SF, Jirstrom K, Doyle EM, Brennan DJ, et al. (2009) JAM-A expression positively correlates with poor prognosis in breast cancer patients. Int J Cancer 125: 1343–1351.EA McSherrySF McGeeK. JirstromEM DoyleDJ Brennan2009JAM-A expression positively correlates with poor prognosis in breast cancer patients.Int J Cancer12513431351
  23. 23. Fuse C, Ishida Y, Hikita T, Asai T, Oku N (2007) Junctional Adhesion Molecule-C promotes metastatic potential of HT-1080 human fibrosarcoma. J Biol Chem 282: 8276–8283.C. FuseY. IshidaT. HikitaT. AsaiN. Oku2007Junctional Adhesion Molecule-C promotes metastatic potential of HT-1080 human fibrosarcoma.J Biol Chem28282768283
  24. 24. Wang Y, Lui WY (2009) Opposite effects of interleukin-1alpha and transforming growth factor-beta2 induce stage-specific regulation of junctional adhesion molecule-B gene in Sertoli cells. Endocrinology 150: 2404–2412.Y. WangWY Lui2009Opposite effects of interleukin-1alpha and transforming growth factor-beta2 induce stage-specific regulation of junctional adhesion molecule-B gene in Sertoli cells.Endocrinology15024042412
  25. 25. Shao M, Ghosh A, Cooke VG, Naik UP, Martin-DeLeon PA (2008) JAM-A is present in mammalian spermatozoa where it is essential for normal motility. Dev Biol 313: 246–255.M. ShaoA. GhoshVG CookeUP NaikPA Martin-DeLeon2008JAM-A is present in mammalian spermatozoa where it is essential for normal motility.Dev Biol313246255
  26. 26. Gliki G, Ebnet K, Aurrand-Lions M, Imhof BA, Adams RH (2004) Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C. Nature 431: 320–324.G. GlikiK. EbnetM. Aurrand-LionsBA ImhofRH Adams2004Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C.Nature431320324
  27. 27. Scheiermann C, Meda P, Aurrand-Lions M, Madani R, Yiangou Y, et al. (2007) Expression and function of junctional adhesion molecule-C in myelinated peripheral nerves. Science 318: 1472–1475.C. ScheiermannP. MedaM. Aurrand-LionsR. MadaniY. Yiangou2007Expression and function of junctional adhesion molecule-C in myelinated peripheral nerves.Science31814721475
  28. 28. Aurrand-Lions M, Johnson-Leger C, Wong C, Du Pasquier L, Imhof BA (2001) Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members. Blood 98: 3699–3707.M. Aurrand-LionsC. Johnson-LegerC. WongL. Du PasquierBA Imhof2001Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members.Blood9836993707
  29. 29. Ebnet K, Aurrand-Lions M, Kuhn A, Kiefer F, Butz S, et al. (2003) The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity. J Cell Sci 116: 3879–3891.K. EbnetM. Aurrand-LionsA. KuhnF. KieferS. Butz2003The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity.J Cell Sci11638793891
  30. 30. Ebnet K, Suzuki A, Horikoshi Y, Hirose T, Meyer Zu Brickwedde MK, et al. (2001) The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J 20: 3738–3748.K. EbnetA. SuzukiY. HorikoshiT. HiroseMK Meyer Zu Brickwedde2001The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).EMBO J2037383748
  31. 31. Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, et al. (2001) Junctional Adhesion Molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol 154: 491–498.M. ItohH. SasakiM. FuruseH. OzakiT. Kita2001Junctional Adhesion Molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.J Cell Biol154491498
  32. 32. Liu Y, Nusrat A, Schnell F, Reaves T, Walsh S, et al. (2000) Human junction adhesion molecule regulates tight junction resealing in epithelia. J Cell Sci 113: 2363–2374.Y. LiuA. NusratF. SchnellT. ReavesS. Walsh2000Human junction adhesion molecule regulates tight junction resealing in epithelia.J Cell Sci11323632374
  33. 33. Mandell KJ, McCall IC, Parkos CA (2004) Involvement of the Junctional Adhesion Molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function. J Biol Chem 279: 16254–16262.KJ MandellIC McCallCA Parkos2004Involvement of the Junctional Adhesion Molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function.J Biol Chem2791625416262
  34. 34. Mandicourt G, Iden S, Ebnet K, Aurrand-Lions M, Imhof BA (2007) JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration. J Biol Chem 282: 1830–1837.G. MandicourtS. IdenK. EbnetM. Aurrand-LionsBA Imhof2007JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.J Biol Chem28218301837
  35. 35. Rehder D, Iden S, Nasdala I, Wegener J, Brickwedde M-KMZ, et al. (2006) Junctional adhesion molecule-A participates in the formation of apico-basal polarity through different domains. Exp Cell Res 312: 3389–3403.D. RehderS. IdenI. NasdalaJ. WegenerM-KMZ Brickwedde2006Junctional adhesion molecule-A participates in the formation of apico-basal polarity through different domains.Exp Cell Res31233893403
  36. 36. Imhof BA, Zimmerli C, Gliki G, Ducrest-Gay D, Juillard P, et al. (2007) Pulmonary dysfunction and impaired granulocyte homeostasis result in poor survival of Jam-C-deficient mice. J Pathol 212: 198–208.BA ImhofC. ZimmerliG. GlikiD. Ducrest-GayP. Juillard2007Pulmonary dysfunction and impaired granulocyte homeostasis result in poor survival of Jam-C-deficient mice.J Pathol212198208
  37. 37. Praetor A, McBride JM, Chiu H, Rangell L, Cabote L, et al. (2009) Genetic deletion of JAM-C reveals a role in myeloid progenitor generation. Blood 113: 1919–1928.A. PraetorJM McBrideH. ChiuL. RangellL. Cabote2009Genetic deletion of JAM-C reveals a role in myeloid progenitor generation.Blood11319191928
  38. 38. Sakaguchi T, Nishimoto M, Miyagi S, Iwama A, Morita Y, et al. (2006) Putative “stemness” gene Jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells. Mol Cell Biol 26: 6557–6570.T. SakaguchiM. NishimotoS. MiyagiA. IwamaY. Morita2006Putative “stemness” gene Jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells.Mol Cell Biol2665576570
  39. 39. Tang T, Li L, Tang J, Li Y, Lin WY, et al. (2010) A mouse knockout library for secreted and transmembrane proteins. Nat Biotechnol 28: 749–755.T. TangL. LiJ. TangY. LiWY Lin2010A mouse knockout library for secreted and transmembrane proteins.Nat Biotechnol28749755
  40. 40. Zimmerli C, Lee BP, Palmer G, Gabay C, Adams R, et al. (2009) Adaptive immune response in JAM-C-deficient mice: normal initiation but reduced IgG memory. J Immunol 182: 4728–4736.C. ZimmerliBP LeeG. PalmerC. GabayR. Adams2009Adaptive immune response in JAM-C-deficient mice: normal initiation but reduced IgG memory.J Immunol18247284736
  41. 41. Powell GT, Wright GJ (2011) Jamb and Jamc are essential for vertebrate myocyte fusion. PLoS Biol 9: e1001216.GT PowellGJ Wright2011Jamb and Jamc are essential for vertebrate myocyte fusion.PLoS Biol9e1001216
  42. 42. Gallardo VE, Liang J, Behra M, Elkahloun A, Villablanca EJ, et al. (2010) Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish. BMC Dev Biol 10: 120.VE GallardoJ. LiangM. BehraA. ElkahlounEJ Villablanca2010Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish.BMC Dev Biol10120
  43. 43. Ruiz-Gomez M, Coutts N, Price A, Taylor MV, Bate M (2000) Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell 102: 189–198.M. Ruiz-GomezN. CouttsA. PriceMV TaylorM. Bate2000Drosophila dumbfounded: a myoblast attractant essential for fusion.Cell102189198
  44. 44. Bour BA, Chakravarti M, West JM, Abmayr SM (2000) Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev 14: 1498–1511.BA BourM. ChakravartiJM WestSM Abmayr2000Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion.Genes Dev1414981511
  45. 45. Strünkelnberg M, Bonengel B, Moda LM, Hertenstein A, de Couet HG, et al. (2001) rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila. Development 128: 4229–4239.M. StrünkelnbergB. BonengelLM ModaA. HertensteinHG de Couet2001rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila.Development12842294239
  46. 46. Artero RD, Castanon I, Baylies MK (2001) The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development 128: 4251–4264.RD ArteroI. CastanonMK Baylies2001The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling.Development12842514264
  47. 47. Galletta BJ, Chakravarti M, Banerjee R, Abmayr SM (2004) SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech Dev 121: 1455–1468.BJ GallettaM. ChakravartiR. BanerjeeSM Abmayr2004SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts.Mech Dev12114551468
  48. 48. Haralalka S, Abmayr SM (2010) Myoblast fusion in Drosophila. Exp Cell Res 316: 3007–3013.S. HaralalkaSM Abmayr2010Myoblast fusion in Drosophila.Exp Cell Res31630073013
  49. 49. Rochlin K, Yu S, Roy S, Baylies MK (2010) Myoblast fusion: when it takes more to make one. Dev Biol 341: 66–83.K. RochlinS. YuS. RoyMK Baylies2010Myoblast fusion: when it takes more to make one.Dev Biol3416683
  50. 50. Postlethwait J, Amores A, Cresko W, Singer A, Yan Y-L (2004) Subfunction partitioning, the teleost radiation and the annotation of the human genome. Trends Genet 20: 481.J. PostlethwaitA. AmoresW. CreskoA. SingerY-L Yan2004Subfunction partitioning, the teleost radiation and the annotation of the human genome.Trends Genet20481
  51. 51. Volff JN (2005) Genome evolution and biodiversity in teleost fish. Heredity (Edinb) 94: 280–294.JN Volff2005Genome evolution and biodiversity in teleost fish.Heredity (Edinb)94280294
  52. 52. Bushell KM, Sollner C, Schuster-Boeckler B, Bateman A, Wright GJ (2008) Large-scale screening for novel low-affinity extracellular protein interactions. Genome Res 18: 622–630.KM BushellC. SollnerB. Schuster-BoecklerA. BatemanGJ Wright2008Large-scale screening for novel low-affinity extracellular protein interactions.Genome Res18622630
  53. 53. Martin S, Söllner C, Charoensawan V, Adryan B, Thisse B, et al. (2010) Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling. Mol Cell Proteomics 9: 2654–2665.S. MartinC. SöllnerV. CharoensawanB. AdryanB. Thisse2010Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling.Mol Cell Proteomics926542665
  54. 54. Söllner C, Wright G (2009) A cell surface interaction network of neural leucine-rich repeat receptors. Genome Biol 10: R99.C. SöllnerG. Wright2009A cell surface interaction network of neural leucine-rich repeat receptors.Genome Biol10R99
  55. 55. Fukasawa H, Bornheimer S, Kudlicka K, Farquhar MG (2009) Slit diaphragms contain tight junction proteins. J Am Soc Nephrol 20: 1491–1503.H. FukasawaS. BornheimerK. KudlickaMG Farquhar2009Slit diaphragms contain tight junction proteins.J Am Soc Nephrol2014911503
  56. 56. Koenen RR, Pruessmeyer J, Soehnlein O, Fraemohs L, Zernecke A, et al. (2009) Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases. Blood 113: 4799–4809.RR KoenenJ. PruessmeyerO. SoehnleinL. FraemohsA. Zernecke2009Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases.Blood11347994809
  57. 57. Rabquer BJ, Amin MA, Teegala N, Shaheen MK, Tsou P-S, et al. (2010) Junctional Adhesion Molecule-C is a soluble mediator of angiogenesis. J Immunol 185: 1777–1785.BJ RabquerMA AminN. TeegalaMK ShaheenP-S Tsou2010Junctional Adhesion Molecule-C is a soluble mediator of angiogenesis.J Immunol18517771785
  58. 58. Wright GJ (2009) Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions. Mol Biosyst 5: 1405–1412.GJ Wright2009Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.Mol Biosyst514051412
  59. 59. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5: 150–163.S. KumarK. TamuraM. Nei2004MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment.Brief Bioinform5150163
  60. 60. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–795.JD BendtsenH. NielsenG. von HeijneS. Brunak2004Improved prediction of signal peptides: SignalP 3.0.J Mol Biol340783795
  61. 61. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580.A. KroghB. LarssonG. von HeijneEL Sonnhammer2001Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J Mol Biol305567580
  62. 62. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310.CB KimmelWW BallardSR KimmelB. UllmannTF Schilling1995Stages of embryonic development of the zebrafish.Dev Dyn203253310
  63. 63. Thisse C, Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protoc 3: 59–69.C. ThisseB. Thisse2008High-resolution in situ hybridization to whole-mount zebrafish embryos.Nature Protoc35969