Funct Integr Genomics DOI 10.1007/s10142-015-0449-9
Identification and expression of an uncharacterized Ly-6 gene cluster in zebrafish Danio rerio Quanyang Guo 1,2 & Dongrui Ji 1,2 & Man Wang 1,2 & Shicui Zhang 1,2 & Hongyan Li 1,2
Received: 31 December 2014 / Revised: 7 June 2015 / Accepted: 16 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The Ly-6/uPAR/CD59/neurotoxin superfamily (Ly-6SF) identified in most metazoan has been shown to play important roles in different biological processes including immunity, cellular adhesion, and cell signaling. Members of this superfamily contain one or more conserved domains known as Ly-6/uPAR (LU) domain, which harbors 8 or 10 conserved cysteine residues forming 4–5 disulfide bonds. In this study, we reported the identification of a novel zebrafish Ly-6 gene cluster on chromosome 21, which consists of seven genes ly21.1, ly21.2, ly21.3, ly21.4, ly21.5, ly21.6, and ly21.7 and their spatiotemporal expression pattern during development. All the seven genes possess features typical of the Ly-6/neurotoxin superfamily, and phylogenetic analysis shows that these genes form a single cluster branching form other members of Ly-6 family, suggesting that the seven genes evolved by an event of intrachromosome gene duplication. However, deduced Ly21.1-7 proteins share little homology with Ly-6 family proteins from other species, no orthologs are identified in vertebrates, including teleosts, hinting that
Electronic supplementary material The online version of this article (doi:10.1007/s10142-015-0449-9) contains supplementary material, which is available to authorized users. * Hongyan Li [email protected]
Institute of Evolution & Marine Biodiversity, Ocean University of China, Room 301, Darwin Building, Qingdao 266003, China
Laboratory for Evolution & Development, Department of Marine Biology, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
ly21.1-7 genes are evolutionarily a novel addition to zebrafish. Expression analyses show that maternal mRNAs of ly21.1-7 genes are detected during early developmental stages, but later in development, they exhibit tissue-specific expression. Except for ly21.2 which is expressed in the skin ionocytes, all the remaining six genes are mainly expressed in the developing brain. Keywords Ly-6 superfamily . Zebrafish . Gene cluster . Expression pattern
Introduction Lymphocyte antigen-6 (Ly-6) proteins are characterized by a conserved Ly-6/uPAR (LU) domain initially identified in the sea-snake erabutoxin b by Tsernoglou and Petsko (1977), which is approximately 70 to 100 amino acids long, and possesses 8 to 10 highly conserved cysteine residues (Cys) in stereotyped positions that form a defined disulfide-bonding pattern (Galat 2008). Almost all Ly-6 family members have an N-terminal signal peptide sequence. They are divided into two broad categories based on whether a C-terminal glycophosphatidyl inositol (GPI)-anchored sequence exists or not. The first family comprises secreted proteins, such as snake neurotoxin, SLURP-1, and rat-secreted urine protein (Fleming et al. 1993; Adermann et al. 1999; Southan et al. 2002), while the second family consists of GPI-anchored proteins, tethered to the membrane by GPI signal, including CD59, Lynx1, and uPAR (Davies et al. 1989; Miwa et al. 1999; Blasi and Carmeliet 2002). A great number of Ly-6 superfamily members have been identified to date in a variety of species such as fruit-fly and mammals (Gumley et al. 1995; Galat 2008; Hijazi et al. 2009). A total of 45 members of the Ly-6 superfamily,
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including 12 transforming growth factor (TGF)-β receptors, have been identified in the human genome, and 36 new Ly-6 members, except for five TGF-β receptors, have been documented in the fly Drosophila melanogaster (Galat 2008; Hijazi et al. 2009). The Ly-6 genes have often been found to be contiguous in the genome, forming six clusters that group 24 genes together, for example, in Drosophila, and so is the case in humans and rats (Hijazi et al. 2009; Galat 2008; Rajesh and Yenugu 2012). Ly-6 superfamily members have been shown to display a diverse and tissue-specific expression pattern, with group of genes often sharing similar expression pattern. In mammals, most Ly-6 superfamily genes are expressed in the cells of the hematopoietic origin, brain, vascular epithelium, kidney tubular epithelium, lung, keratinocytes, stomach, testis, and prostate (Bamezai 2004). For example, in adult mouse, lynx1 is highly expressed in the central nervous system, while lynx2 is mainly expressed in some specific neurons in early stages (Miwa et al. 1999; Dessaud et al. 2006). Similarly, Ly-6 superfamily members also exhibit a dynamic and diverse tissue-specific expression pattern, with subset of genes showing similar tissue expression pattern in Drosophila, like Bou, crok, crim, and cold that are all expressed in the developing ectoderm and trachea (Hijazi et al. 2009; Nilton et al. 2010). Reflecting the differential expression pattern, Ly-6 superfamily members are involved in diverse biological processes including the immune regul at i o n , s i g n al t r a ns d u c t i o n , c e l l a d h e s i o n , a n d neuromodulation. Mammalian Ly-6 proteins such as E48, Ly-6A.2, and uPAR play important roles in cell adhesion, proliferation, and migration (Brakenhoff et al. 1995; Bamezai and Rock 1995; Blasi and Carmeliet 2002), LyA/E is involved in signal transduction in transgenic mice (Stanford et al. 1997), and Lynx2 is associated with anxiety-related behavior via neuronal nicotinic receptors (Tekinaya et al. 2009). In Drosophila, ectoderm-expressed genes Bou, crok, crim, and cold are shown to be linked with septate junction formation (Hijazi et al. 2009). Zebrafish is a widely used model organism for study of developmental genetics and biotechnology, and its embryogenesis and development have been well described (Kimmel et al. 1995). For the past 2 decades, a large number of published literature are related to the expression pattern, activity, and function of genes in early development in zebrafish (Haffter et al. 1996; Abrams and Mullins 2009). However, little information is available to date regarding Ly-6 superfamily genes in embryogenesis and development of zebrafish. As the spatiotemporal expression pattern of genes can often be a foundation for understanding their activity during embryogenesis and development, we thus searched for Ly-6 superfamily genes in zebrafish via bioinformatics approaches, and here, we reported the identification and expression of a novel Ly-6 gene cluster located on chr.21 in zebrafish.
Materials and methods Zebrafish and embryos Zebrafish (Danio rerio) wild-type strain AB were bred and maintained in fish-farming facility, under standard conditions. All fish were maintained in a 14-h on/10-h off light cycle. Artificial spawning and fertilization were performed as previously reported (Kimmel et al. 1995). Gene cloning and sequencing A complete set of reported Ly-6 sequences were used as queries against zebrafish database at NCBI (http://blast. ncbi.nlm.nih.gov/). A gene cluster containing seven putative Ly-6 genes located on chromosome 21 was chosen for detailed study. To obtain their complete ORFs, seven primer pairs were designed using the primer 5 program (accession numbers and seven primer pairs are listed in Tables 1 and 2, respectively) and utilized to amplify complementary DNA (cDNA) fragments using RNAs isolated from D. rerio embryos. Sequence and phylogenetic analyses The assembled cDNAs were analyzed for coding probability with the DNASTAR software package (version 5.0). Their molecular masses and isoelectric points (pI) were predicted by ProtParam (http://www.expasy.ch/tools/protparam.html), and signal peptides and GPI-anchored signal were predicted by SignalP3.0 Server (http://www.cbs.dtu.dk/services/ SignalP/) and Pre-GPI (http://gpcr2.biocomp.unibo.it/gpipe/ index.htm), respectively. Conserved domains were identified using the online program SMART (http://smart.emblheidelberg.de/index2.cgi), and three-dimensional (3D) structures were predicted by homology modeling methods via SWISS-MODEL (http://swissmodel.expasy.org/) (Arnold et al. 2006; Guex and Peitsch 1997; Schwede et al. 2003). The exon-intron structures of the ly21 genes were performed by the online program Gene Structure Display Server (http:// gsds1.cbi.pku.edu.cn/). All the exons and introns were mapped, and then the relative intron lengths were compared. Multiple sequence alignments were performed using the ClustalW method from MegAlign (DNASTAR) software. The phylogenetic tree was constructed by neighbor-joining (NJ) with MEGA6 using 1000 bootstrap replicates. RNA purification and cDNA synthesis Total RNAs were extracted from dechorionated embryos aged at 0.5, 3, 6, 10, and 22 h post-fertilization (hpf), and 1, 2, 3, 4, and 5 days post-fertilization (dpf) using Total-RNA Extract Kit (Sangon Biotech, Shanghai), and cDNAs were reverse
Funct Integr Genomics Table 1
General characteristic features of zebrafish Ly21.1-7 proteins
Accession number (name)
XP_003200436 (Ly21.1) XP_002665293 (Ly21.2) XP_005161541(Ly21.3) XP_005161542(Ly21.4) XP_005161543(Ly21.5) XP_002665292(Ly21.6) XP_005161544 (Ly21.7)
196 198 196 206 199 200 193
20.79 21.04 20.7 20.09 21.25 20.7 20.19
6.49 3.89 5.25 4.59 7.5 7.26 7.17
1–17 1–17 1–17 1–17 1–19 1–19 1–19
−1.23 −11.55 −3.39 −5.56 +1.63 +0.76 +0.39
10C+10C 10C+10C 10C+10C 10C+10C 10C+9C 10C+10C 10C+10C
GPI GPI GPI GPI GPI GPI GPI
The number of domain and position-specific conserved cysteines (e.g., 10C+10C represents that a protein has two domains, and each domain contains 10 conserved cysteines)
transcribed using M-MLV reverse transcriptase in the presence of oligo d(T) primers, following the manufacturer’s instructions. The PCR primers were designed in different exons in each gene transcript to ensure specific amplification from the cDNA templates. The RT-PCR protocols used were as follows: one cycle of 5 min at 94 °C and 33 cycles of 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C, and followed by a final extension of 7 min at 72 °C.
according to the protocol previously described (Wang et al. 2008). The data were analyzed with ABI 7500 SDS software (Applied Biosystems) and quantified with the comparative Ct method (2−ΔΔCt) based on Ct values for ly21.1-7 genes (Livak and Schmittgen 2001). Reaction of each sample was performed in triplicate. Three independent experiments were performed for each gene, and all data were expressed as mean± standard deviation (SD).
Quantitative real-time PCR
Probe synthesis and whole-mount in situ hybridization
Quantitative real-time PCR (qRT-PCR) was performed to examine the expression profile of ly21.1-7 genes. RNAs were extracted from the grinded samples, and cDNAs were synthesized as described above. The seven PCR primer sets specific for ly21.1-7 genes were designed using the primer 5 program based on the sequences we cloned. The β-actin gene was chosen as the reference for internal standardization (the primer sequences are listed in Table 2). After qualification of the cDNA templates and primers, qRT-PCR was performed
Digoxigenin-labeled antisense and sense RNA probes were synthesized using SP6 or T7 RNA polymerase (Roche) and then precipitated with LiCl. Whole-mount in situ hybridization was performed as described previously (Thisse and Thisse 2008). Briefly, digoxigenin-labeled probes were hybridized and then detected with alkaline phosphataseconjugated digoxigenin antibody Fab fragment (1:2000, Roche) and alkaline phosphatase substrate NBT/BCIP (1:50, Roche). Embryos were mounted and observed using a
Primers used for cloning and real-time PCR analysis
PCR cloning primers (5′-3′) Sense Antisense CACAGCACTGGCACTCCTAA TCAGGAGGAAGGCGATCAGA Sense Antisense TGTTATTTTCACCACAGGACACTCT GTGTATAAAGCGTGTCAGATTGTAGA Sense Antisense CTTCTTTTTGTCGCCTTTACTTCAG TCAGAGCATCAGGATGAAGGTGA Sense Antisense AGATTCATGTGCAGACCAAAAGGTA TCAGAGCATCAGGATGAAGGAGATC Sense Antisense TGTGACGATCTGATGGGTTTATGTA TCAGAGGAAAACAGAGAAACAGGAA Sense Antisense TGCATCTGCAAATCTCAGCTTTTC AGAGGAACAGGAAGCTTTGAGTGAT Sense Antisense TCATTCTCATCAGCGCAGGAC CATATA TTGTGTAGAGCAGGTCAGACTG Sense Antisense - -
Real-time PCR primers (5′-3′) AGCTGCTCAGGGACAGAAGAT CCGTTCCT TTGGCATCACAGAA TTATTTTCACCACAGGACACTCTCTCAGC CCATTTCCACAATCAGCAGCACAAC CTGCTGATTGTGAAGTTGGGACCATAAA ACATCTTTCCATTAGGAGTGCCAGTGCT CGGACAGAACTGCTCAACAACTA GAGGCA CAGCCTTGTGTAGTAA CAGGCGAGTCAGTGATTGTAAAAGGATG ATCAGAGGAAAACAGAGAAACAGGAAGC ATGGACTTTAGGAGGCCAGACA CTGACAGC ATGAGATGCCCTTAAC CAGAATCAGGAACTATCGGAAACCAG AAAACAGCAGAGCAATGGCAAACT CCGTGACATCAAGGAGAAGC TACCGCAA GATTCCATACCC
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stereomicroscope. Whole-mount in situ hybridization for all the seven genes was conducted in triplicate.
Results Identification and general characteristics of ly21.1-7 Searching for Ly-6 genes in zebrafish genome identified a novel Ly-6 gene cluster containing seven genes on chromosome 21, with rp137, st8sia5, loxhd1a, and psmd5 located upstream of this gene cluster and stbd1, shroom3, ccni, and ccng2 located downstream (Fig. 1). We then performed RTPCR to amplify their cDNAs, and the seven gene ORFs were obtained and sequenced, indicating that all these genes are expressed in vivo. The seven Ly-6 genes were each designated ly21.1, ly21.2, ly21.3, ly21.4, ly21.5, ly21.6, and ly21.7 according to their location and order on the chromosome (Fig. 1). The general characteristics of all the deduced proteins are presented in Table 1. Each of them includes an N-terminal signal sequence required for its secretion and a hydrophobic C-terminal sequence flanking the consensus site for addition of a glycosyl–phosphatidyl inositol (GPI) anchor (Table 1). SMART prediction revealed that all the proteins share the same organization with two tandemly arrayed LU domains. In specific, the ORFs of ly21.1-7 were 591, 597, 591, 621, 600, 603, and 582 bp in length and coded for proteins with a molecular mass of 20.09∼21.25 kDa and a pI of 3.89∼7.5 (Table 1), respectively. The net charges of the predicted proteins Ly21.1-7 were −1.23, −11.55, −3.39, −5.56, 1.63, 0.76, and 0.39 individually. Genomic structure of ly21.1-7 genes An evolutionary relationship between the Ly-6 genes among different species has been proposed based on conservation of intron-exon boundaries in the coding portions of these genes (Fuse et al. 1990; Chang et al. 1997a; Chang et al. 1997b). Genomic structure analysis revealed that the coding regions of ly21.1-7 share similar exon-intron structures with five exons interspaced by four introns, which resembles the
Fig. 1 Genomic localization of zebrafish ly21.1-7 genes. Arrows indicate direction of transcription. Positions were obtained from the MapView at the National Center for Biotechnology Information (NCBI) website. Distance between genes is not to scale. The accession numbers of
genomic structure of human Lypd3, a homolog of Ly-6 family proteins with two LU domains (Fig. 2), and all the ly21.1-7 genes have an intron dividing the signal peptide into two exons. The seven mature proteins were similarly encoded by four exons. Of note, there are an intron inserted in the first LU domain between the fifth and sixth conserved cysteine residues, and another intron inserted in the first and second LU domains in ly21.1-7 (Fig. 2). In addition, an intron inserted in the second LU domain between the fifth and sixth conserved cysteine residues was observed in ly21.1, ly21.2, ly21.3, ly21.4, ly21.6, and ly21.7, with ly21.5 being an exception in which the intron was inserted in the second LU domain between the fourth and fifth conserved cysteine residues, which may be due to that a conserved cysteine residue is missing in ly21.5 (Fig. 2). Relationships among ly21.1-7 genes Ly21.1-7 have two tandem LU domains, with the first LU domain being longer than the second LU domain. Except for the noncanonical LU2 domain Ly21.5 which contains only nine conserved cysteines, all the other LU domains comprised highly conserved 10 cysteines, which can form five disulfide bridges contributable to their structural similarity. The LU domains also exhibited the characteristics of canonical LU domains with the Cys1 and Cys2 interspaced by two residues, Cys8 and Cys9 closely adjacent, Cys9 and Cys10 separated by four residues, and an Asn residue contiguous to the last cysteine. These provided an additional evidence supporting that they all belongs to Ly-6 family members. Alignment analysis revealed that the N-terminal signal peptides and the C-terminal GPI-anchored signals of Ly21.1-7 proteins both displayed higher homology compared to the LU domains, but their homology outside the canonical featured amino acids in the LU domains are relatively lower. The full length of Ly21.1-7 proteins shared high amino acid sequence identity from 43.6 to 91.4 %. (Fig. S1a–b). By contrast, compared to other Ly-6 members outside chromosome 21 in zebrafish or homologs from other species, Ly21.1-7 exhibited lower homology, especially outside the LU domains (data not shown). The phylogenetic tree showed that Ly21.1-7
zebrafish ly21.1-7 sequences: ly21.1-XP003200436, ly21.2XP002665293, ly21.3- XP005161541, ly21.4-XP005161542, ly21.5XP005161543, ly21.6-XP002665292, and ly21.7-XP002665292
Funct Integr Genomics Fig. 2 Genomic structure of the ly21.1-7 genes in zebrafish and lypd3 (H. sapiens NM_014400) in human. The exons are represented as boxes and are not to scale. 5′-UTR sequence, signal sequence (Sig Seq), LU domain, hydrophobic GPI consensus sequence (GPI), and 3′-UTR sequence are shaded with different colors. Intron breaks are conserved within the superfamily and exons 2, 3 encode LU 1 domain, exons 4, 5 encode LU2 domain as shown in violet (color figure online)
were clustered together, branching form other members of Ly6 family (Fig. S1c). This suggests that ly21.1-7 on chromosome 21 originated from intra-chromosome gene duplication during zebrafish evolution. Sequence comparison also revealed that the homology of LU1 domains of Ly21.1-7 was relatively higher, with identity ranging from 31.4 to 87.8 %, and that of LU2 domains was also higher, with identity being from 38.2 to 97.1 %, whereas the homology between LU1 domains and LU2 domains was rather lower (Fig. S2b). Moreover, both LU1 domains and LU2 domains were branched into two different phylogenetic groups on the phylogenetic tree (Fig. S2c). This implies that the emergence of LU1 and LU2 domains predates the intrachromosome gene duplication. 3D structures Although most sequences of Ly-6 proteins are poorly conserved, the LU domains of Ly-6 proteins all have a similar spatial structure (Galat 2008). The 3D structure prediction using SWISS-MODEL showed that the potential tertiary structures of LU domains of Ly21.1-7 were similar to that of human CD59, fruit-fly boudin, and other Ly-6 family members (Tsetlin 1999), looking like a three finger structure (Fig. S3). The conservation of 3D structures suggests that these proteins either play similar functions or function via similar mechanisms to some extent. Spatiotemporal expression pattern of ly21.1-7 qRT-PCR was used to examine the expression profiles of ly21.1-7 at different developmental stages. The dissociation curve of amplified products in all cases showed a single peak, indicating that the amplifications were specific (data not shown). As shown in Fig. 3, all ly21.1-7 were all expressed in early stages and exhibited different expression patterns in later stages. Generally, the expression pattern of
ly21.1-3 was similar: They were expressed at low level in the early stages examined and upregulated significantly at later stages. To be specific, ly21.1 and ly21.2 peaked at 4 dpf and then deceased less at 5 dpf; ly21.3 peaked at 2 dpf, and then remained stable. Ly21.5, ly21.6, and ly21.7 also showed low level expression at the earliest developmental stages. Interestingly, they had maximum expression at 1 dpf and then decreased at later stages. Compared to other genes, the expression of ly21.4 was distinct: It showed a higher expression level at 1 dpf, and its expression fluctuated continuously until 4–5 dpf (Fig. 3). To analyze the spatiotemporal expression pattern of ly21.1-7, whole mount in situ hybridization was performed. No signals were detected in the sense control (Fig. S4). All the seven genes showed ubiquitously weak maternal expression in the embryos younger than 1 dpf (Fig. S5), which is generally in line with the qRT-PCR results. The expression of ly21.1 was restricted in the brain region of embryos/larvae aged 1 to 5 pdf (Fig. 4). Similarly, the expressions of ly21.3, ly21.4, ly21.5, ly21.6, and ly21.7 were also restricted to the brain region of embryos at 1 dpf (Fig. 4). The positive signals of ly21.4, ly21.5, ly21.6, and ly21.7 were clearly mapped to the dorsal part of the developing brain ventricular zone (Fig. 5), whereas the signal of ly21.1 and ly21.3 was rather weak and thus could not be defined. Interestingly, the expression of ly21.2 was different from that of all the members of ly21 family: No positive signal of ly21.2 was detected in the embryos/larvae before 2 dpf, and the signal became slightly visible only on the surface of yolk sac extended to the branchial region of the larvae aged 3 dpf. The numbers of Ly21.2-positive cells appeared to be not different on the yolk sac surface in the larvae aged from 3 to 5 dpf (Fig. 4). The expression pattern of ly21.2 was reminiscent of the expression of zslc12a10.2, which is a Na, Cl cotransporter-like protein (ZNCC-like 2) specifically expressed on skin ionocytes (Wang et al. 2013). Therefore, ly21.2 may be specifically expressed on skin ionocytes.
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Fig. 3 Expression profile of ly21.1-7 genes at different stages as measured by qRT-PCR. The mRNA expression of ly21.1-7 and β-actin were measured at 0.5, 3, 6, 10, and 22 hpf, and 1, 2, 3, 4, and 5 dpf (hpf hours postfertilization, dpf days postfertilization). Three independent
experiments were performed for each gene. Reaction of each sample was performed in triplicate. Fold difference was calculated as 2−ΔΔCt with zebrafish β-actin as a reference gene. Vertical bars represent the mean±SD (n=3)
Collectively, these data suggest that the seven genes on chromosome 21 evolved by an event of intra-chromosome gene duplication. However, Ly21.1-7 proteins share little homology with Ly-6 family proteins from other species, it is thus difficult to establish orthology relationships between zebrafish Ly21.1-7 and other vertebrate Ly-6 proteins based only on sequence homology. It is also notable that no orthologs of Drsopophila Ly-6 proteins are identified in vertebrates, due to low degree of sequence similarity (Hijazi et al. 2009). In addition, no orthologs of Ly21.1-7 are established in vertebrates, including teleosts, by syntenic analysis, though Ly-6 family proteins containing two LU domains, such as C4.4a, are identified in mammals (mouse and humans) and two uncharacterized Ly-6 members in teleosts (fugu). These suggest that ly21.1-7 genes are evolutionarily a novel addition to zebrafish, which is subjected to independent duplication
We have described here a novel gene cluster consisting of seven genes located on the chromosome 21. Both primary sequence and gene structure analyses reveal that all the seven genes possess features typical of the Ly-6 superfamily, including similar genomic exon-intron structure, cysteine-rich signature motifs, and anchorage to the membrane by a GPI link. In addition, we have identified the transcripts of all the seven genes, indicating that they are actively transcribed and functional in vivo. All the seven genes share similar exon-intron structures, and the deduced proteins Ly21.1-7 all possess the same organization with two tandemly arrayed LU domains. Moreover, phylogenetic analysis shows that these genes form a single cluster independent of other members of Ly-6 family.
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Fig. 4 Expression pattern of ly21.1-7 in the zebrafish at 1–5 dpf by whole mount in situ hybridization. The probes used for in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower right corners. a1–f7
are lateral view with anterior to the left. a1–e6 The six genes except for ly21.2 expressed mainly in the developing brain. f1–f5 ly21.2 gene expression in the skin of the yolk sac from 3 to 5 dpf. f6–f7 High magnification views of f4 and f5 respectively
events from other branches of teleosts (van der Aa et al. 2009), providing a support to the notion that the genes encoding a Ly6 motif are prone to sudden phases of extensive duplication and diversification in different phylogenetic groups (Galat 2008; Hijazi et al. 2009; Bamezai 2004). Maternal mRNAs of ly21.1-7 genes are detected during early developmental stages, but later in development, they clearly show tissue-specific expression. Except for ly21.2, all the
remaining six genes are mainly expressed in the developing brain. Among them, four genes ly21.4, ly21.5, ly21.6, and ly21.7 are expressed specially in brain ventricular zone. It has been known that many members of Ly-6 superfamily genes are expressed in the developing and/or adult nervous system and always detected in both distinct and common neuronal populations, suggesting a specific function of Ly-6 members in the development/differentiation of nervous system (Heintz 2004;
Funct Integr Genomics Acknowledgments This work was supported by Grants of the Natural Science Foundation of China (NSFC) (31272395) and Program for New Century Excellent Talents in the University of Ministry of Education of China (NCET-11-0469) to H. Li.
Fig. 5 Expression analysis of ly21.4, ly21.5, ly21.6, and ly21.7 in the zebrafish brain region at 1 dpf. a1–d1 are lateral view with anterior to the left. a2–d2 are high magnification views of a1–d1, respectively. a3, b3, c3, d3 are frontal view with dorsal to the top. a1–a3 Expression of ly21.4 in zebrafish brain. b1–b3 Expression of ly21.5 in zebrafish brain. c1–c3 Expression of ly21.6 in zebrafish brain. d1–d3 Expression of ly21.7 in zebrafish. The arrow heads indicates the staining signal on the head. ly21.4, ly21.5, ly21.6, and ly21.7 were expressed in the brain ventricular zone
Horie et al. 1998; Mallya et al. 2002; Storstein et al. 2004; Tanaka et al. 1997). It is thus reasonable to deduce that most, if not all, of the ly21 genes identified in this study are involved in the development/differentiation of nervous system in zebrafish. Compared with other members of ly21 family, ly21.2 exhibits a distinct expression pattern: It is specifically expressed in the skin ionocytes. At least three different types of ionocytes, NaR, HR, and NCC cells, have been identified in zebrafish skin/gills. We found that the expression of ly21.2 is similar to that of ZNCC-like 2 encoding a Na, Cl cotransporter-like protein (Wang et al. 2008). Whether ly21.2 and ZNCC-like are coexpressed in the skin ionocytes and whether Ly21.2 is involved in Na, Cl transport remain open and demand further study.
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