zo-3 in the zebrafish, Danio rerio

Gene Expression Patterns 7 (2007) 767–776 www.elsevier.com/locate/modgep

Identification, tissue distribution and developmental expression of tjp1/zo-1, tjp2/zo-2 and tjp3/zo-3 in the zebrafish, Danio rerio Tanja K. Kiener, Inna Sleptsova-Friedrich 1, Walter Hunziker

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Epithelial Cell Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore Received 13 March 2007; received in revised form 19 May 2007; accepted 29 May 2007 Available online 6 June 2007

Abstract The tight junction (TJ) or zona occludens (ZO) proteins TJP1/ZO-1, TJP2/ZO-2 and TJP3/ZO-3 belong to the membrane associated guanylate kinase-like (MAGUK) protein family and link TJ integral membrane proteins to the actin cytoskeleton. TJPs also serve as scaffolds for signaling proteins and transcription factors that regulate vesicular traffic and cell proliferation and differentiation. Here, we report the identification of two tjp1/zo-1 (tjp1.1 and tjp1.2) and tjp2/zo-2 (tjp2.1 and tjp2.2) genes each and one tjp3/zo-3 gene, and characterize their tissue specific distribution and developmental expression in zebrafish embryos. Transcripts for all five TJPs are maternally supplied and localized expression due to embryonic transcription is observed following the midblastula transition stage of development. In addition to a widespread distribution, individual genes show tissue specific expression patterns and a dynamic regulation during the developmental stages from 2 cells to 4 dpf analyzed. The most noticeable differences in expression patterns are observed in the posterior part of the embryo during somitogenesis. While all TJPs are expressed in the pronephric ducts and epidermis by 18 hpf, tjp1.1 is highly expressed in the hypochord and blood island, tjp1.2 in the somites and the posterior part of the notochord, tjp2.1 in the somites and the ventral part of the spinal cord, and tjp2.2 in the somites only. Individual TJPs are also strongly expressed in different layers of the eye and, at later stages, in central nervous system (CNS) tissues. Interestingly, the differential tissue and developmental expression of the two tjp1 and tjp2 genes indicates that the duplicated genes have been adapted for distinct transcriptional regulations during evolution. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Tight junction; Zonula occludens; Tissue distribution; Developmental expression; Teleosts; Zebrafish; ZO-1; ZO-2; ZO-3; TJP1; TJP2; TJP3; Gene expression; Gene duplication

1. Introduction Tight junctions (TJs) play important roles in shielding higher eukaryotes from the surrounding environment. This is especially important for freshwater fish, which live in a hypo-osmotic environment (Davenport, 1985; Varsamos et al., 2005). In addition, TJs regulate the paracellular transport of specific ions and small molecules between the lumenal and serosal compartments of organs, as well

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Corresponding author. Tel.: +65 65 869599; fax: +65 67 791117. E-mail address: [email protected] (W. Hunziker). 1 Present address: Centre of Excellence for Alzheimer’s Disease Research and Care, School of Exercise, Biomedical and Health Sciences Edith Cowan University, Joondalup, 100 Joondalup Drive, WA 6027, Australia. 1567-133X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2007.05.006

as between different tissue compartments (Anderson et al., 2004; Tsukita et al., 2001; Van Itallie and Anderson, 2004, 2006). TJ also restrict the diffusion of integral membrane proteins and outer leaflet lipids between the apical and basolateral plasma membrane domains of epithelial cells, thus playing an important role in maintaining epithelial cell polarity. TJ are predominantly found in epithelial and endothelial cells (Farquhar and Palade, 1963), but TJ-like structures have also been described in other cell types such as myelinated Schwann cells (Dezawa et al., 1996; Poliak et al., 2002). At the molecular level, TJs consist of integral membrane proteins that are linked, via scaffolding proteins, to the actin cytoskeleton (Fanning et al., 1998; Itoh et al., 1999). Integral membrane TJ proteins include claudins, occludin and junctional adhesion molecule and these

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proteins engage in homo and/or heterotypic interactions with cognate proteins on the surface of adjacent cells (Fanning et al., 1999; Gonza´lez-Mariscal et al., 2003). On the cytosolic face, they bind to adaptor or scaffolding proteins, including TJP1, TJP2 and TJP3 (Gumbiner et al., 1991; Haskins et al., 1998; Stevenson et al., 1986). The three TJPs are closely related members of the membrane associated guanylate kinase (MAGUK) family, characterized by the presence of three PDZ (PSD95/Dlg/ZO-1) domains, one SH3 and a guanylate kinase-like (GUK) domain (Gonzalez-Mariscal et al., 2000, 2003). TJPs associate with each other and directly and/or indirectly to actin filaments (Utepbergenov et al., 2006; Wittchen et al., 1999) and also recruit factors involved in signal transduction and the regulation of proliferation and differentiation (Matter and Balda, 2003).

A PhylogeneticTree

fugu tjp1.2 danio tjp1.2 danio tjp1.1 fugu tjp1.1 dog TJP1 human TJP1 danio tjp2.2 fugu tjp2.2 fugu tjp2.1 danio tjp2.1 dog TJP2 human TJP2 dog TJP3 human TJP3 fugu tjp3 danio tjp3

57.6 50

40

30

20

10

The function of different TJ proteins has been extensively studied in mammalian epithelial cell lines and genetic approaches have been used to study the orthologues in the worm and flies (Banerjee et al., 2006; Shin et al., 2006). Little, however, is known about these proteins in fish. Recently, taking advantage of the completion of the fugu genome sequence, the members of the teleost claudin gene family have been identified and their tissue specific expression reported (Loh et al., 2004). Interestingly, the claudin gene family has undergone an unprecedented expansion in teleosts when compared to mammals, possibly reflecting the requirement to adapt the TJ barrier functions to the specialized physiology of aquatic vertebrates. In this paper, we report the identification of five TJPs in teleosts and analyze their tissue specific and developmental expression pattern during zebrafish development.

C humanTJP1 1748 AA zf tjp1.1 1634 AA

zf tjp1.2 1738 AA

Nucleotide Substitutions (x100)

PDZ SH3

MAGUK

α

-

ZU5

Proline rich

DHF 20-108 183-262 96-113 149-172

422-502 619-794 498-572

1632-1738 α

γ

β DHF

11-98 183-261 410-491 85-101 358-398 505-573

679-780

857-1179 907-977 α

+

1304-1325

1498-1610 1585-1604

γ

β

-

DHF 11-98 175-253 84-101 132-162

NLS PDZ

humanTJP2 1190 AA

0

NLS PDZ + PDZ

+

405-486 364-393 500-568

PDZ

PDZ

628-775

SH3

907-979 915-1253 1364-1429 1602-1714 1007-1099 1436-1445 1696-1708

-

MAGUK

β

Pro TEL

33-120 149-302 106-123 307-385

509-590 690-876 604-669

961-1107

Pro

zf tjp2.1 1223 AA

B Percent AA identity Homo vs Danio

tjp1.1

62

Canis vs Danio 63

Fugu vs Danio 66

Homo vs Fugu 59

Canis vs Fugu

zf tjp2.2 1071 AA

60

tjp1.2

63

65

80

61

64

tjp2.1

53

58

68

51

55

tjp2.2

48

51

40

50

52

tjp3

27

28

39

47

49

TEL 33-120 159-308 345-423 557-638 759-926 106-123 484-548 652-720 Pro TEL 30-117 167-294 305-383 479-528 710-811 103-120 432-469 539-607

PDZ

humanTJP3 953AA fugu tjp3 1135 AA zf tjp3 866AA

+

Proline rich PDZ PDZ

SH3

MAGUK

TDL

30-112 214-291 159-182

413-479 508-582

694-794

TLK 63-153

235-313

443-516

722-822 530-598 TEL

16-103

177-254

403-529 551-609

702-792

Fig. 1. Identification, phylogenetic analysis and comparative domain structure of Zf TJPs. (A) Phylogenetic analysis of the TJP family. A rooted phylogenetic tree was created to show the evolutionary relationships between the TJPs from human, dog, fugu and zebrafish. The length of each branch represents the sequence distance between sequence pairs, with the number of nucleotide substitutions indicated on the bottom. (B) Amino acid identities. Pairwise alignment of the amino acid sequences by ClustalW was used to determine amino acid identities between the TJPs of human, dog, fugu and zebrafish. Between human and zebrafish, identities ranged between 65% (tjp1.2) and 27% (tjp3). (C) Domain structure of zebrafish TJPs as compared to their human orthologues. Amino acid sequences were scanned with the MotifScan tool against Prosite and Pfam profile entries. The boxes represent different protein domains, the numbers below these boxes indicate the amino acids that comprise the individual domains. Zebrafish tjp1.1 and tjp1.2 have the same domain organization as their human counterpart but they lack an acidic domain in the C-terminal part of the protein. The known splice variants were also identified among zebrafish ESTs. The zebrafish tjp2.1 and tjp2.2 also have a highly conserved domain organization, but neither the acidic domain nor the b-splice variant was uncovered and the proline rich domain is found between PDZ2 and PDZ3 as opposed to the C-terminal localization in the human orthologue. Intriguingly, fugu tjp3 has the typical MAGUK structure but a cloned zebrafish tjp3 lacks the PDZ3, SH3 and part of the GUK domains. Zebrafish tjp3 has an additional proline rich region in the C-terminus and the C-terminal PDZ-binding motif is conserved (TEL as compared to TDL in human).

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2. Results and discussion 2.1. Identification of TJP genes in the fugu and zebrafish from genome and expressed sequence tag databases 2.1.1. In silico cloning of fugu TJPs The Fugu rubripes genome is the only completely sequenced and extensively annotated teleost genome. We therefore blasted mammalian TJP cDNA sequences against the fugu genome (Aparicio et al., 2002), leading to the in silico identification of five TJP orthologues. The phylogenetic analysis revealed the presence of two tjp1 genes (subsequently referred to as tjp1.1/zo-1.1 and tjp1.2/zo-1.2), two tjp2 genes (tjp2.1/zo-2.1 and tjp2.2/zo-2.2) and a single tjp3 gene (Fig. 1A). The two tjp1 and tjp2 genes probably arose from the whole genome duplication that occurred in the ray finned fish linage (Amores et al., 1998; Christoffels et al., 2004). Whole genome and localized gene duplications have also been implicated in the expansion of the claudin gene family in teleosts (Loh et al., 2004). Two tjp3 genes annotated on the Ensemble fugu genome (v4.0) are partial transcripts of the same locus. The degree of amino acid identity of the fugu TJPs with their human orthologues ranges between 47% and 61%. Fugu tjp1.1 (1740 amino acids) and tjp1.2 (1688 amino acids) are well conserved with their human counterparts (59% and 61% identity, respectively). Tjp2.1 (1183 amino

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acids) and tjp2.2 (1685 amino acids) are less conserved with human TJP2 (51% and 50%, respectively) with the highest divergence in the C-terminal part of the proteins. Fugu tjp3 (1135 amino acids) shows 47% identity to the human orthologue and again, the highest degree of divergence is observed for the C-terminal part of the protein (Fig. 1B). 2.1.2. In silico cloning of zebrafish TJPs The fugu sequences were blasted against the zebrafish genome (Ensemble v41) and five TJP gene family members were located (Fig. 1A). The complete sequences for tjp1.1 (ENSDARG00000059419, Chr 7), tjp1.2 (ENSDARG 00000062597, Chr 25), and tjp2.1 (ENSDARG00000063309, Chr10) are available. All three tjp1 splice forms described in mammals were identified by blasting the mammalian sequences against the expressed sequence tags. An additional TJP gene family member (ENSDARG00000060614) is most likely identical with tjp1.1 since the two share over 96% sequence identity in introns and exons. In case of tjp2.2, although the complete sequence is found in Ensemble, the exons encoding different domains are not annotated to the same gene but are presented as an ab-initio genscan (GENSCAN00000026775, Chr 8), which predicts complete gene structures in genomic DNA. The complete tjp2.2 sequence was assembled in silico from the exons found in Ensemble and confirmed by RT-PCR covering the joining regions. No tjp2 splice forms

Fig. 2. Zebrafish TJP expression patterns during early embryonic development. All figures depicting expression patterns show the embryos in the same orientation: anterior to the left, posterior to the right, dorsal to the top and ventral to the bottom. Expression of tjp1.1 (A,F,K), tjp1.2 (B,G,L), tjp2.1 (C,H,M), tjp2.2 (D,I,N) and tjp3 (E,J,O) was detected by whole mount in situ hybridization of embryos at the 64 cell stage (2 hpf) (A–E), at the sphere stage (4 hpf) (F–J) and at the shield stage (6 hpf) (K–O). Messenger RNA for all TJPs is maternally supplied (A–E). Hybridization signals remain strong after the midblastula transition, when embryonic transcription begins (F–J). Tjp2.1 mRNA is excluded from the nucleus (C, H, arrows), whereas tjp2.2 accumulates in the nucleus (D, I, arrows). At the stage of early gastrulation, TJP expression is weak but uniform (K–O). Tjp3 mRNA shows perinuclear localization (O, inset).

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Fig. 3. Zebrafish TJP expression patterns during early somitogenesis. Expression of tjp1.1 (A,F), tjp1.2 (B,G), tjp2.1 (C,H), tjp2.2 (D,I) and tjp3 (E,J) was detected by whole mount in situ hybridization of embryos at the 6 somite stage (12 hpf) (A–E) and 10 somite stage (14 hpf) (F–J). All TJP genes are expressed in the tail bud region (arrowheads) by 12 hpf (A–D). Tjp3 is expressed in a localized structure, most likely the Kupffer’s vesicle (E, arrow). The most anterior part of the embryo expresses tjp1.2 and tjp2.1 (B, C, arrows). The anterior axial mesoderm expresses lower levels of tjp1.2 (B) compared to other TJPs, whereas the anterior expression of tjp2.2 and tjp3 is restricted to this area (D, E, open arrowheads). In the posterior part of the embryo, the somitic mesoderm expresses high levels of tjp2.1 (C) and moderate levels of tjp1.2 (B, open arrowheads). TJP expression defines the identity of the posterior mesoderm. The axial mesoderm predominantly expresses tjp1.1 (notochord, F), and tjp1.2 (G) while somites express tjp2.1 (H), and tjp2.2 (I). Tjp3 is weakly detected in the posterior axial and paraxial mesoderm (J).

have been identified in zebrafish to date. The complete cDNA of zebrafish tjp3 was cloned by RT-PCR using Nand C-terminal primers corresponding to the Ensemble build of tjp3 (ENSDARG00000002909, Chr 22). Sequencing, however, revealed that the cloned tjp3 cDNA encoded a protein that in the middle portion differed from the protein predicted in silico (see below). Zebrafish tjp1.1 (1634 amino acids) and tjp1.2 (1738 amino acids) show 62% and 63% identity to human TJP1, whereas tjp2.1 (1223 amino acids) and tjp2.2 (1071 amino acids) are only 53% and 48% identical to their human counterpart. Tjp3 shows the lowest degree of identity with 27% only (Fig. 1B). 2.1.3. Domain structure of teleost TJPs TJPs are members of the membrane associated guanylate kinase (MAGUK) protein superfamily, which are characterized by the presence of three PDZ domains, one SH3 domain and one GUK domain. Depending on the particular TJP, acidic domains, proline and arginine rich

regions, ZU5 domains and C-terminal PDZ-binding motifs are also present. The overall domain structure of teleost and mammalian TJPs is well conserved (Fig. 1C). Zebrafish tjp1.1 and 1.2 lack the acidic domain present in the mammalian protein but the C-terminal ZU5 domain and DHF motif are conserved. Zebrafish tjp2.1 and tjp2.2 have both a C-terminal PDZ-binding motif (TEL), which is also present in mammalian TJP2. They lack an acidic domain and their proline rich domain is located between PDZ2 and PDZ3 and not at the C-terminus as in mammalian TJP2. While in silico analysis and ab initio gene scan of the zebrafish tjp3 genomic sequence predict a hypothetical transcript (GENSCAN00000025730) encoding a typical MAGUK protein, a zebrafish tjp3 cDNA that we cloned by RT-PCR lacks PDZ3, the SH3 domain and part of the GUK domain (see Fig. 1C and Supplemental Data 1). Instead, the cloned cDNA contains five additional exons between PDZ2 and the truncated GUK domain, which are found in EST clones but do not all conform to

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splicing using canonical splice donor or acceptor sites. EST clones covering the predicted SH3 and GUK domain, but not PDZ3 of the hypothetical transcript, are also identified (Supplemental Data 1). The primers used to profile the expression of tjp3 (see below) were selected in order to allow detection of both putative tjp3 isoforms. The C-terminal PDZ-binding motif in tjp3 is conserved between zebrafish and mammals (TEL and TDL, respectively), but the zebrafish protein lacks the arginine rich region and contains a second proline rich domain at the C-terminus. 2.1.4. Tissue distribution and expression of TJPs during zebrafish development Despite widespread expression, the individual TJP genes show differences in their expression patterns. Furthermore, their pattern of expression is dynamic during early zebrafish development, which was studied from the 2 cell stage to 4 dpf. Messenger RNA for all TJPs is maternally supplied, with strong in situ hybridization already present at the 2 cell stage (data not shown). Following the midblastula transition, in situ hybridization signals remain strong until gastrulation. At the shield stage, TJP expression levels are weak and uniform (Fig. 2). All TJP genes are expressed in the tail bud region at 12 hpf (Fig. 3). By 4 dpf, expression levels are high in the pronephric ducts, the intestine

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and the central nervous system (CNS), but rather weak in the somites (Fig. 5). Furthermore, hybridization signals are observed in the epidermis, eyes, otic placodes, olfactory pits, branchial arches, and pectoral fin buds (Fig. 5). The most noticeable differences in the expression patterns of individual TJPs are observed in the posterior part of the embryo during somitogenesis. All TJP genes are expressed in the pronephric ducts by 18 hpf, but there is selective labeling for individual TJPs in the hypochord and blood island (tjp1.1), the somites and the posterior part of the notochord (tjp1.2), the somites and the posterior part of the ventral spinal cord (tjp2.1), and somites only (tjp2.2) (Fig. 4). 2.1.4.1. Tjp1.1. Tjp1.1 shows a moderate and uniform expression from the 2 cell stage to the shield stage (Fig. 2). This expression becomes localized to the axial mesoderm at the beginning of somitogenesis (Fig. 3A), with the notochord expressing tjp1.1 at 14 hpf (Fig. 3F). A restricted expression is seen from 18 hpf to 24 hpf in the hypochord and the blood island (Fig. 4A) and at 18 hpf in the medial region of the eyes (Fig. 7A). The pectoral fin buds start to express tjp1.1 at 2 dpf (Fig. 7B). In the CNS, expression is lowest in the forebrain (Fig. 5K). Tjp1.1 is the only family member expressed in the lens,

Fig. 4. Zebrafish TJP expression pattern during the end of somitogenesis. Expression of tjp1.1 (A,F,K), tjp1.2 (B,G,L), tjp2.1 (C,H,M), tjp2.2 (D,I,N) and tjp3 (E,J,O) was detected by whole mount in situ hybridization of embryos at the 18 somite stage (18 hpf) (A–E) and the Prim-5 stage (24 hpf) (F–O). Towards the end of somitogenesis, all TJP genes are expressed in six major domains, including the epidermis, somites, pronephric ducts (A–J) as well as the neural tube, eyes, and otic placodes (asterisk) (K–O). However, there is a selective expression pattern in the posterior part of the embryo, where tjp1.1 is expressed transiently in the hypochord (A, arrow), the blood island (A,F, arrowhead) and the notochord (A,F, open arrowhead). As observed during early somitogenesis, tjp1.2 is expressed both in the notochord (B, G, open arrowheads) as well as in the somites (B,G). Interestingly, the posterior part of the ventral spinal cord is transiently positive for tjp2.1 (C,H, arrowhead). Tjp3 expression is high in the pronephric ducts (E, arrow) and weak in the epidermis (E, arrowheads).

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Fig. 5. Zebrafish TJP expression pattern at the hatching and larval stages. Expression of tjp1.1 (A,F,K,P), tjp1.2 (B,G,L,Q), tjp2.1 (C,H,M,R), tjp2.2 (D,I,N,S) and tjp3 (E,J,O,T) was detected by in situ hybridization on whole mount embryos at the hatching stage (2 dpf) (A–J) and the larval stage (4 dpf) (K–T). Brain regions express high levels of all TJP genes (A–E). Note that tjp1.2 expression is predominant in the hindbrain (B, arrowhead). The epidermis, the somites and the pronephric ducts also express all five TJPs (F–J). The notochord retains tjp1.1 expression (F, open arrowhead). During the hatching period, the spinal cord starts to express tjp1.2 (G, arrow). The pectoral fin buds are tjp3 positive (E, arrowhead). At the beginning of the larval stage, the expression pattern of the five TJP genes converges. In the anterior part of the embryo, all five TJP genes are now expressed in the nose (arrow), otic capsule (asterisk), branchial arches (open arrowhead), and regions of the brain (K–O, see M). Tjp1.2 is the dominant gene expressed in the brain (L). As already observed at 2 dpf, tjp1.1 and tjp1.2 are expressed in the hindbrain (K,L, arrowheads), from where the other isoforms are absent (M,N,O, arrowheads). In the posterior part of the embryo, the pronephric ducts and intestine highly express TJP genes. Expression in the epidermis and somites is low and not readily detected by in situ hybridization (P–T), but the spinal cord now expresses high levels of both tjp1.2 and tjp2.1 (Q,R, arrows).

starting at 24 hpf (Fig. 6D). In addition, the inner nuclear layer of the retina also expresses tjp1.1 by 4 dpf (Fig. 6E). 2.1.4.2. Tjp1.2. Similar to tjp1.1, tjp1.2 is uniformly expressed until gastrulation (Fig. 2). Staining is strong in the most anterior part of the headanlage by 12 hpf (Fig. 3B) and tjp1.2 will remain the dominant TJP gene expressed in the CNS. During somitogenesis, tjp1.2 is detected in the axial and somitic mesoderm (Fig. 3B and G). The posterior part of the notochord expresses tjp1.2 from 18 hpf (Fig. 4B) up to 3 dpf (Fig. 7I). The most posterior immature somites express high levels of tjp1.2 from 18 hpf (Fig. 4B) to 1 dpf (Fig. 4G), after which somite labeling becomes weaker. At 2 dpf tjp1.2 is expressed in the pectoral fin buds (Fig. 7C), in the Duct of Cuvier

(Fig. 7D) and in the heart (Fig. 7G). The spinal cord also starts to express tjp1.2 at 2 dpf (Fig. 5G). By 4 dpf, expression levels are low in the forebrain and high in the mid- and hindbrain (Fig. 5L), as well as in the spinal cord (Fig. 5Q). The eye expresses tjp1.2 in the inner nuclear layer and the ganglion cell layer (Fig. 6F). 2.1.4.3. Tjp2.1. This gene is strongly expressed in embryos until the gastrulation stage. Between 2 hpf and 4 hpf, tjp2.1 is excluded from the nuclei (Fig. 2C and H) whereas the tjp2.2 mRNA shows a nuclear localization at this stage (Fig. 2D and I). Expression levels decrease after 6 hpf to levels comparable to the other TJP genes. Like tjp1.2, tjp2.1 is detected in the anterior part of the headanlage at 12 hpf (Fig. 3C). It is also the first TJP gene to be

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Fig. 6. Zebrafish TJP expression patterns in sensory anlagen. Expression of TJPs in sensory anlagen was detected by whole mount in situ hybridization. (A–C) Olfactory and otic placodes. Expression during sensory organ development is temporally regulated. Tjp3 is the first TJP family member to be expressed in a bell shaped area of the forehead (A, arrowhead), which will give rise to the olfactory placodes, and in the otic placodes (A, arrow) at 14 hpf. At 18 hpf, tjp3 is detected in paired domains of the olfactory placodes (B, arrowhead), the otic placodes (arrow), and the branchial arches vesicles (B, open arrowhead). Tjp2.1 is expressed in the otic placodes at 18 hpf (C, arrow) which is later than tjp3 but earlier than the other TJPs (see Fig. 4K–O). (D–I) Eyes. Tjp1.1 is the only family member expressed in the lens, starting at 1 dpf (D, arrow). By 4 dpf, TJP expression in the retina is mainly observed in the inner nuclear layer as well as in the ganglion cell layer. (E) tjp1.1 is expressed in the inner nuclear layer (arrowheads) and in the lens (arrows). (F) tjp1.2 is also expressed in the inner nuclear layer (arrowheads). In addition, there is a strong signal in the ganglion cell layer (open arrowheads). Tjp2.1 (G) and tjp2.2 (H) are found in the inner nuclear layer only (arrowheads). Tjp2.1 expression appears to be restricted to the outer part of the layer which contains bipolar and horizontal cells (G, arrow). (I) tjp3 is present in the nuclear layer (arrowhead) and also in the ganglion cell layer (open arrowhead).

expressed in the somitic mesoderm (Fig. 3C). The otic placodes express tjp2.1 by 18 hpf (Fig. 6C), which is delayed compared to tjp3 expression but earlier than that of the other TJP family members. Tjp2.1 expression levels are high in the posterior part of the ventral spinal cord from 18 hpf (Fig. 4C) to 24 hpf (Fig. 4H). At later stages, tjp2.1 is detected in the Duct of Cuvier at 2 dpf (Fig. 7E), in the caudal vein at 3 dpf (Fig. 7J) and in the spinal cord at 4 dpf (Fig. 5R). In contrast to the two tjp1 genes, CNS expression levels of tjp2.1 is higher in the fore- and midbrain as compared to the hindbrain region (Fig. 5M). The eye expresses tjp2.1 in the outer part of the inner nuclear layer, which contains bipolar and horizontal cells (Fig. 6G). 2.1.4.4. Tjp2.2. As indicated above, tjp2.2 mRNA is detected in the nuclei at sphere stage (Fig. 2I). At the beginning of somitogenesis tjp2.2 labeling is restricted to the axial part of the headanlage (Fig. 3D), with parasomitic mesoderm expression following at 14 hpf (Fig. 3I). Later in development the expression pattern is similar to that of tjp2.1, with strong signals in the anterior (mature) somites from 18 hpf (Fig. 4I) until 3 dpf (Fig. 5I) and higher expression in the fore- and midbrain than in the hindbrain region (Fig. 5N). Tjp2.2, however, is not expressed in the spinal cord (Fig. 5S). Expression in the retina is restricted to the inner nuclear layer, similar to tjp2.1 (Fig. 6H).

2.1.4.5. Tjp3. Maternal tjp3 mRNA levels are high. At the shield stage, tjp3 mRNA localization is perinuclear (Fig. 2O). Tjp3 shows a localized expression in the tail bud region, most probably in the Kupffer’s vesicle, at 12 hpf (Fig. 3E) and is detected in the axial and paraxial mesoderm by 14 hpf (Fig. 3J). At 14 hpf tjp3 starts to be expressed in the otic and olfactory placodes (Fig. 6A and B), which makes it the earliest TJP expressed in sensory anlagen. Expression levels are high in the pronephric ducts from 18 hpf (Fig. 4E) until 2 dpf (Fig. 5J) but weak in the epidermis (Fig. 4E) and somites (Figs. 4J, 5J). The posterior parts of the pronephric ducts express tjp3 first and the expression then extends along the ducts as they differentiate. The Duct of Cuvier (Fig. 7F) and the heart (Fig. 7H) both express low levels of tjp3 at 2 dpf. At 4 dpf, CNS staining is strong in the midbrain (Fig. 5O) but absent from the spinal cord (Fig. 5T). In the eye, tjp3 is expressed in the inner nuclear layer and in the ganglion cell layer (Fig. 6I), similar to tjp1.2. 2.1.5. Conclusions The characterization of TJP gene expression during vertebrate development has focused on early events in the embryonic development of Xenopus and mouse, where TJP1 is found in the enveloping cell layer and trophectoderm, respectively (Fesenko et al., 2000; Fleming et al., 2000; Merzdorf et al., 1998). Little information is available for the expression pattern of other TJP genes or

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Fig. 7. Zebrafish TJP expression patterns in other selected organs. Expression of TJPs in selected organs was detected by whole mount in situ hybridization. (A) Low expression of tjp1.1 is detected in the medial region of the eyes at 18 hpf (arrowheads). (B,C) Pectoral fin buds. The pectoral fin buds do not only express tjp3 (see Fig. 5E) but also tjp1.1 (A, arrowhead) and tjp1.2 (B, arrowheads) at 2–3 dpf. (D–F) Duct of Cuvier. The Duct of Cuvier expresses tjp1.2 (D), tjp2.1 (E), and tjp3 (F, arrowheads). (G,H) Heart. The newly forming heart transiently expresses high levels of the tjp1.2 (G, arrow) and low levels of tjp3 (H, arrow) at 2 dpf. (I) Notochord. The posterior part of the notochord retains tjp1.2 expression up to 3 dpf (arrowhead). (J) Caudal vein. The caudal vein expresses tjp2.1 at 3 dpf (arrows).

for the expression patterns of TJP genes during organogenesis. In mammals, with the exception of TJP3, TJPs appear to be widely expressed in most adult tissues. Epithelial tissues generally express TJP1, TJP2 and TJP3, whereas endothelial cells apparently lack TJP3 (Inoko et al., 2003). Here, we identify five TJP genes in fugu and zebrafish. The whole genome duplication that occurred in ray finned fish (Amores et al., 1998; Christoffels et al., 2004) probably accounts for the presence of two tjp1 and two tjp2 genes. Despite a broad distribution, the individual TJPs show a tissue specific expression during early organogenesis of the zebrafish embryo. Based on the different spatial and temporal expression pattern, tjp1.1 and tjp1.2 as well as tjp2.1 and tjp2.2 appear to have evolved to be regulated in their unique fashion. It will therefore be of interest to analyze and compare the regulatory sequences of the duplicated genes to identify unique transcription factor binding sites, which could account for the developmental and tissue specific regulation.

Interestingly, the highest degree of expression heterogeneity of the different TJP genes is observed during early development. The expression patterns converge at later stages. In the posterior mesoderm, for example, axial mesoderm is characterized by the expression of tjp1 while somitic mesoderm is characterized by tjp2 expression. Tjp3 is the first TJP gene to be expressed in sensory placodes and tjp1 is predominant in the hindbrain region. It will also be important to analyze if during evolution the different proteins domains of the two forms of tjp1 and tjp2 have undergone changes that may affect their function. For example, subtle modifications in the PDZ domains could allow the recruitment of different sets of claudins, possibly allowing fine-tuning the paracellular permeability properties of different tissues. Several approaches, including targeted inactivation in epithelial cell lines (Umeda et al., 2004), siRNA mediated ablation (McNeil et al., 2006) (Hernandez et al., 2007) and targeted inactivation in mice (Adachi et al., 2006) have recently been used to explore the functional relevance of individual TJPs. Interestingly, mice lacking TJP3/ZO-3 are viable and do not present any obvious phenotype (Adachi et al., 2006). Given the established advantages to elucidate the function of particular genes in development and organ physiology, zebrafish may prove an interesting vertebrate model system to further explore TJP function. 3. Experimental procedures

3.1. In silico cloning and sequence analysis Mammalian TJP1, TJP2 and TJP3 sequences were blasted against the complete fugu rubripes genome (IMCB). The resulting sequences were blasted against the zebrafish genome on Ensemble (v41) (http://www.ensemble.org/Danio_rerio/index.html). Five TJP family members could be located. Analysis of the sequences was done using the DNAstar software modules EditSeq, Seqman, Megalign, and Protean (DNAstar Inc.). Protein alignments and phylogenetic analysis were performed with the MegAlign program using the ClustalW slow/accurate method. Functional domains were uncovered with the Motif Scan tool (http://myhits.isb-sib.ch/cgi-bin/motif-scan or http:// tw.expasy.org), which scans protein sequences against PROSITE and Pfam. Intron–exon boundaries were determined using Genescan (Burge and Karlin, 1997) and putative splice sites using established algorithms (Burge, 1998). Protein domain schemes were drawn with the Protean software module.

3.2. Cloning of zebrafish tjp3/zo-3 and whole mount in situ hybridization Zebrafish TJP cDNA clones were generated by RT-PCR from total RNA of 1 dpf embryos. Primers were designed to span regions of the five TJP family members that show less than 35% identity. Probes had a length of 600–800 bp, except for the tjp1.1 probe, which could not be amplified by RT-PCR. Instead, an EST clone encoding the Nterminus of tjp1.1 was obtained and used to amplify a 240 bp fragment. PCR products were cloned into the pGEM-Teasy vector (Promega) and then subcloned into pBluescript II SK (Stratagene). Plasmids were lin-

T.K. Kiener et al. / Gene Expression Patterns 7 (2007) 767–776 earized with an suitable restriction enzyme and DIG-labeled anti-sense RNA (Roche) generated. Sense RNA was transcribed and used as a negative control. In situ hybridization of whole mount zebrafish embryos was performed as described (Hauptmann and Gerster, 1994). The sequences of the primers and probes used are provided in Supplemental Data 2.

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