Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio) Emmanuelle Cotto 1,2 , Miche ` le Andre ´ 1 , Jean Forgue 1 , Herve ´ J Fleury 2 and Patrick J Babin 1 1 Laboratoire Ge ´ nomique et Physiologie des Poissons, UMR 1067 NUAGE INRA-IFREMER, Universite ´ Bordeaux 1, Talence, France 2 Laboratoire Virologie Syste ´ matique et Mole ´ culaire, E.A. 2968, Universite ´ Victor Segalen Bordeaux 2, Bordeaux, France Transmissible spongiform encephalopathies (TSEs), more commonly called prion diseases, have long been known in mammals, including humans. They are char- acterized by the accumulation of a pathogenic misfolded form (PrP Sc ) of the physiological protein (PrP C ), which is encoded by a single copy of the prion gene (Prnp)in humans [1,2]. Whereas the epidemiological characteris- tics were thought to be totally identified, the variant Creutzfeldt–Jakob disease represents an emerging form of these pathologies, transmitted by the oral route from common food products [3,4]. The species barrier is an important aspect in the prion oral transmission risk. It Keywords brain; duplicated genes; prion; PrP; zebrafish Correspondence P.J. Babin, Laboratoire Ge ´ nomique et Physiologie des Poissons, UMR 1067 NUAGE INRA-IFREMER, Universite ´ Bordeaux I, Avenue des Faculte ´ s, Ba ˆ t. B2, 33405 Talence cedex, France Fax: +33 5 4000 8915 Tel: +33 5 4000 8776 E-mail: p.babin@gpp.u-bordeaux1.fr Note The sequence data presented here have been deposited with the GenBank ⁄ EMBL Data Libraries under the accession numbers AJ850286 for zebrafish PrP1 and AJ620614 for zebrafish PrP2 mRNAs. (Received 19 July 2004, revised 12 November 2004, accepted 18 November 2004) doi:10.1111/j.1742-4658.2004.04492.x Prion diseases are characterized by the accumulation of a pathogenic mis- folded form of a prion protein (PrP) encoded by the Prnp gene in humans. In the present study in zebrafish, two transcripts and the corresponding genes encoding prion proteins, PrP1 and PrP2, related to human PrP have been characterized with a relatively divergent deduced amino acid sequence, but a well preserved overall organization of structural prion pro- tein motifs. Whole-mount in situ hybridization analysis performed during embryonic and larval development showed a high level of PrP1 mRNA spatially restricted to the anterior floor-plate of the central nervous system and in ganglia. Transcripts of prp2 were detected in embryonic cells from the mid-blastula transition to the end of the segmentation period. From 24 h postfertilization up to larval stages, prp2 transcripts were localized in distinct anatomical structures, including a major expression in the brain, eye, kidney, lateral line neuromasts, liver, heart, pectoral fins and posterior intestine. The observed differential developmental expression patterns of the two long PrP forms, prp1 and prp2, and the short PrP form prp3,a more divergent prion-related gene previously identified in zebrafish, should contribute to understanding of the phylogenetic and functional relation- ships of duplicated prion gene forms in the fish genome. Together, the complex history of prion-related genes, reflected in the deduced structural features, conserved amino acid sequence and repeat motifs of the corres- ponding proteins, and the presence of differential developmental expression patterns suggest possible acquisition or loss of prion protein functions dur- ing vertebrate evolution. Abbreviations CNS, central nervous system; dpf, days postfertilization; EST, expressed-sequence tag; gb, GenBank; GPI, glycosyl phosphatidylinositol; HB, hybridization buffer; hpf, hours postfertilization; NGF, nerve growth factor; ORF, open reading frame; PrP, prion protein; PrP C , cellular prion protein; PrP Sc , scrapie prion protein; Prnp, gene for mammalian PrP C ; PrP1, prion protein 1; PrP2, prion protein 2; PrP3, prion protein 3; prp1, prion protein 1 gene; prp2, prion protein 2 gene; prp3, prion protein 3 gene; PTU, 1-phenyl-2-thio-urea; Sho, Shadoo protein; sp., Swiss-Prot; TSE, transmissible spongiform encephalopathie. 500 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS depends in part on the homology between the donor pathogen protein and the natural physiological protein present in the receiver, both in the amino acid sequence of the protein and in its tridimensional conformation [2]. Thus, it is important to compare these parameters in the different vertebrate species to evaluate the risk of a prion passage from one species to another. The exact role and evolutionary origin of human PrP C are still unclear. Genes homologous to the human Prnp have been characterized in different spe- cies of mammals and birds [5,6], and corresponding cDNAs have been identified in turtle [7], and Xenopus [8]. Different cDNAs coding for homologs of tetrapod PrP C have been identified in Fugu [9–11], Atlantic sal- mon [10] and zebrafish (Danio rerio) [9]. These include duplicated protein long forms similar to PrP C in Fugu, initially called PrP-461 ⁄ stPrP-1 and stPrP-2 [10,11] and renamed in this study PrP1 and PrP2, respectively. In Fugu and zebrafish, a cDNA has been identified enco- ding a divergent prion-related protein called PrP- like ⁄ PrPL-P1 [9] and renamed here PrP3. A Shadoo protein (Sho) encoded by the Sprn gene has also been found in mammals [12]. Two duplicated copies of this gene were detected in the fish genome [13]. Although Sho is highly conserved from fish to mammals, it has little overall similarity to human PrP C [12]. In addi- tion, none of the PrP-homologues identified in fish species appeared to resemble doppel, a diverged PrP- related paralogue found in close proximity to human Prnp [14]. These data reflect the complex history of prion-related genes during vertebrate evolution. PrP C mRNA expression sites need to be determined to identify cells that are functionally dependent upon synthesis of this protein. In addition, infected cells must express PrP C to propagate the pathogenic agent and convert the normal form to the pathogenic one [15,16]. The identification of cells that express Prnp is thus the essential starting point to clarify pathogenic and replicative mechanisms of PrP Sc in TSEs. The mammalian Prnp gene has been described as a house- keeping gene with a preferential expression in neurons [2,17]. Transcripts of this gene and PrP C are present in a large variety of adult peripheral tissues [18–20]. In contrast, there is a paucity of data on the spatio- temporal expression of prion proteins and that of related-protein genes during development [9,21–23]. In the present study performed in zebrafish, two transcripts originating from two genes encoding prion- related proteins, PrP1 and PrP2, were characterized with a relatively divergent deduced amino acid sequence but a well preserved overall organization of structural prion protein motifs. The developmental expression profiles of prp1 and prp2 were determined by whole-mount in situ hybridization and compared with the expression of prp3. The observed differential developmental expression patterns of these three genes should help clarify the functional relationships of duplicated forms of the prion-related genes in the fish genome as well as the specific roles and evolution of PrP and related proteins in vertebrates. Results and Discussion Molecular characterization of zebrafish PrP2 The zebrafish dbEST database was screened for poten- tial homologs to tetrapod PrPs using known puffer- fish (Fugu rubripes) and salmon (Salmo salar) prion homologous sequences [10]. Two zebrafish expressed- sequence tags (ESTs) with accession numbers gb|CA470368| and gb|BM071383| were identified. Clone IMAGp998C0911982Q3 corresponding to gb|BM071383| and including the putative initiator methionine was ordered from the Resource for the German Genome Project (RZPD), Berlin, Germany, double strands were sequenced, and the full-length PrP2 mRNA was deposited with the accession number gb|AJ620614|. Using the tblastn program, the zebra- fish genome database (http://www.sanger.ac.uk/ Projects/D_rerio/) was screened (version 22.3b of Ensembl) for the PrP2 cDNA sequence. Two chromo- some 10 DNA contigs, ctg23943 and ctg30140, were recovered using the PrP2 cDNA sequence. The per- fect match obtained on Ensembl zebrafish gene GENSCAN00000028159 (ENSDARG00000028576) of ctg23943 with prp2 transcript indicated that this gene consisted of at least two exons with the coding sequence contained within exon 2. A coding sequence of 1701 bp, from an ATG codon at position 71 of the cDNA to a stop codon starting at position 1772 was contained in a single exon of 3782 bp. The entire 5¢- untranslated region of the characterized prp2 transcript was contained in a single 5¢-noncoding exon with a minimum size of 70 bp, separated from the coding exon by a 3818 bp intron. The position of this 5¢-non- coding exon was confirmed with three additional EST sequences (accession numbers gb|CD604530|, gb|CD600079|, and gb|CD584991|). The sequences at the intron–exon boundaries of zebrafish prp2 were con- sistent with the usual consensus intron–exon splice junction rule (GT ⁄AG). The predicted amino acid sequence of the zebrafish PrP2 was 567 amino acids in length and presented all features previously described for members of the tetra- pod PrP family (Fig. 1), namely a putative signal peptide (amino acids residues 1–19), a long stretch of E. Cotto et al. Expression of prion genes in the developing zebrafish FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 501 Gly-Tyr-Pro-rich repeats (residues 74–246), a hydro- phobic central motif (residues 299–315), two cysteine residues potentially involved in the formation of an intramolecular disulfide bond (residues 399 and 509), two asparagine residues that are significant putative N-glycosylation sites (residues 438 and 443), a poten- tial cleavage site (residue 537), a putative glycosyl phosphatidylinositol (GPI)-anchor site (residue 538), and a predicted hydrophobic C-terminal transmem- brane region (residues 549–567) (Fig. 2). The N-ter- minal signal peptide indicates that the mature protein is located outside the cell. This conserved extracellular localization during vertebrate evolution suggests that PrPs could play a role in interactions with the extracel- lular matrix [24] or act as a receptor for a molecular signal. The disulfide bond should be essential for the conformational protein conservation, and a putative GPI-anchor site found in PrP2 as well as in all other vertebrate PrPs tends to confirm the hypothesis that PrPs must necessarily be located outside the cell, attached to the membrane [25]. Moreover, PrP2 pre- sents two putative N-glycosylation sites, which might protect the extracellular portion of the protein against proteases and nonspecific protein interactions. The secondary structures of the C-terminal region (residues 313–530) of zebrafish PrP2 predicted based upon NMR (PDB identifier 1hjnA) studies of human PrP [26] showed, in the same order as in human PrP C , the two b-sheets and three long a-helices characteristic of the prion protein. This region is the PrP C prion ⁄ doppel alpha-helical C-terminal globular domain (Pfam accession number PF00377). It contained pat- ches of sequence identity between zebrafish PrP2 and tetrapod PrPs that matched with the predicted secon- dary structures of human PrP C (Fig. 2). Hydrophobic cluster analysis [27] predicted the presence of several conserved hydrophobic clusters throughout the com- pared zebrafish PrP2 and human PrP C sequences. This type of similarity is typical of distant but related sequences. In the N-terminal part analogous to the human PrP C globular domain, zebrafish PrP2 con- tained a conserved motif corresponding to PROSITE prion protein signature 1 motif (PS00291), which is held to be a signature of PrPs in vertebrates. This so-called hydrophobic region is rich in small amino acids (Gly, Ala) and is included in a region with simi- larities to viral fusion peptides and reactive loops of serpins [28]. Additional conserved sequence motifs and amino acid positions in zebrafish PrP2 and tetrapod PrPs are included in the corresponding b-sheet ⁄ a-helix structures. This includes helix H1 of human PrP C , which is part of the dimer interface region between Fig. 1. The prion protein (PrP) family in vertebrates. Schematic diagram of tetrapod PrPs, long (PrP1 and PrP2) and short (PrP3) fish PrPs, and vertebrate Shadoo (Sho) proteins. The species abbreviations refer to sequences from human (Hum), chicken (Chi), turtle (Tur), Xenopus (Xen), zebrafish (Zeb), salmon (Sal) and Fugu (Fug). The location and relative size of conserved structural features are shown. These features were initially determined on the structure reported for human PrP C . Domains are indicated by different boxes and ⁄ or letters: S, signal pep- tide sequence; R, repetitive region; H, hydrophobic region; S- -S, disulfide bridge; N, glycosylation site; arrow, GPI anchor residue; T, hydro- phobic tail. Expression of prion genes in the developing zebrafish E. Cotto et al. 502 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS PrP C and PrP Sc [29,30], helix H2 putatively involved in the structural conversion to PrP Sc [30], and helix H3 corresponding to PROSITE prion protein signature 2 motif (PS00706). The alignment of the conserved sequence motifs of the alpha-helical C-terminal domain resulted in 33% amino acid identities between zebra- fish PrP2 and human PrP C , 44% between human and Xenopus PrPs, and 54% between chicken and human PrPs (Fig. 2). Enlargement of the loops between the three helices were observed in zebrafish PrP2 as com- pared to human PrP C sequences, i.e. 47 residues instead of 15 residues, including strand S2, between helices H1 and H2, and 84 residues instead of 10 resi- dues between helices H2 and H3 (Fig. 2). Molecular characterization of zebrafish PrP1 The imperfect match obtained on Ensembl zebrafish gene GENSCAN00000038006 (ENSDARG00000027528) of ctg30140 by screening the Sanger Institute zebrafish genome data for zebrafish PrP2 indicated the presence of an additional copy of a prion-related gene on zebra- fish chromosome 10. Complete genomic sequence (gb|BX640677|), and EST sequences (gb|CK028669|, gb|CK025947|, and gb|CO925322|) extracted from a whole body and the olfactory epithelium cDNA banks, confirmed the existence of this additional expressed prion-related gene in the zebrafish genome. Clone IMAGp998P1614834Q3 corresponding to gb|CK02 5947| was ordered, double strands were sequenced, and the full-length PrP1 mRNA sequence (accession num- ber gb|AJ850286|) was obtained after overlapping with gb|CO925322| EST sequence. The perfect match between prp1 transcript and the genomic sequence extracted from gb|BX640677| indicated that this gene, as zebrafish prp2, consists of at least two exons, the ORF being contained within exon 2. A coding sequence of 1821 bp, from a translation initiator ATG codon at position 86 of the cDNA to a stop codon starting at position 1905, lay within a single exon of 2018 bp. In mammals, PrP C is also encoded by an intronless ORF [1]. The ATG codon was localized Fig. 2. Alignment of the conserved sequence motifs of the C-terminal domain among members of the PrP family. Amino acid numbering starts from the initiator methionine. Gaps inserted to optimize alignments are indicated by dashes. Numbers in parentheses in the align- ments indicate the length of the omitted nonconserved regions. Human PrP C secondary structures, as observed from X-ray (PDB identifier 1I4M) studies [52] are indicated above the human sequence (H1 to H3 for a helices, S1 to S2 for b strands). The horizontal line above the human sequence indicates the fusion-like peptide region of human PrP C . Amino acid residues identical or considered conserved with the human PrP sequence are marked in dark grey and light grey, respectively. Amino acid residues identical (›) or considered conserved (+) in all sequences compared, or in all sequences compared minus one (*) are indicated below the Zeb.PrP2 and Fug.Sho1 sequences. Note that the corresponding S2, H2 and H3 do not exist in fish PrP3 and vertebrate Sho and therefore could not be indicated in the alignment. The allowed conservative substitutions including the hydrophobic amino acid group were defined as follows: A ¼ G; S ¼ T ¼ E ¼ D; R ¼ K ¼ H; Q ¼ N; P; C; V ¼ I ¼ L ¼ M ¼ Y ¼ F ¼ W. Species and sequences abbreviations are the same as in Fig. 1. E. Cotto et al. Expression of prion genes in the developing zebrafish FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 503 7 bp downstream of a 1607 bp intron. The sequences at the intron–exon boundaries of zebrafish prp1 were consistent with the usual consensus sequence (GT ⁄ AG) at intron–exon boundaries. This unique intron was inserted ahead of the ATG initiator codon as in human Prnp [1]. An alternate splice site in the 5¢-untranslated region could give two transcript vari- ants in humans (gb|NM_183079| and gb|NM_000311|), while two 5¢-noncoding exons have been characterized in the sheep and mouse gene [1]. The predicted amino acid sequence of the zebrafish PrP1 was 606 amino acids in length and exhibited, as described for zebrafish PrP2, all features previously described for members of the tetrapod PrP family, namely a putative signal peptide (amino acids residues 1–23), a long stretch of repeats (residues 48–332), a hydrophobic central motif (residues 379–395), two cysteine residues potentially involved in the formation of an intramolecular disulfide bond (residues 463 and 554), two asparagine residues that are significant puta- tive N-glycosylation sites (residues 367 and 445), and a predicted hydrophobic C-terminal transmembrane region (residues 592–606) (Fig. 1). Of note is that a putative GPI-anchor site was predicted in the sequence while no potential cleavage site of the hydrophobic tail could be detected in either zebrafish or Fugu PrP1 sequences. The alignment of the conserved sequence motifs of the alpha-helical C-terminal domain resulted in 25% amino acid identities between zebrafish PrP1 and human PrP C , 62% between zebrafish PrP1 and PrP2, 66% between zebrafish PrP1 and Fugu PrP1, and 57% between zebrafish PrP1 and Fugu PrP2 sequences (Fig. 2). Conserved sequence motifs and amino acid positions (Fig. 2) indicated that zebrafish PrP1 is, as PrP2, a member of the PrP family. Analysis of the N-terminal repeat domain of zebrafish PrP1 and PrP2 The N-terminal domain of zebrafish PrP1 contained nine repeats (residues 53–332) including four highly conserved 37 amino acid-long repeats (residues 100– 247) (Fig. 3). The presence of five Tyr-Pro amino acid conserved motifs inside each long repeat and included in short internal repeats, i.e. [G]-[G]-[Y]-[P] (motifs 3 and 4) and [G]-[G]-[Y]-[P]-[N]-[Q] (motifs 2 and 5), strongly suggests at least two rounds of independent duplications; the first round resulting in the ancestral long repeat unit and the second giving the repeats found in the zebrafish PrP1 sequence. Analysis of the Fig. 3. Alignment of the N-terminal amino acid repeats from human and fish PrP sequences. Amino acid numbering starts from the initiator methionine. Gaps inserted to optimize alignments are indicated by dashes. Numbers in parentheses in the alignments indicate the length of the omitted nonconserved regions. Amino acid residues identical or considered conserved with the human (Hum) PrP sequence are marked in dark gray and light gray, respectively. When the corresponding amino acid position is not available in the human sequence, amino acid res- idues identical or considered conserved in salmon (Sal) PrP and zebrafish (Zeb) PrP1 sequences are marked in dark gray and light gray, respectively. The five Tyr-Pro amino acid conserved positions in fish PrP sequence repeats are indicated by a number below the Zeb.PrP1 sequence. The allowed conservative substitutions are defined as in Fig. 2. Expression of prion genes in the developing zebrafish E. Cotto et al. 504 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS salmon PrP repeats indicated the presence of four almost perfect 36 amino acid-long repeats (residues 106–250) similar to zebrafish PrP1 repeats and included in a total of seven repeats (residues 75–313). However, some supplementary or different amino acid residues found at similar positions between zebrafish PrP1 and salmon PrP repeat sequences strongly sug- gest an independent amplification of the ancestral long repeat unit in each fish lineage. In addition, the N-terminal domain of Fugu PrP1 sequence revealed the presence of imperfect long repeats (residues 66–190) together with six short internal degenerated tandem repeats (residues 219–242) similar to short internal repeat motifs 3 and 4 found in zebrafish PrP1 and sal- mon PrP sequences. The N-terminal part of zebrafish PrP2 contained no long repeats. However, a Tyr-Pro- rich repeat domain (residues 74–246) containing 18 hexapeptide repeats plus seven repeats with an irregu- lar amino acid sequence length was identified (data not shown). The PROSITE consensus pattern of zebrafish PrP2 hexapeptide repeats was [G,N,P,A,S]-[G,N,P, R,S]-[Y]-[P]-[A,N,G,R,V]-[Q,G,A,R], a motif similar to motifs 2 and 5 found in zebrafish PrP1 and salmon PrP sequences. A shorter core motif consisting of [G]- [Y]-[P] or [G]-x-[P] and similar to internal short repeat motifs found in the fish PrP sequences have been iden- tified in Xenopus, turtle, chicken, and human prion sequences, respectively (Fig. 3 and data not shown). This core motif is part of the copper-binding octapep- tide repeat of human PrP (Pfam accession number PF03991). However, the histidine residues, the residues that actually bind the copper, are not conserved in the fish sequences. It should be noted that conserved amino acid residues were identified between mammal PrP sequences proximal to the octapeptide repeats (res- idues 38–56 in human PrP) and zebrafish PrP1 or sal- mon PrP sequences (Fig. 3). This N-terminal part of human PrP might therefore be derived from the ances- tral long repeat unit that was subsequently amplified in the teleost fish lineage. Phylogenetic relationships of zebrafish prion genes Different cDNAs coding for homologs to tetrapod PrP C have been identified in Fugu [9–11], Atlantic sal- mon [10] and zebrafish (Danio rerio) [9]. These include duplicated protein long forms similar to PrP C in Fugu, initially called PrP-461 ⁄ stPrP-1 and stPrP-2 [10,11] and renamed in this study PrP1 and PrP2, respectively. Given that the zebrafish PrP1 sequence could be aligned in its entirety with zebrafish PrP2, Fugu PrP1 and PrP2, and salmon stPrP sequences (data not shown), one can define a fish long-PrP-like sequence group (Fig. 1). Identical amino acid residues at con- served sites between fish PrP1 ⁄ PrP2 and tetrapod PrPs include Pro102 (amino acid numbering refers to human PrP C ), Ala113, Ala116, Ala117, Tyr128, Gly131, Phe141, Glu146, Cys179, Cys214, and Tyr218 (Fig. 2). The functional importance of invariant amino acids of the corresponding alpha-helical C-terminal globular domain of human PrP C can be demonstrated with Pro102, Ala117, and Gly131 variants of human PrP C . A substitution of one of these residues in human PrP C by a hydrophobic amino acid is linked to development of the neurodegenerative Gerstmann– Stra ¨ us-sler–Scheinker disease (Swiss-Prot entry features accession number P04156). A third PrP-like homolog previously identified in zebrafish [9] has been positioned on contig ctg25727 (GENSCAN00000017195, gb|Q7T2P9|) of chromo- some 8 close to a Ras association domain family 2 (RASSF2) homolog (KIAA0168) (GENSCAN- 00000016076). A conserved synteny between a rassf2 homolog and this gene referred to here as prp3 has been previously demonstrated in Fugu [9]. It should be noted that Fugu prp2 [10,13], but not zebrafish prp2 (this study), has been located on the same scaffold in the direct neighborhood of prp3. Alignment of the con- served sequence motifs between fish proteins similar to tetrapod PrPs demonstrated that the fish duplicated PrP long forms, PrP1 and PrP2, are more structurally related to human PrP C than fish PrP3 or Sho sequences (Figs 1 and 2). Ala113, Ala116, Ala117 of the conserved hydrophobic region were the only three conserved amino acid residues that could be identified with confidence in fish PrP3 and tetrapod Sho proteins (Fig. 2). Fish PrP3 could be assigned to the fish short- PrP sequence group (Fig. 1) with lack of some charac- teristic elements of PrPs including the Gly-Tyr-Pro-rich repeat domain before the hydrophobic central motif. No potential glycosylation sites were identified in Fugu PrP3 and only one was predicted in zebrafish PrP3. No cysteine residues included in the corresponding human PrP C helices H2 and H3 have been recorded in Fugu PrP3. However, in the incomplete H3-like sequence of zebrafish PrP3 there was a small conserved hydrophobic motif (amino acid positions 171–175) next to a cysteine residue most certainly corresponding to human Cys214. The evolutionary relationship of genes belonging to the PrP family was evaluated after alignment of the identified conserved sequence motifs and phylogenetic trees were constructed therefrom. The computer- derived phylogenetic trees, derived from alignments encompassing amino acid residues 101–157 and 101– E. Cotto et al. Expression of prion genes in the developing zebrafish FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 505 221 of human PrP C (Fig. 2), grouped with confidence in a separate cluster fish PrP1 ⁄ PrP2 (bootstrap con- fidence level ‡ 97%) from tetrapod PrPs and fish PrP3 ⁄ tetrapod Sho clusters, respectively. The duplicate PrP long forms inside the fish PrP1 ⁄ PrP2 cluster may have arisen during a putative whole-genome duplica- tion in ray-finned fish before the teleost radiation [31]. These proteins seem more closely related to tetrapod PrPs, as suggested by their deduced structural features and conserved amino acid sequences. Fish PrP3 and tetrapod Sho tend to be grouped in the same cluster (bootstrap confidence level ¼ 77%), but they fell into two separate groups. However, the phylogenetic rela- tionships of fish PrP1 ⁄ PrP2, tetrapod PrPs and fish PrP3 ⁄ tetrapod Sho clusters were not decisively resolved, while the gene tree deduced from the globular domain of tetrapod PrP sequences largely agrees with the species tree [11,32]. It should be noted that zebra- fish prp1 is in proximity to a homolog of human RASSF2 on chromosome 10 (GENSCAN00000055419, gb|AAH74035|), a genomic organization conserved between Prnp and RASSF2 at chromosome 20pter-p12 in the human genome. Zebrafish prp1 is around 8.5 kb from a rassf2 homolog in linkage group 10 (gb|BX640477|) and 3.5 kb from sprnb encoding the Sho2 protein, this last gene being inserted between rassf2 and prp1 [13]. Developmental expression pattern of zebrafish prp1 The developmental expression pattern of zebrafish prp1 was characterized from fertilization to 15 days postfertilization (dpf) by using whole-mount RNA in situ hybridization. A very strong hybridization sig- nal was first observed in the central nervous system (CNS) around 48 hours postfertilization (hpf) in an unpaired central structure running from midbrain to hindbrain along the bilateral symmetry axis. The labe- led specialized large and elongated cells of the anter- ior part of the floor-plate were positioned at the base of the commissure separating the two lobes of the mesencephalic tegmentum and above the hypothala- mus (Fig. 4A–E). Situated at the ventral-most part of the neural tube, the floor-plate is a specialized glial structure that controls the regional differentiation of neurons in the nervous system [33]. The prp1 hybrid- ization signal was no longer detected in the floor-plate after 3 dpf. Transcripts of prp1 started to be detected by 48 hpf in cranial ganglia including the trigeminal ganglia and their projections (Fig. 4A,B,D,E). The hybridization signal was maintained in the ganglia up to the larval stages (Fig. 4G–J,L) while an additional prp1 hybridization signal was detected on transverse sections around the cranial cavity by 8 dpf (Fig. 4K). Transcripts coding for PrP C have previously been detected in ganglia and nerves of both the central and peripheral nervous systems during chicken [22] and mouse [21] embryogenesis. The highly spatially restric- ted expression of zebrafish prp1 in the anterior floor- plate and peripheral nervous system could help to clarify the physiological function(s) of the correspond- ing protein that could be relevant to mammalian PrP C . Developmental expression pattern of zebrafish prp2 The embryonic and larval expression pattern of zebra- fish prp2, as evaluated using whole-mount RNA in situ hybridization, is shown in Figs 5 and 6. A high level of PrP2 mRNA was detected in embryonic cells from the mid-blastula transition to the end of the segmenta- tion period (Fig. 5A–D). No hybridization signal was detected in the yolk cell (Fig. 5A,B), the enveloping layer (Fig. 5A) or its derivative, the periderm (Fig. 5D), or in the yolk sac including the yolk syncy- tial layer (Fig. 5C–E,G). The prp2 hybridization signal was intense in the CNS during zebrafish embryonic and larval develop- ment (Fig. 5E,G–J,M,N,R). Starting from a diffuse staining before 24 hpf, sections of hybridized 48 hpf embryos and 8 dpf larvae confirmed that prp2 hybrid- ization signal was localized in areas of telencephalon, mesencephalon and rhombencephalon (Fig. 5H–J,N). The hybridization signal seemed to be prominent in the optic tectum and the rhombencephalon by 8 dpf (Fig. 5M) and was less visible in 15 dpf larvae (Fig. 5R). Mouse and chicken PrP-coding genes are expressed robustly and early in the CNS [21–23]. This suggests an early conserved developmental role of PrPs during brain morphogenesis in vertebrates. The extra- cellular position of PrP2, putatively attached to the cellular membrane by its GPI anchor, suggests that this protein, as human PrP C , might be involved in interactions between cells or with the extracellular mat- rix proteins that it might play a role in the differenti- ation of neurons during CNS development. Moreover, nerve growth factor (NGF) is strongly expressed in the developing mammalian CNS and is known to increase the level of mRNA encoding the prion protein [34,35]. It is important to note that common gene expression sites were found for NGF or brain-derived neuro- trophic factor, a neurotrophin related to NGF [36,37], and prp2 in embryonic nervous system, pectoral fins, and hair cells of the neuromasts (see below). Expression of prion genes in the developing zebrafish E. Cotto et al. 506 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS Fig. 4. Developmental expression pattern of zebrafish prp1 at 48 hpf (A–E), at 3 dpf (F–H), and at 8 dpf (I–L). The animals were raised in water containing PTU to prevent pigment formation. Whole-mount in situ hybridizations with digoxigenin-labeled specific riboprobes are shown on lateral (A,F,G,I), dorsal (B,J), or oblique views (H), with the head on the left. No hybridization signal was obtained with a sense RNA probe (F) (data not shown for other stages). Histological sections of 48 hpf embryos (C–E) and 8 dpf larvae (K,L) were obtained after whole-mount in situ hybridization. Section planes are indicated by dotted lines in A (C–E) and I (K,L). By 48 hpf, a high level of prp1 hybridiza- tion signal is detected in the specialized large and elongated cells (C, insert) of the anterior part of the floor-plate (fp) positioned at the basis of the commissure separating the two lobes of the mesencephalic tegmentum (t) and above the hypothalamus (hy) (A–E). The prp1 hybrid- ization signal is also detected from 48 hpf up to larval stages in cranial ganglia (g) and their projections (gp) (A–E,G–J,L). By 8 dpf, an addi- tional prp1 hybridization signal is detected on transverse sections around the cranial cavity (cc) (K). Notochord (n); optic tectum (ot); otic vesicle (ov); pharynx (p); trabecular cartilage (tc). Scale bars ¼ 100 lm. E. Cotto et al. Expression of prion genes in the developing zebrafish FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 507 Fig. 5. Developmental expression pattern of zebrafish prp2 (A) after the mid-blastula transition (3.5 hpf), (B) at 50%-epiboly (5 hpf), (C) during the segmentation period (15 hpf), (D) at the end of the segmentation period (20 hpf), (E) at 24 hpf, (F–K) at 48 hpf, (L–Q,S) at 8 dpf, and (R) at 15 dpf. Whole-mount in situ hybridizations with digoxigenin-labeled specific riboprobes are shown on lateral views (A–G,L,M,R). In later stages (C–G,L,M,R,S), the head is on the left side. No hybridization signal was obtained with a sense RNA probe (F,L) (data not shown for other stages). Histological sections of 48 hpf embryos (H–K) and 8 dpf larvae (N–Q,S) were obtained after whole-mount in situ hybridization. Section planes are indicated by dotted lines in G (H–K) and M (N–Q). From 3.5 to 48 hpf, the embryos were raised in water containing PTU to prevent pigment formation. (A–D) A high level of PrP2 mRNA is detected in blastomers (bl) and the embryo (em) from the mid-blastula transition to the end of the segmentation period. No hybridization signal is detected in the yolk cell (yc), the enveloping layer (evl) or its deriv- ative the periderm (p), or in the yolk sac (ys). From 24 hpf up to larval stages (E–S), prp2 transcripts are localized in distinct anatomical struc- tures including the pronephric tubules (pt) and ducts (pd), liver (l), heart (h), and intestinal epithelium (ie) (M, insert) of the posterior intestine (pi). In the CNS, the labeled divisions are the telencephalon (t), mesencephalon (m) including the optic tectum (ot), the rhombencephalon (rh), and the eye (e) at the retina level (r). Histological section of a neuromast (n) of the posterior lateral line system (S) detected prp2 tran- scripts in hair cells (hc), but not in supporting cells (sc). Scale bars ¼ 100 lm in A–R, and 5 lminS. Expression of prion genes in the developing zebrafish E. Cotto et al. 508 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS Eyes contained a significant prp2 hybridization signal in embryos raised in water containing 0.2 mm 1-phenyl-2-thio-urea (PTU) to prevent pigment forma- tion (Figs 5E,G and 6C). By 48 hpf, transverse sec- tions confirmed that prp2 hybridization signal was localized in the retina (Fig. 5H). Transcripts of prp2 were detected in lateral line neuromasts of the head region by 3 dpf (data not shown). By 6 dpf, all the neuromasts in both the anterior and posterior lateral line systems expressed prp2 and the hybridization sig- nal was high in 15 dpf larvae neuromasts (Fig. 5R). Transverse histological sections after whole-mount in situ hybridization of 8 dpf larvae demonstrated the presence of prp2 transcripts in mechanoreceptive sen- sory hair cells of the neuromast (Fig. 5S). No hybrid- ization signal could be detected in supporting cells located at the base of the neuromast and peripherally around the neuromast. A high level of prp2 transcripts was observed in kidney during zebrafish embryonic and larval development (Fig. 5E,G,I–K,M,N,P–R). By 24 hpf, pronephric tubules and ducts contained prp2 transcripts, with a more intense hybridization signal in tubules and at the end of the ducts (Fig. 5E). By 48 hpf, tubules and whole ducts were sites of increased prp2 expression (Fig. 5G) as confirmed on transverse sections (Fig. 5I–K). Pronephric tubules and ducts were then highly stained with prp2 antisense probe as demonstrated on transverse sections of 8 dpf larvae (Fig. 5N,P,Q). Information obtained by application of EST numbers to adult kidney cDNA database libraries predicted intense expression of prp2 in zebrafish adult kidney. The presence of prp2-specific hybridization signal was also observed in the developing heart (Figs 5H,M and 6A,C), in pectoral fins (Fig. 6A,C), and liver (Fig. 5M,O,R). A strong prp2 hybridization signal was detected by 8 dpf in enterocytes of the pos- terior part of the intestine (Fig. 5M,Q,R). This last finding should be of interest because in terms of func- tion, the fish intestine is highly regionalized both in the larva and in the adult [38]. The posterior segment of the fish intestine is the absorption site of intact pro- teins, which might thus escape intracellular degrada- tion [39]. In the mammalian gastrointestinal tract, PrP C has been detected in the enteric nervous system [40], in the gut-associated lymphoid system [41,42], and in epithelial cells lining the digestive tract lumen [43,44]. Considering the importance of the intestinal barrier in the process of oral prion infection, this find- ing might help clarify the entry and routing of PrP Sc in the early steps of infection. Our observations of a prp2 hybridization signal in the posterior intestine of zebra- fish larvae call for evaluation of the potential prions uptake of mammalian origin by the fish intestine. Comparison of zebrafish prp1, prp2, and prp3 developmental expression patterns The developmental expression patterns of prp1 and prp2 coding for long zebrafish PrPs were compared with prp3, a gene previously identified in the same species and coding for a short PrP [9]. While prp1 pre- sented a highly spatially restricted expression in the central and peripheral nervous systems, prp2 tran- scripts were found widely distributed within the CNS Fig. 6. Comparison between prp2 and prp3 developmental expression patterns in zebrafish. The expression of prp2 (A,C) and prp3 (B,D) is shown at 48 hpf (A,B) and 3 dpf (C,D). Lateral views, head on the left. Larvae (3 dpf) were raised in water containing PTU to prevent pig- ment formation. The prp2 gene is strongly expressed in the brain (b) (A,C), whereas prp3 hybridization signal is only faintly detected at 48 hpf (B). By 3 dpf, lateral line neuromasts (n) of the head region coexpressed prp2 and prp3 transcripts. The prp3 labeled neuromasts are clearly distinguishable (D) due to the absence of transcripts in the CNS. The heart (h) and the pronephric ducts (pd) contain prp2 transcripts at the two developmental stages (A,C). The prp3 hybridization signal is detected in heart at 48 hpf (B) and in the branchial arches (ba) at 3 dpf (D). Transcripts of prp2 and prp3 are detected in the central part of the pectoral fin (pf) and not in the surrounding epithelial cell layer (inserts in C,D). A stronger hybridization signal is observed in the pectoral fin with prp3. Scale bars ¼ 100 lm. E. Cotto et al. Expression of prion genes in the developing zebrafish FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 509 [...].. .Expression of prion genes in the developing zebrafish and in specific areas outside the CNS of the developing zebrafish The prp1 and prp2-expressing sites in the developing fish system could correlate with the time and sites of PrP1 and PrP2 action(s) during nervous system morphogenesis and also suggest that PrP2 has a pleiotropic role in the course of embryogenesis A high expression of zebrafish. .. demonstrated in the brain, kidney and posterior intestine and was similar to the intense expression demonstrated in the adult brain and peripheral tissues of Fugu prp1 and salmon stPrP homologous genes [10,11], whereas Fugu prp2 transcripts were not detected in the brain [10] In a similar way, numerous expression sites have been previously identified in chicken and mammals for PrP transcripts and proteins during... G & Heinrich G (2001) Brain-derived neurotrophic factor and TrkB tyrosine kinase receptor gene expression in zebrafish embryo and larva Int J Dev Neurosci 19, 569–587 ´ 38 Andre M, Ando S, Ballagny C, Durliat M, Poupard G, Briancon C & Babin PJ (2000) Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis Int J Dev Biol 44, 249–252... prp1 and mammalian Prnp However, differential developmental expression patterns observed among prp1, prp2, and prp3 are much in favour of a functional link between prp2 and tetrapod prion genes due to a widespread localization of both transcripts in the CNS and in peripheral organs Materials and methods Animals Adult zebrafish (Danio rerio) were purchased from local commercial sources Embryos and larvae... eye and patches of embryonic skin and scarcely detectable in the brain or in any other organ [9] In conclusion, the zebrafish genome contains three genes similar to human Prnp Conserved amino acid sequence and repeat motifs and deduced structural features suggest common functionalities among zebrafish PrP1 and PrP2 and mammalian PrPC The genomic context supports a more direct evolutionary link between zebrafish. .. were cloned in pGEM-T FEBS Journal 272 (2005) 500–513 ª 2004 FEBS Expression of prion genes in the developing zebrafish vector (Promega) and used as templates to generate the RNA probes Both antisense- and sense-digoxigenin-labeled RNA probes were obtained using T7 or SP6 RNA polymerase (Promega) and the digoxigenin RNA labeling mix (Roche Diagnostics, Meylan, France) following manufacturer’s instructions... 44, 249–252 39 Sire MF & Vernier JM (1992) Intestinal absorption of protein in teleost fish Comp Biochem Physiol 103A, 771–781 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS Expression of prion genes in the developing zebrafish 40 van Keulen LJ, Schreuder BE, Vromans ME, Langeveld JP & Smits MA (1999) Scrapie-associated prion protein in the gastrointestinal tract of sheep with natural scrapie J Comp Pathol... development [21–23] and in adult animals [19,45,46] As previously observed with prp2 transcripts, a significant level of prp3 hybridization staining was detected in embryonic cells before 24 hpf (data not shown) Differential expression profiles of prp2 and prp3 were exhibited during completion of rapid morphogenesis of primary organ systems During the hatching period, a high level of prp1 and prp2 transcripts... purple color in each figure corresponds to localization of transcript expression Embryos and larvae to be sectioned for light microscopy were postfixed after in situ hybridization, rinsed twice in PBS, twice in 70% ethanol and then embedded in Epon 812 After polymerization at 60 °C, sections were cut 2.5 lm thick with an Ultracut Reichert OM2 511 Expression of prion genes in the developing zebrafish Acknowledgements... PBS-T (10 min), 1 : 3 0.2· SSC-T ⁄ PBS-T (10 min), and PBS-T (2 · 20 min) They were then blocked in blocking solution (PBS-T, 2% sheep serum and 2 mgÆmL)1 BSA) for 1.5 h Embryos and larvae were incubated in preabsorbed sheep antidigoxigenin-AP Fab fragments at 1 : 4000 dilution in blocking solution for 3 h They were rinsed in PBS-T (6 · 15 min) and then in chromogenic buffer (100 mm Tris ⁄ HCl pH 9.5, . Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio) Emmanuelle. fish intestine. Comparison of zebrafish prp1, prp2, and prp3 developmental expression patterns The developmental expression patterns of prp1 and prp2 coding