Tài liệu Báo cáo khoa học: Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio) doc

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:
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 ( />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
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 prp2 has been demonstra-
ted 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 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 mor-
phogenesis of primary organ systems. During the
hatching period, a high level of prp1 and prp2 tran-
scripts were observed in the CNS (Figs 4A–E and
6A,C) while very little prp3 transcript labeling persis-
ted at 48 hpf (Fig. 6B) and no staining signal was
detected in the developing brain at 3 dpf (Fig. 6D).
The hybridization signal for prp3 was detected in heart
(Fig. 6B) as was that of prp2 (Fig. 6A,C), but prp3
transcripts were no longer detected by 3 dpf (Fig. 6D).
Transcripts of prp2 and prp3 were detected starting at
the onset of pectoral fin buds outgrowth and became
restricted to the central part of the fin (Fig. 6C,D),
with a predominance of prp3 transcripts (Fig. 6D).
PrP2 mRNA expression was restricted to two mesen-
chymal central regions parallel to the plane of the
pectoral fin (Fig. 6C, insert). No hybridization signal
was present between them, in the central portion of
the fin, or in the epithelial cell layer surrounding the
fin (Fig. 6C, insert). Lateral line neuromasts of the
head region coexpressed prp2 and prp3 transcripts by

3 dpf. The prp3 labeled neuromasts were clearly distin-
guishable due to the absence of transcripts in the CNS
(Fig. 6D). By 6 dpf, all mature primary neuromasts in
both the anterior and posterior lateral line systems
expressed both prp2 and prp3 transcripts. By 3 dpf, a
specific prp3 signal appeared in the branchial arches
(Fig. 6D). The prp3 expression profile persisted up to
the larval stages and, unlike prp2,noprp3 signal could
be detected by whole-mount in situ hybridization in
kidney, liver, and posterior intestine during embryonic
and larval development. Zebrafish prp3 expression
profile lacks one of the characteristic key features of
tetrapod PrPs, i.e. intense expression in the CNS. Fugu
prp3 transcript has been observed to be confined to
the 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 fea-
tures suggest common functionalities among zebrafish
PrP1 and PrP2 and mammalian PrP
C
. The genomic
context supports a more direct evolutionary link
between zebrafish prp1 and mammalian Prnp. How-
ever, 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 were obtained by
natural mating and raised at 28.5 °C, as described previ-
ously (http://zfin.org/zf_info/zfbook/cont.html). In some
cases, embryo and larvae were raised in water containing
0.15 m m PTU to prevent pigment formation. When used,
this treatment did not affect the developmental expression
patterns of the investigated genes. Developmental stages
were recorded as hours or days postfertilization as des-
cribed previously [47].
Sequence sources and analyses
Accession numbers of tetrapod PrP sequences extracted
from the Swiss-Prot (sp.) or GenBank (gb) databases are
sp.|P04156|, sp.|P27177|, sp.|Q9I9C0|, sp.|Q8QFR0|, and
sp.|Q801J8| for human, chicken (Gallus gallus), turtle (Tra-
chemys scripta), Xenopus (Xenopus laevis) and salmon
(Salmo salar), prion protein precursors, respectively;
sp.|Q801J9| ⁄ sp.|Q8AX89|, sp.|Q800Z8|, and sp.|Q8JIJ1| for
Fugu (Fugu rubripes) PrP1, PrP2, and PrP3, respectively;
gb|AJ850286|, gb|AJ620614|, and sp.|Q7T2P9| for zebrafish
(Danio rerio) PrP1, PrP2, and PrP3, respectively. Accession
numbers of PrP related sequences are sp.|Q9UKY0| for
human prion-like doppel precursor; sp.|Q70YK5| and
gb|BN000523| for zebrafish Shadoo protein and protein 2
precursors, respectively; gb|NP_612393| for human Shadoo
protein precursor.
Multiple alignments were performed with the clustal w
program at followed by manual

adjustments taking into account the results obtained using
the hydrophobic cluster analysis method at http://
smi.snv.jussieu.fr/hca/hca-form.html [27]. EST and genomic
Expression of prion genes in the developing zebrafish E. Cotto et al.
510 FEBS Journal 272 (2005) 500–513 ª 2004 FEBS
databases were screened for potential homologs to tetrapod
PrPs at and http://
www.sanger.ac.uk/. The signal peptide sequence was predic-
ted by using the prediction tool signalp v1.1 at http://
www.cbs.dtu.dk/services/SignalP/ [48]. The putative N-gly-
cosylation sites were analyzed at />services/NetNGlyc/, putative GPI-anchor-sites at http://
129.194.185.165/dgpi/index_en.html, and hydrophobic
regions were identified at />protscale.html with a window size of nine residues [49]. A
model of the repetitive motifs was created using http://
www.expasy.org/prosite/. The predicted secondary and ter-
tiary structures of the C-terminal regions were produced
using the SWISS-PDB viewer at />swissmod/SWISS-MODEL.html. The phylogenetic analysis
was performed using the mega 2 package programs [50].
Tree and branch lengths were obtained by the neighbor-
joining algorithm based on the number of substitutions per
site (Poisson correction distance method, pair-wise-deletion
option for gap sites). Reliability of the neighbor-joining tree
topology was evaluated by bootstrap analysis [51] with
5000 replications.
RNA probes
Reverse-transcriptions were performed with 1 lg of total
RNAs extracted from zebrafish adult brain tissue using an
RNA Wiz extraction kit (Ambion, Austin, TX, USA),
M-MLV Reverse Transcriptase RNase H Minus (Promega,
Charbonnie

`
res les Bains, France), 500 ngÆlL
)1
oligo-dT, and
500 ngÆlL
)1
hexamer random primers (Promega) following
the manufacturer’s instructions. A first primer pair (sense
oligonucleotide ZFC1, 5¢-CAGATTCTCAGTCCACGA-3¢
and antisense oligonucleotide ZFC2 5¢-GCTCACTGTT
TCCTCATCC-3¢) was used to amplify a 606 bp PrP1 cDNA
fragment from nucleotides 1003–1608 (numbered from the
translation initiator codon) in the ORF of prp1 transcript
(accession number gb|AJ850286|). A second primer pair
(sense oligonucleotide ZFA2, 5¢-AGTCAGTGCAGGGCA
TGG3¢ and antisense oligonucleotide ZFA1 5¢ -GCTCA
ACACAACGTCGAGC-3¢) was used to amplify a 696 bp
PrP2 cDNA fragment from nucleotides 839–1534 in the
ORF of prp2 transcript (accession number gb|AJ620614|). A
third primer pair (sense oligonucleotide ZFB1 5¢-AGG
CAGCAGCTAGGGTC-3¢ and antisense oligodeonucleotide
ZFB2 5¢-GTCCAAACACTGTCACGC-3¢) was used to
amplify a 401 bp PrP3 cDNA fragment designed from nucle-
otides 129–529 in the ORF of prp3 transcript (accession
number gb|AJ490524|). These primers were used at 0.5 lM
with 4 lL cDNA, 5 units of Taq DNA polymerase (Prome-
ga), 2.5 mM MgCl
2
, and 2.5 mM dNTP to amplify the
cDNA fragments. The PCR profile contained one denaturing

step at 94 °C for 4 min followed by 30 cycles at 94 °C for
30 s, 56 °C for 30 s, 72 °C for 30 s and one cycle at 72 °C
for 3 min. Both cDNA fragments were cloned in pGEM-T
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. RNA probes were purified using the RNA puri-
fication Nucleospin RNA II kit (Macherey-Nagel, Du
¨
ren,
Germany) and checked for purity by denaturing agarose gel
electrophoresis. Sense probes were synthesized as a control
and did not give any staining after whole-mount in situ
hybridization.
Whole-mount in situ hybridization and sectioning
Embryos and larvae were fixed overnight in 4% parafor-
maldehyde at 4 °C, rinsed twice in phosphate-buffered sal-
ine (PBS: 137 mm NaCl, 2.7 mm KCl, 0.02 m PO
4
),
transferred into 100% methanol and stored at )20 °C. For
in situ hybridization, embryos and larvae were progres-
sively transferred into PBS then PBS-T (PBS plus 0.1%
Tween-20), and digested by 10–20 lgÆmL
)1
proteinase K in
PBS-T for 30 s to 35 min depending on the stage. They
were postfixed in 4% paraformaldehyde and rinsed for

5 · 5 min in PBS-T. Prehybridization was performed in
hybridization buffer plus (HBP) [65% formamide, 5·
sodium-salt citrate (SSC: 0.75 m NaCl, 0.075 m Na
3
cit-
rate.2H
2
O), 100 lgÆmL
)1
heparin, 500 lgÆmL
)1
tRNA,
pH 6.5] for 2 h at 67 °C. After the probes were denatured
for 10 min at 65 °C and added to prewarmed HBP,
embryos and larvae were incubated overnight at 67 °C.
They were rinsed for 5 min in HB (65% formamide,
5· SSC, pH 6.5), and washed at 67 °Cin3:1(v⁄ v)
HB ⁄ 2· SSC ⁄ 0.1% Tween (SSC-T) (10 min), 1 : 1 HB ⁄ 2·
SSC-T (10 min), 1 : 3 HB ⁄ 2· SSC-T (10 min), 2· SSC-T
(20 min), 1 : 1 formamide ⁄ 2· SSC-T (20 min), 2· SSC-T
(2 · 20 min), 0.2· SSC-T (30 min) at 55 °C and once in
0.2· SSC-T at room temperature. They were transferred
into 3 : 1 0.2· SSC-T ⁄ PBS-T (10 min), 1 : 1 0.2· SSC-
T ⁄ 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 preab-
sorbed 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, 100 mm NaCl, 50 mm
MgCl
2
). BM purple (Roche) supplemented with 1 mm
levamisol was used as substrate. The dark blue to purple
color in each figure corresponds to localization of tran-
script 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.
E. Cotto et al. Expression of prion genes in the developing zebrafish
FEBS Journal 272 (2005) 500–513 ª 2004 FEBS 511
Acknowledgements
This manuscript is dedicated to Dominique Dormont.
We thank Frank Bourrat and Mario Wullimann for
their advices. This study was carried out with financial
support from the GIS ‘Infections a
`
Prions’ from the
French Ministry of Research and Education, the Con-
seil Re
´
gional d’Aquitaine and the Fonds Europe
´
en de
De

´
veloppement Re
´
gional (FEDER).
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