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 diver
Trang 1Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes
in zebrafish (Danio rerio)
Emmanuelle Cotto1,2, Miche`le Andre´1, Jean Forgue1, Herve´ J Fleury2and Patrick J Babin1
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 (PrPSc) of the physiological protein (PrPC), 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, Baˆt 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; PrPSc, scrapie prion protein; Prnp, gene for mammalian PrPC; 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.
Trang 2depends 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
PrPC 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
PrPC have been identified in Fugu [9–11], Atlantic
sal-mon [10] and zebrafish (Danio rerio) [9] These include
duplicated protein long forms similar to PrPCin Fugu,
initially called PrP-461⁄ stPrP-1 and stPrP-2 [10,11] and
renamed in this study PrP1 and PrP2, respectively In
Fuguand 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 PrPC [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
PrPCmRNA expression sites need to be determined
to identify cells that are functionally dependent upon
synthesis of this protein In addition, infected cells
must express PrPC 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 PrPSc 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 PrPCare 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
Trang 3Gly-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 PrPC, the two b-sheets and three long a-helices characteristic
of the prion protein This region is the PrPC 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 PrPC (Fig 2) Hydrophobic cluster analysis [27] predicted the presence of several conserved hydrophobic clusters throughout the com-pared zebrafish PrP2 and human PrPCsequences This type of similarity is typical of distant but related sequences In the N-terminal part analogous to the human PrPC 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 PrPC, 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.
Trang 4PrPCand PrPSc[29,30], helix H2 putatively involved in
the structural conversion to PrPSc [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 PrPC, 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 PrPC 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, PrPC 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.
Trang 57 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 PrPC, 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.
Trang 6salmon 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
PrPC have been identified in Fugu [9–11], Atlantic
sal-mon [10] and zebrafish (Danio rerio) [9] These include
duplicated protein long forms similar to PrPCin 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 PrPC), 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 PrPCcan be demonstrated with Pro102, Ala117, and Gly131 variants of human PrPC A substitution of one of these residues in human PrPCby 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 PrPC 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 PrPChelices 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, computer-derived from alignments encompassing amino acid residues 101–157 and 101–
Trang 7221 of human PrPC(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 radiaduplica-tion [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 level¼ 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
RASSF2on 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
rassf2and 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 PrPC 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 PrPC
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 hybridizasegmenta-tion 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 PrPC, 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)
Trang 8Fig 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) posihybridiza-tioned 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.
Trang 9Fig 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 lm in S.
Trang 10Eyes 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, PrPC 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 routfind-ing of PrPScin 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.