Cellular retinol-binding protein type II (CRBPII) in adult zebrafish
(
Danio rerio
)
cDNA sequence, tissue-specific expression and gene linkage analysis
Marianne C. Cameron
1
, Eileen M. Denovan-Wright
2
, Mukesh K. Sharma
1
and Jonathan M. Wright
1
1
Department of Biology, and
2
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
We have determined the nucleotide sequence of a zebrafish
cDNA clone that codes for a cellular retinol-binding protein
type II (CRBPII). Radiation hybrid mapping revealed that
the zebrafish and human CRBPII genes are located in
syntenic groups. In situ hybridization and emulsion autora-
diography localized the CRBPII mRNA to the intestine and
the liver of adult zebrafish. CRBPII and intestinal fatty acid
binding protein (I-FABP) mRNA was colocalized to the
same regions along the anterior-posterior gradient of the
zebrafish intestine. Similarly, CRBPII and I-FABP mRNA
are colocalized in mammalian and chicken intestine.
CRBPII mRNA, but not I-FABP mRNA, was detected in
adult zebrafish liver which is in contrast to mammals where
liver CRBPII mRNA levels are high during development but

Their putative role in cell physiology is in the metabolism of
retinol (vitamin A). Retinol and its derivates are important
for vision, reproduction, metabolism, cellular differentiation
and pattern formation during embryogenesis [4]. After
absorption in the mammalian intestine, the enzyme
b-carotene dioxygenase catalyzes the oxidative cleavage of
b-carotene to retinal. Retinal is reduced to retinol by the
enzyme retinal dehydrogenase. Retinol is then esterified by
the microsomal enzyme lecithin:retinal acyltransferase
(LRAT) to retinoic acid and packaged into chylomicrons
for subsequent uptake by the liver. CRBPI and II bind
retinal and retinol whereas CRBPIII binds retinol, but not
retinal [4–7]. CRABPs bind and transport retinoic acid
[4,5,7]. The CRBPs are thought to participate in ligand
binding and regulate the metabolism of retinal and retinol
while protecting the CRBP-bound ligands from nonspecific
reactions [8]. Biochemical studies have shown that CRBPII-
bound retinol serves as a substrate for the enzyme, LRAT,
implicating CRBPII as a component in directing and
channeling dietary retinol to nascent chylomicroms. Direct
evidence for the role of CRBPI in vitamin A metabolism has
been provided by transgenic knockout studies which
demonstrated that CRBPI knockout mice are phenotypi-
cally normal when fed a vitamin A-enriched diet, but when
the diet is deficient in vitamin A, stores of retinyl esters are
depleted over 5 months and the mice develop abnormalities
consistent with postnatal hypovitaminosis A [9]. To date,
there are no reports of CRBPII and CRBPIII gene
knockouts and therefore direct evidence for their function
in vitamin A metabolism remains speculative. CRBPII is

one of the duplicated copies of the CRBP gene may have
been lost in the amphibian lineage.
The structure and function of fatty acid and retinoid-
binding proteins have been studied extensively in mammals,
but only superficially in other taxa such as the teleost fishes.
Vitamin A and its derivates are clearly important mediators
of normal vertebrate development [4,8,9]. As zebrafish is
promoted as a model experimental system for study of
vertebrate development, an understanding of the function of
CRBPs in vitamin A metabolism during zebrafish embryo-
genesis would be of interest to developmental biologists.
Moreover, comparative studies of CRBP gene expression in
fishes and mammals may provide insight into the role(s) of
these intracellular retinol- and retinal-binding proteins in
vitamin A metabolism. As part of ongoing studies in our
laboratories on the evolution, tissue-specific expression and
gene regulation of members of the intracellular lipid-binding
protein family in zebrafish [18–20], we have determined the
nucleotide sequence of a cDNA clone and deduced the
amino-acid sequence for a zebrafish CRBPII. Furthermore,
we report the tissue-specific distribution of the CRBPII
mRNA in adult zebrafish and assignment of the CRBPII
gene to linkage group 15 in the zebrafish genome.
MATERIALS AND METHODS
Searches of the zebrafish EST database in GenBank
identified a cDNA clone (GenBank accession number
AI544932) that was similar to the 5¢ end of the rat cellular
retinol-binding protein type II. This clone (fb69e02.y1) was
purchased from Incyte Genomics Inc. and the complete
nucleotide sequence was determined [18]. The deduced

4686 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002
dNTPs, 1.25 U Taq DNA polymerase and 100 ng of hybrid
cell DNA. Control reactions contained 100 ng of either
zebrafish or mouse parental cell line DNA or a 1 : 10
mixture of zebrafish and mouse parental cell line DNA.
PCR conditions were 94 °C for 4 min followed by 32 cycles
of 94 °Cfor30s,60°Cfor30s,72°C for 30 s and a final
extension at 72 °C for 7 min Products were separated by
agarose gel electrophoresis and the radiation hybrid panel
was scored and then analyzed according to the directions at
(:8000/zfrh/beta.cgi).
PCR primers (5¢-CCAGCACATCCAGCTTC-3¢)and
(5¢-GCCTGTTTGGAGCATTAG-3¢) (see Fig. 1 for pri-
mer location) were used to amplify a 442-bp product from
DNA of clone fb69e02.y1. This product was used as a
hybridization probe for Northern blot analysis [23]. The size
of the hybridizing mRNA was determined by comparing its
electrophoretic mobility with molecular mass markers
(0.24–9.5 kB RNA ladder, Gibco BRL).
In situ hybridization was performed using an antisense
oligonucleotide probe (see Fig. 1) to determine the pattern
of CRBPII expression in adult zebrafish. Based on
BLASTN
searches of GenBank, the in situ hybridization probe did
not exhibit significant sequence similarity to any other
DNA sequence currently available in GenBank. Fourteen
micrometer transverse, sagittal, and coronal sections of
adult zebrafish were hybridized to DNA probes using
previously described methods [24]. Following hybridization
and post-hybridization washes, the sections were exposed

other species indicate that the cDNA clone codes for the
zebrafish CRBPII (Fig. 2). The zebrafish CRBPII protein
was one amino acid longer than mammalian CRBPII and
equal in length to chicken CRBPII. The molecular mass of
the CRBPII protein in zebrafish, based on the predicted
amino-acid sequence, is 15.8 kDa. The molecular mass of
this zebrafish CRBPII is comparable to other members of
the intracellular lipid-binding protein family which are all
between 14 and 16 kDa [1,2]. The zebrafish CRBPII amino-
acid sequence is most similar to the chicken CRBPII (76%
identity). The zebrafish CRBPII amino-acid sequence
exhibits 73–75% sequence similarity to mammalian
CRBPIIs and less than 40% amino-acid sequence identity
to other intracellular lipid-binding proteins.
Cheng et al. [25] proposed that Arg106 and Arg126
present in some members of the lipid-binding protein family
correspond to Gln109 and Gln129 in CRBPI and CRBPII.
While all FABPs and CRBPs studied to date have the same
tertiary structure, the amino-acid residues at positions 109
and 129 may determine ligand-binding specificity. Both Gln
residues are found in the zebrafish CRBPII sequence at the
comparable positions within the amino-acid alignment of
other intracellular lipid-binding proteins (Fig. 2). Gln109 is
not strictly conserved in all CRBPs, however, as the chicken
CRBPII and mouse CRBPIII have a histidine residue at this
position.
Phylogenetic analyses of 51 intracellular lipid-binding
proteins, from vertebrates and invertebrates, indicate that at
least 14 gene duplications have occurred during the
evolution of this multigene family [16]. As the amino-acid

common ancestral CRBPII gene.
Northern blot-hybridization of the zebrafish CRBPII
cDNA to total RNA extracted from whole adult zebrafish
detected a single mRNA transcript of  720 nucleotides
(Fig. 3A). The difference in size between the mRNA
transcript detected by Northern blot ( 720 nucleotides)
Ó FEBS 2002 Expression of CRBPII in zebrafish (Eur. J. Biochem. 269) 4687
and the size of the cDNA sequence (549 nucleotides) suggests
that the cDNA clone is probably lacking the complete poly A
tail or part of the 5¢ untranslated region, or both.
In situ hybridization analysis of adult zebrafish tissue
sections revealed that the hybridization signal resulting from
the specific annealing of the CRBPII antisense probe was
confined to the intestine and, at relatively lower levels, to the
zebrafish liver (Fig. 3B,C). Hybridization of the CRBPII-
specific probe to the intestine is most evident in the
transverse (Fig. 3B) and coronal (Fig. 3C) sections while
hybridization to the liver is more clearly seen in the coronal
sections (Fig. 3C). The radiolabel associated with the layer
beneath the external skin appears to be non–specific
interaction of the probe with this tissue as it is seen in all
autoradiograms regardless of the hybridization probe
employed, i.e. the CRBPII or I-FABP antisense probes or
the I-FABP negative control sense probe (Figs 3B,C). The
hybridization signal resulting from the specific annealing of
the I-FABP antisense probe was confined to the intestine as
previously reported [18].
As CRBPII and I-FABP mRNA have been colocalized in
the mammalian and chicken proximal portion of the small
intestine [27–29], we examined the distribution of CRBPII

Position Gene
Chromosomal
location
15 gap43 L27645 GAP43 3q13.1-q13.2 Gap43 16 29.5 cM
15 rbp2 (CRBPII) AF363957 RBP2 3q23 Rbp2 9 57.0 cM
15 chd AF034606 CHRD 3q27 Chrd 16 14.0 cM
a
Wood et al. [45];
b
LocusLink ( NCBI.
4688 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002
liver (Fig. 4A,B) [18]. In the transverse sections, the
positional difference of CRBPII mRNA demarcates the
anterior and posterior parts of the intestine (Fig. 4A). In
adjacent sections, the distribution of I-FABP-specific
hybridization signal was the same as that observed for
CRBPII (Fig. 4A). Due to the entwined positioning of the
intestine within the zebrafish, the coronal sections display
three cross-sections corresponding to different regions of the
intestine (Fig. 4B). There was hybridization to only two of
the three cross sections of the intestine for the CRBPII
antisense and I-FABP antisense probes. The radiolabel
associated with the centre of the intestine is present in all
sections labeled with antisense and sense oligonucleotides
indicating nonspecific binding of the probes to the contents
of the gut. Evidence of I-FABP mRNA restricted to just the
anterior of the zebrafish intestine was not previously
observed by us possibly owing to the limited sections
assayed [18]. It is possible that subtle differences in the
amount of CRBPII and I-FABP mRNA exist along the

and I-FABP expression is similar along the anterior-
posterior axis in the intestine of mammals. Therefore, a
corresponding trend for CRBPII and I-FABP expression in
zebrafish is consistent with their expression in mammals.
The abrupt termination of CRBPII expression along the
anterior-posterior axis of the zebrafish intestine, however,
contrasts with the gradual decrease in CRBPII and I-FABP
expression pattern in mammals.
In the 5¢-control regions of the mammalian CRBPII [39]
and I-FABP [35] genes, a closely related cis-element that
consists of nearly perfect tandem repeats, termed retinoid x
response element (RXRE) [40] has been found. It is
conceivable therefore that these two genes may both be
regulated by the action of retinoid x receptor (RXR)
binding to RXRE [41]. The similar distribution of CRBPII
and I-FABP mRNA in the zebrafish intestine reported here
may reflect the co-ordinate regulation of these genes by
common intestinal transcriptional factors in zebrafish.
In addition to being abundant in intestine, CRBPII is
found in neonatal liver hepatocytes of the rat and chick
[10,42]. In rat, however, the levels of CRBPII mRNA in the
Fig. 3. CRBPII mRNA expression in the adult zebrafish. The complete
coding sequence of the zebrafish CRBPII cDNA clone was amplified
by PCR and used as a hybridization probe in Northern blot analysis of
total cellular RNA isolated from adult zebrafish (A). The zebrafish
CRBPII-specific probe hybridized to a transcript of approximately 720
nucleotides. The1.35 kb (upper line) and 0.24 kb (lower line) RNA
molecular mass markers are shown on the left of the panel. In situ
hybridization analysis was performed using a 3¢ end-labelled oligo-
nucleotide complementary to an internal portion of the zebrafish

compared to the levels observed in humans [44]. These
findings suggest that hepatic CRBPII may play a role in
metabolizing hepatic b-carotene to retinal and the subse-
quent esterification of the converted retinol only during the
perinatal period in mammals [42]. The in situ hybridization
and autoradiographic emulsion studies show that CRBPII
mRNA is abundant in the liver of adult zebrafish (Fig. 4).
This pattern of CRBPII expression therefore differs mark-
edly from that observed in rat and chicken [10,42]. Retinol
metabolism of fishes may differ from that of mammals and
chicken in that large amounts of b-carotene continue to be
transported to the adult liver of teleost fishes resulting in the
need for high levels of CRBPII mRNA observed in the liver
of adult zebrafish.
CRBPII and I-FABP mRNA are colocalized in the fish
and mammalian intestine and may be co-ordinately regu-
lated by RXR acting at RXRE within the control regions of
these genes. The differential expression of CRBPII and
I-FABP in the adult zebrafish liver, however, suggests that
other transcription factors may regulate CRBPII gene
expression in the livers of adult zebrafish.
In summary, the zebrafish CRBPII cDNA reported here
has sequence similarity to CRBPs isolated from mammals.
The patterns of gene expression for CRBPII and I-FABP in
fishes and mammals suggest that there is co-ordinate
regulation of these genes in the intestine, but not in the
liver. This may reflect differences in retinol metabolism
between adult teleost fishes and mammals.
ACKNOWLEDGEMENTS
We wish to thank Dr Marc Ekker for providing DNA samples from the

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