Báo cáo khoa học: The evolutionary relationship between the duplicated copies of the zebrafish fabp11 gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes pptx

The evolutionary relationship between the duplicated
copies of the zebrafish fabp11 gene and the tetrapod
FABP4, FABP5, FABP8 and FABP9 genes
Santhosh Karanth
1
, Eileen M. Denovan-Wright
2
, Christine Thisse
3
, Bernard Thisse
3
and Jonathan
M. Wright
1
1 Department of Biology, Dalhousie University, Halifax, Canada
2 Department of Pharmacology, Dalhousie University, Halifax, Canada
3 Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, VA, USA
The multigene family coding for vertebrate intra-
cellular lipid-binding proteins (iLBPs) consists of the
fatty acid-binding protein (FABP), cellular retinoic
acid-binding protein (CRABP) and cellular retinol-
binding protein (CRBP) genes. FABPs bind selectively
to fatty acids, CRABPs bind to retinoic acid, and
CRBPs bind to retinol [1]. For many iLBPs, the
precise physiological function(s) is not completely
understood or remains unknown. However, it is clear
that iLBPs are involved in cellular uptake and intracel-
lular transport of long-chain fatty acids, bile salts and
retinoids, protection of cellular structures from the
detergent effects of fatty acids by sequestering them
until required in various metabolic processes, interaction

Keywords
Fabp11; retinal development; spinal cord;
tandem gene duplication; whole genome
duplication
Correspondence
J. M. Wright, Department of Biology,
Dalhousie University, Halifax, NS, Canada
B3H 4J1
Fax: +1 902 494 3736
Tel: +1 902 494 6468
E-mail:
Website: />(Received 23 January 2008, revised 8 March
2008, accepted 9 April 2008)
doi:10.1111/j.1742-4658.2008.06455.x
We describe the structure of a fatty acid-binding protein 11 (fabp11b) gene
and its tissue-specific expression in zebrafish. The 3.4 kb zebrafish fabp11b
is the paralog of the previously described zebrafish fabp11a, with a deduced
amino acid sequence for Fabp11B exhibiting 65% identity with that of
Fabp11A. Whole mount in situ hybridization of a riboprobe to embryos
and larvae showed that zebrafish fabp11b transcripts were restricted solely
to the retina and were first detected at 24 h postfertilization. In situ hybrid-
ization revealed fabp11b transcripts along the spinal cord in adult zebrafish.
However, the highly sensitive RT-PCR assay detected fabp11b transcripts
in the brain, heart, ovary and eye in adult tissues. By contrast, fabp11a
transcripts had been previously detected in the liver, brain, heart, testis,
muscle, ovary and skin of adult zebrafish. Using the LN54 radiation hybrid
panel, we assigned zebrafish fabp11b to linkage group 16. Phylogenetic
analysis and conserved gene synteny with tetrapod genes indicated that the
emergence of two copies of fabp11 in the zebrafish genome may have
resulted from a fish-specific whole genome duplication event. Furthermore,

we propose that the FABP4–FABP5–FABP8–FABP9 (PERF15) gene
cluster on a single chromosome in the tetrapod genome and the fabp11
genes in the zebrafish genome originated from a common ancestral gene,
which, following their divergence, gave rise to the fabp11 genes of
zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9
genes in tetrapods after the separation of the fish and tetrapod lineages.
Abbreviations
CRABP, cellular retinoic acid-binding protein; CRBP, cellular retinol-binding protein; EST, expressed sequence tag; FABP, fatty acid-binding
protein; hpf, hours postfertilization; iLBP, intracellular lipid-binding protein; LG, linkage group; mya, million years ago; WGD, whole genome
duplication.
FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS 3031
with other enzymes and transport systems, and the
transcriptional regulation of specific genes [2–6]. By
functioning in the transport and metabolism of retinol
and retinoic acid, CRBPs and CRABPs may play an
important role in development, growth and reproduc-
tion, primarily by making retinoids available to recep-
tors in the nucleus to regulate specific gene
transcription [7]. Originally, FABPs and their genes
were named on the basis of the tissue in which they
were first isolated. Later, Hertzel & Bernlohr [8] pro-
posed a different nomenclature, in which FABPs are
numbered according to the temporal order of their
identification (e.g. fabp1 and fabp2). We chose, for the
sake of clarity, to use here the nomenclature adopted
by Hertzel & Bernlohr [8].
Thus far, iLBPs have only been found in species
from the animal kingdom, suggesting that a single
ancestral iLBP gene emerged in animals after their
divergence from plants and fungi [9]. The diversity of

the iLBP multigene family is thought to have arisen
through a series of gene duplication events followed by
their sequence divergence [10]. Estimates of the earliest
iLBP gene duplication vary between 930 and 1000
million years ago (mya) [9–11]. Schaap et al. [9] and
Schleicher et al. [10] have suggested that 600–700 mya,
before the invertebrate and vertebrate lineages split,
the gene(s) that would give rise to the FABP1–
FABP2–FABP6 clade had already diverged from the
gene(s) that would give rise to the FABP4–FABP8
clade. CRBPs and CRABPs, which are absent in inver-
tebrates, might have diverged from the FABP1 clade
after the vertebrates and invertebrates split. To date,
paralogs of 11 genes coding for FABPs have been
described in vertebrates [12].
Zebrafish have attracted the attention of evolu-
tionary molecular biologists partly because of the
abundance of genetic and biological resources for this
model organism for developmental studies, but partic-
ularly for the whole genome duplication (WGD) event
that occurred in ray-finned fishes some 250–400 mya
[13], which has led to investigations on the genesis and
fate of duplicated genes. Gene duplication has been
proposed by Ohno [14] as a major evolutionary force
in driving the increasing complexity of life. In addition
to WGD, tandem duplication of individual genes by
the process of unequal crossing-over during meiosis
may also account for the increase in the number of
genes in eukaryotes [15].
Vayda et al. [16] described a fabp gene from four

Antarctic fishes, termed H
ad
-FABP, which they suggest
is the ortholog of mammalian FABP4. In a previous
communication [17], we described an fabp4 gene from
zebrafish that showed greatest sequence identity to,
and formed a clade in phylogenetic analyses with, the
Antarctic fish H
ad
-FABP. Recently, however, Agulleiro
et al. [12] recognized that the Fabps reported by Vayda
et al. [16] and by us [17] constitute a new FABP clade
that is probably restricted to fishes, and renamed the
novel fish gene and protein fabp11 and Fabp11, respec-
tively [12]. In this article, we report the charac-
terization of a duplicated Fabp11 gene (fabp11b) from
the zebrafish genome, the distribution of fabp11b
mRNA transcripts in adult tissues and during embry-
onic and larval development, and linkage group (LG)
(chromosome) assignment of fabp11b by radiation
hybrid mapping. On the basis of phylogenetic analysis
and conserved gene syntenies of zebrafish fabp11a and
fabp11b with other vertebrate FABP genes, we propose
that the duplicated fabp11 genes in fishes and the
FABP4–FABP5–FABP8–FABP9 gene cluster in tetra-
pods arose from a single progenitor gene.
Results and Discussion
Identification of a duplicated fabp11 gene from
zebrafish
A paralogous gene to the previously described zebra-

fish fabp11 (fabp4, but now referred to as fabp11a) [17]
was identified from a blast search of the zebrafish
genome sequence database at the Wellcome Trust
Sanger Institute (version Zv6, />Danio_rerio/index.html), using as query the GenBank
sequence AY628221. The 3.4 kb duplicated fabp11
(hereafter referred to as fabp11b) consists of four exons
and three introns (Fig. 1), a FABP gene organization
seen for most vertebrate iLBP genes [1]. The four
exons of fabp11b code for 24, 59, 34 and 17 amino
acids, respectively (Fig. 1), identical to what is seen for
zebrafish fabp11a [17]. For fabp11b, the splice junc-
tions for intron 1 and intron 2 conform to the GT ⁄ AG
intron ⁄ exon rule [18], whereas the splice junction for
intron 3 is TA ⁄ AG. A putative binding site for a
GATA transcription factor was identified at position
)471 to )483 in the 5¢-upstream region of fabp11b
(Fig. 1). Divine et al. [19] reported that GATA-4,
GATA-5 and GATA-6 act cooperatively in activating
FABP1 transcription in the murine small intestine. A
putative TATA box is located at position )30 to )24.
Distribution of fabp11b transcripts in the retina
of developing zebrafish embryo and larvae
To determine the spatial and temporal distribution of
fabp11b transcripts during zebrafish embryonic and
larval development, we conducted whole mount in situ
Duplicated fabp11 genes of zebrafish S. Karanth et al.
3032 FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS
hybridization to zebrafish embryos and larvae at
different developmental stages (Fig. 2). fabp11b tran-
scripts were first detected in the retina of developing

embryos at 24 h postfertilization (hpf) in the pig-
mented epithelium, starting in the proximal part of the
retina. fabp11b transcripts spread across this layer of
the retina by 30 hpf (Fig. 2B). A homogeneous distri-
bution of fabp11b transcripts throughout the pig-
mented epithelium was observed at 48 hpf (Fig. 2C).
In contrast to fabp11b, at 24 hpf and 36 hpf the
fabp11a transcripts were detected in the lens and
diencephalon [17]. In 5-day-old larvae, the hybridiza-
tion signal for fabp11b transcripts was still observed in
the same layer of the retina (Fig. 2D). Sellner [20]
reported a similar trend, whereby retinal FABP
appears to be maximally expressed in the chicken ret-
ina around the ninth day of embryonic development
and declines at later stages. Embryonic development in
zebrafish spans 3 days, whereas embryonic develop-
ment in chicken extends over 20–21 days. Two other
FABP transcripts, transcripts for fabp3 and fabp7b,
have been detected in the developing retina of zebra-
fish embryos [17,21]. It has been proposed that FABPs
are involved in sequestering of fatty acids during
retinal differentiation [20].
Tissue-specific distribution of fabp11b transcripts
in adult zebrafish
In situ hybridization was performed on sections of
adult zebrafish to determine the tissue-specific distribu-
tion of fabp11b transcripts. fabp11b transcripts were
detected along the vertebra in the spinal cord of adult
zebrafish (Fig. 3A shows the hybridization signal in a
sagittal section, and Fig. 3B a transverse section).

While describing the distribution of FABP8 expression
in the rabbit spinal cord, Narayanan et al. [22] noted
that the spinal cord has a high rate of fatty acid
biosynthesis. RT-PCR detected fabp11b transcripts in
total RNA extracted from the brain, heart, ovary and
Fig. 1. The sequence of zebrafish fabp11b
and its 5¢-upstream promoter region. Exons
are shown in upper-case letters, with the
coding sequences of each exon underlined
and the deduced amino acid sequence indi-
cated below. The stop codon is indicated by
the diamond symbol. Only partial nucleotide
sequences are shown for introns 2 and 3,
where the dotted lines indicate interruption
in the sequence. +1 indicates the transcrip-
tion start site. A putative polyadenylation
signal, AATAAA, is highlighted in bold and
underlined. A putative TATA box and a site
for GATA-binding factor are highlighted in
bold and underlined. The in situ hybridization
probe (isp) used for detection of fabp11b
transcripts in adult zebrafish tissues, and
PCR primers used for radiation hybrid map-
ping (rhf, rhr) and for RT-PCR detection of
fabp11b transcripts in RNA extracted from
adult zebrafish tissues (rtf, rtr), are either
underlined or overlined.
AB
CD
Fig. 2. Spatiotemporal distribution of fabp11b transcripts during

zebrafish embryonic and larval development was determined by
whole mount in situ hybridization. fabp11b transcripts were first
detected in the pigmented epithelium of the retina (Re) at 24 hpf
(A). The distribution of fabp11b transcripts had spread across the
retina by 30 hpf (B) and 48 hpf (C). In 5-day-old larvae, fabp11b
transcripts were restricted to the circumference of the pigmented
epithelium of the retina (D).
S. Karanth et al. Duplicated fabp11 genes of zebrafish
FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS 3033
eye of adult zebrafish (Fig. 4). fabp11b transcripts were
not detected in total RNA extracted from the liver,
intestine, kidneys, gills or muscle of adult zebrafish. As
a positive control for the quality of RNA in each
tissue, transcripts for the constitutively expressed ef1a
gene were assayed by RT-PCR and detected in all
tissues examined (Fig. 4). The difference observed in
the tissue-specific distribution of fabp11b transcripts
using in situ hybridization and RT-PCR is probably
due to the sensitivities of the two assays; RT-PCR is
far more sensitive than in situ hybridization. In con-
trast to zebrafish fabp11b transcripts, fabp11a tran-
scripts were detected in liver, intestine, brain, heart
and muscle using RT-PCR [17]. Abundant fabp11
transcripts were detected in liver, adipose tissue and
the vitellogenic ovary, transcripts were detected to a
lesser extent in the previtellogenic ovary, heart, kidney,
and muscle, and trace amounts were detected in testis
by RT-PCR using total RNA extracted from tissues of
adult Senegalese sole [12]. In adult Antarctic fishes
[16], fabp11 (H

ad
-FABP) transcripts were detected in
muscle, kidney, heart and brain by the less sensitive
assay of northern blot and hybridization.
Duplicate copies of fabp11 in zebrafish may have
arisen by a fish-specific WGD event
Multiple sequence alignments of selected zebrafish and
mouse FABP amino acid sequences and the prototypic
Fabp11 from the Senegalese sole [12] were performed
using clustalw [23]. Zebrafish Fabp11b showed the
highest sequence identity and similarity (65% and
84%, respectively) with zebrafish Fabp11a, and the
next highest sequence identity and similarity with the
Senegalese sole Fabp11 (63% and 82%, respectively)
(Fig. 5). The sequence identity and similarity of zebra-
fish Fabp11 decreased with paralogous FABP ⁄ Fabps
from zebrafish and mouse (Fig. 5).
Radiation hybrid mapping using the LN54 panel [24]
assigned zebrafish fabp11b to LG (chromosome) 16 at a
distance of 26.59 cR from the marker fc09b04 with
a logarithm (base 10) of odds (LOD) score of 10.
Zebrafish fabp11a had previously been assigned to LG
(chromosome) 19 by the same LN54 radiation hybrid
panel [17]. Conserved gene synteny on zebrafish LGs
(chromosomes) 16 and 19 [17] with genes on human
chromosome 8 (Table 1) suggest that fabp11a and
fabp11b may have arisen by the teleost fish-specific
WGD event that occurred approximately 250–400 mya
[13]. Both fabp11a (LG 19) and fabp11b (LG 16), and
two other duplicated genes on these LGs, showed

conserved gene synteny with the FABP4–FABP5–
FABP8–FABP9 gene cluster on human chromosome 8.
For duplicated genes to be retained in the genome,
Force et al. [25] have proposed that either both dupli-
cated genes undergo subfunctionalization, in which the
functions of the ancestral gene are subdivided between
the sister duplicate genes, or one of the duplicates
acquires a new function, called neofunctionalization.
Force et al. [25] further proposed that subfunctional-
ization of duplicated genes arises owing to the accumu-
lation of mutations in the regulatory elements of
duplicated genes, which leads to divergence in their
tissue-specific patterns of expression. fabp11a mRNA
transcripts were detected in ovary, liver, skin, intestine,
brain, heart, testis and muscle in adult zebrafish [17].
During larval development, fabp11a transcripts were
detected in the lens and diencephalon [17]. In contrast,
fabp11b transcripts were detected in brain, heart, ovary
AB
Fig. 3. Tissue-specific distribution of fabp11b mRNA in adult zebra-
fish sections determined by in situ hybridization. Sagittal (A) and
transverse (B) sections of adult zebrafish were hybridized to an
[
33
P]dATP[aP] 3¢-end-labeled fabp11b antisense probe. The hybrid-
ization signal of the antisense probe was limited to the spinal cord
(Sc) of adult zebrafish.
Fig. 4. RT-PCR detection of fabp11b transcripts in RNA extracted
from adult tissues of zebrafish using fabp11b cDNA-specific prim-
ers. fabp11b transcripts were detected by RT-PCR in RNA

extracted from the brain (B), heart (H), ovary (O), and eye (E). No
fabp11b transcripts were detected in RNA extracted from the liver
(L), gills (G), intestine (I), muscle (M) or kidney (K) of adult zebrafish
(upper panel). As a positive control, constitutively expressed
elongation factor 1a (ef1a) transcripts were detected by RT-PCR in
RNA extracted from all adult tissues examined (lower panel).
Duplicated fabp11 genes of zebrafish S. Karanth et al.
3034 FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS
and eye by RT-PCR (Fig. 4) and in the spinal chord
by in situ hybridization (Fig. 3) in adult zebrafish, and
in the retina during larval development (Fig. 2).
Although fabp11a and fabp11b transcripts exhibit strik-
ingly different patterns of tissue distribution in devel-
oping embryos, larvae, and adult zebrafish, it is not
possible to ascertain whether the duplicated copies of
fabp11 have been retained in the zebrafish genome by
either subfunctionalization or neofunctionalization, as
there is no readily apparent ortholog of fish fabp11 in
tetrapods or other species studied thus far [12].
fabp11, FABP4, FABP5, FABP8 and FABP9 evolved
from a common ancestral gene on a single
progenitor chromosome
We constructed a neighbor-joining phylogenetic tree
using the amino acid sequences of selected vertebrate
FABPs (Fig. 6). Zebrafish Fabp11b and zebrafish
Fabp11a formed a clade with other teleost Fabp11s
(Fig. 6), a clade distinct from the frog, chicken and
mammalian FABP4–FABP5–FABP8–FABP9 clade, as
previously shown by Agulleiro et al. [12]. The zebrafish
Fabp11a and Fabp11b amino acid sequences have

diverged sufficiently from each other that they are not
linked by a common node on the tree. Similarly, the
putative fugu and stickleback Fabp11a and Fabp11b
do not share a common node with their sister
duplicates either, but are all clustered in the same clade
with all other teleost fish Fabp11s.
The sequence identity of zebrafish Fabp11b with
mouse FABP4, FABP5, FABP8 and FABP9 (also
known as PERF15) varied from 43% to 47%
(Fig. 5). The sequence identity of the Senegalese sole
Fabp11 with human FABP4, FABP5, FABP8 and
FABP9 varied from 52.2% to 54.5% [12]. To date,
Fig. 5. Zebrafish (D. rerio, Dr) Fabp11b is aligned with: zebrafish Fabp11a (deduced from AY628221), Fabp3 (AAL40832), Fabp7a
(AAH55621) and Fabp7b (AAQ92970); Senegalese sole (Solea senegalensis, Sos) Fabp11 (CAM58515); mouse (M. musculus, Mm) FABP4
(AAH02148), FABP5 (NP_034764), FABP8 (XP_485204), and FABP9 (NP_035728). Dashes specify gaps and dots indicate amino acid identity.
The percentage sequence identity and similarity of zebrafish, Senegalese sole and mouse FABP sequences with zebrafish Fabp11b are
shown at the end of each sequence.
S. Karanth et al. Duplicated fabp11 genes of zebrafish
FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS 3035
we have no evidence for orthologs of tetrapod
FABP4, FABP5, FABP8 and FABP9 in teleost
fishes. This is based on tblastn searches of com-
plete or nearly complete sequences for several fish
genomes (e.g. Danio rerio, Takifugu rubrifus and Tet-
raodon nigroviridis)inensembl (embl.
org) and in the extensive expressed sequence tag
(EST) databases (e.g. salmonids) in GenBank [26].
Agulleiro et al. [12] were the first to suggest that
fabp11 formed a novel clade among vertebrate
FABPs and that this gene is unique to teleost fishes.

In addition to fabp11 of the Senegalese sole, homol-
ogous fabp genes from the Antarctic fishes [16], and
the zebrafish fabp genes reported here and previously
[17], appear to belong to a sister group of FABP4,
FABP5, FABP8 and FABP9 of mammals, and tetra-
pod FABP4, FABP5, FABP8 and FABP9, and the
teleost fish fabp11 genes might have arisen from a
common ancestral gene [12].
On the basis of the evidence obtained from phylo-
genetic analysis, linkage mapping and conserved gene
synteny, we propose the following model for the
evolution of fabp11 in fishes and the FABP4–
FABP5–FABP8–FABP9 gene cluster in tetrapods
(Fig. 7). First, fabp4, fabp5, fabp8 and fabp9 are
absent in teleost fishes. Second, an ancestral gene
diverged to give rise to the progenitors of fabp11 in
fishes and FABP4, FABP5 , FABP8 and FABP9 in
tetrapods, probably before the fish–tetrapod split
some 450 mya [27]. Third, the FABP4–FABP5–
FABP8–FABP9
gene cluster in tetrapods arose by
successive tandem duplications and divergence of a
single ancestral gene, as has been suggested for the
evolution of some of the globin gene clusters [28].
Last, the ancestral gene of fabp11 and the FABP4–
FABP5–FABP8–FABP9 gene cluster resided on a
chromosome, or a part of it, that was the progenitor
of zebrafish LGs (chromosomes) 16 and 19, chicken
chromosome 2, mouse chromosome 3, human chro-
mosome 8, and frog scaffold 225 (see Experimental

procedures for details of retrieval of genomic
sequence data).
Table 1. Conserved gene synteny of the duplicated copies of zebrafish fabp11 with human FABP4 , FABP5, FABP8 and FABP9.
Gene name
Zebrafish Human
Gene symbol LG position (cM) Mapping panel Gene symbol Chromosomal location
Fatty acid-binding protein 11a fabp11a 19, 50.80–53.10 LN54 – –
Fatty acid-binding protein 11b fabp11b 16, 44.16–45.60 LN54 – –
Fatty acid-binding protein 4 – – – FABP4 8q21
Fatty acid-binding protein 5 – – – FABP5 8q21
Fatty acid-binding protein 8 – – – FABP8 8q21–8q22
Fatty acid-binding protein 9 – – – FABP9 8q21
der1-like domain family, member 1 derl1 16, 46.90 LN54 DERL1 8q24.13
NADH dehydrogenase (ubiquinone)-1beta
subcomplex 9
ndufb9 16, 24.20 T51 NDUFB9 8q13.3
ATPase family, AAA containing 2 atad2 16, 26.70 T51 ATAD2 8q24.13
TAF2 RNA polymerase II, TATA box-binding
protein (TBP)-associated factor
taf2 16, 31.25 LN54 TAF2 8q24.12
RAD21 homolog rad21 16, 61.07 LN54 RAD2 8q24
Hyaluronan synthase 2 has2 16, 31.67 T51 HAS2 8q24.12
Cadherin 17 cdh17 16, 46.90 T51 CDH17 8q22.1
E3 ubiquitin protein ligase edd1 16, 67.71 T51 EDD1 8q22
Eukaryotic translation initiation factor 3,
subunit 3 (gamma)
eif3s3 16, 61.23 HS EIF3S3 8q24.11
Mitochondrial folate transporter ⁄ carrier mftc 19, 46.86 LN54 MFTC 8q22.3
Growth differentiation factor 6 gdf6b 19, 47.30 HS GDF6 8q22.1
Sperm-associated antigen 1 spag1 19, 49.00 T51 SPAG1 8q22.2

Protein tyrosine phosphatase
type IVA, member 3
zfpm2b
19, 50.25 HS PTP4A3 8q24.3
Ribosomal protein L30 rpl30 19, 50.80 LN54 RPL30 8q22
Serine ⁄ threonine kinase 3 stk3 19, 51.88 T51 STK3 8q22.2
Metadherin lyric1 19, 51.95 T51 MTDH (LYRIC) 8q22.1
Antizyme inhibitor 1 azin1 19, 53.30 T51 AZIN1 8q22.2
Angiopoietin 1 angpt1 19, 81.93 T51 ANGPT1 8q22.3–q23
Duplicated fabp11 genes of zebrafish S. Karanth et al.
3036 FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS
Tetrapod FABP5, FABP8, FABP9 and FABP4 are
tandemly arrayed and probably arose by unequal
crossing-over – which gene was duplicated first?
Although it is speculative, it is possible to provide a
parsimonious scheme for the tandem duplication
events that gave rise to the cluster of four FABP
genes on a single mammalian chromosome. In the
frog genome, fabp4, fabp4-like and fabp5 are tan-
demly arrayed on scaffold 225 (Fig. 7). Owing to
sequence identity and their relationship in the phylo-
genetic tree (Fig. 6), fabp4 and fabp4-like may have
arisen in the frog genome as a result of tandem
duplication of an ancestral gene on this chromo-
some. It is not readily apparent, however, whether
the tandem duplication that gave rise to frog fabp5
occurred before or after the tandem duplication that
produced fabp4 and fabp4-like . The phylogenetic
analysis would favor the fabp4⁄ fabp4-like duplication
occurring after the duplication event that generated

fabp5. Orthologs of mammalian FABP8 and FABP9
have not yet been identified, or more likely are not
present, in the frog genome.
Chicken FABP5, FABP8 and FABP4 are also tan-
demly arrayed on chromosome 2; on the basis of data-
base searches, it seems that FABP9 is absent from the
chicken genome. Therefore, chicken FABP8 most
probably arose from the tandem duplication of either
FABP4 or FABP5. Again, the phylogenetic tree
(Fig. 6) suggests that chicken FABP8 may have origi-
nated from tandem duplication of FABP4 rather than
FABP5,asFABP8 and FABP4 form a common clade,
whereas FABP5 was placed in a different clade. Our
model is consistent with the time-scale for these dupli-
cation events based on synonymous ⁄ nonsynonymous
amino acid substitution rates in FABP4, FABP5,
FABP8 and FABP9, and the topology of the phylo-
genetic tree reported by Schaap et al. [9].
Fig. 6. Phylogenetic tree of selected vertebrate FABPs, showing
the relationship between zebrafish Fabp11a and Fabp11b. The
neighbor-joining tree was constructed using H. sapiens LCN1
(NP_002288) as outgroup. The bootstrap values (per 100 duplicates)
are indicated above or under each node. The teleost Fabp11s
formed a common clade, which is indicated by a bracket. Amino
acid sequences used in this analysis include: D. rerio (zebrafish)
(Dr) Fabp11a (derived from AY628221), and Fabp11b (ENS-
DARP0000002311); Gobionotothen gibberifrons (Gog) Fabp11 (H6-
Fabp, AAC60354); Notothenia coriiceps (Nc) Fabp11 (H6-Fabp,
AAC60352); Parachaenichthys charcoti (Pc) Fabp11 (H6- Fabp,
AAC60355); Ta. rubripes (takifugu) (Fr) Fabp11a (deduced from

AL837220), and Fabp11b (deduced from AL836636); Te. nigroviridis
(Tn) Fabp11 (deduced from CR723700); Oryzias latipes (medaka)
(Ol) Fabp11 (deduced from BJ899828); Cyprinus carpio (common
carp) (Cc) Fabp11 (deduced from CF661735); So. senegalensis
(Senegalese sole) (Sos) Fabp11 (CAM58515); Gasterosteus aculea-
tus (stickleback) (Ga) putative Fabp11a (ENSGACP00000004532),
and putative Fabp11b (ENSGACP00000011538); H. sapiens (Hs)
FABP4 (CAG33184), FABP5 (AAH70303), and FABP8 (AAH34997);
FABP9 (PERF15) (Uniprot ID Q0Z7S8); M. musculus (mouse) (Mm)
FABP4 (AAH02148), FABP5 (NP_034764), FABP8 (XP_485204), and
FABP9 (NP_035728); Rattus norvegicus (rat) (Rn) FABP4
(NP_445817), FABP5 (NP_665885), and FABP9 (NP_074045); Sus -
scrofa (pig) (Ss) FABP4 (CAC95166); Gal. gallus (chicken) (Gg)
FABP4 (NP_989621), FABP5 (ENSGALP00000025375), and FABP8
(ENSGALP00000025382); X. tropicalis (African frog) (Xt), putative
FABP4 (ENSXETP00000022878), putative FABP4-like (NP_
001096256.1), and putative FABP5 (ENSXETP00000022879);
Pan troglodytes (chimpanzee) (Pt) FABP9 (PERF15) (ENS-
PTRP00000053126).
S. Karanth et al. Duplicated fabp11 genes of zebrafish
FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS 3037
Fabp5, Fabp8, Fabp9 and Fabp4 are arranged in
sequential order on mouse chromosome 3, and the
FABP5–FABP8–FABP9–FABP4 gene cluster is present
on human chromosome 8. Tandem duplication of
FABP8 may have resulted in the formation of FABP9,
as FABP8 and FABP9 formed a common clade (Fig. 6).
Experimental procedures
Husbandry of zebrafish
Adult zebrafish were purchased from a local aquarium store

and maintained according to established procedures [29].
Experimental protocols were reviewed by the Animal Care
Committee of Dalhousie University in accordance with the
Canadian Committee on Animal Care.
Nucleotide sequence of zebrafish fabp11b cDNA
and gene
We retrieved a novel ensembl gene (ENSDARG000000023
11) from a blastn search of the zebrafish genome sequence
database at the Wellcome Trust Sanger Institute (ver-
sion Zv6; />using the fabp11a cDNA sequence as a query [17]. The
novel ensembl gene (ENSDARG00000002311) exhibited
most sequence identity and similarity to zebrafish fabp11a,
hereafter referred to as zebrafish fabp11b. We confirmed the
sequence of the coding region for fabp11b by comparison
with fabp11b ESTs obtained from the blastn search of
GenBank at the National Center for Biotechnology Infor-
mation. All ESTs were identical to the coding region of
zebrafish fabp11b.
Phylogenetic analysis
blosum62 matrix and clustalw [23] were used to align
FABP sequences from zebrafish and other vertebrates. The
bootstrap neighbor-joining tree was constructed using
mega4 software [30]. Human LCN1 (NP_002288) was used
as the outgroup.
Radiation hybrid mapping of zebrafish fabp11b
A detailed protocol for radiation hybrid mapping of zebra-
fish genes is described by Hukriede et al. [24]. PCR reactions
were carried out using the forward primer rhf (5¢-GT
GTTGTGATTTTCGGTGG-3¢; nucleotide positions 33–51),
and the reverse primer rhr (5¢-TTCTGTCATCTGCTG

TCGTC-3¢; nucleotide positions 396–423), as shown in
Fig. 1. PCR conditions were initial denaturation at 94 °C for
2 min, followed by 30 cycles at 94 °C for 30 s (denaturation),
54.5 °C for 30 s (primer annealing) and 72 °C for 1 min
(elongation), with a final elongation step at 72 °C for 5 min.
In situ hybridization to whole mount embryos
and larvae, and to sections of adult zebrafish
Whole mount in situ hybridization to zebrafish embryos
and larvae was performed using a riboprobe synthesized
Fig. 7. Evolutionary relationship between the duplicated copies of fabp11 in fishes and FABP4, FABP5, FABP8 and FABP9 (PERF15) in frog,
chicken, mouse, and human. In zebrafish, fabp11a is found on LG 19, and fabp11b is on LG 16. In frog (X. tropicalis), fabp4, fabp4-like
(fabp4l) and fabp5 are found on scaffold 225, where fabp4l and fabp5 are immediately adjacent to each other. Chicken (Gal. gallus) FABP5,
FABP8 and FABP4 are located next to each other on chromosome 2. FABP5, FABP8, FABP9 (PERF15) and FABP4 are tandemly arrayed on
human (H. sapiens) chromosome 8 and mouse (M. musculus) chromosome 3. Gaps in white indicate the presence of an additional gene
between two FABP genes on a particular chromosome.
Duplicated fabp11 genes of zebrafish S. Karanth et al.
3038 FEBS Journal 275 (2008) 3031–3040 ª 2008 The Authors Journal compilation ª 2008 FEBS
from fabp11b cDNA clone zgc:110029 (GenBank accession
number BC095142), as described by Thisse & Thisse [31].
In situ hybridization of an oligonucleotide probe to sections
of adult zebrafish followed the protocol of Denovan-Wright
et al. [32]. Briefly, sagittal and transverse sections of adult
zebrafish were hybridized to [
33
P]dATP[aP] 3¢-end-labeled
fabp11b antisense probe, isf (5¢-CACAACACAAGACGTT
TGACAGATAATAGC-3¢; nucleotide positions 11–40),
shown in Fig. 1. Following hybridization and autoradio-
graphy, tissue sections were stained with cresyl violet to
identify specific tissues.

RT-PCR detection of fabp11b transcripts in adult
zebrafish tissues
RT-PCR was employed for the tissue-specific detection of
fabp11b transcripts in RNA extracted from tissues of adult
zebrafish. Following synthesis of cDNA from RNA samples
using the Omniscript RT kit (Qiagen, Mississauga, Ontario,
Canada), fabp11b cDNA was PCR-amplified by the for-
ward primer rtf (5¢-GCTGTCACTACATTCAA
GACCCTGGA-3¢; nucleotide positions 337–363) and the
reverse primer rtr (5¢-ACCATCCGCAAGGCTCATAGTA
GT-3¢; nucleotide positions 1369–1393), shown in Fig. 1.
PCR conditions for the amplification of fabp11b transcripts
comprised an initial denaturation step at 94 °C for 2 min,
followed by 30 cycles at 94 °C for 30 s (denaturation),
56 °C for 30 s (primer annealing) and 72 °C for 1 min
(elongation), with a final elongation step at 72 °C for
5 min. PCR primers used for detection of elongation fac-
tor 1a (ef1a) transcripts in total zebrafish RNA are
described in Pattyn et al. [33]. The PCR conditions were
an initial denaturation step at 94 °C for 2 min, followed by
30 cycles at 94 °C for 30 s (denaturation), 62 °C for 30 s
(primer annealing) and 72 °C for 1 min (elongation), with a
final elongation step of 72 °C for 5 min.
Database searches of genome sequences and
identification of transcription factor-binding sites
The genomic organization of tetrapod FABP4, FABP5,
FABP8 and FABP9 was derived from the Xenopus tropical-
is, Gallus gallus, Mus musculus and Homo sapiens genome
sequence databases at . Putative
transcription factor-binding sites in the 5¢-upstream region

of zebrafish fabp11b were identified using alibaba 2.1 soft-
ware [34].
Acknowledgements
The authors thank Mark Soric and Fernanda Alves-
Costa for technical assistance, and David R. Smith
and Dr Tudor Borza for helpful comments. This work
was supported by funds from the Natural Sciences and
Engineering Research Council of Canada (to
J. M. Wright), Canadian Institutes of Health Research
(to E. Denovan-Wright), and the National Institutes of
Health, the European Commission as part of the
ZF-Models integrated project in the 6th Framework
Programme (to B. Thisse and C. Thisse). S. Karanth is
a recipient of a Faculty of Graduate Studies Scholar-
ship from Dalhousie University.
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