Neurokinin Bs and neurokinin B receptors in zebrafish- potential role ...

Neurokinin Bs and neurokinin B receptors in zebrafishpotential
role in controlling fish reproduction
Jakob Biran a , Ori Palevitch a , Shifra Ben-Dor b , and Berta Levavi-Sivan a,1
a Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel;
and b Department of Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel
Edited by John E. Halver, University of Washington, Seattle, WA, and approved April 27, 2012 (received for review December 2, 2011)
The endocrine regulation of vertebrate reproduction is achieved by
the coordinated actions of several peptide neurohormones, tachykinin
among them. To study the evolutionary conservation and
physiological functions of neurokinin B (NKB), we identified tachykinin
(tac) and tac receptor (NKBR) genes from many fish species,
and cloned two cDNA forms from zebrafish. Phylogenetic analysis
showed that piscine Tac3s and mammalian neurokinin genes arise
from one lineage. High identity was found among different fish
species in the region encoding the NKB; all shared the common Cterminal
sequence. Although the piscine Tac3 gene encodes for two
putative tachykinin peptides, the mammalian ortholog encodes for
only one. The second fish putative peptide, referred to as neurokinin
F (NKF), is unique and found to be conserved among the fish
species when tested in silico. tac3a was expressed asymmetrically in
the habenula of embryos, whereas in adults zebrafish tac3a-expressing
neurons were localized in specific brain nuclei that are
known to be involved in reproduction. Zebrafish tac3a mRNA levels
gradually increased during the first few weeks of life and peaked at
pubescence. Estrogen treatment of prepubertal fish elicited increases
in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic
zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors
via both PKC/Ca 2+ and PKA/cAMP signal-transduction pathways
in vitro. Moreover, a single intraperitoneal injection of NKBa and
NKF significantly increased leuteinizing hormone levels in mature
female zebrafish. These results suggest that the NKB/NKBR system
may participate in neuroendocrine control of fish reproduction.
gonadotropin-releasing hormone | kisspeptin | teleost | gonadotropin
Reproduction is a highly integrated and complex function that
requires synchronized production of gametes by both sexes at
an optimum time for offspring survival. Fish show an enormous
variety of reproductive strategies (1), and were recently chosen as
models for the study of growth, metabolism, and human diseases.
The hypothalamic regulation of gonadotropin secretion in fish is
different from that of mammals, from both endocrinal and anatomical
aspects. In teleosts, the pituitary is innervated directly by
neurons projecting to the vicinity of the pituitary gonadotrophs
(2). Among the neuropeptides released by these nerve endings are
gonadotrophin-releasing hormones (GnRHs) and dopamine,
which act as stimulatory and inhibitory factors on the release of
luteinizing hormone (LH) and follicle-stimulating hormone (3).
However, new actors have recently entered the field of reproductive
physiology: kisspeptins, neurokinin, and dynorphin
have all been implicated in controlling GnRH (4).
Topaloglu et al. (5) found that humans bearing loss-of-function
mutations of the genes encoding either neurokinin B (NKB)
or its cognate receptor, neurokinin receptor 3 (NKBR, Tac3r)
displayed hypogonadotropic hypogonadism; this seminal report
implicated NKB signaling as an essential factor in the onset of
puberty and control of gonadotropin secretion in mammals.
Recent studies provided evidence that, in mammals, a group of
neurons in the hypothalamic arcuate nucleus (ARC) are steroidresponsive
and coexpress NKB, kisspeptin, dynorphin, NKBR
and estrogen receptor α(6). Compelling evidence indicates that
these neurons function in the hypothalamic circuitry regulating
GnRH secretion. The main objective of the present study, using
zebrafish (Danio rerio) as a model, was to examine the involvement
of NKB in fish reproduction.
NKB is a member of the tachykinin (TK) family of peptides.
TKs are characterized by a common carboxyl-terminal amino acid
sequence of FXGLM-NH2 (where X is a hydrophobic residue),
and include substance P, neurokinin A (NKA) and NKB, as well
as neuropeptide K, neuropeptide-γ, and hemokinin-1 (7). NKB is
the only TK synthesized from the preprotachykinin-B gene (8),
which is currently designated as TAC3 in mammals, except for
rodents, where it was named Tac2. Because there are different
names for the gene encoding NKB in different species (TAC3 or
Tac2), in this article we will refer to mRNA products of this gene
as tac3 mRNA and to the peptides as NKB. The receptor that
binds NKB, which is termed NKBR in humans, will be termed
tac3r at the mRNA level and Tac3r at the protein level.
Until now, NKB was not cloned from any fish species, nor was
the NKB/NKBR system shown to be involved in reproduction or
puberty. We report here the identification of previously unidentified
fish NKB/NKBR genes and their possible involvement
in the control of reproduction.
Results and Discussion
Cloning Two Types of tac3 and tac3r and Their Phylogenetic Analysis.
As the first step toward examining the involvement of the NKB/
NKBRs (tac3r) in the control of reproduction in fish, we report
here the identification of the full-length tac3a and tac3b cDNA
from zebrafish brain using real-time PCR with specific primers
(Table S1). Tac3a contains the decapeptide sequence EMH-
DIFVGLM (Fig. S1A) (accession no. JN392856), whereas tac3b
contains a 24-aa peptide (STGINREAHLPFRPNMNDIFVGLL)
(Fig. S1B) (accession no. JN392857), both with the TK signature
motif (FXGLM-NH2) flanked by potential dibasic cleavage sites
and an adjacent glycine at the C terminus for amidation (9). Typically,
following prohormone convertase action, a carboxypeptidase
removes the C-terminal dibasic residues, and a peptidylglycine
a-amidating enzyme converts the exposed glycine into a C-terminal
amide (10). At the protein level, the resulting zfNKBa (Tac3a)
hormone precursor displayed around 25% identity with human or
mouse TAC3, 55% with putative salmon Tac3a, and 52% with
medaka Tac3 identified in this study (accession nos. BK008102 and
BK008114, respectively). zfNKBb (Tac3b) showed only around
18% identity with human and mouse TAC3, 40% and 36% identity
with salmon and medaka Tac3b, respectively. The zebrafish tac3s
shared only a 36% identity (Fig. S1 and Table S2).
In our search for the identification of Tac3 sequences containing
the NKB peptide sequence in fish mRNAs and ESTs
known to date, we encountered 30 previously unidentified
Author contributions: J.B. and B.L.-S. designed research; J.B. and O.P. performed research;
J.B., S.B.-D., and B.L.-S. analyzed data; and J.B., O.P., and B.L.-S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper has been deposited in the GenBank
database [accession nos. JN392856 (zftac3a), JN392857 (zftac3b), JF317292 (zftac3ra),
JF317293 (zftac3rb), and BK008087–BK008126].
1
To whom correspondence should be addressed. E-mail: sivan@agri.huji.ac.il.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1119165109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1119165109 PNAS | June 26, 2012 | vol. 109 | no. 26 | 10269–10274
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piscine Tacs. We generated a phylogenetic tree of all available
vertebrate neurokinin genes (Fig. 1A and Fig. S2A); it showed
that the vertebrate neurokinin genes fall into several distinct
lineage groups. The identified Tac3 precursors from fish were
clustered with all other previously cloned or predicted Tac3
sequences from mammals, frogs, and alligators (Fig. 1A); a second
lineage included Tac1 from both mammals and fish that
were identified in the present study, and the third lineage included
mammalian Tac4 and a unique piscine group, now named
Tac4 (Fig. S2A). No precursors containing the exact NKB sequence
were found in invertebrate species (11).
We cloned the full-length tac3ra and tac3rb cDNA from
zebrafish brain by PCR with specific primers (Table S1). The
predicted tac3ra and tac3rb N termini have features consistent
with a signal peptide, as defined by SignalP program analysis
(Fig. S1). Sequence analysis of the two types of zebrafish
receptors identified distinct potential sites for N-glycosylation,
A
B
C
Fig. 1. Unrooted phylogenetic tree of neurokinin (A) or neurokinin receptor
(B) sequences generated with neighbor-joining (ClustalW 2.1) and maximum
likelihood (Phylip 3.69, ProML) on the basis of alignments performed both by
ClustalW and Muscle (3.8.31), and visualized with FigTree 1.3.1. The Tac1 and
Tac4 (A) and Tacr1 and Tacr2 (B) were grouped (full trees in Fig. S2). The
sequences identified in this study are marked in bold. Gene nomenclature has
been standardized to Tac3, and species are indicated for illustration and
comparison. Numbers at nodes indicate the bootstrap values from 1,000 replicates.
(Scale bar: the substitution rate per residue.) GenBank accession numbers
are detailed in the legend to Fig. S2. (C) Gene organization of zebrafish
tac3a and tac3b. Each gene is consists of seven exons and encode both NKF and
NKB. SP, signal peptide; ATG and TGA, start and stop codon, respectively.
phosphorylation by protein kinase C, protein kinase A, casein
kinase II, tyrosine kinase, and N-myristoylation (Fig. S3). The N
and C termini are, as in other G protein-coupled receptors, the
most divergent regions. In our bioinformatic search for NKB
receptors we found four additional TK receptor genes in zebrafish.
To enable assignment of the genes to the three TK receptor
subfamilies already defined in mammals (12), we identified family
members in additional species, particularly in other fish. The
phylogenetic tree containing vertebrate TK receptors form three
clearly separable groups that corresponded to TAC3R, TAC1R,
and TAC2R (Fig. S2B). Putative orthologs of NK receptor
members were also identified in several nonvertebrate species,
Caenorhabditis elegans, ciona, and octopus; these served as outgroup
sequences in determining the root of these three groups,
which was located between TAC2R and the others, indicating
that these groups split early in the family evolution. The tree
shows that TAC3R and TAC1R are closest to each other, suggesting
that their separation was a more recent evolutionary event
(Fig. 1B and Fig. S2B). Fish have one gene of Tac2r, and two
genes each of Tac1r and Tac3r. Surprisingly, three Tac3r were
found in zebrafish, the only species to have a third receptor of any
one type. However, we were unable to clone this gene from brain
mRNA, so it was excluded from further biological analysis. The
similarity and identity among the various TAC3 receptors is
shown in Table S3.
We found two forms of tac3 genes in zebrafish and salmon, but
more evolved fish contained only one tac3 ortholog; however, all fish
species exhibit two forms of NKB receptors, suggesting that the
piscine NKB/NKBR can provide an excellent model for understanding
the molecular coevolution of the peptide/receptor pairs.
Gene Organization of tac3 and Chromosomal Synteny of Tac3 and
Tac3 Receptor. The in silico analyses of fish genomic structure
verified that the zftac3 consists of seven exons (Fig. 1C). In
mammals the tac3 gene contains seven exons, five of which are
translated to form the prepro-NKB protein (11). Notably, the
NKBa peptide sequence was encoded in the fifth exon [like in
mammals (13, 14)], whereas NKBb spans exons 3–5 (Fig. 1C).
Surprisingly, unlike in mammalian NKBs (11), the deduced
amino acid sequences of both zftac3 genes encoded an additional
putative TK sequence flanked by a Gly C-terminal amidation
signal, and typical endoproteolytic sites at both termini, suggesting
that additional TK peptides (YNDIDYDSFVGLM-NH 2
and YDDIDYDSFVGLM-NH 2, spliced from Tac3a and Tac3b,
respectively) (Fig. 1C and Fig. S1) are produced by the same
precursors. Intriguingly, we found this additional peptide in tac3
not only in zebrafish but in all other fish species identified in this
study (11 species), but not in chicken, lizard, or alligator. These
peptides possess an N-terminal dibasic cleavage site with potential
to release the peptide, and the common NKB motif
FVGLM at their C terminal; therefore, we termed this unique
peptide neurokinin F (NKF) because it has only been found in
fish species to date. As Page et al. (11) anticipated, the vertebrate
TAC3 gene encoded an additional TK in exon 3, in
a similar position to substance P in TAC1, and endokinin A/B in
TAC4. This TK (NKF) still exists in fish but was lost from other
species during evolution. Interestingly, in Tac4 there is a similar
loss of one active peptide in mammals (the C-terminal peptide in
Tac4 as opposed to the N-terminal peptide in Tac3), whereas
most fish species retain putative active peptides in both locations.
Chromosome syntenic analysis revealed that the locus of tac3
is highly conserved between teleosts (Fig. S4). zftac3a is located
on chromosome 23 and tac3b on chromosome 6. The only tac3
found in medaka is located on chromosome 7 (Fig. S4B). For the
zftac3 gene, the nearest neighboring gene (c1galt1a) is nonsyntenic,
whereas the next nearest ones (b4galnt1a and slc6a1)
were found in inverse order in humans (Fig. S4A). Despite nearly
perfect preservation of synteny, we found substantial shuffling of
gene order along corresponding chromosome arms between
zebrafish and human. The neighborhoods of gene loci of tac3a
were conserved in the zebrafish and medaka (Fig. S4B). We then
10270 | www.pnas.org/cgi/doi/10.1073/pnas.1119165109 Biran et al.

explored the genomic locations of tac3 receptors in humans and
various fish species (Fig. S4 C and D). In human, TAC3R is
located on chromosome 4, whereas in zebrafish, fugu (Takifugu
rubripes), medaka (Oryzias latipes), and tetraodon (Tetraodon
nigroviridis) tac3ra are located on chromosome 1, unplaced
(UN), 1, and 18, respectively. The nearest neighboring gene
(cnga2) of the zebrafish tac3ra gene is nonsyntenic with human,
but syntenic with the medaka, fugu, and tetraodon. The nextupstream
neighboring genes (bdh2, nhedc2, and cisd2) were
found in similar locations in all analyzed species (Fig. S4C). The
human genome lacks tac3rb, which is present in tetraodon, fugu,
and medaka. The next-upstream neighboring genes in the
zebrafish (acy3.1, acy3.2, cldnd, and glb1) were found in reverse
order in the tetraodon (Fig. S4D). Tac3rc had no discernible
synteny to any other family members. The presence of two forms
of NKB [in zebrafish and salmon (salmo salar)] and two forms of
cognate receptor genes in lower-vertebrate species supports the
hypothesis of two rounds of genome duplication followed by
degeneration and complementation of the genes. We conclude
that the synteny is better within fish than with mammals; moreover,
fish tac3 and tac3r have conserved synteny with the human
genome, consistent with orthology.
Tissue Distribution of tac3 and tac3 Receptors in Zebrafish. To elucidate
the physiological roles of the NKB/NKBR signaling system,
we next examined the tissue distribution of both ligands
(tac3a, tac3b) and receptors (tac3ra, tac3rb) mRNAs in zebrafish
by means of real-time PCR analysis, according to Biran et al.
(15). We dissected the zebrafish brain into three parts, of which
the anterior part contains the telencephalon, the midbrain contains
the optic tectum, diencephalon, and hypothalamus and the
hindbrain, the medulla oblongata and cerebellum. We mostly
detected tac3a mRNA in the midbrain and tac3b mRNA was
found mainly in the forebrain. Both tac3a and tac3ra were
expressed in the pituitary (Fig. 2A), collaborating findings in
mammals where NKB and NKBR were expressed in the median
eminence, which is missing in fish (6). tac3rb was expressed in the
forebrain and was highest in the ovary. Different types of tac3
and tac3r were expressed in the ovary and testis (Fig. 2A). Different
levels of expression of tac3 and tac3r types were found in
extrabrain tissues (Fig. S5). The expression patterns of these
genes in the brain-pituitary-gonad axis further support the potential
role of the NKB system in fish reproduction.
Gene Expression of the NKB/NKBR System During Sexual Maturation.
Because it is known that the mammalian NKB/NKBR system is
involved in reproduction, and especially in puberty initiation
(16), we aimed to examine whether the piscine system fulfills
a similar role. We used real-time PCR to evaluate the expression
profiles of the zfNKBs and their receptor mRNAs in the brain
during several development stages. Expression of tac3a mRNA
was low at 2–4 wk postfertilization (wpf) (Fig. 2B); it then
gradually increased, peaked at 8 wpf, when the zebrafish go
through puberty, and subsequently decreased by 12 wpf (Fig.
2B), when the gonads contained clear, well-developed oocytes or
A B
spermatozoa (15). The expression of the tac3b, tac3ra, and tac3rb
mRNAs in the zebrafish brain was low and did not change during
sexual maturation. The increase of tac3a mRNA toward puberty,
consistent with kisspeptin signaling initiating puberty (15, 17),
may indicate a possible involvement of the NKB/NKBR system
in controlling puberty.
Localization of Embryonic tac3a Cells by Whole-Mount in Situ Hybridization.
The first appearance of tac3a expression was detected at 3
d postfertilization (dpf) in the right habenula nuclei and the midbrain
(Fig. 3 A, F, andK). The earliest stage that the signal was
observed in the left habenula was at 4–5 dpf, when the signal intensity
in the right habenula and midbrain increased, probably
reflecting increased cell numbers (Fig. 3 B, C, G, H, L, andM). In
addition, there was expression in the hindbrain. Analysis of elderly
larvae (7 and 9 dpf) revealed decreased tac3a signal intensity (Fig. 3
D, E, I, J, N,andO), and at 12 dpf it was barely detected. No signal
was observed at any stage by use of the tac3a sense riboprobe.
During embryogenesis tac3a was dominantly expressed in the righthabenula
nuclei, but in adults it was expressed in both habenular
lobes (Fig. 3P). This asymmetrical expression of tac3a in the
habenula is consistent with previous findings that the habenula in
zebrafish displayed left-right asymmetries in gene expression (18,
19). Interestingly, it was shown that neurons appear sooner in the
left than in the right habenula (20). Neuronal organization asymmetries
in the epithalamus (i.e., habenular nuclei and pineal complex)
are well known among vertebrates (21).
Localization of tac3a mRNA in the Brain of Adult Zebrafish. By using
in situ hybridization (ISH) techniques to determine the localization
of tac3a mRNA in the zebrafish brain, we detected tac3a mRNAexpressing
neurons in the habenula (Fig. 3P), where kisspeptin 1
(kiss1) was previously shown to be expressed (22). Tac3a was also
detected along the periventricular hypothalamus (Fig. 3 Q and R),
in the periventricular nucleus of the posterior tuberculum (Fig. 3Q),
and in the posterior tuberal nucleus (Fig. 3R). These brain nuclei
were previously found to express other important neuropeptides
that regulate reproduction (17, 22), metabolism (23), and stress
(24). In the mammalian ARC, KISS1 neurons that coexpress NKB
and dynorphin were hypothesized to be a central node through
which potential stress, metabolic and photoperiodic signals regulate
GnRH release (25). The nuclear lateralis tuberis is considered as the
piscine structure homologous to the mammalian ARC (23, 26). The
localization of tac3a, kisspeptin2(kiss2), kiss1 receptor b (kiss1rb),
leptin receptor, melanin-concentrating hormone (MCH) 2 and two
MCH1 receptors, Urotensin I, corticotropin-releasing factor, and
corticotropin-releasing factor-binding protein to the ventral zone of
the periventricular hypothalamus (Fig. 3Q) (17,22–24, 26) might
suggest that not all neuropeptide pathways are as conserved as
formerly thought. This suggestion is supported by the recent findings
that kiss2 is not expressed in the zebrafish nuclear lateralis tuberis,
and that kiss2 neurons of the periventricular hypothalamus do not
directly innervate the zebrafish pituitary (22). Finally, the close
similarity between the expression patterns of zebrafish kisspeptin
genes and zebrafish tac3a suggests a possible interaction between
Fig. 2. Expression of zebrafish tac3a, tac3b,
tac3ra, or tac3rb mRNA in various parts of the
brain (A) and changes in zebrafish tac3a at various
ages toward puberty (B) as determined by
real-time PCR. The relative abundance of the
mRNAs was normalized to the amount of elongation
factor 1 α (ef1α) by the comparative
threshold cycle method; the comparative threshold
reflects the relative amount of the transcript.
Results are means ± SEM (n = 11–15). Means
marked with different letters differ significantly
(P < 0.05).
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Fig. 3. (A–O) Localization of tac3a during early stages of development, as
detected by whole-mount ISH. tac3a is dominantly expressed in the right
habenular nuclei, midbrain, and hindbrain. Dorsal view of larva heads, anterior
is to the left (A–E). High magnification of boxed area in Upper panel. Note the
unilateral expression of tac3a to right of the midline (dotted line) in the
habenula (F–J), lateral view of larva heads (K–O). rHbn, right habenula; MB,
midbrain; HB, hindbrain. (P–R) Localization of tac3a in adult zebrafish brain as
indicated by ISH (nomenclature according to ref. 40). Tac3a mRNA-expressing
cells were observed in the ventral (Hav) and medial (Ham) habenula (P) tac3a
mRNA-expressing cells detected in the periventricular nucleus of posterior
tuberculum (TPp), dorsal (Hd), and ventral zone (Hv) of periventricular hypothalamus
(Q) tac3a mRNA-expressing cells observed in the posterior tuberal
nucleus (PTN) and central zone (Hc) of periventricular hypothalamus (R).
(Magnification: A–E and K–O, 40×; F–J, 120×).
these two systems, as previously shown in mammals; however, this
needs further investigation.
Pharmacological Analysis and Signal Transduction Pathways of zfTac3
Receptors. We used functional expression analysis with COS-7
cells to evaluate the response, binding selectivity, and signal
transduction pathways of the unique TK receptors to their agonists.
We previously showed the specificity of the reporter serum
responsive element (SRE)-Luc and cAMP responsive element
(CRE)-Luc, to activation of PKC/Ca 2+ and PKA/cAMP signal
transduction pathways, respectively (15). Graded concentrations
of the zfTKs (NKBa, NKBb, and NKF), and of hNKB and its
agonist senktide were applied to COS-7 cells that expressed
hNKBR, zfTac3ra, or zfTac3rb. The EC 50 values of TKs for each
receptor are summarized (Table S4). Both human and piscine
TKs induced concentration-dependent increases in both SRE-
Luc and CRE-Luc activity (Fig. 4 A–F). For hNKBR, in both
signal transduction systems, zfNKBa and NKF showed high potency,
very similar to human NKB, but zfNKBb peptide exhibited
relatively low potency (Fig. 4 A–D). For both zfTac3 receptors,
zfNKBa and NKF (both derived from tac3a) were similarly
highly potent in both signal transduction pathways (Fig. 4).
These results confirmed that both zfNKBa and NKF were endogenous
ligands of Tac3 receptors, and it is noteworthy that this
report of activation of TK receptors by a second peptide derived
from the NKB gene (zfNKF) is unique. However, NKBb was less
effective than the other forms in eliciting luciferase activity by
both signal transduction pathways. It was previously shown unequivocally
that hNKBR potentially can couple directly to both
phospholipase C and adenylate cyclase, and stimulate both
phosphoinositides hydrolysis and cAMP formation (27). Indeed
both zftac3 receptors, as well as the hNKBR posses both PKC
and PKA phosphorylation sites (Fig. S3). These findings confirm
that the unique zebrafish receptors relay their signal through
both PKC and PKA transduction pathways.
Ligand Models. Fig. 4G is a ribbon representation of the zfNKB
structural model prediction, compared with the hNKB (PDB ID
1p9f). Intriguingly, although the three zfNKBs vary in size—10,
24, and 13 aa for NKBa, NKBb, and NKFa, respectively—all of
the predicted peptides yielded high-resolution, high-quality
structures with typical globular folding. These structures comprised
α-helix-loop motifs (highlighted in red in Fig. 4G). All
three zfNKBs model structures approximated a binding-competent
conformation similar to the human NKB. Our results corroborate
previous ones found for mammals that the formation of
a helical conformation in the mid region of each of the TKs
appears to be crucial for TK-receptor activation (28). Moreover,
mammalian NKB were found to form a helical structure in the
presence of dodecylphosphocholine micelles (29).
In Vivo Effect of Estradiol. Our next aim was to test the involvement
of the NKB system in reproduction. In fish, clear evidence
exists regarding the important role estradiol plays during
the period of reproduction (1). Moreover, ISH was recently used
to show that estradiol increased kisspeptin cell number in the
ventral hypothalamus, where the zfkiss2 neurons are homologous
to the estrogen-sensitive kiss1 hypothalamic neurons in medaka
(17, 22, 30). Similarly to mammals, zebrafish have two forms of
GnRH: GnRH2 is localized to the midbrain tegmentum, and
GnRH3 (considered to be the hypophysiotropic form) is located
at both the olfactory bulb terminal nerve and the preoptic area
(31). Because in mammals kisspeptin and NKB are expressed in
the same neurons in the hypothalamic ARC, and play a key role
in physiological regulation of GnRH neurons (25), we tested the
effect of estradiol on the expression of genes along the GnRHkisspeptin
system. Estradiol treatment of prepubertal zebrafish
enhanced expression of key genes involved in reproduction
(gnrh3, kiss2, and kiss1) concomitantly with significantly increased
tac3a (Fig. 5A). In parallel, we also found significantly increased
expression of tac3ra, tac3rb, and kiss1ra (Fig. 5B). Both Tac3
receptors bound the zfNKBs (Fig. 4 A–F), and Kiss1ra was shown
previously to bind Kiss2, considered to be the more important
form, with higher affinity than Kiss1 (32). In mammals there is
strong evidence of sexual dimorphism of NKB neurons: larger
numbers of NKB neurons have been identified in ARC of ewes
than of rams (33). The transcription of NKB could be directly
altered by estrogen receptors, as sequences corresponding to the
estrogen responsive element and imperfect palindromic estrogen
responsive element have been reported upstream of the TAC3
gene transcriptional start site (8). In fish estradiol is involved in
both early oogenesis and the beginning of the first wave of vitellogenesis
that precede puberty (34), collaborating our finding
that tac3a expression peaked in prepubertal fish (Fig. 2B).
Moreover, increased levels of estradiol are a required characteristic
of both follicular growth and final oocyte maturation in
fish (3, 35), pointing toward the involvement of the piscine NKB
system in control of reproduction, probably in concert with
kisspeptin and GnRH.
In Vivo Effect of NKBs. We next tested the in vivo biological
function of zebrafish NKB peptides. Single intraperitoneal injection
of zfNKBa or zfNKF elicited significant LH secretion in
sexually mature female zebrafish (Fig. 5C). The magnitude of the
induced LH discharge was comparable with that observed in
response to GnRH. LH response to the hNKBR agonist, senktide,
or zfNKBb was less pronounced (Fig. 5C), in a similar way
to the order of potency obtained in the transactivation assay (Fig.
4). Our findings corroborate previous findings that activation of
NKB receptors evoked potent LH-secretory responses in
rodents, sheep, and monkey (4, 36–38).
10272 | www.pnas.org/cgi/doi/10.1073/pnas.1119165109 Biran et al.

A B C
SRE-Luc activity
(fold induction)
CRE-Luc activity
(fold induction)
4
3
2
1
0
. 0
hNKBR zfTac3ra zfTac3rb
-11 -10 -9 -8 -7 -6 -5
peptide (logM)
In conclusion, we provided detailed information on the organization
of the NKB systems in teleosts, including the splice tac3
(NKF). We also show that the zftac3a, unlike zftac3b, expression
increased toward puberty, increased in response to estradiol
treatment, caused increase in LH secretion, and was abundantly
expressed in the brain, notably in the hypothalamus. Tac3
receptors were expressed in the pituitary and gonads, suggesting
that these ligand-receptor pairs are likely involved in the control
of reproductive functions, during puberty or during diverse steps
of reproduction.
Materials and Methods
Animals. Wild-type zebrafish were purchased from a commercial supplier (A &
H). All experimental procedures were approved by the Hebrew University
Administrative Panel for Laboratory Animal Care.
Data Mining, Phylogenetic Analysis, and Chromosomal Synteny. The putative
Tac3 gene sequences were isolated from zebrafish by using a stepwise
evolutionary strategy, with the mouse Tac2 protein (NP_033338.2) as firststep
input, and extension to other genomes and ESTs, as described in the SI
Materials and Methods. Details of sequences, phylogenetic analyses and
synteny are presented in SI Materials and Methods.
4
3
2
1
0
. 0 -11 -10 -9 -8 -7 -6 -5
0
. 0 -11 -10 -9 -8 -7 -6 -5
0
. 0
D 12 E F
10
10
8
8
8
6
6
6
4
4
4
2
2
2
0
. 0
-11 -10 -9 -8 -7 -6 -5
peptide (logM)
3
2
1
0
. 0
-11 -10 -9 -8 -7 -6 -5
-11 -10 -9 -8 -7 -6 -5
peptide (logM)
zfNKBa
zfNKBb
zfNKF
hNKB
Senktide
zfNKBa
zfNKBb
zfNKF
hNKB
Senktide
Fig. 4. Ligand selectivity of the NKB receptors. Human NKBR (A and D) zebrafish Tac3ra (B and E) or zebrafish Tac3rb (C and F), each together with SRE-Luc
(A–C) or CRE-Luc (D–F). The cells were treated with various concentrations of human (hu) NKB: DMHDFFVGLM-NH 2; zebrafish (zf) NKBa: EMHDIFVGLM-NH 2;
zfNKBb: STGINREAHLPFRPNMNDIFVGLL-NH 2; or zfNKF: YNDIDYDSFVGLM-NH 2. Data are expressed as the increase in luciferase activity over basal activity.
Each point was determined in quadruplicate and is given as a mean ± SEM. (G) Ribbon representation of human and zebrafish NKBs structural model. The
PDB ID for the human structure is 1p9f.
A B C
Isolation of Zebrafish tac3 Ligands and Receptors. We designed specific primers
for cloning the putative zfTac3 ligands and receptors (Table S1). The
fragments were PCR-amplified from an adult zebrafish brain cDNA library
and were cloned into pCRII-TOPO vector (Invitrogen).
Tissue Distribution and Expression Profiles. Tissue distributions of zftac3a,
zftac3b, zftac3ra, and zftac3rb were determined by real-time PCR as previously
described (15). Tissue samples were collected from sexually mature postvitellogenic
female and milt-producing male zebrafish, total RNA extraction,
and cDNA samples were prepared as previously described (15). To study gene
expression of the NKB/NKBR system at different ages, 15 fish were randomly
sampled at ages 2, 4, 6, 8, and 12 wpf. The brain was removed, and the pubertal
stage classification was determined as described previously (15). Elongation
factor 1α (39) was used as a reference gene. The primer sequences, R 2 values,
and slopes of the real-time PCR analyses, calculated by linear regression, are
presented in Table S1.
ISH Analysis of Embryos and Adults. ISH was conducted on embryos and adults
according to Mitani et al. (30) and Palevitch et al. (40) respectively, as detailed
in SI Materials and Methods.
Peptide Synthesis. Zebrafish NKBa (EMHDIFVGLM-NH2), NKBb (STGIN-
REAHLPFRPNMNDIFVGLLEMHDIFVGLM-NH2), and NKF (YNDIDYDSFVG-
LM-NH 2) were synthesized by the automated solid-phase method by
Fig. 5. Exposure to estradiol (18 nM) by immersion caused a significant increase in mRNA expression of ligands (A) or receptor (B) genes, in the brain of
juvenile zebrafish. sGnRHa, zfNKBa, zfNKBb, zfNKF, or senktide (see legend to Fig. 4 for details), were injected intraperitoneally to mature zebrafish and
blood was collected 6 h thereafter (C). Hormone values are means ± SEM. Statistical significance vs. corresponding control values: **P < 0.01; *P < 0.05.
Biran et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10273
G
AGRICULTURAL
SCIENCES

applying Fmoc active-ester chemistry, purified by HPLC to >95% purity
(GeneMed), and the carboxyl terminus of each peptide was amidated.
Receptor Transactivation Assay and Protein Structure Modeling. To study the
signaling pathways of the recently identified zfNKBs, the entire coding
regions of zftac3ra and zftac3rb were inserted into pcDNA3.1 (Invitrogen).
The cDNA clone for hNKBR was obtained from the Missouri S&T cDNA Resource
Center (www.cdna.org), and the luciferase assay was conducted (15).
Protein structures of zebrafish NKBa, NKBb, or NKF were predicted on the
I-Tasser server (41, 42).
In Vivo Experiments. Two-month-old prepubertal zebrafish were exposed to
E 2 (Sigma) at a concentration of 5 μg/L (18 nM), or to vehicle (ethanol 100%
to a final concentration of 33 μL/L), by immersion for 3 d. This relatively low
concentration was used because the natural E 2 concentration in the plasma
of adult vitellogenic female zebrafish was determined as 3–4 ng/mL (43).
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Each group of 10 fish was kept in a 3-L aquarium with water maintained at
28 °C and replaced daily. After 3 d, fish were anesthetized and brains were
removed. RNA extraction, reverse transcription of RNA and real-time PCR
were carried out as previously described (15).
Adult female zebrafish were injected intraperitoneally with 20 pmol/g
body weight of either saline, salmon GnRH analog [(D-Ala 6 ,Pro 9 -Net)mammalian
GnRH], zfNKBa, zfNKBb, zfNKF, or senktide (n =8fish per
group). Six hours postinjection the fish were bled (44). Blood was
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−20 °C. Plasma from two animals were pooled (within each treatment)
and analyzed for LH using ELISA for other Cyprinidae, carp, according to
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ACKNOWLEDGMENTS. This research was funded by United States-Israel
Binational Science Foundation Grant 2005096, and by Binational Agricultural
Research and Development Fund MD-8719-08.
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10274 | www.pnas.org/cgi/doi/10.1073/pnas.1119165109 Biran et al.

Supporting Information
Biran et al. 10.1073/pnas.1119165109
SI Materials and Methods
Data Mining, Phylogenetic Analysis, and Chromosomal Synteny. The
putative Tac3 gene sequences were isolated from zebrafish using
a stepwise evolutionary strategy. First, a protein blast was run
using the mouse Tac2 protein (NP_033338.2) as input. The lowest
scoring sequence that still had the neurokinin signature of
FxGLM (XP_001365310, Monodelphis domesitca) was used as
input for a genomic search against the platypus genome. One
region was found (ornAna1, Contig39139:6403–6445), which
translated to GDMHDFFVGLMGKR. This sequence was used
as input to translated blast of fish DNA and EST sequences.
Several ESTs were found that were built into two consensus
contigs (tac3a and tac3b), aligning to two distinct regions in the
zebrafish genome (chr 23 and 6, respectively). The cDNAs were
cloned based on the EST contigs, and the sequences have been
submitted to GenBank (tac3a: JN392856; tac3b: JN392857).
Because of the fact that these genes had more than one putative
active peptide, and the difference in sequence between the
mammalian and fish sequences, it was decided to isolate as many
fish and mammalian sequences as possible to be sure of the
identification of orthology as opposed to paralogy. An additional
27 fish tac genes were found, using the zebrafish protein as an
input to tblastn against the nucleotide and EST databases at the
National Center for Biotechnology Information; 23 of the fish
and 4 of the nonfish (alligator, frog, chicken, and pig) tacs were
built from ESTs, and these have been submitted to GenBank
with the following accession numbers BK008100–BK008126.
The human TAC genes have several isoforms, the ones that
didn’t cause species-specific gaps or extensions in preliminary
alignments were chosen for the final alignments and trees. The
tac3ra and tac3rb sequences were cloned based on the predicted
gene sequences in GenBank. The tac3rc was found in a genomic
search, and once again, more sequences were then sought to
ensure proper classification of the receptors. Additional 19 fish
tac3 receptors were found, many with genomic predictions. The
genomic predictions were manually created, and 13 were improved
and deposited in GenBank (accession nos.: BK008087–
BK008099). Phylogenetic analysis was performed using both
neighbor-joining (ClustalW 2.1) and maximum likelihood (Phylip
3.69, ProML (1) on the basis of alignments performed both by
ClustalW (2) and Muscle (3.8.31) (3). The topologies were the
same in all combinations of multiple alignment and tree construction
programs. Bootstrapping of 1,000 was performed on
the neighbor-joining and of 100 on the maximum-likelihood
trees. Trees were visualized with FigTree 1.3.1 (4). Synteny was
observed using the University of California at Santa Cruz genome
browser and the following genome builds: human: hg19
(5); zebrafish: Zv9/danRer7; medaka: oryLat2; Tetraodon: tet-
Nig2; Fugu: fr2.
In Situ Hybridization Analysis of Embryos and Adults. Adult wild-type
zebrafish were maintained at 27–28 °C on a 14 h:10 h (light:dark)
1. Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. (Distributed by
the author, Department of Genome Sciences) (University of Washington, Seattle, WA).
2. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:
2947–2948.
3. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32:1792–1797.
4. Rambaut A (2007) FigTree, a graphical viewer of phylogenetic trees. Available at:
http://tree.bio.ed.ac.uk/software/figtree/. Accessed July, 2011.
cycle, and fed a range of dry fish food and artemia twice daily.
Embryos were generated from natural crosses by breeding the
male/female pairs.
A fragment of zebrafish tac3a (Table S1) was cloned into
pGEM-T easy vector. Antisense and sense riboprobes were synthesized
(Dig RNA labeling kit; Roche Diagnostics) using SpeI or
NcoI linearized (respectively) plasmids as templates and wholemount
in situ hybridization was conducted according to ref. 6.
We used 0.6–0.8 g sexually mature zebrafish. Fish were first
anesthetized with MS-222 (Sigma) and decapitated. Brains were
removed and fixed with 4% (wt/vol) paraformaldehyde in PBS
for 6 h at 4 °C and immersed in PBS containing 20% (wt/vol)
sucrose and 30% (vol/vol) optimal cutting temperature (OCT)
(Sakura) for about 24 h. Brains were then embedded in OCT,
frozen in liquid nitrogen, sectioned frontally at 12 μm on
a cryostat at −18 °C, and mounted onto Superfrost plus glass
slides (Thermo Scientific).
To detect tac3a and tac3b mRNA, we prepared a specific digoxigenin
(DIG)-labeled riboprobe for tac3a (position 122–360
in GenBank accession no. JN392856), tac3b (position 17–246 in
GenBank accession no. JN392857). Probes were prepared using
DIG RNA labeling kit (SP6/T7; Roche).
In situ hybridization was generally performed as described in ref.
7, with slight modifications. Briefly, sections were washed twice in
PBS, treated with 1 μg/mL protease K for 15 min at 37 °C,
postfixed with 4% paraformaldehyde in PBS for 15 min, and incubated
with 0.25% acetic anhydride in 0.1 M triethanolamine for
10 min. Then the sections were prehybridized at 58 °C for 1 h in
hybridization buffer containing 50% (vol/vol) formamide, 5× saline
sodium citrate (SSC), 0.12 M phosphate buffer (pH 7.4), 100
μg/mL tRNA. Slides were incubated at 58 °C overnight in the
same solution containing 1 μg/mL denatured riboprobe. We used
diethyl pyrocarbonate-treated water for the preparation of all
solutions for treatment before hybridization.
After hybridization, sections were washed twice with 50%
formamide and 2× SSC followed by two washes of 2× SSC and
two washes of 0.5× SSC for 15 min each at 58 °C. Slides were
immersed in DIG-1 (0.1 M Tris-HCl, 0.16 M NaCl, and 0.1%
Tween 20) for 5 min, 1.5% (vol/vol) blocking reagent with DIG-1
for 30 min, and DIG-1 for 15 min, and then incubated with an
alkaline phosphatase-conjugated anti-DIG antibody (diluted
1:1,000 with DIG-1; Roche) for at least 2 h. Sections were
washed with DIG-1 twice for 15 min each, and DIG-3 (0.1 M
Tris-HCl, pH 9.5; 0.1 M NaCl; 0.05 M MgCl2) for 5 min. Sections
were then treated with a chromogenic substrate NBT/BCIP
stock solution (Roche) diluted 1:250 (vol/vol) in DIG-3 until
a visible signal was detected. Sections were immersed in a reaction
stop solution (10 mM Tris-HCl, pH8.0; 1 mM EDTA,
pH8.0) to stop the chromogenic reaction. Sections were then
dehydrated, covered using ClearMount Mounting Solution (Invitrogen)
and examined using light microscopy.
5. Kent WJ, et al. (2002) The human genome browser at UCSC. Genome Res 12:996–1006.
6. Palevitch O, et al. (2007) Ontogeny of the GnRH systems in zebrafish brain: In situ
hybridization and promoter-reporter expression analyses in intact animals. Cell Tissue
Res 327:313–322.
7. Mitani Y, Kanda S, Akazome Y, Zempo B, Oka Y (2010) Hypothalamic Kiss1 but not
Kiss2 neurons are involved in estrogen feedback in medaka (Oryzias latipes).
Endocrinology 151:1751–1759.
Biran et al. www.pnas.org/cgi/content/short/1119165109 1of9

A
1 ccctgtctctgtgtcttgtctgatagatagtccatcacaaaaggatt
107 taactctccaagcagaaactggaactgagctcttttctacagatctacatcctcagctca
167 gagtaatctgtaaagatgtaccgtggacttgtgttactgttcttggttttggtgctggaa
M Y R G L V L L F L V L V L E
247 actcgatggagtgagtcgagctgtcagcagtcagagtctcaaagatcagtttcaagcgag
15
T R W S E S S C Q Q S E S Q R S V S S E 35
307 agtccaagttttcggatgtcgactcataacttgctgaagaggtataatgacatagattat
S P S F R M S T H N L L K R Y N D I D Y 55
367 gacagtttcgtcggattaatggggcgcagaaacgccgaaacagatgatataccaccccaa
D S F V G L M G R R N A E T D D I P P Q 75
427 cgtaaaagggaaatgcacgatatctttgttggactcatgggtcgacgaagcgctgaacct
R K R E M H D I F V G L M G R R S A E P 95
487 gaatccggacgtcaatggaggaaagagtacccagaaccaagcggaggaatcttcttcaac
E S G R Q W R K E Y P E P S G G I F F N 115
547 aaatgcaaactgaggtttcgtcgtgggttatag
K C K L R F R R G L - 125
B
1 ctgaacagcatcctgttaacaccagctcatacgagtacttggataaggtgtgcgaggatg
M
61 tcctgcggctggctgctcgcgctgctcgtccacgtgctgctgctgctcgcgtgcccgaga
1
S C G W L L A L L V H V L L L L A C P R 21
121 ctctcgcggagcgccctcgactactccttcactgacaacagcgacgcccagccggagcgc
L S R S A L D Y S F T D N S D A Q P E R 41
181 tacgacaaacgatatgatgatattgattacgacagtttcgtcggcctgatgggcaggagg
Y D K R Y D D I D Y D S F V G L M G R R 61
241 agcacaggaataaatcgtgaggcacatttgccatttagaccgaatatgaatgacatcttt
S T G I N R E A H L P F R P N M N D I F
301 gtcggactgttaggacggagaaacactttgtcgtctatgagaaaagaaaggagagggaac
81
V G L L G R R N T L S S M R K E R R G N 101
361 attttcttcaaggatggaagactgaggttttgctgtggtgtatga
I F F K D G R L R F C C G V - 115
Fig. S1. Nucleotide and deduced amino acid sequences of the zebrafish tac3a (A) and tac3b (B). Numbering of the deduced amino acid sequences begins with
the first methionine of the ORF to the right of each line. Nucleotide numbers are to the left of each line. The start and stop codons are shaded in gray, signal
peptide amino acids are underlined (as defined by SignalP program analysis http://www.cbs.dtu.dk/services/SignalP/), and the putative secreted peptides are
underlined (nucleotides) and bold (amino acids). These sequences have been deposited in the GenBank nucleotide database under accession numbers JN392856
and JN392857, respectively. Prediction of peptides cleavage sites was conducted using NeuroPred application (1).
1. Southey BR, Amare A, Zimmerman TA, Rodriguez-Zas SL, Sweedler JV (2006) NeuroPred: A tool to predict cleavage sites in neuropeptide precursors and provide the masses of the
resulting peptides. Nucleic Acids Res 34(Web Server issue):W267–W272.
Biran et al. www.pnas.org/cgi/content/short/1119165109 2of9

Fig. S2. Unrooted phylogenetic tree of neurokinin (A) or neurokinin receptor (B) sequences generated with both neighbor-joining (ClustalW 2.1) and
maximum likelihood (Phylip 3.69, ProML) on the basis of alignments performed both by ClustalW and Muscle (3.8.31). Trees were visualized with FigTree
(1.3.1). The sequences identified in this study are marked in bold. Gene nomenclature has been standardized to tac3, and species are indicated for illustration
and comparison. Numbers at nodes indicate the bootstrap values from 1,000 replicates. (Scale bar indicates the substitution rate per residue.) GenBank accession
numbers: Ligands: Danio rerio, zebrafish, tac3a (JN392856); zebrafish, tac3b (JN392857); Pimephales promelas, fathead minnow tac3 (BK008100);
Ictalurus punctatus, channel catfish, tac3 (BK008101); Salmo salar, Atlantic salmon, tac3a (BK008102); Atlantic salmon, tac3b (BK008103); Dissostichus mawsoni,
Antarctic toothfish, tac3 (BK008104); Sebastes rastrelliger, grass rockfish, tac3 (BK008105); Gadus morhua, Atlantic cod, tac3 (BK008107); Boreogadus saida,
Legend continued on following page
Biran et al. www.pnas.org/cgi/content/short/1119165109 3of9

Arctic cod, tac3 (BK008109); Xenopus tropicalis, western clawed frog, tac3 (BK008110); Osmerus mordax, rainbow smelt, tac3 (BK008111); Oryzias latipes,
medaka, tac3 (BK008114); Alligator mississippiensis, American alligator, tac3 (BK008115); Dicentrarchus labrax, European seabass, tac3 (BK008116); zebrafish,
tac1 (BK008124); Gallus gallus, chicken, tac1 (BK008126); Sebastes rastrelliger, grass rockfish, tac1 (K008106); Oncorhynchus mykiss, rainbow trout, tac1
(BK008119); Salvelinus fontinalis, brook trout, tac1 (BK008120); Anoplopoma fimbria, sablefish tac1 (BK008121); Sebastes caurinus, copper rockfish, tac1
(BK008122); Carassius auratus goldfish tac1 (AAB86991.1); rainbow smelt, tac4a (BK008112); rainbow smelt, tac4b (BK008113); Gasterosteus aculeatus, threespined
stickleback, tac4 (BK008117); rainbow trout, tac4 (BK008118); Sus scrofa, pig, Tac4 (BK008123); zebrafish tac4 (BK008125); Arctic cod, tac4 (BK008108);
zebrafish, tac 4 (BK008125); catfish tac1 (NP_001187697); salmon tac1a (ACI67317); salmon, tac1b (ACI68385); frog, tac1 (NP_001165757.1); Japanese medaka,
tac1 (BAH03329); rainbow smelt, tac1 (ACO10148.1); human, TAC1g (NP_054703.1); human, TAC3a (NP_037383.1); human, TAC4a2 (NP_001070974.1); rabbit,
Tac4 (NP_001075634.1); mouse, Tac4 (NP_444323.1); rat, Tac4 (NP_758831.1); mouse, Tac1 (AAI44738.1); cow, Tac1 (AAI42366.1); cow, Tac3 (NP_851360.1); pig,
Tac3 (NP_001007197.1); mouse, Tac3 (NP_033338.2); rabbit, Tac1 (NP_001095168.1). Receptors: zebrafish, tac3ra (JF317292); zebrafish tac3rb (JF317293); zebrafish
tacr3c (XP_002666594); Japanese medaka, tac3ra (BK008087); Japanese medaka, tacr3b (BK008088); Takifugu rubripes, fugu, tacr3a (BK008092); fugu
tacr3b (BK008093); Tetraodon nigroviridis, spotted green pufferfish tacr3a (BK008096); spotted green pufferfish, tacr3b (BK008097); medaka, tacr1a
(BK008089); medaka, tacr1b (BK008090); fugu, tacr1a (BK008095); spotted green pufferfish, tacr1a (BK008099); medaka, tacr2 (BK008091); fugu, tacr2
(BK008094); spotted green pufferfish, tacr2 (BK008098); zebrafish, tacr1a (XP_001343073); zebrafish, tacr1b (XP_692469); zebrafish, tacr2 (XP_001341981.1);
human, TACR1 (NP_001049.1); human, TACR2 (NP_001048.2); human TACR3 (NP_001050); chicken, tacr3 (XM_001232173); chicken, tacr2 (XP_001232177.1);
chicken, tacr1 (NP_990199.1); Neoceratodus forsteri, lungfish, tacr1 (AAZ82194.1); fugu, tacr1b (AAQ02694.1); Octopus vulgaris, octopus, tkr (BAD93354.1);
spotted green pufferfish, tacr1b (CAG05392.1); frog, tacr1 (NP_001106489.1); frog, tacr3 (XP_002934808.1); cow, Tacr3 (NP_001179262.1); cow, Tacr1
(XP_002691234.1); cow Tacr2 (NP_776894.1); rabbit, Tacr3 (NP_001075524.1); rabbit, Tacr1 (XP_002709748.1); rabbit, Tacr2 (NP_001075800.1); mouse, Tacr3
(NP_067357.1); mouse Tacr2 (NP_033340.3); mouse, Tacr1 (NP_033339.2); Caenorhabditis elegans, C. elegans, tkr (NP_500930.1); Ciona intestinalis, ciona, tkr
(NP_001027809.1).
Biran et al. www.pnas.org/cgi/content/short/1119165109 4of9

A
1 tatatctaaatatttctggacatttctggcatggcacagtcacagaacggatctaaccta
M A Q S Q N G S N L 10
61 acggggaactttacgaaccagttcgtgcagccgccgtggcgcgtggcgctgtggtcggtg
T G N F T N Q F V Q P P W R V A L W S V
121 gcgtacagctccatcctggcgatcgcggtgttcgggaatctgatcgtcatgtggatcatt
A Y S S I L A I A V F G N L I V M W I I 50
181 ctggctcataagcggatgcgaaccgtcaccaactactttctgctcaacctggcgttttcg
L A H K R M R T V T N Y F L L N L A F S
241 gacgcctccatggccgccttcaacactttgatcaatttcgtttacgccacacacggagat
D A S M A A F N T L I N F V Y A T H G D 90
301 tggtatttcggagaagcctactgcaaatttcacaactttttccccgtcacctccgtgttt
W Y F G E A Y C K F H N F F P V T S V F
361 gccagcatttactccatgagcgcaatcgcagtcgacaggtacatggccatcatccatcct
A S I Y S M S A I A V D R Y M A I I H P 130
421 ctgaaaccacgactctcggcgacggccaccaaagtgatcattgtgtgtatctgggtgctc
L K P R L S A T A T K V I I V C I W V L
481 gctgtggttttggccttcccgctgtgtttcttttcaaccatcaaaaaactgcccaaacga
A V V L A F P L C F F S T I K K L P K R 170
541 actctctgctatgttgcctggccgagaccttcagaagaccctttcatgtatcatatcatt
T L C Y V A W P R P S E D P F M Y H I I
601 gtggcgatgctggtgtatgttctgccgctggtggtcatgggtatcaactacactattgtc
V A M L V Y V L P L V V M G I N Y T I V 210
661 ggattgaccctttggggaggagagattcctggtgactcctcagacaactatcagggccag
G L T L W G G E I P G D S S D N Y Q G Q 230
721 ctcagggccaagaggaaggtggtgaaaatgatgatcattgtagtggtgacctttgccttc
L R A K R K V V K M M I I V V V T F A F
781 tgctggctgccgtaccatgtgtatttcctggtgacgggattgaacaagcagctggctcga
C W L P Y H V Y F L V T G L N K Q L A R 270
841 tggaagttcattcagcagatctatctgtccatcatgtggcttgccatgagctccaccatg
W K F I Q Q I Y L S I M W L A M S S T M
TM7
901 tataaccccattatttactgctgcctaaacagccggtttcgcgctggcttcaaacgtgtt
Y N P I I Y C C L N S R F R A G F K R V 310
961 ttccgctggtgcccttttgtgcaagtctctgactatgacgagcttgagctgcgggctatg
F R W C P F V Q V S D Y D E L E L R A M 330
1021 aggcataaagtagcgcggcagagcagcatgtacacaatgtcacgaatggagaccaccgta
R H K V A R Q S S M Y T M S R M E T T V 350
1081 gtcaccgtgtgtgacccatcagagccaaacacccagccaggccggaagagcctgcttaac
V T V C D P S E P N T Q P G R K S L L N 370
1141 caccaccaccaccacaacggctgctccaacccagccaagagcaaagaaataacatacatg
H H H H H N G C S N P A K S K E I T Y M 390
1201 caaagcgacccgaaggaggaattctcctgagaaggacttttgatgtaagattcacac
Q S D P K E E F S * 399
1261 tgaagcattaag
TM4
TM3
TM6
TM5
TM2
TM1
30
70
110
150
190
250
290
B
1 tttaagaaggatttcacggttaaatctaccatggctggtcctcagagcggctcaaatgtg
M A G P Q S G S N V 10
61 acgcgtaatttcacaaatcagttcgtgcagccgccgtggcgggtcgccgtctggtcggtc
T R N F T N Q F V Q P P W R V A V W S V
121 gcttacagctcggtgctcgcggtcgccgtgttcggaaacctcattgttatttggatcatt
A Y S S V L A V A V F G N L I V I W I I 50
181 ttggcccataaacggatgcgcaccgtcaccaactattttttgctcaacctggcgttttcc
L A H K R M R T V T N Y F L L N L A F S
241 gacgcgtccatggccgccttcaacacgctcatcaacttcatttacgccacgcacggagag
D A S M A A F N T L I N F I Y A T H G E 90
301 tggtacttcggagaggtttactgcaagttccacaacttcttccctgtgaccgccgtgttt
W Y F G E V Y C K F H N F F P V T A V F
361 gccagcatttactccatgacagcgattgcagtcgacaggtacatggccataatacatcct
A S I Y S M T A I A V D R Y M A I I H P 130
421 ctgaagcctcgtctgtcagccacggctactaaagtggtgattgtctgtatttgggcactg
L K P R L S A T A T K V V I V C I W A L
481 gcagtgattttggctttcccgctgtgtttctactccaccacgagaaccatgcctcgcaga
A V I L A F P L C F Y S T T R T M P R R 170
541 accatttgctacgtcgcctggccaagaccggctgaggattcattcatgtatcacatcata
T I C Y V A W P R P A E D S F M Y H I I
601 gtgacggtgctggtctacatgctgcccctagtggtgatgggcatcacctacactatagtc
V T V L V Y M L P L V V M G I T Y T I V 210
661 ggggttacactttggggaggagagattcctggagactcgtcggacaattatgttggacag
G V T L W G G E I P G D S S D N Y V G Q 230
721 ctacgtgctaagaggaaggtggtgaagatgatgatcgtggtggtggtgactttcgccctc
L R A K R K V V K M M I V V V V T F A L
781 tgctggttgccgtatcacatctatttcatcgtaacaggcctgaacaaacgcctgaacaag
C W L P Y H I Y F I V T G L N K R L N K 270
841 tggaagtccatccagcaggtgtatctgtctgtgctgtggctggccatgagctccaccatg
W K S I Q Q V Y L S V L W L A M S S T M
TM7
901 tacaaccccatcatttactgctgtctgaatggcagatttcgcgcgggcttcaagcgggcc
Y N P I I Y C C L N G R F R A G F K R A 310
961 ttcaggtggtgtcccttcattcaggtgtccagctatgacgaactggaactccgtcccacc
F R W C P F I Q V S S Y D E L E L R P T 330
1021 cggctccatccacgcaaccagagcagcatgtgcaccctgtcccgcgtcgacaccagcctc
R L H P R N Q S S M C T L S R V D T S L 350
1081 catggtgaggacccacgacgcagtcagcggaagagcaccaaatcccaatgtctggtggag
H G E D P R R S Q R K S T K S Q C L V E 370
1141 gtcagagacgaaaacacaccagccacgaaactctgtcttaatagagatcaagcgttcgca
V R D E N T P A T K L C L N R D Q A F A 390
1201 acagagcagctcagctgaagagtgcatgattatagaattaaagcatattctaaaaatgca
T E Q L S * 395
1281 tttaagtgtgcattgagactcaaagctgcagcgtgatgaggttacactgcctccaagt
Fig. S3. Nucleotide and deduced amino acid sequences of the zebrafish tac3ra (A) and tac3rb (B). Numbering of the deduced amino acid sequences begins
with the first methionine of the ORF to the right of each line. Nucleotide numbers are to the left of each line. The start and stop codons are shaded in gray.
These sequences have been deposited in the GenBank nucleotide database under accession numbers JF317292 and JF317293, respectively. Open circles, putative
N-glycosylation sites; open squares, putative protein kinase C phosphorylation sites; open triangle, putative cAMP and cGMP-dependent protein kinase
phosphorylation site; open diamonds, putative Casein kinase II phosphorylation sites; open trapezoid, putative tyrosine kinase phosphorylation site; open
octagons, putative N-myristoylation sites. Predicted transmembrane domains (TM1–TM7) are underlined; arrowheads indicate the exon-intron boundaries.
Biran et al. www.pnas.org/cgi/content/short/1119165109 5of9
TM3
TM4
TM6
TM2
TM1
TM5
30
70
110
150
190
250
290

A B
Zfish chr23
Human chr12 Zfish chr6 Zfish chr23
Synteny to chr 12 con nues
28M
mll2/3
C D
CISD2
NHEDC1
NHEDC2
BDH2
CENPE
TACR3
acvr1b
acvrl1
arhgef25
sp5l
slc26a10
b4galnt1a
c1galt1a
tac3a
28.4MM
znf385a
birc5b
neurod4
myl6b
c1ql4
Synteny to chr x
103.75
104.7 44.25
Chr4
Human
28.7M
44.00
Chr1
zebrafish
suclg1
cisd2
nhedc2 h d 2
bdh2
54.76M
18.97
tac3r tac3r
cnga cnga
19.2
Chr1
medaka
ZNF385A
10 genes
55.41M
NEUROD4
57.4M
TTAC3
29 genes +
14 OR genes
56.54M
MYL6B
12 gen geness
SSTAT2
APOF
16 genes
21 genes
suclg1
cisd2
nhedc2
bdh2
39.07M
39.11M
tac3b
c1galt1b
os9
39.2M
stat2
apof
b4galnt1a
arhgef25
ARHGEF25 A
SLC26A10
B4GALNT1
OS9
58.12M
TAC3
Syntenic gene
Non-syntenic gene
Genes missing in fish in this region
TAC3R
Syntenic gene
Non-syntenic gene
glb1
cldnd
acy3.2
acy3.1
tac3rb
mll2/3
acvr1b
acvrl1
arhgef25
sp5l
52.95M
Chr1
zebrafish
28M
slc26a10
b4galnt1a
c1galt1a
tac3a
28.4M
znf385a
birc5b
neurod4
myl6b
c1ql4
28.7M
acsl6
ccdc111
tac3rb
6.48M
53.05M 6.5M
ccdc111
abca1a aacy3
cldnd
glb1
acsl6
6.51M 306.21M
Chr18
tetroadon
Medaka chr7
12.1M
znf385a
12.25M
306.17M
ChrUn
fugu
c1ql4
myl6b
neurod4
birc5b
tac3
c1galt1a
b4galnt1a b
slc26a10
12.43M
sp5l
arhgef25
acvrl1
acvr1b
mll2/3
glb1
cldnd
acy3
tac3rb
Zfish chr6
39.07M
stat2
apof
39.11M
tac3b
c1galt1b
os9
39.2M
b4galnt1a
arhgef25
TAC3
Syntenic gene
Non-syntenic gene
TAC3R
Syntenic gene
Non-syntenic gene
Fig. S4. Chromosomal locations of zebrafish tac3 and tac3 receptors in various vertebrate species. Genes adjacent to tac3 and tac3r in different genomes are
shown. The genes are named according to their annotation in the human genome. (A) Comparison between zebrafish tac3a and human. (B) Comparison
between zebrafish tac3b and medaka tac3. (C) Comparison between human, zebrafish and medaka tac3ra. Stickleback had identical synteny, and fugu and
green spotted pufferfish differ with dctd in place of suclg1 upstream of tac3ra. (D) Comparison between zebrafish, green spotted pufferfish, and fugu tac3rb.
Biran et al. www.pnas.org/cgi/content/short/1119165109 6of9

Fig. S5. Localization by real-time PCR of zebrafish tac3a, tac3b, tac3ra, and tac3rb mRNA in various tissues. The relative abundances of the mRNAs were
normalized to the amount of elongation factor 1-α (ef1α) by the comparative threshold cycle method, where the comparative threshold reflects the relative
amount of the transcript. Ant. Intest+ panc., anterior intestine and pancreas; post. Intes, posterior intestine. We found low levels of mRNA expression of all
four transcripts in the liver, retina, and adipose tissue. However, relatively high mRNA levels of tac3a and tac3rb were expressed in the gills, tac3a in the
posterior intestine and tac3ra in the muscle.
Table S1. Primers used for cloning, quantitative real-time PCR, and in situ hybridization
Primer Position 5′ to 3′ sequence Slope R 2
Application
zf ef1a-1237F 1,237 aagacaaccccaaggctctca −3.708 0.999 Quantitative
zf ef1a-1419R 1,491 cctttggaacggtgtgattga
real-time PCR
GnRH2-36F 36 gctgatgctgtgtctgagt −3.337 0.994
GnRH2-196R 196 tgtcttgaggatgtttcttc
GnRH3-47F 47 gtgtgttggaggtcagtct −3.103 0.997
GnRH3-208R 208 tccacctcattcactatgtg
kiss1-10F 158 acagacactcgtcccacagatg −3.468 0.991
kiss1-210R 357 caatcgtgtgagcatgtcctg
kiss2-137F 137 gcgttttctgtcaatggag −3.475 0.998
kiss2-317R 317 cgcttcgtttctctttccg
kiss1ra-856F 856 cctaacttcaaggccaac −3.424 0.987
kiss1ra-1095R 1,095 cctctcagtgttgctttc
kiss1rb-755F 755 agacgtcatcggagcgtg −3.305 0.954
kiss1rb-1041R 1,041 cctccttttgaagatcagaggac
zf tac3a-F29 29 tggttttggtgctggaaacc −3.513 0.997
zf tac3a-R191 191 tctgtttcggcgtttctgc
zf tac3b-F86 86 ctccttcactgacaacagcgac −3.239 0.988
zf tac3b-R246 246 gtttctccgtcctaacagtccg
zf tac3ra F154 154 gctcataagcggatgcgaac −3.502 0.969
zf tac3ra R334 334 tggcaaacacggaggtgac
zf tac3rb F343 343 tccatgacagcgattgcagt −3.322 0.964
zf tac3rb R523 523 cgtagcaaatggttctgcgag
zf tac3a F-122 −122 ccctgtctctgtgtcttgtctg In situ
zf tac3a R360 360 gcctataacccacgacgaaac
hybridizaiton/
zf tac3b F-17 −17 ggataaggtgtgcgaggatg
Cloning
zf tac3b stop 382 tcatacaccacagcaaaacctcag
zf tac3Ra start 1 atggcacagtcacagaacgg Cloning
zf tac3Ra stop 1,180 tcaggagaattcctccttcg
zf tac3Rb start 1 atggctggtcctcagagcgg
zf tac3Rb stop 1,161 tcagctgagctgctctgttgc
Biran et al. www.pnas.org/cgi/content/short/1119165109 7of9

Table S2. Percent amino acid sequence identities (black) and similarities (red) among Tac3 of different species as determined by EMBOSS*
Stretcher alignment tool
Zebrafish
Tac3a
Zebrafish
Tac3b
Human
Tac3
Sheep
Tac3
Mouse
Tac2
Salmon
Tac3a
Salmon
Tac3b
European
Seabass
Tac3
Medaka
Tac3a
Rainbow
Smelt
Tac3
Zebrafish Tac3A
(cypriniformes)
— 35.7 25 18.1 24.8 55.3 43.9 27.8 52 59.2 50 31.8 25.4
Zebrafish Tac3B
(cypriniformes)
45.2 — 18.2 19.7 18.9 40.2 40.4 30.4 36.3 40.6 38.3 30.7 20.5
Human Tac3 41.7 34.7 — 55.6 61.1 24.2 24.4 13.2 25.2 25.8 28.6 29.1 39.2
Sheep Tac3 37 38.5 66.7 — 66.7 23 18.9 14.2 24.6 23.4 26 34.9 38.5
Mouse Tac3 45 37.7 73 73.3 — 26.9 27.6 17.2 24.6 26 28.5 36.7 43.9
Salmon Tac3A
(salmoniformes)
71.2 50.8 43.2 37.8 41 — 38.2 23.6 56.1 65.9 57.2 40.2 28.6
Salmon Tac3B
(salmoniformes)
62.1 52.9 34.4 32.6 38.1 55.9 — 35.1 38.9 39.7 34.1 32.1 20.3
European
Seabass Tac3
(perciformes)
42.1 43.2 32.6 30 33.6 41.4 43.3 — 24.4 25.6 26.7 21.8 18
Medaka Tac3A
(beloniformes)
72.8 48.4 40.2 41.5 40.5 72 53.4 43 — 64.5 56.2 31.2 27.2
Rainbow Smelt
Tac3A
(osmeriforms)
74.4 48.4 43 39.1 42.5 77.3 54.2 41.4 78.2 — 63.4 36.4 29.6
Arctic Cod Tac3
(gadiforms)
68.9 49.2 42.1 38.9 43.8 69.6 54.1 40 70.8 73.3 — 34.6 23.4
Frog Tac3 53.5 40.9 48 47.3 50.8 60.6 49.3 39.1 55.5 60.5 54.9 — 36
Aligator Tac3 45.2 37.7 56 54.1 59.3 45.9 38.3 33.1 43.2 47.2 35.9 51.2 —
—, Same species, not applicable.
*http://www.ebi.ac.uk/Tools/psa.
Arctic
Cod
Tac3
Frog
Tac3
Aligator
Tac3
Table S3. Percent amino acid sequence identities (black) and similarities (red) among Tac3r of different species as determined by
EMBOSS* Stretcher alignment tool
Zebrafish
Tac3ra
Zebrafish
Tac3rb
Zebrafish
Tac3rc
Human
TAC3R
cow
Tac3r
Mouse
Tac3r
Chicken
Tac3r
medaka
Tac3ra
Medaka
Tac3rb
Tetraodon
Tac3ra
Tetraodon
Tac3rb
Zebrafish TacR3A
(cypriniformes)
— 74.9 60.7 56.4 57.3 59.2 60.6 73.8 68.4 71.8 65.7 60
Zebrafish TacR3B
(cypriniformes)
85 — 61.3 57.5 57.7 59.7 62 74.5 72.7 73.3 71.1 59.4
Zebrafish TacR3C
(cypriniformes)
75.9 71.7 — 50.5 51.3 53.1 52.2 61.9 61.3 62.4 58.6 52.3
Human TacR3 67.6 67.3 63.8 — 90.1 86 75.3 59.3 54.2 57.6 52.5 68.8
Cow TacR3 67.9 67.4 64.2 93.1 — 86 76.3 60.3 54 57.2 52.1 69.7
Mouse TacR3 70.4 69.5 64.9 90.5 90.3 — 76.6 59.7 54.6 59.7 54 70.3
Chicken TacR3 73.8 72.7 67.4 82.6 83.8 84.1 — 61.7 57.9 60.7 55.1 71.2
Medaka Tac3RA
(beloniformes)
84 84 71.4 69.5 70.2 71.7 74 — 70.4 83.3 67.2 61
Medaka Tac3RB
(beloniformes)
80.5 82 74.7 63.7 63.3 65 68.8 79.9 — 68.8 74.9 54.4
Tetraodon Tac3RA
(tetraodontiformes)
82.5 82.3 75.5 68 68.3 70.4 73.5 92 77.2 — 66.7 57.4
Tetraodon Tac3RB
(tetraodontiformes)
75.2 79 72.3 62.2 61.3 63.3 64.5 75.7 82.7 74 — 53.2
Frog TacR3 73.8 72.4 68.3 78.6 78.2 80.4 83.4 75.9 67.8 73.6 66 —
—, Same species, not applicable.
*http://www.ebi.ac.uk/Tools/psa.
Biran et al. www.pnas.org/cgi/content/short/1119165109 8of9
Frog
Tac3r

Table S4. EC 50 values (nM) of human and unique piscine NKBs
CRE or SRE NKBR zfTac3ra zfTac3rb huNK3R
NKBRs EC 50s CRE-Luc (nM)
zfNKBa 5.75 ± 1.45 4.55 ± 1.57 3.73 ± 1.25
zfNKBb 237.20 ± 134.20 519.20 ± 160.30 605.00 ± 132.00
zfNKF 4.94 ± 1.83 1.80 ± 1.55 4.36 ± 1.25
huNKB 12.94 ± 13.3 8.12 ± 1.56 4.71 ± 2.08
Senktide 48.95 ± 14.89 20.10 ± 15.1 17.41 ± 1.26
NKBRs EC 50s SRE-Luc (nM)
zfNKBa 0.50 ± 0.17 1.47 ± 1.66 0.49 ± 0.18
zfNKBb 8.96 ± 1.19 33.83 ± 14.7 204.40 ± 138.60
zfNKF 0.54 ± 0.13 0.36 ± 0.16 0.52 ± 0.18
huNKB 2.20 ± 1.49 0.82 ± 0.16 0.67 ± 0.16
Senktide 2.67 ± 1.44 2.72 ± 1.54 1.51 ± 1.63
Serum responsive element (SRE)-Luc was used as a reporter gene that follows PKC activation; cAMP responsive
element (CRE)-Luc was used to follow PKA activation. Mean ± SEM.
Biran et al. www.pnas.org/cgi/content/short/1119165109 9of9