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

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