Supplementary data
Materials and Methods
Zebrafish Strains
Embryos and adult fish were raised and maintained under standard laboratory conditions
1
. We used the following mutant and transgenic lines: prts403, prts404, prts416, clos5 2,
casta56 3, Tg(gutGFP)s854 2 and Tg(hs∆Tcf3-GFP) 4.
ENU mutagenesis and Screen
We screened 3578 F3 clutches from 652 ENU-mutagenized F2 families in the
Tg(gutGFP)s854 background for mutations affecting endodermal organ morphogenesis
(E.A.O., H.V., H.A.F., Duc Dong, Pia Aanstad, Takuya Sakaguchi, Michel Bagnat,
Chantilly Munson, Won-Suk Chung, Chong Shin, Silvia Curado, Ryan Anderson, Julie
Frantsve, Dimitris Beis, Thomas Bartman and D.Y.R.S., in preparation). Based on the
number of crosses per F2 family, we calculate that our screen surveyed 1035 mutagenized
genomes. The specific locus test, using the golden mutation, indicated a mutation rate of
approximately 0.3% per gene per mutagenized genome.
Mapping
We mapped the mutation to linkage group 8 using a standard set of SSLP markers
(modified after Knapik 1998). For fine mapping, 1112 single prt mutant embryos were
tested with SSLP markers in the critical interval (Fig. 2a). The primer sequence for zmarkers can be found at http://www.zfin.org/ and the following primers were designed
against CA-repeats within the determined interval: CA-1f ‘5 AAT CAT ACA TTT GGT
CGG TTA TTT-‘3; CA-1r ‘5 TAA GAG ACT GAG CAA CAA AGT TGA-‘3; CA-2f ‘5
GTA AAT CAT TCT TCT CCA GCT GTT-‘3; CA-2r ‘5 TCT ACT ATT TTG GAT
GCA TTT ACG-‘3; CA-3f ‘5 ATA ATT TTT GCT ACA CCT CAG GAC-‘3; CA-3r ‘5
ACA CAC ACT GAT AGT GCT CTT CC-‘3; CA-4f ‘5 CAG GAT GCT AAG TAA
GTG TGT GAG-‘3; CA-4r ‘5 GGC TTC CTG AAC ATC TAA TGT AAC-‘3. For
cloning and sequencing of the wnt2bb gene, total RNA was extracted from wildtype and
prt mutant embryos using Trizol (Invitrogen). cDNAs were generated by performing
RT-PCR using the Superscript Kit (Invitrogen). An initial fragment of about 850 bp was
amplified and extended via 3’RACE. PCR products from at least two independent PCR
reactions per mutant allele were sequenced and analysed.
The prt/wnt2bb nucleotide sequence was deposited at Genbank with the accession
number DQ231559.
Mosaic experiments
Donor embryos were injected with 2.5% rhodamine-dextran, and cells transplanted into
hosts at the 1000 cell stage. To target wild-type cells to the mesoderm, cells were
transplanted to the margin. Two independent groups of cells were transplanted into each
host embryo, 90 degrees apart from each other, to increase the likelihood of contributing
to the LPM. Homozygous prt mutant host embryos were obtained from homozygous prt
mutant fish.
Histochemical methods
Whole-mount in situ hybridisation was performed as previously described 5, using the
following probes: hhex 6, cp 7, sePb 8,9, gsc 10 and foxA1 11. A prt in situ probe (850 bp at
the 3’end of the gene) was cloned into the pCS2+ vector. The antisense probe was
generated by linearizing the plasmid with BamHI, followed by transcription with T3
polymerase.
We used the following antibodies: polyclonal antibody against Prox1 (rabbit, 1:1000;
Chemicon), monoclonal antibody against Islet1/2 (1:15; Developmental Studies
Hybridoma Bank, clone 39.4D5) and fluorescently conjugated antibodies from Molecular
Probes and Jackson Laboratories. Embryos were fixed with 2% PFA overnight at 4C.
Manual removal of the yolk was followed by 60 min incubation with 66 mM Tris pH 7.5,
5 mM MgClB, 1 mM 2-Mercaptoethanol and 50 U/ml DNAseI (Roche) at 37˚C, followed
by whole-mount antibody staining in PBST (1% Triton in PBS pH 7.3) with 5% Sheep
Serum and 1% DMSO added. Stained embryos were embedded in NuSieve GTG low
melting agarose and cut into 200 µm sections with a Leica VT1000S vibratome. Images
were acquired using a Zeiss LSM5 Pascal confocal microscope and a Zeiss/Bio-Rad
Radiance 2100 confocal microscope. The images acquired on the latter microscope were
processed using Volocity software (Improvision).
Injection - Morpholino antisense oligonucleotides and mRNA overexpression
Morpholinos were purchased from GeneTools LLC. We designed a morpholino
oligonucleotide targeted against an ATG upstream of the translational start site with the
following sequence: ‘5 GTGTGCCATATAAAAGTATTCCCCG-3’. Tg(gutGFP)s854
embryos were injected at the one cell stage with ~2ng of morpholino and assayed
between 48 - 54 hpf.
For generation of prt mRNA, a full-length fragment was cloned into pCS2+; the mRNA
was transcribed using the SP6 mMessage mMachine Kit (Ambion) upon linearization
with SacII. One cell stage embryos were injected with 50 – 150 pg of prt mRNA.
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Figure legends
Figure 1 Schematic of the phylogenetic relationship between zebrafish (Dr), mouse
(Mm), rat (Rn) and human (Hs) Wnt2 and Wnt2b genes. For the zebrafish proteins,
Wnt2bb shows 70% identity and 85% similarity with Wnt2, and 84% identity and 92%
similarity with Wnt2ba. Wnt2ba shows 70% identity and 84% similarity with Wnt2.
Figure 2 Linking liver formation and Wnt2bb to canonical Wnt signaling. a–i,
Interference with canonical Wnt signaling using the Tg(hs∆Tcf3-GFP) line and applying
one hour heat-shock at indicated time points led to defects in hepatic development.
Offspring of heterozygous carriers were used for these experiments, thus the observed
phenotype varies in strength between embryos. a-c, Application of a heat-shock at 16
hpf (14 somites) led to a complete absence (asterisk; c) or strong reduction (arrowheads;
b) of the hepatic domain as visualized by hhex expression at 25 hpf. Application of a
heat-shock at 21.5 hpf (25 somites) still led to a complete absence (asterisk; f) or strong
reduction (arrowhead; e) of the liver bud, whereas treating at 25 hpf only resulted in a
reduction of the liver bud (arrowheads; h, i) as assayed by foxA1 expression at 48 hpf.
[The actual stage when the embryos were assayed for marker expression is slightly more
advanced than indicated due to a more rapid development caused by the heat-shock.] a-i
Dorsal views; anterior to the top. j-k, Overexpression of prt mRNA in the early embryo
led to an increase of dorsal fates during gastrulation stages as shown by gsc expression.
Two different experimental embryos are presented (k, m), one dorsal (k) and one animal
pole view (m), respectively; each accompanied by a control embryo (j, l). Arrowheads
delineate the gsc expression domain, which is expanded between two- and threefold in
experimental embryos (k, m).