Supplementary Figure 1. Zebrafish neural tube morphogenesis. Rhodamine-phalloidin
stained transverse sections through the developing neural tube of fixed zebrafish embryos, at
the 1st to 6th somite level. The neural anlage has been outlined on the left, and cell morphologies
have been highlighted on the right. Commencing around the 5-somite stage, the neural plate (a)
begins to fold towards the midline, bringing apical surfaces from opposite sides of the neural
plate into apposition (b). These morphogenetic movements form the neural keel (c). The lumen
of the zebrafish neural tube is not established at this stage. Rather, a clear midline boundary
between apposing neuroepithelial layers is lost (c,d) and cells with medio-lateral polarity can be
observed intercalating across the midline. At the 12-somite stage (e), rhodamine-phalloidin
staining demonstrates the initiation of a midline boundary being established in the ventral region
of the neural primordium. This midline boundary develops in a ventral to dorsal direction during
neural rod stages (f). By the 20-somite stage, cells in apposing neuroepithelial layers begin
retracting their apical surfaces to establish the lumen of the neural tube (g).
2
Supplementary Figure 2. Maternal-zygotic trilobite phenotype. Lateral (a-c) and dorsal (a’c’) views showing WT (a), tri (b) and MZtri (c) embryos at 24 hpf. The tritk50f allele, which
contains a deletion in the tri coding sequence1, was used in these studies. The MZtri phenotype
is more severe than zygotic tri alone, displaying a broader medial-lateral axis (compare b’ and
c’), greater compression of somites, and a more severe yolk-extension phenotype (asterisks in
b and c).
1.
Jessen, J. R. et al. Zebrafish trilobite identifies new roles for Strabismus in gastrulation and
neuronal movements. Nat Cell Biol 4, 610-5 (2002).
2
3
Supplementary Figure 3. Generating slb;ppt germ line-replacement chimeras by 30 hpf
germ cell transplantation. Production of maternal zygotic mutant embryos through use of a
germ line replacement strategy has been previously described1. Donor embryos obtained from
slb/+;ppt/+ intercrosses were injected with 100 pg of GFP-nos1-3’UTR mRNA to mark the
primordial germ cell (PGC) population, and WT host embryos were injected with 2 ng of dead
end MO to eliminate the host PGC population. To increase the efficiency of transferring PGCs
from mutant donor embryos, donors were allowed to develop to 30 hpf and were then screened,
by morphology, for the slb;ppt double mutant phenotype. The presumptive gonad was dissected
from slb;ppt mutants and dissociated through digestion with 0.25% trypsin, 2.5% pancreatin,
1mM EDTA in PBS. GFP-positive slb;ppt germ cells were identified and then transplanted into
the margin of mid-blastula staged host embryos. Despite the heterochronic nature of PGC
transplantation, slb;ppt PGCs were able to repeat normal migration into the presumptive gonad
in 21% of host embryos (n=197). Fertile, adult fish were obtained with germ lines completely repopulated by transplanted slb;ppt PGCs. These germ line-replacement chimeras were mated to
generate large clutches of MZslb;MZppt mutant embryos.
1.
Ciruna, B. et al. Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc
Natl Acad Sci U S A 99, 14919-24 (2002).
3
4
Supplementary Figure 4. MZsilberblick;MZpipetail mutant phenotype. (a) The pptsk13 allele
is an ENU-induced A to T mutation at the second to last nucleotide of intron 2, which changes
the consensus 3’ splice site sequence between exon 2 and exon 3. Improper splicing of the
pptsk13 transcript introduces a stop codon just 4 amino acids beyond the end of exon 2, leading
to early truncation of the Ppt protein. pptsk13 is truncated at least 206 amino acids more Nterminal than any of the other well characterized ppt mutations, and is a presumed null allele.
The pptsk13 allelle was used in all of our studies. (b-g) Lateral and frontal views demonstrating
the 30 hpf phenotypes of (b) a WT embryo, (c) a zygotic slb mutant, (d) a zygotic ppt mutant,
(e) a zygotic slb;ppt double mutant, (f) an MZslb;MZppt mutant embryo, and (g) an
MZslb;MZppt mutant injected with 6 ng of Wnt4 MO. Of interest, maternal-zygotic mutants for
the pptsk13 allele used in this study did not demonstrate dorsal patterning defects previously
reported for MZpptti265 mutants1.
1.
Westfall, T. A. et al. Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator
of Wnt/beta-catenin activity. J Cell Biol 162, 889-98 (2003).
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5
Supplementary Figure 5. Ectopic accumulation of MZtri neural progenitor cells. Confocal
micrographs through the dorsal neural keel of mRFP-labelled1 (a) WT, and (b) MZtri mutant
embryos at 12-15 somite stages, and at the level of the 1st to 6th somite. The lateral extent of
each neural anlage has been highlighted. Note the establishment of a midline in WT embryos
(a), and the ectopic accumulation of MZtri neural progenitors (asterisk in b) at the centre of the
MZtri neural primordium. Scale bars, 50 µm.
1.
Megason, S. G. & Fraser, S. E. Digitizing life at the level of the cell: high-performance laserscanning microscopy and image analysis for in toto imaging of development. Mech Dev 120,
1407-20 (2003).
5
6
Supplementary Figure 6. Rescue of zebrafish Pk1 morphants by Gfp-Pk. (a-c) Phenotypic
analysis of titrated gfp-Pk mRNA injections. Embryos injected with 200 pg of gfp-Pk mRNA (b)
showed no morphological phenotype compared to un-injected controls (a). Injection of >300 pg
of gfp-Pk mRNA resulted in a shortened, curved body axis (c). Less than 200 pg of gfp-Pk
mRNA was therefore injected in all subsequent analyses of PCP signalling requiring gfp-Pk as a
marker of planar polarity. (d-f) Rescue of Pk1 morphant phenotype by gfp-Pk mRNA injection.
Injection of 6 pg of Pk1 MO1 resulted in a shortened, curved body axis (d). Pk1 morphants were
partially rescued by injection of 200 pg of gfp-Pk mRNA (e), and were fully rescued by injection
of 400pg of gfp-Pk mRNA (f).
1.
Carreira-Barbosa, F. et al. Prickle 1 regulates cell movements during gastrulation and neuronal
migration in zebrafish. Development 130, 4037-46 (2003).
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Supplementary Figure 7. Time-lapse analysis of neuroepithelial cell division in
WT and MZtri chimeric embryos. Confocal micrographs taken from time-lapse
analyses of cell division within the neural keel of WT and MZtri chimeric embryos. Time
is indicated as minutes following the completion of cytokinesis. (a-c, d-f) mRFPlabelled1 MZtri neural progenitors within the neural keel of lynGFP-labelled2 WT host
embryos. Apical MZtri daughter cells (arrows in a-f) fail to intercalate across the midline
of the neural keel (a-c), or are severely delayed in their movement (d-f). These results
indicate a cell autonomous requirement for Vangl2 function. (g-i) lynGFP-labelled2 WT
neural progenitors within the neural keel of a mRFP-labelled1 MZtri host embryo. Apical
WT daughter cells (arrow in g-i) also fail to re-integrate into the neuroepithelium
following cell division, suggesting a non-autonomous role for Vangl2 in neurulation.
Scale bars, 50 µm.
1.
2.
Megason, S. G. & Fraser, S. E. Digitizing life at the level of the cell: high-performance laserscanning microscopy and image analysis for in toto imaging of development. Mech Dev 120,
1407-20 (2003).
Koster, R. W. & Fraser, S. E. Tracing transgene expression in living zebrafish embryos. Dev Biol
233, 329-46 (2001).
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Supplementary Figure 8. Blocking cell division rescues MZtri neurulation defects.
WT (a,b,e,f) and MZtri (c,d,g,h) embryos were incubated in 4% DMSO (a,e,c,g), or in a
solution of 150 um aphidicolin and 20 mM hydroxyurea with 4% DMSO (b,f,d,h) in order
to inhibit cell division. Treatment was initiated at 80% epiboly, and embryos were
cultured overnight until 22- to 24-somite stages. (a-b) Lateral and dorsal views of WT
embryos demonstrating that embryogenesis proceeds relatively normally in the
presence of cell division inhibitors. (c-d) Blocking cell division appears to rescue CE
defects associated with the somitic mesoderm (arrowheads in c’ and d’) and neural
plate (arrows in c’ and d’) of MZtri mutants. (e-h) Transverse sections through the trunk
of WT and MZtri embryos stained for sonic hedgehog (shh) expression. shh floor plate
expression (arrowheads) appears normal in WT embryos treated with (f) or without (e)
cell division inhibitors. The expanded shh expression observed in the floorplate of MZtri
embryos (arrowheads in g) is rescued upon blocking cell division (arrowhead in h).
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UNILATERAL
CONTRALATERAL
number of
distribution of
intercalation of
samples
donor cells
donor cells
examined
WT into WT
12%
88%
16
MZtri into MZtri
95%
5%
41
MZtri into WT
62%
38%
37
WT into MZtri
93%
7%
61
WT into WT
64%
36%
42
DONOR into HOST
genotypes
(+ mitotic inhibitor)
Supplementary Table 1. Summary of WT and MZtri chimeric analyses. WT or MZtri donor
cells were transplanted into a single location above the margin of mid-blastula staged WT or
MZtri host embryos to ensure unilateral contribution of donor cell clones to regions of host
embryos that are fated to become spinal cord. To assess the incidence of neural progenitor cell
intercalation across the midline, chimeric embryos were analyzed at 20-somite stages for i) the
unilateral distribution of donor cells within the neural tube, or ii) the presence of donor cells in
the contralateral side of the neural tube.
9