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Maternal nodal and zebrafish embryogenesis
Arising from: A. V. Gore et al. Nature 438, 1030–1035 (2005).
In fish and amphibians, the dorsal axis is specified by the asymmetric
localization of maternally provided components of the Wnt signalling pathway1,2. Gore et al.3 suggest that the Nodal signal Squint (Sqt)
is required as a maternally provided dorsal determinant in zebrafish.
Here we test their proposal and show that the maternal activities of
sqt and the related Nodal gene cyclops (cyc) are not required for
dorsoventral patterning.
Sqt and Cyc induce mesoderm and endoderm1,4. Embryos without
zygotic sqt and cyc (Zcyc;Zsqt) lack all endoderm and most mesoderm5. Gore et al.3 suggest that maternal Sqt might also act as a dorsal
determinant, because injection of antisense sqt morpholinos into
unfertilized eggs induced dorsal defects3. However, we point out
two potential caveats in their study. First, Gore et al.3 do not provide
evidence that their approach eliminates maternal sqt. In particular,
two morpholinos were designed to prevent sqt pre-messenger RNA
splicing, even though there is little, if any, evidence that maternal
pre-mRNAs are deposited in the egg and spliced after oviposition.
Second, regardless of its effect on maternal sqt, the morpholino
approach of Gore et al. also blocks zygotic sqt activity. Hence, these
experiments did not specifically test the requirement for maternal sqt.
We first determined whether fully spliced sqt mRNA is present in
ovaries and unfertilized eggs. Polymerase chain reaction with reverse
transcription (RT–PCR) detected abundant spliced sqt mRNA, consistent with its cytoplasmic localization6 (Fig. 1a). Because we performed the reverse transcription with not only oligo(dT) but also
gene-specific primers, our analysis would have detected unspliced sqt
RNA regardless of its polyadenylation state. The presence of spliced
sqt mRNA in early embryos demonstrates that splice-blocking morpholinos cannot fully block maternal sqt activity, if they have any
effect at all.
To test conclusively the requirement for maternal sqt, one needs
to generate embryos from sqt homozygous mutant mothers and
wild-type fathers. We investigated whether the sqtcz35 allele lacks
a
b
Unfertilized eggs
Oligo(dT) primer: + +
sqt-specific primer: – –
RT: + –
631 nt
Exon1
Exon2
Wild type
a
b
c
d
e
f
g
h
Msqt
–
RT: +
WT M WT M
McycMsqt
968 bp
337 bp
337 bp
1 2 3 4 5
c
hgg1
8 cell
– –
+ +
+ –
Exon1 Exon2
Exon1 Exon2
Sqt activity. Sequencing of genomic DNA confirmed that sqtcz35 contains a 1,848-base-pair (bp) insertion (Fig. 1c)5. Homozygous sqtcz35
embryos contained no detectable sqt mRNA at the 8-cell stage
(Fig. 1b). In the late blastula, all sqt RNA detectable in mutants contained the 1.8-kilobase insertion, which introduces numerous stop
codons in all reading frames (data not shown). Hence, sqtcz35
mutants have strongly reduced levels of sqt mRNA, and the mutant
allele cannot generate wild-type Sqt protein. The presence of an inframe AUG codon 39 to the insertion indicated that sqtcz35 might
produce an amino-terminally truncated Sqt protein (T-Sqt), which
would lack a signal sequence. This would require the ribosome to
bypass 41 upstream AUGs or the initiation of transcription within
the insertion itself (Fig. 1c). Although it is unlikely that T-Sqt is
produced, we tested its activity. Injection of mRNA encoding T-Sqt
did not induce phenotypic abnormalities (Fig. 1d–f). Hence, all available evidence indicates that sqtcz35 completely eliminates Sqt activity,
whereas the splice-site morpholinos3 cannot eliminate the function
of spliced maternal sqt mRNA.
Maternal-zygotic and zygotic sqtcz35 and sqthi975 (a retroviral insertional allele) mutants have similar phenotypes7,8. To investigate
a requirement for maternal Sqt, we generated embryos lacking
In frame AUG
distal to insert T-sqt
N SS
sqtcz35 insertion
(contains 41 AUGs)
1 2
d
Ligand
e
3 4
f
C
MZcycMZsqt
Uninjected
+T-sqt
+sqt
Figure 1 | Splicing and disruption of sqt RNA. a, Primers flanking the first
intron of the sqt gene detected only spliced RNA (337 bp spliced versus
968 bp unspliced) in complementary DNA prepared from total RNA of
unfertilized eggs. Gene-specific primers (lanes 4 and 5) and oligo(dT)
primers (lanes 1 and 2) were used, so that non-polyadenylated, unspliced sqt
RNA could be detected. RT, reverse transcriptase. b, sqt mRNA is detected in
wild-type (WT) embryos (lane 1) but not MZsqtcz35 mutants (M) (lane 2) at
the 8-cell stage. c, Schematic of Sqt protein, showing signal sequence (SS),
prodomain (white), and mature ligand domain (red). The sqtcz35 insertion,
and the N-terminally truncated putative product of sqtcz35 (T-Sqt) are
indicated. d–f, Embryos injected with 50 pg of wild-type sqt mRNA have
ectopic gsc expression (f), but injection of 150 pg of T-Sqt (e) has no effect.
Additional supporting data and details of methods are available from the
authors at http://www.mcb.harvard.edu/Schier/BennettBCASuppl07.pdf.
Figure 2 | The maternal Nodal genes cyclops and squint are not required for
dorsal axis specification. a, c, e, g, Lateral views of live embryos 28 h postfertilization. Genotypes are indicated on the left. Msqt and Mcyc;Msqt
embryos appear phenotypically normal. The hatching gland, an anterior
dorsal mesoderm derivative, is marked by red arrows. MZcyc;MZsqt
embryos lack endoderm and head and trunk mesoderm but retain anterior
neuroectoderm, including a cyclopic eye (black arrow), similar to Zcyc;Zsqt5
and MZoep10 mutants. b, d, f, h, Expression of hgg1, a marker for anterior
dorsal mesoderm, detected by whole-mount in situ hybridization in 10somite-stage embryos (lateral view). We used cycm294, a mutation thought to
eliminate all Cyc activity12. Additional supporting data and details of
methods are available from the authors at http://www.mcb.harvard.edu/
Schier/BennettBCASuppl07.pdf.
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NATURE | Vol 450 | 8 November 2007
maternal but not zygotic sqt, by crossing sqt homozygous mutant
females and wild-type males. Maternal sqt mutants (Msqt) were
viable and phenotypically normal (Fig. 2c). Analysis of markers
expressed in axial, paraxial, intermediate, and lateral mesoderm
did not detect any defects in Msqt embryos (Fig. 2d).
To determine whether maternal sqt acts redundantly with maternal cyc, we analysed Mcyc;Msqt embryos. Mcyc;Msqt embryos generated by germline replacement9 were viable and phenotypically
normal (Fig. 2e). Hence, complete elimination of maternal Nodal
signals does not affect zebrafish embryogenesis. To eliminate all early
Nodal activity, we generated MZcyc;MZsqt embryos. These mutants
developed dorsal derivatives such as the anterior neuroectoderm and
appeared identical to Zcyc;Zsqt embryos and to MZoep10 mutants,
which lack the Nodal co-receptor (Fig. 2g).
These results cannot exclude potential contributions by maternal
cyc or sqt under very particular genetic or environmental conditions8,11, but we have shown that maternal cyc and sqt are not required
for dorsal axis specification or for any other aspect of embryogenesis.
We propose that Nodal signals act primarily as zygotic inducers of
mesendoderm.
James T. Bennett1,2, Heather L. Stickney3, Wen-Yee Choi1,2,4,
Brian Ciruna1,2,5,6, William S. Talbot3 & Alexander F. Schier1,2,4
1
Developmental Genetics Program, Skirball Institute of Biomolecular
Medicine, New York University School of Medicine, New York, New York
10016, USA.
2
Department of Cell Biology, New York University School of Medicine,
New York, New York 10016, USA.
3
Department of Developmental Biology, Stanford University, Stanford,
California 94305, USA.
4
Department of Molecular and Cellular Biology, Harvard Stem Cell
Institute, Center for Brain Science, Broad Institute, Harvard University,
Cambridge, Massachusetts 02138, USA.
e-mail: schier@fas.harvard.edu
5
Department of Molecular and Medical Genetics, University of Toronto,
Toronto, Ontario M5G 1L7, Canada.
6
The Program in Developmental and Stem Cell Biology, The Hospital for
Sick Children, Toronto, Ontario M5G 1L7, Canada.
Received 25 September 2006; accepted 21 August 2007.
1. Schier, A. F. & Talbot, W. S. Molecular genetics of axis formation in zebrafish. Annu.
Rev. Genet. 39, 561–613 (2005).
2. Heasman, J. Patterning the early Xenopus embryo. Development 133, 1205–1217
(2006).
3. Gore, A. V. et al. The zebrafish dorsal axis is apparent at the four-cell stage. Nature
438, 1030–1035 (2005).
4. Schier, A. F. Nodal signaling in vertebrate development. Annu. Rev. Cell Dev. Biol. 19,
589–621 (2003).
5. Feldman, B. et al. Zebrafish organizer development and germ-layer formation require
nodal-related signals. Nature 395, 181–185 (1998).
6. Gore, A. V. & Sampath, K. Localization of transcripts of the zebrafish morphogen
Squint is dependent on egg activation and the microtubule cytoskeleton. Mech. Dev.
112, 153–156 (2002).
7. Aoki, T. O. et al. Regulation of nodal signalling and mesendoderm
formation by TARAM-A, a TGFbeta-related type I receptor. Dev. Biol. 241, 273–288
(2002).
8. Pei, W., Williams, P. H., Clark, M. D., Stemple, D. L. & Feldman, B. Environmental and
genetic modifiers of squint penetrance during zebrafish embryogenesis. Dev. Biol.
308, 368–378 (2007).
9. Ciruna, B. et al. Production of maternal-zygotic mutant zebrafish by germ-line
replacement. Proc. Natl Acad. Sci. USA 99, 14919–14924 (2002).
10. Gritsman, K. et al. The EGF-CFC protein one-eyed pinhead is essential for nodal
signaling. Cell 97, 121–132 (1999).
11. Sirotkin, H. I., Dougan, S. T., Schier, A. F. & Talbot, W. S. bozozok and squint act in
parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish.
Development 127, 2583–2592 (2000).
12. Sampath, K. et al. Induction of the zebrafish ventral brain and floorplate requires
cyclops/nodal signalling. Nature 395, 185–189 (1998).
doi:10.1038/nature06314
Gore et al. reply
Replying to: J. T. Bennett et al. Nature 450, doi: 10.1038/nature06314 (2007).
We presented several lines of evidence indicating that dorso–ventral
asymmetry is apparent in zebrafish embryos by cleavage stages1, one
of which showed that injection of three different morpholino oligonucleotides targeting three different squint (sqt) sequences cause
severe disruption in dorsal structures. We concluded that the dorsal
axis is evident by the 4-cell stage, and suggested that maternal Sqt and
associated factors may function in zebrafish dorsal-axis formation1.
Bennett et al. challenge our results obtained with morpholino oligonucleotides because they do not find a comparable defect in maternal
and zygotic sqt mutants2.
How could these differences be explained? Antisense RNAs targeting short sequences can have off-target effects. One of the three
morpholinos we used1 was a previously described sqt ATG morpholino3, whereas the other two were directed against splice junctions.
Eggs or embryos injected with control morpholinos did not manifest
the same phenotypes. All three sqt morpholinos, when injected into
fertilized embryos, recapitulated the milder-mutant phenotypes4,5,
which argues against off-target effects. Although all three morpholinos might have off-target effects that produce the same phenotype by
chance, this explanation is unlikely.
Bennett et al. also raise a concern related to our sqt splice-junction
morpholinos. They contend that there is little evidence for maternal
pre-messenger RNAs in eggs, and therefore that they cannot be targeted by splice-junction morpholinos. However, for several maternal
transcripts in Xenopus, a pool of unprocessed RNA is present in eggs
and early embryos6–8. Bennett et al. use primers spanning one of the
sqt introns and do not detect unspliced Sqt RNA in ovaries, early
embryos, or at the peak of zygotic sqt expression4,9–11. In further
control experiments (Fig. 1), we consistently detect unspliced and
spliced sqt RNA in zebrafish ovaries, eggs and early embryos, using
primers for both sqt introns (ref. 12, and Fig. 1a–c). Furthermore, we
detect aberrantly spliced and unspliced sqt RNA at the 8-cell stage
(before zygotic sqt expression) on injection of sqt splice-junction
morpholinos, but not of control morpholinos (Fig. 1c). Therefore,
our use of splice-junction morpholinos to block maternal sqt function cannot be excluded as a valid approach.
It is also possible that the mutant used for genetic analysis may not
behave as expected. The sqt insertion mutations are incompletely
penetrant, sensitive to environmental conditions, genetic backgrounds and the age of the mother, and homozygous mutant embryos
frequently survive to adulthood5,13,14. So the sqt alleles may not be
complete nulls. Some maternal zygotic sqtcz35 (MZsqtcz35) embryos
manifest dorso–anterior deficiencies14, similar to our findings after
sqt morpholino injection.
Bennett et al. contend that the sqt alleles must be null because the
insertions should prevent translation of the Sqt protein. However,
some RNAs have functions independent of the protein they encode:
for instance, in Xenopus, removal of VegT transcripts disrupts the
cytoskeleton at the vegetal cortex and prevents formation of germinal
granules15. We find that sqt RNA is present in MZsqt mutant embryos
(Fig. 1d), and could perform a non-coding function. A priori considerations aside, to determine the loss-of-function phenotype
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A
a
ATG
44
1
5'
323
951
Exon 1
280 bp
43 bp
C
TGA
1,930
1,696 1,778
Exon 2
627 bp
746 bp
MO1 / ATG MO MO3 / intronID MO
40–64
314–338
Exon 3
153 bp
81 bp
2,167
237 bp
MO2 / intronIID MO
1,689–1,713
B
3'
D
PCR products
Unspliced
–
RT
Spliced
615 bp
Aberrantly
spliced
469 bp
Unfertilized
egg
Ovary
696 bp
–
+
1 cell
–
+
4 cell
–
+
1,000 cell
–
+
Genomic
DNA
b
402 bp
30% epiboly
–
+
+
100 bp
DNA
ladder
Oligo(dT)
402 bp
Random
hexamer
Ovary
–
RT
Uninjected
Control MO Sqt MO2
Control MO Sqt MO2
30%
30%
30%
Uninjected Uninjected
epiboly
egg
8 cell
8 cell
8 cell
epiboly
epiboly
+
–
+
–
+
–
+
–
+
–
+
–
+
–
+
Genomic
DNA
c
402 bp
100 bp
DNA
ladder
696 bp
615 bp
469 bp
Random
hexamer
MZsqt cz35
–
+
8 cell
–
+
30% epiboly
–
+
100 bp 1 cell
DNA
ladder –
+
8 cell
–
+
30% epiboly
–
+
Genomic
DNA
1 cell
RT
MZsqt hi975
Genomic
DNA
d
696 bp
615 bp
Random
hexamer
Figure 1 | Unspliced sqt RNA is present in zebrafish ovaries, eggs and
embryos. a, The squint locus and polymerase chain reaction (PCR)
products. Red arrows, target sites for the sqt morpholinos. Black arrows,
position of PCR primers. Primer pair AB amplifies part of intron I and exon
II; primer pair CD spans intron II. Numbers in pink indicate nucleotide
positions based on Vega v28 (http://vega.sanger.ac.uk/Danio_rerio/
exonview?transcript5OTTDART00000026522;db5core). b, Reverse
transcription (RT)–PCRs for unspliced sqt intron I. Oligo(dT) or random
hexamer-primed complementary DNAs synthesized from wild-type
zebrafish RNAs was used in PCRs with primer pair AB. A 402-bp unspliced
intron I product is detected in random-hexamer p(dN)6-primed RT–PCRs
at all stages. RT–PCRs on oligo(dT)-primed cDNA using the same primers
detect the 402-bp unspliced product strongly in ovary, 1,000-cell and 30%
epiboly samples, but poorly in unfertilized eggs. Unspliced sqt RNA is not
detected in oligo(dT)-primed cDNA from cleavage-stage embryos. No
product is detected in RT controls for all stages. c, RT–PCRs for unspliced sqt
intron II. Random hexamer-primed cDNA synthesized from wild-type
zebrafish RNAs was used in PCRs with primer pair CD. A 696-bp unspliced
intron II product is detected strongly in whole ovary and unfertilized eggs.
The unspliced intron II product diminishes by the 8-cell stage and is detected
again at 30% epiboly at the peak of zygotic sqt expression. Unfertilized eggs
were injected with control or sqt morpholinos (MOs) and fertilized in vitro
using sperm from wild-type male zebrafish. Only spliced sqt product
(615 bp) is detected in uninjected or control MO-injected embryos at the
8-cell stage, when for sqt MO2-injected embryos, unspliced intron II
containing sqt RNA is still detectable, with an aberrantly spliced sqt RNA
species (469 bp). At 30% epiboly, both the 696-bp and the 469-bp products
are enriched in sqt MO2-injected embryos. The aberrant sqt splice product
should generate truncated Sqt protein lacking the carboxy-terminal 98
amino acids, which include six of seven conserved cysteine residues in the
Sqt mature domain. No product is detected in RT controls. d, RT–PCRs for
sqt transcripts in MZsqt mutant embryos. Random hexamer-primed cDNA
from 1-cell, 8-cell and 30% epiboly MZsqt mutant5,13,14 embryos was used in
PCRs with primer pair CD. Spliced sqt product (blue arrow) is detected in
embryos from both MZsqt mutant alleles5,13,14. Unspliced sqt product (black
arrow) is detected at the 30% epiboly embryonic stage. No product is
detected in RT controls. S. Lim provided these data. Further details (for
example, on the RT–PCR method) are available from the authors.
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NATURE | Vol 450 | 8 November 2007
definitively, a deletion that completely removes the sqt locus is
required.
We therefore stand by our original conclusions1. Although the
mutant analysis by Bennett et al.2 disagrees with our results, we
believe that further investigation is necessary to understand precisely
how maternal Sqt functions.
Aniket V. Gore1{, Albert Cheong1{, Patrick C. Gilligan1 &
Karuna Sampath1,2,3
1
Vertebrate Development Group, Temasek Life Sciences Laboratory, 1
Research Link, National University of Singapore, 117604 Singapore.
2
School of Biological Sciences, Nanyang Technological University, 60
Nanyang Drive, 637551 Singapore.
3
Department of Biological Sciences, National University of Singapore,
117543 Singapore.
e-mail: karuna@tll.org.sg
{Present addresses: Unit on Vertebrate Organogenesis, Laboratory of
Molecular Genetics, The National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892,
USA (A.V.G.); Cell Culture Process Development, Lonza Biologics, 228
Bath Road, Slough, Berkshire, SL1 4DX, UK (A.C.).
1. Gore, A. V. et al. The zebrafish dorsal axis is apparent at the four-cell stage. Nature
438, 1030–1035 (2005).
2. Bennett, J. T. et al. Maternal nodal and zebrafish embryogenesis. Nature 450,
10.1038/nature06314 (2007).
3. Feldman, B. & Stemple, D. L. Morpholino phenocopies of sqt, oep, and ntl mutations.
Genesis 30, 175–177 (2001).
4. Feldman, B. et al. Zebrafish organizer development and germ-layer formation require
nodal-related signals. Nature 395, 181–185 (1998).
5. Aoki, T. O. et al. Regulation of nodal signalling and mesendoderm formation by
TARAM-A, a TGFb-related type I receptor. Dev. Biol. 241, 273–288 (2002).
6. Simon, R., Wu, L. & Richter, J. D. Cytoplasmic polyadenylation of activin receptor
mRNA and the control of pattern formation in Xenopus development. Dev. Biol. 179,
239–250 (1996).
7. Pandur, P. D., Sullivan, S. A. & Moody, S. A. Multiple maternal influences on dorsalventral fate of Xenopus animal blastomeres. Dev. Dyn. 225, 581–587 (2002).
8. Vasudevan, S., Seli, E. & Steitz, J. A. Metazoan oocyte and early embryo development
program: a progression through translation regulatory cascades. Genes Dev. 20,
138–146 (2006).
9. Rebagliati, M. R., Toyama, R., Fricke, C., Haffter, P. & Dawid, I. B. Zebrafish nodalrelated genes are implicated in axial patterning and establishing left-right asymmetry.
Dev. Biol. 199, 261–272 (1998).
10. Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F. & Talbot, W. S. The role of the
zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.
Development 130, 1837–1851 (2003).
11. Erter, C. E., Solnica-Krezel, L. & Wright, C. V. Zebrafish nodal-related 2 encodes an
early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer.
Dev. Biol. 204, 361–372 (1998).
12. Gore, A. V. Localized Molecules and the Establishment of Polarity in Zebrafish. PhD
thesis, National Univ. Singapore (2006).
13. Pei, W., Williams, P. H., Clark, M. D., Stemple, D. L. & Feldman, B. Environmental and
genetic modifiers of squint penetrance during zebrafish embryogenesis. Dev. Biol.
309, 245–258 (2007).
14. Hagos, E. G. Fan, X. & Dougan, S. T. The role of maternal Activin-like signals in
zebrafish embryos. Dev. Biol. 308, 368–378; doi:10.1016/j.ydbio.2007.07.010
(2007).
15. Kloc, M. et al. Potential structural role of non-coding and coding RNAs in the
organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes.
Development 132, 3445–3457 (2005).
doi:10.1038/nature06315
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