1 Department of Biochemistry and Biophysics, Programs in Developmental Biology,
Genetics and Human Genetics, University of California, San Francisco, San
Francisco, CA 94143-0448, USA
2 Abteilung für Entwicklungsbiologie, Biologie I, Universität
Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany
* Author for correspondence (e-mail: didier_stainier{at}biochem.ucsf.edu)
Accepted 12 May 2003
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SUMMARY |
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Key words: Mix-like, nodal, fast1/foxHI, Neural patterning, Zebrafish
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Introduction |
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Nodals belong to the Tgfß superfamily of ligands that bind to and
activate heteromeric type I and type II Activin-like receptors (reviewed by
Whitman, 2001). The founding
member of this Tgfß subgroup, mouse Nodal, was identified from studying a
retroviral insertion that affects node formation
(Zhou et al., 1993
). In
zebrafish, two nodal-related genes, cyclops (cyc)
and squint (sqt), are required for the induction of the
axial and trunk mesoderm, as well as the endoderm
(Feldman et al., 1998
;
Sampath et al., 1998
). Nodal
signaling also appears to be important for neural patterning, as embryos
mutant for both cyc and sqt appear to have expanded anterior
neural fates and loss of trunk spinal cord
(Feldman et al., 2000
).
Additionally, in maternal-zygotic one-eyed pinhead
(MZoep)-mutant embryos, which lack an EGF-CFC cofactor essential for
Nodal signaling, anterior fates appear expanded
(Gritsman et al., 1999
).
However, compound mutant analyses of embryos lacking sqt and
bozozok (boz), a homeobox gene required for axis formation,
indicate that sqt acts in parallel with boz to specify
anterior neuroectoderm, whereas cyc represses anterior neural
development (Sirotkin et al.,
2000
,Sirotkin et al.,
2000
). These data suggest that Nodal signaling can play both
positive and negative roles in neuroectoderm patterning, and that the correct
balance needs to be achieved for the process to occur correctly.
Loss- and gain-of-function analyses indicate that Nodal signaling is
transduced by Smad2 (Madh2 Zebrafish Information Network), and to some
extent Smad3 (Madh3a Zebrafish Information Network). These
receptor-activated Smads are phosphorylated by ligand binding to the receptor
complex (Waldrip et al., 1998;
Tremblay et al., 2000; Brennan et al.,
2001
). Mouse Smad2 mutants, like Nodal mutants,
exhibit defects in the formation of the primitive streak, mesoderm and
endoderm (Waldrip et al.,
1998
; Weinstein et al., 1998). Interestingly, Nodal;Smad2
transheterozygous embryos exhibit anterior neural truncations, further
suggesting that precise levels of Nodal signaling are required for
neuroectoderm patterning (Nomura and Li,
1998
). Upon activation, the receptor-activated Smads form a
complex with Smad4 and translocate to the nucleus. Here, the Smad complex is
recruited to Nodal target genes by its interaction with other DNA-binding
proteins to regulate gene expression
(Derynck et al., 1998
;
Whitman, 1998
).
The first DNA-binding cofactor identified to interact with the Smad complex
is the winged helix transcription factor, Foxh1 (also known as Fast1). Smad2
and Smad4 were shown to form a complex with Foxh1, and to bind to an
activin-responsive element in the Xenopus Mix.2 promoter
(Chen et al., 1996;
Chen et al., 1997
). Cloning and
mutational analysis of the schmalspur (sur) locus in
zebrafish demonstrated that sur encodes Foxh1 and that it is required
for the maintenance of Nodal signaling
(Pogoda et al., 2000
;
Sirotkin et al.,
2000
,Sirotkin et al.,
2000
). Consistent with this model, embryos lacking both maternal
and zygotic sur (MZsur) show defects in axial mesoderm,
although they do not exhibit the defects in endoderm and trunk mesoderm
formation seen in embryos lacking the Nodal ligands Cyc and Sqt
(Feldman et al., 1998
). These
data have led to the proposal that multiple transcription factors can mediate
Nodal signaling in various developmental processes
(Pogoda et al., 2000
;
Stemple, 2000
).
Biochemical studies have shown that members of the Mix family of
homeodomain proteins also function as transcriptional mediators of Nodal
signaling (Germain et al.,
2000), for example, by interacting with a Smad2/Smad4 complex upon
Tgfß signaling and binding the goosecoid (gsc)
promoter. Mapping of the protein-protein interaction domain identified a
common Smad interaction motif within a subgroup of the Mix family members, as
well as in winged helix transcription factors, such as Foxh1
(Germain et al., 2000
).
In zebrafish, the Mix gene bonnie and clyde (bon)
functions downstream of Nodal signaling to regulate endoderm formation
(Kikuchi et al., 2000).
bon expression requires Nodal signaling as it is absent in
cyc/;sqt/
embryos (Alexander and Stainier,
1999
). Additionally, misexpression of a constitutively active form
of the type I Tgfß receptor Tarama promotes ectopic bon
expression (Alexander and Stainier,
1999
). Furthermore, bon overexpression in
cyc/;sqt/
embryos can induce endodermal gene expression
(Kikuchi et al., 2000
).
Finally, bon/ embryos exhibit a severe
reduction in the number of endodermal precursors, which indicates that
bon plays a crucial role in endoderm formation. Here, we show that
Bon also functions in precursors of the axial mesoderm to modulate anterior
neural patterning. We further show that Bon functions cooperatively with the
Nodal signaling components Sqt and Sur (Foxh1) to regulate this process.
Expression analyses in single- and double-mutant embryos show a correlation
between the severity of the neural patterning defects and the level of
dickkopf 1 (dkk1) expression. The defect in dkk1
expression in the mutant embryos is part of an overall defect in dorsal
mesendoderm gene expression.
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Materials and methods |
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Microinjection
For restricted morpholino injection experiments, fluorescein-tagged
morpholino oligonucleotides for bon
(5'-GAT-TCG-CAT-TGT-GCT-GCT-GTC-CTT-C-3') were dissolved in 5 mM
HEPES, pH 7.6, and diluted to 2 ng/nl with 5 mM HEPES/10% Phenol Red.
Rhodamine-dextran (10 kDa, 2.5%) was co-injected into some embryos in order to
enhance the signal for localizing the morpholino. Antibody staining for the
fluorescein-tagged morpholino indicated that the 10 kDa rhodamine-dextran
co-localizes with the morpholino (data not shown). Single cells at the 32-cell
stage were injected with 1 nl of a 2 ng/nl bon MO stock. Following
injections, embryos were fixed for whole-mount in situ hybridization at the
tailbud stage, or photographed using a Zeiss Axioplan microscope. Localization
of the injected clone was visualized with a rhodamine filter, or an
anti-fluorescein antibody following in situ hybridization. Briefly, embryos
were treated with 100 mM glycine, pH 2.2, to inactivate alkaline phosphatase
and washed with PBS-T (phosphate buffered saline + 0.1% Tween).
Anti-fluorescein-alkaline phosphatase conjugated antibody (Boehringer
Mannheim; 1:500) was incubated with embryos overnight at 4°C and detected
with Fast Red (Sigma).
In situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Alexander et al., 1998).
dkk1 anti-sense probe was prepared as described by Hashimoto et al.
(Hashimoto et al., 2000
).
Genotyping
Whole-mount in situ hybridized embryos were genotyped by PCR using
restriction polymorphisms for bonm425 and
surm768, and agarose polymorphism for
sqtcz35 mutant embryos, as described previously
(Feldman et al., 1998;
Kikuchi et al., 2000
;
Sirotkin et al.,
2000
,Sirotkin et al.,
2000
). Genotyping was performed after in situ hybridization as
follows. After photographing, each embryo was washed with 100% methanol and
hydrated with several washes of PBS with 0.1% Tween-20. Genomic DNA was
extracted by digestion overnight in 10 mM Tris, 1 mM EDTA, 0.1% NP40, 0.1%
Tween-20, 50 µg proteinase K at 55°C.
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Results |
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To assess the anterior neural plate during the stages of neural patterning, MO-injected embryos were fixed and examined for otx2 expression. Following in situ hybridization, we also performed anti-fluorescein antibody staining to determine the localization of the bon MO. Embryos with axial mesoderm restriction of the bon MO (n=25) showed a reduction in the otx2 expression domain (Fig. 2A), whereas embryos with bon MO restriction in non-axial mesoderm (n=13) exhibited wild-type otx2 expression (Fig. 2B,C).
|
bon mutant embryos exhibit defects in axial mesodermal gene
expression
To further analyze the requirement of the axial mesoderm during neural
patterning, we examined the expression of the anterior axial mesoderm marker
gsc (Stachel et al.,
1993) at several stages during gastrulation. At the shield stage,
bon/ embryos show gsc expression
that is indistinguishable from that seen in wild-type embryos
(Fig. 3A). At 90% epiboly, the
gsc expression domain is reduced in
bon/ embryos
(Fig. 3C), indicating a
differentiation defect in the anterior axial mesoderm. The same progressive
reduction in anterior axial mesoderm gene expression was also observed with
bmp4. During gastrulation stages, bmp4 is expressed
ventrolaterally, as well as in a discrete domain of the anterior axial
mesoderm (Hwang et al., 1997
;
Martinez-Barbera et al.,
1997
). This expression pattern allowed us to assess dorsoventral
patterning as well as axial mesoderm formation. At 50% epiboly, wild-type and
bon/ embryos show indistinguishable
bmp4 expression ventrolaterally
(Fig. 3D,G), indicating that
dorsoventral patterning is not affected in
bon/ embryos. Dorsal bmp4
expression also appears unaffected at this stage
(Fig. 3D,G; arrowhead). At 90%
epiboly, wild-type and bon/ embryos show a
wild-type pattern of ventrolateral bmp4 expression
(Fig. 3H,I), but the anterior
axial mesoderm bmp4 expression domain is dramatically reduced in
bon/ embryos
(Fig. 3E,F,H,I; arrows). These
data indicate that although the early induction of axial mesoderm occurs
properly in bon/ embryos, its subsequent
differentiation is defective.
|
|
Anteriorly, the emx1 expression domain spreads medially to cover the entire anterior ventral neural plate in sqt/ and bon/;sqt/ embryos (Fig. 4K,L). This expansion appears to be restricted to emx1 expression, as otx2 expression is reduced in sqt/ and bon/;sqt/ embryos (Fig. 4O,P). Consistent with this result, and with the morphological absence of eyes in bon/;sqt/ embryos, the expression of opl (zic1 Zebrafish Information Network) and rxb, markers of the eye field, is dramatically reduced or absent in bon/;sqt/ embryos (data not shown). Together, these data indicate that loss of bon and sqt function leads to synergistic defects in neural patterning.
bon and sqt function in parallel to regulate
mesendodermal gene expression
AP patterning of the neuroectoderm is regulated by posteriorizing signals
and their antagonists (reviewed by
Yamaguchi, 2001). Recent
evidence points to the Wnt signaling pathway as a key regulator of AP
patterning, with Wnt8 as a posteriorizing signal and the Wnt antagonist Dkk1
as promoting anterior neural fates (Glinka
et al., 1998
; Erter et al.,
2001
). The neural patterning defects in
bon/, sqt/
and
bon/;sqt/
embryos were reminiscent of defects caused by an excess of Wnt signaling
(Kim et al., 2000
;
Erter et al., 2001
). Therefore,
we examined the expression of dkk1 in
bon/, sqt/
and
bon/;sqt/
embryos and found that defects in dkk1 expression correlated with the
severity of the neural patterning defects observed in these mutant embryos. At
50% epiboly, dkk1 expression is observed in all marginal blastomeres
(Fig. 5A)
(Hashimoto et al., 2000
;
Shinya et al., 2000
). In
bon/ embryos, there is a dorsal gap in
dkk1 expression (Fig.
5B). This dorsal gap appears more extensive in
sqt/ and
bon+/;sqt+/embryos
(Fig. 5C). In
bon/;sqt/
embryos, dkk1 expression is seen only in the ventral half of the
margin (Fig. 5D). At 70%
epiboly, dkk1 is expressed in cells of the prechordal plate (PCP;
Fig. 5E)
(Hashimoto et al., 2000
;
Shinya et al., 2000
).
Consistent with bon/ embryos exhibiting
defects in anterior axial mesoderm gene expression, the
dkk1-expressing cells appear to coalesce aberrantly in these mutants
(Fig. 5F). In
sqt/ and
bon+/;sqt+/ embryos,
dkk1 expression in the PCP is dramatically reduced
(Fig. 5G), reflecting a defect
in anterior axial mesoderm formation. This reduction is enhanced in
bon/;sqt/
embryos, where dkk1 expression appears to be completely absent in the
PCP region (Fig. 5H). These
data suggest that the defects in dkk1 expression may be responsible,
at least in part, for the neural patterning defects. In order to test this
hypothesis, we overexpressed dkk1 in
bon/ embryos and observed an enlargement of
the forebrain and eyes, suppressing the anterior neural deficiency (data not
shown). However, the cardia bifida phenotype was not rescued, suggesting that
dkk1 functions in neural patterning but not in endoderm
development.
|
bon interacts with sur to regulate neural
patterning and mesendodermal gene expression
The genetic interaction between bon and sqt suggested
that these two genes function in parallel to regulate neural patterning.
However, molecular epistasis analyses have indicated that bon
expression is dependent on Nodal signaling, which places bon
downstream of sqt (Alexander et al., 1999). Thus, additional
signal(s) must function upstream of bon, and additional Nodal
transcriptional mediator(s) must function downstream of sqt. The
foxh1 gene mutant locus sur was a good candidate to be an
additional Nodal transcriptional mediator in neural patterning due to its role
in axis formation (Pogoda et al.,
2000; Sirotkin et al.,
2000
,Sirotkin et al.,
2000
). Therefore, we asked whether
bon/;sur/
embryos exhibit neural patterning defects. Although
bon/ embryos exhibit a slight reduction in
anterior neural structures
(Fig.1B and
Fig. 4B; arrow) and
sur/ embryos exhibit mild cyclopia
(Pogoda et al., 2000
;
Sirotkin et al.,
2000
,Sirotkin et al.,
2000
),
bon/;sur/
embryos exhibit a dramatic reduction of forebrain structures, with the most
severally affected embryos exhibiting an absence of telencephalic and
diencephalic structures, as well as eyes
(Fig. 6B; arrow).
Interestingly,
bon/;sur+/
embryos also exhibited anterior truncations at a low percentage (1.8%,
n=340) when they originated from
bon+/;sur+/ females but
not from bon+/;sur+/
males, indicating that a reduction in maternal Sur (Foxh1) can enhance the
bon neural phenotype.
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Discussion |
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A role for bon in neural patterning
Genetic and embryological analyses indicate that Mix genes are potent
inducers of mesodermal and endodermal gene expression. Ectopic expression of
Mix.1, Milk, Mixer, Bix1, mezzo and bon leads to the
expression of mesodermal and endodermal genes
(Henry and Melton, 1998;
Lemaire et al., 1998
;
Alexander et al., 1999; Latinkic and
Smith, 1999
; Poulain and
Lepage; 2002
). Additionally, a genetic lesion in the zebrafish Mix
gene bon leads to a reduction in endodermal precursors
(Kikuchi et al., 2000
). Our
data point to an essential role for Bon in the axial mesoderm for neural
patterning. We found that a reduction in Bon function in the axial mesoderm
caused by restricted MO injection is associated with anterior neural defects.
In addition, bon/ embryos display defects in
axial mesoderm gene expression. Furthermore, based on the expression pattern
of bon in mesendodermal progenitors before involution, we favor a
model in which Bon regulates the transcription of neural patterning genes that
are expressed in mesendodermal precursors. The finding that dkk1
expression is absent from the dorsal side of
bon/ embryos is consistent with this model.
It is interesting to note that studies in Xenopus had hinted at a
role for Mixer in head formation and Dkk1 expression
(Henry and Melton, 1998
).
Nodal signaling regulates neural patterning through transcriptional
regulation of members of the Wnt signaling pathway
Recent findings have revealed that the spatial variation in the level of
Wnt signal plays a crucial role in the AP patterning of the neuroectoderm
(reviewed by Yamaguchi, 2001;
Erter et al., 2001
;
Kudoh et al., 2002
). Extensive
evidence from genetic and overexpression studies points to the importance of
Wnt antagonism for anterior neural patterning. Specifically, Dkk1
mouse mutant embryos lack head structures anterior to the midbrain, whereas
overexpression of dkk1 in amphibians and zebrafish embryos leads to
enlarged heads (Glinka et al.,
1998
; Hashimoto et al.,
2000
; Mukhopadhyay et al.,
2001
; Shinya et al.,
2000
). Conversely, ectopic expression of wnt8 suppresses
anterior fates, whereas a deficiency in the wnt8 locus or a reduction
of Wnt8 caused by MO injection in zebrafish embryos leads to a loss of
posterior neural fates (Erter et al.,
2001
; Lekven et al.,
2001
). Our data indicate that the precise level of Wnt signaling
required for neural patterning is transcriptionally controlled by Nodal
signaling as well as by Bon and Sur (Foxh1).
Bon and Sqt function in parallel to regulate neural patterning
Overexpression and mutant analyses have indicated that Bon functions
exclusively downstream of Nodal signaling in endoderm formation (Alexander et
al., 1999; Kikuchi et al.,
2000). However the synergistic neural patterning defects seen in
bon/;sqt/
embryos indicate that Bon also functions in parallel to Sqt signaling.
Biochemical analyses indicate that a subset of Mix homeodomain proteins, as
well as winged-helix transcription factors, physically interact with the
Smad2/Smad4 complex through a conserved motif in their C terminus
(Germain et al., 2000
). This
Smad interaction motif is present in Bon and Sur (Foxh1)
(Pogoda et al., 2000
;
Randall et al., 2002
), raising
the possibility that Bon and/or Sur (Foxh1) can interact with the Smad2/Smad4
complex, upon Sqt activation of the Nodal pathway, to activate downstream
targets. The loss of dkk1 expression in
bon/;sqt/
and
bon/;sur/
embryos indicates that dkk1 is one of the genes regulated in this
manner. Whether Bon and Sur (Foxh1) bind directly to the dkk1
promoter needs to be investigated.
In addition, we also found defects in wnt8 expression at the
margin of
bon/;sqt/
embryos suggesting that the neural patterning defect in these double-mutant
embryos may not be solely due to an expansion of Wnt signaling. We do observe
a shortening of the body axis in
bon/;sqt/
and
bon/;sur/
embryos, which may lead to a misplacement of neural organizing centers, such
as the anterior neural boundary cells and the MHB (reviewed by
Liu and Joyner, 2001;
Houart et al., 1998
), which
would further affect AP patterning of the neural plate (see
Fig. 4E-H,
Fig. 6E-F).
Model of genetic network of transcriptional mediators of Nodal
signaling
By combining our results with biochemical
(Germain et al., 2000) and
molecular epistasis data (Alexander et al., 1999), a model emerges in which
the Nodal signal provided by Sqt is transduced by a complex of Smad2/Smad4
that is recruited to specific target genes by either Bon or Sur (Foxh1;
Fig. 7). These two
transcriptional mediators of Nodal signaling have unique functions during the
formation of endoderm and axial mesoderm but have overlapping activities in
neural patterning. The genetic interactions between bon;sqt and
bon;sur indicate that Bon functions in parallel to Sqt and Sur
(Foxh1) to regulate the expression of mesendodermal genes, such as
dkk1, which in turn is required for neural patterning.
In endoderm formation, Bon functions downstream of Nodal signaling in an
Oep-dependent fashion (Alexander et al., 1999;
Kikuchi et al., 2000).
bon expression is unaffected in
MZsur/ embryos (data not shown), suggesting
that an additional Smad-binding transcription factor is involved in regulating
bon expression (Fig.
7; factor Y). A possible candidate for this activity
could be the Mix-like transcription factor, Mezzo that was shown to function
downstream of Nodal signaling. However, bon expression is probably
not regulated by Mezzo as Mezzo lacks a Smad interaction motif and
mezzo MO-injected embryos do not exhibit endoderm defects
(Poulain and Lepage, 2002
).
Thus, we propose that an additional, as yet unidentified, Smad-binding
transcription factor (Y) is involved in the initiation of bon
expression.
Once bon expression is initiated, our model places Bon and Sur
(Foxh1) as the two transcriptional mediators of Sqt signaling in neural
patterning. However, it should be re-emphasized that
MZsqt/ embryos exhibit a less severe neural
patterning defect than that seen in either
bon/;sqt/
or
bon/;sur/
embryos, which indicates that Sqt is not the sole signal regulating Bon
transcriptional activity. Thus, an additional factor (X) may function upstream
of Bon and in parallel to Sqt in neural patterning. In this model, factor X
could correspond to Cyc, as it has been suggested that the ventrolateral
mesoderm, which requires Nodal signaling for its formation, can provide a
secondary posteriorizing signal to the neural plate
(Erter et al., 2001;
Feldman et al., 2000
;
Woo and Fraser, 1997
). The
neural defect seen in
bon/;sqt/
and
bon/;sur/
embryos, but not in
cyc/;sqt/
embryos, may be caused by the presence of ventrolateral mesoderm and its
posteriorizing effect on the neural plate. Further studies should reveal how
the various Nodal ligands, as well as other signals, regulate neural
patterning, either directly, or through their regulation of mesendodermal gene
expression.
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ACKNOWLEDGMENTS |
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