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First published online 14 June 2006
doi: 10.1242/dev.02391
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Biotechnology Center and Center for Regenerative Therapies, University of Technology (TU) Dresden, c/o MPI of Molecular Cell Biology and Genetics, Dresden, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Author for correspondence (e-mail:
brand{at}mpi-cbg.de)
Accepted 4 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish, Spg, Pou2, Oct4, Dorsoventral patterning, Bmp, Epiboly, Maternal effect, Axis formation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gard et al., 1997;
Howley and Ho, 2000;
Rand and Yisraeli, 2001;
Volodina et al., 2003). Cells
are endowed with different sets of these determinants and acquire their fates
according to their position. AV-asymmetry probably prefigures DV polarity, as
removal of vegetal yolk mass leads to lack of dorsal structures in the
zebrafish (Mizuno et al.,
1999; Ober and Schulte-Merker,
1999). The Drosophila AP axis is determined prior to DV
patterning, both of which are executed by the activity of various maternal
factors (reviewed by Roth,
2003). The chordate DV axis is inverted, and ventral specification
involves signaling of Bmps, homologs of invertebrate Dpp
(Arendt and Nübler-Jung,
1994; Holley and Ferguson,
1997). Other axis-defining pathways have not been conserved among
vertebrates and invertebrates during evolution, and vertebrate DV
specification by maternal factors is less well understood.
DV patterning in vertebrates integrates: (1) dorsalizing pathways involving
dorsal stabilization of maternal ß-catenin (reviewed by
Wodarz and Nusse, 1998)
(Schier, 2001); (2)
ventralizing pathways depending on maternal and zygotic TGFß signaling
(Goutel et al., 2000;
Bauer et al., 2001;
Mintzer et al., 2001;
Sidi et al., 2003) (reviewed
by Hammerschmidt and Mullins,
2002) and zygotic Wnt signaling
(Ramel and Lekven, 2004); and
(3) noncanonical Wnt/Ca2+ signaling, which downregulates canonical
Wnt signaling (reviewed in Pandur et al.,
2002).
The zebrafish DV axis is determined prior to zygotic transcription, at the
mid-blastula transition (MBT) (Kane and
Kimmel, 1993; Stachel et al.,
1993; Strähle and
Jesuthasan, 1993). Maternal ß-catenin is a key player to
initiate dorsal identity (reviewed by Hibi
et al., 2002). Specialized cells at the dorsal blastodermal
margin, the prospective shield, become induced by the underlying yolk
syncytial layer (YSL, the functional equivalent of the amphibian Nieuwkoop
center) by maternal factors previously deposited into the egg (reviewed by
Sakaguchi et al., 2002). The
zebrafish shield is functionally equivalent to the Spemann-Mangold organizer
(Shih and Fraser, 1996;
Saude et al., 2000). A key
activity of the shield is the transcriptional repression of ventralizing genes
such as Bmp genes, vox and vent. This repression is mediated
by boz (dharma -Zebrafish Information Network)
(Yamanaka et al., 1998;
Fekany et al., 1999;
Koos and Ho, 1999;
Kawahara et al., 2000a;
Kawahara et al., 2000b;
Leung et al., 2003a). During
gastrulation, the coarse DV pattern is refined and involves formation of a Bmp
gradient (Wilson et al., 1997;
Dosch et al., 1997). This is
achieved by dorsally emanating Bmp antagonists such as chordin and Noggin,
which binds Bmps and prevents them from activating their receptors (reviewed
by Schier, 2001;
Hammerschmidt and Mullins,
2002).
The establishment of ventral identity is also controlled by maternal
factors and involves the TGFß factor Radar, which signals through the
Alk8/Laf receptor to activate zygotic bmp2b and bmp4
expression (Goutel et al.,
2000; Bauer et al.,
2001; Mintzer et al.,
2001; Payne et al.,
2001; Kramer et al.,
2002; Sidi et al.,
2003). Zygotic bmp7 expression is probably activated by a
different, yet unidentified, maternal pathway
(Sidi et al., 2003). The early
embryo is predominantly ventrally specified, except for a dorsal region devoid
of Bmp gene expression, where the organizer is active. Later, during
gastrulation, Bmps autoregulate via the Alk8 receptor
(Bauer et al., 2001;
Mintzer et al., 2001). Dorsal
and ventral factors are crucial to keep the maternally established DV polarity
equilibrated, as evidenced by zebrafish mutants lacking activity of Bmp7
(snh), Bmp2b (swr) or Smad5 (sbn), which fail to
develop proper ventral tissues because of dorsalization of the embryo, or by
ventralized embryos mutated in chd (dino) or
sizzled (ogon/mercedes) (reviewed by
Hammerschmidt and Mullins,
2002).
The zebrafish genome is transcriptionally inactive during the first hours
of embryogenesis, and zygotic transcription starts at MBT. Until this stage,
development relies on maternal factors deposited in the oocyte (reviewed by
Pelegri, 2003). pou2
encodes the POU domain transcription factor Spg/Pou2
(Belting et al., 2001;
Burgess et al., 2002;
Reim and Brand, 2002).
pou2 mRNA is uniformly present during initiation of oogenesis and is
subsequently confined to the animal oocyte region
(Howley and Ho, 2000). After
fertilization, pou2 transcripts are ubiquitously present in the
embryo and overlap spatiotemporally with zygotically transcribed pou2
mRNA from late blastula until midgastrula stages
(Takeda et al., 1994;
Hauptmann and Gerster, 1995;
Reim et al., 2004).
pou2 plays a zygotic role in the establishment of the
midbrain-hindbrain organizer (Schier et
al., 1996; Belting et al.,
2001; Burgess et al.,
2002; Reim and Brand,
2002). An earlier pou2 function for endoderm initiation
was detected through analysis of maternal-zygotic (MZ) spg mutant
embryos (Reim et al., 2004;
Lunde et al., 2004).
By analysis of MZ/Mspg embryos, we describe a novel maternal function of pou2 in establishing DV patterning and, independently, during epiboly. Our analysis indicates that maternal pou2 acts upstream of Alk8 signaling, and is required on the prospective ventral side to initiate and maintain zygotic expression of Bmp genes, vox, vent and eve1. Pou2 is therefore among the earliest maternal factors required for the initiation of ventral-specific fate. Based on the maternal requirement for pou2 in ventral specification, we propose that ventral specification employs an active, pou2-dependent maternal induction step, rather than a default ventralizing program.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); and (2)
MZspg mutant embryos derived from wild-type females carrying
spghi349 mutant germ cells. Primordial germ cells (PGCs)
of spghi349-/- mutant donor embryos were transplanted into
wild-type host embryos at midblastula stages. Mutant donor PGCs were labeled
by injecting a heterologous vasa-3'UTR-gfp mRNA at the
one-cell stage (Wolke et al.,
2002). Transplanted embryos were screened at 1 dpf for GFP
expressing PGCs. To obtain MZspg mutant embryos, transplanted
PGC-chimeric females were crossed to spghi349+/- males.
The progeny consists of MZspg, Mspg, spg+/- and
wild-type embryos. The ratio of these genotypes varied, depending on the total
number of transplanted PGCs. The most successfully transplanted female carrier
produced 80% MZspg embryos per egg lay. Initially, method (1) was not
successful, and method (2) was employed instead to produce MZspg
embryos for the initial experiments. MZspg embryos showed no
difference with respect to their generation strategy.
Injection of mRNAs
mRNAs of ca-alk8 (30 pg), bmp2b (20-30 pg), bmp4
(20-30 pg) and XFD (400 pg) were injected at the one-cell stage. Embryos
depicted in Fig. 6 were
injected with 30 pg pou2 + 100 pg egfp mRNA into one or few
blastomeres at the 16- to 128-cell stage. At the beginning of shield formation
or the beginning of gastrulation, respectively, embryos where photographed
individually with the aid of UV light microscopy to visualize EGFP expression,
subsequently individually processed in single tubes for bmp4 in situ
hybridization analysis.
Cell transplantations
Wild-type cells were injected with rhodamine-dextrane
(Mr 40,000; Molecular Probes), MZspg cells were
injected with 100 pg egfp-mRNA. Labeled wild-type and MZspg
cells were co-transplanted at the shield stage into dorsolateral regions
within the germring of MZspg- and wild-type host embryos,
respectively. Host embryos were analyzed around tb stage, transplanted cells
were monitored by UV light microscopy. Fig.
5: wild-type and MZspg donor cells were rhodamine labeled
as described above and transplanted into wild-type embryos between 50% epipoly
and shield stage. Chimeric embryos were analyzed 24 hpf for several days and
monitored by UV light microscopy.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (reviewed by
Kane and Adams, 2002). In
MZspg embryos, doming is reduced (not shown), and the blastoderm does
not flatten properly (Fig.
1B'). Normal gastrulation commences with the appearance of
the circumferential marginal germring and the shield on the dorsal side,
reflecting involution of cells around the embryonic margin
(Fig. 1C,D). In MZspg,
involution and shield formation starts on schedule; however, the blastoderm
covers only 40%, rather than 50%, of the yolk
(Fig. 1C',D'). The
width of the germring, including the shield, appears thicker when compared
with the wild type (Fig.
1D'; arrowheads). During normal epiboly, the vegetal margin
of the EVL and the YSL `lead the way' (Fig.
1E,G), trailed by the edge of the blastoderm
(Fig. 1E,G). In MZspg,
epiboly of the blastoderm is delayed from the start
(Fig. 1B') and ceases at
the beginning of gastrulation (Fig.
1E'-H'; red arrows indicate the blastoderm margin). By
contrast, epiboly of the underlying YSL continues normally in MZspg
(Fig. 1E'-H'), as
does the EVL (Fig.
1E'-H'; yellow arrows indicate the vegetal margin of
the YSL and EVL; see Fig. S1L-R in the supplementary material). In the wild
type, ventrolateral ectoderm and mesoderm converge to the dorsal side and
extend along the AP axis during gastrulation
(Fig. 1E-H). This process is
disrupted in MZspg (Fig.
1E'-H'), where the prospective head region does not
reach the animal pole on schedule but dives vegetally into the yolk
(Fig. 1H'). Eventually
MZspg mutants develop a dorsalized morphology
(Fig. 1I-I'').
|
). Round
cells line the notochord and the blastoderm margin (arrow in
Fig. 1I'), that may have
detached from the notochord, as judged by their expression of ntl and
foxa1 (Fig. 2D, and
not shown). MZspg embryos are severely shortened along their AP axis,
as their notochord and ntl expression is shortened
(Fig. 1I,I' and
Fig. 2C-H'). After
gastrulation, paraxial somites form dorsally in the wild type
(Fig. 1I). Somites form on
schedule in MZspg, but stretch to the ventral side
(Fig. 2I''). Accordingly,
myod expression is observed on the ventral side of MZspg
embryos (Fig. 2A,B).
pax2.1, a marker for the pronephric duct, a ventrolateral derivative,
is missing in 19 out of 32 MZspg embryos at tailbud (tb) stage
(Fig. 2I,J), or strongly
reduced in 9 out of 32 MZspg embryos (not shown). Pronephric
pax2.1 expression is missing at early somite stages in all of the 37
MZspg embryos examined (Fig.
2B; and data not shown). Like pax2.1, gata1 expression in
ventral mesoderm is either strongly reduced in 14 out of 30 MZspg
embryos, or missing in 16 out of 30 MZspg embryos
(Fig. 2K,L; and data not
shown). Endodermal differentiation also fails in MZspg, as described
elsewhere (Reim et al., 2004;
Lunde et al., 2004).
Dorsalization is also apparent in the neuroectoderm. krox20 is
normally expressed in rhombomeres 3 and 5; rudimentary expression fuses
ventrally in MZspg embryos (Fig.
2E,F, arrows). gbx2 marks the anterior hindbrain
primordium (Fig. 2G, black
arrow) and superimposes mesodermal gbx2 expression (white arrow).
Neuroectodermal expression fails in MZspg
(Reim and Brand, 2002)
(Fig. 2H,H'), while
mesodermal gbx2 expression fuses opposite of ntl expression
in MZspg (Fig.
2H,H', white arrow; inset in H shows a ventroanimal view of
an embryo expressing only gbx2). Dorsalization is also observed for
brain-specific expression of pax2.1; however, expression is strongly
reduced (arrowhead in Fig. 2J)
as its activation requires zygotic pou2
(Reim and Brand, 2002). The
forebrain expression of otx2 is strongly expanded in MZspg
(Fig. 2M,N; chd marks
the dorsal side), which is already manifested at its onset (inset of
Fig. 2M,N). pax6
expression in the fore- and hindbrain-primordium is as well dorsalized in
MZspg (Fig. 2O,P).
Non-neural ectodermal expression of gata2, gata3, p63 and
dlx3 is lacking in MZspg
(Fig. 2Q-V, and data not
shown). At late somitogenesis, MZspg embryos are severely malformed,
and necrotic cells appear throughout the embryo
(Fig. 1J-L), which eventually
dies. In summary, MZspg embryos display a striking fusion of somites
and expansion of anterior neuroectoderm at the expense of non-neural ectoderm
reminiscent of dorsalized phenotypes, as observed (for example) in Bmp-pathway
mutant embryos or in embryos with excess Fgf signaling. In contrast to these
embryos, MZspg embryos show ventrally fused expression of dorsal
markers, which is not completely circumferential, and split notochords, which
might be due to the epiboly defect.
|
). When
transplanted into MZspg embryos, wild-type and mutant cells behave
the same: they do not converge but stay together in clusters
(Fig. 2X), suggesting that cell
movement is impaired in the mutant environment. Conversely, MZspg and
wild-type cells within a wild-type host undergo normal convergence and
extension movements (Fig. 2Y).
These results suggest that abnormal morphogenesis of MZspg cells is
strictly non cell-autonomous, and not due to a cell-intrinsic movement
defect.
|
;
Melby et al., 2000). Both
repress transcription of dorsally expressed organizer genes such as boz,
chd, gsc or axial (foxa2 - Zebrafish Information
Network) in the wild-type blastula (Melby
et al., 2000; Melby et al.,
1999; Kawahara et al.,
2000a; Kawahara et al.,
2000b; Imai et al.,
2001; Leung et al.,
2003a). After normal initiation at sphere or dome stage,
respectively, non-marginal expression of vox and vent are
lost and marginal expression is reduced in MZspg from 40% E (epiboly)
onwards (Fig.
3D',E'). Importantly, MZspg embryos are not
generally delayed, but initiate expression of unaffected markers on schedule
with the wild type at all stages examined. This is substantiated by the proper
timing of the initiation of morphological landmarks such as the shield or
somites (Fig.
1C,C',I,I''; Fig.
2A,B).
Genes required for dorsal identity, including the dorsal shield, are
nog1, chd and gsc. Nog1 antagonizes Bmp signaling by binding
and sequestering Bmp ligands (Zimmerman et
al., 1996). nog1 expression initiates faintly at the
dorsal side at oblong-sphere stage in the wild type
(Fig. 3J)
(Fürthauer et al., 1999).
nog1 expression is stronger and expanded to the ventrolateral side in
MZspg embryos shortly after its activation
(Fig. 3J'). This might
explain the expansion of otx2 we observe in MZspg
(Fig. 2N), as otx2 is
a downstream target of nog in Xenopus
(Gamse and Sive, 2001).
Similarly, chd expression initiates between sphere and dome stage
(Fig. 3K). Initiation of
chd expression is normal in MZspg, although its ventral
limitation is less sharp than in the wild type
(Fig. 3K'). Between dome
stage and 30% E chd strongly expands ventrolaterally in
MZspg (Fig.
3L',M'). gsc expands similarly in
MZspg after normal initiation at sphere-dome stage; however, ectopic
expansion is restricted to the germring
(Fig. 3N-O'). Expansion
of nog1, chd and gsc lasts during blastula stages in
MZspg. A further dorsalizing component is Fgf8, which is expressed
from blastula stages onwards. However, fgf8 expression and activity
is normal at blastula and gastrula stages in MZspg embryos, as Fgf
target genes such as spry4, erm and pea3 are normally
expressed (see Fig. S1 in the supplementary material).
During gastrulation, DV patterning remains disrupted in MZspg embryos. After normal initiation, the ventral marker eve1 is strongly reduced in MZspg from 50% E onwards (Fig. 3F,F'; not shown). bmp2b, bmp4 and bmp7 expression remain lost within the blastoderm; however, bmp2b expression within the YSL is not affected in MZspg, similar to other dorsalized mutants (Fig. 3G-I'). Like gsc, bmp2b and bmp4 are also expressed in the wild-type shield during gastrulation, and are maintained in the shield and prechordal plate, respectively. bmp2b and bmp4 initiation within these domains is not affected in MZspg at shield stage, but expression is not maintained during gastrulation (Fig. 3G',H'; not shown). Other dorsal mesodermal genes expressed in the notochord (nog1 and ntl in the posterior axial mesoderm) or the prechordal plate/hatching gland [hgg1 (ctslb - Zebrafish Information Network) gsc or foxa2 in the anterior axial mesoderm] are ventrolaterally expanded in MZspg (not shown). The prechordal plate did not reach the wild-type level of the animal pole at tb stage (Fig. 1G',H', and not shown). Despite the early and strong dorsalization, specific dorsal features remain intact in MZspg. For example, the shield (Fig. 1C-D'), or ß-catenindependent expression of boz, hhex and sqt was normal in MZspg (Fig. 3P-Q'; not shown).
With regard to DV patterning and epiboly, Mspg embryos look
identical to MZspg embryos until beginning of gastrulation (not
shown), but defects in epiboly always recovers until tb stage in Mspg
embryos (Fig.
1E''-H''). Expressivity and penetrance of dorsalization
is highly variable in Mspg (Fig. 1M,N; see Fig. S1 in the
supplementary material). `Strong' Mspg mutants show severe reduction
of non-neural ectoderm (not shown). A variable proportion of Mspg
embryos even restores to the wild-type phenotype (not shown). Zspg
embryos undergo normal epiboly and display no apparent DV patterning defect
(Reim and Brand, 2002),
suggesting that zygotic pou2 is not required to regulate DV
specification and epiboly.
Establishment of axial asymmetry
Deposition of maternal mRNAs to the unfertilized egg is a common mechanism
to propagate asymmetry to the embryo. In the zebrafish, different mRNA
localization patterns reveal asymmetry established along the AV axis during
oogenesis, including pou2 (Howley
and Ho, 2000). MZspg embryos lack pou2
transcripts (Reim et al.,
2004), and might therefore derive from improperly polarized
oocytes. However, we found that maternally deposited smad3a/3b/5,
radar (gdf6a - Zebrafish Information Network) alk8,
actRIIb (acvr2b - Zebrafish Information Network), foxh1,
zorba and brul (cugbp1 - Zebrafish Information Network)
mRNAs are unaffected in spg-/- mutant oocytes and
MZspg zygotes (not shown). One possibility is therefore that, rather
than controlling asymmetric RNA localization itself, pou2 might
control implementation of asymmetrically deposited information in the
oocyte.
MZspg embryos can respond to Alk8- and Bmpmediated signaling
In MZspg embryos, Bmp gene expression is either not initiated
(bmp2b, bmp4) or is only partially initiated (bmp7), which
prompted us to test if signaling pathway(s) leading to Bmp gene initiation are
affected in MZspg embryos. Maternally expressed
alk8/laf is the Type I TGFß-receptor induced by the
maternal ligand Radar and, presumably via its nuclear effectors Smad5 or
Smad8, activates zygotic expression of bmp2b and bmp4, but
not bmp7 (Hild et al.,
1999; Bauer et al.,
2001; Mintzer et al.,
2001; Payne et al.,
2001; Kramer et al.,
2002; Sidi et al.,
2003). Transcripts encoding components of this maternal signaling
pathway, such as alk8, radar and smad5 are normal in
MZspg embryos at early cleavage and blastula stages (not shown). To
determine at which level pou2 might act during the establishment of
DV patterning, we carried out epistasis experiments by injecting
constitutively active (ca-) alk8 mRNA into MZspg zygotes.
Wild-type embryos are ventralized upon ca-alk8 injection and can be
distinguished from non-injected embryos by their spherical shape at the end of
gastrulation (Fig. 4A,B)
(Bauer et al., 2001). All
injected MZspg embryos (n=45) are similarly ventralized to
the wild type (Fig.
4B,B'). Expression analysis showed suppression of
dorso-axial and neuroectodermal genes, or even ventralization, i.e. loss of
somitic marker expression, depending on the amount of injected mRNA
(Fig. 4D-F').
Alk8 is later required for maintenance of Bmp2b/4/7 signal transduction
(Bauer et al., 2001).
ca-alk8-mediated reversion of dorsalization could reflect its
function to maintain Bmp signaling during gastrulation, which would not allow
a distinction from bypassing its maternal function in the initiation of Bmp
gene expression. Therefore, we analyzed early activity of misexpressed
ca-alk8. We observed an intensified bmp2b expression level
in injected wild-type embryos at the sphere stage
(Fig. 4I,J). MZspg
embryos initiate bmp2b expression upon ca-alk8 misexpression
at the oblong stage, and intensity of expression is comparable with the
wild-type level (Fig. 4I,K).
bmp4 is normally not activated prior to dome stage/30% E
(Fig. 4L). ca-alk8
misexpression therefore elicits premature initiation of bmp4 in
wild-type embryos at sphere stage and MZspg embryos at oblong stage;
however, in both cases the intensity of expression is comparable with the
normal bmp4 initiation level (Fig.
4M,N, compare with Fig.
3C).
|
). Lack
of pou2 prevents bmp2b initiation in MZspg. To test
effects of exogenous Bmp, bmp2b mRNA was injected into MZspg
embryos at the one-cell stage. MZspg embryos (50 of 50) responded to
Bmp gene injection as observed for ca-alk8-injected embryos
(Fig. 4C,C'). mRNA
injection of bmp4 into MZspg rescues and ventralizes
MZspg embryos in a similar fashion
(Fig. 4G; compare with
Fig. 4E-F'). Furthermore,
pou2 mRNA injection cannot rescue Bmp gene mutants, and pou2
is expressed normally in snh/bmp7, sbn/smad5, swr/bmp2b mutant
embryos at blastula and gastrula stages (not shown). Together, mutant in situ
hybridization and epistasis analysis indicates that Alk8-mediated signal
transduction is intact in pou2-deficient embryos. Therefore, maternal
pou2 acts upstream of Bmp gene initiation and upstream of maternal
Alk8 function.
In contrast to the rescue of DV patterning in MZspg embryos,
ca- alk8/Bmp gene misexpression could not rescue endodermal
sox17 expression (Fig.
4H; 40 out of 40 injected embryos), which is never expressed in
MZspg (Reim et al.,
2004). Bmp gene misexpression could not rescue the epiboly defect
(Fig. 4C'); however,
ca-alk8 misexpression led to a slight amelioration of epiboly in 50%
of injected MZspg embryos (Fig.
4B').
|
). With respect to DV patterning the relationship between
pou2 and fgf8 appears rather antagonistic: pou2 is
required to activate Bmp genes (Fig.
3A-C'), while fgf8 misexpression represses
bmp2b, bmp4 and bmp7
(Fürthauer et al., 1997;
Fürthauer et al., 2004).
We therefore asked whether interference with Fgf signaling might cause a
reverse effect in pou2- deficient embryos, by restoring Bmp gene
expression. To analyze the effect of Fgf interference in MZspg
embryos, mRNA encoding XFD, a dominant-negative FGFR
(Amaya et al., 1991), was
injected at the one-cell stage into MZspg. XFD injection caused
different effects: bmp2b expression is restored and even expanded to
the dorsal side at late blastula stages in MZspg embryos, as in
injected wild-type embryos (Fig.
4O). By contrast, XFD injection had no effect on bmp4
expression in the wild type (Fig.
4Q), and bmp4 was not initiated in XFD-injected
MZspg embryos (Fig.
4R). At the same time, ectopic and endogenous expression of
chd, gsc and nog1 was repressed in MZspg by XFD, as
observed in the wild type (not shown)
(Amaya et al., 1993). These
experiments suggest that in the absence of pou2 function,
bmp2b expression, but not bmp4, can be restored by releasing
Fgf-mediated transcriptional repression of bmp2b. This derepression
can bypass the requirement for pou2 to initiate bmp2b
expression. Therefore, bmp2b is probably not directly activated by
pou2.
Dorsalized MZspg cells can be corrected by wild-type environment
pou2-dependent control of proper Bmp gene initiation probably
underlies the dorsalization of MZspg embryos. Therefore, it should be
possible to rescue MZspg cells when transplanted into wild-type
embryos. Rhodamine-dextrane labeled MZspg cells where transplanted
into the ventral region of wild-type host embryos between 50% E and shield
stage. The fate of donor cells was analyzed after 24 hpf. In the wild-type
embryo, cells at ventral positions at late blastula stages give rise to
ventral derivatives such as blood or the ventral tail fin
(Fig. 5A,A'). Similarly,
MZspg cells transplanted into wild-type embryos contribute to blood
and ventral tail fin, but were not found in dorsal mesodermal tissues
(Fig. 5B'-E'). This
indicates an ability of strongly dorsalized MZspg cells to
differentiate into ventral(- most) mesodermal derivatives in wild-type
embryos. This finding illustrates that MZspg cells can show the
maximal possible response to ventralizing signals such as Bmps from the
wild-type environment.
Spatial requirement for pou2 to establish ventral gene expression
During early DV patterning stages, pou2 transcripts are
ubiquitously distributed in the blastula embryo, raising the question of where
pou2 is required. Theoretically, dorsalization in MZspg
might be explained if Pou2 protein would be required for either: (1) induction
of ventral-specifying genes ventrally; (2) repression of dorsalspecifying
genes dorsally; or (3) for both. To analyze the spatial requirement for
pou2 function within the early embryo, we injected pou2 mRNA
into single marginal or non-marginal cells of the 16-32-cell stage of
MZspg embryos, together with egfp mRNA. Coinjected embryos
were fixed at shield initiation (Fig.
6A,B) or the beginning of gastrulation
(Fig. 6C-N), analyzed for EGFP
expression prior to fixation and for bmp4 expression. bmp4
is not initiated in MZspg embryos
(Fig. 3C',H';
compare with Fig. 6B' for
wild-type bmp4 expression) and therefore served as a functional
readout for ventral gene expression in these embryos. EGFP expression allowed
for spatial allocation of pou2-injected cells, in relation to
eventual bmp4 expression domains. We find that bmp4
expression can be restored only in MZspg embryos when ventral
(Fig. 6E-F',I-J) or
ventrolateral blastomeres (Fig.
6A,B,C-D',G-H) were injected with pou2 mRNA (19 out
of 26 injected MZspg embryos, the remaining embryos did not show
bmp4 expression). Restoration of bmp4 expression appears
cell autonomous in MZspg at the beginning of shield formation
(Fig. 6A,B) and at the
beginning of gastrulation (Fig.
6C-J). By contrast, bmp4 expression was never observed in
MZspg embryos injected with pou2 mRNA into
dorsal/dorsolateral or non-marginal blastomeres
(Fig. 6K-N; 16 out of 16
injected MZspg embryos). Therefore, pou2 is required from
early cleavage stages in the prospective ventral blastomeres to initiate
bmp4.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
proportion of MZspg embryos has residual gata1 and
pax2.1 expression within the prospective blood and pronephros,
respectively. Residual expression of these genes was also observed in embryos
devoid of Bmp signaling (Pyati et al.,
2005). Unlike other dorsalized mutants, where somites and dorsal
neuroectodermal markers extend around the circumference of the embryos,
somites in MZspg embryos are displaced from the notochord, which is
split. In this aspect, MZspg embryos resemble epiboly mutants
(Kane et al., 1996), and
altered epiboly may therefore explain the later split notochord and somite
defect in MZspg embryos.
|
). In accordance with this, uncontrolled nog1
expression in MZspg might cause ectopic chd and gsc
expression. nog1 is unlikely to cause repression of bmps
from their onset, as bmp2b/7 expression is already affected before
nog1 becomes expanded in MZspg embryos. nog1
gain-of-function experiments would clarify whether nog1 would be
sufficient to repress Bmp gene initiation.
In contrast to Bmp genes, induction of vox and vent is independent
of zygotic gene expression but becomes partially wnt8 dependent
during late blastula and Bmp gene-dependent during gastrula stages,
respectively (Kawahara et al.,
2000a; Kawahara et al.,
2000b; Ramel and Lekven,
2004). The partial loss of vox/vent expression might
cause ectopic expansion of chd and gsc in MZspg
embryos at late blastula stages. This is similar to loss of both vox
and vent function, which causes ectopic expansion of chd and
gsc (Imai et al.,
2001), and probably explains why embryos mutated in Bmp ligands,
such as swr/bmp2b or snh/bmp7 do not ectopically express
chd or gsc until gastrulation. Taken together, pou2
functions in parallel pathway(s) to activate ventral-specific genes, and is
required to maintain vox, vent and eve1. pou2 might repress
nog1 independently of any relay by bmp genes, as nog1
expression is normal in Bmp gene mutant embryos.
pou2 acts upstream to Alk8/Bmp-mediated signaling
Type I TGFß receptors, including Alk8, mediate TGFß signaling
(reviewed by Hammerschmidt and Mullins,
2002). Alk8 transduces a maternal signal via the nuclear effector
Smad5/8 that is required for initiation of bmp2b and bmp4,
but is dispensable for bmp7 activation. The maternal signal for Alk8
might be Radar, a GDFsubgroup member of Bmps
(Rissi et al., 1995;
Sidi et al., 2003).
alk8/laf, radar and smad5 are maternally and ubiquitously
provided throughout early wild-type development
(Mintzer et al., 2001;
Goutel et al., 2000;
Sidi et al., 2003; Dick et
al., 1999; Hild et al., 1999).
Smad5 is required to relay a maternal signal also to initiate bmp7
expression at blastula stages, and amorphic smad5 mutants exhibit
strong maternal-zygotic effects, where embryos are earlier and more strongly
dorsalized when compared with all thus far described DV patterning mutants,
including compound bmp2b/7- mutant embryos
(Hild et al., 1999;
Schmid et al., 2000;
Kramer et al., 2002). Later,
during gastrulation, Smad5 also mediates auto-/crossregulation of
bmp2b/4/7 (Kishimoto et al.,
1997; Schmid et al.,
2000; Hild et al.,
1999; Kramer et al.,
2002). In case of bmp7 initiation, Alk3 and Alk6 are
putative maternal TGFß receptors; however, the ligand(s) of this
signaling system are so far unknown (reviewed by
Wilm and Solnica-Krezel,
2003).
Similar to the loss of smad5 and radar function, lack of
ventral induction and dorsalization is very early manifested in MZspg
embryos. Furthermore, radar-morphant embryos display ectopic
expression of chd after normal initiation, which is probably due to
reduced Bmp gene expression in these embryos
(Sidi et al., 2003). As
chd and gsc expression is normal in bmp2b mutants,
Radar probably acts either via its zygotic target bmp4 or by other
factor(s) to repress dorsal genes. Although maternal transcripts of alk8,
smad5 and radar are present at normal levels in MZspg
embryos, they are not sufficient to mediate proper Bmp gene activation.
However, we did not analyze their translational or post-translational
expression, which might be affected in MZspg. Injection of an
activated Alk8 receptor or bmp2b/4 is able to rescue and ventralize
MZspg embryos. Therefore, TGFß signaling can function in absence
of Pou2, arguing that pou2 does not act downstream of Alk8 signaling
to activate bmp2b/4. Instead, Pou2 probably acts upstream of the
Alk8, Smad5 and, eventually, Bmp signaling, which would be in accordance with
the strong dorsalization in MZspg embryos.
Ventral requirement for pou2 to initiate Bmp gene expression
pou2 transcripts are ubiquitously present in the early embryo;
however, their uniform distribution does not reveal a distinct spatial
requirement for pou2 in DV patterning. We analyzed the spatial
requirement of pou2 by pou2 mRNA injection into
MZspg embryos at early cleavage stages
(Fig. 6). In the injection
assay, we found that pou2 is needed in ventral or ventrolateral
blastomeres, respectively, from early cleavage stages onwards to restore
bmp4 expression.
Maternal pou2 antagonizes Fgf-mediated Bmp gene repression
Fgfs play an important role on the dorsal side, as dorsal XFD injections
elicit more severe dorsalization than do ventral injections
(Isaacs et al., 1994). In
addition to organizer-derived secreted factors, Fgf signaling also modulates
Bmp activity by transcriptional suppression
(Fürthauer et al., 1997;
Fürthauer et al., 2004).
Antagonism between Egf/Fgf and Bmp signaling is thought to converge on Smad1,
which is phosphorylated by MAPK, further preventing nuclear accumulation of
Smad1 (Kretzschmar et al.,
1997; Koshida et al.,
2002; Pera et al.,
2003). In turn, repression of Bmps at the protein level causes
breakdown of Bmp geneautoregulation in Xenopus and mouse
(Ghosh-Choudhury et al., 2001;
von Bubnoff and Cho, 2001).
However, we did not observe overactivated or ectopic expression of Fgf genes
or their downstream targets spry2/4 and pea3 in
MZspg embryos at any stages, suggesting that the failure of Bmp gene
activation does not involve ectopic Fgf signaling. In spite of normal dorsal
Wnt signaling and elevated expression of organizer molecules such as Nog1 and
Chd, blocking Fgf signaling was sufficient to repress dorsal development in
MZspg embryos, which is reminiscent of interference with dorsal Wnt
signaling in Xenopus (Gerhart et
al., 1989). In particular, expression of bmp2b, but not
bmp4, could be rescued in MZspg embryos. At the same time,
chd, nog1 and gsc could be repressed. This suggests that
dorsalizing pathways crossregulate each other, and removal of one dorsalizing
component causes breakdown of the maintenance of other dorsalizing pathways.
Moreover, in the absence of Fgf signaling, pou2 is dispensable for
bmp2b expression at late blastula stages. By derepression, i.e.
repression of an Fgf-dependent repressor, pou2 might therefore
indirectly activate bmp2b expression at late blastula stages, which
might already apply for bmp2b initiation.
Fig. 7 summarizes the function
of pou2 during establishment of DV patterning.
Dorsal organizer function is normal in MZspg
Cognate dorsalized Bmp gene mutants show normal gene expression within the
dorsal organizer, suggesting that early maternal events in DV patterning and
organizer establishment are normal in these mutants. By contrast, dorsal
expression of gsc, nog1, chd, including the organizer, is aberrant in
the absence of pou2. For example, gsc expansion in
MZspg is similar as in LiCltreated embryos and embryos with
overactivated canonical Wnt signaling, which provoke ectopic axes
(Stachel et al., 1993).
However, dorsalization in MZspg does not include ectopic organizer
activity. boz is among the earliest genes initated dorsally around
MBT by maternal Wnt/nuclear ß-catenin signaling and is normally required
to initiate organizer-specific gene expression
(Koos and Ho, 1999;
Yamanaka et al., 1998;
Fekany et al., 1999;
Ryu et al., 2001;
Leung et al., 2003a;
Leung et al., 2003b).
boz represses bmp2b and mutually represses
vox/vent, thereby defining the extent of the initial dorsal organizer
(Melby et al., 2000;
Kawahara et al., 2000a;
Kawahara et al., 2000b;
Leung et al., 2003a). However,
boz and another ß-catenin-dependent gene, hhex
(Ho et al., 1999), are not
expanded, but are normally expressed in MZspg
(Fig. 3). The reduction of
vox and vent in MZspg embryos seems therefore not
responsible for lateral expansion of boz, as previously indicated
(Imai et al., 2001). Rather,
ved is upregulated upon reduction of vox/vent, and able to
repress boz (Gilardelli et al.,
2004). Downstream of ß-catenin, Nodal signaling is required
for the function of the dorsal organizer, and the Nodal-related factor Sqt
induces chd and nog (reviewed by
Schier, 2001). However,
expression of sqt and cyc, as well as of Nodal signaling is
normal in MZspg embryos (Reim et
al., 2004). Therefore, lack of pou2 does not cause
ectopic function of the dorsal organizer. The broadened shield and various
aberrant forms of axial tissue in MZspg embryos, including split and
incomplete duplications, might be a consequence of reduced convergence
combined with affected epiboly. Altogether, ventral expansion of nog,
chd and gsc at late blastula stages are not sufficient to induce
an ectopic organizer activity in MZspg embryos. Beside the organizer,
gsc and chd are also expressed in non-axial mesendoderm
during blastula stages, which might be predominantly affected in
MZspg embryos. In addition, ectopic organizer activity might require
deregulation of further organizer-specific genes like boz, sqt or
cyc.
|
). Highest
Bmp activity at the ventral side specifies ventral regions and inhibits CE
movements. Lowest Bmp activity at the dorsal side enforces extension, but
reduces convergence movements, and intermediate Bmp activity at lateral
regions of the embryo favor both convergence and extension. Therefore, Bmp
activity tightly links DV patterning to CE movements. Wild-type and
MZspg cells migrate in the same manner, which strictly depends on the
genotype of the host embryo, but not of donor cells
(Fig. 3). The cell
non-autonomous convergence defect of MZspg cells is probably
attributable to loss of Bmp gene function, which also leads to pronounced
extension movements, most obvious at the dorsal axis of wild-type and
dorsalized embryos (Fig. 3)
(Myers et al., 2002). However,
normal extension along the AP axis is probably obscured by stalled
blastodermal epiboly in MZspg embryos.
pou2 is cell-autonomously dispensable at later stages of ventral differentiation
At the beginning of gastrulation, the position of a cell correlates to its
prospective fate (Kimmel et al.,
1990; Ho and Kimmel,
1993). We analyzed the fate of dorsalized MZspg cells
from prospective ventral positions within wild-type embryos
(Fig. 5). The
MZspg/wild-type chimera analysis shows a dispensability of cell
autonomous pou2 for late DV patterning. Therefore, a wild-type
environment is able to bypass non-autonomously, by providing secreted Bmps,
the early requirement of pou2 for Bmp gene activation. This result is
reminiscent to smad5/sbn function: sbn mutant cells
transplanted into wild-type embryos can give rise to ventral tissues,
indicating that in a wild-type environment these cells can differentiate
properly (Hild et al., 1999).
Alternatively, pou2 expression in the wild-type environment could
exert a cell non-autonomous function on transplanted MZspg cells, as
pou2 is expressed in the tail region of wild-type embryos during
somitogenesis (Hauptmann and Gerster,
1995). Similar to Smad5 function, ultimate specification of
ventral fates is independent of cell autonomous pou2, in contrast to
initiation of bmp2b/4 at blastula stages, which is pou2 and
smad5-dependent.
Epiboly is differentially affected in MZspg embryos
Epiboly is a morphogenetic movement of the zebrafish embryo in which the
blastoderm engulfs the spherical yolk (reviewed by
Kane and Adams, 2002).
Movement of the YSL occurs independently of blastoderm epiboly, and the EVL is
linked to the YSL, whereby the yolk may act as a towing motor of epiboly
(Trinkaus, 1951). This is in
accordance with our findings, as EVL and YSL undergo normal epiboly in
MZspg in spite of arrested blastoderm epiboly. It is therefore
unlikely that the blastoderm is merely passively `stretched out' between these
two layers. The epiboly phenotype of MZspg is reminiscent of epiboly
mutants such as hab, ava, law and weg, which eventually
arrest epiboly of deep blastodermal cells
(Kane et al., 1996). These
mutants are allelic series of E-cadherin/hab
(Kane et al., 2005). The gap
between the YSL/EVL and the blastoderm begins to widen at 80% E in
hab, whereas in MZspg a much stronger delay between the
leading EVL/YSL and the lagging deep blastodermal cells is obvious from shield
stage onwards. In ava mutants, deep cells escape into the space
between the YSL and the EVL. We similarly observe scattering of mesodermal
MZspg cells (Fig.
1I', Fig.
2D,H) that might therefore be linked to the epiboly phenotype.
This indicates that cell adhesion is impaired in MZspg and other
epiboly deficient embryos; however, E-cadherin transcription is
normal in MZspg during blastula and gastrula stages (not shown). The
molecular basis of the affected epiboly in MZspg is currently
unknown.
Maternal pou2 and MHB development
Previous analysis of Zspg embryos showed that zygotic
pou2 is required to establish the MHB organizer
(Schier et al., 1996;
Belting et al., 2001;
Burgess et al., 2002;
Reim and Brand, 2002). The MHB
appears slightly more impaired in MZspg when compared with strong
Mspg or Zspg embryos
(Fig. 2F,J, and not shown),
although altered DV patterning and epiboly certainly contribute indirectly to
the phenotype in MHB development, we cannot rule out that residual maternal
Pou2 protein might also have a subtle function in MHB development.
Three independent maternal functions of pou2
MZspg embryos display failure of initial endoderm differentiation
(Reim et al., 2004;
Lunde et al., 2004), of
initial DV patterning and epiboly. These components of the MZspg
mutant phenotype probably reflect independent pou2 functions. (1) In
MZspg embryos DV patterning is disturbed shortly after MBT, whereas
endoderm development is only disrupted later, from the beginning of
gastrulation onwards. (2) Embryos affected in Bmp signaling can differentiate
into endoderm, and endoderm mutants do not fail in DV patterning. (3) DV
patterning mutants, including strongly dorsalized Mspg embryos,
undergo normal epiboly, and epiboly-defective embryos have normal DV and
endoderm differentiation (Kane et al.,
1996; Bruce et al.,
2005). (4) Mutants affected in endoderm formation, including
strongly dorsalized Mspg embryos, have no arrest in epiboly, and vice
versa. (5) Rescue of dorsalization in MZspg restored DV patterning
without any rescue of defective endoderm or epiboly. However, we cannot rule
out a slight improvement of epiboly in ca-alk8 injected
MZspg embryos, which might be Bmp gene-independent. (6)
MZspg cells transplanted into wild-type embryos show similar
morphogenetic movements as wild-type cells and adopt positions according to DV
positional values of the wild-type host, suggesting that MZspg cells
can overcome their epiboly and DV patterning defects. By contrast, in a
similar transplantation experiment, endodermal differentiation remains
defective (Reim et al., 2004).
In summary, we suggest that the phenotype of MZspg mutant embryos
reflects a mosaic of independent functions of pou2 in different
processes, including patterning as well as morphogenesis during early
development.
pou2 and Oct4 in early development
Oct4/Pouf51 is a functional ortholog of pou2 in mammals
(Burgess et al., 2002;
Reim and Brand, 2002), raising
the issue of whether mammalian Oct4 has a similar function as its zebrafish
ortholog. Murine Oct4 is thought to be required for totipotency of
stem cells, and in Oct4 mutant mice, extra-embryonic tissue forms at
the expense of an embryo proper (Pesce and
Schöler, 2000). This seems not to be the case in zebrafish,
at least with respect to embryonic somatic and germ stem cells: embryonic
tissue develops; primordial germ cells develop normally in MZspg
embryos (G.R. and M.B., unpublished). Oct4 is also required for the transition
of the primitive, premigratory into migratory hypoblast
(Botquin et al., 1998) and
functions in early embryonic differentiation of the primitive endoderm and the
germ line (Niwa et al., 2000;
Pesce and Schöler, 2000),
reminiscent of the function of pou2 in controlling endodermal versus
mesodermal fate choice (Lunde et al.,
2004; Reim et al.,
2004). A putative zygotic role in DV patterning in mammals might
be obscured by the requirement for Oct4 before implantation. Recent
observations in mammals suggest the presence of maternally derived asymmetries
determining cleavage patterns
(Zernicka-Goetz, 2004).
Germline inactivation of Oct4 revealed its requirement for germ cell survival
(Kehler et al., 2004), which
so far precluded further analysis of Oct4 in early asymmetry of the mammalian
egg. However, Xenopus XPou-25, which belongs to the same protein
subclass as Pou2 and Oct4, can stimulate transcription of Xvent-2B
and prevents differentiation of neuroectodermal and mesodermal tissues when
misexpressed (Cao et al.,
2004). Although the role of pou2 has been mainly studied
by loss-of-function experiments, the Xeno
