Download Maternal control of the zebrafish organizer

3899
Development 127, 3899-3911 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV3249
Maternally controlled β-catenin-mediated signaling is required for organizer
formation in the zebrafish
Christina Kelly*, Alvin J. Chin*, Judith L. Leatherman, David J. Kozlowski and Eric S. Weinberg‡
Department of Biology, The University of Pennsylvania, Philadelphia, PA 19104, USA
*These two authors contributed equally to the work
‡Author for correspondence ([email protected])
Accepted 2 July; published on WWW 22 August 2000
SUMMARY
We have identified and characterized a zebrafish recessive
maternal effect mutant, ichabod, that results in severe
anterior and dorsal defects during early development. The
ichabod mutation is almost completely penetrant, but
exhibits variable expressivity. All mutant embryos fail to
form a normal embryonic shield; most fail to form a head
and notochord and have excessive development of ventral
tail fin tissue and blood. Abnormal dorsal patterning
can first be observed at 3.5 hpf by the lack of nuclear
accumulation of β-catenin in the dorsal yolk syncytial layer,
which also fails to express bozozok/dharma/nieuwkoid and
znr2/ndr1/squint. At the onset of gastrulation, deficiencies
in expression of dorsal markers and expansion of
expression of markers of ventral tissues indicate a dramatic
alteration of dorsoventral identity. Injection of β-catenin
RNA markedly dorsalized ichabod embryos and often
completely rescued the phenotype, but no measurable
dorsalization was obtained with RNAs encoding upstream
INTRODUCTION
The dorsal signaling center known as the ‘organizer’ was first
recognized in amphibians by Spemann and Mangold (1924) by
its ability to induce a second body axis when transplanted to a
second embryo. This signaling center also exists in other
vertebrate embryos – the teleost ‘embryonic shield’, the avian
‘Hensen’s node’ and the mammalian ‘node’ (reviewed in
Harland and Gerhart, 1997). In amphibians, induction of
mesoderm and formation of the organizer is dependent on the
activity of an earlier vegetal signaling site, often called the
Nieuwkoop center (Nieuwkoop, 1969), which is directly
dependent on maternal gene activity. Establishment of the
Nieuwkoop center requires cortical rotation of the fertilized
egg, resulting in a redistribution of maternal determinants
(Gerhart et al., 1989; reviewed in Moon and Kimelman, 1998).
In Xenopus, formation of the organizer is dependent on at
least two pathways involving maternally encoded products.
The T-box family transcription factor, VegT, is required for the
vegetal-derived signaling that converts cells of the equatorial
zone into mesoderm (Zhang et al., 1998), and β-catenin is
required for embryonic axis formation (Heasman et al., 1994;
Wnt pathway components. In contrast, dorsalization was
obtained when RNAs encoding either Bozozok/Dharma/
Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover,
injection of β-catenin RNA into ichabod embryos resulted
in activation of expression of these two genes, which could
also activate each other. RNA injection experiments
strongly suggest that the component affected by the ichabod
mutation acts on a step affecting β-catenin nuclear
localization that is independent of regulation of β-catenin
stability. This work demonstrates that a maternal gene
controlling localization of β-catenin in dorsal nuclei is
necessary for dorsal yolk syncytial layer gene activity and
formation of the organizer in the zebrafish.
Key words: β-catenin, Maternal effect, Axis formation, Dorsoventral
patterning, Spemann organizer, Nieuwkoop center, Zebrafish, Wnt
signaling, Nuclear localization
Wylie et al., 1996, reviewed in Sokol, 1999). Depletion of
maternal RNA encoding these products results, respectively, in
the inability to form mesoderm and the failure to develop axial
structures (Heasman et al., 1994; Zhang et al., 1998). In both
cases, the Spemann organizer fails to form. Depletion of
Xenopus maternal RNA encoding the GSK3-binding protein
GBP also causes ventralization (Yost et al., 1998). Since GBP
inhibits GSK3 by preventing Axin from binding GSK3 (Farr
et al., 2000), thereby resulting in stabilization of β-catenin
(Yost et al., 1998), this experiment also provides evidence for
maternal factors that regulate β-catenin-mediated dorsal
signaling. In frogs, two changes in protein levels have been
reported after cortical rotation on the prospective dorsal side in
a microtubule-dependent manner: (1) Disheveled accumulates
directly after cortical rotation, indicating the involvement of a
maternal Wnt pathway in dorsal cell fate establishment (Miller
et al., 1999), and (2) GSK3 levels decrease in the dorsal region
in early cleavage stages, possibly by a non-Wnt pathway signal
mediated by GBP (Dominguez and Green, 2000).
Zebrafish and Xenopus share many of the same mechanisms
involved in formation of the organizer and dorsal axis. As in
frogs, the formation of the zebrafish body axis is dependent on
3900 C. Kelly and others
cortical arrays of microtubules (Jesuthasan and Strähle, 1997),
which presumably facilitate the cytoplasmic rearrangements
required for formation of the yolk syncytial layer (YSL).
Additionally, nuclear β-catenin accumulates on the dorsal
side of both zebrafish and Xenopus embryos (Schneider et al.,
1996). In zebrafish, this nuclear localization is first observed
in the dorsal YSL and then later in overlying blastoderm
cells, suggesting that the dorsal YSL may be the functional
equivalent of the Xenopus Nieuwkoop center (Schneider et al.,
1996). Overexpression of β-catenin in zebrafish embryos can
induce a secondary axis (Kelly et al., 1995a), as it does in
Xenopus (Funayama et al., 1995; Guger and Gumbiner, 1995),
indicating a conserved response pathway to β-catenin in these
organisms.
Further evidence of Nieuwkoop center activity of the
zebrafish YSL is its ability to induce and pattern mesoderm
after transplantation to the animal pole region of an intact
blastula (Mizuno et al., 1996) or in a conjugate with an
animal cap (Ober and Schulte-Merker, 1999). Two proteins,
the homeodomain transcription factor Dharma/Nieuwkoid
(Yamanaka et al., 1998; Koos and Ho, 1998; Fekany et al.,
1999) and Nodal-related Znr2/Ndr1 (Erter et al., 1998,
Feldman et al., 1998) have recently been shown to act non-cell
autonomously in the YSL to induce goosecoid, a marker of the
organizer. Dharma/Nieuwkoid is encoded by bozozok (Fekany
et al., 1999), which when mutated leads to a ventralized
phenotype characterized by loss of notochord, prechordal
plate, and anterior and ventral CNS deficiencies (SolnicaKrezel et al., 1996). Znr2/Ndr1 (Erter et al., 1998; Rebagliati
et al., 1998) is encoded by the squint gene (Feldman et al.,
1998), which when mutated results in ventral CNS defects and
cyclopia. Embryos doubly homozygous for mutations in squint
and cyclops (another nodal-related gene) completely lack trunk
mesoderm and endoderm (Feldman et al., 1998). Normally,
LiCl-treated early blastulae are dorsalized by inhibition of
GSK3 (Hedgepeth et al., 1997). Thus, the observations that
LiCl can enhance bozozok/dharma/nieuwkoid expression
(Yamanaka et al., 1998) and that LiCl enhancement of
goosecoid expression is blocked in bozozok embryos (Fekany
et al., 1999) are suggestive that bozozok/dharma/nieuwkoid is
a downstream target of the β-catenin/Tcf pathway in the YSL.
Also in support of this relationship, the dharma/nieuwkoid
promoter has been reported to contain Tcf/Lef-binding sites
(Yamanaka et al., 1998), and nuclear localization of β-catenin
is observed in bozozok mutant embryos (Fekany et al., 1999).
β-catenin was originally shown to be required for axis
formation in Xenopus (Heasman et al., 1994; Wylie et al.,
1996), and it was recently established that mice lacking
functional β-catenin (Huelsken et al., 2000) or Wnt3 (Liu et
al., 1999b) genes also fail to form a normal primary axis. We
report here the characterization of a zebrafish maternal effect
mutant that severely affects formation of the organizer and
subsequent development of dorsal and anterior tissues. We find
that mutant embryos fail to accumulate β-catenin in YSL and
marginal blastoderm nuclei, providing strong evidence for a
requirement for β-catenin-mediated signaling in the zebrafish.
Mutant embryos can be dorsalized and rescued by RNAs
encoding β-catenin and the downstream proteins Znr2/Ndr1/
Squint and Bozozok/Dharma/Nieuwkoid, indicating that the
axis specification pathway downstream of β-catenin is intact in
ichabod mutant embryos. However, since injection of RNAs
encoding proteins that inhibit GSK3 activity or that increase βcatenin stability fail to dorsalize ichabod embryos, the ichabod
gene product most likely functions to ensure nuclear
localization of β-catenin, in parallel to the control of β-catenin
stability, rather than directly on modulating GSK3 activity or
levels.
MATERIALS AND METHODS
Animals
Stocks of Danio rerio were maintained and raised under standard
conditions at 28.5°C (Westerfield, 1993). The flhn1 stock that proved
to harbor the ichabod mutation was obtained from Dr C. Kimmel of
the University of Oregon, and then inbred for several generations in
our laboratory prior to the identification of the ichabod phenotype.
brass zebrafish, particularly suitable for studies involving in situ
hybridization because of delayed and reduced pigmentation and used
in this study as ‘wild-type’ in comparisons with ichabod embryos,
were originally obtained from EkkWill Waterlife Resources,
Gibbonston, FL.
DNA constructs
The following zebrafish probes were used for in situ hybridization:
znr2 (squint/ndr1) (Erter et al., 1998; Rebagliati et al., 1998),
nieuwkoid (bozozok/dharma) (Koos and Ho, 1998), goosecoid
(Stachel et al.,1993), chordin (Miller-Bertoglio et al., 1997), Otx1 (Li
et al., 1994), lim1 (Toyama et al., 1995), bmp4 (Chin et al., 1997), no
tail/Brachyury (Schulte-Merker et al., 1992), eve1 (Joly et al., 1993)
and gata2 (Detrich et al., 1995). The following constructs were used
to prepare RNA for injection: zWnt8 in pT7TS (Kelly et al., 1995b),
zFzA in pT3TS (Nasevicius et al., 1998), XDsh in Sp64T (Sokol et
al., 1995), Xβ-catenin in pCS2 (modified from Funuyama et al.,
1995), zβ-catenin in pZL1 (Kelly et al., 1995a), dominant negative
(dn) GSK3 (Xgsk-3K85R) in pCS2 (Pierce and Kimelman, 1995),
nieuwkoid in pGEM (Koos and Ho, 1998), znr2/squint in pBS(SK−)
(Erter et al., 1998), zebrafish GBP in pCS2+ (Sumoy et al., 1999),
Xenopus Axin GID 2 fragment in pCS2MT (Hedgepeth et al., 1999),
and Xenopus β-Trcp ∆F in pCS2+MT (Liu et al., 1999a).
RNA injections
Capped mRNAs were synthesized from linearized plasmid DNA
either by using 50 units of the appropriate RNA polymerase (T7, T3,
or Sp6) in the presence of 12.5mM m7G (5′)pppGTP cap structure
analog (New England Biolabs), or by using the protocol for the
mMessage mMachine Kit (Ambion). RNA was diluted 1:1 with 0.5%
phenol red/PBS (Sigma) and injected into either the yolk beneath the
blastomeres of 1- to 4-cell-stage embryos (for β-catenin RNAs) or into
a single blastomere of 8- to 16-cell-stage embryos (for all other RNAs
used). Final concentrations of injected RNAs varied from 50-1200
ng/µl, depending on the particular RNA. Embryos were injected with
approximately 1 nl of RNA solution (see Fig. 7 for the amount of
RNA injected for each species of RNA), containing a final
concentration of 0.25% phenol red.
In situ hybridization, immunohistochemistry and
microscopy
In situ hybridization was performed by a modified version (Li et al.,
1994) of the protocol of Schulte-Merker et al. (1992). β-catenin was
detected immunologically with antibody against Xenopus β-catenin
(Schneider et al., 1996) using embryos fixed in 4% paraformaldehyde
in PBS. Immunohistochemical staining was carried out after
incubation with secondary antibody using preincubated AB complex
(ABC Kit, Vector), DAB substrate and hydrogen peroxide. Whole
embryos were observed and photographed under a Leica MZ12 stereo
dissecting microscope.
Maternal control of the zebrafish organizer 3901
RESULTS
ichabod is a maternal effect mutation affecting
anterior and dorsal development
While inbreeding a strain of zebrafish containing the zygotic
recessive mutation floating head (flh) (Talbot et al., 1995), we
found six female fish that yielded embryos of unexpected
phenotype. These embryos lacked anterior structures and
notochords and had excessive development of ventral tail fin
tissue and blood. Most commonly, the embryos completely
lacked heads, and almost all of those embryos that did develop
heads had defective forebrains. When these female fish were
then bred to wild-type or brass males, batches of embryos
entirely abnormal in phenotype were obtained, a result not
expected for the zygotic recessive flh mutation. Although
failure to form a notochord is a characteristic of flh
homozygotes, the other phenotypic characteristics have not
been reported for flh. The presence of a strict maternal effect
mutation unrelated to flh was suggested by two initial findings:
(1) the abnormal embryo phenotype was obtained irrespective
of the male parent used in crosses with these six fish, even
when the females were outcrossed to wild-type fish, and (2)
99-100% of the embryos from these crosses were abnormal.
We set up a breeding scheme to determine if the phenotype
could be transmitted genetically, to test whether it was linked
to the flh mutation, and to discover whether the putative
mutation was dominant or recessive in the female parent (Fig.
1). The six females that transmitted the unusual phenotype
(which we denoted ichabod [ich]) were part of a large brood
of fish from a single tank that had originally been derived from
two pairs of fish (G0 parents), heterozygous for the flh
mutation. Since we had not sequestered these four parents, we
continued the line by breeding the siblings (F1 siblings) of the
six ichabod transmitting females (these original six females
could not be used for further genetic analysis since they yielded
only lethal embryos). Our reasoning was that, if the trait
were recessive, siblings should include females and males
heterozygous for the ichabod mutant allele as well as
homozygous males, and these siblings should be able to
transmit the gene. Alternatively, if the trait were a maternal
effect dominant, it would be recovered only when females
homozygous for the wild-type allele were crossed with male
carriers.
We started over 50 F2 families by breeding 50 sibling pairs
of fish (F1 siblings). In seven of these families, we were able
to recover F2 females that transmitted the ichabod phenotype
to their progeny (F3 embryos). In all cases tested, the
transmission of ichabod phenotype was observed irrespective
of the male to which the females were crossed, confirming that
ichabod is a maternal effect mutation. A dominant mode of
inheritance would be consistent only with ratios of 1/2 or 1/1
of the F2 females within a particular family yielding the
ichabod phenotype. If the mutation were inherited as a
dominant trait, a ratio of 1/4 ichabod transmitting females
would be impossible to obtain since the female F1 parent would
have had to be homozygous for the wild-type allele. A
recessive mode of inheritance is consistent with ratios of 1/4
or 1/2 of the F2 females within a particular family being
ichabod transmitting fish. In this case, the F1 female parent
would be heterozygous and the F1 male parent could be either
heterozygous or homozygous for the mutant ichabod allele.
Fig. 1. ichabod is a recessive maternal-effect mutation.
(Top) Siblings of females that gave rise to ichabod phenotype
embryos were used to generate families to test for the mode of
inheritance of the trait. A brood of F1 fish, bred from two pairs of flh
heterozygote G0 parents, contained six females that transmitted the
ichabod phenotype to >99% of their progeny. Pairs of F1 siblings of
these six transmitting females were used to generate families of F2
fish. (Bottom) Sibling matings were carried out within each family
(e.g., 22 pairwise crosses were performed with progeny of F1 pair no.
2). Matings were also performed between F2 females of family no. 2
and F2 males of family no. 33. In 7 of 33 families, we were able to
recover F2 females that transmitted the ichabod phenotype to their
progeny (F3 embryos). Ratios of ichabod-transmitting females in
four of the families generated from crosses of F1 siblings are
presented. The number of F2 females obtained in the other three
families was too low (<6) to be informative to analyze the mode of
inheritance. In two of the families (no. 7, no. 33), and in a subsequent
cross of F1 female no. 2 with male no. 33, the ratios of affected
families were consistent only with a recessive maternal effect mode
of inheritance. The ratio (A/B) is the number of F2 females that
transmitted the ichabod trait (A) divided by the total number of F2
females tested for that family (B). A χ2 test for significance of the
ratio of 1/4 gave high consistency for crosses within family no. 7
(P=0.65, χ2=0.2), within family no. 33 (P=0.4, χ2=0.79), and
between female no. 2 and male no. 33 (P=0.63, χ2=0.385); but high
inconsistency for the significance of a ratio of 1/2 for crosses within
family no. 7 (P=0.02, χ2=5.4), within family no. 33 (P<0.0001,
χ2=16), and between female no. 2 and male no. 33 (P=0.0015,
χ2=10.28). The mutation described here has received the allelic
assignment p1 (i.e., ichabodp1).
The table in Fig. 1 shows the ratios of ichabod transmitting
females in four of the families generated from crosses of
siblings of the original F1 fish (in three other ichabod
transmitting families, we obtained only two to six adult
females, a number too low to be informative to analyze the
mode of inheritance). In one family (no. 2), the ratio of
transmitting females was close to 1/2, and thus was not
informative in distinguishing between recessive and dominant
3902 C. Kelly and others
Fig. 2. ichabod embryos develop
anterior and dorsal deficiencies with
variable expressivity. Although
greater than 99% of the embryos
bred from ichabod-transmitting
females were larval or embryonic
lethal, the phenotypes of affected
embryos were variable.
Characteristics of classes 1-4 are
described in the text.
modes of inheritance. In another family (no. 28), 26 adult
females were obtained but the ratio of transmitting females was
intermediate between 1/1 and 1/2. However, in two of the
families (no. 7, no. 33), and in a subsequent cross of F1 female
no. 2 with male no. 33, we obtained sufficient numbers of fish
and highly statistically significant ratios to establish that the
ratios of affected families were consistent only with a recessive
mode of inheritance.
By following the transmission of ichabod and flh in the
families generated from crosses of F1 fish, we were able to
show that the two mutations segregated independently, and
were thus unlinked (data not shown). In addition, we have
recently shown that the ichabod locus is on zebrafish linkage
group 19 (C. K. and E. S. W., unpublished results), ruling out
ichabod as an allele of flh, which is located on linkage group
13 (Talbot et al., 1995). The map positions of bozozok and dino
(two other ventralized mutants) on linkage group 15 (bozozok,
Fekany et al., 1999; dino, Fisher et al., 1997) rules out ichabod
as an allele of these genes.
Penetrance and expressivity of the ichabod
mutation
As expected for a maternal effect mutation, over 98% of the
embryos bred from homozygous ichabod females exhibited an
embryonic mutant phenotype and over 99% were embryonic
or larval lethal (7849 embryos were scored in 65 crosses). Less
than 1% survived and were ‘escapers.’ There is variable
expressivity of the mutation and the phenotypes have been
classified into the following groups (Fig. 2). Class 1 (34%) are
headless embryos lacking a notochord, and are also deficient
in trunk development; most embryos in class 1 develop an
excess of blood, have large posterior somites and form multiple
tail fin structures (Fig. 2A,B), but some appear completely
radialized with little trunk development. Class 2 (30%) have
defects similar to class 1, but do form a trunk and at least
some hindbrain is present (Fig. 2C). Class 3 (28%) lack
notochord, have some forebrain and/or midbrain development
but are deficient in anterior forebrain and are either cyclopic
or completely lack eyes (Fig. 2D). Class 4 (6%) lack
notochord, but have recognizable forebrain, midbrain and
hindbrain structure, and eyes (Fig. 2E). Class 5 (2%) are
indistinguishable from wild type, but are larval lethal (not
shown in Fig. 2). This phenotypic series is similar to that
obtained by injection of an RNA encoding dominant negative
Tcf3 (Pelegri and Maischen, 1998; Sumoy et al., 1999) or by
removal of vegetal yolk (Mizuno et al., 1999). The ratio of
phenotypes that we obtained varied in repeated crosses of the
same parental pair. Generally, young homozygous female
parents (3-4 months of age) yielded more severe phenotypes.
As these fish aged, milder embryo phenotypes were seen at an
increased frequency (data not shown). A similar observation,
that severity of embryonic phenotype declines with maternal
age, was reported for the janus mutation, which results in a
splitting of the embryo during cleavage stage (Abdelilah and
Driever, 1997).
ichabod embryos are deficient in formation of the
organizer
Investigation of the morphological development of affected
embryos shows that they are deficient in the formation of the
embryonic shield (Fig. 3). As the wild-type embryo begins
gastrulation at 5-5.5 hpf, a thickening of the germ ring is seen
on the future dorsal side (Fig. 3A). This thickening becomes
the site of the initial involution of mesodermal and endodermal
cells and persists for several hours (Fig. 3B). In ichabod
embryos, a transient slight thickening is seen at 5-5.5 hpf (Fig.
3C), but within 30 minutes, the germ ring is again radially
symmetric in thickness, although with a more irregular border
(Fig. 3D). Although involution or ingression of mesoderm and
endoderm cells does occur, there is no obvious dorsal axial
mesoderm thickening. This is the earliest morphological defect
seen in ichabod phenotype embryos. Curiously, the lack of a
visible shield is observed even in embryos that will go on to
Fig. 3. The embryonic shield forms transiently in ichabod embryos.
The same wild-type (WT) (A,B) and ichabod (ich) (C,D) embryos
were photographed at the 50% epiboly (A,C) and 60% (B,D) epiboly
stages. Arrows point to a dorsal thickening of the germ ring,
characteristic of the forming embryonic shield. In ichabod embryos
of all classes, the thickening was only observed transiently. By 60%
epiboly, the germ ring appeared to be of uniform thickness around
the circumference of the embryo. Irregularities in the shape of the
germ ring border are characteristic of ichabod embryos.
Maternal control of the zebrafish organizer 3903
develop the less severe phenotypes (classes 3, 4 and 5).
Therefore, a morphologically normal embryonic shield is
not required for formation of axial mesoderm and the
formation of a complete neural tube (including anterior
brain). In most ichabod embryos, however, the absence of
a thickened embryonic shield is in fact accompanied by the
development of severe anterior and dorsal defects.
ichabod embryos have reduced dorsal gene
expression and have expanded ventral gene
expression domains
Since ichabod embryos do not form a normal organizer, it
was of interest to determine whether genes usually
expressed within the YSL and embryonic shield are active
in ichabod embryos. In situ hybridization with probes
representing genes with dorsal and ventral expression
patterns indicate that there is an early alteration of
dorsoventral identity in ichabod gastrula embryos. The
earliest changes in gene expression patterns were seen in
the dorsal yolk syncytial layer (YSL) in the sphere stage
blastulae (4 hpf). Transcripts of bozozok/dharma/nieuwkoid
and nodal-related znr2/ndr1/squint, genes normally
expressed at this time in the YSL (Fig. 4A,C) (Yamanaka
et al., 1998; Koos and Ho, 1998; Feldman et al., 1998; Erter
et al., 1998; Rebagliati et al., 1998), were not detected in
the YSL or elsewhere in 4 hpf ichabod embryos (Fig.
4B,D). (The later ring of marginal squint expression,
however, appeared to be normal in ichabod embryos, data
not shown).
Most ichabod embryos also do not express goosecoid and
chordin, which are expressed in wild-type embryos starting
at midblastula and are then characteristically expressed in
the embryonic shield region (Fig. 4E,I) (Stachel et al., 1993;
Schulte-Merker et al., 1994; Miller-Bertoglio et al., 1997).
To determine the distribution of phenotypes in the batch of
embryos tested by in situ hybridization, some sibling
embryos were always allowed to continue development to
24 hpf at which time the distribution of phenotypic classes
was scored. By correlating the percentages of ichabod
embryos that develop into the various phenotypic classes
with the distribution of in situ hybridization expression
patterns, we infer that class 1 and 2 embryos express no
detectable goosecoid at the 50% epiboly stage (Fig. 4F),
that class 3 embryos express the gene only in a few
scattered cells (Fig. 4G), and that class 4 embryos show a
disorganized pattern of reduced transcript (Fig. 4H) (similar
effects were seen at 80% epiboly, data not shown). In the
case of chordin, absence of expression at 50% epiboly was
correlated with the frequency of class 1 embryos (Fig. 4J)
but, unlike goosecoid, expression of chordin was seen in
what are probably class 2 embryos (Fig. 4K), although at
low levels. Another group of embryos, probably of class 3
phenotype, expressed more chordin transcript in the
embryonic shield region (Fig. 4L), but still at lower than
wild-type levels (similar effects were seen at 80% epiboly,
data not shown). lim1, which is normally expressed in the
marginal cells of the embryo from 30-50% epiboly and at
higher levels in the hypoblast at early shield stage (Fig. 4M)
(Toyama et al., 1995; Toyama and Dawid, 1997), is
expressed in the marginal cells of ichabod embryos, but not
in any region suggestive of embryonic shield expression
Fig. 4. ichabod embryos are deficient in formation of axial tissues, but
have expanded expression of ventral markers. Expression of the following
genes was monitored by in situ hybridization as follows:
bozozok/dharma/nieuwkoid in wild-type (A) and ichabod (B) 4 hpf
embryos; znr2/ndr1/squint in wild-type (C) and ichabod (D) 4 hpf embryo;
goosecoid in wild-type (E) and ichabod (F-H) embryos at 50% epiboly;
chordin in wild-type (I) and ichabod (J-L) embryos at 50% epiboly; lim1in
wild-type (M,O) and ichabod (N,P) embryos at 50% epiboly (M,N) and at
95% epiboly (O,P); Otx1 in wild-type (Q) and ichabod (R) embryos at
80% epiboly; no tail/Brachyury in wild-type (S) and ichabod (T) embryos
at 85% epiboly; bmp4 in wild-type (U,W) and ichabod (V,X) embryos at
50% epiboly (U,V) and 70% epiboly (W,X); eve1 in wild-type (Y) and
ichabod (Z) embryos at 50% epiboly; and gata2 in wild-type (A′) and
ichabod (B′) embryos at 50% epiboly. Embryos are shown in the following
orientations: dorsal view with animal pole to the top (A-L, O-T), animal
pole view with dorsal to the right (M,N,U,V,Y-B′), and lateral view with
dorsal to the right (W,X). In the upper right corner of each panel is
indicated whether the embryo is wild type or ichabod. The gene probed for
expression is indicated in the lower right corner of each panel.
3904 C. Kelly and others
of these two markers was expanded to encompass the whole
marginal zone and ectoderm, respectively (Fig. 4Z,B′). The
altered expression pattern of these genes in ichabod embryos
indicates an expansion of ventral identity to the dorsal side of
the embryo.
Fig. 5. β-catenin is not localized in nuclei of ichabod embryos. An
antibody prepared against Xenopus β-catenin (Schneider et al., 1996)
was used to determine the distribution of β-catenin in wild-type
(A,B) and ichabod (C,D) embryos at high (3.5 hpf) (A,C) and sphere
(4.0 hpf) (B,D) stages. Nuclear localization of the protein was
observed in the dorsal YSL of wild-type embryos at both stages and
in cells at the dorsal blastoderm margin (arrows in B) at sphere stage.
Nuclear localization was not observed in ichabod embryos. Arrows
in C indicate YSL nuclei which appear as holes, devoid of β-catenin
staining, whereas the irregularly shaped YSL region does show
staining. These unstained nuclei are also detected at 4 hpf, but are
more difficult to see.
(Fig. 4N). At later stages, when the expression is normally
confined to posterior axial mesoderm (Fig. 4O), ichabod
embryos completely lack expression of the gene (Fig. 4P).
Interestingly, the absence of shield expression at 50% epiboly
and axial mesoderm expression at 95% epiboly is observed in
ichabod embryos of all classes. Similarly, we found an absence
of no tail/Brachyury expression in axial mesoderm of all
ichabod embryos, although expression of this gene at the
margin is present in all ichabod embryos and is even more
pronounced than in wild-type embryos (Fig. 4S,T). Otx1 (Fig.
4Q,R) and Otx2 (data not shown) expression at 80% epiboly,
which marks the future forebrain and midbrain in wild-type
embryos (Li et al., 1994), was absent in class 1 and class 2
embryos (Fig. 4R), and diminished in class 3 phenotypes (data
not shown). These studies of gene expression are consistent
in indicating the absence of a normal organizer, a lack of
axial mesodermal tissue and a failure to induce anterior
neurectoderm in ichabod embryos.
We also studied the expression of a number of ventral
markers. bmp4 is normally expressed in the ventral half of the
shield stage embryo, except for a spot of expression in the
shield (Fig. 4U) (Nikaido et al., 1997; Chin et al., 1997). In
ichabod embryos of all classes, the expression of bmp4 was
expanded to include all of the dorsal side of the embryo (Fig.
4V). The spot of shield expression was absent from all but one
embryo examined (possibly a class 4 or 5 embryo). At later
stages of gastrulation, bmp4 expression continued to be
expressed radially in the marginal half of all ichabod embryos
(Fig. 4X). Results were similar for bmp2b expression (data not
shown). Two other genes normally expressed in ventral regions
at 50% epiboly also showed dorsal expression. eve1 expression
is normally restricted to the ventral and lateral cells of the
marginal zone (Fig. 4Y) (Joly et al., 1993) and gata2 is
normally only expressed in ventral ectoderm (Fig. 4A′)
(Detrich et al., 1995). In all ichabod embryos, the expression
ichabod embryos are deficient in nuclear
localization of β-catenin
Since the formation of the organizer was impaired in ichabod
embryos and early YSL markers were not expressed, we tested
whether β-catenin is appropriately localized in the nuclei on
the dorsal side of the blastula embryo, as had been shown to
occur in wild-type zebrafish and Xenopus embryos (Schneider
et al., 1996; Larabell et al., 1997; Koos and Ho, 1998). As
previously reported (Schneider et al., 1996; Koos and Ho,
1998), we found that nuclear localization of β-catenin in wildtype embryos first occurs in the dorsal YSL nuclei at the high
stage and then in blastoderm cells starting at sphere stage (Fig.
5A,B). In contrast, ichabod embryos (immunohistochemically
stained in parallel) show an absence of any nuclear localization
of β-catenin at these stages (Fig. 5C,D). β-catenin appears to
be present in these embryos, however, since staining of the cell
membrane regions is as intense as in the wild-type embryos
and staining of the YSL cytoplasm is apparent (indicated
by arrows in Fig. 5C). This cytoplasmic staining permits
visualization of the YSL and demonstrates that the layer is very
irregularly shaped in ichabod embryos.
Rescue of the ichabod phenotype by expression of
β-catenin
Since nuclear localization of β-catenin was absent in ichabod
embryos, we tested whether injection of β-catenin RNA could
rescue these embryos. Expression of Xenopus β-catenin
resulted in rescue of goosecoid expression (Fig. 6) and in
dorsalization of embryonic phenotypes (Fig. 7H). Identical
results were also obtained with injection of zebrafish β-catenin
RNA (data not shown). Moreover, phenotypic rescue was
surprisingly effective; in many experiments, over one third of
the injected ichabod embryos developed into fertile adults.
Embryos injected with Xenopus β-catenin RNA showed
nuclear localization of β-catenin and, interestingly, the region
containing the nuclear localized protein was fairly discrete
even though the co-injected GFP RNA lineage tracer was
located throughout the embryo (data not shown). We then used
RT-PCR to amplify β-catenin cDNA from RNA obtained from
2- to 4-cell embryos bred from ichabod homozygous females
and compared its sequence with that from a parental wild-type
strain. We found that there was no difference in the ORF
encoded in the two RT-PCR products; thus, it is highly unlikely
that ichabod is a mutation at the known β-catenin locus. This
conclusion is supported by the finding of a different map
position of the known zebrafish β-catenin gene (chromosome
16, Postlethwait et al., 1998).
Upstream Wnt pathway intermediates fail to rescue
ichabod embryos
Because ichabod embryos could be dorsalized and rescued
with β-catenin RNA, we next carried out a series of injections
with RNAs encoding various proteins in the canonical
Wnt→β-catenin pathway to determine whether a step upstream
of β-catenin in the signaling pathway might be inactive in
Maternal control of the zebrafish organizer 3905
ichabod mutant embryos. Embryos bred from ichabod
homozygous females were injected with RNA and assayed for
expression of the organizer marker goosecoid at shield stage
(Fig. 6), or allowed to develop to 24 hpf and scored for the
degree of anterior and dorsal deficiencies (Fig. 7). For
experiments in which we assayed goosecoid expression, we
also compared the transcript level with that of uninjected
sibling embryos and of injected and non-injected wild-type
embryos. For each experiment in which we scored the
degree of dorsalization, uninjected siblings were scored for
comparison as well. We also tested for the effect of each
injected RNA on development of wild-type embryos.
As shown in Figs 6 and 7A-C, RNAs encoding zebrafish
Wnt8, zebrafish FrizzledA (zFzA) and Xenopus Dishevelled
were all capable of activating ectopic goosecoid expression
in control wild-type embryos, but did not activate goosecoid
expression in ichabod embryos. Each of these RNAs was able
to dorsalize (and in some cases, induce secondary axes in)
wild-type embryos (data not shown), as predicted from
previous studies of Wnt8 and zFzA RNA injections
(Nasevicius et al., 1998; Kelly et al., 1995b), but had no
dorsalizing effects on ichabod embryos. We also injected an
RNA encoding a kinase-dead dominant negative form of
Xenopus GSK3 (dnGSK K85→R, Pierce and Kimelman,
1995), previously shown to dorsalize zebrafish embryos when
injected at high concentration (Nasevicius et al., 1998).
Again, we obtained the expected dorsalizing effects with
wild-type embryos, but not with ichabod embryos (Figs 6,
7E).
We then tested for the dorsalizing effects of RNAs encoding
the GSK3-binding protein GBP (Yost et al., 1998; Sumoy et
al., 1999; Farr et al., 2000) or a fragment of Xenopus Axin
(GID2, containing amino acids 320-429) that binds and inhibits
GSK3 (Hedgepeth et al., 1999). Injected wild-type embryos
were stimulated to produce high amounts of ectopic goosecoid
transcript (Fig. 6) and were markedly dorsalized in phenotype
(data not shown). ichabod embryos, in contrast, failed to
activate goosecoid (Fig. 6) and showed an unchanged ventral
phenotype distribution (Fig. 7D,F) in response to the injection
of zebrafish GBP and Xaxin GID2 RNAs. Since Xaxin GID2
binds to GSK3 and inhibits its kinase activity (Hedgepeth et
al., 1999) and GBP inhibits GSK3 by preventing its binding to
full-length Axin (Farr et al., 2000) and facilitates the depletion
of GSK3 (Dominguez and Green, 2000), it is unlikely that the
wild-type ichabod gene product has a role in the control of
GSK3 phosphorylation of β-catenin and, thus, in the control of
β-catenin stability. We also note that our sequencing of GBP
cDNA derived from ichabod embryos failed to detect any
Fig. 6. β-catenin RNA, but not RNAs encoding upstream Wnt
pathway proteins, can rescue goosecoid expression in ichabod
embryos. Wild-type (wt) or ichabod embryos were injected with
RNAs encoding the indicated proteins (z and X prefixes respectively
indicate zebrafish and Xenopus proteins), or were uninjected
controls. Embryos were allowed to develop to 50% epiboly and then
were assayed for goosecoid expression by in situ hybridization.
These experiments were carried out with batches of ichabod embryos
that developed almost entirely class 1 and class 2 phenotypes. The
lack of goosecoid expression was observed in every ichabod
uninjected and injected embryo, except for those embryos injected
with Xenopus β-catenin RNA.
differences in sequence in the GBP ORF and that GBP maps
to a different chromosome than ichabod (LG 16, Gates et al.,
1999), thus further ruling out GBP as a candidate for ichabod.
A gain-of-function mutation (unlikely from the recessive
pattern of inheritance of ichabod) in Axin1 is also ruled out,
since this is not on LG19 (M. Hibi, T. Hirano and R. Geisler,
personal communication).
3906 C. Kelly and others
Fig. 7. Phenotypic rescue of ichabod embryos
by injection with β-catenin, zrn2/ndr1/squint,
and bozozok/dharma/nieuwkoid RNAs, but not
with RNAs encoding upstream Wnt pathway
proteins. Frequencies of phenotypic classes of
uninjected embryos (black) are compared to
those of injected embryos (gray). Each panel
represents a separate experiment carried out
with a separate batch of embryos. Injected and
uninjected embryos were scored at 24 hpf. For
the Xenopus β-catenin RNA-injected embryos
(H), class 5 embryos include 25 embryos that
developed to adulthood (and are thus ‘class 6’,
17% complete phenotypic rescue). For embryo
batches injected with znr2/ndr1/squint, or
bozozok/dharma/nieuwkoid RNAs, class 5
embryos were only assayed at 24 hpf and we
did not test for survival to adulthood. Injection
of bozozok/dharma/nieuwkoid RNAs resulted
in high percentages of hyperdorsalized
embryos (column marked ‘D’ in [I]). RNAs
encoding Wnt pathway proteins upstream of βcatenin, dn GSK3, GBP, X-axin GID-2 and XβTrcp∆F all failed to significantly ameliorate
the ichabod phenotype. In each case, however,
injection of these RNAs into wild-type
embryos resulted in hyperdorsalized
phenotypes (data not shown). Embryos were
injected with approximately 1 nl of RNA
solution, which varied in concentration from
50-1200 ng/µl, depending on the particular
RNA. The approximate amount of each RNA
(in pg) injected per embryo is indicated at the
top of each panel, and the number of injected
and non-injected control embryos is noted to
the right of each panel.
To test further the idea that the ichabod mutation affects
organizer formation at a step distinct from the regulation of βcatenin stability, we injected mutant embryos with RNA
encoding β-Trcp∆F, a dominant negative form of the Fbox/WD40-repeat protein that recruits phosphorylated βcatenin for degradation by the ubiquitination-proteosome
pathway (Liu et al., 1999a). Injection of this RNA into Xenopus
embryos results in the induction of ectopic axes due to the
stabilization of β-catenin by blocking endogenous β-Trcp (Liu
et al., 1999a). When we injected this RNA into wild-type
zebrafish embryos, we observed the expected expansion of
goosecoid expression (Fig. 6) and dorsalization of embryonic
phenotype (data not shown), but there were no such effects in
injected ichabod embryos (Figs 6, 7G). Thus, under conditions
in which β-catenin would be stabilized by blocking the
degradation pathway, we still observed no amelioration of the
strongly ventralized ichabod phenotype.
Taken together, these results strongly suggest that control of βcatenin stability is not impaired in the mutant. Our examination
of the β-catenin ORF sequence indicates that the ichabod lesion
is not in β-catenin and mapping data rule out the possibility that
ichabod is a mutation in β-catenin cis-regulatory elements. These
results therefore suggest that the ichabod lesion may affect an as
yet poorly understood portion of the signaling pathway affecting
β-catenin release from the Axin-GSK3 complex or in the control
of transport of β-catenin into the nucleus.
Maternal control of the zebrafish organizer 3907
Proteins that act downstream of β-catenin can
dorsalize ichabod embryos
To test whether two genes that are expressed in the zebrafish
dorsal yolk syncytial layer (YSL), znr2/ndr1/squint and
bozozok/dharma/nieuwkoid, are activated by nuclear β-catenin,
we performed β-catenin RNA injection experiments. As also
shown above (Fig. 4B,D), transcripts from these genes were
absent from sphere stage ichabod embryos (Fig. 8E,G).
Following injection of β-catenin RNA, not only did we observe
an increase in both znr2/ndr1/squint and bozozok/dharma/
nieuwkoid transcripts in wild-type embryos (Fig. 8A-D) (as has
also been found by Shimuzu et al., 2000), but both of these
transcripts could now be detected at sphere stage (4.0 hpf) in
ichabod embryos (Fig. 8F,H). These results indicate that
ichabod embryos are capable of znr2/ndr1/squint and
bozozok/dharma/nieuwkoid expression when exogenous βcatenin is supplied, and it can be concluded that β-catenin can
activate the expression of these two genes.
We next took advantage of the absence of nuclear β-catenin
in ichabod embryos to test whether bozozok/dharma/nieuwkoid
could activate znr2/ndr1/squint, and vice versa, without input
from the β-catenin signaling pathway. Wild-type embryos
injected with znr2/ndr1/squint RNA clearly showed activation
of bozozok/dharma/nieuwkoid (Fig. 8I,J) and those injected
with bozozok/dharma/nieuwkoid RNA showed an increase in
expression znr2/ndr1/squint (Fig. 8K,L), but such activation
could have merely enhanced parallel activation by Tcf3/βcatenin in wild-type embryos. When identical experiments
were carried out with ichabod embryos, a striking activation
was observed of bozozok/dharma/nieuwkoid by znr2/ndr1/
squint (Fig. 8M,N) and znr2/ndr1/squint by bozozok/
dharma/nieuwkoid (Fig. 8O,P). These results suggests that the
pathway downstream of β-catenin signaling is not strictly
linear, but that at least two key proteins important in organizer
establishment can crossregulate their expression.
The effects of injection of znr2/ndr1/squint and bozozok/
dharma/nieuwkoid RNAs on expression of goosecoid at shield
stage were also examined. Expression of injected znr2/ndr1/
squint or bozozok/dharma/nieuwkoid RNAs had previously
been shown to result in enlargement of the organizer region or
formation of multiple organizers (Yamanaka et al., 1998; Koos
and Ho, 1998; Erter et al., 1998; Feldman et al., 1998).
Moreover, when injected into the dorsal YSL region, these
RNAs functioned in a non-cell-autonomous fashion to induce
goosecoid in the neighboring blastoderm margin (Yamanaka et
al., 1998; Koos and Ho, 1998; Erter et al., 1998). We found, as
expected, that injection of either of these RNAs markedly
increases and expands goosecoid expression in wild-type
embryos (Fig. 8Q-S). However, injection of bozozok/dharma/
nieuwkoid or znr2/ndr1/squint RNAs into ichabod embryos
only weakly induced goosecoid (Fig. 8U-W). Interestingly,
when the two RNAs were co-injected into ichabod embryos, the
goosecoid expression domain was expanded to the same extent
as when the RNAs were co-injected into wild-type embryos
(Fig. 8T,X). Thus, in the absence of nuclear β-catenin,
Znr2/Ndr1/Squint and Bozozok/Dharma/Nieuwkoid act
synergistically to activate goosecoid.
Further support for positioning znr2/ndr1/squint and
bozozok/dharma/nieuwkoid downstream of ichabod and βcatenin was provided by examining the degree of phenotypic
rescue of ichabod embryos injected with either znr2/ndr1/
squint or bozozok/dharma/nieuwkoid RNAs (Fig. 7I,J). A shift
towards less severe phenotypes was obtained after injection of
either of these RNAs. In either case, significant numbers of
normal-appearing embryos (19-20%) were produced (although
a large number of bozozok/dharma/nieuwkoid RNA-injected
embryos, but none of the znr2/ndr1/squint RNA-injected
embryos, were hyperdorsalized). Phenotypic rescue to class
5/6 embryos was lower, however, than with β-catenin RNA
injections. These results also suggest that normal axis
formation can proceed even when the level of goosecoid
expression in the organizer is absent or very low, since
significant numbers of bozozok/dharma/nieuwkoid RNAinjected ichabod embryos formed normal axes and exhibited
no obvious dorsal or anterior defects, even though goosecoid
transcripts were undetectable. Taken together, our findings
clearly place znr2/ndr1/squint and bozozok/dharma/nieuwkoid
downstream of ichabod and β-catenin in the dorsal axis
pathway.
DISCUSSION
Maternal control of organizer formation in the
zebrafish is mediated by β-catenin
We have found that ichabod gene activity is required in the
maternal parent, for proper control of the zygotic nuclear
localization of β-catenin in the embryo. Phenotypic effects of
a mutation in this gene are manifested in a classic maternal
effect recessive mode of inheritance. The lack of zygotic
requirement for the gene was indicated by our findings that
inheritance of the ichabod phenotype was completely
dependent on the female parental genome, that the male
parental genome had no effect on the distribution of phenotypic
classes of ichabod embryos and that crosses of heterozygous
parents never yielded abnormal embryos.
It has long been appreciated that, in Xenopus, dorsal vegetal
cells from pre-midblastula transition embryos can induce
mesoderm in conjugated ectodermal tissue (Nieuwkoop,
1969), and can induce a complete axis in UV-ventralized
embryos or a second axis in normal embryos (Gimlich and
Gerhart, 1984). Antisense-mediated mRNA-depletion
experiments have identified β-catenin, GBP (an inhibitor of
GSK3) and VegT as maternal factors essential for normal
development (Heasman et al., 1994; Yost et al., 1998; Zhang
et al., 1998). Since depletion of β-catenin RNA results in
failure to form the organizer (Heasman et al., 1994) and GBP
RNA depletion causes varying degrees of ventralization (Yost
et al., 1998), it can be concluded that maternal gene function
is important in regulating β-catenin levels in the Xenopus
embryo. In teleosts as well, mesoderm formation and
dorsoventral axis establishment are dependent on maternal
gene action. In both goldfish and zebrafish, embryos defective
in dorsal development were obtained when vegetal yolk was
removed shortly after fertilization (Tung et al., 1945; Mizuno
et al., 1997, 1999; Ober and Schulte-Merker, 1999),
suggesting that essential maternal determinants are localized
in this region of the egg. The results reported here clearly
show that a consequence of a mutation in ichabod is the failure
to properly localize β-catenin in dorsal YSL and blastoderm
margin nuclei, and that this defect is entirely dependent on
maternal ichabod activity.
3908 C. Kelly and others
Fig. 8. znr2/ndr1/squint and bozozok/dharma/nieuwkoid act
downstream of ichabod and β-catenin in the zebrafish axial signaling
pathway. (A-H) Injection of β-catenin RNA activates expression of
znr2/ndr1/squint and bozozok/dharma/nieuwkoid. Uninjected wildtype embryos express nieuwkoid (A) and squint (C) in the future
dorsal margin (arrows) at 4 hpf, whereas expression of either gene is
not observed in ichabod embryos (E,G). After injection of β-catenin
RNA, expression of these two genes in wild-type embryos is greatly
expanded (B,D), and expression of the two genes in ichabod
embryos is observed in a discrete marginal region (arrows) (F,H).
(I-P) znr2/ndr1/squint and bozozok/dharma/nieuwkoid can
crossactivate each other in both wild-type and ichabod mutant
embryos. Uninjected wild-type embryos express nieuwkoid (I) and
squint (K). Injection of squint RNA results in a marked increase in
nieuwkoid expression (J) and vice versa (L). The cross activation is
also observed in ichabod embryos (M-P). (Q-X) znr2/ndr1/squint
and bozozok/dharma/nieuwkoid act synergistically to activate
goosecoid in ichabod embryos. Injection of either of these RNAs or a
combination of the two RNAs into wild-type embryos causes marked
expansion of the region expressing goosecoid (R,S) in comparison
with the uninjected control (Q). A batch of ichabod embryos that
developed into class 1 and class 2 phenotypes did not express
goosecoid (U). Injection of bozozok/dharma/nieuwkoid or
znr2/ndr1/squint RNAs activated goosecoid only weakly in these
ichabod embryos (V,W), but co-injection of the two RNAs (X)
resulted in expression of goosecoid at the same level as in wild-type
embryos. The labels in the upper right corner indicate whether the
embryo is wild-type or ichabod, the lower left corner, the injected
RNA, and the lower right corner, the gene probed for expression. All
embryos are at 4 hpf sphere stage and are shown in animal pole
views.
ichabod acts upstream of β-catenin but not by
regulation of GSK3 activity or β-catenin stability
We have used RNA injection as a way of testing the epistatic
relationship between ichabod and β-catenin. Injection of βcatenin RNA resulted in effective rescue of wild-type
phenotype in ichabod embryos. We consider it very unlikely
that ichabod is a mutation in the β-catenin gene since the βcatenin ORF is identical in RNAs from eggs of ichabod
homozygous mothers and from embryos of the parental wildtype strain. A mutation in non-coding regulatory regions of a
β-catenin gene is possible, but our immunohistochemical assay
suggests that levels of non-nuclear β-catenin in ichabod and
wild-type embryos are comparable. Moreover, the map
position of ichabod is on chromosome 19 (C. K. and E. S. W.,
unpublished), whereas that of the known β-catenin gene is on
chromosome 16 (Postlethwait et al., 1998).
Nuclear localization and stability of β-catenin can be
modulated by the Wnt/wingless signaling pathway (reviewed
in Cadigan and Nusse, 1997; Sokol, 1999). Although we were
able to dorsalize wild-type embryos by injecting RNAs
encoding zWnt8, zFzA or Xenopus Dishevelled, injection of
these RNAs into ichabod embryos failed to activate goosecoid
(Fig. 6) and had no effect on the degree of dorsalization of
mutant phenotype (Fig. 7). Moreover, injection of RNAs
encoding three proteins that negatively affect endogenous
GSK3 activity similarly were unable to dorsalize ichabod
embryos (Figs 6, 7), although they markedly affected wild-type
embryos. These three RNAs encoded: (1) a kinase-dead
dnGSK3 (Pierce and Kimelman, 1995), which interferes with
GSK3 activity by binding to Axin, thus preventing GSK3
association with Axin (Farr et al., 2000), (2) GBP, which can
function in a similar way (Farr et al., 2000), but which also
facilitates a decrease in GSK3 levels in the embryo
(Dominguez and Green, 2000), and (3) the Xenopus Axin
fragment (GID-2), which rather than enhancing destabilization
of β-catenin by providing a scaffold for GSK3 interaction with
β-catenin, as is the case for full-length Axin, binds to GSK3
and inhibits its kinase activity (Hedgepeth et al., 1999). Thus,
even a depletion of GSK3 activity in ichabod embryos does not
lead to any measurable dorsalization, suggesting that the block
in the mutants is not in the pathway of regulation of GSK3
kinase activity or GSK3 level. Since GSK3 is thought to
directly phosphorylate β-catenin making it more susceptible to
proteolysis (e.g., Yost et al., 1996), our result suggests that βcatenin stability may not be affected in ichabod mutant
embryos.
Further corroboration that ichabod does not function to
regulate β-catenin stability was provided by the failure of
injected Xenopus β-Trcp∆F RNA (Liu et al., 1999a) to
dorsalize ichabod embryos. β-Trcp binds to β-catenin via its
WD40-repeat and binds to the Skp1 ubiquitination complex
through its F-box motif (Liu et al., 1999a). If β-Trcp lacks the
F-box (β-Trcp∆F), the protein acts as a dominant negative and
prevents the recruitment of β-catenin to the ubiquitination-
Maternal control of the zebrafish organizer 3909
proteosome complex (Liu et al., 1999a). Our result showing
that injection of β-Trcp∆F RNA failed to dorsalize ichabod
mutants embryos, while clearly having a dorsalizing effect on
wild-type embryos, strongly suggests that inhibition of βcatenin degradation cannot rescue the ichabod phenotype. If
the ichabod mutation is not located in the β-catenin gene and
ichabod mutants are not impaired in β-catenin stability, then
what step might be blocked in the mutant embryos? We
propose that the wild-type ichabod protein may function in a
step facilitating β-catenin release from the Axin-GSK3-APC
complex, or in the promotion of nuclear transport of β-catenin.
These functions are not yet understood in β-catenin signaling
and it is quite likely that ichabod encodes a novel protein
required for stabilized β-catenin to be utilized as a component
of the dorsal signaling pathway.
The YSL signaling center can form in ichabod
embryos injected with β-catenin RNA
The finding that expression in the YSL of either
Znr2/Ndr1/Squint or Bozozok/Dharma/Nieuwkoid can induce
organizer markers in the overlying blastoderm margin has led
to the conclusion that these proteins are mediators of
Nieuwkoop center activity (Erter et al., 1998; Feldman et al.,
1998; Yamanaka et al., 1998; Koos and Ho, 1998; Fekany et
al., 1999). We found that ichabod embryos fail to express
znr2/ndr1/squint and bozozok/dharma/nieuwkoid in the
dorsal YSL and that injection of β-catenin RNA resulted in
the activation of expression of these two genes. bozozok/
dharma/nieuwkoid has been posited to be a downstream target
of β-catenin signaling in the YSL (Yamanaka et al., 1998;
Fekany et al., 1999). Here, we demonstrate that the absence of
nuclear β-catenin in the YSL results in loss of expression of
this gene and of znr2/ndr1/squint as well, and that injection of
β-catenin RNA can activate expression of both genes in wildtype embryos and in ichabod embryos deficient in nuclear βcatenin. These results not only confirm the enhancement of
expression of these two genes by β-catenin in wild-type
embryos (Shimizu et al., 2000), but also, using the ichabod
mutants blocked in nuclear β-catenin, directly demonstrate the
dependence of bozozok/dharma/nieuwkoid and znr2/ndr1/
squint expression on endogenous β-catenin signaling. A novel
aspect of our studies is the demonstration that, in both wildtype embryos and ichabod mutant embryos, each of these
genes was capable of inducing the other. Thus, even in the
absence of nuclear β-catenin, znr2/ndr1/squint can strongly
induce expression of bozozok/dharma/nieuwkoid (Fig. 8M,N),
and bozozok/dharma/nieuwkoid can induce expression of
znr2/ndr1/squint, albeit more weakly (Fig. 8O,P). A recently
published study failed to detect these activations in
wild-type embryos, probably because far lower amounts of
RNA were injected (Shimizu et al., 2000). Since bozozok/
dharma/nieuwkoid is probably a direct target of Tcf3/β-catenin
as judged by the presence of Tcf3-binding sites in the bozozok
promoter (Yamanaka et al., 1998), one activation pathway is
probably Tcf3/β-catenin→bozozok→squint. Since bozozok
expression is normal in maternal-zygotic one eyed pinhead
mutant blastulae (Gritsman et al., 1999), direct activation by βcatenin in the absence of squint is sufficient for normal blastula
expression of bozozok. However, our results suggest that the
Tcf3/β-catenin→squint→bozozok sequence can also operate,
although it is possible that squint may not be a direct target of
Tcf3/β-catenin. The crossactivation of squint and bozozok
might allow persistence of activation of these genes after a
decrease of β-catenin signal during development.
Injection of either bozozok/dharma/nieuwkoid or znr2/ndr1/
squint RNAs into ichabod mutant embryos only weakly
activated goosecoid, but co-injection of the two RNAs
strikingly activated expression of this gene (Fig. 8U-X). These
results are interesting in two respects. First, although goosecoid
levels were very low (or even absent in one experiment) in
bozozok/dharma/nieuwkoid RNA-injected ichabod embryos,
injection of the RNA strongly dorsalized ichabod embryos and
resulted in many normal appearing embryos (Fig. 7J). This
finding suggests that axis formation and correct dorsoventral
patterning can proceed in zebrafish embryos even with little or
no goosecoid expression. This result is in agreement with the
findings that mouse embryos homozygous for a null goosecoid
allele do not have gastrulation or neural induction defects
(Yamada et al., 1995; Rivera-Perez et al., 1995) and chick
embryos can develop normally even when manipulated so that
their organizer does not express goosecoid (Psychoyos and
Stern, 1996). Second, since co-injection of the two RNAs
strongly resulted in synergistic activation of goosecoid (Fig.
8T,X), a significant interaction of gene pathways dependent
on expression of a TGFβ protein (Znr2/Ndr1/Squint) and
Bozozok/Dharma/Nieuwkoid can occur in the region of the
blastoderm margin. However, since we obtained significant
crossactivation of the two genes (Fig. 8N,P), we might have
expected that levels of both proteins present in ichabod embryos
injected with only one of these RNAs would be sufficient to
synergistically activate goosecoid. Further analysis on the
molecular structure of the induced transcripts produced
endogenously must be carried out before these findings can be
fully understood. The block in β-catenin signaling in ichabod
mutant embryos provides us with an opportunity to further
examine the spatial and temporal aspects of the network of gene
interactions that occur in the YSL and dorsal blastoderm
margin. We plan to extend these studies to investigate whether
other proteins expressed in the YSL are regulated in linear
pathways or whether there is extensive crossactivation of genes
expressed in this early signaling center.
We thank Gianfranco Bellipanni, Mary Mullins, Michael Granato,
Peter Klein, Daniel Kessler and Cecilia Lo for helpful discussions
during the course of this work. We are grateful to Dr C. Kimmel for
sending us the flh stock of zebrafish and Mary Mullins for supplying
us with antibody against β-catenin. cDNA clones were kindly
provided to us by R. Ho, M. Rebagliati, C. Wright, D. Grunwald, S.
Schulte-Merker, R. Toyama, M. Halpern, G. Kelly, S. Ekker, D.
Kimelman, M. Mullins, P. Klein, X. He and D. Kessler. This work
was supported by NIH grant RO1 NS34365 to E. S. W., a University
of Pennsylvania Research Foundation grant to E. S. W., and NIH
training grant fellowships to C. K. and D. J. K.
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