Vanderbilt University, Department of Biological Sciences, VU Station B 351634, Nashville, TN 37235-1634, USA
* Author for correspondence (e-mail: lilianna.solnica-krezel{at}vanderbilt.edu)
Accepted 8 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Blimp1, Chordin, BMP, Gastrula organizer, Convergence and extension
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A wealth of data support the notion that the activity of the gastrula
organizer is highly dynamic, but our understanding of the underlying genetic
hierarchies remains incomplete (Spemann
and Mangold, 2001) (reviewed by
Hibi et al., 2002
). In
zebrafish embryos, many organizer-specific genes, such as goosecoid
(gsc), are expressed in the dorsal blastomeres exclusively, whereas
others, for example boz, are initially expressed in dorsal
blastomeres, somewhat later in both dorsal blastomeres and the dorsal YSL, and
finally exclusively in the dorsal YSL
(Stachel et al., 1993
;
Yamanaka et al., 1998
;
Koos and Ho, 1998
). The
contribution of the dorsal YSL to the specification and function of the
gastrula organizer remains puzzling. Transplantation studies demonstrate that
the yolk cell, including the YSL, is capable of inducing organizer gene
expression in host blastomeres in a non-cell-autonomous fashion
(Mizuno et al., 1999
).
Furthermore, function of boz only in the YSL is sufficient for normal
development (Fekany et al.,
1999
). However, experimental degradation of RNA in the YSL does
not prevent organizer formation (Chen and
Kimelman, 2000
). Therefore, the YSL and the dorsal blastoderm
probably function in a redundant fashion to mediate Spemann-Mangold organizer
formation and function.
The vertebrate gastrula organizer contributes to the elaboration of the
embryonic pattern by secreting factors, including Chordin, Noggin and
Cerberus, that antagonize the ventrally expressed TGF-ß superfamily
cytokines, the BMPs. The resulting BMP activity gradient is thought to specify
a dorsoventral and anteroposterior progression of cell fates, with head and
dorsal midline structures developing at the lowest levels of BMP activity,
trunk structures at intermediate levels, and ventroposterior structures being
specified by the highest BMP activity levels (reviewed by
Hammerschmidt and Mullins,
2002; De Robertis et al.,
2000
). Therefore, understanding of the genetic hierarchy that
regulates BMP activity during vertebrate gastrulation is of particular
importance. In mouse embryos homozygous for mutations that inactivate both
chordin and noggin, axis formation proceeds rather normally,
except that forebrain and notochord are missing
(Bachiller et al., 2000
),
suggesting that yet additional factors operate to limit BMP activity in murine
embryos. In zebrafish, boz and chordin (chd) act in
partially overlapping pathways to limit BMP activity during head and trunk
development (Gonzalez et al.,
2000
). Whereas chd mutant embryos exhibit mild
ventralization and boz embryos lack dorsal structures such as
forebrain and notochord (Schulte-Merker et
al., 1997
; Fekany et al.,
1999
), boz chd double-mutant embryos exhibit synergistic
loss of head and trunk and have an enlarged tail, largely due to excess BMP
activity (Gonzalez et al.,
2000
). Expression of chd is strongly reduced in young
boz gastrulae but, at late gastrulation, returns almost to normal
levels (Fekany-Lee et al.,
2000
). Additionally, chd expression is only mildly
affected in ndr1/sqt mutant embryos
(Dougan et al., 2003
). Thus,
it is likely that several genes play redundant roles in regulating
chd gene expression in zebrafish gastrulae.
The Krüppel-type zinc-finger gene Prdm1/Blimp1 (PR domain
containing 1, with ZNF domain; B lymphocyte-induced maturation
protein 1), a transcriptional repressor known to promote terminal
differentiation of B lymphocytes and macrophages in the mouse embryo
(Turner et al., 1994;
Chang et al., 2000
), is
expressed in multiple tissues during early embryogenesis in Xenopus
laevis, mouse and chick (de Souza et
al., 1999
; Chang et al.,
2002
; Ha and Riddle,
2003
). Notably, Prdm1/Blimp1 is expressed in the
anterior endomesoderm in X. laevis and the visceral endoderm in mice,
tissues hypothesized to possess similar activities as the YSL of the zebrafish
embryo supported by expression of orthologous genes such as
hematopoietically expressed homeobox (hhex) and LIM
homeobox 1a (lhx1a) (reviewed by
Sakaguchi et al., 2002
). Due
to early embryonic lethality of mice with inactivated Prdm1/Blimp1, functional
studies have so far focused on its involvement in B lymphocyte development.
Conditional targeting of prdm1 shows that mature B cells lacking
Prdm1/Blimp1 activity cannot differentiate into immunoglobulin-secreting
plasma cells (Turner et al.,
1994
; Shapiro-Shelef et al.,
2003
). In X. laevis, Prdm1/Blimp1 is hypothesized to
induce anterior endomesoderm and promote head formation by positively
regulating expression of cerberus in cooperation with Chordin and
Mix.1 (de Souza et al.,
1999
).
Recently, zebrafish prdm1 was shown to be affected by a
hypomorphic mutation known as u boottp39 (ubo)
and to be required for slow muscle fiber differentiation
(Roy et al., 2001;
Baxendale et al., 2004
).
Whereas prdm1 expression is dynamic, analyses of the loss-of-function
phenotype have focused on a limited set of developmental events
(Baxendale et al., 2004
). Thus
our understanding of zebrafish Prdm1 during embryogenesis remains
incomplete.
Here we report that zebrafish prdm1 exhibits a dynamic expression pattern shared by the murine, chick and X. laevis Prdm1/Blimp1 encoding genes. Misexpression of prdm1 inhibits the formation of dorsoanterior structures and reduces expression of the BMP antagonist Chordin. Conversely, interference with Prdm1 translation using morpholino oligonucleotides (MOs) increases chordin expression and dorsalizes the embryo. Our studies propose a role for Prdm1 in limiting the function of the gastrula organizer and show that its activity is essential for many cell fate specification and morphogenetic processes in precise correspondence with the intricate expression pattern of this transcription factor.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning
An expressed sequence tag (EST) with sequence similarity to reported
Prdm1/Blimp1 proteins was used to isolate a zebrafish homolog by
3'/5' SMART RACE (BD Biosciences) from a 6.5-hour
postfertilization (hpf) cDNA preparation. Full-length prdm1 was
obtained by proof-reading PCR (PfuUltra, Stratagene), cloned into the
EcoRV site of the pGEM-T Easy vector (Promega) and used for antisense
probe synthesis with SP6 RNA polymerase after ApaI linearization. For
misexpression, the full-length cDNA was cloned into the pCS2+ vector
(Rupp et al., 1994) and used
for capped RNA synthesis with SP6 RNA polymerase after NotI
linearization (mMESSAGE mMACHINE, Ambion). The NCBI accession number is
AY841759.
In situ hybridization
Single and double color whole-mount in situ hybridization was performed
essentially as described (Thisse et al.,
1993); BM Purple (Roche) was used as blue and Magenta phosphate
(175 µg/ml in DMF; Fluka) combined with Tetrazolium Red (350 µg/ml in
70% DMF; Sigma) as red alkaline phosphatase substrates. The following
molecular markers were used: gsc
(Stachel et al., 1993
),
dlx3b (Akimenko et al.,
1994
), six3b
(Kobayashi et al., 1998
;
Seo et al., 1998a
),
pax2a (Krauss et al.,
1991
), egr2b/krox20
(Oxtoby and Jowett, 1993
),
hgg1/ctslb (Vogel and
Gerster, 1997
), boz
(Koos and Ho, 1998
;
Yamanaka et al., 1998
),
chd (Schulte-Merker et al.,
1997
; Miller-Bertoglio et al.,
1997
), szl/ogo
(Martyn and Schulte-Merker,
2003
; Yabe et al.,
2003
), bmp4 (Chin et
al., 1997
;
Martínez-Barberá et al.,
1997
), ntl
(Schulte-Merker et al., 1992
),
wnt8a (Kelly et al.,
1995
), mix (Kikuchi
et al., 2000
), sox17
(Alexander and Stainier, 1999
),
ptc1 (Concordet et al.,
1996
), vox, vent
(Melby et al., 2000
;
Kawahara et al., 2000a
;
Kawahara et al., 2000b
),
six7 (Seo et al.,
1998b
), dkk1
(Hashimoto et al., 2000
),
nog1 (Fürthauer et al.,
1999
), hhex (Ho et
al., 1999
), ndr1
(Rebagliati et al., 1998
),
ndr2 (Erter et al.,
1998
), dlc (Smithers
et al., 2000
), gata1
(Detrich et al., 1995
),
eve1 (Joly et al.,
1993
).
Microinjection and photoactivation of fluorescent lineage tracer
Embryos were microinjected at the 1-cell stage using 50, 100 or 200 pg
doses of synthetic prdm1 capped RNA
(Marlow et al., 1998) or 1, 2
or 4 ng doses of a prdm1-specific morpholino oligonucleotide
(MOprdm1, 5'-TGTGTGATCTCTCCCCTGAGTGTGT-3';
GeneTools, LLC) (Nasevicius and Ekker,
2000
). A mutant form of prdm1
(prdm1mut) predicted to be unable to bind
MOprdm1 was constructed by site-directed mutagenesis
(5'-AC(A/t)CACT(C/a)AGG(G/t)(G/t)AGAGATCA(C/t) ACA-3') and used to
evaluate the specificity of the MOprdm1-induced phenotype.
In co-injection experiments each reagent was microinjected independently at
the 1-cell stage. Injection and photoactivation of anionic dextran DMNB caged
fluorescein (Molecular Probes, D-3310) was performed as described
(Sepich et al., 2000
).
Microscopy
Embryos processed for whole-mount in situ hybridization were mounted in 80%
glycerol/PBT and photographed using a Zeiss Axiophot compound microscope and
an Axiocam digital camera. Live embryos were anesthetized if needed and
mounted in 1.5% or 2.5% methylcellulose.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To place prdm1 in genetic hierarchies that regulate early
development, we examined its expression in several pattern formation mutants.
In bozm168 embryos lacking dorsoanterior tissues such as
forebrain, chorda and prechordal mesoderm
(Solnica-Krezel et al., 1996;
Fekany et al., 1999
), the two
lines of prdm1-expressing slow muscle precursor cells were either
absent or fused, whereas the prechordal plate expression domain of
prdm1 was missing (Fig.
3A,H,B,I). By contrast, injection of synthetic boz RNA
induced ectopic prdm1 expression at early gastrulation (not shown).
Therefore, both loss-of-function and gain-of-function data place mesendodermal
prdm1 expression downstream of boz. Similarly, mesodermal
and endodermal prdm1 expression depends on Nodal signaling, as the
prechordal plate prdm1 expression domain was not detected in
maternal-zygotic one-eyed pinheadtz57 (MZoep)
embryos lacking activity of the EGF-CFC co-factor essential for Nodal activity
(Gritsman et al., 1999
). By
contrast, the ectodermal expression of prdm1 was not affected in
MZoep mutants (Fig.
3A,H,C,J). prdm1 expression is also dependent on BMP
signaling. Embryos homozygous for a null mutation in the bmp2b locus
swirltc300a (swr) lack most ventral cell fates
(Mullins et al., 1996
). We
observed that expression of prdm1 in the non-neural ectoderm was
reduced and confined to a small ventral region of swr embryos
(Fig. 3A,H,D,K). Conversely,
dinott250 (din) mutant gastrulae lacking activity
of the negative BMP regulator Chordin displayed reduced dorsal structures with
a concomitant expansion of the ventral prdm1 expression domain
(Fig. 3E,F)
(Hammerschmidt et al.,
1996
).
|
Misexpression of prdm1 causes deficiency of dorsoanterior structures
In order to investigate the role of Prdm1 during embryogenesis, we
microinjected synthetic prdm1 RNA at the one-cell stage and studied
the effects on embryo morphology. Injection of high doses (400 pg) resulted in
100% embryonic lethality by 24 hpf, whereas most embryos injected with 100 or
200 pg doses completed embryogenesis. Starting at 4 hpf and throughout
gastrulation (6-9.5 hpf), these embryos exhibited irregular epibolic movements
that normally thin and spread the blastoderm around the yolk cell (not shown).
However, the completion of epiboly appeared unaffected. We used 100 pg doses
in all the gain-of-function experiments described below if not indicated
otherwise.
At 1 dpf embryos misexpressing Prdm1 exhibited reduction (45%) or loss (42%) of forebrain, including eyes, anterior midbrain and notochord, while the remainder of the body axis appeared largely unaffected (n=286; Fig. 4A,B,C). A low number of these embryos appeared to be affected much more severely (9%; Fig. 4D) and 4% died by 24 hpf. In support, forebrain expression of six3b was reduced (52%) or missing (42%) and pax2a expression at the mid/hindbrain boundary was reduced (11%), whereas hindbrain expression of krox20 was not affected (n=189; Fig. 4E,F).
|
The similarity of dorsoanterior deficiencies observed in embryos
misexpressing Prdm1 and boz mutants prompted us to test whether
prdm1 RNA-injection could modify the boz phenotype, which
exhibits both variable penetrance and expressivity and decreases with the age
of the female parent (Fekany et al.,
1999). At sub-threshold doses (50 pg), prdm1 RNA
injection affected AP neural patterning mildly in 5.9% of wild-type embryos
(Fig. 4U), indicated by
slightly reduced forebrain six3b expression and unaltered
pax2a and krox20 expression. By contrast, Prdm1
misexpression at the same dose in boz embryos caused increased
penetrance of forebrain defects (66, 67 and 85%, respectively) compared with
untreated siblings (0, 0 and 12%, respectively;
Fig. 4U)
(Fekany et al., 1999
). The
expressivity of the boz phenotype was also increased, as revealed by
further reduction of six3b expression
(Fig. 4S,T)
(Fekany et al., 1999
). Thus,
given that bozm168 is a strong/null allele
(Fekany et al., 1999
;
Koos and Ho, 1999
), Prdm1
misexpression at a sub-threshold dose can impact organizer formation by
regulating expression of other genes in addition and/or parallel to
boz.
The severely affected embryos misexpressing Prdm1
(Fig. 4A,D) suggested that
additional cell types were impaired. Expression of the pan-mesodermal marker
ntl at the blastoderm margin was reduced in 73% of prdm1
misexpressing embryos at late blastula stages (n=188;
Fig. 5A,B). Similarly,
expression of wnt8a, also involved in mesoderm development
(Lekven et al., 2001;
Erter et al., 2001
), was
reduced as well (92%, n=160; Fig.
5C,D). Likewise, expression of two endodermal markers,
mix and sox17, was reduced in prdm1 misexpressing
embryos at late blastula (21%, n=100;
Fig. 5E,F) and early gastrula
stages (91%, n=190; Fig.
5G,H), respectively. In conclusion, Prdm1 can also affect
endodermal and mesodermal cell fate specification when ectopically
expressed.
|
|
|
|
Further supporting the mild dorsalization phenotype, 85% of
MOprdm1-injected embryos displayed a mediolaterally
enlarged neuroectoderm (krox20) and somitic mesoderm (dlc)
at the onset of somitogenesis (10.5 hpf), whereas position of the anterior
prechordal mesoderm (hgg1) was unchanged (n=142;
Fig. 6K-N). In addition, AP
neural patterning (six3b, pax2a krox20) was not affected in
prdm1 morphant embryos (n=167;
Fig. 6O,P). At
mid-somitogenesis (16 hpf), expression of the ventral tissue markers
gata1 in blood precursors and eve1 in ventroposterior cells
in the tail were reduced in 94% (n=139) and 98% (n=135) of
prdm1 morphant embryos, respectively
(Fig. 6Q-T), suggesting a class
C3 dorsalization phenotype according to Mullins et al.
(Mullins et al., 1996).
Nevertheless, at 24 hpf, judged by overall morphology, morphant embryos rather
exhibited class 1 dorsalization characteristics (see Fig. S1A,B in the
supplementary material).
The increase of chd and corresponding decrease of szl
expression in prdm1 morphant embryos
(Fig. 6A-F) prompted us to
investigate whether interference with prdm1 function can suppress the
ventralized phenotypes of din/chd and
ogontm305 (ogo; lacking Szl activity) mutant
embryos. Whereas misexpression of Chd can fully suppress the ventralization
phenotype of ogo/szl mutants (Miller-Bertoglio et al.,
1999), misexpression of Szl does not rescue din/chd mutants,
suggesting that Szl requires Chd function for its dorsalizing activity
(Yabe et al., 2003). At 24
hpf, the ventral fin fold of the tail is characteristically duplicated and
enlarged in both ogo/szl and din/chd
embryos (Fig. 7A,B,D).
Interestingly, when injected with MOprdm1, we observed
partial suppression of this phenotype in ogo/szl (92%,
n=53) but not din/chd (0%, n=69) embryos
(Fig. 7A-E). Accordingly,
bmp4 expression was reduced in 72% of ogo/szl
embryos (n=36; Fig.
7F,G) but not in din/chd embryos (0%,
n=80; Fig. 7H,I)
injected with MOprdm1. Furthermore, the neuroectoderm was
mediolaterally enlarged in ogo/szl but not in
din/chd embryos with impaired prdm1 function
(Fig. 7J-M). However, other
characteristics of the ventralized phenotypes were not affected. The
observation that the dorsalization of prdm1 morphants is associated
with increased chd expression together with the above gain- and
loss-of-function experiments suggest that Prdm1 activity promotes BMP
signaling during zebrafish gastrulation, probably through limiting expression
of chd in the organizer region.
Given that boz negatively regulates bmp2b expression
(Fekany-Lee et al., 2000;
Koos and Ho, 1999
;
Leung et al., 2003
) and
ectopic Prdm1 activity can suppress boz expression
(Fig. 4H), we tested whether
dorsalization of prdm1 morphants is caused by excessive or prolonged
expression of boz and/or its downstream targets. We found that the
boz expression domain was not affected in prdm1 morphant
embryos at late blastula stages, and was correctly downregulated at the onset
of gastrulation (not shown). Similarly, expression of vox and
vent, negatively regulated by Boz
(Kawahara et al., 2000a
;
Kawahara et al., 2000b
;
Melby et al., 2000
;
Imai et al., 2001
), gsc,
six3b and six7 (Kobayashi et
al., 1998
; Seo et al.,
1998a
,b
),
the Wnt8 antagonist dkk1 and the BMP antagonist nog1,
confined to the presumptive anterior prechordal mesoderm
(Hashimoto et al., 2000
;
Fürthauer et al., 1999
),
and hhex, expressed in the dorsal YSL
(Ho et al., 1999
), was normal
in prdm1 morphant embryos at early gastrula stages (not shown).
Furthermore, mesodermal ntl and wnt8a expression and
endodermal mix and sox17 expression were unchanged in
prdm1 morphant embryos (not shown). Hence, although Prdm1 can
suppress boz expression in misexpression experiments, its function
does not appear to be essential for the regulation of boz and its
downstream effectors. We conclude that Prdm1 appears to function in limiting
chd expression via boz- and sqt-independent
mechanisms.
Knockdown of Prdm1 activity increases dorsal extension movements
Dorsalized mutant embryos exhibit an elongated AV and narrowed mediolateral
(ML) axis, reflected by an increased AV/ML ratio
(Myers et al., 2002) and an
increase of the AP embryonic length at the conclusion of gastrulation
(Sepich and Solnica-Krezel,
2004
). Given that prdm1 morphant embryos also showed
altered morphology (Fig. 7N-Q),
we carried out morphometric analyses by capturing micrographs of untreated and
MOprdm1-injected sibling embryos at the end of
gastrulation (Sepich et al.,
2000
). The AV/ML ratio of prdm1 morphant embryos was
significantly increased (1.27±0.05) compared with untreated sibling
embryos (1.1±0.04; Table
2) (Sepich et al.,
2000
). This increase of the AV/ML ratio of prdm1 morphant
embryos prompted us to investigate the length of the nascent embryonic body
(Fig. 7N)
(Sepich et al., 2000
), which
is enlarged in dorsalized mutant embryos at the conclusion of gastrulation
(Myers et al., 2002
;
Sepich and Solnica-Krezel,
2004
). Embryonic length of prdm1 morphant embryos was
significantly increased to 1551.7±50.7 µm, compared with
1415.2±52.3 µm in untreated sibling embryos
(Fig. 7N,O;
Table 2). However, unlike
strongly dorsalized swr/bmp2b or sbn/smad5
embryos, which cease extension of the AP body axis by the end of gastrulation
and do not survive early segmentation
(Myers et al., 2002
),
prdm1 morphant embryos still exhibited increased embryonic length at
early segmentation (Fig. 7P,Q;
Table 2). Furthermore, ML width
of the developing somites was enlarged in prdm1 morphant embryos at
12.5 hpf (Fig. 7R,S).
|
Late phenotypes
Late loss-of-function phenotypes in slow muscle, photoreceptor cell layer,
branchial arches, pectoral fin and cloaca development are presented in Figs S1
and S2 in the supplementary material.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In early mouse and frog gastrulae Prdm1/Blimp1 is expressed in
equivalent structures, the anterior visceral endoderm and the anterior
endomesoderm, respectively, as well as in the presumptive prechordal plate
mesoderm, both of which are implicated in forebrain specification and
patterning (reviewed by de Souza and
Niehrs, 2000; Kiecker and
Niehrs, 2001
). Similarly, prdm1 is expressed in the
entire YSL underlying the blastoderm margin shortly before gastrulation starts
and after onset of gastrulation, in the anterior portion of the prechordal
mesendoderm. Interestingly, in mouse gastrulae, Prdm1/Blimp1
expression was also detected in the posterior visceral endoderm
(de Souza et al., 1999
),
reminiscent of prdm1 expression in ventral and lateral YSL
(Fig. 2A,B).
We have addressed the role of Prdm1 during early development in
gain-of-function and loss-of-function experiments. Misexpression of Prdm1
primarily caused reduction of dorsoanterior structures similar to defects
observed in boz mutants
(Solnica-Krezel et al., 1996).
Furthermore, expression of the dorsal genes boz, gsc and chd
was downregulated. The notion of an important role of Prdm1 in limiting the
gastrula organizer function is further supported by the loss-of-function
experiments, which revealed that prdm1 morphant embryos were mildly
dorsalized at the conclusion of gastrulation. These embryos exhibited an
elongation of the animal-vegetal axis typical of dorsalized zebrafish mutants
(Mullins et al., 1996
;
Myers et al., 2002
).
Accordingly, molecular analyses revealed mediolateral expansion of
chd expression dorsally and reduction of ventral expression of
bmp4 and szl, which encodes another BMP antagonist
positively regulated by BMP signaling
(Yabe et al., 2003
; Martyn et
al., 2003). Furthermore, at midsegmentation ventroposterior gata1 and
eve1 expression was reduced. Additionally, depletion of Prdm1
activity partially suppressed the ventralized ogo mutant phenotype,
but had no such effect on chd mutant embryos. This is also in
agreement with recent work by Yabe et al.
(Yabe et al., 2003
), which
showed that Chd activity is required for the ogo-dependent
dorsalization. We hypothesize that Prdm1 acts directly or indirectly on
chd in limiting gastrula organizer function.
In contrast to gain-of-function experiments, expression of boz and many other dorsal genes downstream of boz, such as gsc, hhex, six3b, six7, dkk1 and nog1, as well as expression of ventral genes such as vox and vent and of the Nodal-related genes ndr1 and ndr2, was not altered in prdm1 morphant embryos. This and the fact that many of the gain-of-function experiments did not reflect endogenous Prdm1 function, led us to propose that although ectopic Prdm1 activity is able to suppress boz expression, its function may not be essential for the regulation of boz and many of its downstream effectors during normal development. We conclude that Prdm1 limits the gastrula organizer function in zebrafish by negatively regulating chd expression, largely via boz independent mechanisms. We hypothesize that Prdm1 regulates boz expression during normal fish development in a functionally redundant manner with other unknown genes.
Our analyses of morphogenetic defects in prdm1-depleted gastrulae
are in support of the molecular data presented above. In embryos deficient in
BMP signaling, the characteristic abnormalities in embryonic shape are a
consequence of specific alteration of convergence and extension gastrulation
movements (Myers et al.,
2002). During normal gastrulation strong extension and moderate
convergence movements are restricted to the dorsal hemisphere, generating
embryos of slightly ovoid shape at the conclusion of gastrulation. Dorsalized
sbn mutant embryos exhibit increased elongation of the animal-vegetal
dimension and of the AP axis, in part due to slightly increased extension
movements of dorsal cell populations
(Myers et al., 2002
; Sepich
and Solnica-Krezel et al., 2004). Our morphometric analyses of prdm1
morphant embryos are consistent with the increased extension movements of
dorsal cell populations causing the increased length of the animal-vegetal and
AP embryonic axes at the end of the gastrula period, typical of dorsalized
mutants.
However, in strongly dorsalized smad5/sbn mutants at
early segmentation, AP axis elongation becomes reduced compared with wild type
and premature tail eversion takes place
(Myers et al., 2002). Both
defects have been attributed to altered cell movements of ventral cell
populations. Whereas cells residing in the ventral regions of wild-type
gastrulae, and experiencing the highest levels of BMP signaling, do not engage
in convergence and extension movements and migrate toward the vegetal pole, in
embryos with strongly reduced BMP signaling these cell populations undergo
strong extension typical of more dorsal cell populations
(Myers et al., 2002
). By
contrast, prdm1 morphant embryos manifest AP axes longer than wild
type at 12.5 hpf, and develop a normal tail. Labeled ventral cell populations
of embryos injected with MOprdm1 underwent relatively
normal movements toward the tailbud (Fig.
7W,X), consistent with a mild dorsalization of the morphant
embryos.
Together our gain- and loss-of-function experiments establish a role for
Prdm1 in limiting organizer function, uncovering noteworthy similarities and
differences to the proposed function of X. laevis Prdm1/Blimp1. In
zebrafish embryos prdm1 gain-of-function impaired primarily
dorsoanterior structures, whereas in X. laevis it affected the entire
anteroposterior axis, without exerting stronger effects on head versus trunk
and tail (de Souza et al.,
1999). While in agreement with our observations, expression of the
BMP antagonist chordin was downregulated in X. laevis
gastrulae overexpressing Prdm1/Blimp1, expression of other
organizer genes, such as gsc and cerberus (cer),
was either unchanged in dorsal marginal zone explants or ectopically induced
in ventral explants (de Souza et al.,
1999
). These data lead to the conclusion that in X.
laevis Prdm1/Blimp1 activity is required for head formation via positive
regulation of cer expression (de
Souza et al., 1999
). Thus specific roles of Prdm1/Blimp1 in the
organizer gene regulatory networks might differ between the two systems.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/2/393/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. and Westerfield, M. (1994). Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J. Neurosci. 14,3475 -3486.[Abstract]
Alexander, J. and Stainier, D. Y. (1999). A molecular pathway leading to endoderm formation in zebrafish. Curr. Biol. 9,1147 -1157.[CrossRef][Medline]
Alexander, J., Rothenberg, M., Henry, G. L. and Stainier, D. Y. R. (1999). casanova plays an early and essential role in endoderm formation in zebrafish. Dev. Biol. 215,343 -357.[CrossRef][Medline]
Bachiller, D., Klingensmith, J., Kemp, C., Belo, J. A., Anderson, R. M., May, S. R., McMahon, J. A., McMahon, A. P., Harland, R. M., Rossant, J. et al. (2000). The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403,658 -661.[CrossRef][Medline]
Barresi, M. J., Stickney, H. L. and Devoto, S. H.
(2000). The zebrafish slow-muscle-omitted gene product
is required for Hedgehog signal transduction and the development of slow
muscle identity. Development
127,2189
-2199.
Baxendale, S., Davison, C., Muxworthy, C., Wolff, C., Ingham, P. W. and Roy, S. (2004). The B-cell maturation factor Blimp-1 specifies vertebrate slow-twitch muscle fiber identity inresponse to Hedgehog signaling. Nat. Genet. 36, 88-93.[CrossRef][Medline]
Chang, D. H., Angelin-Duclos, C. and Calame, K. (2000). BLIMP-1: trigger for differentiation of myeloid lineage. Nat. Immunol. 1,169 -176.[CrossRef][Medline]
Chang, D. H., Cattoretti, G. and Calame, K. L. (2002). The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development. Mech. Dev. 117,305 -309.[CrossRef][Medline]
Chen, S. R. and Kimelman, D. (2000). The role
of the yolk syncytial layer in germ layer patterning in zebrafish.
Development 127,4681
-4689.
Chin, A. J., Chen, J.-N. and Weinberg, E. S. (1997). Bone morphogenetic protein-4 expression characterizes inductive boundaries in organs of developing zebrafish. Dev. Genes Evol. 207,107 -114.[CrossRef]
Clement, J. H., Fettes, P., Knochel, S., Lef, J. and Knochel, W. (1995). Bone morphogenetic protein 2 in the early development of Xenopus laevis. Mech. Dev. 52,357 -370.[CrossRef][Medline]
Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V.,
Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996).
Spatial regulation of a zebrafish patched homologue reflects the
roles of sonic hedgehog and protein kinase A in neural tube and
somite patterning. Development
122,2835
-2846.
De Robertis, E. M., Larraín, J., Oelgeschläger, M. and Wessely, O. (2000). The establishment of Spemann's organizer and patterning of the vertebrate embryo. Nat. Rev. Genet. 1,171 -181.[CrossRef][Medline]
de Souza, F. S. and Niehrs, C. (2000). Anterior endoderm and head induction in early vertebrate embryos. Cell Tissue Res. 300,207 -217.[CrossRef][Medline]
de Souza, F. S. J., Gawantka, V., Gomez, A. P., Delius, H., Ang,
S.-L. and Niehrs, C. (1999). The zinc finger gene
Xblimp1 controls anterior endomesodermal cell fate in Spemann's
organizer. EMBO J. 18,6062
-6072.
Detrich, H. W., III, Kieran, M. W., Chan, F. Y., Barone, L. M., Yee, K., Rundstadler, J. A., Pratt, S., Ransom, D. and Zon, L. I. (1995). Intraembryonic hematopoietic cell migration during vertebrate development. Proc. Natl. Acad. Sci. USA 92,10713 -10717.[Abstract]
Dickmeis, T., Mourrain, P., Saint-Etienne, L., Fischer, N.,
Aanstad, P., Clark, M., Strähle, U. and Rosa, F.
(2001). A crucial component of the endoderm formation pathway,
CASANOVA, is encoded by a novel sox-related gene. Genes
Dev. 15,1487
-1492.
Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F. and
Talbot, W. S. (2003). The role of the zebrafish nodal-related
genes squint and cyclops in patterning of mesendoderm.
Development 130,1837
-1851.
Erter, C. E., Solnica-Krezel, L. and Wright, C. V. E. (1998). Zebrafish nodal-related 2 encodes an early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer. Dev. Biol. 204,361 -372.[CrossRef][Medline]
Erter, C. E., Wilm, T. P., Basler, N., Wright, C. V. and Solnica-Krezel, L. (2001). Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo. Development 128,3571 -3583.[Medline]
Fekany, K., Yamanaka, Y., Leung, T., Sirotkin, H. I.,
Topczewski, J., Gates, M. A., Hibi, M., Renucci, A., Stemple, D.,
Radbill, A. et al. (1999). The zebrafish bozozok
locus encodes Dharma, a homeodomain protein essential for induction of
gastrula organizer and dorsoanterior embryonic structures.
Development 126,1427
-1438.
Fekany-Lee, K., Gonzalez, E., Miller-Bertoglio, V. and
Solnica-Krezel, L. (2000). The homeobox gene bozozok
promotes anterior neuroectoderm formation in zebrafish through negative
regulation of BMP2/4 and Wnt pathways. Development
127,2333
-2345.
Fürthauer, M., Thisse, B. and Thisse, C. (1999). Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo. Dev. Biol. 214,181 -196.[CrossRef][Medline]
Gonzalez, E. M., Fekany-Lee, K., Carmany-Rampey, A., Erter,
C., Topczewski, J., Wright, C. V. E. and Solnica-Krezel, L.
(2000). Head and trunk in zebrafish arise via coinhibition of BMP
signaling by bozozok and chordino. Genes Dev.
14,3087
-3092.
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97,121 -132.[Medline]
Ha, A. S. and Riddle, R. D. (2003). cBlimp-1 expression in chick limb bud development. Gene Expr. Patterns. 3,297 -300.[CrossRef][Medline]
Hammerschmidt, M. and Mullins, M. C. (2002). Dorsoventral patterning in the zebrafish: bone morphogenetic proteins and beyond. Results Probl. Cell Differ. 40, 72-95.[Medline]
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., van
Eeden, F. J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). dino and
mercedes, two genes regulating dorsal development in the zebrafish
embryo. Development 123,95
-102.
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217,138 -152.[CrossRef][Medline]
Hatta, K., Bremiller, R., Westerfield, M. and Kimmel, C. B. (1991). Diversity of expression of engrailed-like antigens in zebrafish. Development 112,821 -832.[Abstract]
Hibi, M., Hirano, T. and Dawid, I. B. (2002). Organizer formation and function. Results Probl. Cell Diff. 40,48 -71.[Medline]
Hild, M., Dick, A., Rauch, G. J., Meier, A., Bouwmeester, T.,
Haffter, P. and Hammerschmidt, M. (1999). The
smad5 mutation somitabun blocks Bmp2b signaling during early
dorsoventral patterning of the zebrafish embryo.
Development 126,2149
-2159.
Ho, C. Y., Houart, C., Wilson, S. W. and Stainier, D. Y. (1999). A role for the extraembryonic yolk syncytial layer in patterning the zebrafish embryo suggested by properties of the hex gene. Curr. Biol. 9,1131 -1134.[CrossRef][Medline]
Imai, Y., Gates, M. A., Melby, A. E., Kimelman, D., Schier, A. F. and Talbot, W. S. (2001). The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish. Development 128,2407 -2420.[Medline]
Joly, J. S., Joly, C., Schulte-Merker, S., Boulekbache, H. and
Condamine, H. (1993). The ventral and posterior
expression of the zebrafish homeobox gene eve1 is perturbed in
dorsalized and mutant embryos. Development
119,1261
-1275.
Kawahara, A., Wilm, T., Solnica-Krezel, L. and Dawid, I. B.
(2000a). Antagonistic role of vega1 and
bozozok/dharma homeobox genes in organizer formation.
Proc. Natl. Acad. Sci. USA
97,12121
-12126.
Kawahara, A., Wilm, T., Solnica-Krezel, L. and Dawid, I. B. (2000b). Functional interaction of vega2 and goosecoid homeobox genes in zebrafish. Genesis 28,58 -67.[CrossRef][Medline]
Keller, A. D. and Maniatis, T. (1991). Identification and characterization of a novel repressor of beta-interferon gene expression. Genes Dev. 5, 868-879.[Abstract]
Kelly, G. M., Greenstein, P., Erezyilmaz, D. F. and Moon, R.
T. (1995). Zebrafish wnt8 and wnt8b share a
common activity but are involved in distinct developmental pathways.
Development 121,1787
-1799.
Kiecker, C. and Niehrs, C. (2001). The role of prechordal mesendoderm in neural patterning. Curr. Opin. Neurobiol. 11,27 -33.[CrossRef][Medline]
Kikuchi, Y., Trinh, L. A., Reiter, J. F., Alexander, J., Yelon,
D. and Stainier, D. Y. (2000). The zebrafish
bonnie and clyde gene encodes a Mix family homeodomain protein that
regulates the generation of endodermal precursors. Genes
Dev. 14,1279
-1289.
Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron,
S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001).
casanova encodes a novel Sox-related protein necessary and sufficient
for early endoderm formation in zebrafish. Genes Dev.
15,1493
-1505.
Kimmel, C. B., Ballard, W. B., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Kishimoto, Y., Lee, K. H., Zon, L., Hammerschmidt, M. and
Schulte-Merker, S. (1997). The molecular nature of zebrafish
swirl: BMP2 function is essential during early dorsoventral
patterning. Development
124,4457
-4466.
Kobayashi, M., Toyama, R., Takeda, H., Dawid, I. B. and
Kawakami, K. (1998). Overexpression of the forebrain-specific
homeobox gene six3 induces rostral forebrain enlargement in
zebrafish. Development
125,2973
-2982.
Koos, D. S. and Ho, R. K. (1998). The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish. Curr. Biol. 8,1199 -1206.[Medline]
Koos, D. S. and Ho, R. K. (1999). The nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the zebrafish pregastrula. Dev. Biol. 215,190 -207.[CrossRef][Medline]
Kramer, C., Mayr, T., Nowak, M., Schumacher, J., Runke, G., Bauer, H., Wagner, D. S., Schmid, B., Imai, Y., Talbot, W. S., Mullins, M. C. and Hammerschmidt, M. (2002). Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling. Dev. Biol. 250,263 -279.[CrossRef][Medline]
Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U. and Fjose, A. (1991). Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. EMBO J. 10,3609 -3619.[Abstract]
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[Medline]
Lekven, A. C., Thorpe, C. J., Waxman, J. S. and Moon, R. T. (2001). Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning. Dev. Cell 1,103 -114.[Medline]
Leung, T., Bischof, J., Söll, I., Niessing, D., Zhang, D.,
Ma, J., Jäckle, H. and Driever, W. (2003).
bozozok directly represses bmp2b transcription and mediates
the earliest dorsoventral asymmetry of bmp2b expression in zebrafish.
Development 130,3639
-3649.
Marlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S. C., Abbas, L., Weaver, M., Driever, W. and Solnica-Krezel, L. (1998). Functional interactions of genes mediating convergent extension, knypek and trilobite, during the partitioning of the eye primordium in zebrafish. Dev. Biol. 203,382 -399.[CrossRef][Medline]
Martínez-Barberá, J. P., Toresson, H., da Rocha, S. and Krauss, S. (1997). Cloning and expression of three members of the zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4.Gene 198,53 -59.[CrossRef][Medline]
Martyn, U. and Schulte-Merker, S. (2003). The ventralized ogon mutant phenotype is caused by a mutation in the zebrafish homologue of Sizzled, a secreted Frizzled-related protein. Dev. Biol. 260,58 -67.[CrossRef][Medline]
Melby, A. E., Beach, C., Mullins, M. and Kimelman, D. (2000). Patterning the early zebrafish by the opposing actions of bozozok and vox/vent. Dev. Biol. 224,275 -285.[CrossRef][Medline]
Miller-Bertoglio, V. E., Fisher, S., Sanchez, A., Mullins, M. C. and Halpern, M. E. (1997). Differential regulation of chordin expression domains in mutant zebrafish. Dev. Biol. 192,537 -550.[CrossRef][Medline]
Mizuno, T., Yamaha, E., Kuroiwa, A. and Takeda, H. (1999). Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish. Mech. Dev. 81,51 -63.[CrossRef][Medline]
Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J.,
Brand, M., van Eedenm, F. J., Furutani-Seiki, M., Granato, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). Genes establishing
dorsoventral pattern formation in the zebrafish embryo: the ventral specifying
genes. Development 123,81
-93.
Myers, D. C., Sepich, D. S. and Solnica-Krezel, L. (2002). Bmp activity gradient regulates convergent extension during zebrafish gastrulation. Dev. Biol. 243, 81-98.[CrossRef][Medline]
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Nguyen, V. H., Schmid, B., Trout, J., Connors, S. A., Ekker, M. and Mullins, M. C. (1998). Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes. Dev. Biol. 199,93 -110.[CrossRef][Medline]
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21,1087 -1095.[Abstract]
Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B.
(1998). cyclops encodes a nodal-related factor involved
in midline signaling. Proc. Natl. Acad. Sci. USA
95,9932
-9937.
Ren, B., Chee, K. J., Kim, T. H. and Maniatis, T.
(1999). PRDI-BF1/Blimp-1 repression is mediated by corepressors
of the Groucho family of proteins. Genes Dev.
13,125
-137.
Roy, S., Wolff, C. and Ingham, P. W. (2001).
The u-boot mutation identifies a Hedgehog-regulated myogenic switch
for fiber-type diversification in the zebrafish embryo. Genes
Dev. 15,1563
-1576.
Rupp, R. A., Snider, L. and Weintraub, H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8,1311 -1323.[Abstract]
Sakaguchi, T., Mizuno, T. and Takeda, H. (2002). Formation and patterning roles of the yolk syncytial layer. Results Probl. Cell Differ. 40, 1-14.[Medline]
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and
Nüsslein-Volhard, C. (1992). The protein product of the
zebrafish homologue of the mouse T gene is expressed in nuclei of the germ
ring and the notochord of the early embryo.
Development 116,1021
-1032.
Schulte-Merker, S., Lee, K. J., McMahon, A. P. and Hammerschmidt, M. (1997). The zebrafish organizer requires chordino. Nature 387,862 -863.[CrossRef][Medline]
Seo, H. C., Drivenes, Ø., Ellingsen, S. and Fjose, A. (1998a). Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia. Mech. Dev. 73,45 -57.[CrossRef][Medline]
Seo, H. C., Drivenes, O., Ellingsen, S. and Fjose, A. (1998b). Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation. Gene 216,39 -46.[CrossRef][Medline]
Sepich, D. S. and Solnica-Krezel, L. (2004). Analysis of cell movements in zebrafish embryos: morphometrics and measuring movement of labeled cell populations in vivo. In Cell migration in Development (ed. J.-L. Guan). Totowa: Humana Press.
Sepich, D. S., Myers, D. C., Short, R., Topczewski, J., Marlow, F. and Solnica-Krezel, L. (2000). Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension. Genesis 27,159 -173.[CrossRef][Medline]
Shapiro-Shelef, M., Lin, K. I., McHeyzer-Williams, L. J., Liao, J., McHeyzer-Williams, M. G. and Calame, K. (2003). Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19,607 -620.[CrossRef][Medline]
Shimizu, T., Yamanaka, Y., Ryu, S., Hashimoto, H., Yabe, T., Hirata, T., Bae, Y., Hibi, M. and Hirano, T. (2000). Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish. Mech. Dev. 91,293 -303.[CrossRef][Medline]
Sirotkin, H. I., Dougan, S. T., Schier, A. F. and Talbot, W.
S. (2000). bozozok and squint act in
parallel to specify dorsal mesoderm and anterior neuroectoderm in zebrafish.
Development 127,2583
-2592.
Smithers, L., Haddon, C., Jiang, Y. J. and Lewis, J. (2000). Sequence and embryonic expression of deltaC in the zebrafish. Mech. Dev. 90,119 -123.[CrossRef][Medline]
Solnica-Krezel, L., Schier, A. F. and Driever, W.
(1994). Efficient recovery of ENU-induced mutations from the
zebrafish germline. Genetics
136,1401
-1420.
Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E.,
Rangini, Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D.
Y., Zwartkruis, F., Abdelilah, S. et al. (1996). Mutations
affecting cell fates and cellular rearrangements during gastrulation in
zebrafish. Development
123, 67-80.
Spemann, H. and Mangold, H. (2001). Induction of embryonic primordia by implantation of organizers from a different species, 1923. Int. J. Dev. Biol. 45, 13-38.[Medline]
Stachel, S. E., Grunwald, D. J. and Myers, P. Z.
(1993). Lithium perturbation and goosecoid expression
identify a dorsal specification pathway in the pregastrula zebrafish.
Development 117,1261
-1274.
Thisse, C., Thisse, B., Schilling, T. F. and Postlethwait, J.
H. (1993). Structure of the zebrafish snail1 gene
and its expression in wild-type, spadetail and no tail
mutant embryos. Development
119,1203
-1215.
Topczewski, J., Sepich, D. S., Myers, D. C., Walker, C., Amores, A., Lele, Z., Hammerschmidt, M., Postlethwait, J. and Solnica-Krezel, L. (2001). The zebrafish glypican Knypek controls cell polarity during gastrulation movements of convergent extension. Dev. Cell 1,251 -264.[Medline]
Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G. and Reuter, G. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13,3822 -3831.[Abstract]
Turner, C. J., Mack, D. H. and Davis, M. M. (1994). Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77,297 -306.[Medline]
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128,3497 -3509.[Medline]
Vogel, A. and Gerster, T. (1997). Expression of a zebrafish cathepsin L gene in anterior mesendoderm and hatching gland. Dev. Genes Evol. 206,477 -479.[CrossRef]
Yabe, T., Shimizu, T., Muraoka, O., Bae, Y. K., Hirata, T.,
Nojima, H., Kawakami, A., Hirano, T. and Hibi, M.
(2003). Ogon/Secreted Frizzled functions as a negative feedback
regulator of Bmp signaling. Development
130,2705
-2716.
Yamanaka, Y., Mizuno, T., Sasai, Y., Kishi, M., Takeda, H., Kim,
C. H., Hibi, M. and Hirano, T. (1998). A novel
homeobox gene, dharma, can induce the organizer in a
non-cell-autonomous manner. Genes Dev.
12,2345
-2353.