1 Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Apartado 14,
2781-901 Oeiras, Portugal
2 Max-Planck-Institut für Entwicklungsbiologie, Abt. Zellbiologie,
Spemannstrasse 35, 72076 Tuebingen, Germany
3 Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, Campus
de Gambelas, 8000-010 Faro, Portugal
Authors for correspondence (e-mail:
jbelo{at}igc.gulbenkian.pt
and
herbert.steinbeisser{at}med.uni-heidelberg.de)
Accepted 4 July 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cerberus, Head induction, Morpholino, Targeted activation, Xenopus laevis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biochemical analysis in Xenopus showed that Cerberus can bind to
Xnr1, BMP4 and Xwnt8 and thereby blocks their function
(Piccolo et al., 1999). These
inhibitory properties of Cerberus are considered essential for the head
inducing activity of this secreted factor.
A gene homologous to cerberus has been isolated in the mouse
(Belo et al., 1997;
Biben et al., 1998
;
Shawlot et al., 1998
). The
expression of mouse cerberus-like (cer-l) and other markers such as
Hesx1, Lim1 and Otx2 in the anterior visceral endoderm
(AVE), led to the hypothesis that this region is the topological mouse
equivalent of the ADE in Xenopus
(Acampora et al., 1995
;
Thomas and Beddington, 1996
;
Belo et al., 1997
;
Bouwmeester and Leyns, 1997
).
Therefore, the AVE was proposed to be the head organizer in the mouse. This
view is supported by the finding that in chimeric mutant mouse embryos
composed of AVE that lacks either Otx2, Lim1 or Hnf3ß,
and wild-type epiblast, the head is not properly induced
(Rhinn et al., 1998
;
Shawlot et al., 1999
;
Dufort et al., 1998
).
Surprisingly, in generated cer-l knockout (KO) mouse lines no
phenotypic head and axis defects were observed, arguing against a role of
cer-l in early embryogenesis (Belo
et al., 2000
; Shawlot et al.,
2000
; Stanley et al.,
2000
).
In Xenopus, the endogenous function of Cerberus in the ADE remains unclear because of the lack of loss-of-function data. In order to characterize the function of Cerberus in head formation, a novel combination of strategies was employed. Endogenous Cerberus was `knocked down' using an antisense morpholino oligonucleotide that specifically blocked the translation of the cerberus mRNA (CerMO). In addition, the relative levels of the signaling molecules BMP4, Xnr1 and Xwnt8, which are antagonized by Cerberus, were raised in the ADE. This was achieved by driving their expression under the control of a mouse cer-l promoter fragment that is specifically activated in the ADE and closely resembles the spatiotemporal expression pattern of endogenous cerberus. Dorsal-vegetal injection of the CerMo does not cause visible head defects in the Xenopus embryo. In contrast, targeted increase of BMP, Nodal or Wnt activity in the ADE resulted specifically in the loss of head, but not trunk-tail structures. These factors synergistically inhibited head structures when simultaneously expressed in the ADE. Remarkably, these phenotypes caused by BMP, Nodal or Wnt were strongly enhanced when, in addition, Cerberus function in the ADE was blocked by the CerMo. The endogenous function of Cerberus in head formation, revealed in this sensitized system, could also be demonstrated in an explant recombination assay. ADE can induce forebrain markers when conjugated with dorsal ectoderm (DE) but not when Cerberus function was knocked down by the morpholino oligo. Furthermore, concomitantly, the ADE represses the expression of more caudal neural markers through the activity of Cerberus.
We demonstrate that endogenous Cerberus can inhibit BMP, Nodal and Wnt in vivo, and that this activity is required in the ADE for proper head induction/patterning in Xenopus.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To obtain the misexpression constructs the CDS from Xnr1, XBMP4 and Xwnt8 cDNAs were amplified by PCR with primers that introduced a NcoI site at the ATG translation start.
Primers used:
Xnr1-F (5'-TTTACTAGTCCATGGCATTTCTGACAGCAGTCC-3') and Xnr1-R (5'-TTTGTCGACTTAACTGCACCCACATTCCTC-3'); XBMP4-F (5'-TTTACTAGTCCATGGGAATTCCTGGTAACCGAATGCTG-3') and XBMP4-R (5'-TTTGTCGACTCAACGGCACCCACACCCTTCC-3'); Xwnt8-F (5'-TTTACTAGTCCATGGGACAAAACACCACTTTGTTCATCC-3') and Xwnt8-R (5'-TTTGTCGACTCATCTCCGGTGGCCTCTG-3').
Each of these amplified CDSs was digested with NcoI and inserted at the ATG of McerP. A 263 bp XhoI-ApaI fragment containing the SV40 poly(A) signal from pCS2+ was inserted downstream of each stop codon, generating McerP-Xnr1, McerP-BMP4 and McerP-Xwnt8. The plasmids CMV.Xnr1, CMV.BMP4 and CMV.Xwnt8 were constructed by cloning the respective CDS PCR fragments at the EcoRI (filled in)-XhoI sites of pCS2+.
The cerberus morpholino oligonucleotide, obtained from Gene Tools LLC, was designed to target the 5' UTR region between bases 35 and 11 upstream of the AUG (5'-CTAGACCCTGCAGTGTTTCTGAGCG-3'). To express the C-terminal HA-tagged Cerberus protein in Xenopus embryos, a 1.4 kb EcoRI-XhoI fragment from pCDNA.Xcer-HA (containing bases from 50 in the 5' UTR) was subcloned in pCS2+. The Xcer-HA rescue construct was generated by subcloning a 1.36 kb EcoRV-XhoI fragment from pCDNA.Xcer-HA, which only includes 11 bases upstream of the ATG, into the EcoRI site of pCS2+.
mRNA synthesis and microinjection
Capped sense mRNAs were synthesized using the Ambion mMessage mMachine kit.
Xenopus eggs were obtained as described by Medina et al.
(Medina et al., 2000) and
staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). In vitro
fertilization and microinjection of X. laevis embryos were performed
as described previously (Bouwmeester et
al., 1996
).
Conjugate assays
Dorsal ectoderm and anterior dorsal endoderm were dissected from stage 10.5
embryos in 1x MBS-H. Conjugates were made by recombining the DE with the
ADE and were grown in 0.5x MBS-H until siblings reached late tailbud
stage 30/31. The conjugates were assayed by RT-PCR for expression of the
neural markers eomes, Xemx1, XBF1, En2 and Krox20.
In situ hybridization and ß-galactosidase staining
Whole-mount and hemi-section in situ hybridization and antisense probe
preparation was carried out as described by Belo et al.
(Belo et al., 1997). The
plasmids containing XBF1, Xotx2 and Xshh fragments were
linearized using XbaI, EcoRI and KpnI respectively,
and transcribed using T3 RNA polymerase. Plasmids containing lacZ, Xcer,
Xhex, XKrox20 and Xnot2 were cut with SalI,
EcoRI, NotI, EcoRI and EcoRI,
respectively, and transcribed using T7 RNA polymerase. Stained embryos (stage
21 and above) were bleached by illumination in 1% H2O2,
4% formamide and 0.5x SSC pH 7.0. For ß-galactosidase staining,
embryos were fixed in MEMFA (room temperature for 1 hour), rinsed in PBS and
stained by using X-gal (Steinbeisser et
al., 1989
).
RT-PCR
Total RNA was prepared from embryos or conjugates with Trizol reagent
(Gibco-BRL) and treated with RNase-free DNase I (Promega). First strand cDNA
primed by random hexamers was synthesized with AMV reverse transcriptase
(Roche) and PCR was performed using standard conditions and the following sets
of primers: Engrailed2-F
(5'-ATGAGCAGAATAACAGGGAAGTGGA-3') and Engrailed2-R
(5'-CCTCGGGGACATTGACTCGGTGGTG-3'), 28 cycles; eomes-F
(5'-GCCTACGAAACAGACTACTCCT-3') and eomes-R
(5'-TAATGGAGGGAGGGGTTTCTAC-3'), 28 cycles; Krox20-F
(5'-AACCGCCCCAGTAAGACC-3') and Krox20-R
(5'-GTGTCAGCCTGTCCTGTTAG-3'), 24 cycles; Nkx2.1-F
(5'-CTGACATATTGAGTCCCCTGGAGG-3') and Nkx2.1-R
(5'-CCAGGTTTCCCAAATTGCCATTGC-3'), 30 cycles; ODC-F
(5'-CAGCTAGCTGTGGTGTGG-3') and ODC-R
(5'-CAACATGGAAACTCACACC-3'), 21 cycles; Xag-F
(5'-CTGACTGTCCGATCAGAC-3') and Xag-R
(5'-GAGTTGCTTCTCTGGCAT-3'), 23 cycles; XBF1-F
(5'-AAAGTGGACGGCAAAGACGGTG-3') and XBF1-R
(5'-CCAATGAACACATCGTCGCTGC-3'), 26 cycles; Xemx1-F
(5'-GCAGAAGCCTTTGTCAGTGG-3') and Xemx1-R
(5'-CCTCCAGTTTCTGCCTCTTG-3'), 31 cycles.
In vitro translation and western blot analysis
For in vitro transcription/translation the TNT®* Coupled Reticulocyte
Lysate System (Promega) was used according to the manufacturer's instructions.
Protein extraction of embryos was carried out as described previously
(Munchberg et al., 1999). Proteins were heated in sample buffer and separated
by denaturing SDS-PAGE using a 13.5% polyacrylamide gel
(Laemmli, 1970). Subsequently,
proteins were transferred to a nitrocellulose membrane
(Townbin et al., 1979
),
detected with monoclonal rabbit anti-
-HA antibody (Santa Cruz) for
Xcer-HA or monoclonal mouse anti-c-myc (Oncogene) for
N Moesin-myc and
developed using a chemiluminescent substrate (Pierce).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Morpholino knockdown of Cerberus in the ADE does not prevent head
formation
Synthetic cerberus mRNA can induce head-like structures when
microinjected in the ventral side of Xenopus embryo
(Fig. 1E)
(Bouwmeester et al., 1996).
This induction was not observed when the 5' UTR cerberus mRNA
and the CerMo were coinjected (Fig.
1F), demonstrating that the morpholino can efficiently inhibit
Cerberus activity in the embryo. In order to assess the phenotypic effect of
knocking down endogenous Cerberus, four- to eight-cell stage embryos were
injected with CerMo in the two dorsal-vegetal blastomeres, the clonal
descendants of which include the ADE cells
(Bauer et al., 1994
). Despite
the ability of the CerMo to block Cerberus activity, we did not observe any
abnormal phenotypes in embryos injected with 3.2 pmol of CerMo
(Fig. 1I). Mild axis defects
were observed when the maximal possible dose (16 pmol) of either CoMo or CerMo
was injected (Fig. 1G,H). Using
this morpholino-mediated knockdown strategy, we conclude that reducing
Cerberus activity in the ADE was not sufficient to impair head formation in
the Xenopus embryo.
Gain of BMP, Nodal or Wnt function in the ADE perturbs head
formation
Cerberus protein can bind to and antagonize BMP4, Xnr1 and Xwnt8 molecules
(Piccolo et al., 1999). We
reasoned that an alternative way to modulate Cerberus activity in the ADE
would be by locally raising the levels of BMP, Nodal and Wnt proteins. This
changes the balance between the agonists (BMP, Nodal and Wnt) and the
antagonist Cerberus. Such a strategy requires that these factors be expressed
strictly in the ADE, as their presence in the dorsal ecto-mesoderm strongly
interferes with axis formation. Unfortunately, according to the available fate
maps, the dorsal-vegetal blastomeres of the eight-cell stage embryo will give
rise not only to the ADE, but also to ectomesodermal cells
(Bauer et al., 1994
). This
compromises the usefulness of the injection of RNA or constitutive expression
constructs in these blastomeres. Therefore, the precise targeting of gene
expression to the ADE, can only be achieved through the use of a promoter,
specific for that region.
A 4.0 kb mouse cerberus-like promoter fragment isolated from a
genomic library, was found to be specifically activated in the AVE of
transgenic mouse lines (M. F., unpublished data). This promoter fragment was
fused to a NLS-lacZ reporter gene (generating McerP-lacZ;
Fig. 2A) and microinjected into
Xenopus embryos. Surprisingly the mouse cer-l promoter was
only activated in the dorsal side of gastrula embryos and ß-galactosidase
(ß-gal) activity could only be detected in the ADE
(Fig. 2B,C). In contrast,
CMV-driven lacZ expression could be detected in both dorsal and
ventral tissues (Fig. 2D,E).
The temporal and spatial specificity of this promoter was confirmed by in situ
hybridization (Fig.
2F,F',G,G') and by RT-PCR (not shown).
Xenopus embryos were injected dorsally at the four- to eight-cell
stage with the McerP-lacZ construct and sagitally sectioned through
the dorsal lip, at stage 10+ and 11. The left halves of these
embryos were hybridized with an antisense lacZ probe
(Fig. 2F,G). The corresponding
right halves were hybridized with a probe against Xcer
(Fig. 2F',G'). The
region of lacZ expression precisely matched the endogenous
cerberus expression domain, detected in the corresponding half
embryos. This finding enabled us to use the mouse cer-l promoter as a
tool to precisely target expression of BMP, Nodal and Wnt proteins to the ADE
of Xenopus embryos. To that end, we fused the cer-l promoter
to BMP4, Xnr1 or Xwnt8 cDNAs generating McerP-BMP4,
McerP-Xnr1 and McerP-Xwnt8, respectively. These constructs were injected in
the two dorsal-vegetal blastomeres of eight-cell stage embryos. When 80 pg of
either McerP-BMP4, McerP-Xnr1 or McerP-Xwnt8 were injected, head development
was markedly affected in stage 35 embryos, whereas the trunk-tail structures
appeared normal (Fig. 3D,G,J). In contrast, the injection of 80 pg of CMV-driven BMP4, Xnr1 or
Xwnt8 expression constructs resulted in severe axial defects
(Fig. 3A-C), leading to either
a complete ventralization (CMV.BMP4; Fig.
3B) or dorsalization (CMV.Xnr1;
Fig. 3B) of the embryo. When
McerP-lacZ was coinjected to monitor targeting efficiency, ß-gal
activity was only detected in the anterior gut/liver/heart region of the
Xenopus embryos (Fig.
3E,H,K). Due to its stability, ß-gal protein can act as a
lineage tracer for the cells where it was originally expressed. Its detection
in the aforementioned tissues, which had already been shown to originate from
the ADE (Bouwmeester et al.,
1996), provides further evidence that the activation of
Mcer-l promoter in the ADE recapitulates the expression pattern of
cerberus.
|
|
BMP, Nodal and Wnt activities synergistically suppress head
formation
Independently raising the levels of Xnr1, BMP4 or Xwnt8 in the ADE led to
defects in head formation. We therefore tested whether those three factors
could synergistically inhibit head structures. Simultaneous microinjection of
the three Mcer-l promoter expression constructs, at a concentration
of 8 pg each per embryo, resulted in loss of cement gland, reduction of the
brain and a small cyclopic eye (Fig.
3N). In embryos injected with a combination of 20 pg of each
construct, the rostral head, including eyes, was completely lost
(Fig. 3M). These experiments
clearly demonstrated that BMP, Nodal and Wnt activity in the ADE synergize to
inhibit head formation.
Next we tested whether the local increase of BMP, Nodal and Wnt activity in the ADE can affect the patterning of this tissue. Such patterning defects could be responsible for the head phenotypes observed in tadpoles. To address this issue, embryos were injected dorsally with a mixture of McerP-BMP4, McerP-Xnr1 and McerP-Xwnt8 (20 pg of each per embryo) and grown until stage 10+ or 12. These embryos, and uninjected siblings, were then hemi-sectioned and analyzed by in situ hybridization for typical ADE markers (Fig. 4). At stage 10+, the expression domains of cerberus and Xhex (Fig. 4A,A') were unaltered in injected embryos (Fig. 4C,C'). Also, no visible changes in cerberus and Xhex expression were observed in stage 12 embryos (Fig. 4B,B',D,D'). These results demonstrated that the ADE patterning is not perturbed by elevated levels of BMP, Nodal and Wnt signaling.
|
|
Similarly, the expression domain of Krox20 was unchanged in
injected embryos, despite the obvious loss of structures rostral to rhombomere
3 (Fig. 5H,I). Xshh
(Stolow and Shi, 1995;
Ekker et al., 1995
), a gene
expressed in the ventral neural tube and notochord along the entire AP axis
(Fig. 5J), was not detected in
the rostral end of the injected embryo
(Fig. 5K), while its expression
in the remaining embryonic regions was identical to the uninjected controls.
This in situ hybridization analysis confirmed and extended the previous RT-PCR
data, demonstrating that elevated levels of BMP, Nodal and Wnt signaling in
the ADE specifically inhibit the formation of forebrain and midbrain
structures.
Cerberus morpholino oligonucleotide specifically enhances the head
defects induced by BMP, Nodal and Wnt
Since CerMo by itself had no visible effect on head formation
(Fig. 1H,I), we tested whether
a possible function of Cerberus could be revealed in a sensitized experimental
system. We simultaneously raised the levels of the agonists BMP, Nodal and Wnt
up to a threshold level, sufficient to titrate their antagonists but without
producing a severe phenotype. Hence, we analyzed whether this phenotype could
be aggravated by simultaneously reducing Cerberus activity in the ADE. Dorsal
injection of low doses (8 pg each) of a mixture of McerP-BMP4, McerP-Xnr1 and
McerP-Xwnt8 caused a partial loss of the head
(Fig. 6B and
Fig. 3N). Remarkably,
coinjection of CerMo strongly increased the head phenotype
(Fig. 6C). The phenotype caused
by CerMo was specific, since it could be rescued by injection of a full-length
Cerberus expression construct that cannot be blocked by the morpholino
oligonucleotide (Fig. 6D).
|
In conclusion, these results clearly demonstrate that endogenous Cerberus protein can inhibit BMP4, Xnr1 and Xwnt8 activities in vivo.
Cerberus function in the ADE is required for the activation of
forebrain markers
The ADE when combined with stage 10.5 DE explants induces dorsal
telencephalic markers (Lupo et al.,
2002). We have shown that in the embryo, modulating Cerberus
activity in the ADE by raising BMP4, Nodal and Xwnt8 levels represses the
expression of forebrain markers, including XBF1 and Xemx1
(Fig. 5A). To test whether the
Cerberus function in the ADE was required for the activation of dorsal
telencephalic markers in the neuroectoderm, we used a modified explant
recombination system (Fig. 7A).
RT-PCR analysis of ADE/DE conjugates revealed that uninjected ADE induced
expression of both a pan telecephalic marker, XBF1, and a dorsal
telencephalic marker, Xemx1 (Fig.
7B). In contrast, ADE explants in which Cerberus function had been
knocked down with CerMo failed to induce both telencephalic markers
(Fig. 7B). The CerMo effect was
specific because expression of XBF1 and Xemx1could be
rescued by coinjection of a cerberus DNA construct that can not be
blocked by the CerMo.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To test in vivo the requirement for this Cerberus-mediated triple
inhibition in the ADE, one cannot rely on dorsal microinjection of RNA or
CMV-driven DNA constructs coding for either BMP4, Xnr1 or Xwnt8 proteins. When
those constructs are microinjected in the dorsal blastomeres, their activation
in the ecto-mesodermal layers leads to strong axial defects, ranging from
strong ventralization (in the case of BMP4 injection;
Fig. 3A) to strong
dorsalization (Xnr1 injection;
Fig. 3B). Using a mouse
cer-l promoter fragment we were able to drive the expression of these
signaling molecules in the ADE of Xenopus embryos. Since the
activation of this promoter closely resembles the spatial and temporal
expression of the Xcer gene, one could use it to very precisely
target the expression of a given molecule to the ADE. Targeted expression of
increasing doses of BMP4 led to head defects with progressive severity
(Fig. 3D-F). Remarkably,
neither the AP axis nor the cement gland were affected. Even at higher doses,
of 80 pg, cement gland tissue was present, although the head was severely
reduced. This phenotype was very different from the one observed after
injection of CMV.BMP4. When the ventralizing and anti-neural activities of
BMP4 (Fainsod et al., 1994;
Wilson and Hemmati-Brivanlou et al., 1995) are spatially restricted to the ADE
only head defects were observed, while the axial structures remained
undisturbed. A similar phenotype had already been reported for the
misexpression of BMP4 in the anterior neural plate, driven by a Pax-6
promoter fragment in transgenic frog embryos
(Hartley et al., 2001
). After
gastrulation, the expression of BMP4 in the Pax-6 domain
downregulated most anterior neural markers, leading to the suppression of
anterior brain and eye formation. This revealed that the interplay between BMP
signaling and localized inhibitors was necessary for the correct patterning of
the anterior neural structures.
Injection of increasing amounts of McerP-Xnr1 resulted in gradual loss of
the eye and reduction of anterior brain structures
(Fig. 3G). This was surprising
because the Nodal proteins are TGF-ß factors with strong dorsalizing
activity. Ectopic Nodal signaling in the entire mesoderm results in expanded
Spemann's organizer tissue and excess of dorsoanterior structures
(Jones et al., 1995;
Smith et al., 1995
;
Joseph and Melton, 1997
)
Similar results were obtained when the Nodal inhibitor Lefty1 was knocked down
in Xenopus by antisense morpholino oligos
(Branford and Yost, 2002
).
This led to the increase of Nodal signaling in the marginal zone causing an
upregulation of Nodal responsive organizer genes. In contrast, expression of
such genes in the ADE was unchanged, indicating that the level of Nodal
signaling was not elevated there.
The cer-l promoter construct drove the expression of Xnr1 in the
ADE but not in the organizer, thereby eliminating the dorsalizing effect on
the mesoderm and, instead, revealing the anti head activity of this protein.
This is in agreement with experiments in zebrafish, where overexpression of
Nodal protein converted forebrain into more posterior neural or mesodermal
tissue. Elevating the level of the Nodal inhibitor Antivin caused the loss of
posterior ectoderm but did not influence forebrain and eye structures
(Thisse et al., 2000).
Microinjection of McerP-Xwnt8 resulted in severe head defects ranging from
cyclopia (at 20 pg; Fig. 3L),
to a severe truncation of the head when a higher dosage of this construct was
used. Interestingly, in this case, and in contrast to the McerP-BMP4 injected
embryos, the cement gland was the first structure to disappear (compare
Fig. 3D,L). Kiecker and Niehrs
(Kiecker and Niehrs, 2001),
have shown that a gradient of Wnt/ß-catenin signaling was involved in the
anteroposterior neural patterning of Xenopus embryos. Wnt activity in
the posterior neural plate is required for the differentiation of posterior
neural cells. Our own results strongly indicate, however, that a targeted
increase of Wnt activity in the ADE also prevented formation of anterior
neural structures, but did not affect more posterior neural tissue. These
observations are supported by genetic data from the zebrafish model. Increased
Wnt signaling in the anterior head due to a mutation in the axin
gene, a negative regulator of the Wnt signaling pathway, resulted in the loss
of forebrain structures (Heisenberg et
al., 2001
).
When the activity of the signaling molecules BMP4, Xnr1 and Xwnt8 was
simultaneously upregulated in the ADE driven by the McerP constructs, a strong
synergistic defect in head structures could be observed
(Fig. 3M,N). Interestingly, the
targeted activation of these molecules in the ADE did not affect the normal
patterning of locally expressed genes such as Xhex and even
cerberus itself (Fig.
4). Although it has been shown that Xnr1 was able to
induce cerberus expression, that seems to occur in a specific time
frame. In particular, cerberus was only induced by the injection of
Xnr1 mRNA, but not by a Xnr1 DNA construct, which is only
expressed after midblastula transition
(Piccolo et al., 1999).
Interestingly, we observed that the adjacent anterior neuroectoderm was
severely affected upon targeted expression of BMP4, Xnr1 and Xwnt8 proteins in
the ADE (Fig. 5). The anterior
neural markers XBF1, Xemx1 and Nkx2.1 and the cement gland
marker Xag showed a marked decrease in the injected embryos, as shown
by RT-PCR and in situ hybridization. However, more posterior markers such as
En2, expressed in the mid and hindbrain, and Krox20,
expressed in rhombomeres 3 and 5, were not affected by the gain of function of
BMP4, Xnr1 and Xwnt8 in the ADE. Nevertheless, these embryos display a severe
truncation of the head region rostral to these structures. In conclusion,
these results strongly indicate that the combined increase of BMP, Wnt and
Nodal activities in the ADE severely compromised the head formation program,
suggesting the necessity for a tight locally controlled inhibition of those
activities.
Correct balance of agonists versus antagonists in the ADE was
essential for head formation
In some cases, the requirement for a given gene during embryonic
development can only be demonstrated by the use of sensitized or compound
system approaches. The mouse cerberus-like gene has been inactivated
in ES cells (Belo et al., 2000;
Shawlot et al., 2000
;
Stanley et al., 2000
), failing
to produce a mutant phenotype during mouse embryogenesis. Mutant mouse embryos
lacking both Nodal inhibitors Cer-l and Lefty1
(cer-l/;Lefty1/)
displayed striking early embryonic phenotypes not observed in the single
mutants (Perea-Gomez et al.,
2002
). Furthermore, in this sensitized compound mutant background,
removal of a single copy of Nodal can partially rescue the
cer-l/;Lefty1/
mutant phenotypes. Therefore, the requirement for the redundant activities of
cerberus-like and Lefty1 at the level of nodal inhibition
could only be assessed using this genetic system. In the
cer-l/;Lefty1/
mice, nodal signaling is enhanced in the entire embryo. This has
profound consequences on the formation of the primitive streak. Similar
results were obtained in chicken embryos where nodal activity was enhanced in
the epiblast, and simultaneously the hypoblast expressing the
cerberus homologue caronte was removed
(Bertocchini and Stern, 2002
).
In our cerberus-like promoter based assay, nodal activity is only
enhanced in the ADE and therefore the formation of the trunk is not affected.
Both the mouse, chick and frog experiments demonstrate that Cerberus function
in vivo can only be revealed in sensitized assay systems.
As in the mouse, suppression of Xenopus Cerberus does not impair
head formation (Fig. 1H,I).
Similar results were obtained when the ADE region was extirpated from DMZ
explants (Schneider and Mercola,
1999) and such explants still developed partial head-like
structures. In order to reveal a putative role of Cerberus in head formation
we established a novel sensitized assay system in the Xenopus
embryo.
We tested the biological relevance of the Cerberus inhibitory activity in the ADE by simultaneously knocking down Cerberus activity and elevating the levels of the agonists BMP4, Xnr1 and Xwnt8. When mild doses of these 3 proteins were targeted to the ADE the resulting weak head phenotype was strongly enhanced when Cerberus was knocked down by coinjection of the CerMo (compare Fig. 6B and C). This indicated that the agonists (BMP, Wnt and Nodal) must reach a critical threshold level in order to inhibit head formation. This threshold level could be lowered through the suppression of the antagonist Cerberus by CerMo, resulting in an aggravation of the phenotype (Fig. 6M). When the relative balance of agonists versus antagonists was restored by coinjection of a full-length cerberus construct that was not targeted by CerMo, the head phenotype was rescued almost completely. This novel approach clearly demonstrated that Cerberus is a functional inhibitor of BMP4, Xnr1 and Xwnt8 activities in vivo (Fig. 6E-L) and that this biological activity in the ADE is required for the correct specification of the head.
Endogenous Cerberus activity and anterior neural patterning
Cerberus was able to induce anterior neural markers including the dorsal
telencephalic markers eomes and Xemx1 in animal cap explants
(Bouwmeester et al., 1996;
Lupo et al., 2002
). Similar
results were obtained when the activities of the BMP inhibitor Chordin and the
Cerberus truncated protein cer-
C1 were combined
(Fetka et al., 2000
;
Lupo et al., 2002
). This
N-terminal fragment of Cerberus can inhibit Wnt activity
(Fetka et al., 2000
) and
retains a residual Nodal inhibiting activity
(Lupo et al., 2002
). In
contrast, the coinjection of chd and cer-S mRNA was unable
to induce the same set of markers, pointing to the simultaneous requirement of
the anti-BMP and anti-Wnt activities of Cerberus in this process. Induction of
XBF1, Xemx1 and eomes expression in dorsal ectoderm explants
(DE) was also observed when they were conjugated with ADE, a tissue where
endogenous cerberus was expressed
(Lupo et al., 2002
).
Knocking down Cerberus function in the ADE with a morpholino oligo,
resulted in a loss of XBF1, Xemx1 and eomes induction in
ADE/DE conjugates (Fig. 7B,C).
Furthermore, uninjected ADE repressed the expression of the more posterior
neural marker Krox20 in the explanted DE, but this marker was
activated in conjugates of DE/ADE injected with CerMo
(Fig. 7C, lane 4). In embryos
injected with CerMo however, eomes and Xemx1 expression in
the brain was not significantly changed (data not shown). This indicates that
in the embryonic context other molecules may compensate for the reduced
Cerberus activity. This could also explain the reported formation of head in
the DMZ explants lacking Cerberus-expressing ADE tissue
(Schneider and Mercola, 1999).
The completeness of these head structures, however, was not demonstrated
because markers identifying only forebrain were not analyzed. Cerberus is the
only known factor expressed in the leading edge of the ADE with anti-BMP,
-Nodal and -Wnt activity. Thus, the anterior neural patterning activity of
Cerberus in ADE/DE conjugates could be revealed through CerMo-mediated
loss-of-function, since no other factors could compensate for it in this
system. When the formation of the AP axis in Xenopus embryos is
perturbed by interfering with gastrulation movements very often neural
patterning defects were observed. It is tempting to speculate that these
defects are the result of the incorrect positioning of the ADE and that
spatially altered Cerberus activity causes aberrant neural patterning.
In conclusion, in the ADE/DE explant system (Fig. 7) a dual novel role for the ADE is described: not only does ADE induce the expression of anterior neural markers but it also represses the expression of more caudal ones through the activity of Cerberus. This clearly demonstrates that the endogenous Cerberus activity in the leading edge of the anterior dorsal endoderm is required for the correct induction and patterning of the brain.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Institute of Human Genetics, University of Heidelberg, Im
Neuenheimer Feld 328, 69120 Heidelberg, Germany
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aberger, F., Weidinger, G., Grunz H. and Richter, K. (1998). Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2. Mech. Dev. 72,115 -130.[CrossRef][Medline]
Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury,
M., Simeone, A. and Brulet, P. (1995). Forebrain and
midbrain regions are deleted in Otx2/ mutants due to
a defective anterior neuroectoderm specification during gastrulation.
Development 121,3279
-3290.
Bauer, D. V., Huang, S. and Moody, S. A.
(1994). The cleavage stage origin of Speman's Organizer: analysis
of the movements of blastomere clones before and during gastrulation in
Xenopus. Development
120,1179
-1189.
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96,195 -209.[Medline]
Belo, J. A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Follettie, M. and De Robertis, E. M. (1997). Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68,45 -57.[CrossRef][Medline]
Belo, J. A., Bachiller, D., Agius, E., Kemp, C., Borges, A. C., Marques, S., Piccolo, S. and De Robertis, E. M. (2000). Cerberus-like is a secreted BMP and Nodal antagonist not essential for mouse development. Genesis 26,265 -270.[CrossRef][Medline]
Bertocchini, F. and Stern, C. D. (2002). The hypoblast of the chick embryo positions the primitive streak by antagonizing Nodal signaling. Dev. Cell 3, 735-744.[Medline]
Biben, C., Stanley, E., Fabri, L., Kotecha, S., Rhinn, M., Drinkwater, C., Lah, M., Wang, C. C., Nash, A., Hilton, D., Ang, S. L., Mohun, T. and Harvey, R. P. (1998). Murine cerberus homologue mCer-1: a candidate anterior patterning molecule. Dev. Biol. 194,135 -151.[CrossRef][Medline]
Blitz, I. L. and Cho, K. W. (1995). Anterior
neurectoderm is progressively induced during gastrulation: the role of the
Xenopus homeobox gene orthodenticle.
Development 121,993
-1004.
Bourguignon, C., Li, J. and Papalopulu, N.
(1998). XBF-1, a winged helix transcription factor with dual
activity, has a role in positioning neurogenesis in Xenopus competent
ectoderm. Development
125,4889
-4900.
Bouwmeester, T., Kim, S. H., Sasai, Y., Lu, B. and De Robertis, E. M. (1996). Cerberus, a head inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382,595 -601.[CrossRef][Medline]
Bouwmeester, T. and Leyns, L. (1997). Vertebrate head induction by anterior primitive endoderm. BioEssays 19,855 -863.[Medline]
Bradley, L. C., Snape, A., Bhatt, S. and Wilkinson, D. G. (1993). The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest. Mech. Dev. 40, 73-84.[CrossRef][Medline]
Branford, W. W. and Yost, H. J. (2002). Lefty-dependent inhibition of Nodal- and wnt-responsive organizer gene expression is essential for normal gastrulation. Curr. Biol. 12,2136 -2141.[CrossRef][Medline]
Cho, K. W., Blumberg, B., Steinbeisser, H. and De Robertis, E. M. (1991). Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67,1111 -1120.[Medline]
De Robertis, E. M., Kim, S. H., Leyns, L., Piccolo, S., Bachiller, D., Agius, E., Belo, J. A., Yamamoto, A., Hainski-Brousseau, A., Brizuela, B., Wessely, O., Lu, B. and Bouwmeester, T. (1997). Patterning by genes expressed in Spemann's organizer. Cold Spring Harb. Symp. Quant. Biol. 62,169 -175.[Medline]
Dufort, D., Schwartz, L., Harpal, K. and Rossant, J.
(1998). The transcription factor HNF3beta is required in visceral
endoderm for normal primitive streak morphogenesis.
Development 125,3015
-3025.
Ekker, S. C., McGrew, L. L., Lai, C. J., Lee, J. J., von
Kessler, D. P., Moon, R. T. and Beachy, P. A. (1995).
Distinct expression and shared activities of members of the hedgehog gene
family of Xenopus laevis. Development
121,2337
-2347.
Fainsod, A., Steinbeisser, H. and De Robertis E. M. (1994). On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13,5015 -5025.[Abstract]
Fetka, I., Doederlein, G. and Bouwmeester, T. (2000). Neuroectodermal specification and regionalization of the Spemann organizer in Xenopus. Mech. Dev. 93, 49-58.[CrossRef][Medline]
Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C. and Niehrs, C. (1997). Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus. Nature 89,517 -519.
Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391,357 -362.[CrossRef][Medline]
Gont, L. K., Steinbeisser, H., Blumberg, B. and de Robertis, E.
M. (1993). Tail formation as a continuation of gastrulation:
the multiple cell populations of the Xenopus tailbud derive from the
late blastopore lip. Development
119,991
-1004.
Hartley, K. O., Hardcastle, Z., Friday, R. V., Amaya, E. and Papalopulu, N. (2001). Trangenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation. Dev. Biol. 238,168 -184.[CrossRef][Medline]
Heasman, J. (2002). Morpholino oligos: making sense of antisense? Dev. Biol. 243,209 214.[CrossRef][Medline]
Heisenberg, C., Houart, C., Take-Uchi, M., Rauch, G., Young,
N., Coutinho, P., Masai, I., Caneparo, L., Concha, M., Geisler, R.,
Dale, T. C., Wilson, S. W. and Stemple, D. L. (2001). A
mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a
fate transformation of telencephalon and eyes to diencephalons.
Genes Dev. 15,1427
-1434.
Hemmati-Brivanlou, A., de la Torre, J. R., Holt, C. and Harland, R. M. (1991). Cephalic expression and molecular characterization of Xenopus En-2. Development 111,715 -724.[Abstract]
Hollemann, T. and Pieler, T. (2000). Xnkx-2.1: a homeobox gene expressed during early forebrain, lung and thyroid development in Xenopus laevis. Dev. Genes Evol. 210,579 -581.[CrossRef][Medline]
Jones, C. M., Kuehn, M. R., Hogan, B. L., Smith, J. C. and
Wright, C. V. (1995). Nodal-related signals induce axial
mesoderm and dorsalize mesoderm during gastrulation.
Development 121,3651
-3662.
Joseph, E. M. and Melton, D. A. (1997). Xnr4: a Xenopus Nodal-related gene expressed in the Spemann organizer. Dev. Biol. 184,367 -372.[CrossRef][Medline]
Kiecker, C. and Niehrs, C. (2001). A morphogen
gradient of Wnt/beta-catenin signalling regulates anteroposterior neural
patterning in Xenopus. Development
128,4189
-4201.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680 -689.[Medline]
Leyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S. and de Robertis, E. M. (1997). Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann Organizer. Cell 88,747 -756.[Medline]
Lupo, G., Harris, W. A., Barsacchi, G. and Vignali, R.
(2002). Induction and patterning of the telencephalon in
Xenopus laevis. Development
129,5421
-5436.
Medina, A., Reintsch, W. and Steinbeisser, H. (2000). Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 92,227 -237.[CrossRef][Medline]
Munchberg, S. R. and Steinbeisser, H. (1999). The Xenopus Ets transcription factor XER81 is a target of the FGF signaling pathway. Mech. Dev. 80, 53-65.[CrossRef][Medline]
Pannese, M., Lupo, G., Kablar, B., Boncinelli, E., Barsacchi, G. and Vignali, R. (1998). The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73,73 -83.[CrossRef][Medline]
Pera, E. M. and de Robertis, E. M. (2000). A direct screen for secreted proteins in Xenopus embryos identifies distinct activities for the Wnt antagonists Crescent and Frzb-1. Mech. Dev. 96,183 -195.[CrossRef][Medline]
Perea-Gomez, A., Vella, F. D., Shawlot, W., Oulad-Abdelghani, M., Chazaud, C., Meno, C., Pfister, V., Chen, L., Robertson, E., Hamada, H., Behringer, R. R. and Ang, S. L. (2002). Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev. Cell 3, 745-756.[Medline]
Piccolo, S., Agius, E., Leyns, L., Bhattacharyya, S., Grunz, H., Bouwmeester, T. and De Robertis, E. M. (1999). The head inducer Cerberus is a multifunctional antagonist of Nodal, BMPand Wnt signals. Nature 397,707 710.[CrossRef][Medline]
Rhinn, M., Dierich, A., Shawlot, W., Behringer, R. R., Le Meur,
M. and Ang, S. L. (1998). Sequential roles for Otx2 in
visceral endoderm and neuroectoderm for forebrain and midbrain induction and
specification. Development
125,845
-856.
Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K. and De Robertis, E. M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79,779 -790.[Medline]
Schneider, V. A. and Mercola, M. (1999). Spatially distinct head and heart inducers within the Xenopus organizer region. Curr. Biol. 9, 800-809.[CrossRef][Medline]
Shawlot, W., Deng, J. M. and Behringer, R. R.
(1998). Expression of the mouse cerberus-related gene,
Cerr1, suggests a role in anterior neural induction and
somitogenesis. Proc. Natl. Acad. Sci. USA
95,6198
-6203.
Shawlot, W., Wakamiya, M., Kwan, K. M., Kania, A., Jessell, T.
M. and Behringer, R. R. (1999). Lim1 is required in
both primitive streak-derived tissues and visceral endoderm for head formation
in the mouse. Development
126,4925
-4932.
Shawlot, W., Min Deng, J., Wakamiya, M. and Behringer, R. R. (2000). The cerberus-related gene, Cerr1, is not essential for mouse head formation. Genesis 26,253 -258.[CrossRef][Medline]
Small, E. M., Vokes, S. A., Garriock, R. J., Li, D. and Krieg, P. A. (2000). Developmental expression of the Xenopus Nkx2-1 and Nkx2-4 genes. Mech. Dev. 96,259 -262.[CrossRef][Medline]
Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70,829 -840.[Medline]
Smith, W. C., McKendry, R., Ribisi, S. Jr and Harland, R. M. (1995). A Nodal-related gene defines a physical and functional domain within the Spemann organizer. Cell 82, 37-46.[Medline]
Spemann, H. and Mangold, H. (1924). Über Induktion von Embryonalanlagen durch Implantation Artfremder Organisatoren. Roux Arch. Entw. Mech. 100,599 -638.
Spemann, H. (1931). Über den Anteil von Implantat und Wirtskeim an der Orientierung und Beschaffenheit der induzierten Embryonalanlage. Roux Arch. Entw. Mech. 123,389 -517.
Stanley, E. G., Biben, C., Allison, J., Hartley, L., Wicks, I. P., Campbell, I. K., McKinley, M., Barnett, L., Koentgen, F., Robb, L. and Harvey, R. P. (2000). Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis. Genesis 26,259 -264.[CrossRef][Medline]
Steinbeisser, H., Alonso, A., Epperlein, H.-H. and Trendelenburg, M. F. (1989). Expression of mouse histone H1(0) promoter sequences following microinjection into Xenopus oocytes and developing embryos. Int. J. Dev. Biol. 33,361 -368.[Medline]
Stolow, M. A. and Shi, Y. B. (1995). Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis. Nucleic Acids Res. 23,2555 -2562.[Abstract]
Thisse, B., Wright, C. V. and Thisse, C. (2000). Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo. Nature 403,425 -428.[CrossRef][Medline]
Thomas, P. and Beddington, R. (1996). Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr. Biol. 6,1487 -1496.[Medline]
Townbin, H., Staehlin, T. and Gordon, J. (1979). Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350 -4354.[Abstract]
Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376,331 -333.[CrossRef][Medline]