RESEARCH ARTICLE 4073
Development 137, 4073-4081 (2010) doi:10.1242/dev.053405
© 2010. Published by The Company of Biologists Ltd
Prohibitin1 acts as a neural crest specifier in Xenopus
development by repressing the transcription factor E2F1
Martina Schneider, Alexandra Schambony* and Doris Wedlich†
SUMMARY
Prohibitin 1 (phb1), which was initially described as an inhibitor of cell proliferation, is a highly conserved protein found in
multiple cellular compartments. In the nucleus it interacts with the transcriptional regulators Rb and E2F1 and controls cell
proliferation and apoptosis. Here we unravel an unexpected novel function for phb1 in Xenopus cranial neural crest (CNC)
development. Xphb1 is maternally expressed; zygotically expressed neurula stage transcripts accumulate in the CNC and the
neural tube. Knockdown of Xphb1 by antisense morpholino injection results in the loss of foxD3, snail2 and twist expression,
whereas expression of c-myc, AP-2 and snail1 remains unaffected. Xphb2, its closest relative, cannot substitute for Xphb1,
underlining the specificity of Xphb1 function. Epistatic analyses place Xphb1 downstream of c-myc and upstream of foxD3, snail2
and twist. To elucidate which subdomain in Xphb1 is required for neural crest gene regulation we generated deletion mutants
and tested their rescue ability in Xphb1 morphants. The E2F1-binding domain was found to be necessary for Xphb1 function in
neural crest development. Gain- and loss-of-function experiments reveal that Xphb1 represses E2F1 activity; suppression of E2F1
through Xphb1 is required for twist, snail2 and foxD3 expression in the CNC. With the Xphb1 dependency of a subset of CNC
specifiers downstream of c-myc, we have identified a new branching point in the neural crest gene regulatory network.
INTRODUCTION
The cranial neural crest (CNC) shapes the vertebrate face. These
cells possess two exceptional properties: pluripotency and motility.
CNC cells differentiate to melanocytes, peripheral neurons, glia,
cartilage, bone and the inflow tracts of the heart, based on an
orchestrated gene regulatory network and their migratory
trajectories (Sauka-Spengler and Bronner-Fraser, 2008). CNC cells
share important characteristics with carcinoma cells in undergoing
epithelial-mesenchymal transition (EMT) and tissue invasion
(Acloque et al., 2009). They express genes involved in tumor
formation, such as c-myc, snail1 and snail2 (slug). They also
upregulate mesenchymal cadherins and metalloproteinases, which
are enriched during metastasis (Kuriyama and Mayor, 2008).
Therefore, deciphering the regulatory network of CNC
development would not only help to understand congenital defects
of craniofacial development but also provide insight into gene
functions in tumor formation and progression.
CNC is induced at the neuroectoderm/ectoderm border under the
influence of defined levels of BMP, canonical Wnt, retinoic acid
(RA), FGF and notch signaling (Basch and Bronner-Fraser, 2006).
These signals activate the neural plate border specifiers pax3, zic1
and dlx, which in turn activate expression of the more restricted,
localized CNC specifiers, including c-myc, foxD3, AP-2, snail1 and
sox9. The latter are thought to initiate CNC fate specification by
controlling CNC proliferation and apoptosis, EMT and emigration
KIT, Campus South, Zoologicak Institute, Cell and Developmental Biology,
Kaiserstrasse 12, D-76131 Karlsruhe, Germany.
*Present address: Department of Biology, University of Erlangen-Nuernberg,
D-91058 Erlangen, Germany
†
Author for correspondence ([email protected])
Accepted 29 September 2010
from the neural fold. In addition, snail2, sox10 and twist are
activated, which serves to maintain CNC pluripotency and survival
but also to regulate the expression of genes required for cell
migration and differentiation (Meulemans and Bronner-Fraser,
2004; Sauka-Spengler and Bronner-Fraser, 2008; Steventon et al.,
2005). This view of hierarchy in neural crest gene activation is
most likely an oversimplification because there is mounting
evidence of a cross-regulation between snail1, snail2 and twist in
the induction of neural crest at the gastrula stage (Carl et al., 1999;
Zhang et al., 2006; Zhang and Klymkowsky, 2009). Although we
have to reconsider the regulatory network at the level of neural
crest specification, the upstream requirement of the neural plate
border specifiers appears correct. In addition to their role in the
CNC, neural plate border specifiers such as pax3 and zic1 also
promote hatching gland and preplacodal development and act
together to stimulate neural crest formation (Hong and SaintJeannet, 2007). In addition, gbx2 collaborates with zic1 in
activating neural crest genes and inhibiting the placodal fate (Li et
al., 2009).
Not all homologs of neural crest specifier genes that are
upregulated in human tumors share common functions with their
orthologs in neural crest cells. snail1 and snail2 behave similarly
by promoting EMT and blocking apoptosis. The proto-oncogene cmyc, however, operates differently. When overexpressed or
mutated, c-myc promotes tumor growth by facilitating cell cycle
entry (Herold et al., 2009; Morrish et al., 2009) and sensitizes cells
for apoptosis (Hoffman and Liebermann, 2008). In neural crest
development, c-myc neither influences cell proliferation nor cell
death; rather, it is required for snail1, snail2 and twist expression
(Bellmeyer et al., 2003).
The human prohibitin homologs, PHB (PHB1) and PHB2, are
tumor suppressors that share 53% amino acid sequence identity;
these proteins are localized to the plasma membrane, nucleus and
mitochondria (Mishra et al., 2006). Studies in C. elegans have
revealed a crucial mitochondrial function for prohibitins in
DEVELOPMENT
KEY WORDS: Snail/slug regulation, c-myc, Cranial neural crest, Xenopus
4074 RESEARCH ARTICLE
MATERIALS AND METHODS
Xenopus embryos, micromanipulation and lineage tracing
Embryos were obtained by in vitro fertilization and staged according to
Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). mRNA for injection
experiments was synthesized in vitro using the mMessage mMachine Kit
(Ambion, Norwalk, CT, USA). Xphb1 (phb1 MO, 5⬘-ATCCCTGTTCTTCCACACGGCTAAT-3⬘) and c-myc [c-myc MO, as previously
described (Bellmeyer et al., 2003)] morpholino antisense oligonucleotides
were purchased from Gene Tools (Philomath, OR, USA). As a control we
used the 3⬘carboxyfluorescein-tagged standard control morpholino
oligonucleotide (co MO) designed by Gene Tools.
Unless indicated otherwise, all injections were performed into the animal
hemisphere of one blastomere of 2-cell stage embryos as follows: 700 pg
RNA, 16 ng phb1 MO, 11 ng c-myc MO. Embryos were co-injected with
lacZ (b-gal) DNA (50 pg) to identify the manipulated side. Embryos were
fixed in MEMFA at stage 18 (unless otherwise noted) and successively
processed for b-gal staining (Sive et al., 2000) using X-Gal (Applichem,
Darmstadt, Germany) as substrate. In the rescue experiment, in which
Hphb1 mRNA was co-injected, dextran-FITC (4 pg; Molecular Probes,
Eugene, OR, USA) was used as a lineage tracer.
Constructs
Xphb1 and Xphb2 were amplified from neurula stage embryos. Xphb1 was
fused C-terminally with six copies of the c-myc epitope in the pCS2+
vector (pCS2+myc). All Xphb1 mutants, Xphb1DN-term (amino acids 116275), Xphb1DRaf-1 (amino acids 1-243) and Xphb1DC-term (amino acids
1-188), were subcloned into pCS2+myc. Primers are listed in Table S1 in
the supplementary material. The mutant Hphb1D185-214, a kind gift of S.
Chellappan (Columbia University, New York, USA), was subcloned into
the pCS2+ vector and the Hphb1 (pcDNA3-PHB) plasmid was kindly
provided by T. Rudel (Julius-Maximilians-Universität, Würzburg,
Germany).
Whole-mount analysis
In situ hybridization was performed as described (Gawantka et al., 1995).
Antisense DIG-labeled probes were synthesized with the DIG RNA
Labeling Kit (Roche, Basel, Switzerland) using template cDNA encoding
Xphb1, twist (Hopwood et al., 1989), snail2 (Mayor et al., 1995), snail1
(Essex et al., 1993), foxD3 (Sasai et al., 2001), c-myc (Bellmeyer et al.,
2003) AP-2 (Luo et al., 2003), Xbra (Smith et al., 1991), chordin (Sasai et
al., 1995), sox2 (Kishi et al., 2000), cytokeratin Xk81A1 (Jonas et al.,
1985), pax3 (Bang et al., 1997), zic3 (Nakata et al., 1997) and meis3
(Salzberg et al., 1999). Images were captured as described previously
(Etard et al., 2005). The probe for cytokeratin Xk81A1 was kindly
provided by R. Mayor (UCL, London, UK). The results of at least three
independent experiments were averaged, and statistical significance was
calculated using Student’s t-test. Proliferation was estimated at stage 18 by
phospho-histone H3 detection, as previously described (Turner and
Weintraub, 1994). Anti-phospho-histone H3 antibody (1:700; Millipore,
Temecula, CA, USA) and anti-rabbit antibody conjugated with alkaline
phosphatase (1:700; Dianova, Hamburg, Germany) were used. TUNEL
staining was carried out at stage 18 as described (Hensey and Gautier,
1998). DIG was detected by anti-DIG Fab fragments conjugated to alkaline
phosphatase (1:2000; Roche) and the chromogenic reaction was performed
using NBT/BCIP in single stainings. In double stainings, BM Purple
(Roche) and BCIP were used.
RT-PCR and real-time PCR
For RT-PCR assays, RNA was extracted from groups of five embryos using
the High Pure RNA Isolation Kit (Roche). Total RNA (500 ng) was reverse
transcribed (M-MLV, Promega) and cDNA was amplified according to
standard protocols. For real-time PCR, embryos were injected animally in
the dorsal blastomeres at the 4-cell stage and total RNA was extracted from
three whole embryos at stage 18 and reverse transcribed. Real-time PCR
was performed using iQ SYBR Green Supermix on an iCycler (BioRad,
Hercules, CA, USA). Expression levels were calculated relative to
ornithine decarboxylase (ODC) and were normalized to uninjected
controls. Results of at least three independent experiments were averaged,
and statistical significance was calculated using Student’s t-test. Primers
are listed in Table S1 in the supplementary material.
Protein analysis
The specificity of phb1 MO was analyzed using the TnT In Vitro
Translation Kit (Promega, Mannheim, Germany) with radioactive
[35S]methionine (GE Healthcare, Little Chalfont, UK). After incubation of
1 mg/ml Xphb1, Xphb2 or Hphb1 DNA alone or together with the phb1 MO
(1 mM), samples were subject to SDS-PAGE and evaluation performed
using a BAS-1500 phosphorimager (Fujifilm, Düsseldorf, Germany).
Western blots were carried out as described (Unterseher et al., 2004).
Lysates corresponding to half an embryo were separated by SDS-PAGE.
Rabbit polyclonal anti-prohibitin antibody was obtained from Abcam
(1:4000; Cambridge, UK) and alkaline phosphatase-conjugated secondary
antibody from Dianova (1:3000). b-catenin (a kind gift of Ralph Rupp,
München, Germany), with Coomassie Brilliant Blue (Serva, Heidelberg,
Germany) staining, was used as a loading control.
Reporter gene assay
For luciferase reporter assays, Xenopus embryos were injected animally
into one blastomere at the 2-cell stage with the pFR-luciferase transreporter plasmid Gal4-DBD-E2F1 and different concentrations of Xphb1
mRNA. As a negative control, only the pFR trans-reporter plasmid (40 pg)
was injected (data not shown). As a positive control, the pFR trans-reporter
plasmid (40 pg) was co-injected with Gal4-DBD-E2F1 (50 pg). This value
was set as 100% in each experiment. The reporter gene assay was
performed at stage 18 as previously described (Gradl et al., 1999). The pFR
trans-reporter plasmid and the Gal4-DBD-E2F1 plasmid, which is a fusion
of the Gal4 DNA-binding domain (DBD) with E2F1, were kind gifts of S.
Kang (Korea University, Seoul, South Korea).
RESULTS
Xenopus prohibitin1 (Xphb1) is expressed in CNC
and is required for twist expression
In a search for genes expressed in migrating cells of the early
Xenopus embryo we identified prohibitin1 (Xphb1) in neural crest
cells by whole-mount in situ hybridization (ISH) (Fig. 1). At
blastula stage, Xphb1 transcripts localized in the animal
hemisphere, accumulated in the posterior dorsal area at late gastrula
and concentrated in CNC and neural tube at neurula stage (Fig. 1AC). When gastrula embryos were cut into two halves (semisections), Xphb1 was detected in the mesendoderm at stage 10 (Fig.
1D,d) and in the posterior neuroectoderm and mesoderm at stage
11.5 (Fig. 1E,e). The sense Xphb1 probe yielded no signal (Fig.
1d⬘,e⬘). Xphb1 expression in CNC was maintained during their
migration, and additional expression areas were found in the eye,
the otic vesicle and the brain (Fig. 1F,G). Double ISH for Xphb1
and twist revealed partial overlap of the expression domains at
stage 18 (Fig. 1H-H⬙). Xphb2, which shares 53% amino acid
identity with Xphb1, was not detected in the neural crest (data not
DEVELOPMENT
development and lifespan control (Artal-Sanz and Tavernarakis,
2009; Artal-Sanz et al., 2003); in mitochondria, the prohibitins
localize predominantly to the inner membrane where they form a
multimeric complex with a chaperone-like function (Osman et al.,
2009). Nuclear prohibitins inhibit cell proliferation and suppress
apoptosis (Fusaro et al., 2002; Wang et al., 1999a). Here, we report
that Xenopus prohibitin1 (Xphb1) acts downstream of c-myc in
neural crest development to control snail2, foxD3 and twist
expression. Whereas PHB-1/PHB1 and PHB-2/PHB2 appear
functionally synonymous in C. elegans and human cell lines, this
is not the case in Xenopus. The role of Phb1 in neural crest
development is not associated with cell proliferation or apoptosis
but involves its ability to bind to and repress the activity of the
transcription factor E2F1.
Development 137 (23)
Fig. 1. Xphb1 is expressed in the presumptive cranial neural crest
(CNC) region. (A-H⬙) In situ hybridization (ISH) for Xphb1 (A-H) and
Xphb1 plus twist (H⬘,H⬙). (A)At blastula (stage 9) Xphb1 is expressed in
the dorsal animal ectoderm (arrowheads; animal pole is towards the top).
(B)At late gastrula stage (stage 11.5) Xphb1 transcripts are found in the
posterior dorsal area (arrowhead). (C)At neurula stage (dorsal view)
Xphb1 is detected in migrating neural crest (arrowhead) and in the neural
tube. (D,E)Semi-sections of stage 10 (D) and stage 11.5 (E) gastrula stage
Xenopus embryos (animal pole towards the top). Insets demonstrate
Xphb1 expression in the mesendoderm (d) and in the posterior
neuroectoderm including mesoderm (e). No Xphb1 mRNA is detectable
in comparable regions of sense control embryos (d⬘,e⬘). (F,G)From
neurulation onwards (F, stage 23; G, stage 28; lateral view) Xphb1
expression is restricted to the neural crest territory (arrowheads), the eye
and brain. (H-H⬙) Double ISH at stage 18 shows that Xphb1 (H, purple) is
partially co-expressed with twist (blue) in the neural crest (H⬘).
(H⬙)Transverse section showing the colocalization of Xphb1 and twist
(arrow); Xphb1 (purple arrowhead) and twist (blue arrowhead) are also
expressed in non-overlapping regions. Scale bars: 400mm. (I)RT-PCR
analysis of Xphb1 expression at the indicated stages. ornithine
decarboxylase (ODC) was used as an internal control. Additional controls
were performed without reverse transcriptase (–RT) and without cDNA
(H2O).
shown). RT-PCR (Fig. 1I) and immunoblots (see Fig. S1 in the
supplementary material) showed that Xphb1 is expressed
throughout early development.
RESEARCH ARTICLE 4075
Fig. 2. Xphb1 is required for CNC formation. (A)The phb1 MO,
showing its binding site in Xphb1. Nucleotides that are identical
between Xphb1 and human PHB1 (Hphb1) or Xphb2 are indicated
(asterisks). A standard control morpholino (co MO) tagged with 3⬘
carboxyfluorescein was used as control. (B)In TnT in vitro translation,
phb1 MO specifically blocks the translation of Xphb1 (lane 2). The phb1
MO had no effect on the translation of Hphb1 (lane 3) or Xphb2 (lane
6). (C)Injection (*) of phb1 MO, but not of co MO, efficiently inhibited
twist expression. This was rescued by co-injection of Hphb1 mRNA or
Xphb1 mRNA lacking the morpholino binding site, whereas Xphb2
mRNA showed no effect. b-gal was used as a lineage tracer (light blue).
The co MO-injected side appears fluorescently labeled. Beneath is
shown the percentage of Xenopus embryos with reduced twist
expression. n, number of embryos analyzed. **, P<0.005. Error bars
indicate standard error.
DEVELOPMENT
Xphb1 specifies neural crest by repressing E2F1
4076 RESEARCH ARTICLE
Development 137 (23)
Fig. 3. Xphb1 regulates CNC formation
independently of proliferation and apoptosis.
(A-C,E,F) Phospho-histone H3 staining (A-C) and
TUNEL assay (E,F) of single side-injected (*)
Xenopus embryos. Dextran-FITC marks the injected
side (C). (D,G)Quantification of phospho-histone
H3 staining (D) and TUNEL assay (G). The average
number of cells counted on the uninjected (n.i.)
side of the embryo was set at 100%; n, number of
embryos analyzed. Neither proliferation nor
apoptosis was influenced after phb1 MO injection.
Error bars indicate standard error.
Xphb1 operates downstream of c-myc in
regulating snail2, foxD3 and twist
Next, we aimed to better allocate Xphb1 into the regulatory
network of neural crest genes by expanding ISH studies of marker
genes and by performing epistatic analyses. As shown in Fig. 4,
depletion of Xphb1 resulted in loss of snail2 and foxD3, in addition
to twist, but did not alter c-myc, snail1 or AP-2 expression.
Reduction of twist, snail2 and foxD3 expression was observed in
nearly 80% of the embryos (see Fig. S3A,B in the supplementary
material). We further confirmed these findings by real-time PCR.
twist mRNA was reduced by 50% and snail2 by 40% in phb1 MOinjected embryos; these effects were rescued by Hphb1 mRNA
injection (see Fig. S4A in the supplementary material). No changes
in the amount of c-myc or snail1 transcripts were found in Xphb1
morphants; a small decrease in AP-2 mRNA was detected by real-
Fig. 4. Xphb1 knockdown inhibits expression of the CNC markers
snail2 and foxD3 but not of c-myc, snail1 and AP-2. Depletion of
Xphb1 mediated by phb1 MO injection (*) specifically inhibited snail2
and foxD3 expression. Neural crest markers c-myc, snail1 and AP-2
were not affected by Xphb1 knockdown.
DEVELOPMENT
The accumulation of Xphb1 in CNC prompted us to examine
whether depletion of Xphb1 affects neural crest development. We
designed an antisense morpholino (phb1 MO) that specifically
blocks Xphb1 translation while Xphb2 synthesis remains unaltered
(Fig. 2A,B). Single-sided injections of phb1 MO at the 2-cell stage
led to the loss of twist expression in the CNC (Fig. 2C). Coinjection of lacZ DNA served to identify the injected side. Neither
lacZ DNA alone nor control morpholino (co MO) injection affected
twist expression, pointing to a specific phb1 MO effect. This was
confirmed by rescue experiments. Both Xphb1 mRNA that lacks
the morpholino binding site and mRNA encoding the human
homolog PHB1 (Hphb1), which shows 90% amino acid identity to
Xphb1, rescued the phb1 MO phenotype. Importantly, Xphb2
mRNA injection did not rescue the depletion of Xphb1 (Fig. 2C).
Phb1 is known from mammalian cell culture studies to control
cell proliferation and apoptosis (Joshi et al., 2003; McClung et al.,
1989; Wang et al., 1999a; Wang et al., 1999b). To address whether
loss of the twist signal is caused by reduced cell proliferation or
increased cell death we performed phospho-histone H3
immunostainings and TUNEL assays. As shown in Fig. 3, phb1
MO injections influenced neither cell proliferation nor apoptosis.
This suggested that Xphb1 might be required in neural crest gene
activation, rather than in controlling CNC proliferation or cell
death.
Neural crest induction takes place during gastrulation and
depends on signals secreted from the dorsolateral mesoderm
(Steventon et al., 2009). To exclude the possibility that Xphb1
knockdown affects mesoderm formation and consequently neural
crest induction, we examined the expression patterns of Xbra and
chordin by ISH. Both genes were expressed normally upon Xphb1
depletion (see Fig. S2 in the supplementary material).
Xphb1 specifies neural crest by repressing E2F1
RESEARCH ARTICLE 4077
Fig. 5. Xphb1 acts downstream of c-myc and upstream of twist,
snail2 and foxD3. (A)Injection of c-myc MO (*) efficiently inhibited
Xphb1 expression (90%, n116). (B)Xenopus embryos injected with cmyc MO and analyzed by quantitative real-time PCR showed a
reduction in relative Xphb1 expression (normalized to the level of ODC
expression). **, P<0.005. (C)c-myc MO-mediated depletion of twist,
snail2 and foxD3 was rescued by co-injection of Xphb1 mRNA (twist,
**, P<0.005; snail2, *, P<0.05; foxD3, **, P<0.005), whereas Xphb2
had no effect. n, number of embryos analyzed. Error bars indicate
standard error.
unique function of Xphb1 (Fig. 5C). Depletion of c-myc also
resulted in loss of snail1 and AP-2 expression; the expression of
these genes was not rescued by Xphb1 mRNA injection (Fig. 6).
Thus, snail1 and AP-2 are regulated by c-myc independently of
Xphb1, whereas c-myc-dependent expression of snail2, foxD3 and
twist requires Xphb1.
To separate Xphb1 clearly from the group of neural plate border
specifiers we investigated whether Xphb1 knockdown alters the
expression of pax3, zic3 and meis3. The expression patterns of
these neural plate border specifiers were unchanged upon Xphb1
depletion (Fig. 7A-C). In line with these results, we did not observe
a broadening or reduction of the epidermal or neural tissue, as
examined by ISH for Xk81A1 (an epidermal marker) and sox2 (a
neural marker) (Fig. 7D,E). Double ISH for sox2 and snail1 or
snail2 further confirmed that the ratio between neural plate and
CNC areas remains stable in the absence of Xphb1 (Fig. 7F,G).
time PCR, but not by ISH (see Fig. S4B in the supplementary
material; compare with Fig. 4).
To examine whether c-myc acts upstream of Xphb1, embryos
were injected with antisense c-myc morpholino (c-myc MO). There
was a decrease in Xphb1 CNC expression in 90% of the injected
embryos, as monitored by ISH (Fig. 5A). Real-time PCR showed
a decrease of ~30% in Xphb1 mRNA (Fig. 5B). As expected,
expression of snail2, foxD3 and twist was lost upon c-myc MO
injection. However, when Xphb1 mRNA was co-injected with cmyc MO, the expression of these genes was rescued. Xphb2 was
unable to rescue the effects of c-myc MO injection, confirming a
Xphb1 controls neural crest gene expression
through repression of E2F1
Next, we aimed to determine whether a specific domain in Xphb1
is required to regulate snail2, foxD3 and twist. Prohibitin1 consists
of a transmembrane domain (TM) required for mitochondrial
localization, as well as binding sites for the nuclear transcription
factors Rb and E2F1 (Mishra et al., 2006; Wang et al., 1999a;
Wang et al., 1999b) (Fig. 8A). Human PHB1 has also been
reported to interact with the ring-finger protein 2 (RNF2) (Choi et
al., 2008) and to bind RAF1 (Rajalingam et al., 2005; Wang et al.,
1999b). Within the Raf-1 binding site, a nuclear export signal
(NES) sequence is located (Mishra et al., 2006). We deleted the N-
DEVELOPMENT
Fig. 6. Expression of snail1 and AP-2 is regulated by c-myc
independently of Xphb1. The expression of snail1 and AP-2 was
reduced by injection (*) of c-myc MO. This was not rescued by coinjection of Xphb1 mRNA. n, number of embryos analyzed. **,
P<0.005. Error bars indicate standard error.
4078 RESEARCH ARTICLE
Development 137 (23)
Fig. 7. Xphb1 depletion does not shift the neural plate border.
(A-C)The expression of zic3, pax3 and meis3 at stage 14 was not
affected after phb1 MO (*) injection. (D,E)cytokeratin Xk81A1 (D,
stage 16) and sox2 (E, stage 18) expression areas remained unchanged
upon phb1 MO injection. (F)In double ISH, the snail1 (purple)
expression area facing the sox2 (red) region showed a normal pattern
after Xphb1 depletion (arrowhead marks b-gal staining). (G)Double ISH
for sox2 (red) and snail2 (purple), which is lost on the phb1 MOinjection side.
terminus containing the TM domain and the Rb binding site
(Xphb1⌬N-term) and generated two different C-terminal
truncations: Xphb1⌬Raf-1, which lacks the Raf-1 binding site and
the NES motif; and Xphb1⌬C-term, which lacks the Raf-1 binding
site, the NES motif and the E2F1-binding sequence (Fig. 8A). All
deletion mutants could be detected by their myc tag in embryo
lysates (Fig. 8B). When these mutants were analyzed for their
ability to rescue the Xphb1 morphant phenotype, we found that
Xphb1⌬C-term was insufficient to restore twist expression (Fig.
DISCUSSION
CNC development requires a complex signaling network, which
includes key regulators that are often found to be dysregulated in
tumors. In this study we identify Xphb1, the Xenopus homolog of
the tumor suppressor gene prohibitin 1, as a novel neural crest
specifier operating downstream of c-myc in regulating snail2,
foxD3 and twist. This distinguishes Xphb1 from the neural plate
border specifiers and places it among the CNC specifiers (Fig. 10).
Importantly, Xphb1 is not required for the activation of snail1 or
AP-2 through c-myc. Bellmeyer et al. have demonstrated that cmyc expression marks CNC already at mid-gastrula (stage 11) and
induces the CNC genes snail2, snail1, sox9, foxD3 and twist
(Bellmeyer et al., 2003). Therefore, the branching in Xphb1dependent and -independent c-myc targets observed here is
surprising and has not been reported previously. Depletion of the
CNC specifiers sox10 and sox9, for example, results in loss of
twist, foxD3, snail2 and snail1 expression (Honore et al., 2003; Lee
et al., 2004; Spokony et al., 2002). More recently, a disjunctive
regulation of snail1 and snail2 has been demonstrated in RhoVdepleted embryos. RhoV is transiently expressed in CNC between
stages 12 and 21. It is essential for the expression of snail2, sox9,
sox10, twist and foxD3, but not for snail1 (Guemar et al., 2007).
The function of this small GTPase, however, is believed to be in
the context of cell rearrangements rather than nuclear activity.
Disturbances in neural fold formation may lead to shifts between
neural plate and neural crest territories, thereby indirectly influencing
gene expression (Guemar et al., 2007). In contrast to RhoV,
overexpression of Xphb1 did not enlarge the CNC area and its
depletion did not alter the border between neural plate and epidermis
(Fig. 7). We conclude that Xphb1 is unable to change the cell fate
from neural or epidermal progenitors to CNC. Furthermore, we
could allocate Xphb1 a nuclear activity because deletion of its
binding site for the transcription factor E2F1 abolished its rescue
ability in morphants (Fig. 8). Neither deletion of the TM domain and
the Rb binding site, nor of the Raf-1 binding domain with the NES
motif, severely affected the rescue capacity, pointing to a pivotal role
DEVELOPMENT
8C). Injections of mRNAs encoding the deletion mutants
Xphb1⌬N-term and Xphb1⌬Raf-1, however, recovered twist
expression in 60% and 50% of injected embryos, respectively (Fig.
8C). These findings point to a crucial role of the E2F1 binding site
for Xphb1 function in CNC. This was confirmed when the rescue
ability of human prohibitin 1 deleted in the E2F1 binding site was
examined, as Hphb1⌬185-214 was unable to reconstitute twist
expression in Xphb1 morphants (Fig. 8C).
These results prompted us to investigate the functional
relationship between Xphb1 and E2F1. We injected a Gal4-DBDE2F1 fusion construct (Choi et al., 2008) into embryos and
measured the response of a pFR-luciferase trans-reporter plasmid
at different concentrations of co-injected Xphb1 mRNA. As shown
in Fig. 9A, the luciferase activity dropped by nearly 50%,
indicating a repressive effect of Xphb1 on E2F1.
We then asked whether expression of E2F1 influences twist
expression. Single-sided injections of human E2F1 DNA led to
downregulation of twist expression, which was restored when
Xphb1 mRNA was co-injected (Fig. 9B). This was also observed
for snail2 and foxD3 (data not shown). We also tested the influence
of E2F1 in Xpbh1-depleted embryos. When phb1 MO was injected
at low doses, an additive effect was observed in the presence of low
concentrations of E2F1 (Fig. 9C). These results indicate that Xphb1
serves to repress the transcription factor E2F1 in order to promote
twist, snail2 and foxD3 expression.
Xphb1 specifies neural crest by repressing E2F1
RESEARCH ARTICLE 4079
for the interaction with E2F1. Strikingly, E2F1 overexpression led to
a decrease in twist, snail2 and foxD3 expression, which was
recovered by overexpression of Xphb1. Depletion of Xphb1,
conversely, promoted a reduction in twist expression through E2F1.
These findings, and the decrease in Gal4-DBD-E2F1 activity in
presence of prohibitin in the pFR-luciferase reporter, suggest that
Xphb1 is required to repress E2F1 function in CNC development.
In Xenopus, an E2F ortholog is maternally expressed. E2F mRNA
specifically localizes during neurula stage in brain, neural tube and
neural crest (Suzuki and Hemmati-Brivanlou, 2000) and E2F seems
to be more widely used in regulating transcription than Xphb1. The
latter is supported by our findings that E2F1 also reduces c-myc,
snail1 and AP-2 expression and that this is not rescued by Xphb1
(data not shown). Expression of dominant-negative E2F constructs
results in disturbances of ventral and posterior cell fates (Suzuki and
Hemmati-Brivanlou, 2000) and in the inhibition of cell cycle
progression from mid-blastula stage onwards (Tanaka et al., 2003).
Based on our results from phospho-histone H3 immunostaining and
TUNEL assay, we can exclude the possibility that Xphb1, together
with E2F1, balances cell proliferation in CNC development (Fig. 3).
Furthermore, cell cycle control by prohibitins via binding E2F1 also
involves Rb (Wang et al., 1999a; Wang et al., 1999b). Our Xphb1
deletion mutants, however, point to an Rb-independent mechanism
because the mutant lacking the Rb binding site rescues the Xphb1
morphant. This is in line with a recent report which demonstrated
that neither overexpression nor depletion of Rb disturbs Xenopus
development (Cosgrove and Philpott, 2007). They argue that in
Xenopus, Rb is hyperphosphorylated and therefore inactive until
stage 30 (Cosgrove and Philpott, 2007).
In mammalian cells, multiple mechanisms of prohibitin-mediated
repression of E2F1 function have been reported, some of which are
Rb independent. Importantly, all of these mechanisms involve
chromatin remodeling. Prohibitin1 is able to recruit histone
deacetylase 1 (HDAC1) and the co-repressor N-CoR into the E2F1
transcription complex, thereby abolishing E2F1 function (Wang et
al., 2002a). Prohibitin1 has also been shown to recruit Bg-1/Brm,
which are part of the SWI/SNF chromatin remodeling complex
(Wang et al., 2002b). Choi et al. reported that prohibitin1 binds
RNF2 and recruits it to E2F1-response promoters (Choi et al., 2008).
RNF2 belongs to the polycomb family. The presence of RNF2 in
polycomb complexes correlates with histone H2A ubiquitylation
(Wang et al., 2004). Therefore, it seems most likely that Xphb1
promotes chromatin rearrangements that are essential for the
activation of those CNC specifier genes that contain E2F1-response
elements in their promoters. XBrg1, the catalytic subunit of the
SWI/SNF complex, is contributed maternally in Xenopus embryos,
but transcripts accumulate in the presumptive CNS, neural crest and
in the otic vesicle (Seo et al., 2005). Loss-of-function studies
revealed a requirement for XBrg1 in neurogenesis. Importantly,
single-sided antisense XBrg1 morpholino injections yield a lateral
DEVELOPMENT
Fig. 8. The E2F1-binding domain is required for CNC formation. (A)Model of prohibitin1 protein domains (according to the human protein
structure). Beneath are shown the various Xphb1 mutants (Xphb1DN-term, Xphb1DRaf-1, Xphb1DC-term) and a human PHB1 mutant that lacks the
E2F1 binding site (Hphb1D185-214). Deletions are indicated by the thin lines. TM, transmembrane domain; NES, nuclear export signal. (B)In western
blots, Xphb1 mutants were detected by anti-myc (9E10) antibody. Coomassie Brilliant Blue (CBB) staining was used to reveal equal loading. (C)Xphb1
mutants and Hphb1D185-214 co-injected with phb1 MO (*) and analyzed by twist ISH showed that both of the mutants that lack the E2F1 binding site
were unable to rescue twist expression. n, number of embryos analyzed. **, P<0.005; *, P<0.05. Error bars indicate standard error.
4080 RESEARCH ARTICLE
Development 137 (23)
Fig. 10. New model of the CNC regulatory gene network in
Xenopus. Based on the present results, Xphb1 was assigned to the
neural crest specifiers. The upstream neural plate border specifiers zic3,
pax3 and meis3 are not affected by Xphb1. Xphb1 acts downstream of
c-myc and upstream of foxD3, snail2 and twist. The c-myc targets
snail1 and AP-2 are regulated independently of Xphb1. Suppression of
the transcription factor E2F1 by Xphb1 is required for twist, snail2 and
foxD3 expression. Black arrows indicate experimental results of this
paper, gray arrows refer to previously published data.
broadening of the sox2 domain (Seo et al., 2005), which might also
affect the CNC domain. The homologs of RNF2 and HDAC1 in
Xenopus have not been cloned. However, two other members of the
HDAC complex, xSin3 and xRBD3, have been reported to coimmunoprecipitate with FoxN3, a protein required in craniofacial
and eye development (Schuff et al., 2007).
Taking our results together with those of previous studies, we
modified the regulatory gene network of CNC development by
adding Xphb1 to the group of CNC specifiers (Fig. 10). We further
demonstrate that Xphb1 separates c-myc target genes into
those that are prohibitin1/E2F-dependent and -independent. As
prohibitin1 is able to recruit a broad set of chromatin modifiers to
the E2F transcription complexes, future studies of epigenetic events
might help to elucidate the molecular mechanism of Xphb1 in
neural crest specification.
Acknowledgements
We thank Drs Chellapan, Kang and Rudel for providing prohibitin and E2F1
constructs; Dr Mayor for the Xk81A1 probe; and Drs Gradl, Koehler, Kashef
and Klymkowsky for comments to the manuscript. M.S. was financed by a
stipend of the Landesgraduiertenfoerderung of Baden-Württemberg.
Fig. 9. E2F1 transcriptional activity is regulated by Xphb1 and
affects neural crest formation. (A)Luciferase reporter assay
performed with Xenopus embryos injected with Gal4-DBD-E2F1, pFR
trans-reporter plasmid (control) and different concentrations of Xphb1
mRNA. The transcriptional activity of E2F1 decreased when Xphb1 was
overexpressed. (B)Injection (*) of human E2F1 DNA inhibited twist
expression in a concentration-dependent manner. This was rescued by
co-injection of Xphb1 mRNA. (C)Knockdown of Xphb1 by phb1 MO (3
ng) together with human E2F1 DNA injection (*) act synergistically in
repressing twist expression. n, number of embryos analyzed. **,
P<0.005; *, P<0.05. Error bars indicate standard error.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053405/-/DC1
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Xphb1 specifies neural crest by repressing E2F1