The F-box protein Cdc4/Fbxw7 is a novel regulator of neural crest development in Xenopus laevis
- Alexandra D Almeida†1,
- Helen M Wise†2,
- Christopher J Hindley1,
- Michael K Slevin3,
- Rebecca S Hartley4 and
- Anna Philpott1Email author
© Almeida et al; licensee BioMed Central Ltd. 2010
Received: 27 January 2009
Accepted: 4 January 2010
Published: 4 January 2010
The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this.
Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4α and xCdc4β. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail.
We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.
During the development of multi-cellular organisms, cells receive signals and must elicit the appropriate response. This involves changes in the level and activity of proteins, and targeted proteolysis represents a rapid and irreversible mechanism to block protein function. During regulated proteolysis, proteins are targeted for degradation by covalent attachment of the 76 amino acid protein ubiquitin, and the polyubiquitin chains assembled on the target protein serve as signals for degradation by the 26S proteasome. Transfer of ubiquitin onto target proteins is catalyzed by a hierarchical multi-enzyme cascade. An E1 (ubiquitin activating) enzyme forms a thioester linkage with the carboxyl terminus of ubiquitin, in an ATP-dependent process. Ubiquitin is then transferred to an E2 (ubiquitin conjugating) enzyme. E3 (ubiquitin ligase) enzymes recruit distinct substrates, allowing ubiquitin transfer, and confer specificity on the ubiquitin proteasome system.
RING (Really Interesting New Gene) E3s are the largest class of E3 ligases, and the human genome encodes approximately 400 proteins with a RING domain . Conserved cysteines and histidines coordinate two zinc ions in the RING domain, which is important for the recruitment and activation of E2 enzymes. Skp1-Cullin1-F-box (SCF) E3 ligases are a large class of modular RING E3 ligases that have the RING component Roc1 (also known as Rbx1 and Hrt1). Cullin1 forms a scaffold to recruit the E2 (via Roc1) and the F-box protein (via binding of the F-box to Skp1) [2, 3]. The F-box component of these E3 ligases is variable, and different F-box proteins recruit different substrates via carboxy-terminal domains, allowing SCF ligases to target a huge number of substrates .
Cdc4 (also known as Fbw7), one of the most extensively studied F-box proteins, was originally identified in Saccharomyces cerevisiae, where it was shown to degrade the cyclin-dependent kinase inhibitor Sic1 [3–8]. In mammals, there are three isoforms of Cdc4: alpha (α), beta (β) and gamma (γ). These are produced by alternative splicing of three unique 5' exons to ten common 3' exons, such that the resulting proteins differ only at their amino termini [9, 10]. In mammals, known Cdc4 substrates include c-Myc, c-Jun, Cyclin E, Notch intracellular domain, c-Myb, sterol regulatory element binding proteins (SREBPs) and steroid receptor coactivator-3 (SRC3) [9, 11–15]. Given these substrates, it is perhaps unsurprising that Cdc4 has been shown to be a haplo-insufficient tumor suppressor gene . This list of substrates also suggests that Cdc4 could regulate developmental events, and attempts to generate knock-out mice led to an embryonic lethal phenotype . We became interested in a role for Cdc4 during neural crest development in particular because several of its substrates have been implicated in the development of this tissue, for example, c-Myc and Notch intracellular domain [18, 19].
The neural crest is a unique population of cells, arising at the neural plate border in response to bone morphogenetic protein, Wnt and fibroblast growth factor signaling (for reviews, see [20, 21]). Neural crest cells are initially multipotent, but subsequently undergo an epithelial to mesenchymal transition and migrate throughout the embryo, where they give rise to a wide range of derivatives (for reviews, see [22, 23]). These include the neurons and glia of the peripheral nervous system, the autonomic nervous system, cartilage, bone, connective tissue, cardiac cells and melanocytes. The induction of the neural crest is often defined according to the expression of neural crest specifier genes, including the transcriptional repressors Snail2 and Snail[24, 25]. A number of proteins involved in neural crest development display dynamic expression patterns, and it is becoming apparent that several are targets of the ubiquitin proteasome system. For example, Snail2 is degraded by the F-box protein Partner of paired (Ppa) .
Here we describe identification of the Xenopus laevis homologues of Cdc4, which are highly conserved at the sequence level, and are also functionally equivalent in terms of their ability to degrade Cyclin E. Two isoforms of Xenopus Cdc4 (xCdc4α and xCdc4β) are found to be dynamically expressed throughout early Xenopus development, with particular enrichment in neural crest and neural crest-derived tissues. Inhibition of xCdc4 activity, using dominant negative F-box mutants, blocks neural crest development, without affecting cell division or cell survival, nor affecting development of the other tissues in which they are expressed. Thus, Cdc4 directly and specifically regulates neural crest formation, independent of a previously described ability to regulate the cell cycle.
X. laevis encodes two isoforms of Cdc4: xCdc4α and xCdc4β
The pseudotetraploid genome of X. laevis presents unique challenges to identifying genes reported in other model systems. In contrast, Xenopus tropicalis is possessed of a diploid genome, making it well suited for genetic manipulation and bioinformatic analysis. The existence of X. tropicalis and X. laevis in the same genus - therefore sharing a high level of evolutionary conservation between their respective genes - suggested to us a method of harnessing the sequenced genome of X. tropicalis to identify potential orthologs of human Cdc4 (hCdc4) present in X. laevis. BLAST of hCdc4α (GenBank accession number AY049984) against the X. tropicalis genome revealed a sequence on scaffold 60:1694203-1694241 with strong nucleotide complementarity to the first exon of hCdc4α. Further downstream (scaffold 60:1696322-1,729,489) were exons 2 to 11, whose sequence corresponded to the conserved regions found within hCdc4.
PCR primers targeting the identified xCdc4 sequence were used on oligo d(T) primed mRNA derived from stage 20 X. laevis embryos. We performed 5' and 3' rapid amplification of cDNA ends (RACE) to isolate a full length product. Although slightly truncated, the protein was most similar to the β isoform of hCdc4 and has been designated xCdc4β (Additional file 1). Analysis of the aligned regions demonstrates that xCdc4β was 98% identical at the amino acid level and 83% identical at the nucleotide level to hCdc4β. In comparison to other X. laevis F-box proteins, xCdc4β was 27% identical and 43% similar to β -TRCP (β -Transducin repeat containing protein; GenBank accession number M98268) , and 17% identical and 20% similar to Skp2 (GenBank accession number DQ228920) .
A second isoform of xCdc4 was similarly cloned from stage 7 X. laevis embryos (GenBank accession number DQ666345). Exon 1 was found on scaffold 60:1646293-1646793. Analysis demonstrated nearly perfect conservation at the amino acid level to the common set of exons, 2 to 11, that occur in hCdc4α and hCdc4β (data not shown). Additionally, the second identified xCdc4 contained an amino terminal exon most similar to the one present in human Cdc4α and is referred to hereafter as xCdc4α. Furthermore, exon 1 of xCdc4β is spliced to the same downstream exons, 2 to 11, used by xCdc4α. Alignment of their amino acid sequences showed strong conservation; differences in amino acid identity being conserved by use of residues with similar characteristics. Furthermore, both the isoform specific nuclear localization signal (amino acids 11 to 14) and common nuclear localization signal (amino acids 169 to 172) found in hCdc4α are present in xCdc4α, which is suggestive of a similar pattern of localization and regulation .
From the region of the gene corresponding to the second exon, xCdc4α and xCdc4β are 99.8% identical (1,623 out of 1,626 nucleotides identical). A single amino acid substitution was made (changing Gly649 to Asp) to the xCdc4α coding sequence used here, as this was conserved in all sequence orthologs. A X. laevis ortholog of hCdc4γ was not detected in either stage 7 or stage 20 embryos.
xCdc4α and xCdc4β proteins are expressed throughout development, including prominent expression in the early neural crest
xCdc4 degrades Cyclin E and xCdc4ΔFbox mutants act as dominant negatives
Mammalian Cdc4, and in particular Cdc4α, is known to target Cyclin E for ubiquitin-mediated proteolysis [35–38]. We wanted to determine whether xCdc4 could target Cyclin E for degradation in X. laevis embryos, both by overexpressing the xCdc4 protein and by knocking out its function. To block xCdc4 function, we initially tried to prevent translation of xCdc4 mRNAs by microinjection of antisense morpholino oligonucleotides. Although xCdc4-directed morpholinos specifically inhibited translation of synthetic mRNA encoding xCdc4α or xCdc4β, demonstrating their functionality, microinjection into embryos had only a small effect on xCdc4α protein levels and no detectable effect on xCdc4β levels, as detected by western blot (data not shown). Morpholinos are only effective when protein levels depend on new translation of mRNAs. Our developmental western blot (Figure 1A, B) indicated that both xCdc4α and xCdc4β are supplied in the egg as maternal stockpiles, and these would not be affected by this antisense strategy.
xCdc4 is a member of the F-box E3 ligase family, and as such, requires an intact F-box to interact with the rest of the SCF complex to target proteins for proteolysis. Deletion of the F-box allows substrate binding but prevents recruitment to the ubiquitination machinery, and this strategy has been successfully used many times to create a dominant negative construct (for example, [9, 39]), whose overexpression results in specific substrate hyper-stability. Therefore, we used an F-box deleted dominant negative form of xCdc4 to block the function of endogenous xCdc4 protein.
Cyclin E migrated as multiple bands in GFP-injected embryos (Figure 6A, lane 2), and these are likely to be phospho-forms of the protein . When co-injected with Cyclin E mRNA, xCdc4 expression resulted in a reduction in the abundance of predominantly the slower-migrating phospho-forms of Cyclin E protein (Figure 6A, lane 3). Densitometry analysis, normalizing to actin expression in the same samples, confirmed that phosphorylated forms of Cyclin E are degraded by xCdc4. This is in agreement with previous results, which demonstrated a requirement for Cyclin E phosphorylation to direct Cdc4-mediated degradation [9, 38, 42]. These results verify that xCdc4 is functionally orthologous to mammalian Cdc4 in its ability to degrade phospho-forms of Cyclin E.
To confirm that the F-box deletion mutant of xCdc4 (xCdc4ΔFbox) possessed dominant negative activity, its ability to inhibit Cyclin E degradation mediated by wild-type xCdc4 was assessed. Cyclin E mRNA was co-injected with xCdc4 along with one of the following: xCdc4ΔFbox, xSkp2ΔFbox, an F-box mutant of the related SCF E3 ligase Skp2, or GFP (Figure 6A, lanes 2 to 5). As expected, overexpression of either xSkp2ΔFbox or GFP had no effect on phospho-Cyclin E degradation by xCdc4. In contrast, degradation of Cyclin E by xCdc4 was inhibited by co-injection of xCdc4ΔFbox (lane 5), confirming that this mutant acts as a dominant negative form of xCdc4 in the embryos. When phospho-Cyclin E levels were normalized to the levels in embryos injected with Cyclin E and GFP, xCdc4 and GFP co-injection led to a 60% reduction in Cyclin E levels (Figure 6B). When xCdc4 was co-injected with xSkp2ΔFbox, a similar reduction in Cyclin E levels was observed (70%). However, coexpression of xCdc4ΔFbox with xCdc4 partially rescued the degradation of Cyclin E. On average, there was a 30% reduction in Cyclin E levels compared to Cyclin E- and GFP-injected embryos. In addition, we also found that xCdc4 targeted endogenous Cyclin E for degradation (data not shown), again predominantly reducing the slower-migrating phospho-form of the protein.
At these early developmental stages, Cyclin E is expressed throughout the ectoderm , so may act as a target for xCdc4. Since Cdc4 is known to regulate the stability of other proteins that regulate cell proliferation (for a review, see ), we investigated whether xCdc4 modulated cell cycle progression in the early embryo. As xCdc4α and xCdc4β share the same substrate binding regions, xCdc4βΔFbox (hereafter known as xCdc4ΔFbox) would be expected to inhibit the activity of both xCdc4 isoforms, and was used throughout in this study. Indeed, xCdc4αΔFbox gave similar results to xCdc4βΔFbox (data not shown).
xCdc4ΔFbox does not affect cell cycle in the embryonic ectoderm
xCdc4ΔFbox inhibits neural crest development
To quantify the reduction in expression of neural crest markers brought about by overexpression of xCdc4ΔFbox, the area of staining of Snail2, Snail and c-Myc on the injected side of each embryo was measured and expressed as a ratio of the area on the uninjected side. A ratio less than 1 indicated a reduction in staining on the injected side compared to the uninjected side. The mean ratio was then calculated for at least two independent experiments, and compared with the ratio for GFP-injected embryos (which had minimal effect).
Overexpression of xCdc4ΔFbox blocked neural crest development, as determined by expression of Snail2, Snail and c-Myc, resulting in an average 57% reduction in Snail2 staining on the injected side of the embryo, compared with embryos injected with GFP alone. Overexpression of xCdc4 had essentially no effect on Snail2 staining (n = 58 to 122). Similarly, overexpression of xCdc4ΔFbox reduced Snail and c-Myc staining on the injected side of the embryo by 80% and 67% respectively, when compared to embryos injected with GFP alone (n = 30 to 52 for both).
As our sections revealed rather broad expression of xCdc4 in ectodermal and mesodermal derivatives, we investigated whether xCdc4ΔFbox affected patterning and/or specification of other tissues. We saw that anterior-posterior patterning and mesoderm development of the embryos were unaffected by overexpression of either wild-type xCdc4 or xCdc4ΔFbox, as determined by Otx2, Engrailed2, Krox20, MyoD, Heavy chain myosin (HCM) and epidermal keratin expression (Additional files 2 and 3). Moreover, Sox2, a marker of neural plate neuronal precursors, and Neural beta-tubulin (NβT), a marker of neuronal differentiation, were also unaffected (Figure 8S; Additional file 3). This suggested that the effect of xCdc4ΔFbox overexpression on neural crest development was not dependent on either early patterning events or secondary effects resulting from induction of mesoderm, and was indeed specific to the neural crest. This effect is also specific to xCdc4ΔFbox; the Skp2ΔFbox mutant had no effect on Snail2 expression (Additional file 4).
We investigated whether xCdc4 activity was required for expression of other regulators of neural crest formation lying upstream of Snail2 and Snail (Figure 8). At stage 15, Pax3 (53%) and Msx1 (100%) showed significant reduction in the presence of xCdc4ΔFbox but not wild-type xCdc4, indicating that xCdc4 plays a broad role in controlling expression of regulators of neural crest. Interestingly, xCdc4ΔFbox inhibited neural crest expression of Opl in some embryos (62%), but did not have a significant effect on its placodal expression (Figure 8M). This is supported by the failure of xCdc4ΔFbox to affect the expression of the placodal marker Six1 (Additional file 5). Thus, xCdc4 does not play an essential role in specification of all the tissues in which it is expressed, but does have an essential role in formation of the neural crest.
We also noted that embryos that were expressing xCdc4ΔFbox show other developmental abnormalities, including oedema and a high frequency of reduced eyes (Figure 11A). These phenotypes, which we have not been characterized further, may be secondary to loss of neural crest or may result from a later requirement of xCdc4 activity in other tissues where it is expressed, for example, the eye field (Figure 1I). As injected mRNA is not thought to persist much beyond tailbud stages, these defects most likely result from a requirement for xCdc4 at these earlier stages.
We were surprised at the low frequency of TUNEL-positive cells detected in this assay, although embryos that had been wounded were used as a positive control and were highly TUNEL-positive (data not shown). Therefore, an alternative assay for apoptosis was also used. We took advantage of the fact that hBclXL can block apoptosis in X. laevis[19, 40]. We checked whether Snail2 expression in xCdc4ΔFbox injected embryos could be rescued by co-expression of hBclXL. xCdc4ΔFbox, xCdc4 or GFP (1 ng) were injected unilaterally into two-cell-stage embryos. In addition, 1 ng of hBclXL or GFP were co-injected as appropriate. Whole mount ISH for Snail2 was performed on stage 18 embryos, and Snail2 staining in xCdc4ΔFbox/hBclXL-injected embryos was compared to xCdc4ΔFbox/GFP-injected embryos (n = 57 to 82; Figure 12E-J). In these experiments, xCdc4ΔFbox/GFP-injected embryos displayed an average 32% reduction in Snail2 staining, compared to GFP-injected embryos (mean ± SEM ratios were 0.66 ± 0.05 and 0.97 ± 0.02, respectively). However, no rescue of Snail2 staining was seen when hBclXL was co-injected with xCdc4ΔFbox, with an average 41% reduction in Snail2 staining on the injected side compared to embryos injected with GFP/hBclXL. Thus, loss of neural crest after inhibition of xCdc4 activity is not due to tissue loss by apoptosis.
In summary, we have identified two homologues of Cdc4 in X. laevis that show dynamic expression in the early embryo, in particular in the developing neural crest and its derivatives. While unexpectedly having no effect on cell cycling, blocking xCdc4 function by overexpression of a dominant negative form of the protein resulted in inhibition of specification and formation of the neural crest. This had long-term consequences for the formation of neural crest derivatives such as melanocytes and cartilage. Thus, regulated and specific proteolysis by xCdc4 plays an essential function in early development distinct from its well-established role in regulating cell division.
This work has identified a novel role for the F-box protein xCdc4 in neural crest development in X. laevis. Cdc4 orthologs in vertebrates have attracted considerable interest due to the plethora of substrates they degrade, and the fact that Cdc4 is a haplo-insufficient tumor suppressor protein [10, 16]. However, the embryonic lethality of Cdc4 knock-out mice precluded a detailed analysis of the role of this protein in development .
X. laevis Cdc4 is likely to be orthologous to vertebrate Cdc4 for several reasons. Firstly, xCdc4 is highly conserved compared to human Cdc4, both in terms of sequence and apparent gene organization. Secondly, xCdc4α and xCdc4β are capable of degrading Cyclin E, a known substrate of mammalian Cdc4. This validates the use of X. laevis to study the developmental function of Cdc4. In contrast, recovered Cdc4-/- mouse embryos show profound vascular defects , precluding study in other developmental processes. Developing amphibian embryos do not require a functional vasculature until later embryonic stages, making this an excellent system for studying other functions of Cdc4.
In this work, we report the isolation of two xCdc4 isoforms, xCdc4α and xCdc4β. The genes encoding them are almost identical from the region corresponding to the second exon (1,622 out of 1,625 nucleotides identical). This strongly suggested that the structure of the Xenopus gene was conserved compared to mammals. In humans and mice, the Cdc4 locus encodes three isoforms of Cdc4; designated α, β and γ. These are produced by alternative splicing of three unique 5' exons to ten common 3' exons, yielding proteins that differ only at their amino termini. Although our data suggest that this gene structure is preserved, no xCdc4γ isoform was detected during this work. In addition, in silico analysis, using the Ensembl genome browser, failed to detect sequences corresponding to the γ specific exon.
To check if xCdc4 was functionally orthologous to vertebrate Cdc4, the ability of xCdc4 to degrade Cyclin E was examined. xCdc4 overexpression reduced the abundance of Cyclin E, compared to embryos co-injected with GFP, preferentially reducing the abundance of the slower migrating hyper-phosphorylated form of Cyclin E (Figure 6A). This is in agreement with previous findings that Cdc4 only interacts with phosphorylated Cyclin E [35–38]. xCdc4 also degrades Cyclin E in vivo, while xCdc4ΔFbox does not (Figure 6).
The spatio-temporal expression of xCdc4 was examined by immunoblotting for xCdc4 isoforms, and by whole mount ISH (Figure 1). Using antibodies that detected xCdc4, both isoforms were expressed at all stages tested. xCdc4α migrated at an apparent molecular weight of 100 kDa, despite having a predicted molecular weight of 79 kDa (Figure 1A). This is in keeping with a previous report that showed that human Cdc4α migrated with an apparent molecular weight of 110 kDa, although its predicted molecular weight was 80 kDa . In contrast, xCdc4β consistently migrated slightly faster than its predicted molecular weight of 62 kDa (Figure 1B).
Although expressed across the embryo, the neural crest and placodes were the first sites where xCdc4 transcripts accumulated most prominently (Figures 1, 2 and 4). Subsequently, transcripts were detected in the branchial arches and fin mesenchyme, which are both neural crest derived tissues, as well as in the somites and brain, and co-localized in many areas with other known markers of neural crest and placodes (Figures 1 and 2). In order to examine the function of xCdc4 during development, gain and loss of function experiments were performed. Translation-blocking morpholinos did not inhibit expression of endogenous xCdc4α and xCdc4β (data not shown). One possible explanation for this was the maternal expression of the proteins. Therefore, an F-box deletion mutant was used as an alternative way to inhibit the function of xCdc4. xCdc4ΔFbox acted as a dominant negative inhibitor of xCdc4's ability to degrade Cyclin E (Figure 6) because it retains its ability to bind to substrate but cannot recruit the substrate to the rest of the SCF complex. The specificity of F-box proteins resides in their substrate binding site, and F-box deletion mutants, which bind specifically to their substrate but not to the Skp1 component of the SCF E3 ligase complex, have been widely employed as reagents to block degradation of specific F-box targets. Confirming this, xSkp2ΔFbox did not inhibit xCdc4-mediated degradation of the protein. As xCdc4 is expressed strongly in the neural crest, we investigated the effect of overexpression of xCdc4ΔFbox on development of this tissue.
Inhibition of xCdc4, using xCdc4ΔFbox, inhibited neural crest development, as determined by Snail2 and Snail ISHs (Figures 8, 9 and 10). This inhibition of neural crest development occurred at an early stage, upstream of c-Myc, reducing expression of a number of proteins acting as regulators of Snail2, Snail and c-Myc (Figure 8). This indicates that xCdc4 is required for establishment of neural crest identity, rather than simply maintenance of that identity. Although expressed more broadly in ectodermal and mesodermal derivatives, we did not detect a requirement for xCdc4 function in specification or differentiation of other tissues, nor in patterning of the neural tube (Additional files 2, 3 and 5), strongly indicating a tissue-autonomous role in the neural crest.
The most well characterized role of Cdc4 is in regulating the cell cycle, and we investigated whether xCdc4 regulates cell cycling in the early X. laevis embryo. xCdc4ΔFbox overexpression did not perturb the cell cycle as determined by phH3 staining in these early embryos (Figure 7). The results from phH3 staining are consistent with previous observations that cell proliferation has a minor role in neural crest development; neural and neural crest induction has been reported to proceed normally when cell division was blocked from mid-gastrula stages onwards [19, 45].
xCdc4ΔFbox does not inhibit neural crest development through activation of an apoptotic program. The evidence for this is threefold: xCdc4ΔFbox overexpression did not lead to a loss of β-gal staining expressed from co-injected mRNA; there was no change in TUNEL staining (Figure 12A-D); and finally, co-injection of hBclXL did not affect the ability of xCdc4ΔFbox to block neural crest development (Figure 12E-J). X. laevis embryos can undergo apoptotic cell death from stage 10.5 onwards [46–48]. TUNEL analysis during embryonic development showed that only 50 to 60% of embryos had TUNEL-positive cells in the ectoderm prior to stage 18 . After this stage a higher percentage of embryos were TUNEL-positive, but individual embryos displayed less TUNEL staining. In terms of neural crest development, Sox10 and Id3 depletion have been reported to increase TUNEL staining in the neural crest, but this was coupled to a reduction in cell proliferation [50, 51]. However, it has been reported that inhibition of the cell cycle reduces clearance of apoptotic cells, meaning that cell cycle inhibition in these embryos may have led to increased TUNEL staining .
Previous studies have reported increased levels of apoptosis occurring in neural folds rather than other areas of the embryonic ectoderm [47, 49, 53], and a number of genes involved in neural crest development possess anti-apoptotic activity (for example, [53, 54]). It is possible that apoptosis regulates neural crest development in a stage-specific manner. For example, it may be important in defining the neural crest boundary, rather than for neural crest induction. However, as xCdc4 acts at an early stage of neural crest development, regulation of apoptosis may not be involved.
The identification of Cdc4 as a regulator of neural crest development adds to existing evidence that the ubiquitin proteasome system plays a role in the development of this tissue. Overexpression of dominant negative Cullin1, which is predicted to inhibit all SCF E3 ligases, expanded the neural crest in X. laevis. This was primarily due to stabilization of the Wnt pathway component β-catenin, although other substrates are likely to have been involved . Recently, the F-box protein Ppa was shown to regulate neural crest development by degradation of Snail2 in X. laevis. Overexpression of Ppa inhibited neural crest development . The related protein Snail is degraded by the F-box protein β-TRCP in tissue culture cells . Thus, a number of proteins involved in neural crest development display dynamic expression patterns, and regulation by ubiquitin-mediated proteolysis is emerging as a method to achieve this through activity of different E3 ligases that target distinct substrates.
What substrates is Cdc4 targeting to influence neural crest formation? c-Myc regulates neural crest development [19, 57] and can be degraded by Cdc4 [11, 29], but we saw that xCdc4ΔFbox decreased c-Myc mRNA (Figure 8), indicating that xCdc4 also acts upstream of c-Myc expression. Indeed, xCdc4 acts upstream of early regulators of neural crest, such as Pax3 and Msx1 (Figure 8). We hypothesized that xCdc4 was degrading a negative regulator of neural crest development. c-Jun is another known target of Cdc4  and is expressed at the right time and place to be regulating neural crest formation, although its exact role is poorly understood [58, 59]. We saw that overexpression of c-Jun did indeed inhibit Snail2 expression (Additional file 6). However, when we tested whether xCdc4 could target c-Jun protein for degradation in embryos by co-injection of mRNAs encoding these proteins, and performing immunoblotting against HA-tagged c-Jun, we saw no effect of xCdc4 on c-Jun levels (data not shown). From these results, it is not clear whether c-Jun is a target for Cdc4 in this developmental context, and in any case, an essential role for c-Jun in regulating neural crest formation in X. laevis has not been clearly demonstrated.
Identification of E3 ligase targets is challenging, and co-factors required for protein degradation may be active only in certain contexts. For example, it has been noted that Cdc4-mediated degradation of c-Jun in tissue culture cells required co-expression of Glycogen synthase kinase 3β (GSK3β) . It will be important to now identify the in vivo targets of Cdc4 that regulate formation of the neural crest if we are to have a fuller understanding of the role of selective protein degradation in the development of this tissue.
Here we identify xCdc4 as a novel regulator of neural crest development in X. laevis, acting early in neural crest formation, potentially by regulation of c-Jun. These results demonstrate that Cdc4's role as a tumor suppressor protein may extend beyond its ability to regulate the cell cycle to an ability to directly regulate tissue differentiation.
Materials and methods
Plasmids and constructs
Expression of constructs was verified by immunoblotting embryo lysates after microinjection of capped mRNA.
X. laevis Cdc4β (xCdc4β) was cloned by PCR from stage 20 cDNA. Primers used to obtain the full length coding sequence were: forward, 5'-ATGGGCTTCTACGGCAC-3'; reverse, 5'-CCTTCACTTCATGTCCACGTC-3'. xCdc4β was cloned into pCS2+repaired to synthesize capped mRNA for microinjection. An F-box deletion mutant of xCdc4β (xCdc4βΔFbox) was produced by PCR and triple ligation strategy. The F-box region of xCdc4β (nucleotides 379 to 516 inclusive) was replaced with an Ase I site (ATTAAT, amino acids Ile and Asn).
Primers 5'-GCAACCGAATTCACCACCATGGGCTTCTACGGCAC-3' and 5'-GGTTGCATTAATAAAGTCCCGCTGAAACTGG-3' were used to amplify xCdc4β upstream of the F-box, and 5'-GCAACCATTAATGAAGATGGGATCGATGAGC-3' and 5'-GGTTGCCTCGAGTCACTTCATGTCCACGTC-3' were used to amplify xCdc4β downstream of the F-box.
X. laevis Cdc4α (xCdc4α) was similarly cloned using cDNA derived from stage 7 embryos. Primers used to obtain the full length coding sequence were: forward, 5'-GCTGGCTTTTGGAAATGAATCAGG-3'; reverse, 5'-CTTCACTTCATGTCCACATCAAAGTCC-3'. xCdc4α was cloned into pGEM-T Easy (Promega, Madison, WI, USA) downstream of the SP6 promoter. The GenBank accession number is DQ666345. A single point mutation was introduced into this sequence (nucleotide G1946A in the coding sequence, resulting in G649D in the protein), to introduce an Asp residue that is conserved amongst vertebrates, to correct what was likely a cloning error; xCdc4α (G) was much less potent at inhibiting formation of neural crest compared to xCdc4α (D) (data not shown). XCdc4α (D) also degraded Cyclin E more efficiently than xCdc4α (G) (data not shown). Site directed mutagenesis was performed using a QuikChange® Multi Site Directed Mutagenesis kit (Stratagene, La Jolla, California) according to the manufacturer's instructions. xCdc4αΔFbox was produced by an identical method to that described for xCdc4βΔFbox. X. laevis Skp2ΔFbox has been described elsewhere .
X. laevis c-Jun in pCS2+ (GenBank accession number AJ243954) was a kind gift of Professor Walter Knochel (Institute of Biochemistry, University of Ulm, Germany).
Anti-Cdc4 monoclonal antibodies (3B7 and 3A9) were a kind gift from Dr Axel Behrens (CRUK). Anti-FLAG M2 conjugated to horse radish peroxidase (A8592; used at 1:1,000), anti-tubulin B512 (T5168; used at 1:2,000) and anti-rabbit IgG conjugated to alkaline phosphatase (A9919; used at 1:1,000) were from Sigma (St Louis, MO, USA). Horse radish peroxidase conjugated anti-rabbit and mouse IgG (NA943 and NA931; used at 1:5,000) were from Amersham (GE Healthcare, Uppsala, Sweden), rabbit anti-phH3 (06570; used at 1:1,000) was from Upstate (Millipore, Billerica, MA, USA) and alkaline phosphatase conjugated anti-digoxigenin fab fragments (11093274910; used at 1:5,000) were from Roche (Basel, Switzerland).
Xenopus laevis embryo manipulation
X. laevis embryos were obtained by hormone-induced egg laying and in vitro fertilization by standard methods. Unilateral injections into the animal pole of two-cell-stage embryos (unless otherwise indicated) were carried out with in vitro transcribed capped mRNA (Ambion, Austin, TX, USA). mRNA was injected at doses of up to 2 ng in a volume of 10 nl, and 0.5 ng of β-gal mRNA was co-injected as a lineage tracer. GFP mRNA was injected as a control. Embryos were staged according to  and grown to the required stage. They were fixed in MEMFA (4% formaldehyde, 100 mM MOPS, 2 mM EGTA, 1 mM MgSO4, pH 7.4), washed in phosphate-buffered saline (PBS)/2 mM MgCl2, and stained with 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) in X-gal mixer (5.35 mM K3Fe(CN)6, 5.35 mM K4Fe(CN)6, 1.2 mM MgCl2, 0.1% sodium deoxycholate, 0.2% NP-40 in PBS). Alternatively, embryos were stained with 1.7 mg/ml Red-gal® (6-chloro-3-indolyl-beta-D-galactopyranoside) in X-gal mixer. Embryos were washed in PBS, dehydrated in methanol and stored at -20°C.
In situ hybridization
Whole mount ISH was performed using a BioLane™ HTI in situ robot (Holle and Huttner (Tubingen, Germany). The washes and composition of solutions were as described in , with some modifications in the protocol. The RNase step was omitted, and embryos were blocked with 2% Blocking Reagent (Roche) and 20% heat inactivated lamb serum in maleic acid buffer. Incubation with 1:5,000 anti-digoxigenin was performed in the same solution. The color reaction was terminated using PBS washes and embryos were re-fixed in MEMFA. Embryos were bleached as described in . xCdc4 in pBSK+ was linearized with Pst I and transcribed T3 for antisense, and linearized with Not I and transcribed T7 for sense. The following probes were used: c-Myc, MsxI, Opl/Zic1, Pax3, Snail, Six1, Sox2, Sox10 and Snail2.
To quantify the area of Snail2 expression, Openlab™ software (Improvision/Perkin Elmer, Waltham, MA, USA) was used to select the area of Snail2 staining on the injected (I) and uninjected (U) side, using all injected embryos unless they were damaged. For each experiment the average ratio of I/U was determined for each injection to determine the change in Snail2 staining on the injected side, a ratio of <1 indicating Snail2 reduction. For at least two experiments, a mean ratio ± standard error of the mean (SEM) for each injection was calculated by taking the mean of the average ratios, and a Student's t-test performed. The ratios were compared to GFP-injected control embryos.
ISH on sectioned embryos was performed as described in . Stage 15 to 22 embryos were fixed in MEMFA, embedded in a paraffin and beeswax solution and sectioned using a Leica microtome. The ISH was performed on 14-μm sections using xCdc4 and Snail2 probes. The slides were mounted using Aquamount (BDH-Merck, VWR International, West Chester, PA, USA).
Whole mount antibody staining
Whole mount antibody staining, using anti-phH3, was performed as described in . The chromogenic reaction was developed as described, using 0.45 mg/ml Nitroblue tetrazolium chloride (NBT; Roche) and 0.2 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche) in alkaline phosphatase buffer.
TUNEL staining to detect apoptotic cells
Embryos were devitellinized, fixed in MEMFA, stained for β-gal and bleached. They were washed in 1× Terminal Deoxynucleotidyl Transferase (TdT) buffer (Invitrogen, Paisley, UK); 100 mM potassium cacodylate pH 7.2, 2 mM CoCl2 and 0.2 mM dithiothreitol) and incubated overnight at room temperature in TdT buffer with 0.5 μM alkaline stable digoxigenin-11-dUTP (Roche) and 150 U/ml recombinant TdT (Invitrogen). The TdT reaction was stopped by washing embryos at 65°C with PBST (phosphate-buffered saline, 0,1% Tween 20)/1 mM EDTA. Overnight incubation of the embryos in PBST/20% heat inactivated goat serum with anti-digoxigenin was performed at 4°C. Embryos were washed with PBS, and staining was developed using NBT/BCIP.
Analysis of melanocyte distribution
Embryos were injected with 0.5 ng of xCdc4ΔFbox, xCdc4 or control GFP mRNA into each of the two animal dorsal cells of four-cell-stage embryos. At stage 43, the embryos were fixed in MEMFA and dehydrated overnight in ethanol. For the examination of melanocytes, individual embryos were photographed, the anterior region selected and the number of melanocytes counted using Image J software. For each condition, the number of melanocytes was averaged, the mean ± SEM was calculated and a Student's t-test performed.
Embryos were lysed in 100 mM NaCl, 5 mM EDTA, 0.1% Triton X-100 and 50 mM β-glycerophosphate. Cleared supernatants were mixed with an equal volume of 2× SDS gel loading buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol and 0.2% bromophenol blue), and dithiothreitol was added to a final concentration of 100 mM. One embryo equivalent per lane was loaded. In order to quantify protein levels, blots were imaged by using infrared fluorescence of appropriately tagged secondary antibodies and quantified by using a LiCOR Biosciences (Lincoln, Nebraska, USA) Odyssey scanner and software.
green fluorescent protein
in situ hybridization
phosphorylated histone H3
Partner of paired
Really Interesting New Gene
standard error of the mean
Terminal Deoxynucleotidyl Transferase
terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling.
The authors gratefully acknowledge Dr Axel Behrens (CRUK) for supplying the anti-Cdc4 antibodies, and Therese Mitchell, Alison Jones and Dr Ian Horan for technical assistance. HMW was funded by a Wellcome Trust studentship. ADA was funded by a Fundacao Ciencia e Tecnologia studentship and CJH by a CRUK studentship. Work in AP's lab is funded by MRC Research Grants G0500101 and G0700758. Work in RSH's lab is funded by a grant from the National Cancer Institute-National Institutes of Health (R01CA095898).
- Semple CA: The comparative proteomics of ubiquitination in mouse. Genome Res. 2003, 13: 1389-1394. 10.1101/gr.980303.PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M: Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 2002, 416: 703-709. 10.1038/416703a.View ArticlePubMedGoogle Scholar
- Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW: F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell. 1997, 91: 209-219. 10.1016/S0092-8674(00)80403-1.View ArticlePubMedGoogle Scholar
- Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ: SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996, 86: 263-274. 10.1016/S0092-8674(00)80098-7.View ArticlePubMedGoogle Scholar
- Hartwell LH, Mortimer RK, Culotti J, Culotti M: Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973, 74: 267-286.PubMed CentralPubMedGoogle Scholar
- Verma R, Feldman RM, Deshaies RJ: SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. Mol Biol Cell. 1997, 8: 1427-1437.PubMed CentralView ArticlePubMedGoogle Scholar
- Verma R, Annan RS, Huddleston MJ, Carr SA, Reynard G, Deshaies RJ: Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science. 1997, 278: 455-460. 10.1126/science.278.5337.455.View ArticlePubMedGoogle Scholar
- Feldman RM, Correll CC, Kaplan KB, Deshaies RJ: A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell. 1997, 91: 221-230. 10.1016/S0092-8674(00)80404-3.View ArticlePubMedGoogle Scholar
- Strohmaier H, Spruck CH, Kaiser P, Won KA, Sangfelt O, Reed SI: Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature. 2001, 413: 316-322. 10.1038/35095076.View ArticlePubMedGoogle Scholar
- Welcker M, Clurman BE: FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer. 2008, 8: 83-93. 10.1038/nrc2290.View ArticlePubMedGoogle Scholar
- Yada M, Hatakeyama S, Kamura T, Nishiyama M, Tsunematsu R, Imaki H, Ishida N, Okumura F, Nakayama K, Nakayama KI: Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7. Embo J. 2004, 23: 2116-2125. 10.1038/sj.emboj.7600217.PubMed CentralView ArticlePubMedGoogle Scholar
- Nateri AS, Riera-Sans L, Da Costa C, Behrens A: The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling. Science. 2004, 303: 1374-1378. 10.1126/science.1092880.View ArticlePubMedGoogle Scholar
- Moberg KH, Bell DW, Wahrer DC, Haber DA, Hariharan IK: Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature. 2001, 413: 311-316. 10.1038/35095068.View ArticlePubMedGoogle Scholar
- Oberg C, Li J, Pauley A, Wolf E, Gurney M, Lendahl U: The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem. 2001, 276: 35847-35853. 10.1074/jbc.M103992200.View ArticlePubMedGoogle Scholar
- Kitagawa K, Hiramatsu Y, Uchida C, Isobe T, Hattori T, Oda T, Shibata K, Nakamura S, Kikuchi A, Kitagawa M: Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb. Oncogene. 2009, 28: 2393-2405. 10.1038/onc.2009.111.View ArticlePubMedGoogle Scholar
- Mao JH, Perez-Losada J, Wu D, Delrosario R, Tsunematsu R, Nakayama KI, Brown K, Bryson S, Balmain A: Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene. Nature. 2004, 432: 775-779. 10.1038/nature03155.View ArticlePubMedGoogle Scholar
- Tsunematsu R, Nakayama K, Oike Y, Nishiyama M, Ishida N, Hatakeyama S, Bessho Y, Kageyama R, Suda T, Nakayama KI: Mouse Fbw7/Sel-10/Cdc4 is required for notch degradation during vascular development. J Biol Chem. 2004, 279: 9417-9423. 10.1074/jbc.M312337200.View ArticlePubMedGoogle Scholar
- Cornell RA, Eisen JS: Notch in the pathway: the roles of Notch signaling in neural crest development. Semin Cell Dev Biol. 2005, 16: 663-672. 10.1016/j.semcdb.2005.06.009.View ArticlePubMedGoogle Scholar
- Bellmeyer A, Krase J, Lindgren J, LaBonne C: The protooncogene c-myc is an essential regulator of neural crest formation in xenopus. Dev Cell. 2003, 4: 827-839. 10.1016/S1534-5807(03)00160-6.View ArticlePubMedGoogle Scholar
- Meulemans D, Bronner-Fraser M: Gene-regulatory interactions in neural crest evolution and development. Dev Cell. 2004, 7: 291-299. 10.1016/j.devcel.2004.08.007.View ArticlePubMedGoogle Scholar
- Steventon B, Carmona-Fontaine C, Mayor R: Genetic network during neural crest induction: from cell specification to cell survival. Semin Cell Dev Biol. 2005, 16: 647-654. 10.1016/j.semcdb.2005.06.001.View ArticlePubMedGoogle Scholar
- Le Douarin NM, Creuzet S, Couly G, Dupin E: Neural crest cell plasticity and its limits. Development. 2004, 131: 4637-4650. 10.1242/dev.01350.View ArticlePubMedGoogle Scholar
- Huang X, Saint-Jeannet JP: Induction of the neural crest and the opportunities of life on the edge. Dev Biol. 2004, 275: 1-11. 10.1016/j.ydbio.2004.07.033.View ArticlePubMedGoogle Scholar
- Aybar MJ, Nieto MA, Mayor R: Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. Development. 2003, 130: 483-494. 10.1242/dev.00238.View ArticlePubMedGoogle Scholar
- Mayor R, Morgan R, Sargent MG: Induction of the prospective neural crest of Xenopus. Development. 1995, 121: 767-777.PubMedGoogle Scholar
- Vernon AE, LaBonne C: Slug stability is dynamically regulated during neural crest development by the F-box protein Ppa. Development. 2006, 133: 3359-3370. 10.1242/dev.02504.View ArticlePubMedGoogle Scholar
- Spevak W, Keiper BD, Stratowa C, Castanon MJ: Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats. Mol Cell Biol. 1993, 13: 4953-4966.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin HR, Chuang LC, Boix-Perales H, Philpott A, Yew PR: Ubiquitination of cyclin-dependent kinase inhibitor, Xic1, is mediated by the Xenopus F-box protein xSkp2. Cell Cycle. 2006, 5: 304-314.View ArticlePubMedGoogle Scholar
- Welcker M, Orian A, Jin J, Grim JE, Harper JW, Eisenman RN, Clurman BE: The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc Natl Acad Sci USA. 2004, 101: 9085-9090. 10.1073/pnas.0402770101.PubMed CentralView ArticlePubMedGoogle Scholar
- Hong CS, Saint-Jeannet JP: The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border. Mol Biol Cell. 2007, 18: 2192-2202. 10.1091/mbc.E06-11-1047.PubMed CentralView ArticlePubMedGoogle Scholar
- Aoki Y, Saint-Germain N, Gyda M, Magner-Fink E, Lee YH, Credidio C, Saint-Jeannet JP: Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. Dev Biol. 2003, 259: 19-33. 10.1016/S0012-1606(03)00161-1.View ArticlePubMedGoogle Scholar
- Nakata K, Nagai T, Aruga J, Mikoshiba K: Xenopus Zic family and its role in neural and neural crest development. Mech Dev. 1998, 75: 43-51. 10.1016/S0925-4773(98)00073-2.View ArticlePubMedGoogle Scholar
- Schlosser G, Ahrens K: Molecular anatomy of placode development in Xenopus laevis. Dev Biol. 2004, 271: 439-466. 10.1016/j.ydbio.2004.04.013.View ArticlePubMedGoogle Scholar
- Pandur PD, Moody SA: Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines. Mech Dev. 2000, 96: 253-257. 10.1016/S0925-4773(00)00396-8.View ArticlePubMedGoogle Scholar
- Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW, Elledge SJ: Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science. 2001, 294: 173-177. 10.1126/science.1065203.View ArticlePubMedGoogle Scholar
- Welcker M, Singer J, Loeb KR, Grim J, Bloecher A, Gurien-West M, Clurman BE, Roberts JM: Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation. Mol Cell. 2003, 12: 381-392. 10.1016/S1097-2765(03)00287-9.View ArticlePubMedGoogle Scholar
- Ye X, Nalepa G, Welcker M, Kessler BM, Spooner E, Qin J, Elledge SJ, Clurman BE, Harper JW: Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase. J Biol Chem. 2004, 279: 50110-50119. 10.1074/jbc.M409226200.View ArticlePubMedGoogle Scholar
- Hao B, Oehlmann S, Sowa ME, Harper JW, Pavletich NP: Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol Cell. 2007, 26: 131-143. 10.1016/j.molcel.2007.02.022.View ArticlePubMedGoogle Scholar
- Boix-Perales H, Horan I, Wise H, Lin HR, Chuang LC, Yew PR, Philpott A: The E3 ubiquitin ligase Skp2 regulates neural differentiation independent from the cell cycle. Neural Develop. 2007, 2: 27-10.1186/1749-8104-2-27.PubMed CentralView ArticleGoogle Scholar
- Richard-Parpaillon L, Cosgrove RA, Devine C, Vernon AE, Philpott A: G1/S phase cyclin-dependent kinase overexpression perturbs early development and delays tissue-specific differentiation in Xenopus. Development. 2004, 131: 2577-2586. 10.1242/dev.01121.View ArticlePubMedGoogle Scholar
- Rempel RE, Sleight SB, Maller JL: Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase. J Biol Chem. 1995, 270: 6843-6855. 10.1074/jbc.270.28.16918.View ArticlePubMedGoogle Scholar
- Sangfelt O, Cepeda D, Malyukova A, van Drogen F, Reed SI: Both SCF(Cdc4alpha) and SCF(Cdc4gamma) are required for cyclin E turnover in cell lines that do not overexpress cyclin E. Cell Cycle. 2008, 7: 1075-1082.View ArticlePubMedGoogle Scholar
- Vernon AE, Philpott A: The developmental expression of cell cycle regulators in Xenopus laevis. Gene Expr Patterns. 2003, 3: 179-192. 10.1016/S1567-133X(03)00006-1.View ArticlePubMedGoogle Scholar
- Saka Y, Smith JC: Spatial and temporal patterns of cell division during early Xenopus embryogenesis. Dev Biol. 2001, 229: 307-318. 10.1006/dbio.2000.0101.View ArticlePubMedGoogle Scholar
- Harris WA, Hartenstein V: Neuronal determination without cell division in Xenopus embryos. Neuron. 1991, 6: 499-515. 10.1016/0896-6273(91)90053-3.View ArticlePubMedGoogle Scholar
- Anderson JA, Lewellyn AL, Maller JL: Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus. Mol Biol Cell. 1997, 8: 1195-1206.PubMed CentralView ArticlePubMedGoogle Scholar
- Hensey C, Gautier J: A developmental timer that regulates apoptosis at the onset of gastrulation. Mech Dev. 1997, 69: 183-195. 10.1016/S0925-4773(97)00191-3.View ArticlePubMedGoogle Scholar
- Stack JH, Newport JW: Developmentally regulated activation of apoptosis early in Xenopus gastrulation results in cyclin A degradation during interphase of the cell cycle. Development. 1997, 124: 3185-3195.PubMedGoogle Scholar
- Hensey C, Gautier J: Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev Biol. 1998, 203: 36-48. 10.1006/dbio.1998.9028.View ArticlePubMedGoogle Scholar
- Honore SM, Aybar MJ, Mayor R: Sox10 is required for the early development of the prospective neural crest in Xenopus embryos. Dev Biol. 2003, 260: 79-96. 10.1016/S0012-1606(03)00247-1.View ArticlePubMedGoogle Scholar
- Kee Y, Bronner-Fraser M: To proliferate or to die: role of Id3 in cell cycle progression and survival of neural crest progenitors. Genes Dev. 2005, 19: 744-755. 10.1101/gad.1257405.PubMed CentralView ArticlePubMedGoogle Scholar
- Yeo W, Gautier J: A role for programmed cell death during early neurogenesis in xenopus. Dev Biol. 2003, 260: 31-45. 10.1016/S0012-1606(03)00222-7.View ArticlePubMedGoogle Scholar
- Tribulo C, Aybar MJ, Sanchez SS, Mayor R: A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest. Dev Biol. 2004, 275: 325-342. 10.1016/j.ydbio.2004.07.041.View ArticlePubMedGoogle Scholar
- Vega S, Morales AV, Ocana OH, Valdes F, Fabregat I, Nieto MA: Snail blocks the cell cycle and confers resistance to cell death. Genes Dev. 2004, 18: 1131-1143. 10.1101/gad.294104.PubMed CentralView ArticlePubMedGoogle Scholar
- Voigt J, Papalopulu N: A dominant-negative form of the E3 ubiquitin ligase Cullin-1 disrupts the correct allocation of cell fate in the neural crest lineage. Development. 2006, 133: 559-568. 10.1242/dev.02201.View ArticlePubMedGoogle Scholar
- Yook JI, Li XY, Ota I, Fearon ER, Weiss SJ: Wnt-dependent regulation of the E-cadherin repressor snail. J Biol Chem. 2005, 280: 11740-11748. 10.1074/jbc.M413878200.View ArticlePubMedGoogle Scholar
- Barembaum M, Bronner-Fraser M: Early steps in neural crest specification. Semin Cell Dev Biol. 2005, 16: 642-646. 10.1016/j.semcdb.2005.06.006.View ArticlePubMedGoogle Scholar
- Knochel S, Schuler-Metz A, Knochel W: c-Jun (AP-1) activates BMP-4 transcription in Xenopus embryos. Mech Dev. 2000, 98: 29-36. 10.1016/S0925-4773(00)00448-2.View ArticlePubMedGoogle Scholar
- Peng Y, Xu RH, Mei JM, Li XP, Yan D, Kung HF, Phang JM: Neural inhibition by c-Jun as a synergizing factor in bone morphogenetic protein 4 signaling. Neuroscience. 2002, 109: 657-664. 10.1016/S0306-4522(01)00526-7.View ArticlePubMedGoogle Scholar
- Wei W, Jin J, Schlisio S, Harper JW, Kaelin WG: The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase. Cancer Cell. 2005, 8: 25-33. 10.1016/j.ccr.2005.06.005.View ArticlePubMedGoogle Scholar
- Nieuwkoop PD, Faber J: Normal table of Xenopus laevis. 1994, New York: Garland PublishingGoogle Scholar
- Sive HL, Grainger RL, Harland RM: Early Development of Xenopus laevis. A Laboratory Manual. 2000, Cold Spring Harbour Laboratory PressGoogle Scholar
- Butler K, Zorn AM, Gurdon JB: Nonradioactive in situ hybridization to xenopus tissue sections. Methods. 2001, 23: 303-312. 10.1006/meth.2000.1142.View ArticlePubMedGoogle Scholar
- Zhang W, Koepp DM: Fbw7 isoform interaction contributes to cyclin E proteolysis. Mol Cancer Res. 2006, 4: 935-943. 10.1158/1541-7786.MCR-06-0253.View ArticlePubMedGoogle Scholar
- Welcker M, Clurman BE: Fbw7/hCDC4 dimerization regulates its substrate interactions. Cell Div. 2007, 2: 7-10.1186/1747-1028-2-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang X, Orlicky S, Lin Z, Willems A, Neculai D, Ceccarelli D, Mercurio F, Shilton BH, Sicheri F, Tyers M: Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. Cell. 2007, 129: 1165-1176. 10.1016/j.cell.2007.04.042.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.