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First published online 5 January 2006
doi: 10.1242/dev.02201

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A dominant-negative form of the E3 ubiquitin ligase Cullin-1 disrupts the correct allocation of cell fate in the neural crest lineage

The Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge, CB2 1QR, UK and Department of Anatomy, Downing Site, University of Cambridge, Cambridge, CB2 3DY, UK.

^* Author for correspondence (e-mail: np209{at}mole.bio.cam.ac.uk)

Accepted 7 November 2005

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Selective protein degradation is an efficient and rapid way of terminating protein activity. Defects in protein degradation are associated with a number of human diseases, including potentially DiGeorge syndrome, which is characterised by abnormal development of the neural crest lineage during embryogenesis. We describe the identification of Xenopus Cullin-1, an E3 ubiquitin ligase, and show that blocking the function of endogenous Cullin-1 leads to pleiotropic defects in development. Notably, there is an increased allocation of cells to a neural crest fate and within this lineage, an increase in melanocytes at the expense of cranial ganglia neurons. Most of the observed effects can be attributed to stabilisation of ß-catenin, a known target of Cullin-1-mediated degradation from other systems. Indeed, we show that blocking the function of Cullin-1 leads to a decrease in ubiquitinated ß-catenin and an increase in total ß-catenin. Our results show that Cullin-1-mediated protein degradation plays an essential role in the correct allocation of neural crest fates during embryogenesis.

Key words: Cullin-1, SCF, Neural crest, ß-catenin, Ubiquitin, Protein degradation, Xenopus

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During embryonic development the molecular profile of a cell has to be regulated in order to achieve cell specialisation. A large proportion of this regulation is carried out at the transcriptional level. However, it is becoming increasingly clear that proteins have to be actively removed from a cell if their function is no longer required. This removal is achieved by protein degradation via the 26S proteasome, which can degrade proteins down to peptides.

As not all proteins have to be degraded, an active selection mechanism is in place that marks the degradation targets by adding multiple ubiquitin molecules. This ubiquitination is catalysed by an enzyme cascade involving the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), the ubiquitin ligase (E3) (reviewed by Deshaies, 1999) and the still controversial E4, which is thought to facilitate the transition from monoubiquitination to polyubiqutination (Koegl et al., 1999).

The E3 ligases, which bind to a substrate and attach the ubiquitin molecule, are believed to provide substrate specificity. Structurally, E3 ligases can be subdivided into Ring-domain or HECT-domain based ligases. Both are protein motifs that enable the binding of the E2-conjugating enzyme, the protein that provides the ubiquitin molecules. Some E3 ligases, e.g. Smurf, Neuralized, Mind Bomb or Greul are single proteins. They usually contain the substrate-binding domain in the N terminus and the Ring or HECT domain in the C terminus. However, the majority of the E3 ligases consist of a protein complex where the tasks of substrate- and E2 binding are subdivided between separate proteins (Deshaies, 1999).

A well-known group of multi-subunit E3 ligases are the Cullin-Ring ubiquitin ligases (CLRs), which are based around a Cullin core. The function of Cullin proteins is to bind other proteins with its N and C termini; hence, acting as a scaffold for the CLR complex. The archetypal CLR is the Cullin-1-based SCF (Skp1, Cullin-1, a Ring protein and an F-box protein) complex. The N terminus of Cullin-1 binds to a linking protein such as Skp1 and via this to an F-box protein that binds the substrate. It is believed that the substrate specificity of SCF stems from the F-box protein; examples of F-box proteins are cyclin F, cdc4, ß-TrCP and Skp2 (Bai et al., 1996; Latres et al., 1999; Lisztwan et al., 1998; Skowyra et al., 1997; Yu et al., 1998). These are involved in the ubiquitination of cell cycle regulators such as p27^Kip1, p21 and cyclin E (Nakayama et al., 2000; Wang et al., 1999; Yu et al., 1998), and transcription factors such as ß-catenin, Gli, IB and E2F (del Pozo et al., 2002; Ou et al., 2002; Jiang and Struhl, 1998; Yaron et al., 1998). The C terminus of Cullin protein binds to a Ring protein, e.g. Rbx-1, which in turn binds the E2 enzyme. There are at least seven known Cullin proteins in humans, which can assemble into different CLRs.

Many E3 ligases have been found to be involved in developmental processes. Among the single protein E3 ligases, GREUL1 (Borchers et al., 2002) anteriorises ectoderm when overexpressed in Xenopus, while Smurf degrades Smad1 in Xenopus (Zhu et al., 1999) and Drosophila (Podos et al., 2001), thereby limiting BMP signalling. Recently, ectodermin, a Smad4 ubiquiting ligase has been shown to limit TGF-ß signalling in early Xenopus embryos and in human adult cells (Dupont et al., 2005). Interestingly, not all ubiquitination events necessarily result in degradation. For example Neuralized and Mind Bomb are E3 ligases that ubiquitinate the Notch ligand Delta, which subsequently leads to its internalisation and enhanced Notch signalling (Chen and Casey Corliss, 2004; Deblandre et al., 2001; Itoh et al., 2003; Le Borgne and Schweisguth, 2003). Among the multi-protein E3 ligases, Ozz promotes ß-catenin degradation in myofibres (Nastasi et al., 2004), while APC/C (an E3 ligase complex whose subunit APC2 has Cullin protein homology), controls synapse size at the neuromuscular junction in Drosophila (van Roessel et al., 2004).

Although the structure of the SCF (the archetypal multiprotein CLR) is well understood (Zheng et al., 2002) and many of its targets have been identified, large gaps remain in our understanding of its function (Deshaies, 1999), particularly with regards to its role in a developmental context. Knockout studies of its core component Cullin-1, which should lead to the abolishment of all canonical SCF activity, have been carried out in C. elegans, mouse and Arabidopsis (Dealy et al., 1999; Kipreos et al., 1996; Shen et al., 2002; Wang et al., 1999). The resulting phenotypes were hyperplasia with a shortened G1 phase in C. elegans, and arrested embryogenesis in all cases. Similarly, loss of function of the Ring protein Roc1a, which provides the link between Cullin-1 and the E2-conjugating enzyme, leads to embryonic lethality in Drosophila (Noureddine et al., 2002). In parallel, a role of the SCF complex in the Drosophila eye development is well documented (reviewed by Ou et al., 2003). Finally, RNAi against Skp1 (the linking protein between Cullin proteins and the F-box protein) in C. elegans causes tissue hyperplasia and embryonic arrest (Nayak et al., 2002). Loss-of-function studies have also been carried out on the F-box components of the SCF. Mice with a mutation for cyclin F die at midgestation (Tetzlaff et al., 2004). In Skp2-deficient mice there was an increased accumulation of Cyclin E and p27 in the mutant cells but the mice were viable although slightly smaller (Nakayama et al., 2000). Finally, hypomorphic loss of function of the F-box protein Slimb (Drosophila ß-TrCP) in a tissue-restricted manner leads to an accumulation of Ci (Gli) and Arm (ß-catenin), which in turn results in supernumerary limbs (Jiang and Struhl, 1998).

Some evidence for a role of the SCF in early vertebrate development comes from studies on the F-box protein ß-TrCP (Latres et al., 1999). Overexpression of a dominant-negative ß-TrCP in early Xenopus embryos caused secondary axes (Lagna et al., 1999; Marikawa and Elinson, 1998). This is consistent with the proposed role of ß-TrCP in ß-catenin degradation, which is a well known mode of regulating ß-catenin during the establishment of the axis in Xenopus and mouse (reviewed by Huelsken and Birchmeier, 2001; Weaver and Kimelman, 2004). Although these studies indicate the importance of the SCF during very early vertebrate embryonic development, its role in later developmental events remains to be characterised.

This characterisation is especially important as it has been recognised that protein degradation deficiencies underlie many human diseases (reviewed by Sakamoto, 2002). A number of cancers appear to be caused by the deregulation of protein degradation. For example, cervical cancer is thought to be due to the excessive destruction of the tumour suppressor p53 (reviewed by Sakamoto, 2002) and in breast cancer cells high levels of cyclin E coincided with a mutation in the F box protein Cdc4 (Rajagopalan et al., 2004; Strohmaier et al., 2001). In addition, neurodegenerative diseases such as Parkinson's disease (Staropoli et al., 2003) and Angelman syndrome (Kishino et al., 1997; Matsuura et al., 1997) also appear to be the consequence of mutations in E3 ligases. DiGeorge syndrome, a birth defect characterised by misregulation of neural crest cells during early development, has been associated with a deletion of UFD1 (Yamagishi et al., 1999) (reviewed by Baldini, 1999). This protein appears to be involved in the linking step between polyubiquitination and proteasome-mediated degradation (Bays and Hampton, 2002). A definitive causal relationship between UFD1 and DiGeorge syndrome has not been proven yet, and there are other good candidates deleted in this syndrome, including TBX1 (reviewed by Baldini, 2005). In addition, several of the syndrome's neural crest defects can be recapitulated by inactivation of TGF-ß signalling (Wurdak et al., 2005). Nevertheless, the large variety of defects caused by aberrant protein degradation (reviewed by Sakamoto, 2002) indicates that this process is vital in many tissues and stages during development. The investigation of protein degradation during development should contribute towards our understanding of the basis of such diseases known as `ubiquitinopathies'.

Here, we present the functional analysis of Xenopus Cullin-1 in a developmental context. We show that Cullin-1 is expressed throughout early Xenopus development and is enriched in neural tissue. Overexpression of a truncated dominant-negative form leads to an accumulation of ß-catenin, one of its degradation targets, and a reduction of ubiquitinated ß-catenin, suggesting that it prevents ß-catenin degradation. Embryos where the dominant-negative Cullin-1 is overexpressed, display a range of embryological defects most noticeably an upregulation of melanocytes, a neural crest derived tissue. Analysis with molecular markers at different stages in development suggests that this increase is due both to an increased allocation of ectodermal tissue to the neural crest and to an increased allocation of neural crest progenitors to the melanocyte lineage. Our findings demonstrate for the first time a link between Cullin-1 function and neural crest development. Moreover, these findings demonstrate that there is a requirement for protein degradation that goes beyond the very early embryonic stages and is essential for the correct cell fate allocation throughout development.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Embryo culture and injections
X. laevis embryos were obtained by hormone induced egg laying and in vitro fertilisation by standard methods. Injections were carried out with in vitro transcribed capped RNA animally into one blastomere at the two-cell stage. RNA was injected in a volume of 10 nl, at doses ranging from 10 pg to 3 ng, and 200-300 pg of lacZ RNA was co-injected as a lineage tracer. As a control, lacZ RNA was injected alone at the highest dose employed for the experimental RNA. Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967) and grown to the desired stage. They were then fixed in MEMFA and stained with X-gal as described (Bourguignon et al., 1998).

In situ hybridisation
In situ hybridisation was performed as previously described (Harland, 1991). Probes were made from digoxigenin-labelled antisense RNA. At neurula stages, embryos were analysed with the X. laevis molecular marker Sox3 (Penzel et al., 1997). Moreover, the X. tropicalis Sox9 (Spokony et al., 2002), Slug (Mayor et al., 1995), Sox10 (Aoki et al., 2003; Honore et al., 2003) and Zic3 (Nakata et al., 1997) were also used. At tadpole stages, embryos were analysed for N-tubulin, a marker for differentiated neurons (Chitnis and Kintner, 1995; Oschwald et al., 1991). The chromogenic reaction was carried out with either nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) or with 5-bromo-6-chloro-3-indolyl-phosphate (magenta phos). In order to obtain sections, embryos were embedded in gelatine albumin and 30 µm sections were cut using a vibratome.

Western blot analysis
Embryos were snap-frozen at the desired stage. Proteins from 20 embryos were extracted in 150 µl 15 mM Tris-HCl pH 7.5 containing the Complete Protease Inhibitor (Roche). Excessive yolk was removed by the addition of an equal volume of Freon (1,1,2-Trichlorotriofluoroethane, Sigma F5217). Proteins were separated in 8% SDS acrylamide gels and transferred onto nitrocellulose membranes. Blots were hybridised with a rabbit polyclonal anti ß-catenin (1/500, Santa Cruz-H102), a mouse monoclonal Cullin-1 (1/200, Santa Cruz-Cul1-D5) or a mouse monoclonal anti alpha tubulin (1/5000, clone DM1A, Sigma) antibody. Between different antibody stainings the blots were stripped for 1 hour at 55°C in 100 mM ß-mercaptoethanol containing stripping solution (62.5 mM Tris-HCl pH 6.7, 2% SDS). Goat-anti-mouse-HRP or goat-anti-rabbit-HRP secondary antibodies were used (1/2500, Jackson Immunoresearch Laboratories). The blots were processed with ECL or ECL plus (Amersham), and the chemiluminescent signal was detected on medical film.

Immunoprecipitations
H293T cells were cultured in DMEM + 10% foetal calf serum. Confluent cultures were transfected with DNA using Lipofectamine 2000 (Invitrogen). Empty pCS2 vector DNA was used when not all of the constructs were employed. The following CMV expression constructs were used for the transfections. X. laevis ß-catenin was N-terminally HA-tagged and cloned into the expression vector pCS107. pCW7 Myc-His₆-Ubiquitin (a gift from M. Goldberg) and either the truncated pCS107 Cullin-1 (Cul1-C75 and Cul1-C477) or the full-length pCS107 Cullin-1 (TEgg108l17) or cullin-3 (TNeu078f05) were used. Thirty-six hours after transfection, 10 µM proteasome inhibitor MG132 was added to allow accumulation of ubiquitinated proteins. After 48 hours, cells were harvested (10 cm dish/transfection) and proteins were extracted. To reduce non-specific binding, lysates were pre-cleared with mouse IgG and Protein G beads for 1 hour. They were then incubated with anti-Myc-antibody-coupled agarose (Santa Cruz, 9E10, sc40-AC) overnight. The next day, the beads were washed thoroughly and the bound proteins were eluted in sample buffer. The eluate was separated on 8% SDS acrylamide gels and westerns were performed with anti-HA-HRP (1/5000 Roche). Approximately 10% of the lysate was retained as a control. Those samples were separated on 8% SDS acrylamide gels and westerns were performed as described above.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cullin-1 is a very conserved protein among vertebrate species
Cullin-1 was identified during a gain-of-function screen in X. tropicalis for genes affecting neural development (Voigt et al., 2005). The active clone in the screen was a C-terminally truncated form, missing the last 75 amino acids (clone TGas038c21, thereafter referred to as Cul1-C75; Fig. 1A,B). A full-length Xenopus tropicalis Cullin-1 clone was subsequently identified in an EST database that allows full coding sequence predictions (TEgg108l17) (Gilchrist et al., 2004) and was fully sequenced. Xenopus tropicalis Cullin-1 is 99.6% identical to mouse and human Cullin-1 at the protein level (Fig. 1A). Sequence comparison showed that the truncated clone lacks a large region of the Rbx-1 binding domain, the SCF subunit that binds to the E2 conjugating enzyme and provides the SCF with ubiquitin molecules (Zheng et al., 2002) (Fig. 1A-C). This truncation is very likely to inhibit the interaction between Cullin-1 and Rbx-1 (Fig. 1C). Moreover, the neddylation site, which is thought to activate the SCF functionally by attaching a Nedd8 molecule to Cullin-1 (Lammer et al., 1998; Morimoto et al., 2000; Osaka et al., 1998) (star in Fig. 1A), is located in the missing area.

Cullin-1 is expressed during embryonic development and is enriched in neural tissue
Cullin-1 RNA is ubiquitously found throughout the blastula and gastrula embryo. At neural plate stages, it is enriched in the area of the neural ectoderm, particularly in the anterior region. In the tadpole, expression is enriched in the neural tube, eye and branchial arches (Fig. 2A) (see also Voigt et al., 2005).

Western blot analysis showed that there is a large supply of maternal Cullin-1 protein in the egg and that the protein level remains near constant throughout early embryonic development. A slight increase is observed during late neurula/early tailbud stages (stage 19-28) (Fig. 2B).

Ectopic truncated and full-length Cullin-1 leads to an increase of ß-catenin levels in Xenopus embryos
A number of embryos that were injected with Cul1-C75 displayed secondary axes, a telltale sign of ectopic ß-catenin stabilisation. As Cul1-C75 lacks a large part of the interaction domain with Rbx-1, we anticipated that it would act as dominant negative of endogenous Cullin-1, i.e. inhibit the formation of active complexes by titrating out the remaining proteins in the complex. This in turn would lead to a stabilisation of ß-catenin. Indeed, an increase in ß-catenin was observed in Cul1-C75 embryos at stage 8 and stage 19 (Fig. 3A,C). One might expect that overexpression of the full-length clone would have the opposite effect, i.e. it would enhance the degradation of ß-catenin. Contrary to our expectations, overexpression of the full-length clone had the same effect as the truncated form, leading to an increase of ß-catenin levels at stage 10 and stage 19 (Fig. 3B,C). Both constructs produced proteins of the expected molecular weight when overexpressed in the embryo (Fig. 3C). To find out whether a very low dose of overexpressed Cullin-1 might lead to a downregulation of ß-catenin, we injected doses of Cullin-1 ranging from 10 pg to 3 ng into embryos. A decrease in endogenous ß-catenin was not observed in any of the doses, whereas an increase was observed with doses as low as 50 pg (Fig. 3D).

Overexpression of truncated and full-length Cullin-1 in H293T cells leads to a decrease of ubiquitinated ß-catenin
If the endogenous SCF complex was inhibited, we would expect that the increase in ß-catenin would be due to a reduction of its ubiquitination. Therefore, we examined the levels of ubiquitinated ß-catenin when Cullin-1 or Cul1-C75 were overexpressed. We transfected H293T cells with HA-ß-catenin and Myc-His-Ubiquitin in the presence of 10 µm MG132, a proteasome inhibitor (for the last 12 of 48 hours). The addition of the inhibitor allowed us to detect ubiquitinated forms of ß-catenin that are normally rapidly degraded (Fig. 4). We then immunoprecipitated ubiquitinated proteins with an anti-Myc antibody, separated the bound proteins on an SDS/Page gel and used and anti HA antibody to detect ubiquitinated ß-catenin levels. We found that co-expression of Cullin-1 or Cul1-C75 led to a decrease of ubiquitinated ß-catenin (Fig. 4).

The increase of total ß-catenin, together with the decrease of ubiquitinated ß-catenin, suggests that the ß-catenin ubiquitination machinery is inhibited, indicating that ectopic full-length or truncated Cullin proteins inhibit the SCF complex, most probably acting as dominant negatives of endogenous Cullin proteins. However, the Cul1-C75 form of Cullin-1 lacks only part of the Rbx1 interaction domain; therefore, it is formally possible that it retains wild-type activity. In this scenario, neither Cul1-C75 nor Cullin-1 act as dominant negatives when overexpressed, but they both act as wild-type instead. To address this possibility, we have constructed a severely truncated form of Cullin-1 in the C-terminus that lacks the entire Rbx1 interacting domain, Cul1-C477 (Fig. 1B). When overexpressed in H293T cells, Cul1-C477 reduced the level of ubiquitinated ß-catenin and indeed did so to a greater extent than either the full-length Cullin-1 or the Cul1-C75 construct (Fig. 4). Taken together, these results suggest that both the truncated and full length Cullin-1 act as dominant negatives when overexpressed, most likely by inhibiting the activity of endogenous Cullin-1 by titrating out the N-terminal components of the SCF complex into inactive or partial complexes.

Thus, these constructs provided us with an opportunity to study the effect of inhibiting endogenous Cullin proteins in development. Alternative means of inhibiting endogenous Cullin proteins such as ATG or splice site directed morpholinos in X. tropicalis were not effective, perhaps owing to the abundance of maternally supplied protein (data not shown).

Inhibition of endogenous Cullin-1 in Xenopus embryos causes a complex phenotype
Having established that ectopic Cullin-1 acts as a dominant negative, we carried out all of the following experiments with the full-length and the truncated Cullin-1 protein (Cul1-C75). The same phenotypes were observed with both forms. The phenotypes were complex but several distinct features could be observed. Duplicated axes, a known consequence of ectopic ß-catenin/Wnt signalling, were seen with low frequency (around 10%), presumably owing to targeting the injections to the animal rather than to the marginal area of the embryo (Fig. 5, Fig. 6D). The next most frequent phenotype were eye defects that ranged from malformations to complete absence of eye development (missing eyes scored in Fig. 5, shown in Fig. 6C). Ectopic tissue, particularly in the form of epidermal folds, was also observed with high frequency (Fig. 5, Fig. 6G). Finally, the most frequent phenotype was an increase in melanocytes, seen even at the lowest doses injected (250 pg) (Fig. 5, Fig. 6B,E,G).

View larger version (58K): [in this window] [in a new window]   Fig. 1. Sequence and constructs for Xenopus Cullin-1. (A) Protein alignment of the mouse, human and X. tropicalis Cullin-1. The full-length protein is 99.6% identical at the amino acid level. The Rbx-1- and Skp1-binding domains are boxed in red and blue, respectively. (B) Diagrammatic representation of Cullin-1 constructs. Two C-terminally truncated versions of Cullin-1 were used. Cul1-C75 and Cul1-C477 are missing 75 and 477 amino acids from the C terminus respectively (C-terminal truncations indicated by purple bars in A). As a consequence, Cul1-C477 contains none and Cul1-C75 only contains a subset of the Rbx-1-binding sites. Moreover, lysine 720, the neddylation site (green star in A) is missing in both constructs. These constructs should lose or have reduced binding ability to Rbx-1, one of the components of the SCF complex, and are likely to interfere with the correct formation of the endogenous SCF. A fourth construct (N148-Cul1-C477), missing the 148 N-terminal and the 477 C-terminal amino acids, was also created. This deletes both the Skp-1- (yellow) and the Rbx-1- (red) binding domains and should not interfere with the endogenous SCF complex. (C) A wild-type SCF complex (with its substrate ß-catenin) versus a complex based on the truncated Cullin proteins (Cul1-C75). In the wild-type scenario, Cullin proteins provide the bridge between the F-box protein that binds ß-catenin and the E2 conjugating enzyme that supplies ubiquitin molecules. The truncation is likely to inhibit the interaction between the Cul1-C75 and Rbx-1/E2, hence preventing SCF function. Polyubiquitination of ß-catenin is shown by a series of grey circles, which are missing in the Cul1-C75-based SCF complex.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Expression analysis of Cullin-1. (A) RNA expression pattern of Cullin-1 during early X. tropicalis development. Cullin-1 is ubiquitous at blastula (1, lateral view) and gastrula (2, vegetal, 3, animal view) stages. At neurula stages (4, anterior view) it becomes enriched in neural tissue. At tadpole stages, it becomes enriched in the neural tube, eye and branchial arches (5+6, anterior towards the left, dorsal upwards). (B) Cullin-1 protein presence during development. Westerns with an anti-Cullin-1 antibody were carried out on protein extracts of whole X. laevis embryos at different developmental stages. Cullin-1 protein is found throughout development. Cullin-1 protein levels are slightly increased during late neurula/early tadpole stages.

As a test for the specificity of the Cullin-1 phenotype, we overexpressed the Xenopus tropicalis Cullin-3, which is thought to be part of an E3 ligase complex that is similar to the SCF, but with different degradation targets. Although Cullin-3 is also expressed ubiquitously during embryonic development, we did not detect a phenotype when it was overexpressed (data not shown).

Inhibition of Cullin-1 leads to an increase of melanocytes at the expense of cranial ganglia
As mentioned above, the most frequent phenotype at tadpole stages were ectopic melanocytes (91%, n=228). We quantitated the increase of melanocytes in transverse sections and found that there is an average 4.4-fold increase. As melanocytes are derived from neural crest tissue, which also gives rise to the cranial ganglia, we examined the formation of cranial nerves, using N-tubulin as a marker (Chitnis and Kintner, 1995; Oschwald et al., 1991). We found that N-tubulin was not disrupted in the brain or in the spinal cord, except for defects as a consequence of secondary axes. However, N-tubulin-positive cranial ganglia were severely disrupted or absent in 74% (n=35) of the embryos (Fig, 6B,C,E,F, arrows). This finding suggests that increased melanocytes are produced at the expense of cranial neurons.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Cul1-C75 and Cullin-1 RNA injections increase the level of ß-catenin in the Xenopus embryo. (A) 1.6 or 2.5ng of Cul1-C75 (truncated) capped RNA were injected animally into each blastomere of two-cell stage X. laevis embryos. Proteins were extracted at stage 10 and analysed for ß-catenin levels. Injection led to an upregulation of ß-catenin levels. Blots were stripped and analysed against -tubulin as a loading control. (B) The same was carried out for full-length Cullin-1 RNA. Embryos were analysed at stage 8 and surprisingly an upregulation of ß-catenin levels was also observed. (C) ß-Catenin levels were also analysed at stage 19 (neurula stage), a time during which neural crest tissue becomes specified. As at stage 8 and 10, injections of 1.6 and 2.5 ng of either the Cul1-C75 and the full-length Cullin-1 lead to an increase in ß-catenin levels. This was also found for stage 24 (data not shown). To determine whether both constructs are translated correctly, western blots were stripped and subsequently analysed for Cullin-1 protein levels (with an antibody raised towards Cullin-1's N-terminus). The full-length Cullin-1 RNA (lane 4 and 5) results in higher levels of proteins of the correct size when compared with uninjected embryos (lane 1). A clear size difference between Cul1-C75 and Cullin-1 is also visible. (D) A wide range of Cullin-1 RNA doses were injected (10 pg-3 ng into each blastomere at the two-cell stage) and embryos were analysed at stage 10. A decrease in ß-catenin was never observed; indeed the increase of ß-catenin correlates with the increased dose of Cullin-1 injection.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Cullin-1, Cul1-C75 and Cul1-C477 decrease ß-catenin ubiquitination in cell lines. H293T cells were transfected with Myc-His-Ubiquitin, HA-ß-catenin and either Cul1-C477, Cullin-1 or Cul1-C75. After 36 hours, 10 µM MG132 (a proteasome inhibitor) was added for 12 hours to accumulate ubiquitinated protein. All ubiquitinated protein was immunoprecipitated (IP) via Myc. Ubiquitinated ß-catenin was visualised with an anti-HA antibody. Co-expression of any of the Cullin-1 gene constructs led to a reduction of the levels of ubiquitinated ß-catenin. The severity of reduction corresponded to the amount of truncation, with the most truncated construct Cul1-C477 causing the most severe reduction (lane 3 IP), the Cul1-C75 (lane5) causing a strong reduction and full-length Cullin-1 (lane 4) causing some reduction. This suggests that Cullin1, Cul1-C75 and Cul1-C477 inhibit ß-catenin ubiquitination, probably by inhibiting the endogenous SCF complex. As a control, 10% of the lysate was removed previous to the IP and analysed for total levels of ß-catenin by anti-HA staining. Co-transfection of Cullin-1 led to increased ß-catenin levels in the lysate control in 4/6 experiments (not shown here). An increase in total ß-catenin is not always detected in these experiments because of the high levels of exogenous ß-catenin and because the ubiquitinated form is only a fraction of the total ß-catenin.

Interestingly, these markers showed that the expansion of neural crest often occurred in the anterior most neural plate of experimental embryos, an area that is normally crest free. When the injected RNA was localised to the anterior neural plate (the area adjacent to where neural crest tissue is found) Sox3, a neural plate marker, was often inhibited in that area (Fig. 8B). These findings suggest that inhibition of endogenous Cullin-1 function leads to ectopic neural crest at the expense of neural plate tissue in the anterior neural plate.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Comparative dose response of Cul1-C75 and Cullin-1 in inducing developmental abnormalities. Embryos were injected with different doses of Cul1-C75 or Cullin-1 RNA animally into one blastomere at two-cell stage and grown to tadpole stages (stage 39/40). Morphological analysis was carried out by comparing the uninjected with the injected side and embryos were scored for defects. The phenotype predominantly consisted of ectopic melanocytes, ectopic tissue (such as epidermal folds), eye defects and secondary axes. The most robust phenotype was ectopic melanocytes, which could be seen in almost all of the injected embryos. The dose of RNA injected correlates with the penetrance of the phenotype. The truncated Cullin-1 construct (Cul1-C75) was more potent at causing a phenotype than the full-length construct.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We attribute these effects to the inhibition of the activity of the endogenous SCF complex. The C-terminally truncated Cullin-1 is missing the Rbx1-binding site and therefore would act as a dominant negative by titrating out endogenous components into inactive complexes (Fig. 1C). This was substantiated by an increase in one of the Cullin-1 degradation targets, ß-catenin, accompanied by a decrease of the ubiquitinated form. Interestingly, the same phenotypic and biochemical effects were observed after overexpressing the wild type Cullin-1. Obtaining the same effect with a truncated and a full-length clone is not without precedent: Neuralized is another case of a ubiquitin ligase where overexpression of a truncated, dominant-negative form and a full-length clone have the same effect (Deblandre et al., 2001). In another study, Mistry et al. (Mistry et al., 2004) found that overexpression and loss of Cullin-3 leads to an accumulation of Ci155, the Drosophila homologue of the Gli genes.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Phenotypes observed in Cul1-C75 and Cullin-1 injected embryos. (A) Wild-type embryo for comparison. The most striking phenotype observed was an increase in melanocytes (B), but eye defects (C, star represents where the eye should be) and secondary axes (D) were also commonly observed (see Fig. 5 for quantification). Embryos were hybridised in situ with a probe for N-tubulin, a marker for differentiated neurons (purple). Ectopic N-tubulin can be observed in the secondary axis (D). The lineage tracer lacZ (light-blue staining) indicates the area of injection. Lateral view with anterior towards the left and dorsal towards the top. The injected side is shown. Transverse sections of the embryos show clearly the large upregulation of melanocytes (E-G) and the ectopic tissue (white arrowhead) in the injected side. Neural differentiation in the neural tube is not affected (as marked by N-tubulin); however, cranial ganglia neurons (N-tubulin positive, arrows) are absent (compare B,C with A and E,F with control side). Transverse view with dorsal towards the top and the injected side on the right.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Phenotype of a severely C-terminally truncated form of Cullin1, Cul1-C477. In order to ensure that the C-terminally truncated Cul1-C75 does not act as wild-type form of Cullin-1, we made a Cullin-1 construct (Cul1-C477) that is lacking all of the C terminus (Fig. 1B). A 250 pg dose of this construct together with the lineage tracer lacZ (light blue) were injected animally at the two-cell stage into X. laevis embryos. Embryos were analysed at tadpole stages for morphological defects. Injected embryos (A-C,E) showed an accumulation of melanocytes (A-C, arrow in C), secondary axes (B) and ectopic tissue (C), all phenotypes previously observed with the other Cullin-1 constructs. For comparison, uninjected control embryos are shown (D,F). This indicates that all of these constructs act in the same manner, which is most likely to be a dominant-negative mechanism. All embryos are shown laterally. In A-D, anterior is towards the left; in E,F, embryos are facing each other.

View larger version (59K): [in this window] [in a new window]   Fig. 8. Cul1-C75 RNA injections increase early neural crest markers. (A) Embryos were injected with 500 pg of Cul1-C75 or Cullin-1 (same phenotype, but not shown) plus the lineage tracer lacZ (light blue) into one blastomere at the two-cell stage. As a control, 500 pg lacZ RNA was injected alone. Embryos were grown to neurula stage and analysed for the early neural crest markers Sox9, Sox10, Slug and Zic3 (purple). Expression of all markers was expanded, predominantly into the anterior neural plate. In some cases, expression of Sox9 and Sox10 were reduced (not shown). Dorsal view with anterior towards the top. (B) When injections were targeted to the anterior neural plate, expression of Sox3 (a marker for proliferating neural tissue) was reduced. This was in the same region where the expansion of neural crest markers (as shown for Sox9) was seen. Dorsal view with anterior towards the top.

We suggest that in truncated or full-length Cullin-1 injected embryos increased ß-catenin signalling is responsible, directly or indirectly, for the observed increased expression of the early neural crest markers Slug, Sox9, Sox10 and Zic3. In turn, these would mediate the increased allocation to neural crest fate and in particular the melanocyte lineage at the expense of cranial neuron fate. Indeed, overexpression of ß-catenin expands Slug expression at the expense of the pan-neural Sox2 (LaBonne and Bronner-Fraser, 1998) and overexpression of Sox10 leads to a massive increase of melanocytes, a phenotype that can also be induced by ectopic Wnt1 (Honore et al., 2003). Overexpression of Zic3 leads to neural and neural crest hyperplasia, concomitant with increased Slug expression, an increase in melanocytes, and loss of or deformed eyes (Nakata et al., 1997). This resembles the Cullin-1 phenotype very closely. Within the neural crest lineage, Wnt/ß-catenin signalling may promote directly the melanocyte fate by activating genes such as the transcription factor MITF/nacre, which is required for the development of melanocytes (reviewed by Yanfeng et al., 2003). In addition, Sox10 transcriptionally regulates mitfa (Dutton et al., 2001; Elworthy et al., 2003).

Finally, the expansion of the neural crest domain in the anterior neural plate observed in truncated and full-length Cullin-1 injected embryos is very similar to phenotypes observed in the intracellular Wnt signalling repressor headless/tcf3 zebrafish mutant (Dorsky et al., 2003; Kim et al., 2000). Therefore, this effect can also be accounted for by increased/ectopic Wnt/ ß-catenin signalling in the anterior neural plate.

There are several Wnt genes expressed in the dorsal neural tube and neural crest that could mediate ß-catenin activation (reviewed by Wu et al., 2003; Yanfeng et al., 2003) and several Wnt inhibitors could limit Wnt activity in this area. For example, extracellular Wnt inhibitors are expressed in the dorsal neural tube and these could limit Wnt signalling during neural and neural crest development (Baranski et al., 2000; Duprez et al., 1999; Jin et al., 2001; Ladher et al., 2000). Furthermore, intracellular Wnt inhibitors such as tcf3 are thought to prevent Wnt signalling in the anterior neural plate (Dorsky et al., 2003; Kim et al., 2000). Here, we have shown that alongside these inhibitors, deactivation of Wnt signalling by ubiquitination and degradation of ß-catenin is important for neural crest development.

Although protein degradation is a recognised means for limiting ß-catenin signalling in early Xenopus development, where no zygotic transcription takes place, it was not known whether it is equally important in later vertebrate development. To date, most studies that implicate protein degradation in later developmental events come from Drosophila (Jiang and Struhl, 1998; Ou et al., 2003; van Roessel et al., 2004). Our results show that inhibition of Wnt and possibly other signalling pathways by Cullin-1-mediated proteolysis is a major contributor in the correct development of the neural crest lineage. If this process is misregulated, serious defects in the development of the neural crest can occur, such as, for example, in DiGeorge syndrome. Therefore, understanding how protein degradation contributes to the specification of neural crest fates will lead to a better understanding of the aetiology of such disorders.

	ACKNOWLEDGMENTS

 
We thank Jon Pines and Mark Jackman for helpful discussions, and Enrique Amaya and Eva Asscher for comments of the manuscript. J.V. was a Wellcome Trust 4 year program PhD student and N.P. is a Wellcome Trust Senior Research Fellow. This work was supported by the Wellcome Trust.

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