RESEARCH ARTICLE 3057
Development 136, 3057-3065 (2009) doi:10.1242/dev.036855
Vegetally localized Xenopus trim36 regulates cortical
rotation and dorsal axis formation
Tawny N. Cuykendall and Douglas W. Houston*
Specification of the dorsoventral axis in Xenopus depends on rearrangements of the egg vegetal cortex following fertilization,
concomitant with activation of Wnt/β-catenin signaling. How these processes are tied together is not clear, but RNAs localized to
the vegetal cortex during oogenesis are known to be essential. Despite their importance, few vegetally localized RNAs have been
examined in detail. In this study, we describe the identification of a novel localized mRNA, trim36, and characterize its function
through maternal loss-of-function experiments. We find that trim36 is expressed in the germ plasm and encodes a ubiquitin ligase
of the Tripartite motif-containing (Trim) family. Depletion of maternal trim36 using antisense oligonucleotides results in ventralized
embryos and reduced organizer gene expression. We show that injection of wnt11 mRNA rescues this effect, suggesting that Trim36
functions upstream of Wnt/β-catenin activation. We further find that vegetal microtubule polymerization and cortical rotation are
disrupted in trim36-depleted embryos, in a manner dependent on Trim36 ubiquitin ligase activity. Additionally, these embryos can
be rescued by tipping the eggs 90° relative to the animal-vegetal axis. Taken together, our results suggest a role for Trim36 in
controlling the stability of proteins regulating microtubule polymerization during cortical rotation, and subsequently axis
formation.
INTRODUCTION
Understanding the establishment of the embryonic body axis from
a symmetrical egg has been a long-standing problem in
developmental biology. In amphibian eggs, axial determination
requires a dorsally directed rotational movement of the egg cortex
(cortical rotation) during the first cell cycle (Elinson and Rowning,
1988; Vincent et al., 1986). Cortical rotation is driven by a parallel
array of microtubules beneath the cortex (Elinson and Rowning,
1988), and the degree of rotation is correlated with the size and
extent of Spemann organizer formation, the main determinant of
dorsal fate in the embryo (Gerhart et al., 1989). Cortical rotation is
thought to transport axial determinants to the dorsal side (Kageura,
1997; Marikawa et al., 1997) along the microtubule array, resulting
in activation of the Wnt/β-catenin signaling pathway and in the
dorsal stabilization of β-catenin (reviewed by Weaver and
Kimelman, 2004).
Wnt/β-catenin signaling is clearly sufficient for axis formation
(McMahon and Moon, 1989; Sokol et al., 1995; Yost et al., 1996).
The requirement for Wnt activity in vertebrates was first
demonstrated through the depletion of maternal β-catenin mRNA
using antisense oligonucleotides (Heasman et al., 1994). β-catenindepleted embryos are ventralized, lacking dorsal tissues of all three
germ layers, including the neural tube, notochord and somites.
Additional maternal depletion studies showed that other Wnt/βcatenin components are also required for proper axis formation
(Belenkaya et al., 2002; Houston et al., 2002; Kofron et al., 2001;
Sumanas et al., 2000; Yost et al., 1998). Despite the importance of
both cortical rotation and Wnt/β-catenin signaling in axis
determination, how these two processes are initiated and their interrelationships are not clearly understood.
The University of Iowa, Department of Biology, 257 BB, Iowa City, IA 52242, USA.
*Author for correspondence ([email protected])
Accepted 23 July 2009
Recent studies have shown that the Wnt ligand Wnt11 plays a
crucial role in axis formation and maternal Wnt/β-catenin signaling
(Tao et al., 2005). wnt11 mRNA is vegetally localized during
oogenesis to the region of the germ plasm (Kloc and Etkin, 1995;
Ku and Melton, 1993), which is a subcellular aggregation of
mitochondria, membranous organelles and ribonuceloproteins that
is required for establishing the germline (reviewed by Houston and
King, 2000b). Germ plasm is anchored in the cortex and is displaced
dorsally by cortical rotation, thus distributing wnt11 mRNA to the
dorsal side (Tao et al., 2005). Depletion of maternal wnt11 mRNA
using antisense oligonucleotides results in ventralized embryos, and
molecular epistasis experiments demonstrate that Wnt11 lies
upstream of β-catenin (Tao et al., 2005).
An alternative model suggests that Wnt/β-catenin signaling is
achieved by intracellular transport of β-catenin stabilizing agents
dorsally along vegetal microtubules (Weaver and Kimelman, 2004).
Evidence supporting this model comes from cytoplasmic
transplantation experiments (Kageura, 1997), the visualization of
Dishevelled (Miller et al., 1999) and Gbp (also known as Frat1)
(Weaver et al., 2003) movement during cortical rotation and the
ventralization phenotype observed upon depletion of maternal gbp
(Yost et al., 1998). However, these models are not mutually
exclusive, as the enrichment of Wnt activators on the dorsal side
might sensitize these cells to ongoing Wnt signaling. Either model
would require the vegetal microtubule array; however, the
mechanisms that form this array and the extent of its involvement in
Wnt signaling are not well understood.
The formation of vegetal microtubule arrays during the first
cell cycle is conserved in many organisms (Eyal-Giladi, 1997),
whereas cortical rotation per se and wnt11 localization and
function in axis formation might be features specific to basal fish
and amphibians. Recent studies have shown that the germ-plasmlocalized mRNA fatvg (adipophilin), which encodes a lipidvesicle-associated protein, is required for axis formation by
regulating the movement of vesicles during cortical rotation
(Chan et al., 2007). These data, together with the role of localized
wnt11, suggest that germ-plasm-localized molecules might play
DEVELOPMENT
KEY WORDS: Xenopus, Dorsal axis, Wnt, Microtubules, Cortical rotation, Trim, Ubiquitylation
3058 RESEARCH ARTICLE
MATERIALS AND METHODS
Oocytes and embryos
Oocytes were manually defolliculated and cultured in a modified oocyte
culture medium [OCM; 70% L-15 media, 0.04% BSA, 1 mM GlutaMAX
media, 1.0 μg/ml gentimicin, pH 7.6-7.8 (Heasman et al., 1991)] at 18°C.
Embryos were obtained as described (Sive et al., 2000; Kerr et al., 2008).
For tipping experiments, eggs were de-jellied and sorted within 20 minutes
of fertilization; the colored eggs were then transferred to a Nitex mesh dish
containing 5% Ficoll in 0.3⫻ MMR. Individual eggs were oriented with the
sperm entry point uppermost and were left in place until the first cleavage.
The buffer was gradually changed to 0.1⫻ MMR and embryos were cultured
to the tailbud stage.
Plasmids
A full-length cDNA for trim36 in the vector pCMV-SPORT6 was obtained
commercially (Open Biosystems) and the insert size was confirmed by
restriction enzyme digestion. The coding region (CDS) for trim36 was
amplified by PCR and cloned into pCR8/GW/TOPO (Invitrogen). Clones
were verified by sequencing and selected clones in the 5⬘-L1 to L2-3⬘
orientation were inserted via recombination into a pCS2+ Gatewayconverted vector (custom vector conversion kit; Invitrogen). Details of the
Gateway plasmid are available upon request. Template DNA for sense
transcription from trim36-pCS2+ was prepared by NotI digestion and was
cleaned by proteinase K digestion. Capped messenger RNA was synthesized
using SP6 mMESSAGE mMACHINE Kits (Ambion).
Antisense oligos and host transfer
Antisense oligodeoxynucleotides (oligos) used were HPLC-purified
phosphorothioate-phosphodiester chimeric oligos (Integrated DNA
Technologies) with the following sequences (* indicates phosphorothioate
linkages): trim36-3, 5⬘-C*T*TCCAGGTGTCGATG*C*T-3⬘ (nt 408-426);
trim36-4, 5⬘-G*C*CTATACTTCTTCGC*C*C-3⬘ (nt 506-523); trim36MO-A, 5⬘-CCCGTCTCCTCCATCTGCGCTTGTT-3⬘ (nt 198-222); βcatenin 303, 5⬘- T*G*C*CTTTCGGTCTGG*C*T*C-3⬘ (Heasman et al.,
1994).
Oocytes were injected in the vegetal pole (doses of oligos are indicated
in the text) and cultured for 24 hours at 18°C before being matured by
treatment with 2.0 μM progesterone. For rescue experiments, mRNAs were
injected immediately prior to progesterone treatment. Matured oocytes were
colored with vital dyes, transferred to egg-laying host females, recovered
and fertilized essentially as described (Heasman et al., 1991).
Analysis of gene expression using real-time RT-PCR
Total RNA was prepared from oocytes and embryos using proteinase K and
then treated with RNase-free DNase as described (Houston and Wylie,
2005). Real-time RT-PCR was carried out using the LightCycler 480 System
(Roche Applied Science). Samples were normalized to ornithine
decarboxylase (odc) levels and relative expression values were calculated
against a standard curve of control cDNA. Samples lacking reverse
transcriptase in the cDNA synthesis reaction failed to give specific products.
The data shown are representative individual experiments using unreplicated
samples; experiments were repeated at least twice with similar results.
Primer sequences and detailed protocols are available upon request.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed essentially as described
(Sive et al., 2000; Kerr et al., 2008). Template DNAs for in vitro
transcription were prepared by digestion, followed by transcription, with
appropriate restriction enzymes and polymerases: trim36-pcmvsport6
(SalI/T7), nr3 (a gift from R. Harland, University of California-Berkeley,
Berkeley, CA, USA; EcoRI/T7), eomes and myod (gifts from J. Gurdon, The
Gurdon Institute, Cambridge, UK; EcoRI/T3 and BamHI/SP6, respectively),
and sizzled (from M. Kirschner, Harvard Medical School, Boston, MA,
USA; Addgene plasmid 16688, BamHI/T3). Antisense RNA probes labeled
with digoxigenin-11-UTP (Roche) were synthesized using polymerases and
reaction buffers from Promega.
Immunostaining
Whole-mount immunostaining for microtubules and cytokeratins was
performed using previously described methods (Elinson and Rowning,
1988). Fluorescence was visualized on a Leica DMI3000B inverted
microscope using a 63⫻ dry objective (Leica Microsystems). Antibodies
were α-tubulin mouse antibody, clone DM1A (ascites 1:200, Sigma) and
cytokeratin mouse antibody, clone 1h5 (supernatant 1:5, DSHB). Secondary
antibodies were goat anti-mouse conjugated with Alexa-488 or -564
(Invitrogen/Molecular Probes) diluted in PGT (1⫻PBS, 1% goat serum,
0.5% Triton X-100; 1:500). Whole-mount staining for β-catenin was
performed essentially as described (Schneider et al., 1996), using a rabbit
anti β-catenin antibody (C2206, Sigma, 1:200).
For immunostaining of sections, samples were fixed and embedded in
polyethylene glycol 400 distearate (PEDS wax, Polysciences) with 10%
cetyl alcohol (1-hexadecanol), modified from Godsave et al. (Godsave et al.,
1984). Sections were immunostained as described (Houston and Wylie,
2003). Tor70 (a gift from Richard Harland) was used at a 1:6 dilution;
12/101 (DSHB) was used at a 1:6 dilution. Detection was performed as
described above. For histology, sections were stained with Hematoxylin and
Eosin (H&E).
Guinea pig Trim36 antibodies were raised against purified Xenopus
Trim36 protein (Cocalico Biological). The trim36 CDS was cloned into the
pDEST17 vector (Invitrogen) by Gateway recombination to generate a
fusion protein N-terminally tagged with six histidines (6-His). Recombinant
protein was produced in bacteria following the manufacturer’s instructions,
and refolded from inclusion bodies as described (Houston and King, 2000a).
The 6-His-tagged Trim36 protein was then purified over a nickel column
(His GraviTrap, GE Healthcare), eluted with 300 mM imidazole and
dialyzed against PBS.
Ubiquitylation assays
Embryos were injected at the 2-cell stage with full-length or mutant trim36
mRNAs, HA-ubiquitin (a gift from S. Piccolo, University of Padua, Italy),
or both (1 ng of each), and cultured to stages 9-10. Samples were lysed in
cell lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1
mM EGTA (pH 8.0), 1% Triton X-100, with protease and/or phosphatase
inhibitors) and immunoprecipitated using Trim36 antisera. The resulting
complexes were electrophoresed and immunoblotted as described (Yokota
et al., 2003). Samples were blotted against HA-ubiquitin (mouse anti-HA
clone 3F10, 1:500, Roche Applied Science) and against Trim36 (1:2000).
DEVELOPMENT
significant roles in axis formation as well as in germ-cell
formation. To better understand the functions of localized
mRNAs, we undertook the identification and functional analysis
of novel vegetally localized mRNAs in Xenopus. In this work, we
describe one such novel localized mRNA, the Xenopus homolog
of tripartite motif-containing 36 (trim36).
Trim36 is a member of the large Tripartite motif-containing
protein family [known as the Trim or RBCC family (reviewed by
Meroni and Diez-Roux, 2005)]. Trim proteins are defined by a
conserved domain architecture, consisting of an N-terminal RING
finger domain, in combination with adjacent B-box-type zinc fingers
and a coiled-coil motif. Mammalian Trim36 localizes to
microtubules in cultured cells and is thought to have a role in
acrosome exocytosis (Kitamura et al., 2003; Kitamura et al., 2005).
Here we show that, in Xenopus, trim36 mRNA is localized vegetally
in the oocyte and is enriched in the germ plasm and adult germ cells.
Interestingly, we find that depletion of maternal trim36 results in
ventralized embryos. We show that Trim36 does not regulate Wnt
signal transduction directly, that Trim36 is required for
polymerization of cortical microtubules during cortical rotation and
that ubiquitin ligase activity is likely to be required for this function.
These data provide evidence that Trim36 is essential for positioning
the dorsalizing Wnt signal in early development and further suggest
that proteins encoded by vegetally localized mRNAs are important
in the control of microtubule polymerization or stabilization in the
vegetal cortex.
Development 136 (18)
RESULTS
Trim36 is encoded by a vegetally localized mRNA
in Xenopus
We set out to identify novel mRNAs localized to the vegetal cortex
of Xenopus oocytes. To accomplish this, we isolated total mRNA
from hand-dissected vegetal cortex pieces (Elinson et al., 1993) and
intact whole oocytes, generated labeled cDNA and probed
Affymetrix GeneChip arrays in duplicate. These data were analyzed
for RNAs enriched in the cortex relative to the whole oocyte. The
details and full results of this screen will be described elsewhere. We
selected one of the most highly enriched genes from this screen for
further functional analysis, tripartite motif-containing 36 (trim36;
UniGene ID Xl.6926), which is enriched by ~25-fold in the cortex
compared with the whole oocyte (data not shown).
We verified the enrichment of trim36 in the cortex by RT-PCR
(data not shown) and obtained a full-length EST through a
commercial source. This full-length trim36 EST (from Xenopus
laevis) encodes a cDNA of ~4000 nucleotides (nt), with a 2200 nt
coding region (CDS) preceded by several in-frame stop codons (see
Fig. S1 in the supplementary material). Conceptual translation of the
cDNA identified a methionine residue in a favorable initiation
context and yielded a putative protein of 733 amino acids. Amino
acid sequence analysis revealed characteristic domains classifying
this protein as a member of the large tripartite motif-containing
family (Trim), namely an N-terminal RING-finger domain followed
by two B-box zinc-finger domains and a coiled-coil domain
(reviewed by Meroni and Diez-Roux, 2005) (see Fig. S1 in the
supplementary material). The C-termini of Trim proteins can include
a variety of different functional domains, such as bromo- or NHLdomains, or in the case of Trim36, a B30.2/SPRY domain of
uncertain function. Trim36 also contains a COS motif, which groups
this protein within a subclass of Trim proteins that localize to
microtubules (Short and Cox, 2006). Amino acid sequence
alignments using the conceptual Trim36 protein sequence indicated
strong similarity to human and mouse Trim36 proteins (also known
as mouse Haprin, and human RBCC728 or RNF98; ~75% identity)
(see Fig. S1 in the supplementary material) and revealed that it is
95% identical to the Xenopus tropicalis Trim36 protein.
Comparisons across species using the Metazome database
(http://www.metazome.com/) found that the Xenopus tropicalis
trim36 gene occupies a conserved position with respect to
surrounding genes compared to other tetrapods, suggesting
orthology (data not shown).
Analysis of trim36 mRNA expression during early development
further confirmed its vegetal cortex localization and also showed
that trim36 is enriched in the germ plasm of oocytes and early
embryos. Temporally, trim36 is abundant maternally, but decreases
throughout development, as assessed by RT-PCR (Fig. 1A).
Spatially, trim36 mRNA is enriched in the mitochondrial
cloud/Balbiani body of stage I oocytes (Fig. 1B), the origin of germplasm material (Heasman et al., 1984). In fully grown stage VI
oocytes, trim36 is localized in a compact expression domain in the
vegetal cortex (Fig. 1C). trim36 RNA was further detected in the
germ plasm of fertilized eggs and 4- to 8-cell-stage embryos,
clustered in yolk-free islands along vegetal cleavage furrows (Fig.
1D). During neurula stages, trim36 RNA is expressed outside the
germ plasm in the developing neural tube and is enriched at the
midbrain-hindbrain boundary (Fig. 1E).
In situ hybridization on sectioned blastulae and gastrulae revealed
trim36 transcripts associated with the germ plasm during
gastrulation, including perinuclear localization in stage 11 embryos
(Fig. 1F,G), a hallmark of the germ plasm. trim36 RNA was
RESEARCH ARTICLE 3059
Fig. 1. Expression of trim36 in Xenopus. (A) RT-PCR for trim36 at
different Nieuwkoop and Faber (NF) stages; ‘–RT’ was processed in the
absence of reverse transcriptase. ornithine decarboxylase (odc) was
included as a loading control. (B-E) Whole-mount in situs using
antisense trim36 probe. (B) Stage I oocytes, (C) stage VI (left) and stage
IV (right) oocytes, (D) 4-cell embryos (vegetal view) and (E) neurula
embryos (dorsal/anterior view). (F-H) In situs for trim36 on sections;
insets in F and G are low-power views, inset in H is trim36 sense probe.
(F) Stage 7 sagittal section, (G) stage 11 sagittal section and (H) adult
testis.
undetectable in the primordial germ cells (PGCs) of postgastrulation embryos, including migrating PGCs at the tailbud stage
(data not shown). We also examined the testis, as mammalian trim36
homologs are implicated in acrosome function, and found robust
trim36 staining in spermatogenic cells of the adult testis (Fig. 1H).
Overall, our expression data show that trim36 is localized to the
germ plasm of oocytes and early embryos, and is expressed in the
developing nervous system and in adult germ cells.
Depletion of maternal trim36 causes reduced
dorsal axis structures
Although the enrichment of trim36 in the germ plasm was
suggestive of a role in PGC formation, other germ-plasm RNAs,
such as wnt11 (Tao et al., 2005) and fatvg (Chan et al., 2007), have
been implicated in dorsoventral axis formation. To examine the role
of trim36 in early development, we injected antisense
oligodeoxynucleotides (oligos) into full-grown oocytes to deplete
maternal trim36 mRNA. We identified two different oligos against
trim36, trim36-3 and trim36-4, that were effective in reducing
trim36 mRNA in a dose-dependent manner. These oligos were
synthesized in a phosphorothioate-modified form and used in
maternal loss-of-function experiments. We generated embryos
deficient in trim36 by transferring oligo-injected oocytes into egglaying host females and fertilizing the recovered experimental eggs.
Both oligos gave comparable results, but oligo trim36-3 gave the
most consistent results and was used in the following experiments.
A representative RT-PCR analysis of trim36 mRNA levels in control
and oligo-injected oocytes is shown in Fig. 2A. The doses used in
these experiments (3-4 ng trim36-3) typically depleted 80-90% of
maternal trim36 mRNA.
Embryos derived from the fertilization of oocytes injected with
oligos against trim36 developed normally up to gastrulation, but
were then delayed in blastopore lip formation compared with
DEVELOPMENT
Trim36 in axis formation
Fig. 2. Depletion of maternal trim36. (A) Quantitative real-time PCR
of trim36 expression in oligo-injected oocytes. Samples were
normalized to odc levels and displayed as a percentage relative to the
uninjected oocyte (Un oo) sample. Oocytes were injected with 3.0 or
4.0 ng of trim36-3 oligo. (B) Phenotype of an uninjected stage 24
embryo. (C) Phenotypes of sibling trim36-depleted embryos. (D) In situ
hybridization of sizzled (szl) in an uninjected control embryo. (E) szl
expression in a trim36-depleted embyro. (F) H&E-stained section of a
control embryo, with the neural tube (n.t.) and notochord (n.o.)
indicated. (G) H&E-stained section of a trim36-depleted embryo;
notochord and neural tube are absent, and a somite muscle mass
persists in the midline (arrow). (H,I) Notochord marker Tor70 (green)
and somite marker 12/101 (red) in control (H) and trim36-depleted (I)
embryos. (J) Histogram showing the distribution of phenotypes (see
key) in trim36-depleted embryos (from two experiments).
embryos derived from uninjected sibling oocytes. These trim36depleted embryos eventually completed gastrulation, but in the
majority of cases failed to form neural plates and formed large cysts
beneath the animal pole. By the late neurula and early tailbud
stages, trim36-depleted embryos were radially ventralized in the
extreme cases, or formed partial axes lacking anterior structures in
the less severe cases (Fig. 2B,C). The effects of trim36 oligo
injection were highly consistent over many experiments, resulting
in ~82% of injected embryos with ventralized phenotypes (80/98;
81.6%), compared with a very low incidence of ventralization in
uninjected embryos (1/117; 0.9%). We counted the distribution of
phenotypes in two experiments (Fig. 2J) and found that over twothirds of the embryos with ventralized phenotypes were severely
affected (no axis). The remaining embryos were less severely
affected (partial axis), and only one embryo was unaffected in these
two experiments. We confirmed the presence of excess ventral
tissue by performing in situ hybridization on early tailbud-stage
embryos for sizzled (szl) mRNA (Fig. 2D,E), a marker of belly
tissue (Salic et al., 1997). In line with these results, trim36-depleted
embryos also lacked well-formed neural tubes, somites and
notochords, in some cases forming unorganized lumps of somite
tissue in the midline (Fig. 2F,G). The absence of notochord in
trim36-depleted embryos was verified by double immunostaining
for notochord (Tor70 antibody) and somite (12/101 antibody)
markers on sectioned embryos (Fig. 2H,I).
Development 136 (18)
Fig. 3. Dorsal marker expression in trim36-depleted embryos.
Dorsal genes and β-catenin protein in control (A,C,E,G) and trim36depleted (B,D,F,H) embryos. (A,B) In situs for eomes in stage 12
embryos. Dorsal view, anterior is to the top. Arrow in A indicates eomes
expression in the anterior notochord. (C,D) In situs for myod in stage 12
embryos. Dorsal view, anterior is to the top. (E,F) In situs for nr3 in
stage 9.5 embryos. Vegetal view, dorsal is to the top.
(G,H) Immunostaining for β-catenin in stage 8 embryos. Animal pole
view of cleared embryos, dorsal is to the right. Arrow in G indicates
nuclear β-catenin. (I) Quantitative real-time PCR of dorsal (sia and nr3)
and ventral (szl) markers in control (Un) and trim36-depleted (trim36–)
embryos at stage 10.5.
Since differentiated dorsal structures require signals from the
organizer, we examined the extent of organizer specification in
gastrula-stage trim36-depleted embryos using in situ
hybridization and RT-PCR (Fig. 3). In trim36-depleted embryos,
the notochord expression domain of eomesodermin (eomes) was
lost (Fig. 3A,B), and the expression of myod, an early molecular
marker for somites, was reduced (Fig. 3C,D). Organizer markers
nodal-related 3 (nr3) (Fig. 3E,F) and noggin (not shown) were
severely reduced in early gastrula-stage embryos. Since nr3 is a
direct target of Wnt signaling in the early embryo (Smith et al.,
1995), we also examined the dorsal stabilization of β-catenin
directly by immunostaining. β-catenin was enriched in the dorsal
nuclei of control embryos (Fig. 3G; see Fig. S2A,B in the
supplementary material), whereas this staining was reduced or
absent in trim36-depleted embryos (Fig. 3H; see Fig. S2C,D in the
supplementary material). Consistent with these results, real-time
RT-PCR analysis of gene expression in trim36-depleted embryos
showed much reduced levels of direct Wnt target genes siamois
and nr3, and a concomitant increase in szl expression, which is a
ventral marginal zone marker at this stage (Fig. 3I). Furthermore,
activity of the Wnt/β-catenin reporter, TOPflash, was reduced by
~50% in trim36-depleted gastrulae, further supporting the
deficiency in Wnt/β-catenin signaling at this stage (see Fig. S2E
in the supplementary material).
To confirm the specificity of these phenotypes, we injected a
subset of trim36-depleted oocytes with trim36 mRNA prior to their
maturation and fertilization. Injection of trim36 mRNA reduced the
proportion of ventralized embryos to ~35% (15/43; 34.9%; Fig. 4AC). These rescued embryos also showed partial restoration of wildtype levels of organizer markers sia and nr3 at the gastrula stage
DEVELOPMENT
3060 RESEARCH ARTICLE
Trim36 in axis formation
RESEARCH ARTICLE 3061
Fig. 4. Specificity of trim36 depletion. (A-C) Phenotypes of
representative control uninjected (Un, A), trim36-depleted (trim36–, B)
and rescued (trim36–; + trim36 RNA, C) embryos. (D) Quantitative realtime PCR of dorsal (sia and nr3) and ventral (szl) markers in rescue
experiments (green bars). N.D., not determined.
Wnt/β-catenin signal transduction is not
dependent on Trim36
We next tested the hypothesis that Trim36 might be required within
the Wnt signal transduction cascade. Preliminary experiments
showed that ubiquitous expression of high doses of trim36 (>300 pg)
caused defects in embryo integrity, leading to disaggregation of the
embryos during gastrulation and to epidermal lesions (see Fig.
S4A,B in the supplementary material). Lower doses had no visible
effect on development, and we failed to induce secondary axis
formation by injecting trim36 into ventral vegetal blastomeres, a
well-characterized effect of Wnt ligands and signaling molecules
(data not shown). Trim36 was not sufficient to activate Wnt
signaling in animal cap assays (see Fig. S4C in the supplementary
material) and also failed to rescue β-catenin-depleted embryos (see
Fig. S4D in the supplementary material).
We next stimulated Wnt/β-catenin signaling in trim36-depleted
embryos to determine the activity of Wnt ligands and effectors in the
absence of trim36. Oocytes were injected with the trim36-3 oligo
and were subsequently injected with wnt11 mRNA prior to host
transfer and fertilization. The resulting embryos were analyzed at
the gastrula stage for Wnt target gene expression by RT-PCR and in
situ hybridization, and at the tailbud stage by phenotype
comparisons. In at least three separate experiments, injection of
Fig. 5. trim36-depleted embryos are rescued by wnt11 mRNA.
trim36 was depleted by the injection of antisense oligos into oocytes
and 50 pg wnt11 mRNA was injected prior to fertilization by the hosttransfer method. gsc (A,C,E) and nr3 (B,D,F) expression in stage 10.5
embryos; (A,B) uninjected; (C,D) trim36-depleted (trim36–); and (E,F)
trim36-depleted + 50 pg wnt11 mRNA (trim36–; + wnt11).
(G) Histogram showing the distribution of phenotypes (see key) in
uninjected embryos, trim-36-depleted embryos and trim36-depleted
embryos rescued by wnt11 injection (from two experiments).
(H,I) Quantitative real-time PCR analysis of nr3 expression in control,
depleted and rescued embyros injected with 50 pg wnt11 (H) or 1.0 ng
dngsk3b (I) (green bars).
wnt11 restored the expression of dorsal markers (gsc and nr3; Fig.
5A-F,H), as well as reducing the incidence of axis deficiency (Fig.
5G). Some embryos were mildly dorsalized at a moderate wnt11
dose, whereas higher doses caused severe dorsalization (see Fig. S5
in the supplementary material). wnt11 also rescued notochord
formation in two out of three embryos examined by histology (see
Fig. S5 in the supplementary material). Similar results were seen
using wnt8 (data not shown) and dominant-negative gsk3β
(dngsk3β; Fig. 5I), further supporting the idea that Trim36 acts
upstream of Wnt pathway activation. The expression of dorsal
markers could also be rescued by injection of the BMP antagonist
noggin (see Fig. S6 in the supplementary material), which functions
downstream of Wnt/β-catenin signaling, suggesting that other
pathways controlling dorsoventral patterning are not affected by
trim36 depletion. Overall, these data suggest that Trim36 is not
required for Wnt/β-catenin signal transduction per se, but that it
might lie upstream of or parallel to Wnt/β-catenin signaling.
Trim36 is required for vegetal microtubule array
formation and cortical rotation
Since Trim36 and other Trim proteins with a COS motif associate
with microtubules (Short and Cox, 2006), we investigated a possible
role for Trim36 in microtubule function during cortical rotation. We
generated trim36-depleted eggs through the host-transfer procedure
and immunostained against α-tubulin at ~80 minutes post
DEVELOPMENT
(Fig. 4D). Ventral markers were not rescued, possibly owing to the
complex and indirect regulation of ventral genes, sizzled in
particular, by the organizer (Lee et al., 2006; Collavin and Kirschner,
2003).
Additionally, we obtained similar results using a translationblocking morpholino oligo (see Fig. S3 in the supplementary
material), providing further evidence for specificity. Because
morpholinos do not target the degradation of mRNAs, we can argue
against a structural role for trim36 RNA in axis formation, as has
been suggested for other classes of localized RNAs (Kloc et al.,
2005; Heasman et al., 2001). To summarize, these results show that
trim36 is required, either directly or indirectly, for the stabilization
of β-catenin and for Wnt target gene activation, leading to normal
dorsal tissue formation in Xenopus.
3062 RESEARCH ARTICLE
Development 136 (18)
Fig. 6. Trim36 regulates vegetal microtuble
formation and cortical rotation. (A-C) Immunostaining
of microtubules at 80 minutes post fertilization in control
(A, uninjected), trim36-depleted (B, trim36–) and βcatenin-depleted (C, βcat–) eggs. (D-F) Immunostaining of
cytokeratin at 80 minutes post fertilization in control (D,
uninjected), trim36-depleted (E, trim36–) and β-catenindepleted (F, βcat–) eggs. (G-I) Immunostaining of
microtubules at 80 minutes post fertilization in control (G,
uninjected), trim36-depleted (H, trim36–) and rescued (I,
trim36–; + trim36 RNA) eggs. Vegetal views are shown in
A-I, viewed with a 63⫻ objective. (J-O) Representative
phenotypes of uninjected (J,K), trim36-depleted (trim36–;
L,M) and β-catenin-depleted (βcat–; N,O) embryos. (J,L,N)
Normal orientation, (K,M,O) tilted 90°. (P-S) Frames from
time-lapse movies of cortical rotation in DiOC6(3)-stained
eggs. Controls (P,Q) and trim36-depleted (R,S) embryos at
time 0 (t0) and +10 minutes (t+10⬘). Arrows indicate the
starting points of indicated germ-plasm islands,
arrowheads indicate the final position of the same islands.
Images were taken with a 10⫻ objective.
movies. In movies of uninjected eggs, the germ-plasm islands were
seen moving across the oocyte (see Movie 1 in the supplementary
material; Fig. 6P,Q shows individual frames), whereas in time-lapse
videos of trim36-depleted eggs, germ-plasm islands remained
stationary or moved slowly (see Movie 2 in the supplementary
material; Fig. 6R,S shows individual frames). These results were
seen in all of the videos of individual trim36-depleted eggs (n=4),
from two separate experiments, demonstrating that normal cortical
rotation does not occur in the absence of trim36.
Trim36 depletion is rescued by tipping/gravity
Inclining, or tipping, eggs by 90° can rescue the deficiencies in
dorsal axis formation resulting from microtubule disruption during
the period of cortical rotation (Scharf and Gerhart, 1980; Scharf and
Gerhart, 1983). In order to determine whether the effect of trim36
depletion on microtubule array formation is sufficient to explain the
loss of the dorsal axis, we tested whether orienting trim36-depleted
eggs by 90° could rescue normal dorsal development. As controls,
we included uninjected eggs and β-catenin-depleted eggs within the
same host-transfer experiment. β-catenin depletion does not affect
microtubule formation and is required for the ultimate outcome of
cortical rotation; we thus expected that these eggs would remain
ventralized following inclination. We further expected that if Trim36
were primarily required for microtubule polymerization, then tilting
would be able to rescue dorsal development.
We obtained uninjected, trim36- and β-catenin-depleted eggs by
host transfer and quickly sorted them into dishes containing Ficoll
buffer. Eggs were then manually tipped 90° relative to the animalvegetal axis and left in this position until the first cleavage. A subset
of each experimental group was placed in the same Ficoll buffer
without tipping. These untipped eggs developed as expected;
resulting in normal uninjected embryos and ventralized trim36- and
β-catenin-depleted embryos (10/12 and 8/8 ventralized,
respectively; Fig. 6J,L,N). From two separate tipping experiments,
we recovered a lower proportion of affected trim36-depleted
DEVELOPMENT
fertilization, a time when robust microtubule arrays are present and
cortical rotation is occurring (Elinson and Rowning, 1988). As
controls, we included uninjected sibling oocytes, as well as βcatenin-depleted oocytes, which are also ventralized (Heasman et
al., 1994) but should have normal cortical rotation. These controls
showed robust, normal organization of microtubule arrays (Fig.
6A,C; ~90% normal; n=30 for each). By contrast, trim36-depleted
eggs either lacked microtubule organization completely or exhibited
fragmented microtubules in a loose organization (Fig. 6B; 77%
affected; n=22). As an additional control to ensure that trim36depleted eggs were indeed activated and were otherwise developing
normally, we stained for cytokeratin in the vegetal cortex.
Cytokeratins form parallel arrays similar to microtubules upon
activation (Klymkowsky et al., 1987) and, importantly, these arrays
appear independently of microtubules (Houliston and Elinson,
1991). Consistent with this idea, we found that uninjected, βcatenin- and trim36-depleted eggs all exhibited normal cytokeratin
arrays (Fig. 6D-F; 100% normal; n=16 for each). In a separate series
of experiments, we tested the specificity of this microtubule effect
by examining trim36-depleted eggs that were rescued by injection
of trim36 mRNA. In these experiments, the incidence of aberrant or
absent microtubules was reduced in rescued eggs (46% affected;
n=15; Fig. 6I) compared with trim36-depleted siblings from the
same experiment (74% affected, n=15; Fig. 6H), indicating that the
disruption of microtubules is indeed specific to trim36 depletion.
We next examined the extent of cortical rotation more directly
using time-lapse imaging. For simplicity, we made short time-lapse
movies of prick-activated oocytes stained with DiOC6(3), which
labels the mitochondria associated with the germ plasm and can be
used to follow cortical rotation movements in living eggs (Quaas and
Wylie, 2002; Savage and Danilchik, 1993). We treated uninjected
and trim36-depleted oocytes with progesterone to induce maturation
and 20 hours later we stained them with DiOC6(3) and prickactivated them. At 50 minutes after pricking, we collected images
over 10 minutes from different eggs and assembled these into
Fig. 7. Trim36 ubiquitin ligase activity is required to rescue the
dorsal axis in trim36-depleted embryos. (A) Auto-ubiquitylation of
Trim36. Immunoblotting of Trim36 immune complexes from lysates of
control uninjected embryos (Un), trim36-injected embryos (1 ng
trim36), HA-ubiquitin-injected embryos (1.0 ng HA-ub) or embryos
injected with both trim36- and HA-ub. Top panel shows blotting using
anti-HA, lower panel shows blotting using Trim36 antisera. (B) Trim36
CA/HA is deficient in auto-ubiquitylation. Immunoprecipitation and
blotting as in A. (C-F) Immunostaining of microtubules at 80 minutes
post fertilization. (C) Uninjected eggs (Un), (D) trim36-depleted eggs
(trim36–) and (E,F) trim36-depleted embryos injected with either 200
pg wild-type trim36 RNA (E) or 200 pg trim36 CA/HA (F).
(G) Quantitative real-time PCR analysis of nr3 expression in control
embryos, trim-36-depleted embryos and trim36-depleted embyros
injected with wild-type trim36 RNA or with trim36 CA/HA RNA.
Embryos in G are siblings of the embryos in C-F.
embryos (3/9 ventralized; Fig. 6M) in the tipped group than in the
untipped groups. Tipped β-catenin eggs remained ventralized (5/5
ventralized; Fig. 6O) and tipped uninjected eggs were normal (6/7
normal; Fig. 6K). As an independent marker for dorsal development,
we scored for the presence of notochord in putatively rescued
trim36-depleted embryos. Embryos were fixed at the tailbud stage,
and H&E-stained sections were examined. Both tipped and untipped
uninjected embryos had notochords, and tipped and untipped βcatenin-depleted embryos lacked notochords and somite tissue in
each case (3/3 embryos sectioned in each case; data not shown).
Untipped trim36-depleted embryos lacked notochords, as seen in
earlier experiments. However, tipped trim36-depleted embryos had
clearly distinguishable notochords in all of the embryos sectioned
(n=3; data not shown). These data show that gravity-driven
rearrangements in the egg are sufficient to restore the dorsal axis and
notochord development in the absence of Trim36.
Trim36 ubiquitin ligase activity is required for
microtubule polymerization
Many Trim proteins have ubiquitin ligase activity (Meroni and DiezRoux, 2005), and this has recently been shown for human TRIM36
(Miyajima et al., 2009). To gain insight into Trim36 function, we
asked whether Xenopus Trim36 has ubiquitin ligase activity and
whether this activity is required for microtubule regulation. Since
RESEARCH ARTICLE 3063
the putative substrates for Trim36 are unknown, we first tested the
ability of Trim36 to catalyze its own ubiquitylation, a property of
many ubiquitin ligases. Trim36 mRNA was overexpressed in
embryos in the presence or absence of an mRNA encoding
hemagglutinin (HA)-tagged ubiquitin. Trim36 protein was
immunoprecipitated and the resulting complexes were
immunoblotted with HA antibodies. We detected numerous high
molecular weight bands in immunoprecipitates from co-injected
embryos probed against HA-ubiquitin (Fig. 7A), indicating that
ubiquitin conjugation had occurred. Blotting of the immune
complexes with Trim36 antisera confirmed that equivalent amounts
of Trim36 protein were recovered in each case (Fig. 7A).
To determine the role of Trim36-dependent ubiquitin ligase
activity during cortical rotation, we compared the ability of wild-type
and ubiquitylation-deficient Trim36 forms to rescue the depletion of
maternal trim36. The RING finger and B-box 2 domains have been
implicated in mediating the substrate recognition and ubiquitin ligase
activity of Trim proteins (Meroni and Diez-Roux, 2005). We made
point mutations in conserved cysteine and histidine residues within
the B-box 2 domain of Trim36 (C217A/H220A) that were implicated
in the activity of a related Trim protein, Trim5α (Javanbakht et al.,
2005). This mutant was inactive in gain-of-function assays. We
confirmed that the C217A/H220A mutant was deficient in autoubiquitylation activity, as shown by a reduced amount of ubiquitin
immunoprecipitated along with mutant Trim36 (Fig. 7B) compared
with wild-type Trim36.
We next depleted oocytes of trim36 by antisense oligo injection,
followed by rescue with either trim36 or trim36-C217A/H220A
mRNAs. In two separate experiments, fertilized eggs rescued with
wild-type trim36 showed normal microtubule array formation in at
least half the cases, and had normal nr3 levels. By contrast, depleted
embryos injected with trim36-C217A/H220A did not show either
normal microtubule polymerization (Fig. 7C-F) or nr3 levels (Fig.
7G). Overall, these data suggest that the ability of Trim36 to
ubiquitylate target proteins is necessary for the normal
polymerization and alignment of microtubules during cortical
rotation.
DISCUSSION
trim36 is localized to the germ plasm and encodes
a ubiquitin ligase
We identified trim36 in a microarray screen for RNAs enriched in
the vegetal cortex of Xenopus oocytes. The RNA for trim36 localizes
to the mitochondrial cloud of stage I oocytes and is found in a pattern
identical to that of germ plasm up to stage 11 of embryogenesis.
Although we did not specifically address germ-cell formation in this
study, it is possible that Trim36 also functions in specifying
primordial germ cells. Targeted interference with Trim36 function
in germ-plasm-containing cells could address these questions,
although morpholino inhibition of zygotic Trim36 function disrupts
morphogenesis and somite arrangement (Yoshigai et al., 2009),
making the study of primordial germ cells problematic.
RING finger domain-dependent E3-ubiquitin ligase function is
characteristic of Trim proteins (reviewed by Meroni and Diez-Roux,
2005). Our results, and those of a recent study on human TRIM36
(Miyajima et al., 2009), show that Trim36 has ubiquitin ligase
activity both in vivo and in vitro, although the targets of Trim36
ubiquitylation have not yet been identified. Interestingly, a mutant
Trim36 with substitutions in the B-box domain, which mediates
substrate recognition, was unable to rescue dorsal axis formation,
suggesting that Trim36 could not be recruited to the relevant protein
complexes.
DEVELOPMENT
Trim36 in axis formation
A subset of Trim proteins (including Trim36 and Trim18) contain
a COS motif, which confers localization to microtubules (Short and
Cox, 2006). Interestingly, TRIM18 mutations in Opitz syndrome
patients result in the accumulation of PP2A in the microtubule
fraction, leading to the dysregulation of microtubule dynamics
(Trockenbacher et al., 2001). By analogy, Trim36 could regulate the
microtubule-associated fraction of a protein involved in microtubule
stability or organization, and potentially Wnt signaling.
Trim36 is required for cortical rotation and axis
determination
Trim36-depleted embryos do not form a normal vegetal microtubule
array, and do not undergo cortical rotation. These effects of Trim36
depletion can be rescued by re-orienting the eggs prior to the first
cleavage by 90°, a treatment that overcomes the effects of UV
irradiation. Our results thus suggest that Trim36 is not required for
the synthesis of a component needed for dorsal axis formation.
Furthermore, Wnt11 rescues trim36 depletion, ruling out a role for
Trim36 in the regulation of the core Wnt/β-catenin signaling
components.
It is intriguing that injection of Wnt11 into trim36-depleted
oocytes, which would be expected to generate spatially unrestricted
expression, results in the rescue of regional organizer gene
expression and a single axis (Fig. 5). Wnt signaling induces a
number of autoregulatory molecules, so it is possible that small
differences in the abundance of injected Wnt could be regulated to
result in a single active domain. Alternatively, Brown et al. (Brown
et al., 1994) have shown that mature eggs have cryptic asymmetry,
which could bias where the axis forms in the presence of a uniform
signal. Taken together, our data suggest a model in which Trim36 is
required to stabilize or align microtubules in the vegetal cortex
region, resulting in the proper distribution of wnt11 mRNA and/or
β-catenin stabilizing agents in the embryo.
Our results are reminiscent of data obtained by depletion of
another germ-plasm-localized mRNA, fatvg (Chan et al., 2007), but
with several important differences. fatvg-depleted embryos fail to
undergo cortical rotation (microtubules were not examined) and
exhibit stabilized β-catenin at the vegetal pole, which is essentially
a phenocopy of UV irradiation (Darras et al., 1997; Medina et al.,
1997). Our results differ, as we see an overall loss of β-catenin and
Wnt target gene expression, in addition to a loss of cortical rotation.
It is widely assumed that microtubules have a role solely in
transporting dorsalizing molecules; however, our results suggest that
the transport and activation of dorsalizing molecules might be
closely coupled. A more thorough and consistent comparison of the
phenotypic effects of disrupting microtubules and/or directional
transport is needed to better understand how these processes are
linked.
Is the role of Trim36 in axis formation conserved? The
polymerization of microtubules at the vegetal pole of fertilized eggs
has been observed in many chordate species (Eyal-Giladi, 1997).
The Trim36 protein sequence is well conserved among vertebrates;
however, our work is the first to describe its function in early
development. It will be of interest to discover whether Trim36 has a
wider role in early embryonic patterning, or whether it is required
only in animals with cortical rotation. In either case, comparison of
Trim36 function will help to better understand the complex problem
of symmetry breaking in eggs.
Acknowledgements
The authors would like to thank J. Gurdon, R. Harland and S. Piccolo for
reagents; I. Dawid and S. Piccolo for discussions on ubiquitin ligases; and
members of the Houston lab for critical reading of the manuscript. The
Development 136 (18)
monoclonal antibodies 12/101 and 1h5, developed by J. P. Brockes and M.
Klymkowsky, respectively, were obtained from the Developmental Studies
Hybridoma Bank (DSHB), developed under the auspices of the NICHD and
maintained by The University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242, USA. This work was supported by The University of Iowa and
by NIH grant 5R01 GM083999-02 to D.W.H. Deposited in PMC for release
after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/18/3057/DC1
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DEVELOPMENT
Trim36 in axis formation