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PLOS One logoLink to PLOS One
. 2007 Feb 14;2(2):e213. doi: 10.1371/journal.pone.0000213

Xnrs and Activin Regulate Distinct Genes during Xenopus Development: Activin Regulates Cell Division

Joana M Ramis 1, Clara Collart 1, James C Smith 1,*
Editor: Thomas Zwaka2
PMCID: PMC1790703  PMID: 17299593

Abstract

Background

The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type β family such as Vg1, activin B, the nodal-related proteins and derrière.

Methodology and Principle Findings

Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17α/β, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited.

Conclusions/Significance

These observations reveal distinct functions for these two classes of the TGF-β family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.

Introduction

The mesoderm of the amphibian embryo arises through an inductive interaction in which cells of the vegetal hemisphere act on overlying equatorial cells [1]. Of the several mesoderm-inducing factors that have been discovered, most are members of the transforming growth factor type β family. These include activin [2][4], Vg1 [5], [6], five nodal-related proteins [7][9], and derrière [10]. Although these factors have similar abilities to induce gene expression in isolated animal pole regions, they are differently expressed in the embryo (see above references) and under some experimental conditions have different abilities to exert long-range effects [11], [12]. In addition, each exerts different effects at different concentrations [7], [13]. The challenge now is to elucidate the individual roles of these proteins within the embryo and to ask how their actions are coordinated.

Some attempts along these lines have been made, and it proves that although each of the factors is essential for normal development, their roles differ. For example, ablation of the maternal transcripts encoding Vg1 causes a reduction in anterior and dorsal development and the down-regulation of genes such as chordin, cerberus and noggin [6]. Of the zygotically-expressed inducing factors, depletion of activin also causes axial defects [3], [14], [15], although these are less severe than those caused by loss of Vg1, and inhibition of derrière activity causes just posterior defects [10]. Simultaneous inhibition of the activities of all the nodal related proteins, by expression of Cerberus-short, causes loss of mesoderm [16], [17] and the down regulation of genes such as Chordin and Pintallavis [18]. The requirements of the individual nodal related proteins have not been studied in detail, although injection of antisense morpholino oligonucleotides directed against Xnr1 causes defects in left-right axis determination [19].

Here we perform microarray analyses of gene expression in embryos in which activin or nodal-related signalling has been inhibited. We find that activin and the nodal-related proteins regulate distinct and almost completely non-overlapping sets of genes, with those regulated by the nodal-related genes tending to be expressed in a more restricted pattern than those regulated by activin. It further proved that the nodal-related proteins often regulate the expression of genes involved in regional specification, while activin particularly regulates genes involved in the control of the cell cycle. Consistent with this observation, we find that inhibition of activin B in the early embryo causes dorsal axial mesodermal cells to fail to exit the cell cycle: the results of others [20][22] suggest that it is the continued proliferation of these cells that underlies the gastrulation defects observed in such embryos.

Results

Microarray results

In an effort to understand the different requirements for activin B and the nodal-related genes during Xenopus development, we have carried out microarray analyses of gene expression in embryos in which signalling by the two classes of factor has been disrupted. Activin signalling was blocked using an antisense morpholino oligonucleotide [3], and nodal-related signalling by Cerberus-short, a truncated form of Cerberus [17]. Our microarray slides comprise 10,898 probes designed to recognise sequences derived from a large scale Xenopus tropicalis EST project [23]. These arrays also recognise X. laevis transcripts [24].

For each series of experiments Xenopus laevis embryos from three different spawnings were injected with RNA encoding Cerberus-short (150 pg into each blastomere at the four-cell stage) or with antisense morpholino oligonucleotide MO3 (50 ng into the one-cell stage) (samples), or with water or antisense morpholino oligonucleotide mMO1 (50 ng) (controls). These doses of Cerberus-short RNA and MO3 were based on previous work [3], [16] and were chosen so as to yield a strong phenotype in which gastrulation was substantially or completely inhibited. In an effort to identify early and perhaps direct targets of activin and the nodal-related proteins, embryos were cultured to stage 10.5 for RNA isolation and some were allowed to develop to later stages to confirm that embryos displayed the expected phenotypes (Fig. 1A–F). Each microarray slide was hybridised with a 1∶1 mixture of sample and control cDNAs, each labelled with a different dye. Each hybridisation was repeated with the Cy3 and Cy5 dyes ‘swapped’, so that six microarray slides were hybridised for each experiment.

Figure 1. Inhibition of activin B and nodal-related protein function causes distinct phenotypes and results in the differential regulation of different classes of gene.

Figure 1

(A,D) Control embryos (here injected with water; those injected with mMO1 look identical) at stage 11 (A) and 26 (D). (B,E) Embryos injected with MO3, and which therefore lack activin B activity. (B) Stage 11; (E) stage 21. Note the delay in gastrulation and the failure to form a proper axis. (C,F) Embryos injected with Cerberus-short RNA, and which therefore lack nodal-related activity. Note the failure to involute and the formation of a radially symmetrical structure. (G,H). Correlation between microarray and PCR results.

Transcripts recognised by the oligonucleotides were considered to be differentially expressed when (i) they showed at least a two fold difference (sample versus control) in expression levels in at least four out of the six microarrays and (ii) were significantly different (q = 0; see Experimental procedures). In embryos in which activin B signalling was inhibited, 40 oligonucleotides fulfilled these rigorous criteria, of which 8 were down regulated, and in those in which nodal signalling was inhibited, 20 oligonucleotides (representing 18 genes) were differentially expressed, of which 17 were down regulated (Table 1). The up regulation of Cerberus in the latter experiment is probably due to the introduction of Cerberus-short mRNA into these embryos. Only Sizzled, which encodes an inhibitor of the Tolloid Proteinase [25], was differentially expressed in both types of embryo.

Table 1. Genes regulated by activin B and nodal-related proteins in the Xenopus embryo.

Qiagen Xt oligo name Gene name Accession No. X. tropicalis Accession No. X. laevis Log2 (sample/control) microarray Log2 (MO3/mMO1)RT-PCR Log2 (CerS/control)RT-PCR Expression pattern at stage 10.5
Genes regulated by activin B
Xt_10009473 PBK CR761713 BC088936 2.3 2.7 −0.1 Ubiquitous (this paper)
Xt_10009228 RPN2 CR848133 BC046727 2.2 1.4 −0.1 Ubiquitous (this paper)
Xt_10000757 GADD45G CR761710 BC078567 1.9 2.3 0.2 Ubiquitous (this paper)
Xt_10004273 TPX2 CX840441 AF244546 1.8 0.2 −0.2 Ubiquitous (this paper)
Xt_10000182 MAPKBP1 AL853880; AL901553 BC076779 1.6 1.9 −0.3 ND
Xt_10006424 unknown CT030473 no homology 1.6 ND ND Ubiquitous (this paper)
Xt_10005146 H2BFS CR760086 XLHISH3A 1.6 0.2 0.2 Ubiquitous (this paper)
Xt_10009545 ADIPOQ DT438351; DT438350 BC094476 1.5 1.3 −0.1 ND
Xt_10006410 unknown DR834759; DR834758 no homology 1.5 ND ND ND
Xt_10000971 Serpina3 BC087988 BC084845 1.5 ND ND ND
Xt_10000346 H1FOO CR761180 X13855 1.4 0.2 0.4 Ubiquitous (this paper)
Xt_10004307 unknown AL790455; BX693495 no homology 1.4 ND ND ND
Xt_10010739 BC052883 CX363787; CX363786 AF035443 1.3 0.6 0.0 Ubiquitous (this paper)
Xt_10008973 DNMT1 CT025477 BC072774 1.3 0.2 0.3 Ubiquitous (see Fig. 2)
Xt_10005756 GPR4 CR761039 AY766161 1.3 0.4 −0.5 Ubiquitous (this paper)
Xt_10000636 unknown AL956096; BX753658 BC085023 1.3 ND ND ND
Xt_10005344 RASD1 DR842169; DR842168 BC081268 1.2 1.2 −0.1 ND
Xt_10000635 PCOLN3 BX708936 BC068657 1.2 1.5 −0.1 Ubiquitous (this paper)
Xt_10005362 Sizzled AL639345 AF136184 1.2 1.4 1.0 Restricted [25]
Xt_10001337 nucleoplasmin NM_001016938 BC072778 1.2 1.2 −0.1 Ubiquitous (this paper)
Xt_10008633 KRT24 CX745012; CX745011 BC043901 1.2 1.0 0.1 Ubiquitous (this paper)
Xt_10003301 unknown DR871833; DR871832 no homology 1.2 ND ND ND
Xt_10008086 PCQAP CX911575; CX911574 BC070536 1.2 0.0 0.2 ND
Xt_10007252 unknown CX961230; CX961229 BC054976 1.2 ND ND Ubiquitous (this paper)
Xt_10004134 TUBA1 CT030272 Z31591 1.1 1.1 −0.1 Ubiquitous (this paper)
Xt_10002761 unknown DR880099; DR880098 no homology 1.1 ND ND Ubiquitous (this paper)
Xt_10000615 unknown CR761187 BC097911 1.1 3.3 −0.2 Ubiquitous (this paper)
Xt_10008957 FAM3A CR761057 BC108550 1.1 0.7 0.1 Ubiquitous (this paper)
Xt_10004044 Eomesodermin CX814795; CX814794 BC084243 1.1 1.4 −0.7 Restricted [41]
Xt_10002067 C2orf28 CF591510 BC094117 1.0 0.8 −0.1 Ubiquitous (this paper)
Xt_10008956 Cdc6 CR761778 AY222352 1.0 1.1 0.1 Ubiquitous (this paper)
Xt_10004730 unknown BQ390504; BQ390503 no homology 1.0 ND ND Ubiquitous (this paper)
Xt_10002154 MPDU1 CR761821 BC108439 −1.5 −1.4 0.0 Ubiquitous (this paper)
Xt_10009727 DHCR7 BQ394956; BQ394955 BC054203 −1.2 −1.7 −0.5 Ubiquitous (this paper)
Xt_10000076 cyclin D1 BQ522031; BQ522030 BC041525 −1.2 −2.0 0.3 Ubiquitous (this paper)
Xt_10002938 MRPL12 CR855493 BC084828 −1.2 −0.9 −0.2 Ubiquitous (this paper)
Xt_10010347 EMP2 CT025318 BC106297 −1.2 −1.5 0.1 Ubiquitous (this paper)
Xt_10009006 SOX2 CR760314 AF005476 −1.1 −1.0 0.3 Ubiquitous [42]
Xt_10008667 ATP1A1 CR926442 U49238 −1.1 −1.3 −0.6 Restricted [42]
Xt_10005487 FKBP1B CT025367 AB006678 −1.0 −1.0 0.2 Ubiquitous (this paper)
Genes regulated by nodal-related proteins
Xt_10003376 unknown DN089489; DN089488 no homology 3.2 ND ND ND
Xt_10002006 Cerberus NM_203515 BC081277 2.5 −0.4 3.6 Restricted [43]
Xt_10005362 Sizzled CR761702 AF059570 1.0 1.4 1.0 Restricted [25]
Xt_10010306 unknown DN030301; DN030300 no homology −3.0 ND ND ND
Xt_10008637 darmin CX493718 BC055979 −2.8 ND ND Restricted [44]
Xt_10000401 HEX CR761571 U94837 −2.6 0.1 −2.3 Restricted [45]
Xt_10005916 GATA4 NM_001016949 DQ096869 −2.4 −0.2 −2.2 Restricted [42]
Xt_10001409 Xsox17-beta BX762953 BC070615 −2.2 0.0 −2.3 Restricted [46]
Xt_10000180 Xsox17-beta CR848411 BC070615 −1.8 0.0 −2.3 Restricted [46]
Xt_10009950 Xdkk-1 NM_001016283 AF030434 −2.1 0.3 −2.3 Restricted [47]
Xt_10009377 GATA6 CT030595 BC082349 −1.9 −0.8 −1.9 Restricted [48]
Xt_10009394 Xsox17-alpha BC074527 BC106403 −1.8 0.1 −2.3 Restricted [46]
Xt_10000572 Xiro3 BC067972 AF027175 −1.7 −1.4 −1.7 Restricted [49]
Xt_10010791 Xiro3 BC067972 AF027175 −1.6 −1.4 −1.7 Restricted [49]
Xt_10000293 Frzb precursor CR761513 U78598 −1.7 −0.4 −1.5 Restricted [42]
Xt_10006059 ApoB BC075459 BC074467 −1.5 −1.1 −1.6 Restricted (http://Xenopus.nibb.ac.jp/)
Xt_10003855 chordin CR761722 BC077767 −1.3 −0.6 −1.6 Restricted [50]
Xt_10010647 PDGFRA CR761598 M80798 −1.3 −0.4 −1.9 Restricted [51]
Xt_10004020 unknown NM_001015997 BC097726 −1.3 −1.0 −1.3 ND
Xt_10006733 XFz8 DT402720; DT402719 AF017177 −1.3 1.2 −1.5 Restricted [52]

Up regulated genes are shown in green and down regulated genes in red. The Table also shows the RT-PCR data plotted in Fig. 1 and used to validate the microarray results. The data confirm that genes regulated by activin signalling are not regulated by nodal-related signalling, and vice-versa. A description of the expression pattern of each gene is indicated (‘ubiquitous’ or ‘restricted’).

ND: Not determined.

Our experiments identify fewer nodal-regulated genes than the recent microarray study of Sinner and colleagues [26]. This difference probably derives from the facts that Sinner and colleagues harvested embryos at stage 11 rather than 10.5, and defined genes as being differentially expressed if expression levels differed by a factor of 1.4 rather than 2.0. Like Wessely and colleagues, who used a macroarray approach [18], we note that both Chordin and Xsox-17beta are down regulated by Cerberus-short. We also note that some genes that are down regulated following interference with activin signalling, such as Xbra and goosecoid [3], were not identified in the present screen. The most likely explanation for this apparent discrepancy is that the expression of such genes is frequently reduced by only 50% or thereabouts [3], and our criteria for defining genes as being differentially expressed (see above) is so stringent that such differences might be regarded as insignificant. RT-PCR analysis of the RNA samples used on the microarrays confirmed previous observations [3] that the expression of these genes is indeed reduced in embryos in which activin signalling is inhibited (data not shown).

Real-time RT-PCR validation

Our microarray results were validated by real-time RT-PCR The X. laevis homologues of the X. tropicalis cDNAs recognised by the oligonucleotides (http://informatics.gurdon.cam.ac.uk/cgi-bin/public.exe) were identified by BLAST searches (Table 1), and PCR primers were designed for the great majority of the transcripts that were considered to be differentially expressed. In the case of the activin B experiment, we were unable to identify X. laevis homologues for six of the cDNAs, and two primer pairs did not yield a product; in the case of the Cerberus-short experiment, X. laevis homologues could not be identified for two cDNAs.

Our RT-PCR analysis used the same RNA samples that were used for microarray experiments. Of the genes tested, 80% of those identified in the activin B experiment were confirmed as being differentially expressed, and all of those identified in the Cerberus-short experiment were similarly verified. Bilateral correlation analysis of the results obtained by microarray hybridization and those obtained by real-time RT-PCR showed a Pearson Correlation of 0.848 (p = 0.000) for the activin B experiment and of 0.975 (p = 0.000) for the Cerberus-short experiment (Fig. 1G,H). RT-PCR experiments confirmed that genes regulated by activin signalling are not regulated by nodal-related signalling, and vice-versa (Table 1). Together, these experiments show that activin and the nodal-related genes regulate distinct genes during early Xenopus development.

Classification of genes regulated by activin and nodal-related genes

The expression pattern of each differentially expressed gene was determined from the literature, where possible, or by carrying out in situ hybridisations using Xenopus tropicalis embryos with probes generated by the polymerase chain reaction (PCR). Consistent with the different expression patterns of activin B and of the nodal-related genes [3], [7][9], [27], the expression patterns of the genes regulated by the two types of signalling molecules differed (see Table 1). Thus, of the 15 different genes regulated by nodal-related signalling whose expression patterns we know, all are expressed in a restricted fashion (for example, see Fig. 2A,B), and of the 31 genes regulated by activin B, 28 are expressed ubiquitously (for example, see Fig. 2C–F) and three in a restricted fashion.

Figure 2. Expression patterns of genes regulated by activin and nodal-related proteins.

Figure 2

(A,B) Expression pattern of Chordin, a gene that is mis-regulated following inhibition of Xnr signalling. Note that Chordin transcripts are restricted to the dorsal marginal zone. (C–F) Expression pattern of DNMT1, a gene that is mis-regulated following inhibition of activin signalling. (C) and (D) show embryos hybridised using a sense probe; (E) and (F) show embryos hybridised using an antisense probe. Note that DNMT1 is expressed ubiquitously.

Genes were then manually classified according to the annotation of their human homologues (NCBI databases, http://www.ncbi.nih.gov/). Interestingly, this analysis also revealed differences between embryos lacking activin B and those in which nodal related signalling is inhibited (Fig. 2G). In particular, while several of the genes regulated by the nodal-related genes are involved in signal transduction or the regulation of transcription, several of the genes whose expression is affected by lack of activin B activity are involved in cell cycle regulation; this is not the case for embryos in which nodal signalling is inhibited.

Activin regulates cell division in the involuting mesoderm

Both our microarray experiments and our real-time RT-PCR analyses show that down-regulation of activin B, but not loss of nodal-related activity, causes the mis-regulation of genes involved in cell cycle control. One of the effects of the loss of activin B function is a disruption of gastrulation [3], and in this connection we note that the mitotic index of involuting dorsal mesoderm is significantly decreased during gastrulation [28] and that arrest of the cell cycle is required for both bottle cell formation [20] and for convergent extension movements [21], [22]. We therefore asked whether loss of activin B affects cell division during early embryogenesis.

Embryos injected with control oligonucleotide mMO1 or specific antisense oligonucleotide MO3 were fixed at the mid gastrula stage and stained using an antibody recognising phosphorylated histone H3, which marks mitotic chromosomes [28]. Inspection of such embryos revealed that the down-regulation of the cell cycle that normally takes place in dorsal axial mesoderm does not occur (Fig. 3). In three control embryos stained as sections the mean mitotic index in dorsal axial mesoderm was 0%; in six embryos injected with MO3 the mitotic index was 12.7±2.7% (mean±standard deviation). Similarly, in a control embryo stained as a whole-mount and then sectioned, the mitotic index was 0%; in an embryo injected with MO3 it was 20%. This failure of the dorsal axial mesoderm to undergo cell cycle arrest is consistent with the observed mis-regulation of cell cycle genes, and it may explain why embryos lacking activin function fail to gastrulate properly [see refs 20][22].

Figure 3. Inhibition of activin B function prevents dorsal axial mesoderm from exiting the cell cycle.

Figure 3

(A) Diagram illustrating from which part of the embryo sections in (B–E) are derived. (B,C) Composite images of 10 serial sagittal sections of representative embryos stained with an antibody recognising phosphorylated histone H3 as whole mounts and then sectioned at 12 µm. (B) Control embryo injected with mMO1. Note absence of mitotic cells in involuting mesoderm. (C) Embryo injected with specific antisense oligonucleotide MO3. Involution is perturbed and mitotic cells are visible in dorsal tissue. (D,E) Frozen sections of embryos stained with an antibody recognising phosphorylated histone H3. (D) Control embryo injected with mMO1. Note absence of mitotic cells in involuting mesoderm. (E) Embryo injected with specific antisense oligonucleotide MO3. Involution is perturbed and mitotic cells are visible in dorsal tissue.

Discussion

Our experiments show that activin B and the nodal-related proteins regulate distinct sets of genes in the early Xenopus embryo. In the future it will be interesting to investigate the molecular basis of this difference. One difference between activin and the nodal-related proteins is that their expression patterns differ, with activin B being expressed ubiquitously [3], [27] and the nodal-related proteins being restricted to the vegetal and equatorial regions of the embryo [7][9]. Consistent with these observations, we note that nodal-regulated genes tend to be expressed in more restricted patterns than do activin-regulated genes (Fig. 2A–F). Another difference is that signalling by the nodal-related proteins, but not activin, requires responding cells to express EGF-CFC family members such as XCR1, 2 and 3 [29][32]. This difference between activin and the nodal-related proteins may underlie the ability of activin to activate Smad2 earlier than does Xnr1 or derrière [33]. We note that other studies have also noted differences between activin and nodal signalling; for example, continuous treatment of P19 cells with activin causes only transient activation of Smad2 while treatment with nodal causes sustained activation [32].

Of the genes that are exclusively regulated by activin, several have been implicated in cell cycle regulation (Fig. 2G), and embryos that lack activin B function fail to arrest the cell cycle in dorsal axial mesoderm (Fig. 3). These observations indicate that the role of activin B differs from that of the nodal-related proteins in the early Xenopus embryo, and that one of its functions is to control the cell cycle during this critical phase of early Xenopus development. This is of importance because axial mesodermal cells arrest the cell cycle after involution [28], and if they are forced to proliferate, this results in a severe disruption of gastrulation [20][22]. Interestingly, we note that the ability of activin to inhibit cell division is not restricted to the early Xenopus embryos; activin also causes cell growth arrest in human breast cancer cells and in human hepatocytes [34], [35].

We note no effect of the loss of activin on the cell cycle elsewhere in the Xenopus embryo; there is no acceleration of cell division in the animal hemisphere, for example, in embryos injected with MO3. It is likely that the cell cycle in the dorsal marginal zone is regulated through locally-acting mRNAs or proteins that require activin signalling for their expression or appropriate post-translation modification.

Finally, what do our results say about the role of activin in mesodermal patterning? Although we emphasise here the role of activin in controlling the expression of genes involved in the regulation of the cell cycle, our previous data, confirmed in the course of the present work (data not shown), indicates that in the absence of activin the expression of genes such as goosecoid, chordin and Xbra is reduced by 20–80%, depending on stage and dose of antisense morpholino oligonucleotide [3]. These observations suggest that activin and the nodal-related proteins (together with Vg1 and derrière) cooperate to specify mesodermal pattern in the embryo, although the results described in this paper argue that the role of activin in this process is less significant than is the role of the Xnrs.

Materials and Methods

Xenopus embryo manipulations and microinjection

Embryos of Xenopus laevis were obtained by artificial fertilisation, maintained in 10% normal amphibian medium [36], and staged as described [37]. For inhibition of nodal-related protein function, embryos were injected at the one cell stage with 600 pg Cerberus-short RNA [17] or, as a control, water. For inhibition of activin B, embryos were injected with 50 ng antisense morpholino oligonucleotide MO3 [3] or, as a control, mMO1 [3]. Embryos were harvested at stage 10.5 for microarray analysis or stage 12 for immunocytochemistry.

Microarray construction, RNA isolation, labelling and microarray hybridisation

These were performed as described [24].

Microarray data analysis

Microarray results were imported into Acuity (Axon) and normalised using Lowess normalisation. Data files were created for points which satisfied the following filter: (Sum of Medians) ≥500 AND (Flags) ≥0 AND (%>B532+1SD)≥55 OR (%>B635+1SD)≥55. This filter eliminates data points flagged as bad by GenePix, or that had the sum of media less than 500, or which had fewer than 55% of pixels above background. Points passing these criteria for at least four out of the six microarrays were used for further analysis. Oligonucleotides were considered to be differentially expressed when they showed at least a two fold difference in expression levels in four out of the six microarrays and had a q value of 0 as assessed by the Significance Analysis of Microarrays software [38]. The microarray datasets were deposited in the GEO data repository (http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi) (accession numbers GSE4771 and GSE4777).

Real time RT-PCR

Differential expression was validated by real-time RT-PCR using the Roche LightCycler 480. Primers specific for ornithine decarboxylase (ODC) were as described [3]; others are listed in Table 2.

Table 2. RT-PCR primers used in this study.

Qiagen Xt oligo name Forward primer Reverse primer
Xt_10000076 CCAGACATTTGTTGCCCTCT GTTGTGTTGCTGCTGTGCTT
Xt_10000180 TTATGGTGTGGGCAAAGGAC CTCTTCCCTCTTCATCCTCTTC
Xt_10000182 CCACAGAGTGAAGCACCTGA AAAACTCAAAAAGAGCCACACTT
Xt_10000293 TGTACCATCGATTTCCAGCA TCACATGCCAGGCTCTCTG
Xt_10000346 TAAGAAGGCAGTTGCTGCAC CCTTCTCTAGCCCTTTGTTCA
Xt_10000401 GCGAGAGACAGGTCAAAACC TTCAATGTCCACCTCCTGGT
Xt_10000572 AGGTGTCCACCTGGTTTGCT TCAGTGTCTGGGTCATCCAA
Xt_10000615 GCCCCAGAACCACTAAGTAAC CCTGGACCACCATCTCTGAA
Xt_10000635 AATGGCTTCACGGGTAGATG AAGCTTTGTCCAGTGCCTTG
Xt_10000757 AGCCCTTCAGATCCACTTCA GCATCCTCATTTGGATTCGT
Xt_10000971 CCTGAACTGGGAAAAATCCA AATTCCCATTCCCATGTCAG
Xt_10001337 TCCCTTATATGGGGGTGTGA GGAACTCATCCTTTGCCTTG
Xt_10002006 GAATGGAGCCCCACAGAATA TTGCTGATTTGGAACATGGA
Xt_10002067 CTGGACCTGTGGAACTGCTC CAACAAGCCACGGAAAAACT
Xt_10002154 TCGGATTCCTTATCCAGCAC GCCTGCATAGCCGTAATCAT
Xt_10002938 GAGATATCCACGGTCAGGTTG AGCAGAGTAAGGCTGGCAAT
Xt_10003855 AACTGCCAGGACTGGATGGT GGCAGGATTTAGAGTTGCTTC
Xt_10004020 TCGTCTTGATGGCTGTGTTC GTGGAGACCTGCATTTCGTT
Xt_10004044 CCTACCCAAGGACAAGGTCA TGAAAGGCAAACCCACTTTT
Xt_10004134 AGAGTTCCAAACAAACTTGGTG CTGGCACAGATAGCTGCTCA
Xt_10004273 AAGCCCAAGCTCGTAGAACA CGGCTGAGCCTTGAATTTAG
Xt_10005146 GATACCGGCATCTCTTCCAA ATGGTGGAGCGCTTGTTGTA
Xt_10005344 ATGTGGATGTTCCCATCGTT GTCTGGGCTCATCTCACTGG
Xt_10005362 AACAAGGTCTGCTCCTTCCA ATGGTGTCTCCACCTCCTTG
Xt_10005487 ACAGATGAGTGTGGGGCAGA GCTCCACATCAAAGGTCAGG
Xt_10005756 GTGGGCTTCTTCTTCAATGC GAGTGAGTGCCCAGGATGAT
Xt_10005916 GCTTAAAACTCTCGCCACAGA TGCTTTAAGCTAAGACCAGGTTG
Xt_10006059 CTTTACATCTGTCCTGCCTCA TAGTCAGCACCCCTCATCAT
Xt_10006733 GGTGCCCAGCATCAAATCTA GAACATGCTGCCAATGAACA
Xt_10008086 CACACCAAGTCAAGCAAGGA TCCTTGCCCACCAACTACAG
Xt_10008633 AGTTCTGCAGGTGGTTTTGG GCAAGACGGTCATTGAGGTT
Xt_10008637 AAGTTCTGTTATCCCCTGTGC TTTCTATTGCCACCCAGTCC
Xt_10008667 CGACATGATCCTGTTGGATG TCTGTGCCCAGATCGATACA
Xt_10008956 CAGAAACTGCTGGTCTGTGC ATCCCGCTCCTCTATCTTGA
Xt_10008957 GGACTCAATGTGGCTCTTGT GCCCAACTGTCTCTGAAACC
Xt_10008973 TTTGGAGAGGGATCAGGATG AGGTATCCTTCCTCAGACAGTTC
Xt_10009006 GTCAAGTCGGAATCCAGCTC TTCTGCCCCAGGTAGGTACA
Xt_10009228 ATCCGCTCCAATGTTGACTC GTGAGCAAGGCTTCAATGGT
Xt_10009377 CCTTTCTGACTTTTGCACAGC GGCAAAGTCTGTTGGATGGT
Xt_10009394 GGTTACAGTTTGCCCACTCC GTAGGGCATCATCTGGCACT
Xt_10009473 GGCAGAGAACATGGCAAGAG AGGCCGAATGCATAGATGTC
Xt_10009545 CCAGTCGATGGGCTGTATTT TTTGTCACCGACAACCTGAA
Xt_10009727 TGGGTCTCCTTCCAGGTGTT AGGTGGGTGATGGTCCAG
Xt_10009950 CACGGGCTAGAGATTTTCCA GGCCTCGCTTAGTGTCTTTG
Xt_10010347 AGACAATGCCTGGTGGGTAG GTTGCCTGGATGGTCTGAAT
Xt_10010647 GAATGGCAAAACCTGACCAT GCGAGTAACTGCAGGGTGAT
Xt_10010739 CCCCTTATACCCCAAAGAGC ATGTTGGTCTCCCGTAACAC

In situ hybridisation

This was carried out on embryos of Xenopus tropicalis, essentially as described [39], [40]. Probes were made by use of T7 RNA polymerase; substrates were PCR products obtained using T7 and SP6 primers applied to cDNA clones derived from a large scale Xenopus tropicalis EST project [23].

Immunocytochemistry and Image Acquisition

Embryos to be subjected to frozen sectioning were fixed in 3.7% formaldehyde, 10% DMSO, 100 mM MOPS pH7.4, 2 mM EGTA, 1mM EDTA for 2 hr at room temperature and embedded in 25% sucrose, 15% cold water fish gelatin (Sigma) at room temperature for 24 hr. Sections (14 µm) were cut at −17°C and stored at −80°C. They were incubated overnight at 4°C with anti-phosphohistone H3 antibody (Upstate Biotechnology, 1∶1000) and then with anti rabbit IgG antibody coupled to Alexa 568 (Molecular Probes, A11011, 1∶200). Nuclei were counterstained with DAPI.

Whole-mount immunostaining using anti-phosphohistone H3 antibody was performed as described [28].

Acknowledgments

We thank our colleagues James Smith, Martin Roth, Mike Gilchrist and Rick Livesey for advice. We are also grateful to Eddy De Robertis for Cerberus-short and Roger Pedersen and Derek Stemple for helpful discussions.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work is supported by the Wellcome Trust, an EC Marie Curie Individual Fellowship to JMR, and the EC Network of Excellence ‘Cells into Organs’.

References

  • 1.Heasman J. Patterning the Xenopus blastula. Development. 1997;124:4179–4191. doi: 10.1242/dev.124.21.4179. [DOI] [PubMed] [Google Scholar]
  • 2.Asashima M, Nakano H, Shimada K, Kinoshita K, Ishii K, et al. Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). Roux's Arch Dev Biol. 1990;198:330–335. doi: 10.1007/BF00383771. [DOI] [PubMed] [Google Scholar]
  • 3.Piepenburg O, Grimmer D, Williams PH, Smith JC. Activin redux: specification of mesodermal pattern in Xenopus by graded concentrations of endogenous activin B. Development. 2004;131:4977–4986. doi: 10.1242/dev.01323. [DOI] [PubMed] [Google Scholar]
  • 4.Smith JC, Price BM, Van Nimmen K, Huylebroeck D. Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A. Nature. 1990;345:729–731. doi: 10.1038/345729a0. [DOI] [PubMed] [Google Scholar]
  • 5.Weeks DL, Melton DA. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell. 1987;51:861–867. doi: 10.1016/0092-8674(87)90109-7. [DOI] [PubMed] [Google Scholar]
  • 6.Birsoy B, Kofron M, Schaible K, Wylie C, Heasman J. Vg1 is an essential signaling molecule in Xenopus development. Development. 2006;133:15–20. doi: 10.1242/dev.02144. [DOI] [PubMed] [Google Scholar]
  • 7.Jones CM, Kuehn MR, Hogan BL, Smith JC, Wright CV. Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development. 1995;121:3651–3662. doi: 10.1242/dev.121.11.3651. [DOI] [PubMed] [Google Scholar]
  • 8.Joseph EM, Melton DA. Xnr4: a Xenopus nodal-related gene expressed in the Spemann organizer. Dev Biol. 1997;184:367–372. doi: 10.1006/dbio.1997.8510. [DOI] [PubMed] [Google Scholar]
  • 9.Takahashi S, Yokota C, Takano K, Tanegashima K, Onuma Y, et al. Two novel nodal-related genes initiate early inductive events in Xenopus Nieuwkoop center. Development. 2000;127:5319–5329. doi: 10.1242/dev.127.24.5319. [DOI] [PubMed] [Google Scholar]
  • 10.Sun BI, Bush SM, Collins-Racie LA, LaVallie ER, DiBlasio-Smith EA, et al. derrière: a TGF-beta family member required for posterior development in Xenopus. Development. 1999;126:1467–1482. doi: 10.1242/dev.126.7.1467. [DOI] [PubMed] [Google Scholar]
  • 11.Jones CM, Armes N, Smith JC. Signalling by TGF-beta family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin. Curr Biol. 1996;6:1468–1475. doi: 10.1016/s0960-9822(96)00751-8. [DOI] [PubMed] [Google Scholar]
  • 12.Williams PH, Hagemann A, Gonzalez-Gaitan M, Smith JC. Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo. Curr Biol. 2004;14:1916–1923. doi: 10.1016/j.cub.2004.10.020. [DOI] [PubMed] [Google Scholar]
  • 13.Green JB, New HV, Smith JC. Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell. 1992;71:731–739. doi: 10.1016/0092-8674(92)90550-v. [DOI] [PubMed] [Google Scholar]
  • 14.Dyson S, Gurdon JB. Activin signalling has a necessary function in Xenopus early development. Curr Biol. 1997;7:81–84. doi: 10.1016/s0960-9822(06)00030-3. [DOI] [PubMed] [Google Scholar]
  • 15.Marchant L, Linker C, Mayor R. Inhibition of mesoderm formation by follistatin. Dev Genes Evol. 1998;208:157–160. doi: 10.1007/s004270050167. [DOI] [PubMed] [Google Scholar]
  • 16.Agius E, Oelgeschlager M, Wessely O, Kemp C, De Robertis EM. Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development. 2000;127:1173–1183. doi: 10.1242/dev.127.6.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Piccolo S, Agius E, Leyns L, Bhattacharyya S, Grunz H, et al. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature. 1999;397:707–710. doi: 10.1038/17820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wessely O, Kim JI, Geissert D, Tran U, De Robertis EM. Analysis of Spemann organizer formation in Xenopus embryos by cDNA macroarrays. Dev Biol. 2004;269:552–566. doi: 10.1016/j.ydbio.2004.01.018. [DOI] [PubMed] [Google Scholar]
  • 19.Toyoizumi R, Ogasawara T, Takeuchi S, Mogi K. Xenopus nodal related-1 is indispensable only for left-right axis determination. Int J Dev Biol. 2005;49:923–938. doi: 10.1387/ijdb.052008rt. [DOI] [PubMed] [Google Scholar]
  • 20.Kurth T. A cell cycle arrest is necessary for bottle cell formation in the early Xenopus gastrula: integrating cell shape change, local mitotic control and mesodermal patterning. Mech Dev. 2005;122:1251–1265. doi: 10.1016/j.mod.2005.09.002. [DOI] [PubMed] [Google Scholar]
  • 21.Leise WFI, Mueller PR. Inhibition of the cell cycle is required for convergent extension of the paraxial mesoderm during Xenopus neurulation. Development. 2004;131:1703–1715. doi: 10.1242/dev.01054. [DOI] [PubMed] [Google Scholar]
  • 22.Murakami M, Moody SA, Daar IO, Morrison DK. Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation. Development. 2004;131:571–580. doi: 10.1242/dev.00971. [DOI] [PubMed] [Google Scholar]
  • 23.Gilchrist MJ, Zorn AM, Voigt J, Smith JC, Papalopulu N, et al. Defining a large set of full-length clones from a Xenopus tropicalis EST project. Dev Biol. 2004;271:498–516. doi: 10.1016/j.ydbio.2004.04.023. [DOI] [PubMed] [Google Scholar]
  • 24.Chalmers AD, Goldstone K, Smith JC, Gilchrist M, Amaya E, et al. A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis. Mech Dev. 2005;122:355–363. doi: 10.1016/j.mod.2004.09.004. [DOI] [PubMed] [Google Scholar]
  • 25.Lee HX, Ambrosio AL, Reversade B, De Robertis EM. Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. Cell. 2006;124:147–159. doi: 10.1016/j.cell.2005.12.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sinner D, Kirilenko P, Rankin S, Wei E, Howard L, et al. Global analysis of the transcriptional network controlling Xenopus endoderm formation. Development. 2006;133:1955–1966. doi: 10.1242/dev.02358. [DOI] [PubMed] [Google Scholar]
  • 27.Dohrmann CE, Hemmati-Brivanlou A, Thomsen GH, Fields A, Woolf TM, et al. Expression of activin mRNA during early development in Xenopus laevis. Dev Biol. 1993;157:474–483. doi: 10.1006/dbio.1993.1150. [DOI] [PubMed] [Google Scholar]
  • 28.Saka Y, Smith JC. Spatial and temporal patterns of cell division during early Xenopus embryogenesis. Dev Biol. 2001;229:307–318. doi: 10.1006/dbio.2000.0101. [DOI] [PubMed] [Google Scholar]
  • 29.Dorey K, Hill CS. A novel Cripto-related protein reveals an essential role for EGF-CFCs in Nodal signalling in Xenopus embryos. Dev Biol. 2006;292:303–316. doi: 10.1016/j.ydbio.2006.01.006. [DOI] [PubMed] [Google Scholar]
  • 30.Onuma Y, Yeo CY, Whitman M. XCR2, one of three Xenopus EGF-CFC genes, has a distinct role in the regulation of left-right patterning. Development. 2006;133:237–250. doi: 10.1242/dev.02188. [DOI] [PubMed] [Google Scholar]
  • 31.Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol. 2003;19:589–621. doi: 10.1146/annurev.cellbio.19.041603.094522. [DOI] [PubMed] [Google Scholar]
  • 32.Kumar A, Novoselov V, Celeste AJ, Wolfman NM, ten Dijke P, et al. Nodal signaling uses activin and transforming growth factor-beta receptor-regulated Smads. J Biol Chem. 2001;276:656–661. doi: 10.1074/jbc.M004649200. [DOI] [PubMed] [Google Scholar]
  • 33.Lee MA, Heasman J, Whitman M. Timing of endogenous activin-like signals and regional specification of the Xenopus embryo. Development. 2001;128:2939–2952. doi: 10.1242/dev.128.15.2939. [DOI] [PubMed] [Google Scholar]
  • 34.Ho J, de Guise C, Kim C, Lemay S, Wang XF, et al. Activin induces hepatocyte cell growth arrest through induction of the cyclin-dependent kinase inhibitor p15INK4B and Sp1. Cell Signal. 2004;16:693–701. doi: 10.1016/j.cellsig.2003.11.002. [DOI] [PubMed] [Google Scholar]
  • 35.Burdette JE, Jeruss JS, Kurley SJ, Lee EJ, Woodruff TK. Activin A mediates growth inhibition and cell cycle arrest through Smads in human breast cancer cells. Cancer Res. 2005;65:7968–7975. doi: 10.1158/0008-5472.CAN-04-3553. [DOI] [PubMed] [Google Scholar]
  • 36.Slack JM. Regional biosynthetic markers in the early amphibian embryo. J Embryol Exp Morphol. 1984;80:289–319. [PubMed] [Google Scholar]
  • 37.Nieuwkoop PD, Faber J. Normal Table of Xenopus Laevis. Amsterdam, North Holand: Daudin; 1975. [Google Scholar]
  • 38.Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98:5116–5121. doi: 10.1073/pnas.091062498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Harland RM. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 1991;36:685–695. doi: 10.1016/s0091-679x(08)60307-6. [DOI] [PubMed] [Google Scholar]
  • 40.Khokha MK, Chung C, Bustamante EL, Gaw LW, Trott KA, et al. Techniques and probes for the study of Xenopus tropicalis development. Dev Dyn. 2002;225:499–510. doi: 10.1002/dvdy.10184. [DOI] [PubMed] [Google Scholar]
  • 41.Ryan K, Garrett N, Mitchell A, Gurdon JB. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell. 1996;87:989–1000. doi: 10.1016/s0092-8674(00)81794-8. [DOI] [PubMed] [Google Scholar]
  • 42.Pollet N, Muncke N, Verbeek B, Li Y, Fenger U, et al. An atlas of differential gene expression during early Xenopus embryogenesis. Mech Dev. 2005;122:365–439. doi: 10.1016/j.mod.2004.11.009. [DOI] [PubMed] [Google Scholar]
  • 43.Bouwmeester T, Kim S, Sasai Y, Lu B, De Robertis EM. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature. 1996;382:595–601. doi: 10.1038/382595a0. [DOI] [PubMed] [Google Scholar]
  • 44.Pera EM, Martinez SL, Flanagan JJ, Brechner M, Wessely O, et al. Darmin is a novel secreted protein expressed during endoderm development in Xenopus. Gene Expr Patterns. 2003;3:147–152. doi: 10.1016/s1567-133x(03)00011-5. [DOI] [PubMed] [Google Scholar]
  • 45.Jones CM, Broadbent J, Thomas PQ, Smith JC, Beddington RS. An anterior signalling centre in Xenopus revealed by the homeobox gene XHex. Curr Biol. 1999;9:946–954. doi: 10.1016/s0960-9822(99)80421-7. [DOI] [PubMed] [Google Scholar]
  • 46.Hudson C, Clements D, Friday RV, Stott D, Woodland HR. Xsox17alpha and -beta mediate endoderm formation in Xenopus. Cell. 1997;91:397–405. doi: 10.1016/s0092-8674(00)80423-7. [DOI] [PubMed] [Google Scholar]
  • 47.Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature. 1998;391:357–362. doi: 10.1038/34848. [DOI] [PubMed] [Google Scholar]
  • 48.Fletcher G, Jones GE, Patient R, Snape A. A role for GATA factors in Xenopus gastrulation movements. Mech Dev. 2006;123:730–745. doi: 10.1016/j.mod.2006.07.007. [DOI] [PubMed] [Google Scholar]
  • 49.Bellefroid EJ, Kobbe A, Gruss P, Pieler T, Gurdon JB, et al. Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. Embo J. 1998;17:191–203. doi: 10.1093/emboj/17.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont LK, et al. Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell. 1994;79:779–790. doi: 10.1016/0092-8674(94)90068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Jones SD, Ho L, Smith JC, Yordan C, Stiles CD, et al. The Xenopus platelet-derived growth factor alpha receptor: cDNA cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression. Dev Genet. 1993;14:185–193. doi: 10.1002/dvg.1020140305. [DOI] [PubMed] [Google Scholar]
  • 52.Deardorff MA, Tan C, Conrad LJ, Klein PS. Frizzled-8 is expressed in the Spemann organizer and plays a role in early morphogenesis. Development. 1998;125:2687–2700. doi: 10.1242/dev.125.14.2687. [DOI] [PubMed] [Google Scholar]

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