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The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Xenopus Oct25 is a POU family subclass V (POU-V) transcription factor with a distinct domain structure. To investigate the contribution of different domains to the function of Oct25, we have performed gain of function analyses. Deletions of the N- or C-terminal regions and of the Hox domain (except its nuclear localization signal) result in mutants being indistinguishable from the wild type protein in the suppression of genes promoting germ layer formation. Deletion of the complete POU domain generates a mutant that has no effect on embryogenesis. However, disruption of the alpha-helical structures in the POU domain, even by a single amino acid mutation, causes reversal of protein function. Overexpression of such mutants leads to dorsalization of embryos and formation of secondary axial structures. The underlying mechanism is an enhanced transcription of genes coding for antagonists of the ligands for ventralizing bone morphogenetic protein and Wnt pathways. Corresponding deletion mutants of Xenopus Oct60, Oct91, or mouse Oct4 also exhibit such a dominant-negative effect. Therefore, our results reveal that the integrity of the POU domain is crucial for the function of POU-V transcription factors in the regulation of genes that promote germ layer formation.

Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.

Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss-of-function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N-tubulin and Islet-1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior-posterior pattern formation, but they also regulate N-tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos.

The balance between differentiation signals and signals maintaining the undifferentiated state of embryonic cells ensures proper formation of germ layers. The nodal/activin pathway represents one of the major signaling chains responsible for the differentiation of embryonic cells into mesodermal and endodermal germ layers, while Oct4 is one of the major players in the maintenance of an undifferentiated state. Here we show that Oct25, an Oct4 homologue in Xenopus, antagonizes the activity of nodal/activin signaling by inhibiting the transcription of its target genes, Gsc and Mix2. The inhibitory effect is achieved by forming repression complexes on the promoters of Gsc and Mix2 between Oct25 and the signal transducers of the nodal/activin pathway, WBSCR11, FAST1, and Smad2. We have analyzed the significance of the Oct binding site for its inhibitory effect within the Gsc promoter. Albeit VP16-Oct25 fusion protein demonstrated a stimulating effect and EVE-Oct25 revealed a repression effect on an artificial reporter that is composed of eight repeats of Oct binding motifs, both fusions, like wild-type Oct25, inhibited mesendoderm formation and the activity of Gsc and Mix2 promoters. These results suggest that the regulatory effect of Oct25 on the expression of Gsc and Mix2 is mediated by specific protein/protein interactions. Furthermore, we demonstrate that histone deacetylase activities are not required for the inhibitory effect of Oct25. Our results provide a novel view in that Oct25 controls the nodal/activin pathway and thus maintains the undifferentiated state of embryonic cells in preventing them from premature differentiation.

VegT and beta-Catenin are key players in the hierarchy of factors that are required for induction and patterning of mesendoderm in Xenopus embryogenesis. By descending the genetic cascades, cells lose their pluripotent status and are determined to differentiate into distinct tissues. Mammalian Oct-3/4, a POU factor of subclass V (POU-V), is required for the maintenance of pluripotency of embryonic stem cells. However, its molecular function within the early embryo is yet poorly understood. We here show that the two maternal Xenopus POU-V factors, Oct-60 and Oct-25, inhibit transcription of genes activated by VegT and beta-Catenin. Maternal POU-V factors and maternal VegT show an opposite distribution along the animal/vegetal axis. Oct-25, VegT and Tcf3 interact with each other and form repression complexes on promoters of VegT and beta-Catenin target genes. We suggest that POU-V factors antagonize primary inducers to allow germ layer specification in a temporally and spatially coordinated manner.

Three POU factors of subclass V, Oct-25, Oct-60 and Oct-91 are expressed in Xenopus oocytes and early embryos. We here demonstrate that vegetal overexpression of Oct-25, Oct-60, Oct-91 or mammalian Oct-3/4 suppresses mesendoderm formation in Xenopus embryos. Oct-25 and Oct-60 are shown to inhibit activin/nodal and FGF signaling pathways. Loss of Oct-25 and Oct-60 function results in elevated transcription of mesendodermal marker genes and ectopic formation of endoderm in the equatorial region of gastrula stage embryos. Within the ectoderm, Oct-25 promotes neural fate by upregulating neuroectodermal genes, such as Xsox2, which prevent differentiation of neural progenitors into neurons. We also show that mouse Oct-3/4 and Xenopus Oct-25 or Oct-60 behave as functional homologues. We conclude that Xenopus Oct proteins are required to control the levels of embryonic signaling pathways, thereby ensuring the correct specification of germ layers.

A functional knockdown of FoxN3, a member of subclass N of fork head/winged helix transcription factors in Xenopus laevis, leads to an abnormal formation of the jaw cartilage, absence or malformation of distinct cranial nerves, and reduced size of the eye. While the eye phenotype is due to an increased rate of apoptosis, the cellular basis of the jaw phenotype is more complex. The upper and lower jaw cartilages are derivatives of a subset of cranial neural crest cells, which migrate into the first pharyngeal arch. Histological analysis of FoxN3-depleted embryos reveals severe deformation and false positioning of infrarostral, Meckel's, and palatoquadrate cartilages, structural elements derived from the first pharyngeal arch, and of the ceratohyale, which derives from the second pharyngeal arch. The derivatives of the third and fourth pharyngeal arches are less affected. FoxN3 is not required for early neural crest migration. Defects in jaw formation rather arise by failure of differentiation than by positional effects of crest migration. By GST-pulldown analysis, we have identified two different members of histone deacetylase complexes (HDAC), xSin3 and xRPD3, as putative interaction partners of FoxN3, suggesting that FoxN3 regulates craniofacial and eye development by recruiting HDAC.

Transcription factors of the Fox (fork head box) family have been found in all metazoan organisms. They are characterised by an evolutionary conserved DNA-binding domain of winged helix structure. In the South African clawed frog, Xenopus laevis, more than 30 Fox genes have been found so far. This review summarises our present knowledge regarding the general structure and common features of the fork head box and will then characterise Fox genes that have been described in Xenopus. Special attention was paid to the temporal and spatial expression patterns during early embryonic development. For some of these genes, the molecular mechanisms leading to their regulation after the onset of zygotic transcription are known. We also report on functional aspects including target gene regulation, cell or tissue specification and interference with the cell cycle. Finally, Fox proteins serve as mediators of signalling pathways and they might function as checkpoint molecules for the cross-regulatory interactions of different intracellular signal transduction chains.

The HIVEP gene family encodes for very large sequence-specific DNA binding proteins containing multiple zinc fingers. Three mammalian paralogous genes have been identified, HIVEP1, -2 and -3, as well as the closely related Drosophila gene, Schnurri. These genes have been found to directly participate in the transcriptional regulation of a variety of genes. Mammalian HIVEP members have been implicated in signaling by TNF-alpha and in the positive selection of thymocytes, while Schnurri has been shown to be an essential component of the TGF-beta signaling pathway. In this study, we describe the isolation of Xenopus HIVEP1, as well as partial cDNAs of HIVEP2 and -3. Analysis of the temporal and spatial expression of the XHIVEP transcripts during early embryogenesis revealed ubiquitous expression of the transcripts. Assays using Xenopus oocytes mapped XHIVEP1 domains that are responsible for nuclear export and import activity. The DNA binding specificity of XHIVEP was characterized using a PCR-mediated selection and gel mobility shift assays.

We report on the temporal and spatial expression pattern of two novel genes of the Xenopus fork head/winged helix family, xFoxB2 and xFoxI1c. xFoxB2 is activated at the late blastula stage and first expressed within the dorsolateral ectoderm except for the organiser territory. During gastrulation, xFoxB2 is found in two ectodermal stripes adjacent to the dorsal midline. Expression is completely down-regulated during neurulation. However, two distinct sets of cells expressing xFoxB2 re-appear in the rhombencephalon of swimming tadpoles. xFoxI1c is initially expressed at the early neurula stage in an epidermal ring around the neural field. Subsequent expression is found to be increased, and is exclusively localised to placodal precursor cells. The placodal expression remains until stage 40, when it is restricted to a distinct region in the lateral body wall behind the gills.

The Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast to Xvent-2B and other members of the Xvent-2 subfamily, Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show that GATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activate Xvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of beta-catenin before midblastula transition results in a suppression of Xvent-1B transcription.

The Xenopus tropicalis BMP-2 and BMP-4 genes have been cloned and sequenced. A comparison with the corresponding genes from X. laevis reveals that the BMP-4 genes are conserved at a higher extent than the BMP-2 genes. This is especially evident for the intron sequences, but is also reflected by the exon sequences. While the amino acids of X. tropicalis and X. laevis BMP-4 proteins diverge at about 4%, those of BMP-2 proteins diverge at about 7%. By reverse transcriptase polymerase chain reaction analyses and whole mount in situ hybridizations, we demonstrate the temporal and spatial expression patterns of X. tropicalis BMP-2 and BMP-4 genes. BMP-2 is found to be expressed maternally, and later in development, in migrating heart progenitor cells as well as in the final heart, within the pineal gland and the olfactory placodes. BMP-4 is zygotically activated within the ventral marginal zone and later found in the eye, the otic vesicle, the heart and within blood islands. Although the overall patterns are very similar to those found in X. laevis, there is some distinct difference which might result from the accelerated development in X. tropicalis.

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (beta-catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, which depend on Wnt/beta-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-beta superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed.

Xenopus FoxD3 (XFD-6) is an intron-less gene initially expressed within the Spemann organizer and later in premigratory neural crest cells. Based upon sequence and expression pattern comparisons, it represents the Xenopus orthologue to zebrafish fkd6, chicken CWH-3 and mammalian HFH-2 (genesis). Early expression of FoxD3 is activated by the Wnt-pathway and inhibited by BMP signalling. Ectopic overexpression of FoxD3 leads to an enlargement of the neural plate concomitant with a failure in neural crest formation, loss of anterior structures, lack of closure of the neural tube and severe defects in somitogenesis. Phenotypic variation is accompanied by down-regulation of neural crest markers, including Xslug, Xtwist and Xcadherin-11. FoxD3 also inhibits its own expression, thereby acting in a negative autoregulatory loop. By injections of VP16 and engrailed fusions we can demonstrate that FoxD3 acts as a negative transcriptional regulator; this repressive function strictly requires the presence of the winged helix domain. Transplantation experiments show that FoxD3 overexpressing cells from the prospective neural crest do neither differentiate nor migrate.

Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-b genes, we have analysed the potential of Xenopus cJun (AP-1) as a mediator of BMP-4 expression during Xenopus development. RNA injection experiments revealed that both heteromeric cFos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling was found to activate the Xenopus cJun gene only at a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, we find a spatial overlap of c-Jun and BMP-4 transcripts which suggests that AP-1 might serve as an additional activatory component for the autoregulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5 0 flanking region and two in the second intron of the BMP-4 gene. We further demonstrate that AP-1 acts independently of Xvent-2 which has recently been shown to mediate the early expression of BMP-4 in gastrula stage embryos. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

Previous reports have shown that the Gi-protein-coupled CXC chemokine receptor 4 is activated by stromal cell-derived factor 1 (SDF-1). The receptor is present in many cell types and regulates a variety of cellular functions, including chemotaxis, adhesion, hematopoiesis, and organogenesis. To examine the role of CXCR4 as a regulator of organogenesis in the vertebrate embryo, we have isolated a cDNA encoding the Xenopus laevis homologue of CXCR4 (xCXCR4). The encoded polypeptide was functionally reconstituted with recombinant Gi2 in baculovirus-infected insect cells. Although xCXCR4 shares only 42% of its extracellular residues with mammalian CXCR4, it is indistinguishable from human CXCR4 in terms of its activation by human SDF-1 and SDF-1 . The fact that only 19 of these residues are specifically present in the extracellular portions of CXCR4 suggests that these residues may be involved in recognizing SDF-1 and/or mediating CXCR4 activation by SDF-1. Xenopus CXCR4 mRNA expression was up-regulated during early neurula stages and remained high during early organogenesis. Whole mount in situ hybridization analysis showed abundant expression of xCXCR4 mRNA in the nervous system, including forebrain, hindbrain, and sensory organs, and in neural crest cells. xCXCR4 mRNA was also detected in the dorsal lateral plate, the first site of definitive hematopoiesis in the amphibian embryo corresponding to aorta-gonadmesonephros or para-aortic splanchnopleura in mammals. This observation suggests that SDF-1 and CXCR4 are involved in regulating the migratory behavior of hematopoietic stem cells colonizing the larval or fetal liver. The hematopoietic defects observed in mice lacking SDF-1 or CXCR4 may, at least in part, be explained by a disturbance of this migration.

We investigated the Xenopus winged helix gene XFD-4, its cDNA, and a pseudoallelic cDNA, termed XFD-4', representing Xenopus orthologues to chicken CWH-2 and mammalian MFH-1. XFD-4/4' genes are activated after midblastula transition in dorsolateral mesoderm but not within the dorsal lip. Later, expression is found in two segmented lines of cells bordering the somites, in head mesenchyme, in ventral abdominal muscle, and in the tail tip. Smad2 RNA injection leads to ectopic expression of XFD-4'. Since activation is also observed in activin A treated animal cap explants in the presence of cycloheximide, XFD-4/4' genes represent direct targets of activin signaling. Note that the future nomenclature for XFD-4 will be FoxC2a and for XFD-4' will be FoxC2b (Fox Nomenclature Committee).

We have isolated the gene, its corresponding cDNA and a closely related cDNA encoding the Xenopus winged helix factors XFD-13' and XFD-13, respectively. XFD-13/13' are regarded as pseudo-alleles and, based upon a comparison of sequences and genomic structures, represent the Xenopus orthologues to mammalian FREAC-1/HFH-8. XFD-13/13' genes are not transcribed during oogenesis, zygotic transcription starts at late gastrula/early neurula and transcripts persist throughout embryogenesis. Expression is found within head derived neural crest cells and the dorsolateral plate (DLP). At later developmental stages, cell populations of the DLP migrate to the ventral region but exclude the most posterior part. Since they are subsequently found to accumulate in vessel like structures, we suggest that these cells represent hematopoietic/endothelial progenitor cells.

Members of the TGF β superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGF β signalling, smad1 , 2 and 5 , from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGF β signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGF β signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGF β ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGF β members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.

The Xvent homeobox multigene family is essential for the patterning of the ventral mesoderm in Xenopus embryos. We have identified two novel members of this family, Xvent-1B and Xvent-2B, and have characterized their genomic structures. These two genes show a clustered organization and have probably arisen by gene duplication with subsequent inversion. Cis-regulatory elements within the promoters of both genes have been identified which contribute to their spatial activation. Xvent-2B is activated by BMP-2/4 in the absence of de novo protein synthesis, suggesting that this gene is a direct target of BMP-signalling. In contrast, Xvent-1B does not directly respond to BMP-2/4, but is activated by Xvent-2B. This activation is documented by Xvent-1B promoter/reporter studies, Xvent-2B overexpression and loss-of-function analysis using a dominant-negative Xvent-2 mutant. However, cycloheximide experiments reveal that Xvent-2B by itself is not sufficient to activate transcription of the Xvent-1B gene, but that there is a requirement for additional factor(s) being synthesized after midblastula transition.

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.

The Xenopus laevis BMP-4 gene shows an evolutionary conserved structure containing two coding exons and a leader exon. The transcripts which are detected after zygotic activation of the gene in ventral mesoderm of late blastula stage embryos do either contain the leader exon or begin within the first intron. Luciferase reporter/promoter studies revealed multiple elements being required for the activation and for the spatial control of transcription. These elements are located within the upstream region and within the second intron and they interact with a most proximal located basal promoter being indispensable for transcriptional activation. The auto-activatory capacity of BMP-4 is mediated by several enhancer elements being responsive not only to BMP-4 but also to BMP-2 signaling. BMP-2 might thus function as a natural activator of the BMP-4 gene in the early embryo. Since reporter activity obtained with distinct BMP-2/4 responsive promoter deletion mutants is simultaneously inhibited by the dominant negative BMP receptor as well as by chordin, we suggest that down-regulation of the BMP-4 gene by chordin results from an interference with the auto-regulatory loop at the level of protein–protein interactions.

Studies with amphibian embryos have contributed major insights into the molecular basis of induction processes and the formation of germ layers during vertebrate embryogenesis. Primary signals that have been identified as growth factors or growth factor-related ligands act as inducing factors on their target cells and, by a change of the genetic program, evoke a specification of the cellular differentiation pathways. While at present the signal transduction mechanisms leading from the ligands via cognate receptors to the nuclei are still poorly understood, there is growing information on transcription factors which are activated upon induction. They govern the expression of other regulatory molecules and co-ordinate the expression of cell type-specific structural genes. Meanwhile, it is generally accepted that development and cellular differentiation in all multicellular organisms depends upon a cascade of evolutionarily conserved transcription factors. Striking structural similarities within their DNA-binding domains allow many of these factors to be subdivided into different transcription factor families. Most of the basic knowledge on these factors emerged from the pioneering work done with Drosophila embryos which was greatly facilitated by the availability of numerous mutants. Despite the fact that Drosophila development until the blastoderm stage proceeds in a multinuclear syncytium and thus is significantly different from that in vertebrate organisms, the primary structures of many embryonic transcription factors have been conserved in higher organisms. This especially holds true for the various DNA binding motifs and it facilitated the isolation and characterization of vertebrate homologues to factors previously identified in lower organisms.

Transcription of the early response gene XFD-1' (XFKH1) in the dorsal lip (Spemann organizer) of Xenopus embryos is activated by dorsal mesoderm inducing factors. Promoter studies revealed the presence of an activin A response element (ARE) which is both necessary and sufficient for transcriptional activation of reporter genes in animal cap explants incubated with activin A. Surprisingly, this ARE is also active within vegetal explants in the absence of exogenously added inducers, but an additional inhibitory response element prevents transcription of the XFD-1' gene in the ventral/vegetal region of the embryo in vivo. This element is located upstream of the ARE, it responds to bone morphogenic proteins 2 and 4 (BMP-2/4) triggered signals and it overrides the activating properties of the ARE. Expression patterns of BMP-2 and BMP-4 in the late blastula stage embryo and, especially, their absence from the dorsal blastopore lip may thus control the spatial transcription of the XFD-1' gene. Accordingly, the temporal activation and the spatial restriction of XFD-1' gene activity to the Spemann organizer is regulated by antagonistic actions of two distinct members of the TGF-beta family (activin and BMP) which act on different promoter elements.

Determination (inducing) factors, the extracellular matrix, signaling pathways, transcription factors and genes interact in pattern formation and neural induction. Genes can either be activated or repressed. The animalvegetal and dorso–ventral polarities are determined in very early developmental stages. Factors of the TGF-β superfamily in a graded distribution are involved in the determination of endoderm, mesoderm and ectoderm. The differentiation of mesoderm also depends on the animal ectoderm. Neural inducing factors have been partially purified.

Since its discovery five years ago the conserved family of fork head/HNF-3-related transcription factors has gained increasing importance for the analysis of gene regulatory mechanisms during embryonic development and in differentiated cells. Different members of this family, which is defined by a conserved 110 amino acid residues encompassing DNA binding domain of winged helix structure, serve as regulatory keys in embryogenesis, in tumorigenesis or in the maintenance of differentiated cell states. The purpose of this review is to summarize the accumulating amount of data on structure, expression and function of fork head/HNF-3-related transcription factors.

The fork head (fkh) domain defines the DNA-binding region of a family of transcription factors which has been implicated in regulating cell fate decisions across species lines. We have cloned and molecularly characterized the crocodile (croc) gene which encodes a new family member from Drosophila. croc is expressed in the head anlagen of the blastoderm embryo under the control of the anterior, the dorsoventral and the terminal maternal organizer systems. The croc mutant phenotype indicates that the croc wild-type gene is required to function as an early patterning gene in the anterior-most blastoderm head segment anlage and for the establishment of a specific head skeletal structure that derives from the non-adjacent intercalary segment at a later stage of embryogenesis. As an early patterning gene, croc exerts unusual properties which do not allow it to be grouped among the established segmentation genes. A single-site mutation within the croc fkh domain, which causes a replacement of the first out of four conserved amino acid residues thought to be involved in the coordinate binding of Mg2+, abolishes the DNA binding of the protein in vitro. In view of the resulting lack-of-function mutant phenotype, it appears likely that metal binding by the affected region of the fkh domain is crucial for proper folding of the DNA-binding structure.

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.

Zn finger proteins (ZFPs) of the C2/H2 type in Xenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XlcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites for in vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activity in vivo is discussed.

Two novel fork head related cDNA sequences, termed XFD-2 and XFD-2', have been isolated from a Xenopus laevis gastrula stage cDNA library. XFD-2 and XFD-2' proteins share 88% sequence identity; a comparison of their fork head domains yields 96% identity. Such close homology suggests that the two genes represent pseudo-allelic variants of a common ancestor and probably arose by the ancient tetraploidization event in this species. Both genes are activated at midblastula transition. Main transcriptional activity is found during blastula and gastrula stages of development; thereafter, there is a gradual decrease of transcripts until somite segregation stages. Whole mount in situ hybridisation of blastula stage embryos reveals that the genes are initially transcribed within the animal hemisphere. Subsequently, we observe their transcription in a circumferential mode along the marginal zone, i.e., within the forming mesoderm. During gastrulation, these cells enter the blastoporus at the ventral, lateral and dorsal sites. At the end of gastrula and during neural stages transcripts are localized within somitogenic mesoderm, notochord, lateral and ventral mesoderm, neural floor plate, spinal cords and in the developing brain.

The zinc finger motif defines a large superfamily of nucleic acid binding proteins. Conserved amino acid sequence elements associated with structurally variant zinc finger clusters define subfamilies of zinc finger proteins (ZFPs). The FAR domain (Finger Associated Repeats) is a novel type of repeat element found at the amino-terminus in a subfamily of Xenopus laevis ZFPs. Northern blot analyses of three different members of the FAR subfamily (XFO 6, XFO 9-3 and XFG 68) revealed that each of these genes is transcribed during oogenesis, embryogenesis and in all investigated tissues of adult animals thereby indicating a ubiquitous distribution of transcripts. All FAR-ZFPs tested so far have specific RNA homopolymer binding activity; they associate preferentially with poly(U). The FAR repeats possess limited primary sequence homology with a sequence in the nucleolar shuttling protein NO38, within a region that contains a casein kinase II phosphorylation site.

POU-V class proteins like Oct4 are crucial for keeping cells in an undifferentiated state. An Oct4 homologue in Xenopus laevis, Oct25, peaks in expression during early gastrulation, when many cells are still uncommitted. Nevertheless, extensive morphogenesis is taking place in all germ layers at that time. Phenotypical analysis of embryos with Oct25 overexpression revealed morphogenesis defects, beginning during early gastrulation and resulting in spina-bifida-like axial defects. Analysis of marker genes and different morphogenesis assays show inhibitory effects on convergence and extension and on mesoderm internalization. On a cellular level, cell-cell adhesion is reduced. On a molecular level, Oct25 overexpression activates expression of PAPC, a functional inhibitor of the cell adhesion molecule EP/C-cadherin. Intriguingly, Oct25 effects on cell-cell adhesion can be restored by overexpression of EP/C-cadherin or by inhibition of the PAPC function. Thus, Oct25 affects morphogenesis via activation of PAPC expression and subsequent functional inhibition of EP/C-cadherin.

Nanog is a homeodomain transcription factor associated with the acquisition of pluripotency. Genome analyses of lower and higher vertebrates revealed that the existence of Nanog is restricted to gnathostomata but absent from agnatha and invertebrates. To elucidate the function of Nanog in nonmammalia, we identified the Danio rerio ortholog of Nanog and characterized its role in gain and loss of function experiments. We found Nanog to be crucial for survival of early zebrafish embryos, because depletion of Nanog led to gastrulation defects with subsequent lethality. Mouse Nanog overexpression could rescue these defects. Vice versa, zebrafish Nanog was found to promote proliferation and to inhibit differentiation of mouse embryonic stem cells in the absence of leukemia inhibitory factor. These findings indicate functional conservation of Nanog from teleost fishes to mammals. However, Nanog was lost in the genome of the anurans Xenopus laevis and Xenopus tropicalis. Phylogenetic analysis revealed that deletion probably occurred in a common anuran ancestor along with chromosomal translocations. The closest homologs of Nanog in Xenopus are the Vent proteins. We, therefore, investigated whether the Xvent genes might substitute for Nanog function in Xenopus. Although we found some similarities in phenotypes after overexpression and in the regulation of several marker genes, Xvent1/2 and Nanog cannot substitute each other. Depletion of Nanog in zebrafish cannot be rescued by ectopic expression of Xvent, and Xvent depletion in Xenopus cannot be overcome by ectopic expression of zebrafish Nanog.

We describe the fine-structure of the Xenopus laevis XFG 5-1 gene which codes for an RNA homopolymer binding Zn finger protein of the FAR (Finger Associated Repeat) subfamily. The gene contains six exons, i.e., a leader exon (I), four exons (II-V), each of them encoding one individual copy of the FAR repeat, and one exon (VI) encoding the linker as well as the complete multifinger-region of the corresponding protein. Isolation and characterization of distinct cDNAs revealed that primary transcripts are alternatively spliced, thereby leading either to mRNAs containing different copy numbers of the FAR repeat or, by utilization of an alternative splice acceptor site in front of exon VI, to an extension of the linker region between the FAR repeats and the multifinger domain. We also describe the fine-structure of a closely related gene, termed XFG 5-2, which is located downstream to the XFG 5-1 gene. The general structural organization in both genes is identical, but point mutations should give rise to a XFG 5-2 protein with a different number of Zn finger units.

Suramin, a polyanionic compound which is known to interact with the receptors of growth factors inhibits the expression of dorsal marker genes in whole embryos and isolated dorsal blastopore lips. Suramin also prevents activin A induced dorsalization of animal cap explants from blastula stage embryos, but it simultaneously evokes a shift of the differentiation pattern from dorsal mesodermal structures (notochord, somites) to ventral mesodermal derivatives (mesothelium and erythroid precursor cells). The results are consistent with the assumption that the dorsal vegetal zone (Nieuwkoop center) primarily releases more general/ventral mesodermalization signals. They further suggest a dual role of activin A in early embryogenesis. While the maternal component may contribute to a more general/ventral type of induction, increasing concentrations of the zygotic component along with the activation of primary response genes may contribute to the dorsalization of the organizer.

We have isolated a cDNA clone from a Xenopus laevis tadpole cDNA library which probably codes for the large subunit of glutathione synthetase. The corresponding protein comprises 474 amino acids and shows a significant homology with the large subunit of glutathione synthetase of Schizosaccharomyces pombe . RNase protection experiments revealed that the gene is transcribed during oogenesis and that zygotic expression starts after midblastula transition. Transcripts are also detected in varios adult tissues suggesting an ubiquitous distribution of the corresponding protein.

Finger associated box-zinc finger proteins (FAX-ZFPs) constitute a subfamily of the many C2H2 type ZFPs in Xenopus laevis. FAX is a highly conserved protein domain connected to variable zinc finger clusters. Three different FAX-ZFPs encoding genomic fragments were isolated and subjected to a detailed structural characterization. All three exhibit a common, highly conserved exon/intron organization, with the variant zinc finger clusters organized in a single exon for each gene analysed. Two of the three genomic fragments contain a second FAX-ZFP encoding (partial) transcription unit each. Further evidence for a clustered organization of FAX-ZFP transcription units is provided by Southern blot analysis of large genomic restriction fragments separated by transverse field gel electrophoresis, and by in situ hybridization on intact chromosomes. Comparative sequence analysis of the genes isolated reveals an exceptional degree of DNA sequence conservation in both exon and intron regions in one part of the FAX encoding region, suggesting that recent gene conversion has led to the combination of these sequence elements with DNA segments including regions encoding variant zinc finger clusters. Overexpression of the FAX domain by itself or of a full-length FAX-ZFP in X. laevis embryos by means of mRNA injection does not interfere with the normal developmental program, suggesting general and not cell specific/regulatory functions for X. laevis FAX-ZFPs.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

A gene family encoding the fork head/HNF-3 domain has been identified in the South African clawed frog, Xenopus laevis. Screening of genomic DNA and gastrula stage derived cDNA libraries with a DNA fragment encoding the Drosophila fork head domain led to the isolation of a number of different clones encoding this motif. While one of the Xenopus fork head sequences, XFD-3, represents the Xenopus counterpart to rat HNF-3 beta, all other sequences encode novel types of fork head related proteins. Here we report on XFD-1, a DNA binding protein which can bind to the HNF-3 alpha target sequence. Analysis of temporal and spatial expression revealed that the gene is activated at blastula stage and that transcripts are localized in a rather thin stripe of cells invaginating the dorsal blastopore lip (organizer) during gastrulation. XFD-1 mRNA is localized within the notochord and, by the end of neurulation, is no longer detectable. In the animal cap assay the gene is activated by incubation with the vegetalizing factor (activin A) but not with bFGF.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity.

We have screened a Xenopus ovary cDNA library using a synthetic oligonucleotide derived from that part of the inhibin beta A sequence, which is highly conserved within the TGF-beta family. Out of several clones yielding autoradiographic signals four turned out to represent Xenopus counterparts to the human bone morphogenetic protein 4 (BMP-4). Each two of the four sequences are nearly identical and probably account for different alleles whereas the two pairs showing 5% divergence may have arisen by genome duplication in this tetraploid species. The amino acid sequence of the Xenopus protein is 80% homologous to the human sequence showing no single exchange within the last 100 amino acids at the C-terminus. This region, which constitutes the main part of the mature, biologically active protein, also exhibits substantial homologies to other representatives of the TGF-beta family, especially to the Drosophila DPPC protein. Transfection of COS-1 cells with the Xenopus BMP-4 sequence under control of the CMV-promoter leads to the secretion of a protein which exhibits mesoderm inducing activity when tested with animal cap explants from Xenopus blastula stage embryos.

Two related but clearly different cDNA clones corresponding to elongation factor 1 alpha (EF-1 alpha) mRNAs were isolated from a Xenopus laevis gastrula-stage library. Whereas the nucleotide sequences of these two cDNAs differ within the coding region at 49 out of 1386 positions (3.5%), the derived amino acid sequences are completely identical, thereby indicating a substantial evolutionary constraint on this translation factor. Southern-blot analysis of genomic DNA suggests that, besides the two closely related EF-1 alpha genes investigated in this study, other more-distantly related genes may exist in the X. laevis genome. Transcription of EF-1 alpha genes during oogenesis and embryonic development was studied by Northern-blot analysis and by in situ hybridizations. A high amount of EF-1 alpha mRNA was detected in previtellogenic oocytes. At later stages of embryonic development, EF-1 alpha mRNA was found to be accumulated in translationally active tissues.

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult beta I-globin gene and to the two coordinately expressed larval beta I- and beta II-globin genes have been determined and compared to those of the adult alpha-genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult alpha- and beta-genes. The two larval beta-genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.

Maintenance of endoplasmic reticulum (ER) homeostasis is essential for correct protein targeting and secretion. ER stress caused by accumulation of unfolded or misfolded proteins leads to disruption of cellular functions. We have investigated the effect of ER stress on Xenopus embryogenesis. ER stress induced by tunicamycin (TM) treatment of embryos resulted in defects affecting germ layer formation. We observed up-regulation of ER stress response genes, enhanced cytoplasmic splicing of xXBP1 RNA, and increased rate of apoptosis. In animal cap assays, TM treatment inhibited mesoderm formation induced by overexpression of activin/nodal RNA but did not affect mesoderm formation induced by functional activin protein, suggesting that dysfunction of ER caused a failure in activin/nodal processing and/or secretion. The observation that activin protein renders mesoderm formation under ER stress strengthens the role of activin/nodal for mesoderm induction. The results underline the functional significance of ER homeostasis in germ layer formation during Xenopus embryogenesis. Developmental Dynamics 236:2844–2851, 2007 © 2007 Wiley-Liss, Inc.

IRE1 is an atypical serine/threonine kinase transmembrane protein with RNase activity. In the unfolded protein response (UPR), they function as proximal sensor of the unfolded proteins in the endoplasmic reticulum (ER). Upon activation by ER stress, IRE1 performs an unconventional cytoplasmic splicing of XBP1 pre-mRNA and thus allows the synthesis of active XBP1, which activates UPR target genes to restore the homeostasis of the ER. IRE1/XBP1 signaling is hence essential for UPR but its function during embryogenesis is yet unknown. The transcripts of the two isoforms of IRE1 in Xenopus, xIRE1alpha and xIRE1beta are differentially expressed during embryogenesis. We found that xIRE1beta is sufficient for cytoplasmic splicing of xXBP1 pre-mRNA. Although gain of xIRE1beta function had no significant effect on Xenopus embryogenesis, overexpression of both, xIRE1beta and xXBP1 pre-mRNA, inhibits activin A induced mesoderm formation, suggesting that an enhanced activity of the IRE1/XBP1 pathway represses mesoderm formation. Surprisingly, while loss of XBP1 function promotes mesoderm formation, the loss of IRE1beta function led to a reduction of mesoderm formation, probably by action of IRE1 being different from the IRE1/XBP1 pathway. Therefore, both gain and loss of function studies demonstrate that IRE1 is required for mesoderm formation in Xenopus embryos.

Lineage labeling is one of the most important techniques in developmental biology. Most recently, a set of photoactivatable fluorescent proteins originating from marine cnidarians became available. Here, we introduce the application of the green to red photoconvertible protein EosFP as a novel technique to analyze early vertebrate development. Both injection of EosFP mRNA and purified, recombinant EosFP followed by a light-driven green to red conversion allow lineage labeling in virtually any temporal and spatial dimension during embryonic development for at least 2 weeks. Specific staining of cells from nonsurface layers is greatly facilitated by light-driven conversion of EosFP compared with traditional methods. Therefore, green to red photoactivatable proteins promise to be a powerful tool with the potential to satisfy the increasing demand for methods enabling detailed phenotypical analyses after manipulations of morphogenetic events, gene expression, or signal transduction. Developmental Dynamics 236:473–480, 2007. © 2006 Wiley-Liss, Inc.

Using RT-PCR and in situ hybridisation, we have analysed the temporal and spatial expression patterns of Xenopus Fox genes of subclass N. By screening cDNA libraries and by RT-PCR using embryonic RNA and primers derived from EST analyses, we could isolate FoxN2, FoxN4, FoxN5 and different isoforms of FoxN3. FoxN2 and FoxN3 transcripts were found during all developmental stages including early cleavage and tailbud stages. FoxN5 transcripts were only present at early cleavage stages, while FoxN4 expression began after midblastula transition. Spatial expression of FoxN2 was first detected in the early eye field and later, in the branchial arches, the vagal ganglion and in the developing retina. FoxN3 transcripts were found within the animal cap. In post-gastrula embryos, neural crest cells and the early eye field showed strong expression of FoxN3. At late tadpole stages, the branchial arches were stained. FoxN4 was expressed in the early eye field and later in the developing retina cells, the nephrostomes of the pronephric kidney and in the midbrain. A ubiquitous expression of FoxN5 was found in early cleavage stage embryos.