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SummaryMesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low-throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data comprised of more than two data types is challenging. Here, we use linked self-organizing maps to combine ChIP-seq/ATAC-seq with temporal, spatial and perturbation RNA-seq data fromXenopus tropicalismesendoderm development to build a high resolution genome scale mechanistic GRN. We recovered both known and previously unsuspected TF-DNA/TF-TF interactions and validated through reporter assays. Our analysis provides new insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly-dimensional multi-omic data sets.HighlightsBuilt a generally applicable pipeline to creating GRNs using highly-dimensional multi-omic data setsPredicted new TF-DNA/TF-TF interactions during mesendoderm developmentGenerate the first genome scale GRN for vertebrate mesendoderm and expanded the core mesendodermal developmental network with high fidelityDeveloped a resource to visualize hundreds of RNA-seq and ChIP-seq data using 2D SOM metaclusters.

Background hes genes are chordate homologs of Drosophila genes, hairy and enhancer of split, which encode a basic helix-loop-helix (bHLH) transcriptional repressor with a WRPW motif. Various developmental functions of hes genes, including early embryogenesis and neurogenesis, have been elucidated in vertebrates. However, their orthologous relationships remain unclear partly because of less conservation of relatively short amino acid sequences, the fact that the genome was not analyzed as it is today, and species-specific genome duplication. This results in complicated gene names in vertebrates, which are not consistent in orthologs. We previously revealed that Xenopus frogs have two clusters of hes5, named “the hes5.1 cluster” and “the hes5.3 cluster”, but the origin and the conservation have not yet been revealed. Results Here, we elucidated the orthologous and paralogous relationships of all hes genes of human, mouse, chicken, gecko, zebrafish, medaka, coelacanth, spotted gar, elephant shark and three species of frogs, Xenopus tropicalis (X. tropicalis), X. laevis, Nanorana parkeri, by phylogenetic and synteny analyses. Any duplicated hes5 were not found in mammals, whereas hes5 clusters in teleost were conserved although not as many genes as the three frog species. In addition, hes5 cluster-like structure was found in the elephant shark genome, but not found in cyclostomata. Conclusion These data suggest that the hes5 cluster existed in the gnathostome ancestor but became a single gene in mammals. The number of hes5 cluster genes were specifically large in frogs.

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes.

SUMMARY Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA., Graphical Abstract, In Brief How do maternal transcription factors interact with chromatin regions to coordinate the endodermal gene regulatory program? Paraiso et al. demonstrate that combinatorial binding of maternal Otx1, Vegt, and Foxh1 to select enhancers and super-enhancers in the genome controls endodermal cell fate specification during zygotic gene activation.

The mechanism of intercellular transport of Wnt ligands is still a matter of debate. To better understand this issue, we examined the distribution and dynamics of Wnt8 in Xenopus embryos. While Venus-tagged Wnt8 was found on the surfaces of cells close to Wnt-producing cells, we also detected its dispersal over distances of 15 cell diameters. A combination of fluorescence correlation spectroscopy and quantitative imaging suggested that only a small proportion of Wnt8 ligands diffuses freely, whereas most Wnt8 molecules are bound to cell surfaces. Fluorescence decay after photoconversion showed that Wnt8 ligands bound on cell surfaces decrease exponentially, suggesting a dynamic exchange of bound forms of Wnt ligands. Mathematical modeling based on this exchange recapitulates a graded distribution of bound, but not free, Wnt ligands. Based on these results, we propose that Wnt distribution in tissues is controlled by a dynamic exchange of its abundant bound and rare free populations., 動く分子と動かない分子が協調して、安定した位置情報を素早く作り出す. 京都大学プレスリリース. 2021-06-04.

Background hes genes are chordate homologs of Drosophila genes, hairy and enhancer of split, which encode a basic helix-loop-helix (bHLH) transcriptional repressor with a WRPW motif. Various developmental functions of hes genes, including early embryogenesis and neurogenesis, have been elucidated in vertebrates. However, their orthologous relationships remain unclear partly because of less conservation of relatively short amino acid sequences, less conserved synteny, and species-specific gene duplication. This results in complicated gene names in vertebrates, which are not consistent in orthologs. In a previous study, we revealed that Xenopus frogs have two clusters of hes5, named “the hes5.1 cluster” and “the hes5.3 cluster.” The origin has not yet been revealed. Results Here, we elucidated the orthologous and paralogous relationships of all hes genes of human, mouse, chicken, gecko, zebrafish, medaka, coelacanth, spotted gar, elephant shark, and Xenopus frogs (X. tropicalis and X. laevis) by phylogenic and synteny analysis. Any clusters of hes5 were not found in amniotes, whereas duplicated hes5 clusters in teleost were found although not as many genes as Xenopus. In addition, hes5 cluster-like structure was found in the elephant shark genome, but not found in cyclostomata. Conclusion These data suggest that the hes5 cluster existed in the gnathostome ancestor, but was lost in amniotes.

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes.

SummaryHow morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. “HA trap” is based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, while “Dpp trap” is based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show thatdpptranscriptional refinement from an initially uniform to a localized expression and persistent signaling in transientdppsource cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement ofdppfor the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

Xenopus laevis is a classic developmental model, but its allotetraploid genome has limited our ability to perform genetic manipulations. The advance of... Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus. The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.

Wnt proteins direct embryonic patterning, but the regulatory basis of their distribution and signal reception remain unclear. Here, we show that endogenous Wnt8 protein is distributed in a graded manner in Xenopus embryo and accumulated on the cell surface in a punctate manner in association with “N-sulfo-rich heparan sulfate (HS),” not with “N-acetyl-rich HS”. These two types of HS are differentially clustered by attaching to different glypicans as core proteins. N-sulfo-rich HS is frequently internalized and associated with the signaling vesicle, known as the Frizzled/Wnt/LRP6 signalosome, in the presence of Wnt8. Conversely, N-acetyl-rich HS is rarely internalized and accumulates Frzb, a secreted Wnt antagonist. Upon interaction with Frzb, Wnt8 associates with N-acetyl-rich HS, suggesting that N-acetyl-rich HS supports Frzb-mediated antagonism by sequestering Wnt8 from N-sulfo-rich HS. Thus, these two types of HS clusters may constitute a cellular platform for the distribution and signaling of Wnt8., Wnt proteins mediate embryonic development but how protein localization and patterning is regulated is unclear. Here, the authors show that distinct structures with different heparan sulfate modifications (‘N-sulfo-rich’ and ‘N-acetyl-rich’) regulate cellular localization and signal transduction of Wnt8 in Xenopus.

Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization.

Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species.

Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals' health. Also, another gene coding CDKn1b is lost besides CDKn1c and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c.S expression is higher than that of CDKn2c.L, and a conserved noncoding sequence (CNS) of CDKn2c genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus.

Background In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. Results To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5′ upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5′ upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5′ region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. Conclusions On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5′ upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate. Electronic supplementary material The online version of this article (10.1186/s40851-019-0143-1) contains supplementary material, which is available to authorized users.

hox genes are found as clusters in the genome in most bilaterians. The order of genes in the cluster is supposed to be correlated with the site of expression along the anterior-posterior body axis and the timing of expression during development, and these correlations are called spatial and temporal collinearity, respectively. Here we studied the expression dynamics of all hox genes of the diploid species Xenopus tropicalis in four Hox clusters (A–D) by analyzing high-temporal-resolution RNA-seq databases and the results showed that temporal collinearity is not supported, which is consistent with our previous data from allotetraploid Xenopus laevis. Because the temporal collinearity hypothesis implicitly assumes the collinear order of gene activation, not mRNA accumulation, we determined for the first time the timing of when new transcripts of hox genes are produced, by detecting pre-spliced RNA in whole embryos with reverse transcription and quantitative PCR (RT-qPCR) for all hoxa genes as well as several selected hoxb, hoxc and hoxd genes. Our analyses showed that, coinciding with the RNA-seq results, hoxa genes started to be transcribed in a non-sequential order, and found that multiple genes start expression almost simultaneously or more posterior genes could be expressed earlier than anterior ones. This tendency was also found in hoxb and hoxc genes. These results suggest that temporal collinearity of hox genes is not held during early development of Xenopus., Summary: qPCR analysis for de novo transcription of hox genes suggest that temporal collinearity is not held for all hox genes during early development of Xenopus tropicalis.

Introducing exogenous DNA into an embryo can promote misexpression of a gene of interest via transcription regulated by an attached enhancer-promoter. This protocol describes plasmid DNA microinjection into

The homeodomain transcription factor Otx2 has essential roles in head and eye formation via the negative and positive regulation of its target genes, but it remains elusive how this dual activity of Otx2 affects cellular functions. In the current study, we first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible cyclin-dependent kinase (Cdk) sites and one Akt site, and analyzed the biological activities of phosphomimetic (4E) and nonphosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not the 4A mutant downregulated the Cdk inhibitor gene p27xic1 (cdknx) and posterior genes, and promoted cell proliferation, possibly forming a positive-feedback loop consisting of Cdk, Otx2 and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, resulting in enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation dependent. These data suggest that Otx2 orchestrates cell proliferation, anteroposterior patterning and eye formation via its phosphorylation state.

The gastrula organizer, an embryonic tissue, has a central role in early development of all eumetazoans, from cnidarians to vertebrates. In amphibians, the organizer—also known as the Spemann–Mangold organizer—is located in the dorsal blastopore lip of the gastrula embryo. This organizer is capable of inducing a secondary body axis when transplanted into the ventral region of a blastula embryo. In sea anemone embryos, the organizer has recently been recognized in the blastoporal lip, implying its ancient origin among eumetazoans. Here we review the molecular basis of vertebrate and cnidarian organizers, which have been widely studied using Xenopus, zebrafish, and mice for more than 20 years, and have relatively recently been studied using Nematostella. Recent genome-wide investigations have provided a comprehensive overview of transcription factor (TF) binding sites and regulatory principles of the gene regulatory network (GRN) in the Xenopus organizer. These analyses provide a platform for genome-wide evolutionary study of organizer-equivalent tissues in other organisms, including cnidarians. Here, we discuss an evolutionary prospective for the organizer, focusing on (1) the formation of the organizer by combinatorial signaling pathways such as Wnt, Nodal, and Bmp; and (2) the GRN regulating organizer formation and activity by TFs such as Lim1/Lhx1, Otx, Goosecoid, Brachyury, and FoxA.

Xenopus laevis is a classic developmental model, but its allotetraploid genome has limited our ability to perform genetic manipulations. The advance of...

From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-β signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied byFoxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.

SummarySHP2 is a ubiquitously expressed protein tyrosine phosphatase, deregulation of which is associated with malignant neoplasms and developmental disorders. SHP2 is required for full activation of RAS-Erk signaling in the cytoplasm and is also present in the nucleus, where it promotes Wnt target gene activation through dephosphorylation of parafibromin. SHP2 is distributed both to the cytoplasm and nucleus at low cell density but is excluded from the nucleus at high cell density. Here, we show that SHP2 physically interacts with transcriptional coactivators YAP and TAZ, targets of the cell-density-sensing Hippo signal. Through the interaction, nonphosphorylated YAP/TAZ promote nuclear translocalization of SHP2, which in turn stimulates TCF/LEF- and TEAD-regulated genes via parafibromin dephosphorylation. Conversely, YAP/TAZ phosphorylated by Hippo signaling sequester SHP2 in the cytoplasm, thereby preventing nuclear accumulation of SHP2. Hence, YAP/TAZ serve as a rheostat for nuclear SHP2 function, which is switched off by the Hippo signal.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi , Epipedobates machalilla , Gastrotheca riobambae , and Eleutherodactylus coqui , as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt ( zvegt ) mRNA was detected as a minor isoform besides the major maternal ( mvegt ) isoform of the X. laevis egg. In addition, α-amanitin–insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1 , have been evolutionary conserved in frogs.

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.

Xenopus laevis has an allotetraploid genome of 3.1 Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.

Inductive interactions mediated by the TGF-β and FGF–MAPK pathways are essential for specification of the germ layers and embryonic body axes during early vertebrate embryogenesis. TGF-β and FGF ligands signal through receptor Ser/Thr and Tyr kinases, respectively, and these signaling pathways cross-talk to regulate transcription and cell behavior. The allotetraploid Xenopus laevis and its ancestral diploid Xenopus tropicalis are versatile model organisms with which to study the inductive interactions and mechanisms of these signal transduction pathways. Here we have analyzed the draft genome of X. laevis with respect to the genomic organization and differential expression of genes in the TGF-β and FGF pathways. Genomic structure and gene expression analyses of pathway components in X. laevis revealed that genetic modulations, including deletions resulting in singletons and differential expression of homeologs, have occurred frequently among extracellular regulatory factors of the TGF-β pathway after allotetraploidization. Moreover, differential gene expression was found for factors regulating various cellular responses including co-receptors, decoy receptors, and intracellular negative regulators in both the TGF-β and FGF–MAPK pathways. We summarize the patterns of genetic alterations in the allotetraploid frog X. laevis and discuss the importance of these changes with regard to developmental processes.

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann–Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.

Summary Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region [1–3]. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment [4–6]. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin ( Da ), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes ( zic1/zic4 ) in Da , is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da . Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

Gradient formation and signaling ranges of secreted proteins are crucial problems to understand how morphogens work for positional information and patterning in animal development. Yet, extracellular behaviors of secreted signaling molecules remain unexplored compared to their downstream pathways inside the cell. Recent advances in bioimaging make it possible to directly visualize morphogen molecules, and this simple strategy has, at least partly, succeeded in uncovering molecular behaviors of morphogens, such as Wnt (wingless-type MMTV integration site family member) and BMP (bone morphogenetic protein) as well as secreted Wnt binding proteins, sFRPs (secreted Frizzled-related proteins), in embryonic tissues. Here, we review the regulation of Wnt signaling by sFRPs, focusing on extracellular regulation of Wnt ligands in comparison with other morphogens. We also discuss evolutionary aspects with comprehensive syntenic and phylogenetic information about vertebrate sfrp genes. We newly annotated several sfrp genes including sfrp2-like 1 (sfrp2l1) in frogs and fishes and crescent in mammals.

Secreted Frizzled-related proteins (sFRPs) are thought to negatively modulate Wnt signalling. Although Wnt proteins are thought to diffuse extracellularly and act as morphogens, little is known about the diffusibility of either Wnts or sFRPs. Here we show that Frzb and Crescent (Cres), which are members of the sFRP family, have the ability to regulate the diffusibility and signalling areas of the Wnt ligands Wnt8 and Wnt11. We found, using the Xenopus embryo, that Wnts do not diffuse effectively, whereas Frzb and Cres spread very widely. Interestingly, Frzb and Cres substantially promoted the diffusion of Wnt8 and Wnt11 through extracellular interactions. Importantly, we show that Wnt8 conveyed by sFRPs can activate canonical Wnt signalling despite the function of sFRPs as Wnt inhibitors, suggesting a novel regulatory system for Wnts by sFRPs.

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.