Takayoshi Yamamoto

Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor

Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B–Cdk1. Here we show that cyclin B–Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B–Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B–Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B–Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B–Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B–Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B–Cdk1.

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis

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.

Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus

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.

The medaka dhc2 mutant reveals conserved and distinct mechanisms of Hedgehog signaling in teleosts

BackgroundPrimary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction.ResultsTo precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling.ConclusionsCiliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.

FRET-based tension measurement across actin-associated mechanotransductive structures using Lima1

During embryogenesis, mechanical forces play important roles in morphogenesis and tissue differentiation. To measure these forces, we developed a new Förster resonance energy transfer (FRET)-based tension sensor that uses the actin-associated protein, Lima1. The sensor was validated in HeLa cells where we found the FRET index decreased or increased in response to changes in the cellular environment. Introducing the sensor into Xenopus embryos enabled us to observe tension distributions and sequential changes over the entire embryo. We found that during neural tube closure, neural ectodermal cells showed a gradual decrease in their FRET index, indicating they generate higher tension with the progression of neural tube closure. With appropriate controls and application, our Lima1 tension sensor has the ability to uncover and define other tension distributions and their functions in development and differentiation.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos

BackgroundUnderstanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this “mid-embryonic lethality hypothesis” by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations.ResultsBy subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species.ConclusionIn opposition to the “mid-embryonic lethality hypothesis,” our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

Fam46a regulates BMP-dependent pre-placodal ectoderm differentiation in Xenopus

ABSTRACT The pre-placodal ectoderm (PPE) is a specialized ectodermal region which gives rise to the sensory organs and other systems. The PPE is induced from the neural plate border during neurulation, but the molecular mechanism of PPE formation is not fully understood. In this study, we examined the role of a newly identified PPE gene, Fam46a, during embryogenesis. Fam46a contains a nucleoside triphosphate transferase domain, but its function in early development was previously unclear. We show that Fam46a is expressed in the PPE in Xenopus embryos, and Fam46a knockdown induces abnormalities in the eye formation and the body color. At the neurula stage, Fam46a upregulates the expression of PPE genes and inhibits neural crest formation. We also show that Fam46a physically interacts with Smad1/Smad4 and positively regulates BMP signaling. From these results, we conclude that Fam46a is required for PPE formation via the positive regulation of BMP signaling. Our study provides a new mechanism of ectodermal patterning via cell-autonomous regulation of BMP signaling in the PPE. Summary: The newly identified placode gene Fam46a is required for the differentiation of the pre-placodal ectoderm (PPE) in early Xenopus development through its interaction with Smad1/Smad4 and its regulation of BMP signaling.

Comprehensive analyses of hox gene expression in Xenopus laevis embryos and adult tissues

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.