Force-dependent remodeling of cytoplasmic ZO-1 condensates contributes to cell-cell adhesion through enhancing tight junctions.

The physiological importance of biomolecular condensates is widely recognized, but how it is controlled in time and space during development is largely unknown. Here, we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass because they receive weaker tensile forces. Furthermore, analyses using MDCK cells demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to the establishment of robust cell-cell adhesion during early development.

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/ß-Catenin Signaling in Mouse Spermatogenic Stem Cells.

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/ß-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/ß-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

Genome evolution in the allotetraploid frog Xenopus laevis.

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.

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping.

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

Conservation of structure and function in vertebrate c-FLIP proteins despite rapid evolutionary change.

Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development.

Transgenic Xenopus laevis for live imaging in cell and developmental biology.

The stable transgenesis of genes encoding functional or spatially localized proteins, fused to fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), is an extremely important research tool in cell and developmental biology. Transgenic organisms constructed with fluorescent labels for cell membranes, subcellular organelles, and functional proteins have been used to investigate cell cycles, lineages, shapes, and polarity, in live animals and in cells or tissues derived from these animals. Genes of interest have been integrated and maintained in generations of transgenic animals, which have become a valuable resource for the cell and developmental biology communities. Although the use of Xenopus laevis as a transgenic model organism has been hampered by its relatively long reproduction time (compared to Drosophila melanogaster and Caenorhabditis elegans), its large embryonic cells and the ease of manipulation in early embryos have made it a historically valuable preparation that continues to have tremendous research potential. Here, we report on the Xenopus laevis transgenic lines our lab has generated and discuss their potential use in biological imaging.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Cell movements of the deep layer of non-neural ectoderm underlie complete neural tube closure in Xenopus.

In developing vertebrates, the neural tube forms from a sheet of neural ectoderm by complex cell movements and morphogenesis. Convergent extension movements and the apical constriction along with apical-basal elongation of cells in the neural ectoderm are thought to be essential for the neural tube closure (NTC) process. In addition, it is known that non-neural ectoderm also plays a crucial role in this process, as the neural tube fails to close in the absence of this tissue in chick and axolotl. However, the cellular and molecular mechanisms by which it functions in NTC are as yet unclear. We demonstrate here that the non-neural superficial epithelium moves in the direction of tensile forces applied along the dorsal-ventral axis during NTC. We found that this force is partly attributable to the deep layer of non-neural ectoderm cells, which moved collectively towards the dorsal midline along with the superficial layer. Moreover, inhibition of this movement by deleting integrin ß1 function resulted in incomplete NTC. Furthermore, we demonstrated that other proposed mechanisms, such as oriented cell division, cell rearrangement and cell-shape changes have no or only minor roles in the non-neural movement. This study is the first to demonstrate dorsally oriented deep-cell migration in non-neural ectoderm, and suggests that a global reorganization of embryo tissues is involved in NTC.

Inference of the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes from comparative gene mapping.

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.

Dual in vitro effects of cortisol on cell turnover in the medaka esophagus via the glucocorticoid receptor.

AIMS: Cortisol is a glucocorticoid in mammals, but has both gluco- and mineralocorticoid activities in teleost fish. Our previous in vivo studies on osmoregulatory esophagi of euryhaline fish showed that epithelial apoptosis for the simple epithelium in seawater and cell proliferation for the stratified epithelium in fresh water are both induced by cortisol. The aim of the present study was to examine the mechanism of these dual cortisol effects on esophageal cell turnover. MAIN METHODS: We developed a tissue culture method for the esophagus from euryhaline medaka (Oryzias latipes) and assessed cell proliferation and apoptosis in vitro in response to cortisol and 11-deoxycorticosterone (DOC), a recently identified agonist of the teleostean mineralocorticoid receptor. KEY FINDINGS: Epithelial apoptosis, a well-established glucocorticoid function, was stimulated by treatment of the esophagus culture with 10nM cortisol for 8 days, but no effects were seen at higher doses (100 and 1000 nM). In contrast, cell proliferation was induced by 1000 nM cortisol treatment for 8 days and this response was dose-dependent. Both effects were blocked by RU-486, a glucocorticoid receptor antagonist. DOC showed no significant effects at 10-1000 nM. SIGNIFICANCE: In the esophageal epithelium in euryhaline fish, cortisol induces either apoptosis or cell proliferation via the glucocorticoid receptor, depending on the cortisol concentration. The glucocorticoid signaling may play a more important role than mineralocorticoid signaling in differentiation of the osmoregulatory esophagus in euryhaline fishes.

MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization.

Closure of the neural tube requires both the change and maintenance of cell shape. The change occurs mainly through two coordinated morphogenetic events: cell elongation and apical constriction. How cytoskeletal elements, including microtubules, are regulated in this process in vivo is largely unknown. Here, we show that neural tube closure in Xenopus depends on orthologs of two proteins: MID1, which is responsible for Opitz G/BBB syndrome in humans, and its paralog MID2. Depletion of the Xenopus MIDs (xMIDs) by morpholino-mediated knockdown disrupted epithelial morphology in the neural plate, leading to neural tube defects. In the xMID-depleted neural plate, the normal epithelial organization was perturbed without affecting neural fate. Furthermore, the xMID knockdown destabilized and caused the disorganization of microtubules, which are normally apicobasally polarized, accounting for the abnormal phenotypes. We also found that the xMIDs and their interacting protein Mig12 were coordinately required for microtubule stabilization during remodeling of the neural plate. Finally, we showed that the xMIDs are required for the formation of multiple epithelial organs. We propose that similar MID-governed mechanisms underlie the normal morphogenesis of epithelial tissues and organs, including the tissues affected in patients with Opitz G/BBB syndrome.

Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos.

Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified approximately 450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within approximately 3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region approximately 0.5-1.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates.

Xenopus Rnd1 and Rnd3 GTP-binding proteins are expressed under the control of segmentation clock and required for somite formation.

The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor-8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP-binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant-negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation-related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis.

High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases.

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1-Smad4 and Smad2-Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.

Prolactin inhibits osteoclastic activity in the goldfish scale: a novel direct action of prolactin in teleosts.

In teleosts, prolactin is involved in calcium regulation, but its role in scale/bone metabolism is unclear. Using the in-vitro system with goldfish scales developed recently, we explored the effects of teleost prolactin, growth hormone, and somatolactin on osteoclasts and osteoblasts. Addition of prolactin at concentrations of 0.01-100 ng/ml reduced osteoclastic activity, partly via osteoclast apoptosis, after 6-18 h incubation. Conversely, growth hormone and somatolactin at a concentration of 100 ng/ml increased osteoclastic activity after 18 h incubation, indicating the specificity of the inhibitory effect of prolactin on osteoclastic activity. On the other hand, these three hormones promoted osteoblastic activity at concentrations of 10-100 ng/ml. The results from this study are the first demonstration of direct effects of prolactin on scale/bone metabolism and osteoclastic activity in a teleost.

The initiator caspase, caspase-10beta, and the BH-3-only molecule, Bid, demonstrate evolutionary conservation in Xenopus of their pro-apoptotic activities in the extrinsic and intrinsic pathways.

Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10beta), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10beta and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10beta and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10beta mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

Transgenic frogs expressing the highly fluorescent protein venus under the control of a strong mammalian promoter suitable for monitoring living cells.

To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.

The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity.

FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.