Search

Summary: 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.

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.

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., Highlights • c-FLIP/CFLAR is specific to the vertebrate lineage. • A rapid evolutionary divergence of the c-FLIP subfamily members has occurred. • The pseudocatalytic triad is formed by evolutionarily conserved amino acids. • Vertebrate c-FLIP proteins work on both apoptotic inhibition and NF-κB activation. • c-FLIP proteins play a developmental role during embryogenesis of frog and fish.

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.

Summary 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/β-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/β-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., Graphical Abstract, Highlights • Wnt/β-catenin signaling promotes the differentiation of GFRα1+ spermatogonia • SHISA6 is a cell-autonomous Wnt inhibitor expressed in subset GFRα1+ cells • SHISA6 confers resistance to differentiation induction by Wnt/β-catenin signaling • SHISA6+ spermatogonia show stem cell-related properties, In mouse testicular seminiferous tubules, Yoshida and colleagues questioned how sperm stem cells follow different fates (to differentiate or to self-renew) in response to homogeneously distributed extracellular signals. They showed that heterogeneous expression of Shisa6, a cell-autonomous Wnt inhibitor, confers resistance to homogeneously distributed differentiation-promoting Wnt signaling. This may be generic for stem cell regulation in a facultative niche.

Objective: To examine the effects of fasting and refeeding on intestinal cell proliferation and apoptosis in an opportunistic predator, hammerhead shark (Sphyrna lewini) of elasmobranch fishes which are among the earliest known extant groups of vertebrates to have the valvular intestine typical for the primitive species. Methods: Animals were euthanized after 5-10 d of fasting or feeding, or after 10-day fasting and 5-day refeeding. Intestinal apoptosis and cell proliferation were assessed by using oligonucleotide detection assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry of proliferating cells nuclear antigen. Results: Plasma levels of cholesterol and glucose were reduced by fasting. Intestinal apoptosis generally decreased during fasting. Numerous apoptotic cells were observed around the tips of the villi, primarily in the epithelium in the fed sharks, whereas fewer labeled nuclei were detected in the epithelium of fasted sharks. Refeeding returned intestinal apoptosis to the level in the fed sharks. Proliferating cells were observed in the epithelium around the troughs of the villi and greater in number in fed sharks, whereas fewer labeled nuclei were detected in fasted sharks. Conclusions: The cell turnover is modified in both intestinal epithelia of the shark and the murines by fasting/feeding, but in opposite directions. The difference may reflect the feeding ecology of the elasmobranchs, primitive intermittent feeders.

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.

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 β1 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.

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. Developmental Dynamics 238:2867–2876, 2009. © 2009 Wiley-Liss, Inc.

An investigation was undertaken on the adsorption and desorption properties of the expanded poly (tetrafluoroethylene) (ePTFE) films grafted with 2-(dimethylamino)ethyl methacrylate (DMAEMA) to anionic dye anions with one to three sulfonic groups in response to temperature changes. The amount of adsorbed metanil yellow (MY) anions increased with the grafted amount and most of the dimethylamino groups appended to the grafted PDMAEMA chains worked as an adsorption site to MY anions for the DMAEMA-grafted ePTFE (ePTFE-g-PDMAEMA) films with the grafted amounts of higher than 1.1 mmol/g. When the dye-anion-adsorbed ePTFE-g-PDMAEMA films were alternately immersed in water at two different temperatures, dye anions were desorbed from the ePTFE-g-PDMAEMA films at higher temperatures without any chemical agents. The amount of desorbed dye anions increased with an increase in the temperature of water from 40 to 80°C. Desorption of dye anions is caused by either deprotonation of dimethylamino groups appended to the grafted PDMAEMA chains or thermosensitive contraction of the grafted PDMAEMA chains. These results indicate that the ePTFE-g-PDMAEMA films can be applied as a regenerative ion-exchange membrane for adsorption and desorption processes of anionic compounds in response to the temperature change. The thermally regenerative ion-exchange properties of the ePTFE-g-PDMAEMA films was superior to that of the PE-g-PDMAEMA films reported in our previous article in the fact that the total degree of desorption was higher for the ePTFE-g-PDMAEMA films. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007

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.

An investigation was undertaken on the adsorption and desorption properties of 2-(dimethylamino)ethyl methacrylate grafted polyethylene (PE-g-PDMAEMA) films to anionic dye anions with one to three sulfonic groups in response to pH and temperature changes. The amounts of dye anions adsorbed on the PE-g-PDMAEMA films passed through the maximum values at about pH 3 because of an increase in the protonation of dimethylamino groups caused by a decrease in the pH value. The amounts of adsorbed dye anions decreased below pH 3 because the ionic strength increased with the addition of HCl to adjust the initial pH values of the aqueous dye solutions. The amounts of adsorbed dye anions decreased with an increase in the number of sulfonic groups in the dye molecules at the same pH value because electrostatic repulsion was generated between free sulfonic groups of the dye anions adsorbed onto the PE-g-PDMAEMA films and free dye anions in the medium. A large number of dye anions adsorbed were desorbed from the PE-g-PDMAEMA film with initial pH values above 11.0. The cyclic processes of adsorption at pH 3.0 and desorption at pH 11.0 were repeated without considerable fatigue. The PE-g-PDMAEMA films showed practically regenerative adsorption and desorption behavior in response to the pH changes. In addition, when the dye-anion-adsorbed PE-g-PDMAEMA films were alternately immersed in water at two different temperatures, dye anions were desorbed in water at higher temperatures without any chemical agents because of the deprotonation of dimethylamino groups and thermosensitive contraction of grafted PDMAEMA chains. These results indicate that PE-g-PDMAEMA films can be applied as regenerative ion-exchange membranes for adsorption and desorption processes of anionic compounds in response to the pH and temperature. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 381–391, 2006

ウンシュウミカンの摘果時期の違いが食味に及ぼす影響を調査した結果,慣行の摘果時期より遅い摘果で食味が向上した.'上野早生'では9月摘果区,'宮川早生'では8月摘果区で有意に"甘さ"と"口当たり"の評価値が高かった.

It is well established that in Xenopus, bone morphogenetic protein (BMP) ventralizes the early embryo through the activation of several target genes encoding homeobox proteins, some of which are known to be necessary and sufficient for ventralization. Here, we used an inhibitory form of Xmsx-1, one of BMP’s targets, to examine its role in head formation. Interestingly, ventral overexpression of a dominant Xmsx-1 inhibitor induced an ectopic head with eyes and a cement gland in the ventral side of the embryo, suggesting that Xmsx-1 is normally required to suppress head formation in the ventral side. Supporting this observation, we also found that wild-type Xmsx-1 suppresses head formation through the inhibition of nodal signaling, which is known to induce head organizer genes such as cerberus, Xhex and Xdkk-1. We propose that negative regulation of the BMP/Xmsx-1 signal is involved not only in neural induction but also in head induction and formation. We further suggest that the inhibition of nodal signaling by Xmsx-1 may occur intracellularly, through interaction with Smads, at the level of the transcriptional complex, which activates the activin responsive element.

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

© 2013 Takahashi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In vitro Effects of the Prolactin, Growth Hormone and Somatolactin on Cell Turnover in Fish Esophagus: Possible Mode of Opposite Osmoregulatory Actions of Prolactin and Growth Hormone

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.

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.

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.

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.

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 10 nM 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.

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.

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.

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 β-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. Developmental Dynamics 233:562–569, 2005. © 2005 Wiley-Liss, Inc.

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.

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

Molecular chaperones are a group of proteins that bind to newly synthesized polypeptides or nonnative proteins caused by various stresses such as heat shock and which assist them to fold to the native structure by protecting them from aggregation. Chaperonin (Hsp 60) is one of the members of the molecular chap eron group and has been extensively studied. Chaperonin binds nonnative pro teins and folds them to the native form in an ATP-dependent fashion. In this chapter we show kinetic analysis of the interaction between chaperonin GroEL (from E. coli) and reduced α-lactalbumin as an example in an experiment with the BIACORE system. As determined by X-ray crystallography, the shape of GroEL is a porous cylinder of 14 identical 57K subunits and is made of 7-fold rotationally symmetrical rings stacked back-to-back with a dyad symmetry (Fig.1.1)[1,2]. Co-chaperonin, GroES, is a heptameric ring of identical 10K subunits. In the presence of ATP (ADP), GroEL and GroES oligomers form asymmetric 1:1 complexes in vitro, where GroES caps the cavity of one ring of GroEL, leaving the cavity in the other ring open [3,4]. Chaperonin has been found to bind many substrate proteins of which the structures are considered to be in an unfolded state. If substrate proteins unfolded in denaturant such as guanidine hydrochloride were diluted into denaturant free buffer, almost all the substrate protein will fold (or aggregate) rapidly and spontaneously.

The role of the optic vesicle in lens development was reinvestigated in Cynops pyrrhogaster. To study the necessity for the optic vesicle in early lens development, the optic anlages of stage 17-27 embryos were ablated and the frequency of free lens formation was examined with lens specific markers. Free lens formation was not observed when operations were performed prior to contact between the head surface epidermis and the optic vesicle (stages 17-18). On the contrary, free lens formation occurred in all cases where the optic vesicles were removed after the initiation of lens placode formation in the head surface epidermis (stage 27). However, no lens fiber formation was observed in these free lenses as judged by the absence of lens fiber specific gene expression, namely gamma-crystallin, at stages when secondary lens fiber formation could be found in the control lenses of the unoperated sides. The pattern of expression of alpha A-crystallin in the developing free lens also differed from that of the normally developing lens. This paper is the first report to indicate that the coordinated and sequential expression of crystallin genes are influenced by the optic vesicle; the optic vesicle is required for proper regulation of the alpha A- and gamma-crystallin but not beta B1-crystallin genes.

Brachyury , a T-box transcription factor, is expressed exclusively in primordial notochord cells of ascidian embryos, and plays a pivotal role in differentiation of notochord cells. Previously, we identified ~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 cis -regulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating ~3-kb upstream regions of the genes fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancers for each of these eight genes were narrowed to ~0.5–1.0-kb long regions. 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.

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.

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.

The kidney and vasculature are intimately linked functionally and during development, where nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms underlying the differentiation of kidney vs. blood/vascular lineages are unknown. The odd skipped related1 (osr1) gene encodes a zinc finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a segmentspecific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Kidney glomeruli and proximal tubules were absent while the cardinal vein and posterior venous plexuswere expanded. Altered kidney vs. vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function with blockade of endoderm development by knockdown of sox32/ casanova rescued kidney proximal tubule but not glomerular development. Absence of glomerular development correlated with osr1 co-expression with wt1 in podocyte progenitor cells. The results indicate that osr1 affects kidney proximal tubule and angioblast development by a non-cell autonomous effect mediated by endoderm while glomerular development is likely to be due to a cell-autonomous role for osr1 in podocyte progenitors.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor β superfamily, have been identified by their ability to induce cartilage and bone from nonskeletal cells and have been shown to act as a ventral morphogen inXenopusmesoderm. We isolated a murine homeobox-containing gene, distal-less 5 (mDlx5), as a BMP-inducible gene in osteoblastic MC3T3-E1 cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA showed increase in various osteogenic markers, a fourfold increase in alkaline phosphatase activity, a sixfold increase in osteocalcin production, and appearance in mineralization of extracellular matrix. Furthermore, mDlx5 was induced orthotopically in mouse embryos treated with BMP-4 and in fractured bone of adult mice. Consistent with these observations, we also found that injection of mDlx5 mRNA into dorsal blastomeres enhanced the ventralization ofXenopusembryos. These findings suggest that mDlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorsoventral patterning of embryonic axis.