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Among the five human senses, light, sound, and force perceived by eye, ear, and skin, respectively are physical actions, and therefore can be easily measured and expressed as objective, univocal, and simple digital data with physical quantity. However, taste and odor molecules perceived by tongue and nose are chemical actions, it has been difficult to express them as objective and univocal digital data, since no reference chemicals can be defined. Therefore, while the recording, saving, transmitting to remote locations, and replaying human visual, auditory, and tactile information as digital data in digital devices has been realized (this series of data flow is defined as DX (digital transformation) in this review), the DX of human taste and odor information is not yet in the realization stage. Particularly, since there are at least 400,000 types of odor molecules and an infinite number of complex odors that are mixtures of these molecules, it has been considered extremely difficult to realize "human olfactory DX" by converting all odors perceived by the human olfaction into digital data. In this review, we discuss the current status and future prospects of the development of "human olfactory DX," which we believe can be realized by utilizing odor sensors that employ the olfactory receptors (ORs) that support human olfaction as sensing molecules (i.e., human OR sensor).

Migration of cell populations is a fundamental process in morphogenesis and disease. The mechanisms of collective cell migration of epithelial cell populations have been well studied. It remains unclear, however, how the highly motile mesenchymal cells, which migrate extensively throughout the embryo, are connected with each other and coordinated as a collective. During gastrulation in chick embryos, the mesoderm cells, that are formed by an epithelial-to-mesenchymal transition (EMT), migrate in the 3D space between ectoderm and endoderm of the embryo. Using live imaging and quantitative analysis, such as topological data analysis (TDA), we found that the mesoderm cells undergo a novel form of collective migration, in which they form a meshwork structure while moving away from the primitive streak. This meshwork is supported by N-cadherin-mediated cell-cell adhesion, which undergoes rapid reorganization. Overexpressing a mutant form of N-cadherin decreases the speed of tissue progression and the directionality of the collective cell movement, whereas the speed of individual cells remains unchanged. To investigate how this meshwork arises and how it contributes to the cell movement, we utilized an agent-based theoretical model, showing that cell elongation, cell-cell adhesion, and cell density are the key parameters for the meshwork formation. These data provide novel insights into how a supracellular structure of migrating mesenchymal cells forms and how it facilitates efficient migration during early mesoderm formation.

The pluripotent epiblast gives rise to all tissues and organs in an adult body. Its differentiation starts at gastrulation when the epiblast generates mesoderm and endoderm germ layers through a process called epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast’s cellular morphology and its pluripotency is not well understood. In this work, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit; whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and its developmental function is to mediate planar symmetry-breaking within an epithelialized epiblast, taking place after epiblast MET but before gastrulation EMT.

Epiblast is composed of pluripotent cells which will give rise to all cell lineages in a human body. It forms a single-cell layered epithelium conserved among all amniotic vertebrates (birds, reptiles and mammals) and undergoes complex morphogenesis both before and during gastrulation. Our knowledge of the amniote epiblast is based on data acquired through cellular and molecular analyses of early chick and mouse embryos in vivo and mammalian pluripotent stem cells (PSCs) in vitro. Very few studies have been published on biomechanical characteristics of the amniote epiblast, largely due to lack of experimental tools for measuring and perturbing biomechanical properties. Also missing is a conceptual framework that can integrate both biomechanical and molecular parameters of the epiblast. This review is aimed at providing a background based on which epiblast morphogenesis, including its transition between the epithelial and mesenchymal states, can be understood from a biomechanical perspective. This simple developmental biology system is suitable for testing a multitude of theoretical models in biomechanics, leading to a better understanding of biomechanical logics and constraints governing multicellular organization.

Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation., Summary: Early chick embryos share previously unappreciated features with anamniote embryos such as the timing of zygotic gene activation and yolk syncytial layer formation.

Disruption of the CLASP- and Dystroglycan-mediated cortical microtubule anchoring reduces epiblast–basement membrane interactions and initiates gastrulation., Amniote epiblast cells differentiate into mesoderm and endoderm lineages during gastrulation through a process called epithelial-to-mesenchymal transition (EMT). Molecular regulation of gastrulation EMT is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules to the basal cortex via CLIP-associated protein (CLASP), a microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediated through the CLASP-binding partner LL5. Live-imaging using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilized basal microtubules. We further show that Dystroglycan is localized to basolateral membrane in epiblast cells. Basal but not lateral localization of Dystroglycan was regulated by CLASP. We propose that epiblast–BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which initiates gastrulation EMT.

The neural crest is a transient structure unique to vertebrate embryos that gives rise to multiple lineages along the rostrocaudal axis. In cranial regions, neural crest cells are thought to differentiate into chondrocytes, osteocytes, pericytes and stromal cells, which are collectively termed ectomesenchyme derivatives, as well as pigment and neuronal derivatives. There is still no consensus as to whether the neural crest can be classified as a homogenous multipotent population of cells. This unresolved controversy has important implications for the formation of ectomesenchyme and for confirmation of whether the neural fold is compartmentalized into distinct domains, each with a different repertoire of derivatives. Here we report in mouse and chicken that cells in the neural fold delaminate over an extended period from different regions of the cranial neural fold to give rise to cells with distinct fates. Importantly, cells that give rise to ectomesenchyme undergo epithelial-mesenchymal transition from a lateral neural fold domain that does not express definitive neural markers, such as Sox1 and N-cadherin. Additionally, the inference that cells originating from the cranial neural ectoderm have a common origin and cell fate with trunk neural crest cells prompted us to revisit the issue of what defines the neural crest and the origin of the ectomesenchyme.

Gastrulation is a developmental process to generate the mesoderm and endoderm from the ectoderm, of which the epithelial to mesenchymal transition (EMT) is generally considered to be a critical component. Due to increasing evidence for the involvement of EMT in cancer biology, a renewed interest is seen in using in vivo models, such as gastrulation, for studying molecular mechanisms underlying EMT. The intersection of EMT and gastrulation research promises novel mechanistic insight, but also creates some confusion. Here we discuss, from an embryological perspective, the involvement of EMT in mesoderm formation during gastrulation in triploblastic animals. Both gastrulation and EMT exhibit remarkable variations in different organisms, and no conserved role for EMT during gastrulation is evident. We propose that a ‘broken-down’ model, in which these two processes are considered to be a collective sum of separately regulated steps, may provide a better framework for studying molecular mechanisms of the EMT process in gastrulation, and in other developmental and pathological settings.

Molecular and cellular mechanisms of epithelial-mesenchymal transition (EMT), crucial in development and pathogenesis, are still poorly understood. Here we provide evidence that distinct cellular steps of EMT occur sequentially during gastrulation. Basement membrane (BM) breakdown is the first recognizable step and is controlled by loss of basally localized RhoA activity and its activator neuroepithelial-transforming-protein-1 (Net1). Failure of RhoA downregulation during EMT leads to BM retention and reduction of its activity in normal epithelium leads to BM breakdown. We also show that this is in part mediated by RhoA-regulated basal microtubule stability. Microtubule disruption causes BM breakdown and its stabilization results in BM retention. We propose that loss of Net1 before EMT reduces basal RhoA activity and destabilizes basal microtubules, causing disruption of epithelial cell-BM interaction and subsequently, breakdown of the BM.

The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, “tet-on” and “tet-off”, works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes.

A series of heterodinuclear germanium−ruthenium complexes having sulfido/oxo bridges, Dmp(Dep)Ge(μ-E1)(μ-E2)Ru(η6-arene) (E1, E2 = S, O; arene = benzene, p-cymene; Dmp = 2,6-dimesitylphenyl, Dep = 2,6-diethylphenyl) were synthesized by the reaction of [Ru(η6-arene)Cl2]2 and the corresponding diarylgermanedichalcogenoles, Dmp(Dep)Ge(E1H)(E2H). The reaction with tertiary phosphines gave the corresponding adducts Dmp(Dep)Ge(μ-S)(μ-E)Ru(PR3) (E = S, O; R = Ph, Et), in which the arene ligand on the ruthenium was replaced by a mesityl group of Dmp. When Dmp(Dep)Ge(μ-S)2Ru(PPh3) was treated with the Bronsted acids H(OEt2)2BArF4 and HOTf, a sulfido bridge was protonated to afford [Dmp(Dep)Ge(μ-S)(μ-SH)Ru(PPh3)]X (X = BArF4, OTf). Likewise, the methylation reaction with Me3OBF4 proceeded at a μ-S, generating [Dmp(Dep)Ge(μ-S)(μ-SMe)Ru(PPh3)](BF4). On the other hand, protonation of Dmp(Dep)Ge(μ-S)(μ-O)Ru(PPh3) gave a μ-OH complex, [Dmp(Dep)Ge(μ-S)(μ-OH)Ru(PPh3)]+, while the analogous methylation afforded the cationi...

Mesenchymal-epithelial transitions (MET) are crucial for vertebrate organogenesis. The roles of Rho family GTPases in such processes during actual development remain largely unknown. By electroporating genes into chick presomitic mesenchymal cells, we demonstrate that Cdc42 and Rac1 play important and different roles in the MET that generates the vertebrate somites. Presomitic mesenchymal cells, which normally contribute to both the epithelial and mesenchymal populations of the somite, were hyperepithelialized when Cdc42 signaling was blocked. Conversely, cells taking up genes that elevate Cdc42 levels remained mesenchymal. Thus, Cdc42 activity levels appear critical for the binary decision that defines the epithelial and mesenchymal somitic compartments. Proper levels of Rac1 are necessary for somitic epithelialization, since cells with activated or inhibited Rac1 failed to undergo correct epithelialization. Furthermore, Rac1 appears to be required for Paraxis to act as an epithelialization-promoting transcription factor during somitogenesis.

A S/SH bridged hetero-dinuclear Ru/Ge complex cation reacted with H(2) to afford the μ-S/μ-H complex. The reaction was considerably slower compared to that of the μ-S/μ-OH complex. Thus, the μ-S/μ-SH and μ-S/μ-OH complexes might provide models for the unready and ready states, respectively, of [NiFe] hydrogenase.

We synthesized a new affinity gel (PKSI-Toyopearl) using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as an affinity ligand, and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-Toyopearl column. Adsorbed protein was eluted with 50 mM glycine-hydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma. Further purification was performed by chromatography on a DEAE-Toyopearl 650M column. Plasma kallikrein was finally purified 1720-fold with a 63% yield by these procedures. On sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, a band was observed at approximately 88 kDa. These findings indicate that PKSI-Toyopearl is a valuable tool for the purification of plasma kallikrein from human plasma.

Epithelial–mesenchymal transition (EMT) is a process in which epithelial cells are converted into mesenchymal cells. It plays crucial roles in the formation of complex organs and in tissue repair during animal development. After EMT, cells acquire migratory and invasive properties, which can contribute to organ fibrosis and cancer metastasis. However, the precise cellular mechanisms that govern EMT in various in vivo contexts are poorly understood. Gastrulation is a process through which the three embryonic germ layers are formed. During this process, epithelial cells in the epiblast layer dynamically change their shape and ingress through the primitive streak to become mesoderm or endoderm. EMT occurs during this gastrulation process, and in this chapter, I describe the key principles of EMT and characterize EMT in the primitive streak during chicken gastrulation. Gastrulation EMT is a multistep process that includes the dissociation of cell–cell contact, the loss of epithelial polarity, and the degradation of the basement membrane (BM). Our recent studies indicate that epiblast/BM interaction requires basally localized RhoA activity, and CLASP- and dystroglycan-mediated cortical microtubule anchoring, the disruption of which causes BM degradation during gastrulation EMT.

The primitive streak is where the mesoderm and definitive endoderm precursor cells ingress from the epiblast during gastrulation. It is often described as an embryological feature common to all amniotes. But such a feature has not been associated with gastrulation in any reptilian species. A parsimonious model would be that the primitive streak evolved independently in the avian and mammalian lineages. Looking beyond the primitive streak, can one find shared features of mesoderm and endoderm formation during amniote gastrulation? Here, we survey the literature on reptilian gastrulation and provide new data on Brachyury RNA and laminin protein expression in gastrula-stage turtle (Pelodiscus sinensis) embryos. We propose a model to reconcile the primitive streak-associated gastrulation in birds and the blastopore-associated gastrulation in extant reptiles. © 2012 Japanese Society of Developmental Biologists.

Carcinomas, cancers of epithelial origin, constitute the majority of all cancers. Loss of epithelial characteristics is an early step in carcinoma progression. Malignant transformation and metastasis involve additional loss of cell-cycle control and gain of migratory behaviors. Understanding the relationships among epithelial homeostasis, cell proliferation, and cell migration is therefore fundamental in understanding cancer. Interestingly, these cellular events also occur frequently during animal development, but without leading to tumor formation. Can we learn anything about carcinomas from developmental biology? In this review, we focus on one aspect of carcinoma progression, the Epithelial–Mesenchymal Transition (EMT), and provide an overview of how the EMT is involved in normal amniote development. We discuss 12 developmental and morphogenetic processes that clearly involve the EMT. We conclude by emphasizing the diversity of EMT processes both in terms of their developmental context and of their cellular morphogenesis. We propose that there is comparable diversity in cancer microenvironment and molecular regulation of cancer EMTs.

The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time-lapse imaging and molecular techniques. Emu embryos in general take 2-3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50-56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27- to 30-somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed.

Regulated disruption of the basement membrane (BM) is a critical step in many epithelial-mesenchymal transition (EMT) processes. Molecular mechanisms controlling the interaction between the BM and the basal membrane of epithelial cells and its subsequent disruption during EMT are poorly understood. Using chick embryos as a model, we analyzed the molecular complexity of this interaction during gastrulation EMT. Transcriptome data indicated that the BM of the gastrulation stage chick epiblast contains a full range of BM component proteins with unique subtype combinations. Integrins and dystroglycan are 2 major groups of basal membrane proteins involved in BM interaction. We provide evidence that dystroglycan gene expression is restricted to the epiblast during early development and its expression is downregulated in cells undergoing gastrulation EMT. The β-dystroglycan protein is localized to the basolateral membrane in epiblast cells and the basal localization is lost in cells undergoing EMT. Disruption of actin filaments leads to a decrease in the lateral membrane localization of β-dystroglycan and a relative increase in basal membrane localization, whereas disruption of microtubules leads to the loss of BM/basal membrane interaction and basal membrane β-dystroglycan localization. Overall, these data suggest an involvement of dystroglycan, especially the regulation of its expression and localization, in gastrulation EMT.

Epithelial to mesenchymal transition (EMT) is a morphogenetic process in which cells lose their epithelial characteristics and gain mesenchymal properties, and is fundamental for many tissue remodeling events in developmental and pathological conditions. Although general cell biology of EMT has been well-described, how it is executed in diverse biological settings depends largely on individual context, and as a consequence, regulatory points for each EMT may vary. Here we discuss developmental and cellular events involved in chick gastrulation EMT. Regulated disruption of epithelial cell/basement membrane (BM) interaction is a critical early step. This takes place after molecular specification of mesoderm cell fate, but before the disruption of tight junctions. The epithelial cell/BM interaction is mediated by small GTPase RhoA and through the regulation of basal microtubule dynamics. We propose that EMT is not regulated as a single morphogenetic event. Components of EMT in different settings may share similar regulatory mechanisms, but the sequence of their execution and critical regulatory points vary for each EMT.

Benzodiazepines are a class of psychoactive drugs widely used for their anxiolytic, anticonvulsant, muscle relaxant and hypnotic properties. Although the benzodiazepine receptor in the central nervous system has been well studied, the role of peripheral type benzodiazepine receptor, PBR, remains elusive. Here, we show that there are two PBR homologous genes in amniotes, PBR and PBRL , based on phylogenetic analysis. In chickens, PBRL is exclusively expressed during early development in differentiating primitive erythrocytes and this expression is tightly correlated with that of hemoglobin genes. PBR is not expressed in hematopoietic system during this period and is weakly expressed in developing central nervous system. Because one of PBRs’ known functions is to regulate heme transport between the mitochondria and cytoplasm, we investigated expression profiles of heme biosynthesis genes. Seven of the eight enzymes involved in heme biosynthesis, with the exception of protoporphyrinogen oxidase , are present in chicken genome. Five of them, δ-aminolevulinate synthase , δ-aminolevulinic acid dehydrogenase , porphobilinogen deaminase , coproporphyrinogen decarboxylase and ferrochelatase , show stage-specific increase in gene expression correlated with primitive hematopoiesis, but not with primitive erythrocyte differentiation. PBRL protein is localized to the mitochondria in culture cells, and pharmacological inhibition of PBRL activity results in a decrease in globin protein levels during primitive erythropoiesis. Our data suggest a developmental role of PBRs in erythropoiesis in chickens, possibly via the regulation of heme availability for the assembly of functional hemoglobins.

During early development in vertebrates, cells change their shapes dramatically both from epithelial to mesenchymal and also from mesenchymal to epithelial, enabling the body to form complex tissues and organs. Using somitogenesis as a novel model, Rho family GTPases have recently been shown to play essential and differential roles in individual cell behaviors in actual developing embryos. Levels of Cdc42 activity provide a binary switch wherein high Cdc42 levels allow the cells to remain mesenchymal, while low Cdc42 levels produce epithelialization. Rac1 activity needs to be precisely controlled for proper epithelialization through the bHLH transcription factor Paraxis. Somitogenesis is expected to serve as an excellent model with which one can understand how the functions of developmental genes are resolved into the morphogenetic behavior of individual cells.

The S/OH bridged dinuclear Ru−Ge complex cation, [Dmp(Dep)Ge(μ-S)(μ-OH)Ru(PR3)](BArF4) (3), which was synthesized quantitatively by the protonation of Dmp(Dep)Ge(μ-S)(μ-O)Ru(PR3) (1) with H(OEt2)2BArF4 (ArF = 3,5-(CF3)2C6H3), was found to promote facile and reversible H2 activation leading to H2O and [Dmp(Dep)Ge(μ-S)(μ-H)Ru(PR3)](BArF4) (4) under mild conditions. The μ-hydride of 4 exhibits protonic behavior, and the acidity of the μ-hydride is sufficiently high to transform 1 into 3. Thus, when a C6D6 solution of 1 with a catalytic amount of H(OEt2)2BArF4 (1 mol %) was heated to 70 °C for 24 h under an H2 atmosphere, [Dmp(Dep)Ge(μ-S)Ru(PR3)] (5) and H2O were formed. The molecular structures of 4 and 5 were determined by X-ray analysis. In the catalytic conversion of 1 to 5, H2 is consumed formally as two protons generating H2O, and two electrons which are stored in 5 as the Ge−Ru bond or as Ru d-electrons. Activation of H2 using the Ge−Ru complexes provides an intriguing functional model of hydrogenases,...

The first 1,2,3,4,5,6,7-hexathiagermepanes Dmp(Ar)GeS6 [Dmp=2,6-dimesitylphenyl, Ar = Mes, Dep (2,6-diethylphenyl), Tip (2,4,6-triisopropylphenyl)] were synthesized along with 1,2,3,4,5-tetrathiagermolanes by sulfurization of the corresponding dihydrogermanes. The hexathiagermepane Dmp(Tip)GeS6 slowly converted to the tetrathiagermolane with release of elemental sulfur owing to the steric hindrance.