Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica)

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 3²‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 18²‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.     

Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites

In extracts of senescent leaves of the tobacco plant Nicotiana rustica, two colorless compounds with UV/VIS characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively identified as Nr-NCCs. These two polar NCCs were found in similar amounts in the fresh extracts, and their constitutions could be determined by spectroscopic analysis. The data showed both of the two Nr-NCCs to have the same tetrapyrrolic core structure, as reported previously for all other NCCs from senescent higher plants. In the less polar catabolite, named Nr-NCC-2, this core structure was conjugated with a glucopyranose unit, as similarly discovered earlier in Bn-NCC-2, an NCC from oilseed rape (Brassica napus). The more polar NCC from tobacco leaves, Nr-NCC-1, carried an additional malonyl substituent at the 6'-OH group of the glucopyranosyl moiety. Partial (enzyme-catalyzed) hydrolysis of Nr-NCC-1 gave Nr-NCC-2, while enzyme-catalyzed malonylation of Nr-NCC-2 gave Nr-NCC-1, establishing the identity of their basic tetrapyrrole structure. In earlier work (on the polar NCCs from oilseed rape), only separate glucopyranosyl and malonyl functionalities were detected. Nr-NCC-1, thus, represents a further variant of the structures of NCCs from senescent higher plants and exhibits an unprecedented peripheral refunctionalization in chlorophyll catabolites.     

Chlorophyll breakdown in spinach: on the structure of five nonfluorescent chlorophyll catabolites*

In extracts of senescent leaves of spinach (Spinacia oleracea), five colourless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named So-NCCs. The most abundant polar NCC in the leaves of this vegetable, So-NCC-2, had been isolated earlier and its constitution was determined by spectroscopic means. The catabolite So-NCC-2 was found to be an epimer of a polar NCC from barley (Hordeum vulgare), the first non-green chlorophyll catabolite from a higher plant to be structurally analyzed. Here, we report on the isolation of four additional So-NCCs from the extracts of senescent leaves of Sp. oleracea by two- (or multi-)stage chromatographic purification and on their structural characterization. The constitution of So-NCC-3 could be determined by spectroscopic analysis in combination with chemical correlation with a known NCC from Cercidiphyllum japonicum (Cj-NCC): So-NCC-3 was identified as the hydrolysis product of the methyl ester function of Cj-NCC. The less polar catabolite So-NCC-4 could be directly identified with Cj-NCC. Two further So-NCCs, So-NCC-1 and So-NCC-5, were detected only in trace amounts. Five structurally related nonfluorescent chlorophyll catabolites (So-NCCs) are thus present in senescent leaves of spinach. The structures of this set of So-NCCs indicate several peripheral refunctionalization reactions and inform on the late catabolic transformations during leaf senescence. The transformation of the tetrapyrrolic skeleton in chlorophyll catabolism in spinach and in C. japonicum is revealed to exhibit a common stereochemical pattern. This revised version was published online in June 2006 with corrections to the Cover Date.     

On the Nature of Isomeric Nonfluorescent Chlorophyll Catabolites in Leaves and Fruit – A Study with a Ubiquitous Phylloleucobilin and its Main Isomerization Product

The typical main products of chlorophyll (Chl) breakdown in higher plants are non‐fluorescent, colorless phyllobilins, named phylloleucobilins. These long elusive Chl‐catabolites are linear tetrapyrroles, whose structure elucidation has required thorough spectroscopic analyses. Interestingly, in recent LC/MS studies of leaf extracts, isomeric forms of phylloleucobilins were detected. The existence of isomeric phyllobilins may suggest incomplete stereo‐selectivity of catabolic processes, or isomerization processes in plant cells or in the analytes. Here we report a study with the phylloleucobilin NCC‐1, a basic Chl‐catabolite in extracts of leaves and fruit. NCC‐1 and its main isomerization product in aqueous solution were identified as 8²‐epimers. Formation of 8²‐epi‐NCC‐1 from NCC‐1 implies an unstable enol(ate)‐intermediate, which reverts to NCC‐1 or converts to 8²‐epi‐NCC‐1. Such reversible epimerization reactions are a non‐biological in vitro feature of typical phylloleucobilins, and probably also take place in vivo.     

Control of neural crest cell behavior and migration: Insights from live imaging

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this Review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.Key words: live imaging, neural crest, EMT, Rho GTPase, ephrin, PCP signaling, cadherin, VEGFNeural crest cells (NCCs) are a pluripotent population of cells that migrate from the dorsal neuroepithelium and give rise to multiple cell types including neurons and glia of the peripheral nervous system, pigment cells and craniofacial bone and cartilage.¹ An important hallmark of NCCs is their remarkable ability to migrate over long distances and along specific pathways through the embryo. NCC migration begins with an epithelial to mesenchymal transition (EMT), in which NCCs lose adhesions with their neighbors and segregate from the neuroepithelium.^2,3 Following EMT, NCCs acquire a polarized morphology and initiate directed migration away from the neural tube. While migrating along their pathways to their target tissues, NCCs are guided by extensive communication with one another and by other cues from the extracellular environment. Each of these aspects of NCC migration requires precise regulation of cell motile behaviors, although the mechanisms controlling them are still not well understood. A critical step toward understanding the molecular control of NCC motility is characterization of NCC behaviors as they migrate in their native environment. In the past 15 years, multiple studies have analyzed specific behaviors associated with NCCs along the various stages of their journey and have begun to identify molecules controlling these behaviors. In this review we will focus specifically on these studies that employ live imaging and will highlight the strength of live imaging to reveal mechanisms regulating NCC motility and migration pathways.     

Dual origin of melanocytes defined by Sox1 expression and their region‐specific distribution in mammalian skin

Melanocytes are pigment‐producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1⁺ dorsal neuroepithelial cells but are derived from Sox1^? cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1^? cells clearly segregate from cells that originated from Sox1⁺ dorsal neuroepithelial cell‐derived NCCs. The possible derivation of Sox1^? cells from epidermal cells also strengthens their non‐neuroepithelial origin.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Neural crest cells (NCCs) are a multipotent embryonic cell population that contributes to the formation of various craniofacial structures including teeth. It has been generally believed that dental enamel is an ectodermal derivative, whereas the dentin–pulp complex and the surrounding supporting tissues originate from NCC-derived mesenchyme. These traditional concepts stem mainly from several early studies of fishes and amphibians. Recently, Wnt1-Cre/R26R mice, a mouse model for NCC lineage analysis, revealed the contribution of NCCs to mammalian tooth development. However, the discrepancy of expression patterns between different NCC-specific transgenic mouse lines makes it compulsory to revisit the cell lineage in mammalian tooth development. Here, we reevaluated the NCC lineage during mouse tooth development by using P0-Cre/R26R mice, another NCC-specific transgenic mouse line. Inconsistent with the traditional concepts, we observed the potential contribution of NCCs to developing enamel organ and enamel formation. We also demonstrated that the P0-Cre transgene was specifically expressed in migrating NCC in the hindbrain region, where NCC contributes to tooth, validating their applicability for NCC lineage analysis. Our unanticipated finding may change the general understanding of tooth development and provide new insights into dental stem cell biology.     

Syntheses and Flavors of Four Possible Optical Isomers of Marmelo Lactones

We have isolated a tetracycline-resistant (Tc^r) Bacillus species (named HE-1) which carries multiple plasmids. HE-1 was identified as Bacillus cereus and found to bear four plasmids. Tetracycline resistance could be attributed to one of four plasmids (designated as pTIT β2 (4.7 kb)) indistinguishable from pBC16, a Tc^r plasmid formerly found in B. cereus [K. Bernhard, H. Schrempf, and W. Goebel, J. Bacteriol., 133, 897 (1978)]. All the other three plasmids (named pTITα (4.0 kb), pTIT β1 (4.7 kb) and pTIT γ (12.4 kb)) were cryptic and did not correlate with bacterial phenotypic traits such as antibiotic resistance or antibiotic and bacteriocin production. B. cereus HE-1 also showed resistance to penicillin, but this seemed very likely to be chromosomally determined in B. cereus. Of interest was the fact that pTITα, pTIT β1, and pTITγ had a noticeable DNA homology among them in blot hybridization. pTIT β2 alone did not shared sequence homology with the other three plasmids.     

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)^pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.     

Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo

The neural crest is an excellent model to study embryonic cell migration, since cell behaviors can be studied in vivo with advanced optical imaging and molecular intervention. What is unclear is how molecular signals direct neural crest cell (NCC) migration through multiple microenvironments and into specific targets. Here, we tested the hypothesis that the invasion of cranial NCCs, specifically the rhombomere 4 (r4) migratory stream into branchial arch 2 (ba2), is due to chemoattraction through neuropilin-1-vascular endothelial growth factor (VEGF) interactions. We found that the spatio-temporal expression pattern of VEGF in the ectoderm correlated with the NCC migratory front. RT-PCR analysis of the r4 migratory stream showed that ba2 tissue expressed VEGF and r4 NCCs expressed VEGF receptor 2. When soluble VEGF receptor 1 (sVEGFR1) was injected distal to the r4 migratory front, to bind up endogenous VEGF, NCCs failed to completely invade ba2. Time-lapse imaging revealed that cranial NCCs were attracted to ba2 tissue or VEGF sources in vitro. VEGF-soaked beads or VEGF-expressing cells placed adjacent to the r4 migratory stream caused NCCs to divert from stereotypical pathways and move towards an ectopic VEGF source. Our results suggest a model in which NCC entry and invasion of ba2 is dependent on chemoattractive signaling through neuropilin-1-VEGF interactions.     

Loss of Xenopus cadherin-11 leads to increased Wnt/β-catenin signaling and up-regulation of target genes c-myc and cyclin D1 in neural crest

Xenopus cadherin-11 (Xcadherin-11) is an exceptional cadherin family member, which is predominantly expressed in cranial neural crest cells (NCCs). Apart from mediating cell–cell adhesion it promotes cranial NCC migration by initiating filopodia and lamellipodia formation. Here, we demonstrate an unexpected function of Xcadherin-11 in NCC specification by interfering with canonical Wnt/β-catenin signaling. Loss-of-function experiments, using a specific antisense morpholino oligonucleotide against Xcadherin-11, display a nuclear β-catenin localization in cranial NCCs and a broader expression domain of the proto-oncogene cyclin D1 which proceeds c-myc up-regulation. Additionally, we observe an enhanced NCC proliferation and an expansion of specific NCC genes like AP2 and Sox10. Thereby, we could allocate NCC proliferation and specification to different gene functions. To clarify which domain in Xcadherin-11 is required for early NCC development we tested different deletion mutants for their rescue ability in Xcadherin-11 morphants. We identified the cytoplasmic tail, specifically the β-catenin binding domain, to be necessary for proper NCC development. We propose that Xcadherin-11 is necessary for controlled NCC proliferation and early NCC specification in tuning the expression of the canonical Wnt/β-catenin target genes cyclin D1 and c-myc by regulating the concentration of the nuclear pool of β-catenin.     

Genomically integrated transgenes are stably and conditionally expressed in neural crest cell-specific lineages

Neural crest cells (NCCs) are a transient embryonic structure that gives rise to a variety of cells including peripheral nervous system, melanocytes, and Schwann cells. To understand the molecular mechanisms underlying NCC development, a gene manipulation of NCCs by in ovo electroporation technique is a powerful tool, particularly in chicken embryos, the model animal that has long been used for the NCC research. However, since expression of introduced genes by the conventional electroporation method is transient, the mechanisms of late development of NCCs remain unexplored. We here report novel methods by which late-developing NCCs are successfully manipulated with electroporated genes. Introduced genes can be stably and/or conditionally expressed in a NCC-specific manner by combining 4 different techniques: Tol2 transposon-mediated genomic integration (Sato et al., 2007), a NCC-specific enhancer of the Sox10 gene (identified in this study), Cre/loxP system, and tet-on inducible expression (Watanabe et al., 2007). This is the first demonstration that late-developing NCCs in chickens are gene-manipulated specifically and conditionally. These methods have further allowed us to obtain ex vivo live-images of individual Schwann cells that are associated in axon bundles in peripheral tissues. Cellular activity and morphology dynamically change as development proceeds. This study has opened a new way to understand at the molecular and cellular levels how late NCCs develop in association with other tissues during embryogenesis.     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

Hyaluronan (HA) is a major extracellular matrix component whose tissue levels are dynamically regulated during embryonic development. Although the synthesis of HA has been shown to exert a substantial influence on embryonic morphogenesis, the functional importance of the catabolic aspect of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in neural crest development and the morphogenesis of neural crest derivatives, as evidenced by the presence of severe craniofacial abnormalities in Wnt1-Cre–mediated Tmem2 knockout (Tmem2^CKO) mice. Neural crest cells (NCCs) are a migratory population of cells that gives rise to diverse cell lineages, including the craniofacial complex, the peripheral nervous system, and part of the heart. Analysis of Tmem2 expression during NCC formation and migration reveals that Tmem2 is expressed at the site of NCC delamination and in emigrating Sox9-positive NCCs. In Tmem2^CKO embryos, the number of NCCs emigrating from the neural tube is greatly reduced. Furthermore, linage tracing reveals that the number of NCCs traversing the ventral migration pathway and the number of post-migratory neural crest derivatives are both significantly reduced in a Tmem2^CKO background. In vitro studies using Tmem2-depleted mouse O9-1 neural crest cells demonstrate that Tmem2 expression is essential for the ability of these cells to form focal adhesions on and to migrate into HA-containing substrates. Additionally, we show that Tmem2-deficient NCCs exhibit increased apoptotic cell death in NCC-derived tissues, an observation that is corroborated by in vitro experiments using O9-1 cells. Collectively, our data demonstrate that TMEM2-mediated HA degradation plays an essential role in normal neural crest development. This study reveals the hitherto unrecognized functional importance of HA degradation in embryonic development and highlights the pivotal role of Tmem2 in the developmental process.     

Elucidating timing and function of endothelin-A receptor signaling during craniofacial development using neural crest cell-specific gene deletion and receptor antagonism

Cranial neural crest cells (NCCs) play an intimate role in craniofacial development. Multiple signaling cascades participate in patterning cranial NCCs, some of which are regulated by endothelin-A receptor (Ednra) signaling. Ednra^−/− embryos die at birth from severe craniofacial defects resulting from disruption of neural crest cell patterning and differentiation. These defects include homeotic transformation of lower jaw structures into upper jaw-like structures, suggesting that some cephalic NCCs alter their “identity” in the absence of Ednra signaling. To elucidate the temporal necessity for Ednra signaling in vivo, we undertook two strategies. We first used a conditional knockout strategy in which mice containing a conditionally targeted Ednra allele (Ednra^fl) were bred with mice from the Hand2-Cre and Wnt1-Cre transgenic mouse strains, two strains in which Cre expression occurs at different time periods within cranial NCCs. In our second approach, we used an Ednra-specific antagonist to treat wild type pregnant mice between embryonic days E8.0 and E10.0, a time frame encompassing the early migration and proliferation of cranial NCCs. The combined results suggest that Ednra function is crucial for NCC development between E8.25 and E9.0, a time period encompassing the arrival of NCCs in the arches and/or early post-migratory patterning. After this time period, Ednra signaling is dispensable. Interestingly, middle ear structures are enlarged and malformed in a majority of Ednra^fl/fl;Wnt1-Cre embryos, instead resembling structures found in extinct predecessors of mammals. These observations suggest that the advent of Ednra signaling in cranial NCCs may have been a crucial event in the evolution of the mammalian middle ear ossicles.