Time-lapse tracing of mitotic cell divisions in the early Xenopus embryo using microscopic MRI

Mitotic cell division is a highly regulated cellular event in all organisms, but its direct visualization in the vertebrates is limited to animals with transparent embryos. Here, we report on the use of microscopic magnetic resonance imaging (mMRI) to noninvasively observe mitotic cell division of early blastomeres in the optically opaque Xenopus laevis embryo. Due to intrinsic tissue contrast, cell nuclei can be directly visualized without the need for contrast enhancing labeling. By taking two-dimensional in vivo time-lapse image sequences, the karyokinesis of a blastomere is followed. Timing and orientation of the cleavages can be traced for five cell divisions to establish a cell lineage tree, including orientation and timing of the mitosis. This work demonstrates for the first time the use of MRI for the visualization of cell divisions and expands the experimental scope of the Xenopus embryo

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). “Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.” Development. 126, 3703–3713.]. We present a revised model of gastrulation movements in Xenopus laevis

Two-Dimensional and Three-Dimensional Time-Lapse Microscopic Magnetic Resonance Imaging of Xenopus Gastrulation Movements Using Intrinsic Tissue-Specific Contrast

The amphibian embryo undergoes radical tissue transformations during blastula and gastrula stages, but live observation of internal morphogenetic events by optical microscopy is not feasible due to the opacity of the early embryo. Here, we report on the use of microscopic magnetic resonance imaging (MRI) to directly follow morphogenetic movements during blastula and gastrula stages of the Xenopus laevis embryo. We compare three different MRI modalities that take advantage of the intrinsic contrast present in embryonic tissues: three-dimensional (3D) fat-imaging, 3D water-imaging, and 2D high-speed high-resolution imaging of early embryonic stages. We show that the features revealed by the intrinsic contrast correlate with the histological structure of the embryo. Using this tissue specific intrinsic contrast, the main embryonic tissues and internal tissue movements as well as archenteron invagination can be differentiated without cell labeling. We present 2D and 3D time-lapse sequences of early Xenopus embryonic development, spanning the stages from early blastula to the end of gastrula, which show the complex internal rearrangements of gastrulation in essentially real-time

Differential mobility separation of leukotrienes and protectins.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageDifferential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to μLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.Prof. Jan Veltkamp fond

Systematic Screening for Novel Lipids by Shotgun Lipidomics

A commonly accepted LIPID MAPS classification recognizes eight major lipid categories and over 550 classes, while new lipid classes are still being discovered by targeted biochemical approaches. Despite their compositional diversity, complex lipids such as glycerolipids, glycerophospholipids, saccharolipids, <i>etc.</i> are constructed from unique structural moieties, <i>e.g.</i>, glycerol, fatty acids, choline, phosphate, and trehalose, that are linked by amide, ether, ester, or glycosidic bonds. This modular organization is also reflected in their MS/MS fragmentation pathways, such that common building blocks in different lipid classes tend to generate common fragments. We take advantage of this stereotyped fragmentation to systematically screen for new lipids sharing distant structural similarity to known lipid classes and have developed a discovery approach based on the computational querying of shotgun mass spectra by LipidXplorer software. We applied this concept for screening lipid extracts of <i>C. elegans</i> larvae at the <i>dauer</i> and L3 stages that represent alternative developmental programs executed in response to environmental challenges. The search, covering more than 1.5 million putative chemical compositions, identified a novel class of lyso-maradolipids specifically enriched in <i>dauer</i> larvae

Dissociation of Fatty Liver and Insulin Resistance in I148M PNPLA3 Carriers: Differences in Diacylglycerol (DAG) FA18:1 Lipid Species as a Possible Explanation

Fatty liver is tightly associated with insulin resistance and the development of type 2 diabetes. I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene is associated with high liver fat but normal insulin sensitivity. The underlying mechanism of the disassociation between high liver fat but normal insulin sensitivity remains obscure. We investigated the effect of I148M variant on hepatic lipidome of subjects with or without fatty liver, using the Lipidyzer method. Liver samples of four groups of subjects consisting of normal liver fat with wild-type PNPLA3 allele (group 1); normal liver fat with variant PNPLA3 allele (group 2); high liver fat with wild-type PNPLA3 allele (group 3); high liver fat with variant PNPLA3 allele (group 4); were analyzed. When high liver fat to normal liver fat groups were compared, wild-type carriers (group 3 vs. group 1) showed similar lipid changes compared to I148M PNPLA3 carriers (group 4 vs. group 2). On the other hand, in wild-type carriers, increased liver fat significantly elevated the proportion of specific DAGs (diacylglycerols), mostly DAG (FA18:1) which, however, remained unchanged in I148M PNPLA3 carriers. Since DAG (FA18:1) has been implicated in hepatic insulin resistance, the unaltered proportion of DAG (FA18:1) in I148M PNPLA3 carriers with fatty liver may explain the normal insulin sensitivity in these subjects

Ganglioside lipidomics of CNS myelination using direct infusion shotgun mass spectrometry

Gangliosides are present and concentrated in axons and implicated in axon-myelin interactions, but how ganglioside composition changes during myelin formation is not known. Here, we present a direct infusion (shotgun) lipidomics method to analyze gangliosides in small amounts of tissue reproducibly and with high sensitivity. We resolve the mouse ganglioside lipidome during development and adulthood and determine the ganglioside content of mice lacking the St3gal5 and B4galnt1 genes that synthesize most ganglioside species. Our results reveal substantial changes in the ganglioside lipidome during the formation of myelinated nerve fibers. In sum, we provide insights into the CNS ganglioside lipidome with a quantitative and sensitive mass spectrometry method. Since this method is compatible with global lipidomic profiling, it will provide insights into ganglioside function in physiology and pathology

Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants

10.1038/srep15926SCIENTIFIC REPORTS5