Podcasts about 3d fish

The team takes an incredible journey into the world of 3D scanned specimens. Guests include #Scanallfish star Dr. Adam Summers of the University of Washington, Thadeus Beuser of Oregon State University, and Ben Frable of the Scripps Institution of Oceanography.

Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge and achievement of this doctorate thesis was the development of a novel method to perform 3D-SIM on mammalian embryos (“3D structured illumination microscopy of mammalian embryos and spermatozoa” published in BMC Developmental Biology). The development and fine-tuning of this method took over two years due to the time-intense generation of embryos and the subsequent two day long embryo staining, embedding and scanning with steps that required novel techniques such as micromanipulation which was not associated with sample preparation prior to this protocol. Problem identification was time-intensive since each of the numerous steps necessary could negatively affect the image quality. This method was fine-tuned during three studies. The first study “Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos” (published in Nucleus) investigates the profound changes of nuclear architecture during cattle preimplantation development of embryos generated by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Fibroblast nuclei in embryos generated by SCNT go through similar changes in nuclear architecture as embryos generated by IVF. In both embryo types the occurrence of a large, chromatin-free lacuna in the center of nuclei around major embryonic genome activation (EGA) was noted. Similarly, the chromosome territory-interchromatin compartment (CT-IC) model applied to both types of embryos, featuring a lacuna or not, with an enrichment of RNA polymerase II and H3K4me3, a histone modification for transcriptionally competent chromatin, in less concentrated chromatin and an enrichment of H3K9me3, a transcriptionally restrictive histone modification, in more concentrated chromatin. However, large, highly concentrated H3K4me3 and H3K9me3 clusters were noted in both embryo types at chromatin concentrations that did not fit to the model. The chromatin-free lacunas were highly enriched in newly synthesized mRNA. The second study “Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications” (published in PLOS ONE) presents the changes of the nuclear envelope and lamina during bovine preimplantation development. Before major EGA, chromatin-free areas of the nuclear periphery were also free of nuclear pore complexes (NPCs), whereas after major EGA, the entire nuclear periphery was equipped with at least a fine layer of chromatin and associated NPCs. Three types of nuclear invaginations were predominant at different stages. The most common invagination was lamin B and NUP153 positive and was most prominent between the 2-cell and 8-cell stages until the onset of major EGA. Lamin B positive, but NUP153 negative invaginations were most prominent during stages with large nuclear volume and surface reductions. The least common invagination was lamin B negative but NUP153 positive and occurred almost exclusively at the morula stage. RNA-Seq and 3D-SIM data showed large deposits of spliced NUP153 mRNA and cytoplasmic NUP153 protein clusters until shortly after major EGA. NUP153 association with chromatin was initiated at metaphase. The third study “Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development” (published in the Journal of Reproduction and Development) demonstrated that rabbit embryonic nuclei feature a nuclear invagination type containing a large volume of cytoplasm that provides cytoplasmic proximity to nucleoli in addition to the small volume invaginations that were previously observed in bovine nuclei. The underlying mechanism for these two invaginations must differ from each other since small volume invaginations were frequently emanating from large volume invaginations emanating from the nuclear border but large volume invaginations were never emanating from small volume invaginations emanating from the nuclear border. Abundance of import/export competent invaginations featuring NPCs peaked at the 4-cell stage, which is the last stage before a drastic nuclear volume decline and also the last stage before major EGA is initiated at the 8- to 16-cell stage. Import/export incompetent invaginations positive for lamin B but not NUP153 peaked at the 2-cell stage. This was the stage with the largest variability in nuclear volumes. This may hint at an interphase nuclear surface reduction mechanism. Additionally, previously generated but unpublished 3D-FISH data about the localization changes of a stably inserted reporter gene upon activation in cloned bovine embryos was analyzed and documented in the study “Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos” (published in Nucleus). This study showed that the stably inserted OCT-4 reporter gene “GOF” in bovine fetal fibroblasts was initially moved towards the nuclear interior in day 2 bovine embryos generated by SCNT of bovine fetal fibroblasts. However, in day 4 SCNT embryos the localization of GOF had moved towards the periphery while it was still activated. Its carrier chromosome territory did not significantly move differently compared with the non-carrier homolog. Constant proximity of GOF to its carrier chromosome territory ruled out a movement by giant loops. In cooperation with the Department of Histology and Embryology of the Ege University (Izmir, Turkey) the destructive effects of cryopreservation on blastomere integrity were analyzed in the study “Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification” (published in Anatomia, Histologia, Embryologia). The cryopreservation method slow freezing caused more damage to blastomeres and to the zona pellucida than its fast freezing alternative vitrification. This was most likely caused by ice crystal formation and the longer exposure to the toxic side effects of cryoprotectants before freezing was complete.

Im Zellkern einer jeden Zelle besteht eine gewisse Ordnung der darin vorhandenen DNA und Proteine. Diese Ordnung wird unter dem Begriff „Zellkernarchitektur“ zusammengefasst. In der vorliegenden Arbeit ging es um die nähere Betrachtung einiger Aspekte der Zellkernarchitektur. Diese Aspekte betrafen 1. die Anordnung von Genen, 2. die Anordnung von Chromatin mit Hilfe unterschiedlicher Histonmodifikationen und 3. die Anordnungen von Chromosomenabschnitten, die mit komplexen messenger RNA-Sonden hybridisiert werden. Im ersten Teil der vorliegenden Arbeit wurde mittels 3D FISH die dreidimensionale Positionierung von drei auf dem Chromosom 1 lokalisierten Genen in Zellkernen der Burkitt- Lymphom Zelllinie DG75 bestimmt. Diese Zelllinie wurde von Stefan Bohlander zur Verfügung gestellt und enthielt einen induzierbaren episomalen Vektor für das CALM-AF 10 Gen. Messungen der Genexpression, die in der Bohlander Gruppe mit Hilfe eines Affymetrix- Chips durchgeführt wurden, zeigten das die Induktion des Transgens zu genomweiten Veränderungen der Expressionsmuster hunderter Gene in dieser Zelllinie führten. Die für die 3D FISH Experimente ausgewählten Markergene zeigten nach der Induktion eine signifikant veränderte Expression. Dennoch änderte sich die radiale Positionierung dieser Gene, darunter versteht man die mehr innere oder mehr periphere Position der Gene, nicht. Dieses Ergebnis schien zuerst darauf hinzuweisen, dass die Transkriptionsstärke keine bedeutsamer Faktor im Hinblick auf die radiale Positionierung ist. Die Befunde der Affymetrix-Chip Analyse für diese Gene konnten jedoch in einer anschließende Untersuchungen der Genexpression mit Real-Time-PCR nicht bestätigt werden, obwohl der Vergleich von Affymetrix-Chip und Real- Time-PCR Daten insgesamt eine klare Korrelation zwischen den Datensätzen zeigte. Bei Diskrepanzen gehen wir davon aus, dass Real-Time-PCR die zuverlässigeren Ergebnisse liefert. Bei der hier durchgeführten Real-Time-PCR Untersuchung wurden auch die Expressionsstärken aller in einer Nachbarschaft von etwa 1 Mbp um die Markergene annotierten Gene ermittelt. Dieses Fenster wurde gewählt, weil Untersuchungen in der Arbeitsgruppe von Thomas Cremer und anderen Gruppen gezeigt haben, dass ~1 Mbp Chromatindomänen die Basisstruktur der Chromatinorganisation darstellen. Als Maß für die gesamte Genexpression einer Chromatindomäne wurde eine „Total Expression Strength“ (TES) berechnet. Dieser Wert basiert auf den Real-Time-PCR Werten der annotierten Gene und berücksichtigt auch die Länge der ungespleissten RNA, die von einem Gen transkribiert wird. Dabei zeigte sich, dass das Markergen in der Domäne mit dem höchsten TES Wert am weitesten innen im Zellkern lokalisiert ist. Dieser Befund unterstützt Befunde aus der wissenschaftlichen Literatur, dass die radiale Positionierung von individuellen Genen von Eigenschaften der lokalen Umgebung abhängt. Da sich die Nachbarschaft der untersuchten Markergene nicht nur im Hinblick auf die TES Werte sondern auch im Hinblick auf die Dichte der dort annotierten Gene und den GC-Gehalt unterscheidet, bleibt offen, welcher dieser Parameter als Prädiktor für die zu erwartende radiale Position individueller Gene eine entscheidende Rolle spielt. Möglich ist auch, dass alle Parameter zusammenwirken oder dass je nach den speziellen Umständen einer Untersuchung verschiedene Parameter die radiale Positionierung eines Gens bevorzugt beeinflussen. Die Stabilität der radialen Positionierung der Markergene trotz einer genomweiten Veränderung des Genexpressionsmusters nach CALM-AF 10 Induktion stimmt mit Befunden verschiedener Arbeitsgruppe überein, die für einen hohen Grad an räumlicher Stabilität der Chromatinanordnung während der Interphase sprechen; ~1 Mbp Chromatindomänen zeigen dementsprechend meist nur sehr begrenzte lokale Bewergungen (

In mammalian cell nuclei chromosome territories (CTs) occupy positions correlating with their gene-density and chromosome size. While this global radial order has been well documented, the question of whether a global neighborhood order is also maintained has remained a controversial matter. To answer this question I grew clones (of HeLa, HMEC and human diploid fibroblast cells) for up to 5 divisions (32 cells) and performed 3D FISH experiments to visualize the nuclear positions of 3 different CT pairs. Using different landmark-based registration approaches I assessed the similarity of CT arrangements in daughter cells and cousins. As expected from a symmetrical chromatid movement during mitotic anaphase and telophase, I was able to confirm previous findings of a pronounced similarity of CT arrangements between daughter cells. However, already after two cell cycles the neighborhood order in cousins was nearly completely lost. This loss indicates that a global neighborhood order is not maintained. Further, I could show in the present thesis that a gene density correlated distribution of CTs, which has already been shown in different cell types of various species appears to be independent of the cell cycle. Moreover I could provide evidence that the nuclear shape plays a major role in defining the extent of this gene-density correlated distribution, as nuclei of human, old world monkey and bovine fibroblasts showed an increased difference in the radial distribution of gene poor/dense CTs when their nuclei were artificially reshaped from a flat ellipsoid to a nearly spherical nucleus. The observation that a gene-density correlated distribution of CTs has been found in nuclei from birds to humans argues for a significant, yet undiscovered functional impact. So far CTs have been investigated mainly in cultured cells and to some extent in tissues, yet little is known about the origin and fate of CTs during early development. To gain insights into the very early organization of CTs in preimplantation embryos I have developed a fluorescence in situ hybridization (FISH) protocol, which enables the visualization of CTs in three dimensionally preserved embryos. Using this protocol I have investigated CTs of bovine chromosomes 19 and 20, representing the most gene-rich and gene-poor chromosomes, respectively. Equivalent to the distributions described in other species I could confirm a gene density related spatial CT arrangement in bovine fibroblasts and lymphocytes with CT 19 being localized more internally and CT 20 more peripherally. Importantly, I did not find a gene density related distribution of CTs 19 and 20 in early embryos up to the 8-cell stage. Only in embryos with more than 8 cells a significant difference in the distribution of both chromosomes became apparent that increased upon progression to the blastocyst stage. Since major genome activation in bovine embryos occurs during the 8- to 16-cell stage, my findings suggest an interrelation between higher order chromatin arrangements and transcriptional activation of the embryonic genome. Using another experimental set up I analyzed the topology of a developmentally regulated transgene utilizing bovine nuclear transfer (NT) embryos derived from fetal fibroblasts, which harbored a mouse Oct4/GFP reporter construct integrated at a single insertion site on bovine chromosome 13. I analyzed the intranuclear distribution of the transgene as well as its position in relation to its harboring chromosome in donor cell nuclei and day 2 NT embryos, where the transgene is still inactive as well as in day 4 NT embryos, where transgene expression starts, and day 7 NT embryos, where expression is highly increased. Compared to donor cell nuclei I found a more peripheral location of both BTA 13 CTs and the Oct4/GFP transgene in day 2, day 4 and day 7 NT embryos, although there was a trend of the transgene and both BTA 13 CTs to re-localize towards the nuclear interior from d2 to d7 embryos. Moreover, I found the transgene located at the surface of its harboring CT 13 in donor fibroblasts, whereas during preimplantation development of NT embryos it became increasingly internalized into the chromosome 13 territory, reaching a maximum in d7 NT embryos, i.e. at the developmental stage when its transcription levels are highest. These latter experiments show that the transfer of a somatic nucleus into a chromosome depleted oocyte triggers a large scale positional change of CTs 13 and of an Oct4/GFP transgene and indicate a redistribution of this developmentally regulated Oct4/GFP transgene during activation and upregulation in developing NT embryos.

Recently it has been shown that the gene-density correlated radial distribution of human 18 and 19 homologous chromosome territories (CTs) is conserved in higher primates in spite of chromosomal rearrangements that occurred during evolution. However, these observations were limited to apes and New World monkey species. In order to provide further evidence for the evolutionary conservation of gene-density-correlated CT arrangements, we extended our previous study to Old World monkeys. They comprise the remaining species group to be analyzed in order to obtain a comprehensive overview of the nuclear topology of human 18 and 19 homologous CTs in higher primates. In the present study we investigated four lymphoblastoid cell lines from three species of Old World monkeys by three-dimensional fluorescence in situ hybridization (3D-FISH): two individuals of Japanese macaque ( Macaca fuscata), crab-eating macaque ( Macaca fascicularis), and an interspecies hybrid individual between African green monkey (Cercopithecus aethiops) and Patas monkey ( Erythrocebus patas). Our data demonstrate that gene-poor human 18 homologous CTs are located preferentially close to the nuclear periphery, whereas gene-dense human 19 homologous CTs are oriented towards the nuclear center in all cell lines analyzed. The gene-density-correlated positioning of human 18 and 19 homologous CTs is evolutionarily conserved throughout all major higher primate lineages, despite chromosomal inversions, fusions, fissions or reciprocal translocations that occurred in the course of evolution in these species. This remarkable preservation of a gene-density-correlated chromatin arrangement gives further support for a functionally relevant higher-order chromatin architecture. Copyright (C) 2005 S. Karger AG, Basel.

Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more. To address possible long-range movements of chromosome territories (CTs) during an entire interphase, fluorescence labeling of a small number of CTs was performed in living HeLa cells stably expressing histone H2B-GFP. This was achieved by in vivo scratch replication labeling with fluorescent nucleotides. Labeled cells were cultivated for several cell cycles until labeled chromatids had segregated. Such cells were followed by time-lapse confocal microscopy over time-scales of up to 20 hours covering major parts or the complete cell cycle. Positional changes of the intensity gravity centers of labeled CTs in the order of several µm were observed in early G1, thereafter, the positions remained within a range of ~1 µm till the end of G2. In conclusion, CT arrangements were highly constrained from mid G1 to late G2 / early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. More extended movements observed in early G1 might play a role when CTs “home in” to establish a non-random radial CT arrangement. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase whereas unbleached chromosome segments were often observed to become located at distant sites in the metaphase plates. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus, indicating that CT neighborhoods were not preserved during mitosis. The variability of CT neighborhoods during clonal growth was further confirmed by 3D-FISH experiments. A series of experiments of a more preliminary character looked at the influence of the nuclear lamina in constraining and determining a higher order nuclear architecture by selectively interacting with mid-to-late replicating chromatin. Simultaneous immunodetection of lamin B on two-color replication labeled neuroblastoma cell nuclei revealed specific attachment of the mid-to-late replicating chromatin compartment not only along the periphery but also inside the nucleus along invaginations of the lamina. 4D-live cell observation of lamin C-GFP expressing CHO cells with mid-to-late replicating chromatin labeled simultaneously revealed concomitant movements of replication foci attached to lamin invaginations. Moreover, a functional essay was employed which uses injection of a dominant negative lamin A mutant protein (ΔNLA) to cause a reversible disruption of the nuclear lamina. Initial results point to concomitant distortion of the mid-to-late replication pattern and a preferential attachment of the respective chromatin sites to partially disrupted lamin B (as compared to lamin A and nuclear pore complex). Finally, a model is presented on the chromosome positioning in mammalian nuclei depending on cell cycle and nuclear shape.