Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol

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

Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol

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

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background: Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass. Results: Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa. Conclusions: Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background: Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass. Results: Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa. Conclusions: Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa

Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications

<div><p>The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided <i>NUP153</i> mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of <i>NUP153 </i>at MGA.</p></div

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Combined and Fast Computable Thermal Models for Situationally Optimal Tempering of Machine Tool Components

The focus of this paper is on the modeling of an existing machine frame with its cooling structures. An overview is given of all the modeling methods used, their respective sub-targets and their interactions with each other. The modeling workflow encompasses both the mechanical and fluidic properties of the machine frame, including the fluid circuits and their control. The study results are the different requirements for modeling and the achievable result accuracy with regard to the temperature and deformation field. It forms the basis for the subsequent investigation of various temperature control variants, which are not part of this paper

The 4D nucleome: Evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments

AbstractRecent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC). The INC comprises the compact, transcriptionally inactive core of chromatin domain clusters (CDCs). The ANC is formed by the transcriptionally active periphery of CDCs, called the perichromatin region (PR), and the interchromatin compartment (IC). The IC is connected to nuclear pores and serves nuclear import and export functions. The ANC is the major site of RNA synthesis. It is highly enriched in epigenetic marks for transcriptionally competent chromatin and RNA Polymerase II. Marks for silent chromatin are enriched in the INC. Multi-scale cross-correlation spectroscopy suggests that nuclear architecture resembles a random obstacle network for diffusing proteins. An increased dwell time of proteins and protein complexes within the ANC may help to limit genome scanning by factors or factor complexes to DNA exposed within the ANC