Dual spindle formation in zygotes keeps parental genomes apart in early mammalian embryos

AbstractAt the beginning of mammalian life the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible to spatially combine the two genomes and then segregate them to create the two-cell embryo. Utilizing light-sheet microscopy, we showed that two bipolar spindles form in the zygote, that independently congress the maternal and paternal genomes. These two spindles aligned their poles prior to anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.One Sentence Summary: After fertilization, two spindles form around pro-nuclei in mammalian zygotes and keep the parental genomes apart during the first division.

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Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth

The Journal of Cell Biology ◽

10.1083/jcb.201805044 ◽

2019 ◽

Vol 218 (7) ◽

pp. 2150-2168 ◽

Cited By ~ 23

Author(s):

Ana F. David ◽

Philippe Roudot ◽

Wesley R. Legant ◽

Eric Betzig ◽

Gaudenz Danuser ◽

...

Keyword(s):

Spindle Assembly ◽

Light Sheet ◽

Spindle Pole ◽

Directional Bias ◽

Fiber Assembly ◽

Light Sheet Microscopy ◽

Sister Chromatids ◽

Spindle Dynamics ◽

Dividing Cells ◽

Microtubule Growth

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole–derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

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Non-rodent mammalian zygotes assemble dual spindles despite the presence of paternal centrosomes

10.1101/2020.10.16.342154 ◽

2020 ◽

Cited By ~ 1

Author(s):

Isabell Schneider ◽

Marta de Ruijter-Villani ◽

M. Julius Hossain ◽

Tom A. E. Stout ◽

Jan Ellenberg

Keyword(s):

Self Assembly ◽

Spindle Assembly ◽

Light Sheet ◽

Self Organization ◽

Parental Genome ◽

Mammalian Embryo ◽

Light Sheet Microscopy ◽

Assembly Pathway ◽

Spindle Structure ◽

Human Ivf

AbstractThe first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded and, instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In non-rodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether non-rodent zygotes assemble a single centrosomal spindle around both genomes, or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form around the parental genomes despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in non-rodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.SummaryThis study investigates spindle assembly during the first embryonic division in bovine zygotes that, like human, inherit centrosomes from the sperm. It shows that two independent microtubule arrays form by self-organization around parental genomes with only loosely connected centrosomes.

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A robust ex vivo system to study cellular dynamics underlying mouse peri-implantation development

10.1101/2021.08.22.457265 ◽

2021 ◽

Author(s):

Takafumi Ichikawa ◽

Hui Ting Zhang ◽

Laura Panavaite ◽

Anna Erzberger ◽

Dimitri Fabrèges ◽

...

Keyword(s):

Ex Vivo ◽

Light Sheet ◽

Cell Segmentation ◽

Mouse Development ◽

Cellular Dynamics ◽

Light Sheet Microscopy ◽

Embryonic Tissues ◽

Mammalian Embryos ◽

3D Architecture ◽

Embryonic Ectoderm

Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to 6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of cases, its compatibility with light-sheet microscopy enables study of cellular dynamics through automatic cell segmentation. We find that upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D-ex vivo system thus offers an unprecedented access to peri-implantation development for in toto monitoring, measurement and spatio-temporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues.

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Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.202010106 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Isabell Schneider ◽

Marta de Ruijter-Villani ◽

M. Julius Hossain ◽

Tom A.E. Stout ◽

Jan Ellenberg

Keyword(s):

Self Assembly ◽

Spindle Assembly ◽

Light Sheet ◽

Self Organization ◽

Parental Genome ◽

Mammalian Embryo ◽

Light Sheet Microscopy ◽

Assembly Pathway ◽

Spindle Structure ◽

Human Ivf

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

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Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

10.1101/501445 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ana F. David ◽

Philippe Roudot ◽

Wesley R. Legant ◽

Eric Betzig ◽

Gaudenz Danuser ◽

...

Keyword(s):

Spindle Assembly ◽

Light Sheet ◽

Spindle Pole ◽

Directional Bias ◽

Fiber Assembly ◽

Light Sheet Microscopy ◽

Sister Chromatids ◽

Spindle Dynamics ◽

Dividing Cells ◽

Microtubule Growth

AbstractDividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle-pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at non-centrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus-ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubules. By ultra-fast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth towards individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore-fiber assembly.

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Immunoprobe localization in mammalian embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157693 ◽

1990 ◽

Vol 48 (3) ◽

pp. 32-33

Author(s):

Patricia G. Calarco ◽

Margaret C. Siebert

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Thin Sections ◽

Relative Lack ◽

Large Size ◽

Mammalian Embryos ◽

Antigen Localization ◽

Difficult Challenge ◽

Lowicryl K4m ◽

Fertilized Egg

Visualization of preimplantation mammalian embryos by electron microscopy is difficult due to the large size of the ircells, their relative lack of internal structure, and their highly hydrated cytoplasm. For example, the fertilized egg of the mouse is a single cell of approximately 75μ in diameter with little organized cytoskelet on and apaucity ofor ganelles such as endoplasmic reticulum (ER) and Golgi material. Thus, techniques that work well on tissues or cell lines are often not adaptable to embryos at either the LM or EM level.Over several years we have perfected techniques for visualization of mammalian embryos by LM and TEM, SEM and for the pre-embedding localization of antigens. Post-embedding antigenlocalization in thin sections of mouse oocytes and embryos has presented a more difficult challenge and has been explored in LR White, LR Gold, soft EPON (after etching of sections), and Lowicryl K4M. To date, antigen localization has only been achieved in Lowicryl-embedded material, although even with polymerization at-40°C, the small ER vesicles characteristic of embryos are unrecognizable.

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Faculty Opinions recommendation of Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1123365.584346 ◽

2008 ◽

Author(s):

Kees Jalink

Keyword(s):

Embryonic Development ◽

Light Sheet ◽

Early Embryonic Development ◽

Light Sheet Microscopy

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Achromatic terahertz Airy beam generation with dielectric metasurfaces

Nanophotonics ◽

10.1515/nanoph-2020-0536 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Qingqing Cheng ◽

Juncheng Wang ◽

Ling Ma ◽

Zhixiong Shen ◽

Jing Zhang ◽

...

Keyword(s):

Biomedical Imaging ◽

Frequency Dispersion ◽

Light Sheet ◽

Photonic Devices ◽

Self Healing ◽

Beam Focusing ◽

Light Sheet Microscopy ◽

Airy Beam ◽

Potential Applications ◽

Airy Beams

AbstractAiry beams exhibit intriguing properties such as nonspreading, self-bending, and self-healing and have attracted considerable recent interest because of their many potential applications in photonics, such as to beam focusing, light-sheet microscopy, and biomedical imaging. However, previous approaches to generate Airy beams using photonic structures have suffered from severe chromatic problems arising from strong frequency dispersion of the scatterers. Here, we design and fabricate a metasurface composed of silicon posts for the frequency range 0.4–0.8 THz in transmission mode, and we experimentally demonstrate achromatic Airy beams exhibiting autofocusing properties. We further show numerically that a generated achromatic Airy-beam-based metalens exhibits self-healing properties that are immune to scattering by particles and that it also possesses a larger depth of focus than a traditional metalens. Our results pave the way to the realization of flat photonic devices for applications to noninvasive biomedical imaging and light-sheet microscopy, and we provide a numerical demonstration of a device protocol.

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