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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2014)
Many, if not most, embryos begin development with extremely short cell cycles that exhibit unusually rapid DNA replication and no gap phases. The commitment to the cell cycle in the early embryo appears to preclude many other cellular processes that only emerge as the cell cycle slows just prior to gastrulation at a major embryonic transition known as the mid-blastula transition (MBT). As reviewed here, genetic and molecular studies in Drosophila have identified changes that extend S phase and introduce a post-replicative gap phase, G2, to slow the cell cycle. Although many mysteries remain about the upstream regulators of these changes, we review the core mechanisms of the change in cell cycle regulation and discuss advances in our understanding of how these might be timed and triggered. Finally, we consider how the elements of this program may be conserved or changed in other organisms.
    Cover page: From Egg to Gastrula: How the Cell Cycle Is Remodeled During the Drosophila Mid-Blastula Transition
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)

    Background

    In Drosophila embryos, the midblastula transition (MBT) dramatically remodels the cell cycle during the 14(th) interphase. Before the MBT, each cycle is composed of only a short S phase and mitosis. At the MBT, S phase is dramatically lengthened by the onset of late replication, and a G2 phase is introduced. Both changes set the stage for gastrulation and require downregulation of Cdc25 phosphatase, which was previously attributed to the elimination of its transcripts at the MBT.

    Results

    Premature removal of cdc25 transcripts by RNAi did not affect progression to the MBT. Instead, an antibody against the Cdc25 isoform Twine showed that Twine protein was abundant and stable until the MBT, when it was destabilized and rapidly eliminated. Persistence of pre-MBT levels of Twine was sufficient to prevent cell-cycle slowing. Twine protein destruction was timed by the nucleocytoplasmic ratio and depended on the activation of zygotic transcription at the MBT, including expression of the gene tribbles, whose activity was sufficient to trigger Twine destruction and was required for prompt Twine disappearance.

    Conclusions

    We propose that the developmentally regulated destruction of Twine protein is a critical switch that contributes to the cell-cycle change at the MBT, including the addition of a G2 phase and onset of late replication. Moreover, we show that this destruction is triggered by the nucleocytoplasmic ratio-dependent onset of zygotic transcription of tribbles and other unknown genes.
      Cover page: Mechanism and Regulation of Cdc25/Twine Protein Destruction in Embryonic Cell-Cycle Remodeling
      • Thesis
      • Peer Reviewed
      UC San Francisco Electronic Theses and Dissertations (2013)

      In Drosophila embryos, the mid-blastula transition (MBT) dramatically remodels the cell cycle during interphase 14. Before the MBT, each cycle is composed of only a short S-phase and mitosis. At the MBT, S-phase is dramatically lengthened by the onset of late replication, and a G2 phase is introduced. These changes set the stage for morphogenesis, including the cellularization of the syncytial embryo and the onset of gastrulation. The introduction of the G2 phase was known to result from the downregulation of Cdc25 phosphatase and the inactivation of Cdk1 kinase; however, the signals modifying S-phase were unknown.

      We injected cdc25 mRNA to bypass the developmentally programmed downregulation of Cdc25 at the MBT. Introduction of either Cdc25 isoform (String or Twine) or enhanced Cdk1 activity triggered premature replication of late-replicating sequences, even after their specification, and thereby shortened S-phase. Reciprocally, reduction of Cdk1 activity by knockdown of mitotic cyclins extended pre-MBT S-phase. These findings suggest that high Cdc25 and Cdk1 contribute to the speed of the rapid, pre-MBT S-phases.

      Previous reports suggested that downregulation of Cdc25 activity at the MBT resulted from elimination of cdc25 transcripts. However, premature removal of cdc25 transcripts by RNAi did not affect progression to the MBT. Instead, a new antibody against the Cdc25 isoform Twine showed that Twine protein was abundant and stable until the MBT, when it was destabilized and rapidly eliminated. Twine protein destruction was timed by the nucleo-cytoplasmic ratio and depended on the activation of zygotic transcription at the MBT, including expression of the gene tribbles, whose activity was sufficient to trigger Twine destruction and was required for prompt Twine disappearance.

      Thus, we propose that the nucleo-cytoplasmic ratio triggers the remodeling of the cell cycle at the MBT by triggering the onset of transcription of a group of genes that inactivate Cdk1 (including through the destruction of Twine), which results in the lengthening of S-phase and addition of the G2 phase.

      • 14 supplemental videos
      Cover page: Regulation of the embryonic onset of late DNA replication in Drosophila melanogaster
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2009)
      Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.
        Cover page: Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo
        • Article
        • Peer Reviewed
        LBL Publications (2018)
        During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.
          Cover page: Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.
            Cover page: Single-cell reconstruction of developmental trajectories during zebrafish embryogenesisCreative Commons 'BY-NC-ND' version 4.0 license
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2019)
            The adult Hydra polyp continually renews all of its cells using three separate stem cell populations, but the genetic pathways enabling this homeostatic tissue maintenance are not well understood. We sequenced 24,985 Hydra single-cell transcriptomes and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified gene modules and putative regulators expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. In addition, we built a gene expression map of the Hydra nervous system. Our work constitutes a resource for addressing questions regarding the evolution of metazoan developmental processes and nervous system function.
              Cover page: Stem cell differentiation trajectories in Hydra resolved at single-cell resolution
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