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Open Access Publications from the University of California

Scholarly Works (21 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2015)
A requirement that an animal be able to feed to grow constrains how a cell can grow into an animal, and it forces an alternation between growth (increase in mass) and proliferation (increase in cell number). A growth-only phase that transforms a stem cell of ordinary proportions into a huge cell, the oocyte, requires dramatic adaptations to help a nucleus direct a 10(5)-fold expansion of cytoplasmic volume. Proliferation without growth transforms the huge egg into an embryo while still accommodating an impotent nucleus overwhelmed by the voluminous cytoplasm. This growth program characterizes animals that deposit their eggs externally, but it is changed in mammals and in endoparasites. In these organisms, development in a nutritive environment releases the growth constraint, but growth of cells before gastrulation requires a new program to sustain pluripotency during this growth.
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2016)
    Metazoans start embryogenesis with a relatively naïve genome. The transcriptionally inert, late-replicating heterochromatic regions, including the constitutive heterochromatin on repetitive sequences near centromeres and telomeres, need to be re-established during development. To explore the events initiating heterochromatin formation and examine their temporal control, sequence specificity, and immediate regulatory consequence, we established a live imaging approach that enabled visualization of steps in heterochromatin emergence on specific satellite sequences during the mid-blastula transition (MBT) in Drosophila. Unexpectedly, only a subset of satellite sequences, including the 359-base-pair (bp) repeat sequence, recruited HP1a at the MBT. The recruitment of HP1a to the 359-bp repeat was dependent on HP1a's chromoshadow domain but not its chromodomain and was guided by maternally provided signals. HP1a recruitment to the 359-bp repeat was required for its programmed shift to later replication, and ectopic recruitment of HP1a was sufficient to delay replication timing of a different repeat. Our results reveal that emergence of constitutive heterochromatin follows a stereotyped developmental program in which different repetitive sequences use distinct interactions and independent pathways to arrive at a heterochromatic state. This differential emergence of heterochromatin on various repetitive sequences changes their replication order and remodels the DNA replication schedule during embryonic development.
      Cover page: TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2016)
      Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.
        Cover page: Selfish drive can trump function when animal mitochondrial genomes compete
        • 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
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2015)
            Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.
              Cover page: Selections that isolate recombinant mitochondrial genomes in animals
              • Thesis
              • Peer Reviewed
              UC San Francisco Electronic Theses and Dissertations (2018)

              In preparation for the dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila embryos the MBT involves the onset of key properties that distinguish the highly repetitive satellite sequences composing the heterochromatin from the rest of the genome. In early embryos the satellite DNA lacks the characteristic heterochromatic features of late replication and repressive chromatin modifications, like H3K9 methylation and HP1a. My thesis work has explored how the embryo controls the restoration of these conserved features to the heterochromatin. I describe and analyze the results of experiments employing a combination of fly genetics and real time microscopy on developing embryos. At the MBT, down-regulation of Cyclin:Cdk1 activity initiates cell cycle slowing by delaying replication of satellite DNA and extending S phase. I show that Cdk1 activity inhibits the chromatin association of Rif1, a repressor of origin firing. Following Cdk1 down-regulation at the MBT, Rif1 bound selectively to satellite sequences. In the next S phase, Rif1 dissociated from different satellites in a schedule that anticipated their replication. A mutant version of Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. In contrast, loss of Rif1 shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of Cdc7. Following the Rif1 mediated introduction of late replication, satellite sequences begin acquiring H3K9 methylation and recruit the protein HP1a. By using a novel protein inactivation approach called JabbaTrapping, I show that the K9 methyltransferase Eggless plays a major role in the establishment of this repressive chromatin state. Additionally, live imaging of Eggless suggests that the rapid early cell cycles prevent this enzyme from introducing heterochromatic marks before the MBT. Experimental manipulation of interphase duration showed that cell cycle speed regulates Eggless. By focusing on the initial events in the de novo formation of heterochromatin my work emphasizes how heterochromatin is integrated into the development of the embryo. Through regulation of the two proteins Rif1 and Egg, the embryonic cell cycle program times the appearance of late replication and repressive chromatin modifications, two widely conserved features of heterochromatin.

              • 15 supplemental videos
              Cover page: The developmental control of heterochromatin formation in the Drosophila embryo
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2015)
              The timing mechanism for mitotic progression is still poorly understood. The spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase [1-3], is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins-Cyclin A, Cyclin B, and Cyclin B3-influence mitotic progression and are degraded in a stereotyped sequence [4-11]. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. Our results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins-Cyclins A and B-restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. We propose that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost.
                Cover page: Cyclin B3 Is a Mitotic Cyclin that Promotes the Metaphase-Anaphase Transition
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2008)
                Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)(Pbl)-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF(Pbl)-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEF(Pbl)-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF(Pbl)-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability.
                  Cover page: Rho-dependent control of anillin behavior during cytokinesis
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2018)
                  In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase-promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.
                    Cover page: Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition
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