The Nuclear-to-Cytoplasmic Ratio: Coupling DNA Content to Cell Size, Cell Cycle, and Biosynthetic Capacity.

Though cell size varies between different cells and across species, the nuclear-to-cytoplasmic (N/C) ratio is largely maintained across species and within cell types. A cell maintains a relatively constant N/C ratio by coupling DNA content, nuclear size, and cell size. We explore how cells couple cell division and growth to DNA content. In some cases, cells use DNA as a molecular yardstick to control the availability of cell cycle regulators. In other cases, DNA sets a limit for biosynthetic capacity. Developmentally programmed variations in the N/C ratio for a given cell type suggest that a specific N/C ratio is required to respond to given physiological demands. Recent observations connecting decreased N/C ratios with cellular senescence indicate that maintaining the proper N/C ratio is essential for proper cellular functioning. Together, these findings suggest a causative, not simply correlative, role for the N/C ratio in regulating cell growth and cell cycle progression.

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities.

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Histone concentration regulates the cell cycle and transcription in early development.

The early embryos of many animals, including flies, fish and frogs, have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. Here, we examine the effects of under- and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone-sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda, and lack of SIN3A and associated histone deacetylases. Our findings uncover a crucial regulatory role for histone concentrations in ZGA of Drosophila.

Modeling the role for nuclear import dynamics in the early embryonic cell cycle.

Nuclear composition determines nuclear function. The early embryos of many species begin life with large pools of maternally provided components that become rapidly imported into an increasing number of nuclei as the cells undergo repeated cleavage divisions. Because early cell cycles are too fast for nuclei to achieve steady-state nucleocytoplasmic partitioning, the composition of cleavage stage nuclei is likely dominated by nuclear import. The end of the rapid cleavage stage and onset of major zygotic transcription, known as the mid-blastula transition (MBT), is controlled by the ratio of nuclei/cytoplasm, indicating that changes in nuclear composition likely mediate MBT timing. Here, we explore how different nuclear import regimes can affect protein accumulation in the nucleus in the early Drosophila embryo. We find that nuclear import differs dramatically for a general nuclear cargo (NLS (nuclear localization signal)-mRFP) and a proposed MBT regulator (histone H3). We show that nuclear import rates of NLS-mRFP in a given nucleus remain relatively unchanged throughout the cleavage cycles, whereas those of H3 halve with each cycle. We model these two distinct modes of nuclear import as "nucleus-limited" and "import-limited" and examine how the two different modes can contribute to different protein accumulation dynamics. Finally, we incorporate these distinct modes of nuclear import into a model for cell-cycle regulation at the MBT and find that the import-limited H3 dynamics contribute to increased robustness and allow for stepwise cell-cycle slowing at the MBT.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition.

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

Bellymount-Pulsed Tracking: A Novel Approach for Real-Time In vivo Imaging of Drosophila Abdominal Tissues.

Quantitative live imaging is a valuable tool that offers insights into cellular dynamics. However, many fundamental biological processes are incompatible with current live imaging modalities. Drosophila oogenesis is a well-studied system that has provided molecular insights into a range of cellular and developmental processes. The length of the oogenesis coupled with the requirement for inputs from multiple tissues has made long-term culture challenging. Here, we have developed Bellymount-Pulsed Tracking (Bellymount-PT), which allows continuous, non-invasive live imaging of Drosophila oogenesis inside the female abdomen for up to 16 hours. Bellymount-PT improves upon the existing Bellymount technique by adding pulsed anesthesia with periods of feeding that support the long-term survival of flies during imaging. Using Bellymount-PT we measure key events of oogenesis including egg chamber growth, yolk uptake, and transfer of specific proteins to the oocyte during nurse cell dumping with high spatiotemporal precision within the abdomen of a live female.

Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation.

Early embryos often have relatively unstructured chromatin that lacks active and inactive domains typical of differentiated cells. In many species, these regulatory domains are established during zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 fast, reductive, syncytial nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. These divisions incorporate maternally-loaded, cytoplasmic pools of histones into chromatin. Previous work found that chromatin incorporation of replication-coupled histone H3 decreases while its variant H3.3 increases in the cell cycles leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription as well as heterochromatin, suggesting a link between H3.3 incorporation and ZGA. Here, we examine the factors that contribute to H3.3 incorporation at ZGA. We identify a more rapid decrease in the nuclear availability of H3 than H3.3 over the final pre-ZGA cycles. We also observe an N/C ratio-dependent increase in H3.3 incorporation in mutant embryos with non-uniform local N/C ratios. We find that chaperone binding, not gene expression, controls incorporation patterns using H3/H3.3 chimeric proteins at the endogenous H3.3A locus. We test the specificity of the H3.3 chaperone pathways for H3.3 incorporation using Hira (H3.3 chaperone) mutant embryos. Overall, we propose a model in which local N/C ratios and specific chaperone binding regulate differential incorporation of H3.3 during ZGA.

Versatile roles for histones in early development.

The nuclear environment changes dramatically over the course of early development. Histones are core chromatin components that play critical roles in regulating gene expression and nuclear architecture. Additionally, the embryos of many species, including Drosophila, Zebrafish, and Xenopus use the availability of maternally deposited histones to time critical early embryonic events including cell cycle slowing and zygotic genome activation. Here, we review recent insights into how histones control early development. We first discuss the regulation of chromatin functions through interaction of histones and transcription factors, incorporation of variant histones, and histone post-translational modifications. We also highlight emerging roles for histones as developmental regulators independent of chromatin association.

Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo.

The DNA damage checkpoint is crucial to protect genome integrity.1,2 However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio)3-7 and the activation of the checkpoint kinase, Chk1.8-17 How Chk1 activity is coupled to the N/C ratio has remained poorly understood. Here, we show that dynamic changes in histone H3 availability in response to the increasing N/C ratio control Chk1 activity and thus time the MBT in the Drosophila embryo. We show that excess H3 in the early cycles interferes with cell-cycle slowing independent of chromatin incorporation. We find that the N-terminal tail of H3 acts as a competitive inhibitor of Chk1 in vitro and reduces Chk1 activity in vivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, we show that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression.

Dynamics of Free and Chromatin-Bound Histone H3 during Early Embryogenesis.

During zygotic genome activation (ZGA), the chromatin environment undergoes profound changes, including the formation of topologically associated domains, refinements in nucleosome positioning on promoters, and the emergence of heterochromatin [1-4]. In many organisms, including Drosophila, ZGA is associated with the end of a period of extremely rapid, exponential cleavage divisions that are facilitated by large maternally provided pools of nuclear components. It is therefore imperative that we understand how the supply of chromatin components relative to the exponentially increasing demand affects nuclear and chromatin composition during early embryogenesis. Here, we examine the nuclear trafficking and chromatin dynamics of histones during the cleavage divisions in Drosophila using a photo-switchable H3-Dendra2 reporter. We observe that total H3-Dendra2 in the nucleus decreases with each cleavage cycle. This change in nuclear composition is due to depletion of large pools (>50%) of free protein that are present in the early cycles. We find that the per nucleus import rate halves with each cycle and construct a mathematical model in which increasing histone demand determines the dynamics of nuclear H3 supply. Finally, we show that these changes in H3 availability correspond to a large (∼40%) reduction in global H3 occupancy on the chromatin, which is compensated by the increased incorporation of H3.3. The observed changes in free nuclear H3 and chromatin composition may contribute to the cell-cycle slowing, changes in chromatin structure, and the onset of transcription associated with this developmental stage.

Cell-Size Control.

Cells of a given type maintain a characteristic cell size to function efficiently in their ecological or organismal context. They achieve this through the regulation of growth rates or by actively sensing size and coupling this signal to cell division. We focus this review on potential size-sensing mechanisms, including geometric, external cue, and titration mechanisms. Mechanisms that titrate proteins against DNA are of particular interest because they are consistent with the robust correlation of DNA content and cell size. We review the literature, which suggests that titration mechanisms may underlie cell-size sensing in Xenopus embryos, budding yeast, and Escherichia coli, whereas alternative mechanisms may function in fission yeast.