Coordination of cell growth and division with development, with particular focus on the oocyte-to-embryo transition, control of cell size, and regulation of metazoan DNA replication.
Proper development from a single-cell fertilized embryo to an adult requires the generation of the right number and size of cells of the appropriate type, arranged with correct patterning. Our research goal is to understand the developmental control of cell division and cell size. We are investigating the transition through which an oocyte becomes an embryo: how the proteome is altered to permit this profound change by alteration of mRNA translation and protein stability, the regulators responsible, and how this leads to cell cycle changes from meiosis to embryonic mitosis. Other goals are to delineate the coordination of cell size between tissue layers and the regulation of replication origin activation and replication fork progression.
Left. Model of replication fork (green rings) collisions following repeated origin firing. Such collisions would produce double-strand breaks.
Right. The presence of double-strand breaks (gamma H2Av staining, green) at multiple replication forks (EdU nucleotide label, red) in a single follicle cell nucleus (DNA, blue). Image from Jessica Alexander.
The oocyte-to-embryo transition
This transition marks the onset of development as the differentiated oocyte changes to a totipotent embryo. It involves the completion of meiosis in the oocyte, fertilization, and restart of mitosis in the embryo. In most animals these profound changes in cell cycle and developmental potency occur in the absence of transcription, thus are controlled by changes in mRNA translation and protein stability. We exploited the ability to isolate mature oocytes and activated eggs in Drosophila to define the changes in mRNA translation that accompany the oocyte-to-embryo transition by polysome profiling and ribosome footprinting. These studies revealed that hundreds of mRNAs become activated for translation, while hundreds of others become repressed. Quantitative proteomics identified proteins whose abundance changes during this transition. These datasets demonstrated that there is a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. A meiosis-specific form of the Anaphase Promoting Complex, APCCort, contributes to downregulation of proteins as meiosis is completed.
Quantitative proteomics was applied to identify proteins whose abundance is altered during an earlier developmental transition: oocyte maturation. At oocyte maturation the meiotic arrest in prophase I that permits oocyte differentiation is released, and the oocyte progresses into the meiotic divisions. Comparisons of the proteome changes at oocyte maturation with those at egg activation showed dynamic regulation of proteins needed for late oogenesis, completion of meiosis, and early embryogenesis. The proteomic studies identified new candidate regulators for these events whose crucial roles have been confirmed by mutant analysis as well as RNAi studies. These will be the focus of future research efforts.
We identified the PAN GU (PNG) kinase by the requirement of its function for the onset of mitosis in the fertilized Drosophila embryo. Active PNG kinase is composed of a catalytic ser/thr kinase subunit (PNG) and two activating subunits (PLU and GNU). The mutant phenotype is a consequence of PNG promoting translation of cyclin B mRNA, leading to active Cyclin B/CDK1 kinase and the onset of mitosis in the embryo. The role of PNG in regulating translation was evaluated by ribosome footprinting in the mutant, which revealed that PNG has a global function in controlling the translation of hundreds of mRNAs but does so uniquely at the oocyte-to-embryo transition. Developmental control of PNG kinase activity is exerted by regulation of the association of the GNU subunit with the complex via phosphorylation. This regulation links activation of translation of maternally provided mRNAs to the completion of meiosis in the egg.
Control of cell size in organogenesis
The size of tissue layers must be properly scaled during the development of organs. This can involve coordination of cell division, except in organs such as the placenta, skin, and Drosophila nervous system in which one tissue layer acts as an envelope to seal adjacent layers. In these cases, the enveloping layer must grow as the organ grows but without cell division, as cytokinesis would disrupt the seal. To solve this conundrum, the trophoblast giant cells of the placenta, the skin keratinocytes, and the subperineurial glia cells of the Drosophila nervous system employ a widely used strategy to produce large cells: growth by increased DNA content and ploidy, in the absence of cell division. In this manner, the subperineurial glia are able to increase in size to accommodate increasing numbers of neurons during development, while maintaining their septate junctions that comprise the blood-brain barrier. We have demonstrated not only that increased ploidy of the subperineurial glia is necessary for their growth but that ploidy levels, and thus size, are actively controlled by the underlying neuronal mass. In addition, we have found that the Drosophila wrapping glia, which ensheath the axons in the peripheral nervous system, have increased ploidy in proportion to the length of the axon they ensheath. Future goals are to define the regulatory networks through which adjacent tissue layers in the nervous system coordinate their cell size.
Regulation of metazoan DNA replication
Proper regulation of DNA replication is essential to ensure gene copy number and genome stability, yet technical difficulties in capturing initiation of replication at origins and isolating replication forks have limited our understanding of metazoan DNA replication. We identified genomic regions in Drosophila that in response to developmental cues become refractory to replication or undergo repeated rounds of initiation of replication to generate amplified gene copy number. These have permitted us to track replication forks directly and to decipher the control of replication origins. We demonstrated that a chromatin protein called Suppressor of Underreplication (SUUR) tracks with replication forks in specific genomic intervals and impedes fork progression, leading to unreplicated DNA and chromosome fragile sites.
It was proposed in addition to causing increased DNA copy number, repeated firing of a replication origin can lead to colliding replication forks that cause double-strand breaks. Thus both defects could contribute to genome instability in cancer cells in which replication is not regulated properly. We exploited developmental gene amplification in Drosophila to test directly whether origin refiring leads to double-strand breaks and found that indeed the presence of multiple replication forks results in double-strand breaks. Continued fork progression requires signaling of DNA damage and repair through nonhomologous end joining.
Six regions in the Drosophila ovarian follicle cells undergo amplication as a consequence of repeated activation of defined replication origins. Multiple mechanisms are employed for replication initiation at these origins, and origin activity also is subjected to control by surrounding chromatin. We identified additional sites in the genome in which the chromatin can inhibit or activate a replication origin at a distance. Mechanistic analysis of these chromatin effects has uncovered several steps in replication activation vulnerable to chromatin influence.
Alexander, J.L., Barrasa, M.I., Orr-Weaver, T.L. (2015) Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair. Curr. Biol. 25, 1654-1660.
Nordman, J.T., Kozhevnikova, E.N., Verrijzer, P., Pindyurin, A.V., Andreyeva, E.N., Shloma,V.V., Zhimulev, I.F., and Orr-Weaver, T.L. (2014) DNA copy number control through inhibition of replication fork progression. Cell Reports 9, 841-849.
Unhavaithaya, Y. and Orr-Weaver, T.L. (2012) Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes and Dev. 26, 31-36.
Kronja, I., Whitfield, Z.J., Yuan, B. Dzeyk, K., Kirkpatrick, J., Krijgsveld, J., and Orr-Weaver, T.L. (2014) Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition. Proc. Natl. Acad. Sci. USA 111, 16023-16028.
Kronja, I., Yuan, B., Eichhorn, S., Dzeyk, K., Krijgsveld, J., Bartel, D.P., and Orr-Weaver, T.L. (2014) Widespread changes in the posttranscriptional landscape at the Drosophila oocyte-to-embryo transition. Cell Reports 7, 1495-1508.
Whitfield, Z., Chisholm, J.A., Hawley, R.S., and Orr-Weaver, T.L. (2013) A meiosis-specific form of the APC/C promotes the oocyte-to-embryo transition by decreasing levels of the Polo kinase inhibitor Matrimony. PLoS Biology 11, e1001648.
Orr-Weaver, T.L. (2015) When bigger is better: the role of polyploidy in organogenesis. Trends in Genetics 31, 307-315.