Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin.
In budding yeast, the Start checkpoint integrates multiple internal and external signals into an allor-none decision to enter the cell cycle. Here, we show that Start behaves like a switch due to systems-level feedback in the regulatory network. In contrast to current models proposing a linear cascade of Start activation, transcriptional positive feedback of the G1 cyclins Cln1,2 induces the near-simultaneous expression of the ~200-gene G1/S regulon. Nuclear Cln2 drives coherent regulon expression, while cytoplasmic Cln2 drives efficient budding. cln1,2-deleted cells frequently arrest as unbudded cells, incurring a large fluctuation-induced fitness penalty due to both the lack of cytoplasmic Cln2 and insufficient G1/S regulon expression. Thus, positivefeedback-amplified expression of Cln1,2 simultaneously drives robust budding and rapid, coherent regulon expression. A similar G1/S regulatory network in mammalian cells, comprised of nonorthologous genes, suggests either the conservation of regulatory architecture or convergent evolution.Positive feedback in genetic control networks can ensure that cells do not slip back and forth between either cell cycle phases or developmental fates. For example, commitment to sporulation in budding yeast is driven by transcriptional positive feedback of the meiotic inducer IME1 [1][2][3] . In Xenopus laevis, positive feedback underlies the all-or-none characteristics of oocyte maturation 4, 5 and mitotic entry 6,7 , suggesting the frequent use of positive feedback to regulate cellular transitions.Absent from this list of examples is the well-studied Start checkpoint controlling cell cycle commitment in budding yeast. Nutrient limitation and pheromone exposure arrests cells prior to DNA replication, while size control extends G1 in small daughter cells [8][9][10][11] . Beyond Start, cells proceed through division almost independently of size and environment 9 . Previous experiments suggested that Start represents a feedback-free cascade of events 12 (see schematic in Fig. 1a; omitting red arrows). The transition is initiated by the G1-cyclin Cln3 [13][14][15] , which in complex with Cdc28 activates the transcription of about 200 genes 16 by phosphorylating promoter-bound protein complexes that include the transcription factors SBF and MBF 17 and the transcriptional inhibitor Whi5 18,19 . Phosphorylation and inactivation of Whi5 is rate-limiting, and phosphorylated Whi5 rapidly exits the nucleus. The G1/S regulon, which includes two additional G1-cyclins, CLN1,2, contributes to the activation of B-type cyclins, DNA replication, spindle pole body duplication, and bud emergence. Mitotic B-type cyclins then inactivate SBF 20 and, with NRM1, inactivate MBF 21 , thus turning off the G1/S regulon.Correspondence and requests for materials should be addressed to J.M.S. (e-mail: jskotheim@rockefeller.edu). Any one of the three G1-cyclins suffices to activate the regulon, suggesting the potential for transcriptional positive feedback of CLN1,2 on their own expression 22,23 . Howev...
Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.
SUMMARY Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast (EPI) or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell resolution image analysis pipeline, to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE, and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling, and propose a model where interactions between NANOG, GATA6 and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos, and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.
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