Thomas Gregor

Probing the limits to positional information.

The reproducibility and precision of biological patterning is limited by the accuracy with which concentration profiles of morphogen molecules can be established and read out by their targets. We consider four measures of precision for the Bicoid morphogen in the Drosophila embryo: the concentration differences that distinguish neighboring cells, the limits set by the random arrival of Bicoid molecules at their targets (which depends on absolute concentration), the noise in readout of Bicoid by the activation of Hunchback, and the reproducibility of Bicoid concentration at corresponding positions in multiple embryos. We show, through a combination of different experiments, that all of these quantities are approximately 10%. This agreement among different measures of accuracy indicates that the embryo is not faced with noisy input signals and readout mechanisms; rather, the system exerts precise control over absolute concentrations and responds reliably to small concentration differences, approaching the limits set by basic physical principles.

Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient

Patterning in multicellular organisms results from spatial gradients in morphogen concentration, but the dynamics of these gradients remain largely unexplored. We characterize, through in vivo optical imaging, the development and stability of the Bicoid morphogen gradient in Drosophila embryos that express a Bicoid-eGFP fusion protein. The gradient is established rapidly (approximately 1 hr after fertilization), with nuclear Bicoid concentration rising and falling during mitosis. Interphase levels result from a rapid equilibrium between Bicoid uptake and removal. Initial interphase concentration in nuclei in successive cycles is constant (+/-10%), demonstrating a form of gradient stability, but it subsequently decays by approximately 30%. Both direct photobleaching measurements and indirect estimates of Bicoid-eGFP diffusion constants (D < or = 1 microm(2)/s) provide a consistent picture of Bicoid transport on short ( approximately min) time scales but challenge traditional models of long-range gradient formation. A new model is presented emphasizing the possible role of nuclear dynamics in shaping and scaling the gradient.

The Onset of Collective Behavior in Social Amoebae

All Together Now In the social amoeba Dictyostelium discoideum, periodic synthesis and release of cyclic AMP (cAMP) guides the cellular aggregation required to form fruiting bodies. It has been unclear whether the initiation of this behavior is owing to synchronization of autonomously oscillating cells or whether individual cells remain nonoscillatory unless the entire population becomes oscillatory. Gregor et al. (p. 1021, published online 22 April; see the Perspective by Prindle and Hasty) used live-cell imaging to show that cAMP pulses originate from a specific location in space and that individual cells move in and out of these signaling centers. The observations suggest that oscillations do not originate from autonomous activities of specialized cells. However, individual cells do display stochastic cAMP-pulsing below a threshold external concentration of cAMP, and the generation of synchronized oscillations could only be modeled accurately when this random pulsing was taken into account. Stochastic pulsing of individual cells plays a critical role in initiating cyclic adenosine monophosphate pulses. In the social amoebae Dictyostelium discoideum, periodic synthesis and release of extracellular cyclic adenosine 3′,5′-monophosphate (cAMP) guide cell aggregation and commitment to form fruiting bodies. It is unclear whether these oscillations are an intrinsic property of individual cells or if they exist only as a population-level phenomenon. Here, we showed by live-cell imaging of intact cell populations that pulses originate from a discrete location despite constant exchange of cells to and from the region. In a perfusion chamber, both isolated single cells and cell populations switched from quiescence to rhythmic activity depending on the concentration of extracellular cAMP. A quantitative analysis showed that stochastic pulsing of individual cells below the threshold concentration of extracellular cAMP plays a critical role in the onset of collective behavior.

Precise Developmental Gene Expression Arises from Globally Stochastic Transcriptional Activity

Early embryonic patterning events are strikingly precise, a fact that appears incompatible with the stochastic gene expression observed across phyla. Using single-molecule mRNA quantification in Drosophila embryos, we determine the magnitude of fluctuations in the expression of four critical patterning genes. The accumulation of mRNAs is identical across genes and fluctuates by only ∼8% between neighboring nuclei, generating precise protein distributions. In contrast, transcribing loci exhibit an intrinsic noise of ∼45% independent of specific promoter-enhancer architecture or fluctuating inputs. Precise transcript distribution in the syncytium is recovered via straightforward spatiotemporal averaging, i.e., accumulation and diffusion of transcripts during nuclear cycles, without regulatory feedback. Common expression characteristics shared between genes suggest that fluctuations in mRNA production are context independent and are a fundamental property of transcription. The findings shed light on how the apparent paradox between stochastic transcription and developmental precision is resolved.

The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA.

New quantitative data show that the Bicoid morphogen gradient is generated from a dynamic localized source and that protein gradient formation requires protein movement along the anterior-posterior axis.

Quantitative Imaging of Transcription in Living Drosophila Embryos Links Polymerase Activity to Patterning

Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting macroscopic domains have been mainly characterized by their levels of gene products. However, the establishment of such patterns results from differences in the dynamics of microscopic events in individual cells such as transcription. It is unclear how these microscopic decisions lead to macroscopic patterns, as measurements in fixed tissue cannot access the underlying transcriptional dynamics. In vivo transcriptional dynamics have long been approached in single-celled organisms, but never in a multicellular developmental context. Here, we directly address how boundaries of gene expression emerge in the Drosophila embryo by measuring the absolute number of actively transcribing polymerases in real time in individual nuclei. Specifically, we show that the formation of a boundary cannot be quantitatively explained by the rate of mRNA production in each cell, but instead requires amplification of the dynamic range of the expression boundary. This amplification is accomplished by nuclei randomly adopting active or inactive states of transcription, leading to a collective effect where the fraction of active nuclei is modulated in space. Thus, developmental patterns are not just the consequence of reproducible transcriptional dynamics in individual nuclei, but are the result of averaging expression over space and time.

Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.

Significance There is considerable information about the spatial regulation of gene expression during pattern formation in animal development. Significantly less is known about temporal control, in part due to our inability to analyze gene activity in real time. Using a recently developed approach for the visualization of gene expression in living Drosophila embryos, we examined the well-known even-skipped stripe 2 expression pattern. Surprisingly, we observe that this classic pattern is quite transient and generated by discontinuous surges of transcriptional activity in individual cells. These results challenge a purely static framework for dissecting developmental programs and emphasize the importance of the dynamic features of pattern formation. We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4–10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.

Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos

The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.

A comparison of methods for the calculation of NMR chemical shifts

A theory (MPL) to compute the NMR chemical shifts in condensed matter systems using periodic boundary conditions was presented by F. Mauri, B. Pfrommer, and S. G. Louie [Phys. Rev. Lett. 77, 5300 (1996)]. The MPL method has been implemented so far within a pseudopotential formulation in which the wave functions are expanded in plane waves. In this paper, we compare analytically the MPL approach within the density functional theory to existing methods for the calculation of the chemical shifts such as GIAO (gauge-including atomic orbitals), CSGT (continuous set of gauge transformations), and IGAIM (individual gauges for atoms in molecules). To this end we apply the MPL approach to molecules since the latter methods are conceived only for finite systems. We show theoretically the equivalence between a variant of the CSGT and the MPL method applied to finite systems. Moreover, we analyze numerically the efficiency of the different methods when atomic orbital basis sets are employed, by comparing the basis-se...

Accurate measurements of dynamics and reproducibility in small genetic networks

Quantification of gene expression has become a central tool for understanding genetic networks. In many systems, the only viable way to measure protein levels is by immunofluorescence, which is notorious for its limited accuracy. Using the early Drosophila embryo as an example, we show that careful identification and control of experimental error allows for highly accurate gene expression measurements. We generated antibodies in different host species, allowing for simultaneous staining of four Drosophila gap genes in individual embryos. Careful error analysis of hundreds of expression profiles reveals that less than ∼20% of the observed embryo‐to‐embryo fluctuations stem from experimental error. These measurements make it possible to extract not only very accurate mean gene expression profiles but also their naturally occurring fluctuations of biological origin and corresponding cross‐correlations. We use this analysis to extract gap gene profile dynamics with ∼1 min accuracy. The combination of these new measurements and analysis techniques reveals a twofold increase in profile reproducibility owing to a collective network dynamics that relays positional accuracy from the maternal gradients to the pair‐rule genes.