Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics

Lucchetta, Elena M.; Lee, Ji Hwan; Fu, Lydia A.; Patel, Nipam H.; Ismagilov, Rustem F.

doi:10.1038/nature03509

Cited by 433 publications

(410 citation statements)

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“…48 Lack of water, food, and nutrients is among the reasons for the fatal effect observed on pure PDMS substrates. 41,49 With the understanding of flies' viability and oviposition on pure PDMS and agar substrates, additional experiments could be designed to precisely study the effects of exposure area and size of the oviposition site on these behaviors. In addition, understanding the effect of exposure to PDMS is becoming more and more important in design and development of microfluidic devices for Drosophila assays.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, microfluidic devices and Micro-Electrical-Mechanical Systems (MEMS) have been successfully used in many Drosophila assays, [38][39][40][41][42][43][44][45][46] mainly because of their capability to provide scalable and controllable environments for quantitative embryonic [38][39][40][41][42][43] and larval [44][45][46] investigations in flies. These miniaturized devices that are made primarily from the elastomer polydimethylsiloxane (PDMS) have significantly enhanced the throughput, accuracy, and reproducibility of these assays.…”

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confidence: 99%

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Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

et al. 2015

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Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila's oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surfacemodified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations. V C 2015 AIP Publishing LLC. [http://dx

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

et al. 2015

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“…Although further experimental research is needed, we assume that scaling of the Bicoid profile with the embryo length is a required feature of any model. Several possibilities for the mechanism producing this scaling have been proposed, including oppositely directed morphogen gradients (29)(30), RNA-binding proteins (31), or nuclear degradation (13). Our flow model can improve scaling provided that the flow magnitude also scales with the embryo length.…”

Section: Discussionmentioning

confidence: 99%

Determining the scale of the Bicoid morphogen gradient

Hecht

Rappel

Levine

2009

Proc. Natl. Acad. Sci. U.S.A.

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Bicoid is a morphogen that sets up the anterior-posterior axis in early Drosophila embryos. Although the form of the Bicoid profile is consistent with a simple diffusion/degradation model, the observed length scale is much larger than should be expected based on the measured diffusion rate. Here, we study two possible mechanisms that could, in principle, affect this gradient and, hence, address this disagreement. First, we show that including trapping and release of Bicoid by the nuclei during cleavage cycles does not alter the morphogen length scale. More crucially, the inclusion of advective transport due to cytoplasmic streaming can have a large effect. Specifically, we build a simple model based on the (limited) experimental data and show that such a flow can lead to a Bicoid profile that is consistent with various experimental features. Specifically, the observed length scale is obtained, a steady profile is established, and improved scaling between embryos of different lengths is demonstrated.Drosophila ͉ embryonic development ͉ flow ͉ length scale D uring embryonic development, originally identical cells need to appropriately differentiate into a large variety of cell fates. This differentiation is position-dependent and is regulated by diffusible signaling molecules, known as morphogens. Morphogens are produced in a specific region of a tissue and move away from their source to form long-range concentration gradients. Cells subsequently differentiate in response to the morphogen concentration (1-2); thus, the pattern of morphogen concentration is crucial to the functional formation of tissues and organisms.In Drosophila melanogaster, Bicoid is a maternally transcribed gene that organizes anterior development (3-6). Its mRNA is localized at the anterior pole of the oocyte and is translated shortly after egg deposition. As a consequence of the anterior localization of mRNA, a gradient of Bicoid is formed along the anterior-posterior (AP) axis, simultaneously with nuclear cleavage cycles (7). This gradient determines the boundary of expression of downstream effectors such as Hunchback, eventually leading to the establishment of the segmented body plan. The dynamics of Bicoid pattern formation were recently studied in several experiments (8-15). The gradient was found to have, to a good approximation, an exponential decay with distance from the anterior. The exponential profile is consistent with the general model of diffusion and constant degradation rate which gives a length scale of ͌ D/␦, where D is the diffusion constant and ␦ is the degradation constant.This simple diffusion/degradation picture, however, turns out to be inconsistent with several recent experiments. In one such experiment (13), the Bicoid diffusion constant was measured using fluorescence measurements of Bicoid-eGFP protein in the nuclei. Upon photobleaching, the recovery rate of nuclear Bicoid was determined, and a diffusion constant of D ϭ 0.27 m 2 /s was obtained. With no degradation, this would lead to a length scale of ϭ ͌ DT where...

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“…Even-skipped expression was faster in the warm half of the embryo, but both embryos resolved the same final expression pattern. 27 Fig. 4 Time-series (min) response of microfluidic local Rhodamine-EGF stimulation through visualization of Ras activation.…”

Section: Exploiting Microfluidic Flow For Unique Opportunities In Expmentioning

confidence: 99%

Microfluidics-based systems biology

2006

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Systems biology seeks to develop a complete understanding of cellular mechanisms by studying the functions of intra-and inter-cellular molecular interactions that trigger and coordinate cellular events. However, the complexity of biological systems causes accurate and precise systems biology experimentation to be a difficult task. Most biological experimentation focuses on highly detailed investigation of a single signaling mechanism, which lacks the throughput necessary to reconstruct the entirety of the biological system, while high-throughput testing often lacks the fidelity and detail necessary to fully comprehend the mechanisms of signal propagation. Systems biology experimentation, however, can benefit greatly from the progress in the development of microfluidic devices. Microfluidics provides the opportunity to study cells effectively on both a single-and multi-cellular level with high-resolution and localized application of experimental conditions with biomimetic physiological conditions. Additionally, the ability to massively array devices on a chip opens the door for high-throughput, high fidelity experimentation to aid in accurate and precise unraveling of the intertwined signaling systems that compose the inner workings of the cell.

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Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics

Cited by 433 publications

References 19 publications

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

Determining the scale of the Bicoid morphogen gradient

Microfluidics-based systems biology

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