Kim Y et al. (2011), Detection of dynamic spatiotemporal response to...

XB-ART-42823

PLoS One 2011 Jan 07;61:e14624. doi: 10.1371/journal.pone.0014624.

Show Gene links Show Anatomy links

Detection of dynamic spatiotemporal response to periodic chemical stimulation in a Xenopus embryonic tissue.

Kim Y , Joshi SD , Messner WC , LeDuc PR .

???displayArticle.abstract???
Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions--continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.

???displayArticle.pubmedLink??? 21305055
???displayArticle.pmcLink??? PMC3031512
???displayArticle.link??? PLoS One
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: cfd gnl3 nucb1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Spatiotemporal control of dexamethasone over Xenopus Animal Cap explants with the biosensor GR-nuc-GFP using a pressure feedback microfluidic approach.(A) Construction of DEX biosensor GR-nuc-GFP. GR-nuc-GFP resides in the cytoplasm, but moves into the nucleus after DEX is added. Dark areas in the cells indicate accumulation of GR-nuc-GFP. (B) Tissue explants from different frogs attached to the substrate in the microfluidic channels. Tissue explants spreading at the begining (left panel), 3 hours (middle panel), and 6 hours (right panel) after attachment in the microfluidic channel. (C) Ratio of the area of the tissue explants normalized by the initial area versus time (nâ=â3). Error bars represent standard deviations. (D) Microfluidic interface control system consisting of feedback control loop and modular microfluidics (see Materials and Methods). (E) Pressure modulation mechanism that allows long-term and high-speed control of the flow rate in a microfluidic channel [28]. (F) Schematic showing the confocal microscopic imaging of the cross-typed microfluidic channel. (G) Simulations showing flow pathlines over and around a single explant through a Computational Fluid Dynamics (CFD) simulation indicating no flow disruption around the explant. (H) Laminar flow interface between the stream of DEX (upper inlet; black) and the stream DFA (lower inlet) before the experiment. (I) Regulated laminar flow interface covers quarter of the AC explant. (J) Laminar flow interface moves to the center of the channel, exposing DEX to the half of the AC explant.
	Figure 2. Localized response to spatially defined continuous stimulation.(A) Laminar flow interface profile over time. (B) Schematic depicting two regions within a single AC explant subject to different stimulation conditions: constant stimulation (CS; upper region; DEX & DFA) and no stimulation (NS; lower region; DFA). (C) Images of the explant subjected to CS and NS depicted in (B) at the beginning (left), 60 minutes (middle), and 120 minutes (right). The dotted line marks the interface, which correlates to the line between CS and NS regions in (B). (D) Intensity ratios of GFP levels in nucleus relative to cytoplasm. Error bars represent standard deviations for 20 cells (** indicates p<0.01). Variable expression of GR-nuc-GFP biosensor across the animal cap is due to the uneven inheritance of mRNA encoding GR-nuc-GFP into 1 or 2-cell stage embryo.
	Figure 3. Localized responses to spatiotemporal periodic stimulations with 50% duty cycles.(A) The stream of DEX is controlled by directing a laminar flow interface over the explant allowing periodic stimulation profiles to be applied. Circles on the top of the periodic pattern of the interface represent initial interface positions while squares on the bottom of the pattern indicate repositioned interfaces. (B) Laminar flow interface profile over time. (C) Schematic depicting three regions of a single AC explant exposed to different stimulation conditions: CS (upper region), 2-minute 50% duty cycle PS (middle), and NS (lower). (D) AC explants exposed to CS, PS, and NS regions of (C) at 0 minutes (left), 60 minutes (middle), and 120 minutes (right). The dotted lines mark the interfaces, which correlate to the lines between CS, PS, and NS regions in (C). (E) High resolution views of explants shown in (D) in the area indicated by the rectangular shape in (C) at the beginning (left), 60 minutes (middle), and 120 minutes (right). The dotted lines represent the interfaces, which correlate to the lines between CS, PS, and NS regions in (C) and (D). (F) Intensity ratios of GFP in the nucleus versus cytoplasmic intensities. Error bars represent standard deviations for 20 cells (** indicates p<0.01). Variable expression of GR-nuc-GFP biosensor across the animal cap is due to the uneven inheritance of mRNA encoding GR-nuc-GFP into 1 or 2-cell stage embryo.
	Figure 4. Responses of the tissue explant to four different stimulation profiles: continuous stimulation (CS), and 50% duty cycle periodic stimulation (PS); 2 min-, 10 min-, and 40 min-period.(A) Input stimulation profiles. (B) Responses of 30 individual tracked cells from 3 different tissue explants to four different stimulation cases with different duty cycles: CS, 2-minute 50% duty cycle PS, 10-minute 50% duty cycle PS, and 40-minute 50% duty cycle PS. Error bars indicate standard deviations. Dotted lines represent the response to CS (left panel). (C) Mathematical model recapitulated GR-nuc-GFP movements after various periodic stimulation profiles. This model was constructed using a first-order differential equation (see Materials and Methods). The parameters reproducing the response to CS were applied to the other PS cases to predict their response without any additional parameters (modeled CS; modeled 2-minute PS; modeled 10-minute PS; and modeled 40-minute PS). Dotted lines represent the modeled response to CS (left panel). The modeled results well approximate experimental results in (B).

References [+] :

Ariizumi, In vitro induction systems for analyses of amphibian organogenesis and body patterning. 2001, Pubmed