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

Abstract

Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions--a temperature step--in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100 min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development.

Figure 1

Experimental set up. a, A schematic drawing of a PDMS microfluidic device with a D. melanogaster embryo developing in a temperature-step (T-step). b, A microphotograph illustrating the T-step around the embryo, visualized using a suspension of thermochromic liquid crystals (Image Therm Engineering) flowing at a flow rate of 50 mm s⁻¹. The temperature of the green stream is 21 °C and the temperature of the red stream is 24 °C. Scale bar, 400 µm.

Figure 2

The rate of development in each half of the embryo exposed to a T-step is affected by temperature. Each half of the embryo is in a different cell cycle, as demonstrated by difference in nuclear density. Number of nuclei in enlarged areas shown underneath in yellow numbering. a, b, Embryos exposed to a T-step of 20 °C/27 °C for 140 min. a, Anterior half 20 °C, posterior half 27 °C. b, Anterior half 27 °C, posterior half 20 °C. c, d, Embryos exposed to a T-step of 17 °C/27 °C for 150 min. c, Anterior half 17 °C, posterior half 27 °C. d, Anterior half 27 °C, posterior half 17 °C. In all images, higher nuclear density was observed in the warmer half of the embryo.

Figure 3

Even-skipped expression in embryos exposed to a T-step of 20 °C/27 °C. a–c, Embryos with a cool anterior half and warm posterior half. d–f, Embryos with a warm anterior half and cool posterior half. Even-skipped stripes were consistently expressed in the warm half first (a and b, d and e), but resolved in the correct positions (c, f). g, Intensity profile of Even-skipped expression in embryos exposed to the T-step in panels c (red) and f (blue), compared to an average intensity profile (green) of Even-skipped expression in four control embryos that developed at room temperature.

Figure 4

Hunchback expression in embryos exposed to a T-step and time-dependent T-step. a, Hunchback intensity profiles of embryos with anterior half at 27 °C and posterior half at 20 °C. Hunchback position was normal, varying over 5% EL (46–51% EL). b, Hunchback intensity profiles of embryos exposed to a time-dependent T-step with anterior half at 27 °C and posterior half at 20 °C, with the exception of a brief temperature reversal (anterior 20 °C/posterior 27 °C) between 65 and 100 min. The position of Hunchback was variable over 18% EL (35–53% EL).