(464g) Microfluidics Approach for Systems Level Analysis of Dorsoventral Patterning in Drosophila

Spatial control of cell differentiation in embryos can be provided by morphogen gradients, defined as concentration fields of molecules that act as dose-dependent regulators of gene expression. Some of the first morphogens were identified in Drosophila, one of the leading models for the analysis of developmental patterning. The dorsoventral (DV) axis of the Drosophila embryo is patterned by the nuclear localization gradient of Dorsal (Dl), a Rel-family transcription factor, which subdivides the embryo into three germ layers [1]. The regions exposed to the highest, intermediate, and lowest levels of Dl, respectively, contribute to the formation of the mesoderm, the nervous system, and the skin of the embryo.

The DV patterning system is dominated by feedforward loops, a network motif in which a gene is controlled both by the primary input and by one of its more proximal targets. For instance, Snail, a transcription factor expressed in the future mesoderm, is activated both by Dorsal and by Twist, a transcription factor that is, in turn, activated by Dorsal [2]. The DV patterning network has been deduced based on a combination of mutant analyses, transgenesis experiments, and postgenomic approaches. Given the DV network structure, it now becomes possible to explore its dynamics, function, and evolution using quantitative approaches.

Quantitative analysis of any system controlled by morphogen gradients requires the information about the regulatory regions of genes comprising the network and about the spatial distribution of patterning signals. The DV patterning in Drosophila is arguably one of the best understood networks with regard to its sequence-specific transcriptional regulation, but the information on the distribution of patterning signals is currently lacking, mainly due to the technical difficulties associated with imaging the spatial distribution of proteins and transcripts along the DV axis embryo [3]. To enable high throughput analysis of the DV patterning signals, we have developed a microfluidic device in which hundreds of embryos are oriented vertically. Such "end-on" orientation allows for the entire DV axis data to be collected from each embryo. Previously, end-on imaging has been possible only for small numbers of embryos, which had to be individually placed into an upright position [3]. We describe the design and the physical principles behind this device and demonstrate how it can be successfully used to quantify the spatial distribution of nuclear Dl, as well as its cell signaling and transcriptional targets.

References

[1] Hong, JW., Hendrix, DA., Papatsenko, D., and Levine, MS. (2008) How the Dorsal gradient works: Insights from postgenome technologies. Proc Natl Acad Sci USA 105, 20072?20076.

[2] Ip, YT., Park, RE., Kosman, D., Yazdanbakhsh, K., and Levine, M. (1992). dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes Dev 6, 1518?1530.

[3] Kanodia, JS. Rikhy, R., Kim, Y., Lund, V., DeLotto, R., Lippincott-Schwartz, J., and Shvartsman, SY. Dynamics of the Dorsal morphogen gradient. Proc. Natl. Acad. Sci. 106:1087-92.