Microsystems for controlled genetic perturbation of live Drosophila embryos: RNA interference, development robustness and drug screening

Abstract

A combination of genetic regulation and microenvironmental perturbation might prove especially useful for identifying the fundamental mechanisms responsible for development biology and critical biochemical networks. This could transform our understanding of genomics, proteomics, and drug discovery and lead to personalized drugs and molecular diagnostics for improved healthcare. With the complete sequencing of the Drosophila melanogaster, there has been a growing interest in correlating its genetics to human disease. The Drosophila embryo is an excellent whole-animal model, and is ideal for high throughput analysis. This review highlights research on the recent development of miniature tools to study critical cellular processes in Drosophila embryogenesis. First, we discuss the use of micro-injectors to conduct chip-level RNA interference experiments on self-assembled Drosophila embryos in order to determine how genes contribute to the growth and function of a developing organism. Second, we review recent progress on perturbing the microenvironment as a complementary approach to perturbing the genetic components of the development network. We designed a bio-compatible microfluidic device that automatically positions live embryos within a fluid-filled imaging channel and exposes embryos to constant or biphasic temperature or different drug concentrations. We have demonstrated that a thermal gradient applied across live Drosophila embryos results in asynchronous cellularization. Additionally, preliminary drug screening experiments were carried out on live Drosophila embryos in both 96-well plates and microfluidic channels. Time-lapse differential interference contrast microscopic images were taken to show the effects of colchicine (399.40 Da) and cytochalasin D (507.62 Da) on cellularization.

Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms

“…Fakhoury et al were able to use a simple microfluidic device for the analysis of drug effects on Drosophila embryo development. 19 A Y-channel design was used to introduce one or two drugs into the main channel. In this main channel, a number of embryos were immobilized using interfacial tension created by oil, water/alcohol and SAM-modified surfaces, and a single or combination of drugs was flowed over the embryos.…”

“…For example, positioning a large number of embryos in well-ordered arrays and orient them in a predetermine manner is a prerequisite for automating high-throughput microinjection or image processing. 19,[22][23][24][25][26] Due to the size of the embryos, there is considerable diffraction and optical distortion when acquiring images with particular orientations. To obtain high quality data, the embryos are required to be precisely aligned.…”

Characterization of microfluidic clamps for immobilizing and imaging of Drosophila melanogaster larva's central nervous system

“…Microfluidic-based immobilization techniques for C. elegans have been well developed using chemical (CO 2 ) or mechanical [tapering microchannels or encapsulation using deflectable polydimethylsiloxane (PDMS) membranes] approaches. [15][16][17][18][19] Furthermore, various microfluidic devices have also been developed for improving the throughput of the assays performed on embryos [20][21][22][23][24][25][26][27] such as microinjection, [20][21][22][23][24] self-assembly of eggs and morphogenesis, 25,26 and developmental studies. 27 However, very few devices have been developed for on-chip larval studies.…”

On-chip immobilization of planarians for in vivo imaging

“…The use of microfluidic systems has emerged as a powerful approach to enable immobilization for high-resolution imaging, direct in vivo manipulation, and high-throughput screens of a variety of organisms including Drosophila melanogaster embryos and larvae 18 19 20 21 22 , Caenorhabditis elegans 21 23 24 25 26 27 28 29 30 31 , and zebrafish ( Danio rerio ) embryos and larvae 32 33 34 35 . Immobilization of freshwater planarians, however, presents several unique challenges: Planarians are about an order of magnitude larger than C. elegans , have a squishy, readily deformed body, vary in size depending on the species, feeding state, last asexual reproduction event, and other factors 36 , and are significantly more photophobic than C. elegans or D. melanogaster larvae.…”