An Integrated Microfluidic Device For C-Elegans Early Embryogenesis Studies And Drug Assays

The Caenorhabditis elegans embryo is a widely used model for the functional analysis of dynamic cellular processes, such as chromosome segregation, cytokinesis or lineage analysis. However, the conventional embryo preparation method that relies on manually dissecting gravid worms to extract embryos is somewhat time consuming and does not lend itself to high throughput assays. Here, we report a fully integrated microfluidic approach for C. elegans early embryogenesis assays with unprecedented accuracy and throughput. The device consists of a compressible microfluidic pillar-array chamber for robust and fast on-chip extraction of embryos from the uterus of gravid nematodes. Subsequently, embryos are immobilized by automated fluidic transfer in a microtrap array for individual tracking of a large number of embryos, including fragile mutants with drug-permeable eggshells. Our device allows high-resolution live imaging of very early events in embryogenesis, starting from the one-cell stage. We also demonstrate the feasibility of well-controlled compound application in versatile microfluidic pharmacological assays performed on early embryos.

Mutual Inhibition Between the Anaphase-promoting Complex and the Spindle Assembly Checkpoint in C. Elegans Embryos

Alexandra Bezler

The cell cycle orchestrates timely duplication and distribution of cellular components to daughter cells. Checkpoints operate to survey the faithful completion of specific steps during the cell cycle, thus ensuring the equal segregation of genetic material. During mitosis, the anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and the exit from mitosis. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. The principal line of work of this thesis is the characterization of a novel allele of the APC5 component SUCH-1 in C. elegans. We find that some such-1(t1668) embryos lack paternally-contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/Mad1 or MDF-2/Mad2 rescues proper timing in these embryos. Taken together, our findings support a model whereby the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos. In a second line of work, we set out to ask whether the difference in the pace of cell cycle progression at the two-cell stage in early C. elegans embryos is important for proper development. The pace of cell cycle progression changes in specific lineages during development of metazoan organisms, and in C. elegans this is already apparent at the two-cell stage, since the anterior blastomere AB divides 2 min before the posterior blastomere P1. Since cell cycle duration is temperature-dependent in nematodes, we set out to induce a synchronous division between the two cells by heating preferentially P1, and thus accelerating specifically its cell cycle. To this end, in collaboration with the Renaud laboratory, we developed a microfluidic chip that can accommodate C. elegans embryos and generate a transient temperature gradient between AB and P1. So far, we have managed to achieve a temperature difference of ∼2 °C over the 50 µm that correspond to the length of a C. elegans embryo. Analysis of embryonic development at different temperatures indicates that this should be increased to ∼3-5 °C by further optimization to achieve the desired synchronous division.

EPFL2011

Single-Cell Analysis of Mycobacterial Persistence Using Microfabricated Tools

Meltem Elitas

Bacterial cells behave as individuals despite being genetically identical and subject to the same environment. Although the underlying mechanisms of cellular individuality are not well understood, temporal variation of gene expression and protein levels in individual cells could act as sources for phenotypic heterogeneity of populations. Bacterial persistence in fluctuating environments relies on such diversity, allowing some individuals, by chance, to survive a challenge that would kill an unadapted and phenotypically homogeneous population. Bacterial persistence is a non-genetic, metastable phenomenon that is readily lost upon subculture of surviving cells in the absence of the environmental stress. A medically relevant example of persistence is the fractional killing of bacteria by antibiotics, whereby a subpopulation of cells survives indefinitely despite prolonged exposure to a bactericidal drug. It is hypothesized in the case of tuberculosis (TB) that drug therapy may require administration of multiple drugs for 6-9 months in order to eliminate a subpopulation of "persister" cells. The focus of this thesis is to understand how persisters tolerate antibiotics that kill their genetically identical siblings. To address this question, automated time-lapse fluorescence microcopy was applied in conjunction with microfluidic and microelectromechanical systems (MEMS) to study bacterial responses to antibiotics in real time at the single-cell level. A genetic approach was employed to identify mutants of Mycobacterium smegmatis with altered rates of persistence against the first-line anti-TB drug isoniazid (INH). A microfluidic platform was designed for high-throughput screening of transposon (Tn) mutant libraries. A pilot screen of 1,100 Tn mutants revealed 11 mutants that showed increased (persister-up, PU) or decreased (persister-down, PD) survival when exposed to isoniazid. These mutants were further characterized in comparison to wild-type cells using the single-cell microfluidics platform. This single-cell analysis revealed distinct modes of persistence within each category, which were otherwise masked at the population level. The results indicate that persistence behavior was the cumulative result of the dynamics of cell division and cell lysis. Mutants with decreased persistence exhibited behavior at single-cell level that challenges the accepted mechanism of isoniazid-mediated killing. Therefore, these results yield new insights into antibiotic persistence and isoniazid's mode of action. Persisters cannot be easily studied in non-fractionated populations of cells where they represent a small percentage of the total. As a complementary approach, a method was developed based on dielectrophoretic (DEP) purification of naturally occurred low frequency persister cells from an antibiotic-treated bacterial culture. Purification of persister cells would allow for downstream analysis using conventional bulk/population level transcriptomic and proteomic approaches. The crossover frequencies for live and dead cells were determined to enable selective trapping or release from the device. This approach enabled a proof-of-concept enrichment of live bacteria in the mixture of live and dead cells after INH treatment, based on their dielectric properties. Quantitative confirmation of the enriched/isolated cell populations was attempted using flow cytometry. The high-throughput microfluidic screening and single-cell approaches developed in this thesis could, in principal, be applied to the genetic analysis of a wide variety of dynamic cellular processes that can be imaged by time- lapse fluorescence microscopy, such as gene expression, cell division, chromosome replication and segregation, and protein localization. The biochemical analysis of live cells isolated by dielectrophoresis-based separation would provide useful information about the underlying persistence mechanism. Dielectrophoresis could also be a valuable analytical tool for mutant characterization based on their intrinsic dielectric properties, either at single-cell or population levels. In conclusion, this thesis presents several new microfabricated tools to identify and understand bacterial persistence. The thesis illustrates how integration of microfluidic tools with automated time-lapse microscopy allows hidden characteristics that are present but invisible in the macro- world of batch cultures to be examined in a quantitative and high-throughput manner in the micro-world of single-cell analysis.

EPFL2012

Development and application of experimental tools to elucidate the gene regulatory network underlying adipogenesis

Carine Delattre-Gubelmann

Excess fat mass in obese individuals, which is characterized by an increase of white adipocyte cell size and number, significantly raises the risk of developing metabolic syndrome symptoms as well as cancer. A detailed understanding of the transcriptional mechanisms mediating white adipocyte differentiation (i.e., adipogenesis) is required to counteract the growing obesity epidemics. In this thesis, we have implemented two combinatory approaches to detect new protein-DNA interactions in the mouse, one of which targets the adipogenic gene regulatory network (GRN) that underlies the white adipocyte differentiation process. As a first approach to shed light on GRNs, we have developed and validated a powerful approach that combines a high-throughput yeast one-hybrid-based (Y1H) system to detect transcription factors (TFs)-DNA element interactions, together with a novel microfluidics-based technique, enabling the identification and characterization of individual binding sites for detected TFs within the respective regulatory sequences at unprecedented throughput and resolution. Using well-described regulatory elements as well as an uncharacterized enhancer which can play also a role in adipogenesis, we show that our approach in a first phase uncovers many known as well as novel TF-DNA interactions, of which a large proportion were independently validated. In addition, we further demonstrate how our approach can be used in a second phase to characterize detected interactions by enabling the generation of a relative DNA occupancy landscape over the length of the respective DNA element. Furthermore, we provide evidence that this system enables the detection of novel interactions that may have in vivo relevance. Given the strict requirements for direct and well-characterized protein-DNA interaction data to generate and model gene regulatory networks, the presented two-tiered approach should be of great value to enhance our understanding of the regulatory principles underlying mouse gene expression. To identify transcription factors involved in white adipocyte differentiation, we overexpressed TFs in preadipocytes and quantified their effect on differentiation. To this end, we have created a mouse open-reading frame resource consisting of more than 750 fully sequence-verified TF clones. We transferred these TF clones to a Tet-On lentiviral vector and used them to infect white preadipocytes. Based on microscopy-assisted quantification of lipids after induction of differentiation, we identified putatively novel TF candidates that strongly enhanced differentiation based on the increased lipid accumulation, indicating a potential role in adipogenesis. Gene expression and ChIP-seq experiments have been performed for the three top candidates (ZEB1, ZFP30 and ZFP277) to further study their function within the adipogenic regulatory network. To assess whether the effect of these TFs on adipogenesis could be reproduced in vivo, murine adipose stromal-vascular fraction containing adipocyte precursor cells were implanted into mice fed a high-fat diet, after each of the three TF candidates were knockdown or overexpressed in these cells. This experiment and the RNA-seq analyses are currently being processed while this thesis is being written. Furthermore, due to the absence of quantitative real-time PCR (qPCR) primer designing tools that would allow for gene-specific expression profiling in a high-throughput fashion, we design a user-friendly platform: GETPrime. This platform uniquely combines and automates several features critical to optimal qPCR primer design for all annotated Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio genes in the Ensembl database. GETPrime primers have been extensively validated experimentally, demonstrating their high quality as well as high transcript specificity in complex samples.

EPFL2013