An automated microfluidic platform for C. elegans embryo arraying, phenotyping, and long-term live imaging

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population's statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPR(mt)) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Metabolic control of muscle and muscle stem cell function

Hongbo Zhang

Skeletal muscle composed of myofibers and a small amount of muscle stem cells (MuSCs). It plays important roles in energy metabolism. Some of the key metabolites, such as NAD+ and acetyl-CoA were recently found to regulate muscle and MuSCs function. Most of these functions are related to the cellular mitochondria. In my PhD thesis projects, we aimed at exploring the link between NAD+ levels, mitochondrial function, muscle structural homeostasis and MuSCs senescence. We also investigated the impact of acetyl-CoA on MuSCs activation and proliferation, through the control of protein acetylation. These projects mainly focus on three aspects: Rejuvenation of adult stem cells by NAD+ repletion. Aging is accompanied by SCs senescence. However, the initial signaling events mediating SCs senescence are still not well defined. We demonstrate the importance of mitochondrial activity as a pivotal modulator of MuSCs senescence. MuSCs from aged mice have reduced NAD+ levels, mitochondrial content and capacity for oxidative respiration. Importantly, the induction of the mitochondrial unfolded protein response (UPRmt), subsequent to increasing cellular NAD+ with the precursor nicotinamide riboside (NR), prevents MuSCs senescence and improves muscle function and regeneration in aged mice. NR also prevents MuSCs dysfunction in the mdx mouse, a known mouse model of accelerated MuSCs senescence. Extending these observations to other SC pools and on the organism as a whole, we demonstrate that NR delays neural and melanocyte SCs senescence, while also increasing mouse lifespan. Control of MuSCs function by acetyltransferase KAT2A. MuSCs are essential for skeletal muscle homeostasis and repair. The complete mechanism controlling MuSCs maintenance and differentiation, however, remains elusive. We found that the acetyltransferase KAT2A expression is essential for myogenic differentiation of MuSCs. KAT2A loss-of-function in cells blocks MuSCs differentiation, and in mice impairs muscle regeneration after damage. The regulation of KAT2A in MuSCs function might rely on its ability to enzymatically acetylate other proteins, and in particular the transcription factor PAX7. NAD+ repletion improves muscle function in muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations that lead to progressive muscle degeneration. Our bioinformatics analysis in mice and DMD patients suggests a role for NAD+ in protecting the muscle from metabolic and structural degeneration. Validating these findings, we reveal that the mdx mouse is characterized by reductions in muscle NAD+ levels, concurrent to increased PARP activity and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the key enzymes for NAD+ consumption and biosynthesis. Replenishing the NAD+ pool by dietary NR supplementation benefits muscle function in mdx and mdx/Utr-/- mice, an effect replicated in C. elegans models of DMD. The beneficial effects of NAD+ repletion in muscular dystrophy are pleiotropic and rely on the improvement in mitochondrial function, structural protein expression and on reductions in general PARylation, inflammation and fibrosis. In summary, our work demonstrates the critical role of NAD+ and Acetyl-coA in maintaining muscle and MuSCs function. Repletion of NAD+ levels, by NR administration, and/or boosting the function of acetyltransferase KAT2A might be attractive strategies to maintain muscle homeostasis and MuSCs function in aging and muscular dystrophy.

EPFL2016

Long-term culture and high-resolution imaging of C. elegans using an automated microfluidic platform

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Govindan Narayanan Krishnamani, Laurent Mouchiroud

The nematode Caernohabditis elegans is one of the most employed small model organisms in biology. Studies on transgenic animals often require the accurate observation of highly localized fluorescent signals inside the worms at high magnification, hence demanding the full immobilization of the animals. Moreover, in order to observe the dynamics of biological processes over the lifespan of a worm, the same worm has to be immobilized repeatedly, in a reversible manner and under normal physiological conditions. We describe a microfluidic platform for the automated culture, treatment and long-term high-resolution imaging of C. elegans. Our device features: (i) a microfluidic design tailored for the isolation of L4 larvae from a mixed larval population and for their successive culture and treatment; (ii) a worm immobilization method, based on the thermoreversible sol-gel transition of the biocompatible triblock copolymer Pluronic F127 inside the microfluidic chip, thereby enabling high-resolution imaging; (iii) an integrated temperature control system, both to ensure viable environmental conditions for C. elegans culture and to steer the worm immobilization/release process. We apply this device to observe mitochondrial dynamics in muscle cells during aging at single worm resolution. We expect our platform to enable the simultaneous study and repeated observation of multiple phenotypes in single worms or specific worm populations, such as lifespan and motility assays, in addition to high-resolution imaging.

2015

Multi-dimensional imaging and phenotyping of C. elegans embryos via an automated microfluidic device

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Virginija Jovaisaite, Thomas Lehnert, Alexis Pierre Henri Marette, Laurent Mouchiroud, Shreya Narasimhan

We introduce a microfluidic platform for automated on-chip worm culture, creation of synchronized embryo microarrays, and long-term parallel live imaging of Caenorhabditis elegans embryos. Our device allows studying well-defined embryo populations at unprecedented spatio-temporal resolution, via fully automated multi-dimensional imaging, covering six independent dimensions: the 3 spatial coordinates, development time, exposure duration/type (brightfield, fluorescent), and embryo number in the microarray. We successfully employed our platform to investigate the impact of perturbations of the mitochondrial functions on C.elegans embryogenesis. Our analyses revealed that specific mitochondrial stresses trigger a mitochondrial unfolded protein response (UPRmt) in the embryos. These observations are the first proof that the UPRmt molecular pathway is functional during C.elegans embryogenesis.

2015

Metabolic control of muscle and muscle stem cell function

Hongbo Zhang

EPFL2016