Supervisor: Dr Valérie Wittamer
1st project:
Deciphering the molecular determinants of microglia ontogeny during vertebrate development using the zebrafish model
The goal of our research is to provide novel insights into the ontogeny and functions of microglia, the resident-tissue macrophages of the central nervous system (CNS). Understanding these key aspects of microglia biology are of major importance since these cells serve multiple functions in physiology and disease and represent major targets for therapeutic intervention in a wide variety of neurological disorders, including neuroinflammation and neurodegeneration.
A major breakthrough in our understanding of microglia biology was the demonstration over the last few years that microglia exhibit specific differentiation and homeostatic features, which distinguish them from other populations of myeloid cells. Indeed, in contrast to blood monocytes which are constantly replenished from bone marrow-derived committed progenitors, microglia are maintained in the nervous tissue throughout life, independently from any bone marrow input. Because these findings have fundamental consequence on how to address microglia ontogeny and function for therapeutic purpose, they have opened new avenues for delineating the developmental program of microglia and its regulation. However, despite progress, the molecular mechanisms that control microglia specification and maintenance in the CNS are still poorly understood and remain to be determined.
Our group takes advantage of the unique attributes of the zebrafish model system to address microglia development in ways not possible in other vertebrates. Indeed, because the first steps of microglia ontogeny occur early during embryogenesis, transparent transgenic zebrafish embryos offer great opportunities to characterize these processes in a non invasive way. Through gene profiling, we have determined the molecular signature of zebrafish microglia, and have identified several candidate genes required for microglia development. In the proposed project, we will use state-of-the-art molecular, genetic and live imaging approaches to functionally characterize their specific contribution to microglia biology in vivo. The project will rely on targeted genome editing gene manipulation using the CRISPR/Cas9 technology to manipulate microglia gene functions in zebrafish and examine the subsequent effects on microglia biology through live imaging analyses on fluorescent transgenic embryos.
Insights into the molecular events that instruct microglial cell fates will ultimately advance current reprogramming protocols for generation of microglia-like cells in vitro for clinical use or to model disease in a dish.
Techniques:
Zebrafish breeding and embryo/larvae handling, in situ hybridization, molecular biology, transgenesis, microinjection, fluorescence microscopy, live imaging, flow cytometry….
2nd project:
Evolutionary Aspects of Microglia Self-Renewa using the Zebrafish Model
The goal of our research is to provide novel insights into the ontogeny and functions of microglia, the resident-tissue macrophages of the central nervous system (CNS). Understanding these key aspects of microglia biology are of major importance since these cells serve multiple functions in physiology and disease and represent major targets for therapeutic intervention in a wide variety of neurological disorders, including neuroinflammation and neurodegeneration.
A major paradigm shift in the field was the demonstration a few years ago that microglia in the mouse are maintained in the nervous tissue throughout life, independently from any contribution from bone marrow-derived progenitors. These findings have fundamental consequence on how to address microglia ontogeny and function for therapeutic purpose and have thus opened up new avenues for research. However, the molecular mechanisms that regulate microglia self-renewal, expansion and density in the CNS have remained almost completely unexplored. It is also unclear whether microglial self-renewal is a universal feature in vertebrates, or unique to mammals.
Interestingly, we have recently shown that, in contrast to the mouse, embryonic microglia in zebrafish are completely replaced in the adult by a second wave originating from larval hematopoietic stem cell (HSC)-derived progenitors. Do these observations imply that embryonic microglia are unable to self-maintain? Additionally, are adult microglia self-renewing cells or do they require continuous input from HSC-derived progenitors? In this project, we will address these fascinating questions using new cutting-edge technologies. By combining live imaging, fate mapping, microglial transplantation, single-cell omics and CRISPR/Cas9 mutagenesis, we will elucidate the self-renewing potential of the various ontogenetically distinct microglial populations, both in the brains of embryos and adult fish. The results from these experiments will then shape the focus of downstream comparative transcriptome analysis, which will help defining the genetic program of microglia self-renewal in mammals.
3rd project:
Neuronal Regulation of Microglia during Vertebrate Development
Microglia represent a distinct population of resident macrophages that act as immune sentinels and serve multiple functions in central nervous system (CNS) physiology and disease. In addition, microglia are also key regulators of CNS development, and the recent discovery that defective microglia can be directly causative in several neurodevelopmental disorders have highlighted their therapeutic potential. Over recent years, increasing evidence have pointed to the existence of crucial microenvironmental interactions of microglia in the developing brain, including with neurons and different classes of glia. However, despite considerable advances in the field, the physiological consequences of such communications are still poorly understood.
The proposed project will focus on investigating the bidirectional crosstalk that takes place specifically between microglia and neurons, and whose importance in physiology and disease is increasingly supported. In particular, our goal is to provide new insights into how neurons regulate microglia behavior and functions during embryogenesis. These fundamental questions will be addressed using zebrafish, a model system that offers unique experimental approaches for the study of microglia in live conditions, owing to the transparency of its embryo and powerful genetics. The project will rely on state-of-the-art approaches that include, among others, confocal and light-sheet microscopy, transgenesis, CRISPR/Cas9 mutagenesis, chemo- and opto-genetics, and comparative genomic and transcriptomic analyses.