Periodic Reporting for period 1 - Glial Patterning (The genetic basis for glial differentiation in the developing zebrafish retina.)
Berichtszeitraum: 2016-03-01 bis 2018-02-28
For more than 150 years glia have been recognised as a significant constituent of neural tissue, but their role as chief regulators of nervous system development and function has only recently emerged. In recent years it has become clear that glia have an essential role in maintaining a healthy brain. Glial defects are now commonly associated with neurological disease including autism, schizophrenia, neurodegeneration and chronic pain. Linkage studies have found many gliopathy genes, but our understanding of how any of these genes function in glia or the nervous system is unknown. With so little known about glial genes, the goal of this project is to enhance our understanding of glial differentiation with a focus on conserved genetic programs. This research will is done using a combination of comparative genomics; novel reverse genetic screening and glial physiology studies with the goal of improving our understanding of the glia differentiation process. In the upcoming years, this research program will expand to include studies in glial differentiation, glial physiology and developing animal models of human glial disease.
The overall objectives of this study were:
1) To defining the retinal glia differentiation temporally. The morphological and physiological changes that retinal glia go through over the course of their development have not yet been defined. Here we carefully documented the retinal glial differentiation over time to test the if glial differentiation can be temporally segregated into distinct developmental stages.
2) To develop a candidate CRISPR screen to identify factors and generate stable mutant lines to comprehensively document their developmental functions in MG and their requirement for retinal glial patterning.
3) To generate the first temporal transcriptomic analysis of glial cell population over the course of their development and in turn, provide novel insights into the distinct regulatory networks control retinal glial patterning during development.
The overall objectives of this study were:
1) To defining the retinal glia differentiation temporally. The morphological and physiological changes that retinal glia go through over the course of their development have not yet been defined. Here we carefully documented the retinal glial differentiation over time to test the if glial differentiation can be temporally segregated into distinct developmental stages.
2) To develop a candidate CRISPR screen to identify factors and generate stable mutant lines to comprehensively document their developmental functions in MG and their requirement for retinal glial patterning.
3) To generate the first temporal transcriptomic analysis of glial cell population over the course of their development and in turn, provide novel insights into the distinct regulatory networks control retinal glial patterning during development.
We have extensively studied the morphological and physiological changes of zebrafish MG over their development which has allowed us to categorise these cells into at least six progressive stages. These studies have indicated that, topographically, retinal glia have at least five independent regions (apical foot process, inner plexiform processes, soma region, outer plexiform processes, and the basal end foot process) each developing independently during different temporal windows.
We conducted transcriptomics on each defining stage of MG development and identified the genetic programs that give rise to each of the defining features (topographical regions) of these cell morphologies. These large transcriptomic databases serve as blueprints of the cell-specific differentiation process.
Recent advances in CRISPR technology that we and others have made have allowed us to use this dataset to conduct large-scale reverse genetic screens and for the first time test the functions of these differentiation factors in-mass. To date, we have screened over 130 genes using this approach and identified morphological defects in all MG compartments. Thus, we have developed a novel strategy for identifying distinct genetic pathways that drive morphological differentiation events that can experimentally be validated.
In combination with our current reverse genetic approaches, these studies will give us tremendous insights into a currently undefined area of glial biology. The implications of this research also provide novel insights into cellular evolution and have the potential to impact studies on many neural disease-related genes.
We conducted transcriptomics on each defining stage of MG development and identified the genetic programs that give rise to each of the defining features (topographical regions) of these cell morphologies. These large transcriptomic databases serve as blueprints of the cell-specific differentiation process.
Recent advances in CRISPR technology that we and others have made have allowed us to use this dataset to conduct large-scale reverse genetic screens and for the first time test the functions of these differentiation factors in-mass. To date, we have screened over 130 genes using this approach and identified morphological defects in all MG compartments. Thus, we have developed a novel strategy for identifying distinct genetic pathways that drive morphological differentiation events that can experimentally be validated.
In combination with our current reverse genetic approaches, these studies will give us tremendous insights into a currently undefined area of glial biology. The implications of this research also provide novel insights into cellular evolution and have the potential to impact studies on many neural disease-related genes.
We have generated many tools for performing transcriptomics and reverse genetics, however, the appropriate testing paradigms for using these tools in-vivo is more restricted. Thus, we have embarked on a project to develop new tools for testing glial physiology in-vitro. For instance, two well-known functions of retinal glia are providing an energy reserve and recycling neurotransmitters for the highly active and metabolically demanding neurons surrounding them. We recently developed fluorescent-resonance-energy-transfer (FRET) sensor transgenic animals that we can use to measure metabolism components in the retina. With these transgenics, we can evaluate how light activation of the photoreceptors effects both the production and depletion of these essential metabolic components. Indeed, these studies will play a critical role in furthering our understanding of glial evolution, function, and human glial disorders.