Microglia are the dominant immune cells in the central nervous system (CNS). They play a critical role in brain development by both trimming and promoting synapse formation, which makes our brains more efficient as we transition out of childhood. Additionally, microglia clear apoptotic neurons, which is an essential task both in brain development and disease. One outstanding question in developmental neuroscience research is whether there are distinct subsets of microglia that perform these diverse functions.
Dr. Anna Molofsky is an Associate Professor at the Weill Institute for Neurosciences at the University of California, San Francisco, USA. Her lab’s publication in Nature Communications studied microglia in zebrafish using confocal microscopy along with sequencing methods to identify two phenotypically and functionally distinct subtypes of microglia in the developing zebrafish brain.
We spoke with Dr. Nicholas Silva, a postdoctoral researcher in Dr. Molofsky’s lab and a primary author on the paper about their work.
Dr. Anna Molofsky (right) with Dr. Nicholas Silva with their ZEISS confocal microscope.
What are the research goals of the Molofsky lab?
We are interested in understanding how innate immune cells known as microglia shape the healthy brain during development and disease states. Our goal is to identify key cellular and molecular processes that regulate postnatal synaptic development in hopes to inform new immune-based therapies for psychiatric and neurodevelopmental illnesses.
Tell us about your recent discoveries and your use of confocal microscopy.
Our recent work published in Nature Communications identifies two unique subtypes of microglia during brain development in zebrafish; one subtype is located within the midline optic tectum (OT) and the other from the hindbrain. We found phenotypic, functional, and transcriptional differences associated with these two microglia subtypes.
Microglia uptake of synaptic proteins within the zebrafish hindbrain at 28 days post fertilization (dpf). Zebrafish microglia; mpeg-GFP (Green), presynaptic marker; SV2 (Red), and DNA (Blue). Acquisition was done with a ZEISS confocal microscope.
Confocal microscopy was instrumental throughout our work; we specifically used it to:
- Identify phenotypic differences of OT microglia versus hindbrain microglia
- Perform in situ hybridization studies to confirm transcriptome differences of OT versus hindbrain microglia
- Provide evidence of functional differences between OT microglia (high cathepsin activity using the biomarker Prosense680 and higher number of engulfed neuronal corpses) versus hindbrain microglia (higher number of engulfed synapses)
Image 1: An image showing the enriched gene cd74a (MHC class II) expressed in ramified microglia within the hindbrain region of the zebrafish brain. Zebrafish microglia (4C4: White), cd74a (Red), and DNA (Blue). Image 2: Amoeboid microglia enriched in the optic tectum (OT) with catalytic activity of cathepsin b and l, which is indicated by fluorescence probe Prosense 680. Zebrafish microglia (mpeg: Green), Prosense 680 (white), and DNA (Blue). Images acquired with a ZEISS confocal microscope.
In summary, we show that microglia found in the OT are condensed and amoeboid in shape, remove apoptotic neurons, and express lysosomal cathepsin genes. In contrast, microglia in the hindbrain are branched out in shape, engulf synapses, and express synaptic pruning genes.
These findings suggest that not all microglia are created equal and their function is dictated by their location within the brain. The identification of microglia subpopulations within the brain will aid in our understanding of biological functions for future immune-based therapies.
Where will this work go next?
Currently we aim to interrogate the molecular mechanisms of the top differentially expressed genes (DEGs) associated with the synaptic-enriched microglia subpopulation. Using cell-type specific CRISPR/Cas9 and live imagining strategies, we hope to identify novel immune and extracellular matrix (ECM) genes that are critical for regulating microglia synaptic interactions during development and disease.