Abstract
To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 °C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 °C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) > 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.
Highlights
Knowledge on brain development and brain diseases has significantly improved in the last decade since the first quantification of mouse brain transcriptomes[1]
We developed an experimental method to isolate cells like neurons, macrophages and microglia from 3 to 8 days post-fertilization larval zebrafish brains
For the establishment of the protocol, we worked with transgenic fish lines that express green fluorescent protein (GFP) in macrophages/microglia under the macrophage-expressed gene promoter and DsRed in neurons under the neural ß-tubulin promoter (NBT:DsRed)[7,8,9]
Summary
Knowledge on brain development and brain diseases has significantly improved in the last decade since the first quantification of mouse brain transcriptomes[1]. To gain a detailed understanding of these processes we need to understand changes in gene expression in the respective cell types To this aim, we developed an experimental method to isolate cells like neurons, macrophages and microglia from 3 to 8 days post-fertilization (dpf) larval zebrafish brains. The protocol, described here in detail, shows the isolation of neurons, macrophages and microglia from zebrafish larval brains, but virtually, it can be adapted to any other cell present within the brain - either by using transgenic fish lines or labelled with specific antibodies This method will allow a better characterization of CNS cells through their genome wide gene expression analyses and will help to understand their role during development and brain diseases
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