Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

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.

Gene expression profiling reveals a conserved microglia signature in larval zebrafish

“…We chose 3 dpf, when macrophages start colonizing the brain and differentiate into early microglia (Figure a,b), 5 dpf when differentiation based on morphological criteria (ramification) is apparent (Figure a, b) and 7 dpf when microglia differentiation has further proceeded (Figure a, b). Microglia were isolated from dissociated brains using the microglia‐specific 4C4 antibody to perform immunohistochemistry followed by fluorescence‐activated cell sorting (FACS) (Figure c, Figure S1; Mazzolini et al, ). As shown in Figure and in recent publications, the 4C4 antibody is highly specific for zebrafish microglia and does not detect other cell types in the brain (Chia, Mazzolini, Mione, & Sieger, ; Ohnmacht et al, ; Tsarouchas et al, ).…”

“…Microglia were isolated by FACS from heads of 3, 5, and 7 dpf Et(Zic4:Gal4TA4 , UAS:mCherry) hmz5 larvae as previously described (Mazzolini, Chia, & Sieger, ) whereas macrophages were isolated from whole 28 hpf Tg(mpeg1:EGFP) larvae. FACS allowed cell separation from debris in function of their size (FSC‐A) and granularity (SSC‐A).…”

“…To understand the changes in gene expression during development in larval zebrafish microglia, we isolated microglia at three different time points. We chose 3 dpf, when macrophages start colonizing the brain and differentiate into early microglia (Figure 1a Figure S1; Mazzolini et al, 2018). As shown in Figure 1 and in recent publications, the 4C4 antibody is highly specific for zebrafish microglia and does not detect other cell types in the brain (Chia, Mazzolini, Mione, & Sieger, 2018;Ohnmacht et al, 2016;Tsarouchas et al, 2018).…”

RNA-seq analysis and compound screening highlight multiple signalling pathways regulating secondary cell death after acute CNS injuryin vivo

“…Cephalic mpeg1 + macrophage-lineage cells were isolated from larval heads by fluorescence-activated cell sorting (FACS) following an established protocol (Mazzolini et al, 2018). The gating parameters for FACS were adjusted with the help of wild-type Fig.…”

“…To obtain RNA from macrophage-lineage cells for RNA-seq analysis and RT-qPCR, GFP + cells were isolated from mpeg1:GFP larvae using FACS, and RNA was extracted from sorted cells as previously described (Mazzolini et al, 2018). All steps were performed at 4°C to preserve RNA integrity.…”