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

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.


Introduction
Knowledge on brain development and brain diseases has significantly improved in the last decade since the first quantification of mouse brain transcriptomes 1 . Indeed, genome wide gene expression analysis gives us access to detailed genetic information on brain tissue and cells that can complement and improve observations made with other techniques and tools.
The zebrafish is a potent biological model, easy to breed and to modify genetically; its optical transparency at larval stages allows live imaging observations 2 . Unfortunately, compared to human and mouse, the number of available antibodies to perform immuno-staining is rather low. To remedy this, transgenic zebrafish fish lines are easily made by genetically modifying fish to express fluorescent proteins under cell type specific promoters. Transgenic zebrafish lines have been used in the past to study the role of macrophages and microglia during central nervous system (CNS) development and disease 3,4,5,6 . However, 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 specifically isolate cells like neurons, macrophages and microglia from 3 to 8 days post-fertilization (dpf) 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 (mpeg1:eGFP) and DsRed in neurons under the neural ß-tubulin promoter (NBT:DsRed) 7,8,9 . Furthermore, we performed immuno-staining of microglia using 4C4, a mouse monoclonal antibody that specifically stains zebrafish microglia 10,11 . Afterwards, ribonucleic acid (RNA) is extracted from these cells for further quantitative polymerase chain reaction (qPCR) or transcriptome analyses. This protocol has been designed to efficiently homogenize brain tissue from zebrafish larvae; collect neurons, macrophages/microglia and microglia without alteration of their plasma membrane integrity and finally extract RNA from these cells in high quality (RIN > 7) and quantity to perform genomic analysis. Unlike previously published studies that use trypsin treatment at 37 °C to digest brain tissue 12,13 , this protocol promotes work at 4 °C till the RNA extraction step to reduce modifications of the gene expression profile. This step is crucial as microglia and macrophages are highly sensitive cells which respond to changes in their microenvironment immediately by altering their gene expression profile and polarization 14,15,16 .
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.

Representative Results
The described protocol is a straightforward approach to isolate neurons, macrophages and microglia from zebrafish larval brains. From these isolated cells, significant amounts of high quality (RIN > 7) RNA were extracted. The aim of this protocol is to isolate different types of cells from the CNS, with minimal modification of their gene expression profile to analyze and characterize cell properties and functions. Therefore, the entire protocol is performed at 4 °C with a mechanical brain tissue homogenization. This method has been successfully used for two studies performed in the laboratory. In the first study, neurons and macrophages/microglia were isolated from 8 dpf mpeg1:eGFP + /NBT:DsRed + larvae ( Figure 1). FACS allowed cell separation from debris in function of their size (FSC-A) and granularity (SSC-A) ( Figure 1A). Single cells were then separated from doublets or cell agglomerates ( Figure 1B). From the single cell population, a gate was drawn to eliminate dead cells (DAPI + ). The corresponding dot plot revealed that this experimental protocol preserves cell plasma membrane integrity, as the rate of dead cells is only 26.7% ( Figure 1C). Finally, neurons (DsRed + ) and macrophages/microglia (GFP + ) were easily segregated from the live cell population gates. The neuron population (23.1 %) appeared to be more prominent than the macrophages/microglia population (1.56 %) within the brain ( Figure 1D). This protocol has allowed to isolate RNA from those cells to perform subsequent qPCR analyses to compare the expression of specific genes between neurons and macrophages/microglia. For the second study this method focused on microglia isolation from 3, 5 and 7 dpf larval brains. In contrast to the experiment described above, cells were isolated by immuno-staining using 4C4, an antibody which specifically labels microglia (Figure 3 A-D). As previously described, microglia (4C4 + ) were selected from live cells and collected ( Figure 3D). Microglia numbers within zebrafish larval brains are variable ( Table 1), and very low at 3 dpf (# 25 per fish). Quality and quantity of extracted RNA from those cells were measured using a micro-capillary electrophoresis based system. Results obtained of extracted RNA from microglia of 5 dpf larval zebrafish brains have been provided to illustrate an example of RNA analysis (Table 1 (5 dpf; experiment 4)). Figure 4 shows the electrophoresis trace and its graphic representation obtained for this sample with a clear visualization of ribosomal RNA (28s and 18s). This data is necessary to calculate sample RIN and to determine RNA concentration. Table 1 summarizes the number of isolated microglia per fish, the amount of RNA per microglia and the RIN score obtained for each different experiment at 3, 5 and 7 dpf. The amount and the quality of extracted RNA from isolated microglia using this method allowed us to amplify the RNA into cDNA using a kit. Quality and quantity tests provided by Edinburgh Genomics confirm that the amplified cDNA is of sufficient quality for library preparation and subsequent sequencing. Figure 5 shows the size distribution of cDNA fragments and their amount measured using an electrophoretic system. In this sample, the cDNA had a mean size of 299bp at a concentration of 36100 pmol/l. Table 2 illustrates respectively quality and quantity tests made on amplified cDNA from RNA samples ( Table 1 ( 5 dpf; experiment 4)). The amplified cDNA has been used successfully for sequencing.
Several studies performed in the laboratory confirmed that the quality and quantity of extracted RNA from neurons, macrophages and microglia can be used for subsequent qPCR and genome wide gene expression analyses. Therefore, this experimental protocol can be used to reliably isolate different types of CNS cells without altering their membrane integrity and limiting modification of their gene expression profile.

Discussion
The experimental protocol described here represents a robust and efficient method to isolate brain cells from zebrafish larvae from 3 to 8 dpf. Importantly, this is the first protocol that allows the specific isolation of microglia from larval zebrafish brains. The protocol is designed to preserve cell membrane integrity and to minimize potential modifications of gene expression occurring during the processing. This last point is crucial for the relevance of results based on the analysis of those isolated cellular genomic profiles. Indeed, microglia and macrophage polarization are strongly influenced by their microenvironment. At 37 °C, these cells would have changed their gene expression profile in response to experimental conditions (injury (transection) response). Therefore, it was crucial to perform this experiment at 4 °C prior to RNA extraction, to slow down cellular processes and metabolic activities. Furthermore, the mechanical brain tissue homogenization at 4 °C was chosen instead of enzymatic tissue digestion at 37 °C to avoid any impact on gene expression profiles.
It is important to highlight that this method is very quick; within a day several brain cell populations can be isolated from at least two different experimental conditions and their RNA extracted. The total length of the protocol depends on the number of larvae used for each condition as the transection of larval heads is the limiting step (# 350 heads/h). In general, to work with microglia from 3, 5 and 7 dpf it is recommended to transect # 600 heads per test to get enough RNA to extract ( Table 1). As microglia represent the cell type with the lowest yield (approx. 112 cells per head at 7 dpf), the number of heads can be reduced for other cell types including macrophages (approx. 170 cells per head at 7 dpf).
Another advantage of this protocol is that once settings on the FACS sorter have been established, the same settings can be used for different experiments. It has been observed that cell populations fit perfectly from one experiment to another with gates previously designed, showing the reproducibility of experiments using this method.
A slight disadvantage of this method is the relatively low amount of RNA that is harvested. However, this limitation is more a biological issue than a technical issue, as the number of microglia is very low at early stages of brain development ( Table 1). Because of this low quantity of RNA collected, amplification steps need to be considered to perform genome wide gene expression analysis. Fortunately, these amplification steps produce sufficient amounts of high quality cDNA. Thus, global changes in the gene expression profiles of isolated cells can be studied.
In conclusion, this protocol provides a robust method to isolate and study various CNS cell types from larval zebrafish brains. This can be applied to gain a deeper understanding of these cells during development as well as to study their role in disease.

Disclosures
The authors have nothing to disclose.