Microglia are resident leukocytes in the vertebrate central nervous system (CNS), with various roles in health and disease. In the healthy state, microglia contribute to CNS function through the clearance of dead/dying cells and debris (Blume et al., 2020; Diaz-Aparicio et al., 2016; Herzog et al., 2019; Mazaheri et al., 2014; Neumann et al., 2009; Peri and Nüsslein-Volhard, 2008; Sieger et al., 2012), synaptic pruning (Bilimoria and Stevens, 2015; Milinkeviciute et al., 2019; Paolicelli et al., 2011; Schafer et al., 2012; Scott-Hewitt et al., 2020; Weinhard et al., 2018), and regulation of neuronal populations through various mechanisms (Anderson et al., 2019; Brown and Neher, 2014; Cunningham et al., 2013; Neher et al., 2011; Sierra et al., 2010; Vilalta and Brown, 2018). It is now appreciated that nearly all CNS diseases show evidence of activation and often dysregulation of microglia, but the mechanisms by which microglia contribute to disease are not well understood. For example, in mouse models of neurodegenerative disease, a transcriptional signature has been described and attributed to so-called ‘disease associated microglia’ (Deczkowska et al., 2018; Keren-Shaul et al., 2017). This transcriptional signature shows upregulation of certain genes, many with poorly understood function, with downregulation of homeostatic genes (Deczkowska et al., 2018; Holtman et al., 2015; Keren-Shaul et al., 2017). Further, it is not clear whether the function of these disease-associated genes represents dysregulated microglial function in the diseased state, or if these genes could represent a transcriptional program that is important in controlling disease conditions.

Importantly, the function of many genes expressed by microglia, many of which have been identified in disease or degenerative conditions, remains to be determined. There is a need to better understand baseline regulation and function of microglia expressed genes, in order to understand how microglia contribute to neurodegenerative disease. Along these lines, our recent transcriptome analysis of microglia isolated from regenerating zebrafish retinal tissue (Mitchell et al., 2019) found that apoc1 was the top hit for microglia-enriched genes. Further, apoc1 was among the top enriched genes in phagocytic microglia isolated from adult zebrafish brain analyzed by RNA-seq (Wu et al., 2020), and is also highly enriched in microglia during acute damage response in the zebrafish brain (Oosterhof et al., 2017). At the protein level, zebrafish APOC1 was differentially regulated during retinal damage and regeneration (Eastlake et al., 2017). Together, such results indicate that this gene is crucial to some aspect of microglial function, though this function remains unknown. Interestingly, human APOC1 variants may be associated with increased Alzheimer's disease (AD) risk (Bertram et al., 2007; Drigalenko et al., 1998; Poduslo et al., 1998; Zhou et al., 2014, 2019). Such a genetic link is also apparent, and well-appreciated, for APOE (Cervantes et al., 2011; Jun et al., 2012; Saunders et al., 1996; Strittmatter et al., 1993; Verghese et al., 2011), which lies just upstream of APOC1 on chromosome 19 in humans (Mak et al., 2002; Smit et al., 1988). One report suggests an anti-inflammatory function for APOC1, however this function may in some way be linked to certain APOE alleles (Cudaback et al., 2012). In contrast, other work suggests possible APOE-independent effects of APOC1 (Prendecki et al., 2018; Zhou et al., 2019). Somewhat paradoxically, both over expression and knock-out of Apoc1 in mouse models appear to result in cognitive defects (Abildayeva et al., 2008; Berbée et al., 2011).

Similar to findings in zebrafish by transcriptome analysis, relatively strong microglial expression of APOC1 has also been described in RNA-seq analyses of human microglia (Gosselin et al., 2017). In addition, APOC1 is one of the most highly upregulated genes in microglia isolated from brains of human AD patients (Mathys et al., 2019; Srinivasan et al., 2020) and among the upregulated genes in aged human microglia (Olah et al., 2018). In contrast, microglial expression of Apoc1 in mouse models is comparatively much lower (Gosselin et al., 2017). This discrepancy in expression of Apoc1 by microglia between humans and mouse models may, at least in part, explain our current lack of understanding of Apoc1 function as it relates to baseline microglial function in the CNS. This could also be at least part of the reason that this gene is under-studied in the CNS relative to Apoe and suggests that alternative models could be appropriate for studying this gene. Some previous work in zebrafish has studied apoc1 in the early embryo during epiboly (Wang et al., 2013), but expression and regulation of this gene in the animal, after microglia colonize the developing CNS, has not yet been explored.

Although the above referenced RNA-seq experiments, including our own, indicate that microglia are a prominent cell type expressing Apoc1 in the CNS, to our knowledge this has not been demonstrated in situ. Though Apoc1 mRNA has been detected in cultured astrocytes (Petit-Turcotte et al., 2001), it is not clear if or when this is the case in vivo, and localization of the protein appears to occur at other locations in the CNS (Abildayeva et al., 2008; Evangelou et al., 2019; Petit-Turcotte et al., 2001). In addition, although apolipoprotein expression by peripheral macrophages has been studied in terms of lipoprotein metabolism (Fuior and Gafencu, 2019), Apoc1 has received little attention compared to other apolipoprotein genes, most notably Apoe. Further, the regulation of Apoc1 expression in microglia in vivo has not been studied. We considered that RXR heterodimers could be important in this regard, and that modulation of these receptors could affect Apoc1 expression by microglia in vivo, given that published in vitro studies have examined the role of LXR-RXR and PPAR-RXR receptors in the regulation of the apolipoprotein gene cluster in macrophages (Chawla et al., 2001; Dahabreh and Medh, 2012; Mak et al., 2002; Subramanian et al., 2017). Also notable, there are reports of retinoic acid (RA) regulation of apolipoprotein genes in astrocytes (Zhao et al., 2014), indirect effects of RA on apoc1 in the zebrafish embryo (Wang et al., 2015), as well as LXR regulation in both astrocytes and macrophages (Laffitte et al., 2001b; Liang et al., 2004; Mak et al., 2002). Considering these reports and the advantages of the zebrafish model for pharmacological manipulations via immersion and in situ imaging, as well as transcriptome analyses indicating conserved expression of Apoc1 by microglia in both human and zebrafish as discussed above, the zebrafish could provide an excellent model organism to probe this gene.

Here, we confirm orthology of human APOC1 and zebrafish apoc1. We show that apoc1 expression is indeed localized to microglia in the developing zebrafish CNS and in the adult zebrafish retina. We determine that the onset of apoc1 in a subset of microglia begins by 3 days post fertilization (dpf) and by 5 dpf most microglia express high levels of this gene. To further understand the regulation of apoc1 expression in microglia, and to compare to that of apoeb, we used an in vivo pharmacological approach with compounds that modulate RARs, RXRs, PPAR, and LXR receptors and examined their effects on microglial expression of apoc1 and apoeb. The use of zebrafish for this work allowed us to selectively modulate activity of these receptors using pharmacological immersion treatments during early CNS development. We show evidence that microglial expression of these two apolipoprotein transcripts is differentially regulated by LXR versus PPAR modulators, and that RXR receptors could be involved in endogenous regulation of microglial expression of apoc1. In particular we show evidence that in microglia, apoc1 is more significantly influenced by LXR-RXR agonists than apoeb. In contrast, expression of apoeb is more significantly influenced by PPAR-RXR modulation. We show that microglia express transcripts for both nr1h3 (lxra, the only lxr gene in zebrafish) and nr1c3 (pparg), suggesting that microglia could directly respond to modulation of these receptors. This suggests that future therapeutic approaches could potentially extend this work towards selective targeting of APOC1 separate from APOE, if such a goal is found to be appropriate, to modulate human neurodegenerative disease in the CNS. In addition, our findings further justify the use of zebrafish as a model for future studies into the regulation and function of microglia-expressed apoc1 in the CNS.

We compared the chromosomal regions containing the human APOC1 gene, mouse Apoc1 gene, and zebrafish apoc1 gene in the three species (Fig. 1). All three species show similar organization including chromosomal clustering of apolipoprotein genes with the apoeb (zebrafish; APOE: human, Apoe: mouse) gene upstream of apoc1 in all three species (Fig. 1A–C). Other similarities include other apolipoprotein genes (apoc2 and apoc4: zebrafish; APOC2 and APOC4: human; Apoc2 and Apoc4: mouse), found downstream of apoc1 in all three species. Tomm40 (mouse) and TOMM40 (human) are upstream of Apoc1 in mice and humans, but in zebrafish tomm40 lies roughly 2Mb downstream of apoc1, and in the opposite orientation (Fig. 1A–C). In humans, there is also a pseudogene (APOC1P) downstream of APOC1 (Lauer et al., 1988) that is not found (or not annotated) in mouse or zebrafish. Another difference in apolipoprotein gene clustering is that apoa4b.2 is found upstream of apoc1 in zebrafish, but in humans and mice, respectively, APOA4 and Apoa4 are found on a different chromosome (chromosome 11, chromosome 9).

Fig. 1.

Orthology of zebrafish apoeb and apoc1 to human and mouse genes. (A–C) Organization of the apolipoprotein gene clusters in zebrafish (A), human (B), and mouse (C). (D) Amino acid alignment of zebrafish APOC1 to human APOC1. (E) Phylogenetic relationship of zebrafish apoc1 to the shown species as determined by ensembl.org.

Fig. 1.

Orthology of zebrafish apoeb and apoc1 to human and mouse genes. (A–C) Organization of the apolipoprotein gene clusters in zebrafish (A), human (B), and mouse (C). (D) Amino acid alignment of zebrafish APOC1 to human APOC1. (E) Phylogenetic relationship of zebrafish apoc1 to the shown species as determined by ensembl.org.

To further examine orthologous relationship of human APOC1 and zebrafish apoc1, we used the DRSC Integrative Ortholog Prediction Tool (DIOPT). DIOPT is an ortholog and paralog search tool that compares ortholog predictions from multiple algorithms, such as Compara, eggnog and OrthoDB (Hu et al., 2011). The DIOPT score was 10 for apoc1 when comparing human and zebrafish genes (Fig. S1), indicating the human and zebrafish genes as orthologs. DIOPT analysis also showed that the orthology was ranked ‘high’ meaning that the pairs had the best scores for either forward or reverse searches and had an overall score of above 2 (Fig. S1). An amino acid alignment (UniProt) was also performed between the two species showing similarity of 56%, and conserved identity of 35% (Fig. 1D). We also used Ensembl to create a gene tree for apoc1, to further investigate the relationship of the zebrafish apoc1 gene to other species. Based on this gene tree, there is a common ancestral apoc1 gene that gave rise to both the mammalian and zebrafish genes (Fig. 1E). Collectively, we conclude that human APOC1 and zebrafish apoc1 are orthologs. This orthologous relationship supports that the zebrafish is an appropriate model organism to study this gene.

In our previous report describing the transcriptome of zebrafish microglia isolated from regenerating retinas, apoc1 was our top hit for differentially expressed genes in microglia (Mitchell et al., 2019). We therefore used the tools at zfregeneration.org (Nieto-Arellano and Sánchez-Iranzo, 2018) to re-examine apoc1 expression in our own work as well as another published study (Oosterhof et al., 2017), which described the transcriptome of zebrafish brain microglia. For comparison, we also examined expression of apoeb in this manner, a disease-associated apolipoprotein gene with well-appreciated expression by microglia in several species. In microglia isolated from regenerated retinas, apoc1 is also highly abundant and enriched in microglia compared to other retinal cells (Fig. 2A). In the zebrafish brain, apoc1 and apoeb transcripts are highly enriched in microglia when compared to other brain cells (Fig. 2B). Both microglia and other retinal cells express apoeb (Fig. 2C); these other apoeb+ retinal cells are most likely the Müller glia, for which apoeb expression has previously been described (Raymond et al., 2006). In the brain, apoeb is highly enriched in microglia with some less abundant expression in other brain cells (Fig. 2D).

Fig. 2.

Expression of apoc1 and apoeb in the zebrafish CNS measured by RNA-seq. (A,B) Normalized expression (fpkm, fragments per kilobase million reads) of apoc1 in sorted populations of microglia compared other cell types isolated from regenerating zebrafish retina (A; Mitchell et al., 2019) or zebrafish brain (B; Oosterhof et al., 2017). (C,D) Normalized expression (fpkm) of apoeb in sorted populations of microglia compared other cell types isolated from regenerating zebrafish retina (A; Mitchell et al., 2019) or zebrafish brain (B; Oosterhof et al., 2017).

Fig. 2.

Expression of apoc1 and apoeb in the zebrafish CNS measured by RNA-seq. (A,B) Normalized expression (fpkm, fragments per kilobase million reads) of apoc1 in sorted populations of microglia compared other cell types isolated from regenerating zebrafish retina (A; Mitchell et al., 2019) or zebrafish brain (B; Oosterhof et al., 2017). (C,D) Normalized expression (fpkm) of apoeb in sorted populations of microglia compared other cell types isolated from regenerating zebrafish retina (A; Mitchell et al., 2019) or zebrafish brain (B; Oosterhof et al., 2017).

In order to confirm and demonstrate unique expression of apoc1 by microglia in the zebrafish CNS, we extracted mRNA from adult zebrafish retinas and generated cDNA by reverse transcription. To amplify cDNA corresponding to apoc1 mRNA transcripts, we designed three primer pairs for PCR. These primer pairs hybridize in the 5′UTR/first exon and 3′UTR/last exon of apoc1 and are expected to detect both previously described transcript variants of zebrafish apoc1 (Wang et al., 2013). Gel electrophoresis revealed RT-PCR products at the expected size from each primer pair (Fig. S2). The cDNA amplicons were cloned and sequenced revealing identity comparisons to be 99% for all three primer pairs. We chose the product from primer pair 2 to serve as a template for generation of in-house sense and anti-sense DIG-labeled RNA probes to detect apoc1 transcripts in situ. No signal was obtained from the sense probe in adult retina or embryos (Fig. S3).

We first confirmed microglial expression of apoc1 in adult zebrafish retinas, as this was the source of mRNA for cDNA cloning. We were also interested in determining if microglia, or other cell types, express detectable apoc1 in the undamaged adult zebrafish retina since the gene was identified in our study of retinal regeneration (Mitchell et al., 2019). Expression of apoc1 was confirmed in adult mpeg1:mCherry retinas using in situ hybridizations followed by immunofluorescence (Fig. S4). In the adult retina, microglia express mpeg1-driven reporters (Mitchell et al., 2019, 2018). Nearly all mpeg1:mCherry+ cells co-expressed apoc1 (Fig. S4), though the expression of apoc1 in each individual cell appears to be somewhat heterogenous.

We next examined expression of apoc1 in situ in embryonic zebrafish at 3 and 5 dpf, to determine if microglia express apoc1 during early brain and retinal development. We chose these time points because microglia colonize the brain and retina by 3 dpf (Blume et al., 2020; Casano et al., 2016; Herbomel et al., 2001; Xu et al., 2016). In order to confirm that apoc1 was expressed by microglial cells, in situs were first performed using mpeg1:mCherry fish. Co-expression of mpeg1:mCherry and apoc1 confirmed that microglial cells in the developing brain and retina at both 3 and 5 dpf express apoc1, and apoc1 transcripts were localized mainly to microglia (Fig. 3A–C). At 3 dpf, some mpeg1:mCherry+ microglia did not co-localize with apoc1 (Fig. 3B–B″), but most microglia expressed apoc1 by 5 dpf (Fig. 3C–C″).

Fig. 3.

Expression of apoc1 in the zebrafish embryo visualized by in situ hybridization. (A) Region of imaging of embryos at 3 and 5 dpf is indicated by the red box. (B–C″) In situ hybridization for apoc1 using in-house generated RNA probes (green) in mpeg1:mCherry (magenta) transgenic embryos at the indicated ages. (D) DIC image of 3 dpf embryo. (E) Visualization of apoc1 transcripts in the 3 dpf embryo. (F–H) Visualization of apoc1 transcripts in situ in whole embryos at 5 dpf, using HCR in situ hybridization. (F) Fluorescent image of apoc1 HCR probe signal showing transcripts in the head (brain and eyes). (G) DIC image of whole embryo. (G′) Fluorescent image of apoc1 HCR probe signal showing transcripts in the head, eyes, region of remaining yolk sac (ys), and region of developing liver(lv)/gut. (F) Enlarged region indicated by dashed box in G′; transcripts in the developing CNS are consistent with microglia pattern and morphology. (H) Merged image of DIC and fluorescent HCR probe signals to detect mpeg1 and apoc1 in the tail. Transcripts for apoc1 are not observed in mpeg1+ macrophages. Images are representative of n=6 embryos per timepoint.

Fig. 3.

Expression of apoc1 in the zebrafish embryo visualized by in situ hybridization. (A) Region of imaging of embryos at 3 and 5 dpf is indicated by the red box. (B–C″) In situ hybridization for apoc1 using in-house generated RNA probes (green) in mpeg1:mCherry (magenta) transgenic embryos at the indicated ages. (D) DIC image of 3 dpf embryo. (E) Visualization of apoc1 transcripts in the 3 dpf embryo. (F–H) Visualization of apoc1 transcripts in situ in whole embryos at 5 dpf, using HCR in situ hybridization. (F) Fluorescent image of apoc1 HCR probe signal showing transcripts in the head (brain and eyes). (G) DIC image of whole embryo. (G′) Fluorescent image of apoc1 HCR probe signal showing transcripts in the head, eyes, region of remaining yolk sac (ys), and region of developing liver(lv)/gut. (F) Enlarged region indicated by dashed box in G′; transcripts in the developing CNS are consistent with microglia pattern and morphology. (H) Merged image of DIC and fluorescent HCR probe signals to detect mpeg1 and apoc1 in the tail. Transcripts for apoc1 are not observed in mpeg1+ macrophages. Images are representative of n=6 embryos per timepoint.

We examined apoc1 expression throughout the entire embryo, using hybridization chain reaction wholemount in situ hybridization (HCR WISH) (Choi et al., 2018). In 3 dpf embryos, we observed signal from the apoc1 probe in the brain and eyes, consistent with localization of this transcript within microglia, and in the yolk sac (Fig. 3D,E) where apoc1 expression has previously been described (Wang et al., 2013). In 5 dpf embryos, apoc1 transcripts were also present in the region of the developing liver (Fig. 3G,G′); liver expression of apoc1 has been described (Fuior and Gafencu, 2019). However, only brain localized macrophages (i.e. microglia) express apoc1 (Fig. 3G′,F), as apoc1 signal was not observed in locations of other macrophages present in the developing embryo such as the tail fin (Fig. 3H). Interestingly, this indicates that of the macrophage populations in the embryo, apoc1 expression is restricted to microglia.

To visualize expression of apoc1 simultaneously with apoeb in the normally developing CNS, we used multiplex HCR WISH of wild-type embryos with probe sets specific for apoc1, apoeb, and mpeg1 (Fig. 4). Both apoc1 and apoeb localized with mpeg1 in the optic tectum and retina at 3 dpf. Signal from apoc1 was consistent with microglial morphology and almost always co-localized with mpeg1 (Fig. 4). Consistent with apoeb expression both microglia and other CNS cells (Fig. 2), in addition to mpeg1+ cells, we also observed expression of apoeb in mpeg1- cells (Fig. 4). In the optic tectum, apoeb+ signal was observed in a ring-like, possibly perinuclear, pattern in cells of the optic tectum that did not co-localize with mpeg1 (Fig. 4C,E), likely representing other glial cells or possibly neurons. Also observed and expected, apoeb signal was present in cells with morphological and spatial characteristics consistent with the Müller glia (Fig. 4H,J), for which apoeb expression has previously been described (Raymond et al., 2006). RT-qPCR revealed a nearly 100-fold increase in apoc1 transcript levels in the heads of zebrafish between 3 and 5 dpf (Fig. 4L). This indicates that from 3 to 5 dpf, while more microglia may begin to express apoc1, apoc1 transcript levels are strongly increased on a per microglial cell basis. In contrast, apoeb transcripts in heads increased approximately twofold (Fig. 4K) from 3 to 5 dpf.

Fig. 4.

Multiplex detection of mpeg1, apoc1, and apoeb transcripts in the developing zebrafish CNS. HCR probe sets were used to detect mpeg1, apoc1, and apoeb transcripts in whole zebrafish embryos at 3 dpf. (A) Region and orientation of imaging of the zebrafish brain. Orientation markers: C, caudal; R, rostral. (B–D) Signal from each probe set detected within the optic tectum. (E) Merge of all three probe set signals. The region indicated by the dotted lines pertains to the optic tectum. (F) Region and orientation of imaging of the zebrafish eye/retina. Orientation markers: D, dorsal; N, nasal; V, ventral. (G–I) Signal from each probe set detected within the eye/retina. (J) Merge of all three probe set signals. The region indicated by the dotted lines pertains to the embryonic eye boundary (outer circle) as well as the lens (inner circle). In embryos, the eye is comprised nearly entirely of lens and retina. (K,L) Fold change in expression in heads from 3 to 5 dpf of apoeb (K) and apoc1 (L) measured by RT-qPCR. Images in A and F were generated in BioRender. Images are representative of n=6 embryos.

Fig. 4.

Multiplex detection of mpeg1, apoc1, and apoeb transcripts in the developing zebrafish CNS. HCR probe sets were used to detect mpeg1, apoc1, and apoeb transcripts in whole zebrafish embryos at 3 dpf. (A) Region and orientation of imaging of the zebrafish brain. Orientation markers: C, caudal; R, rostral. (B–D) Signal from each probe set detected within the optic tectum. (E) Merge of all three probe set signals. The region indicated by the dotted lines pertains to the optic tectum. (F) Region and orientation of imaging of the zebrafish eye/retina. Orientation markers: D, dorsal; N, nasal; V, ventral. (G–I) Signal from each probe set detected within the eye/retina. (J) Merge of all three probe set signals. The region indicated by the dotted lines pertains to the embryonic eye boundary (outer circle) as well as the lens (inner circle). In embryos, the eye is comprised nearly entirely of lens and retina. (K,L) Fold change in expression in heads from 3 to 5 dpf of apoeb (K) and apoc1 (L) measured by RT-qPCR. Images in A and F were generated in BioRender. Images are representative of n=6 embryos.

Given the strong induction of apoc1 during early CNS development (Fig. 4K,L), we were interested in determining how microglial expression of apoc1 may be induced, and to investigate how that may be similar or different from that of apoeb. Coordinate regulation of the apolipoprotein gene cluster has been reported (Cudaback et al., 2012; Evangelou et al., 2019; Mak et al., 2002). Various in vitro studies have examined the role of LXR-RXR and PPAR-RXR receptors in the regulation of the apolipoprotein gene cluster in macrophages and other cell types in vitro (Chawla et al., 2001; Dahabreh and Medh, 2012; Mak et al., 2002; Subramanian et al., 2017). Further, there are reports of RA regulation of these genes in astrocytes (Zhao et al., 2014), indirect effects of RA on apoc1 in the zebrafish embryo (Wang et al., 2015), as well as LXR regulation in both astrocytes and macrophages (Laffitte et al., 2001b; Liang et al., 2004; Mak et al., 2002). Using in silico analysis of the ∼5 kb upstream region of zebrafish apoc1, we found predicted binding sites for RAR and RXR receptors, as well as a predicted PPAR-RXR site (