Lxr regulates lipid metabolic and visual perception pathways during zebrafish development
Introduction
Liver X receptors (LXRs) belong to the nuclear receptor subfamily of ligand-activated transcription factors. Two LXR genes are present in mammals, LXRα (NR1H3) and LXRβ (NR1H2), encoding highly related proteins that share ∼78% amino acid sequence identity in both DNA and ligand-binding domains. Both LXR proteins form permissive heterodimers with retinoid X receptor (RXR) and control gene expression through binding to LXR response elements (LXREs) in the promoter region of target genes (Baranowski, 2008, Teboul et al., 1995). LXRs are key regulators of multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism, and they also have regulatory roles in inflammatory responses and immunity (Jakobsson et al., 2012). The endogenous LXR agonists are oxidized derivatives of cholesterol referred to as oxysterols. Synthetic ligands, such as T0901317 (T0) and GW3965 (GW), can also modulate LXR transcriptional activity (Jakobsson et al., 2012, Gabbi et al., 2009, Steffensen et al., 2013); however, T0 is also known to be a ligand for other nuclear receptors, such as farnesoid X receptor (FXR) and pregnane X receptor (PXR) in humans (Krasowski et al., 2011).
During embryonic development in rodents, LXRα is expressed in the liver, yolk sac, small intestine and other tissues involved in lipid metabolism. LXRβ is also detected in the embryonic liver and endocrine tissues, but is additionally expressed early on in several structures of the CNS, including the retina (Annicotte et al., 2004, Sakamoto et al., 2007). In adulthood, LXRα transcript remains high in tissues involved in metabolism, whereas LXRβ is ubiquitously expressed (Annicotte et al., 2004).
In addition to their role in regulation of lipid metabolism in mammals, LXRs also have functions in the CNS (Wang et al., 2002). In vivo studies have shown that LXRs promote ventral midbrain neurogenesis and dopaminergic neuron development (Sacchetti et al., 2009), and that they are implicated in the differentiation process of Bergmann glia cells, and migration of neurons during cerebellar development (Xing et al., 2010). LXRβ is also important for cerebral cortex lamination and neocortical neuron migration in mice (Fan et al., 2008). However, a potential function for LXR in the retina and other neuronal structures remains obscure.
The role of Lxr has also been studied in zebrafish. While there are two LXR genes in mammals, only one lxr gene with higher similarity to the mammalian LXRα is present in zebrafish. During zebrafish development, lxr transcripts are detected in several tissues, such as the liver, intestine, brain, neural retina and lens, and its expression pattern remains ubiquitous at the adult stage (Archer et al., 2012, Archer et al., 2008). Similarly to its role in mammalian systems, lxr also seems to play a crucial role in the control of lipid homeostasis in zebrafish. Previous studies have indicated that Lxr activation with synthetic ligands induced the expression of genes related to cholesterol transport and lipid synthesis in the liver, brain and eye of adult zebrafish (Archer et al., 2012, Archer et al., 2008, Sukardi et al., 2012). Additionally, treatment with the Lxr agonist T0 induced hepatic lipid accumulation in adult zebrafish, an effect that has also been shown in mammalian models (Sukardi et al., 2012, Schultz et al., 2000).
Studies on the role of Lxr in zebrafish during embryonic development are scarce (Archer et al., 2012, Archer et al., 2008). The expression of genes important for cholesterol transport and lipid synthesis has been shown to be affected in zebrafish embryos exposed to GW, and morpholino knockdown of Lxr resulted in impaired lipid deposition in the head and eye region of the zebrafish embryos, malformation of the pharyngeal skeleton and in elevated levels of cholesterol in the yolk of the Lxr morphant (Archer et al., 2012), suggesting an important function of Lxr in controlling lipid homeostasis in the developing zebrafish. However, more studies are needed to assess the molecular networks affected by Lxr activation in zebrafish at early developmental stages.
In this report, we investigated whether Lxr has a primary function in regulating lipid metabolic processes during zebrafish development, similarly to the role of LXR in mammals. We also set out to discover new functions of Lxr during zebrafish development. We first characterized the ability of the synthetic human LXR ligands T0 and GW to activate zebrafish Lxr by generating a stable reporter cell line expressing the zebrafish Lxr ligand binding domain. Next, we performed a whole-genome microarray analysis on zebrafish larvae treated with the synthetic ligands to evaluate organism wide transcriptional effects induced by Lxr activation during zebrafish larval development. Assessment of enriched biological processes provided insights into the molecular pathways altered by Lxr activation during zebrafish development, and tissue enrichment analysis inferred the tissues most affected by Lxr-mediated signaling in zebrafish. The expressions of genes not previously shown to be altered by Lxr activation in vertebrates were revealed in zebrafish. This study supports zebrafish as an alternative in vivo model for Lxr-related studies and demonstrates a novel role for Lxr during development.
Section snippets
Cell culture conditions and generation of stably transfected reporter cell lines expressing GAL4-zfLxr and GAL4-zfPparγ
The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with phenol red and supplemented with 5% fetal calf serum (FCS), 1% glutamine, 1% penicillin/streptomycin and the selection agents puromycin (0.5 μg/mL) and G418 (1 mg/mL) in a 5% CO2 humidifed atmosphere at 37 °C. All cell culture reagents were obtained from Gibco (Grand Island, NY).
The generation of the reporter cell line expressing the zebrafish Lxr (HG5LN-zfLxr cells) was performed in two steps. First, HG5LN cell line
The mammalian LXR agonists T0901317 (T0) and GW3965 (GW) activate zebrafish Lxr
The LXR ligands T0 and GW are potent synthetic agonists of mammalian LXRs (Schultz et al., 2000, Collins et al., 2002). We constructed a stable zebrafish Lxr (zfLxr) reporter cell line, in which the lxr ligand binding domain was fused to the GAL4 DNA binding domain, and luciferase expression was driven by upstream UAS elements. Both T0 and GW activated zfLxr in a dose-dependent manner with EC50s of 39.15 nM for T0 and 58.01 nM for GW (Fig. 1A), in the same range of EC50 values published for the
Effects of Lxr activation on cholesterol transport and lipid metabolism in zebrafish
Several studies demonstrate that LXRs have key physiological functions in the control of cholesterol transport and homeostasis in mammals (Baranowski, 2008). LXRs protect against cholesterol overload by directly upregulating the expression of the ATP-binding cassette cholesterol transporters Abca1 and Abcg1, which are involved in reverse cholesterol transport from peripheral tissues to the liver. Additionally, LXR activation stimulates hepatic cholesterol excretion by inducing the transcription
Conclusions
In conclusion, our data demonstrate a primary role of Lxr in regulating lipid metabolic processes during zebrafish development. Several LXR-mediated transcriptional effects observed in mammalian systems were conserved in zebrafish, supporting the use of zebrafish as an alternative in vivo model for Lxr-related studies involving lipid metabolism and cholesterol homeostasis. Moreover, pharmacological Lxr activation and morpholino knockdown studies indicated that Lxr controls the expression of
Acknowledgments
We thank Drs. Wanfu Wu and Yu-Bing Dai for advice on sectioning and staining. This study was funded by grants from the Environmental Protection Agency (R834289), the National Institute of Health/National Institute of Environmental Health Sciences (R21ES020036), the Swedish Research Council (H2416223), and the Robert A. Welch Foundation (E-0004). The views expressed in the article do not necessarily reflect the views of the funders.
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