Jennifer L. Anderson

Zebrafish Lipid Metabolism: From Mediating Early Patterning to the Metabolism of Dietary Fat and Cholesterol

Lipids serve essential functions in cells as signaling molecules, membrane components, and sources of energy. Defects in lipid metabolism are implicated in a number of pandemic human diseases, including diabetes, obesity, and hypercholesterolemia. Many aspects of how fatty acids and cholesterol are absorbed and processed by intestinal cells remain unclear and present a hurdle to developing approaches for disease prevention and treatment. Numerous studies have shown that the zebrafish is an excellent model for vertebrate lipid metabolism. In this chapter, we review studies that employ zebrafish to better understand lipid signaling and metabolism.

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

Visualization of Lipid Metabolism in the Zebrafish Intestine Reveals a Relationship between NPC1L1-Mediated Cholesterol Uptake and Dietary Fatty Acid

The small intestine is the primary site of dietary lipid absorption in mammals. The balance of nutrients, microorganisms, bile, and mucus that determine intestinal luminal environment cannot be recapitulated ex vivo, thus complicating studies of lipid absorption. We show that fluorescently labeled lipids can be used to visualize and study lipid absorption in live zebrafish larvae. We demonstrate that the addition of a BODIPY-fatty acid to a diet high in atherogenic lipids enables imaging of enterocyte lipid droplet dynamics in real time. We find that a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment distinguishable from lipid droplets. We also show that dietary fatty acids promote intestinal cholesterol absorption by rapid re-localization of NPC1L1 to the intestinal brush border. These data illustrate the power of the zebrafish system to address longstanding questions in vertebrate digestive physiology.

Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake

Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4–6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and serves as a springboard for future investigations to elucidate their roles in development and disease in the larval zebrafish model.

Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewings sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Pediatric sarcomas: translating molecular pathogenesis of disease to novel therapeutic possibilities

Pediatric sarcomas represent a diverse group of rare bone and soft tissue malignancies. Although the molecular mechanisms that propel the development of these cancers are not well understood, identification of tumor-specific translocations in many sarcomas has provided significant insight into their tumorigenesis. Each fusion protein resulting from these chromosomal translocations is thought to act as a driving force in the tumor, either as an aberrant transcription factor (TF), constitutively active growth factor, or ligand-independent receptor tyrosine kinase. Identification of transcriptional targets or signaling pathways modulated by these oncogenic fusions has led to the discovery of potential therapeutic targets. Some of these targets have shown considerable promise in preclinical models and are currently being tested in clinical trials. This review summarizes the molecular pathology of a subset of pediatric sarcomas with tumor-associated translocations and how increased understanding at the molecular level is being translated to novel therapeutic advances.

Long-Chain Acyl-CoA Synthetase 4A Regulates Smad Activity and Dorsoventral Patterning in the Zebrafish Embryo

Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl-CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated bone morphogenetic protein (Bmp) signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 mitogen-activated protein kinase and the Akt-mediated inhibition of glycogen synthase kinase 3, critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a-deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes.

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

Phosphoproteomic Profiling Reveals IL6-Mediated Paracrine Signaling within the Ewing Sarcoma Family of Tumors

Members of the Ewing sarcoma family of tumors (ESFT) contain tumor-associated translocations that give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass spectrometry–based phosphoproteomics to quantify global changes in phosphorylation. This unbiased approach identified hundreds of unique phosphopeptides enriched in processes such as regulation of cell cycle and cytoskeleton organization. In particular, phosphotyrosine profiling revealed a large upregulation of STAT3 phosphorylation upon EWS/FLI1 knockdown. However, single-cell analysis demonstrated that this was not a cell-autonomous effect of EWS/FLI1 deficiency, but rather a signaling effect occurring in cells in which knockdown does not occur. Conditioned media from knockdown cells were sufficient to induce STAT3 phosphorylation in control cells, verifying the presence of a soluble factor that can activate STAT3. Cytokine analysis and ligand/receptor inhibition experiments determined that this activation occurred, in part, through an IL6-dependent mechanism. Taken together, the data support a model in which EWS/FLI1 deficiency results in the secretion of soluble factors, such as IL6, which activate STAT signaling in bystander cells that maintain EWS/FLI1 expression. Furthermore, these soluble factors were shown to protect against apoptosis. Implications: EWS/FLI1 inhibition results in a novel adaptive response and suggests that targeting the IL6/STAT3 signaling pathway may increase the efficacy of ESFT therapies. Mol Cancer Res; 12(12); 1740–54. ©2014 AACR.

Evaluation of In Vitro Activity of the Class I PI3K Inhibitor Buparlisib (BKM120) in Pediatric Bone and Soft Tissue Sarcomas

Pediatric bone and soft tissue sarcomas often display increased Akt phosphorylation through up regulation of insulin-like growth factor (IGF1) signaling. Additionally, Akt signaling has been linked to resistance to IGF1 receptor (IGF1R) and mTOR (mammalian target of rapamycin) inhibitors in sarcoma, further demonstrating the role of Akt in tumor survival. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the pan-class I PI3K inhibitor buparlisib (BKM120) in pediatric bone and soft tissue sarcomas. Buparlisib inhibited activation of Akt and signaling molecules downstream of mTORC1 (mTOR complex 1) in Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines. Anti-proliferative effects were observed in both anchorage dependent and independent conditions and apoptosis was induced within 24 hours of drug treatment. Buparlisib demonstrated cytotoxicity as a single agent, but was found to be more effective when used in combination. Synergy was observed when buparlisib was combined with the IGF1R inhibitor NVP-AEW541 and the mTORC1 inhibitor rapamycin. The addition of NVP-AEW541 also further reduced phospho-Akt levels and more potently induced apoptosis compared to buparlisib treatment alone. Additionally, the combination of buparlisib with the MEK1/2 inhibitor trametinib resulted in synergy in sarcoma cell lines possessing MAPK pathway mutations. Taken together, these data indicate buparlisib could be a novel therapy for the treatment of pediatric bone and soft tissue sarcomas.