👤 Tori Hensley

Forward genetic screening is a powerful approach to assign functions to genes and can be used to elucidate the many genes whose functions remain unknown. A key step in forward genetic screening is mapping: identification of the gene causing the phenotype. Existing mapping methods use a bioinformatic mapping-by-sequencing approach based on allelic frequency calculations that often identify large genomic regions which contain an intractable number of candidate genes for testing. Here, we describe WheresWalker, a modern mapping-by-sequencing algorithm that identifies a mutation-containing interval and then supports positional cloning to shrink the interval, which drastically reduces the number of potential candidates, allowing for extremely rapid mutation identification. We validated this method using mutants from a forward genetic mutagenesis screen in zebrafish for modifiers of ApoB-lipoprotein metabolism. WheresWalker correctly mapped and identified novel zebrafish mutations in mttp, apobb.1, and mia2 genes, as well as a previously published mutation in maize. Further, we used WheresWalker to identify a previously unappreciated ApoB-lipoprotein metabolism-modifying locus, slc3a2a. Show less

Oxidative stress perturbs lipid homeostasis and contributes to metabolic diseases. Though ignored when compared with mitochondrial oxidation, the endoplasmic reticulum (ER) generates reactive oxygen species requiring antioxidant quality control. Using multi-organismal profiling featuring Drosophila, zebrafish, and mammalian hepatocytes, here we characterize the paraoxonase-like C20orf3/adipocyte plasma-membrane-associated protein (APMAP) as an ER-localized antioxidant that suppresses ER lipid oxidation to safeguard ER function. APMAP-depleted cells exhibit defective ER morphology, ER stress, and lipid peroxidation dependent on ER-oxidoreductase 1α (ERO1A), as well as sensitivity to ferroptosis and defects in ApoB-lipoprotein homeostasis. Similarly, organismal APMAP depletion in Drosophila and zebrafish perturbs ApoB-lipoprotein homeostasis. Strikingly, APMAP loss is rescued with chemical antioxidant N-acetyl-cysteine (NAC). Lipidomics identifies that APMAP loss elevates phospholipid peroxidation and boosts ceramides-signatures of lipid stress. Collectively, we propose that APMAP is an ER-localized antioxidant that promotes lipid and lipoprotein homeostasis in the ER network. Show less

Lipoproteins are essential for lipid transport in all bilaterians. A single Apolipoprotein B (ApoB) molecule is the inseparable structural scaffold of each ApoB-containing lipoprotein (B-lps), which are responsible for transporting lipids to peripheral tissues. The cellular mechanisms that regulate ApoB and B-lp production, secretion, transport, and degradation remain to be fully defined. In humans, elevated levels of vascular B-lps play a causative role in cardiovascular disease. Previously, we have detailed that human B-lp biology is remarkably conserved in the zebrafish using an Show less

Cardiomyopathy (CM) is associated with atrial remodeling and atrial fibrillation (AF), often complicating rhythm management. Ventricular dysfunction contributes to AF through pressure and volume overload, while AF worsens ventricular function via tachycardia and irregular activation. Evidence suggests catheter ablation improves outcomes in CM patients, though success is influenced by the extent of atrial and ventricular remodeling. Patients undergoing their first catheter ablation for AF were divided into hypertrophic (HCM), ischemic (ICM), non-ischemic (NICM), and no-CM groups. Pre-ablation late-gadolinium enhancement cardiac magnetic imaging (LGE-MRI) was used to assess left atrial (LA) fibrosis burden and anatomical distribution. Patients were followed prospectively for arrhythmia recurrence. A total of 552 patients, 39 HCM (69% obstructive), 39 ICM, 115 with NICM, and 359 without CM were included between January 2015 and December 2022. LA fibrosis was significantly higher in patients with CM (19.1 ± 7.5% vs. 16.5 ± 6.9%; P = 0.01). HCM and ICM had the greatest LA fibrosis among the different CM subtypes (21.3 ± 8.7% and 21.9 ± 9.1%, respectively). There was no significant difference in the regional distribution of fibrosis among the various groups. AF recurrence was observed in 321 (58.2%) after 456 (175-1204) days. Multivariate analysis revealed that compared to no CM, HCM was associated with a three-fold increase in AF recurrence (HR = 3.07, 95% CI 2.06-4.58, P < 0.001), followed by ICM (HR 1.61, 95%, CI 0.95-2.72; P = 0.07) and NICM (HR of 1.53, 95% CI 1.14-2.06; P = 0.05). LA fibrosis and volume index were independently associated with recurrence (HR = 1.03, 95% CI 1.01-1.06, P = 0.01 and HR = 1.02, 95% CI 1.01-1.03, P = 0.01). Genetic testing revealed key distinctions between HCM and NICM, with MYBPC3 and MYH7 as prominent genes in HCM and a heterogeneous genetic basis in NICM. Hypertrophic cardiomyopathy is associated with the highest risk of AF recurrence followed by ischemic and non-ischemic cardiomyopathy after catheter ablation. LA fibrosis regional patterns did not differ between cardiomyopathy types, while overall fibrosis and volume predicted recurrence. Show less

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). The export of lipids from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Although triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile, and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways. Show less

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. Unlike murine models, to which various standardized high lipid diets such as a high-cholesterol diet (HCD) are available, there has yet to be a uniformly adopted zebrafish HCD protocol. In this study, we have developed an improved HCD protocol and thoroughly tested its impact on zebrafish lipid deposition and lipoprotein regulation in a dose- and time-dependent manner. The diet stability, reproducibility, and fish palatability were also validated. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LPs) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae for 8 days produced hepatic steatosis that became more stable and sever after 1 day of fasting and was associated with an opaque liver phenotype (dark under transmitted light). Unlike larvae, adult fish fed HCD for 14 days followed by a 3-day fast did not develop a stable fatty liver phenotype, though the fish had higher ApoB-LP levels in plasma and an upregulated lipogenesis gene fasn in adipose tissue. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease. Show less

Liver hepatocellular carcinoma (LIHC) is the major form of liver cancer that is the fourth most common cause of cancer death worldwide. It has been reported that the multifunctional protein p62 (also known as SQSTM1) plays a cancer-promoting role in LIHC, but the detailed mechanisms underlying p62 interaction with LIHC remains unclear. To gain a comprehensive understanding of p62 interaction with LIHC in clinical settings, we performed bioinformatic analyses using various online algorithms derived from high throughput profiling. Our results indicate that p62 expression is significantly upregulated, partially due to its promoter demethylation, rather than p62 gene mutation, in LIHC. Mutation of TP53, CTNNB1, or ALB significantly correlates with, and mutation of AXIN1 reversely correlates with, the p62 expression level. Its upregulation occurs as early as liver cirrhosis, and go through all stages of the carcinogenesis. HCV infection makes a significant contribution to p62 upregulation in LIHC. We further identified p62-associated molecular signatures in LIHC, including many genes that are involved in antioxidant stress and metabolism, such as SRX1 and TXNRD1. Regarding to the clinical outcome, p62 expression level reversely correlates with the survival of LIHC patients (p<0.01). Importantly, we experimentally validated that p62 depletion in liver cancer cell lines downregulates the expression of SRX1 and TXNRD1 at both transcriptional and translational levels, and reduces cell proliferation. As the potential mechanisms underlying the tumor-promoting role of p62, we show that p62 upregulation is remarkably associated with reprogramming of pathways mediated by p53, Wnt/β-catenin, and Keap1-NRF2, which are crucial for oncogenesis in many contexts. Our findings provide a comprehensive insight into the interaction between p62 and LIHC, offering valuable information for understanding of LIHC pathogenesis. Show less

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell. Show less