Steven A Farber

Johns Hopkins University | JHU · Department of Biology

Project BioEYES celebrated 20 years in K12 schools during the 2022-2023 school year. Using live zebrafish (Danio rerio) during week-long science experiments, sparks the interest of students and teachers from school districts, locally and globally. Over the past two decades, BioEYES has been replicated in different ways based on the interest and cap...

Zebrafish have become a powerful model of mammalian lipoprotein metabolism and lipid cell biology. Most key proteins involved in lipid metabolism, including cholesteryl ester transfer protein, are conserved in zebrafish. Consequently, zebrafish exhibit a human-like lipoprotein profile. Zebrafish with mutations in genes linked to human metabolic dis...

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but i...

Hyperlipidemia predisposes individuals to cardiometabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apolipoprotein B-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intesti...

Even though many experimental approaches benefit from tracking individual juvenile animals, there is yet to be a commercial zebrafish rack system designed to accomplish this task. Thus, we invented playpens, an acrylic, and screen container, to raise 12 individual zebrafish juveniles per standard 10 L tank on an existing recirculating fish system....

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is it...

Even though many experimental approaches benefit from tracking individual larval animals, there is yet to be a commercial zebrafish rack system designed to accomplish this task. Thus, we invented playpens, an acrylic and screen container, to raise 12 individual zebrafish juveniles per standard 10 L tank on an existing recirculating fish system. Dur...

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide an improved protocol to assay the impact of a high-cholesterol diet (HCD) on zebrafish lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as...

The intestine is responsible for efficient absorption and packaging of dietary lipids before they enter the circulatory system. This review provides a comprehensive overview of how intestinal enterocytes from diverse model organisms absorb dietary lipid and subsequently secrete the largest class of lipoproteins (chylomicrons) to meet the unique nee...

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes ( 1 )...

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with i...

Triglyceride-rich lipoproteins (TRLs) are micelle-like particles that enable efficient transport of lipids throughout the bloodstream, but also promote atherosclerotic cardiovascular disease. Despite this central relevance to cardiovascular disease, very little is known about how lipids are loaded onto nascent TRLs prior to secretion. Here we show...

Background The ability of animals and their microbiomes to adapt to starvation and then restore homeostasis after refeeding is fundamental to their continued survival and symbiosis. The intestine is the primary site of nutrient absorption and microbiome interaction, however our understanding of intestinal adaptations to starvation and refeeding rem...

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities avai...

This study examined the feasibility, acceptability, and efficacy of a video game-based digital therapeutic combining applied behavior analysis techniques and gaze-contingent eye tracking to target emotion recognition in youth with autism spectrum disorder (ASD). Children aged 4–14 years with ASD were randomized to complete Lookware™ (n = 25) or a c...

Cytoplasmic lipid droplets are highly dynamic storage organelles; their rapid synthesis, expansion, and degradation, as well as their varied interactions with other organelles allow cells to maintain lipid homeostasis. While the molecular details of lipid droplet dynamics are currently a very active area of investigation, this work has been primari...

Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. While much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is unknown. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an...

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1,200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression...

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression s...

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression s...

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology,...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Here we present a new animal model that recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a mitochon...

Apolipoprotein B (apoB) exists as apoB100 and apoB48 which are mainly found in hepatic very low density lipoproteins (VLDL) and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive...

Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular dama...

Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant ( mttp c655 ) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, tha...

BioEYES is a K-12 science outreach program that develops self-sustaining teachers as a replication strategy to address high demand for the program while promoting long-term school partnerships. This paper explores the practices of “model teachers” from multiple grades, who are empowered over a three-year period to deliver BioEYES’ hands-on science...

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression d...

Apolipoprotein-B (APOB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular dama...

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structu...

The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we p...

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 compensa...

Exons 2–10 of zebrafish coding genes are divisible by 3 (symmetrical) more frequently than one-third of the time. Length of exons 1–10 of all coding genes in Ensembl genome assembly GRCz10 were divided by 3 and remainder (0, 1, or 2) noted. For each exon, percentage of remainders 0, 1, and 2 were calculated and displayed. (EPS)

Sequence alignments of slc27a2 and creb3l3a. The shaded yellow region indicates the skipped exon (2). For slc27a2, the shaded green region indicates the ATP/AMP motif and the shaded blue region indicates the FATP/VLACS motif [47]. For creb3l3a, the shaded green region indicates the basic region and the shaded blue region indicates the leucine zippe...

Mutations analyzed in this study and their predicted outcome. (TIF)

List of primers and methods used in this study. (TIFF)

6-dpf pla2g12bsa659/sa659 larvae (bottom) display a darkened yolk sac compared to wildtype siblings (top). (EPS)

Three lines with ESS mutations do not reveal shorter amplicons expected with an exon skip. abca1asa9624 (ATP-binding cassette, sub-family A, member 1A), cd36sa30701 (cluster of differentiation cd), and pla2g12bsa659 (phospholipase A2 Group XIIB) yield PCR products (using pooled larvae from heterozygous incrosses) that match the predicted length of...

RNA expression profiling reflect a 3-fold decrease in pla2g12bsa659/sa659 larvae compared to wildtype siblings (adj. p = 3.68 x 10−8). (XLSX)

Two lines with ESS mutations have a shorter amplicon, supporting an exon skip. slc27a2asa30701 (solute carrier family 27, member 2a) and abca1bsa18382 (ATP-binding cassette transporter, sub-family A, member 1B) had a shorter amplicon (210 and 116 bp shorter, respectively) that matches the predicted length of amplicons of these cDNAs (pooled from he...

The developing zebrafish is a well-established model system for studies of energy metabolism, and is amenable to genetic, physiological, and biochemical approaches. For the first 5 days of life, nutrients are absorbed from its endogenous maternally deposited yolk. At 5 days post-fertilization, the yolk is exhausted and the larva has a functional di...

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, allow 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 compensa...

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach we found that lipids lab...

Caveolae and their structural protein caveolin 1 (CAV1) have roles in cellular lipid processing and systemic lipid metabolism. Global deletion of CAV1 in mice results in insulin resistance and increases in atherogenic plasma lipids and cholesterol, but protects from diet-induced obesity and atherosclerosis. Despite the fundamental role of the intes...

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites...

BioEYES, a nonprofit outreach program using zebrafish to excite and educate K–12 students about science and how to think and act like scientists, has been integrated into hundreds of under-resourced schools since 2002. During the week-long experiments, students raise zebrafish embryos to learn principles of development and genetics. We have analyze...

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipidrich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that l...

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, incl...

The zebrafish model facilitates the study of lipid metabolism and transport during development. Here, we outline methods to introduce traceable fluorescent or radiolabeled fatty acids into zebrafish embryos and larvae at various developmental stages. Labeled fatty acids can be injected into the large yolk cell prior to the development of digestive...

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...

It is well known that consistent excess alcohol consumption promotes the accumulation of abnormally high amounts of triglyceride in hepatocytes. However, the relative contributions of dietary and newly synthesized fatty acids to alcoholic hepatic steatosis are still under investigation, and the effect of ethanol metabolism on metabolic channeling o...

Elevated high‐density lipoprotein (HDL) cholesterol is incontrovertibly correlated with protection from cardiovascular disease in humans. The main structural component of HDL isapolipoprotein A‐I (APOA‐I). Cellular APOA‐I dynamics (e.g., transport, degradation, recycling) directly influence circulating APOA‐I and HDL levels. APOA‐Icell biology has...

Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. A key component of lipoproteins are apolipoproteins, proteins that structure these particles and regulate lipid metabolism through control of cellular lip...

Dyslipidemias are a major cause of morbidity and mortality in the world, particularly in developed nations. Investigating lipid and lipoprotein metabolism in experimentally tractable animal models is a crucial step towards understanding and treating human dyslipidemias. The zebrafish, a well-established embryological model, is emerging as a notable...

Dyslipidemias are a major cause of morbidity and mortality in the world, particularly in developed nations. Investigating lipid and lipoprotein metabolism in experimentally tractable animal models is a critical step toward understanding and treating human dyslipidemias. The zebrafish, a well-established embryological model, is emerging as a notable...

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...

Active compounds derived from primary and secondary screening assays. (0.06 MB DOC)

Challenges in imaging lipid-processing events in live, intact vertebrate models have historically led to reliance on cultured cell studies, thus hampering our understanding of lipid metabolism and gastrointestinal physiology. Fluorescently-labeled molecules, such as BODIPY-labeled lipids, can reveal lipid-processing events in live zebrafish (Danio...

Regulation of intestinal dietary fat absorption is critical to maintaining energy balance. While intestinal microbiota clearly impact the host's energy balance, their role in intestinal absorption and extraintestinal metabolism of dietary fat is less clear. Using in vivo imaging of fluorescent fatty acid (FA) analogs delivered to gnotobiotic zebraf...

sox9a in situ hybridization in wild-type and sox9b mutant larvae at 80 hpf. sox9a does not appear to be expressed in endodermal tissues in wild-type or sox9b mutant animals. Dorsal views, anterior (A) to the left. (TIF)

sox9b mutants show defects in bile secretion and transport as assessed by BODIPY-FL analog feeding. (A–B) Fluorescent micrographs of 7 dpf live wild-type (A) and sox9b mutant (B) larvae after BODIPY feeding showing lack of filling of the gallbladder (arrowhead) in sox9b mutants (B). Lateral views, anterior (A) to the left. (C–F) Confocal images of...

The intrahepatic biliary cells in sox9b mutants are more susceptible to Notch signaling inhibition. (A–D) Confocal images of livers in Tg(Tp1bglob:VenusPest); Tg(Tp1bglob:H2B-mCherry) wild-type (A, C) and sox9b mutant (B, D) larvae treated with DMSO (A–B) or 50 µM DAPT (C–D) from 106 to 154 hpf. DAPT treatment caused an increase in the relative pro...

Time course analyses of the pancreas (A–F) and liver (G–L) in wild-type and sox9b mutant larvae at 2, 3, and 4 weeks. (A–F) Tg(Tp1bglob:H2B-mCherry);TgBAC(neurod:GFP) double transgenic fish from sox9b heterozygote incrosses were raised together and 40 of them were fixed and genotyped at each time point. The pancreas, along with the gut, of 6 to 11...

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-re...

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic...

The challenge of studying complex protein networks in whole animals has driven the development of new methods for manipulating protein function with spatial and temporal precision. A novel combination of chemical and genetic protein regulation (Rodriguez and Wolfgang, in this issue of Chemistry & Biology) achieves levels of control that will revolu...

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...

Lipids are essential for cellular function as sources of fuel, critical signaling molecules and membrane components. Deficiencies in lipid processing and transport underlie many metabolic diseases. To better understand metabolic function as it relates to disease etiology, a whole animal approach is advantageous, one in which multiple organs and cel...

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migr...

Research involving model organisms necessitates recording and archiving many types of animal maintenance and use data. We developed a comprehensive inventory system using FileMaker Pro® to incorporate, record, and archive data on zebrafish stocks, tank organization, husbandry, and fish usage. Our relational database is constructed of tables contain...

Ezetimibe inhibits cholesterol metabolism in zebafish larvae: Mean percent inhibition of intestinal and gallbladder fluorescence in ezetimibe treated larvae (6.25 uM, 12.5 uM, 25 uM and 37.5 uM). N = 6 larvae for each dose. Error bars indicate standard deviation. (0.06 MB DOC)

Swallowing assay in live zebrafish larvae: (A–F) Bright field and corresponding fluorescent images of control and representative compound treated 6 dpf zebrafish larvae following ingestion of fluorescent microscphreres. (G, H) Quantification of intestinal fluorescence in compound treated and sibling control larvae. Compound 1 strongly inhibits swal...

Conserved ezetimibe binding domain in human and zebrafish NPC1L1 protein: The 62 amino acid tract of NPC1L1 shown to mediate binding of ezetimibe (amino acids 510 to 572). 41 of the amino acids in the human and zebrafish proteins are identical, 11 are conserved and 10 and non-conserved. Conserved phenylalanine (F) and methionine (M) residues requir...

Lipid reporter metabolism in ezetimibe and compound treated larvae: Values represent intestinal and gallbladder fluorescence. 4 larvae analyzed at each time point. Error bars are standard deviation from the mean of 3 independent experiments. (2.27 MB DOC)

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lip...

Many fundamental questions remain regarding the cellular and molecular mechanisms of lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in the mouse, yet each appr...

Prenylation of G protein gamma (gamma) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Ggamma subunits mutated so that they can no l...

Many fundamental questions remain regarding the cellular and molecular mechanisms of digestive lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in mice, yet each...

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...