US20020162124A1

US20020162124A1 - High throughput genetic screening of lipid and cholesterol processing using fluorescent compounds

Info

Publication number: US20020162124A1
Application number: US10/062,225
Authority: US
Inventors: Steven Farber; Michael Pack
Original assignee: Individual
Current assignee: Thomas Jefferson University; University of Pennsylvania Penn
Priority date: 2001-01-30
Filing date: 2002-01-30
Publication date: 2002-10-31
Also published as: US8124056B2; WO2002031202A3; US20020049986A1; US20090136428A1; US20030135869A1; US7488467B2; WO2002031202A2; AU2002213117A1

Abstract

The present invention utilizes fluorescent lipids, particularly quenched phospholipid or cholesterol analogues, to facilitate screening for phenotypes representing perturbations of lipid processing; screening for genetic mutations that lead to disorders of phospholipid and/or cholesterol metabolism; and screening of compounds designed to treat disorders of phospholipid and/or cholesterol metabolism. The present invention is a mutant fish containing a metabolic defect affecting phospholipid and cholesterol processing. The mutant fish is useful for studying phospholipid and cholesterol metabolism and for screening compounds designed to treat disorders of phospholipid and/or cholesterol metabolism.

Description

CONTINUING APPLICATION DATA

This application claims priority under 35 U.S.C. §120 based upon U.S. [0001]
Non Provisional Application No. 09/974,550 filed Oct. 10, 2001 and upon U.S. Provisional Application No. 60/264,977 filed Jan. 30, 2001.[0002]

GOVERNMENT RIGHTS TO THE INVENTION

[0003] The invention was made with government support under grant F32 NS103265 awarded by the National Institute of Health. The government has certain rights to the invention.

FIELD OF THE INVENTION

The present invention generally relates to the fields of biochemistry and pharmacology and to a mutant fish having a metabolic defect affecting phospholipid and cholesterol processing, and more particularly to the use of a mutant fish labeled with fluorescent lipids to screen for drugs and genetic alterations related to phospholipid and/or cholesterol metabolism and, more particularly, to the use of a mutant zebrafish in conjunction with tagged or quenched lipids for studying lipid metabolism in vivo,.

BACKGROUND OF THE INVENTION

Genetic analysis in zebrafish is a powerful approach for identifying genes that direct vertebrate development (1-3). Since the completion of the large-scale chemical mutagenesis screens in 1997, the phenotypic and molecular characterizations of many mutations have been reported (4-16). Analyses of mutations that affect early developmental processes, such as the specification of the embryonic axes and germ layers, have been particularly rewarding (7, 10, 17-27). Recently, related work with mutations that affect organogenesis has led to the recognition that the zebrafish is an important model system for biomedical research (28-31). Given the many aspects of organ physiology that have been conserved during vertebrate evolution, genetic screening to assay organ function in the optically transparent zebrafish is a valuable approach to understanding a variety of metabolic processes and disorders in vertebrates.

By zebrafish chemical mutagenesis screening, nine recessive lethal mutations that perturb development of the digestive organs were identified (2, 31). Although the mutants were identified using morphological criteria, their phenotypic analysis suggests that in some cases the affected genes regulate developmental processes that are relevant to digestive physiology and other aspects of vertebrate metabolism.

Through the analysis of these and other zebrafish mutants, the limitations inherent to genetic screens that are based solely on morphological criteria became apparent. First, not all organs are readily distinguished in zebrafish larvae, and mutations that perturb organ morphology are often overlooked. Second, since it is difficult to visualize specific cell populations within many larval organs, mutations that affect the development or function of these cells can be overlooked as well. Third, despite the transparency of the zebrafish larva, the function of few organs can be effectively assayed by visual inspection alone.

For these reasons, it was concluded that, in most instances, morphology-based screens are best suited for the identification of genes that regulate specification and patterning of embryonic structures. By contrast, screens designed to address biomedical concerns are most effective when they assay physiological processes directly.

Within the past few years, the discovery and analysis of zebrafish mutants affecting organogenesis has confirmed an important role for the zebrafish in biomedical research. The ability to apply high throughput genetic analyses to vertebrate organ physiology using this model system is unprecedented and will undoubtedly, over time, lead to the discovery of many genes that regulate vertebrate organ development and physiology. Such zebrafish research will complement research in other vertebrate model systems.

By conducting a mutagenesis screen using fluorescent lipids, an undertaking not feasible with standard zebrafish screening strategies, the power of high throughput genetic analysis can be applied to lipid metabolism. This has important implications for human diseases such as, but not limited to, cancer, inflammatory and cardiovascular diseases, and congenital and acquired diseases of the intestine and liver.

The fluorescent phospholipase A ₂(PLA₂) substrates described in the present invention are the first prototypes in this class of reagents. Although lipid metabolism in the digestive tract is complex and involves multiple organs the present invention discloses a method of assaying this pathway since gall bladder fluorescence represents one of the last steps in lipid processing. Because they serve as reporters of lipid processing, the fluorescently-tagged reagents of the instant invention provide a sensitive assay for a wide range of digestive developmental and physiological processes including, but not limited to, swallowing; lipid digestion, absorption, and transport; esophageal sphincter function; intestinal motility; organogenesis of the mouth and pharynx, esophagus, intestine, liver, gallbladder and biliary system, and exocrine pancreas and ducts; and the cellular and molecular biology of PLA₂regulation, polarized transport, and secretion.

Given the shared features of lipid processing in mammals and teleosts (82, 105), zebrafish mutagenesis screens using lipid reporters can be used to identify genes with functions relevant to human lipid metabolism and disease. Moreover, since both mammals and teleosts metabolize lipids in an analogous manner, the high throughput screens and fluorescent lipids disclosed in the instant invention can be employed using a variety of vertebrate model systems, including but not limited to, rodents, amphibia, and fish. The present invention involves utilizing fluorescent lipids to screen for phenotypes representing perturbations of lipid processing; to screen for mutations of specific genes that lead to disorders of phospholipid and/or cholesterol metabolism; and to screen for compounds designed to treat disorders of phospholipid and/or cholesterol metabolism, such as, but not limited to cancer, inflammatory and cardiovascular disease, and congenital and acquired diseases of the intestine and liver.

Because the precise mechanism of phospholipid and cholesterol processing is unknown at this time, there exists a need in the art for models to study such metabolism. In order to study phospholipid and cholesterol metabolism, especially in vivo, one of the best approaches is to generate genetic mutant organisms, such as fish, that have a metabolic defect affecting phospholipid and cholesterol processing. This approach allows the creation of new fish strains for physiological study as well as tissues and cell lines for in vitro examinations. The present invention is further directed to a mutant fish having a metabolic defect affecting phospholipid and/or cholesterol processing. The invention is useful for the study of phospholipid and/or cholesterol metabolism and, hence, for the development of treatments of diseases or disorders related to phospholipid and/or cholesterol metabolism. In addition, this invention relates to isolated cells derived from these fish and to the use of these cells in assays for assessing treatments of diseases or disorders related to phospholipid and/or cholesterol metabolism.

Abbreviations

“PLA ₂” means “phospholipase A₂”

“PLA ₁” means “phospholipase A₁”

“PLB” means “phospholipase B”

“PLD” means “phospholipase D”

“PED6” means “N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine”

“hpf” means “hour post-fertilization”

“dpf” means “day post-fertilization”

“FRET” means “fluorescence resonance energy transfer”

“PC” means “phosphatidylcholine”

“TLC” means “thin layer chromatography”

“cPLA ₂” means “cytoplasmic PLA₂”

“sPLA ₂” means “secretory PLA₂”

“COX” means “cyclooxygenase”

“APC” means “adenomatous polyposis coli”

“EP” means “early pressure”

“ENU” means “ethylnitrosourea”

“WT” means “wild-type”

“SLR” means “single locus rate”

“IVF” means “in vitro fertilization”

“BAC” means “bacterial artificial chromosome”

“PAC” means “P1-derived artificial chromosome”

“YAC” means “bacterial artificial chromosome”

“SSR” means “simple sequence report”

“CSGE” means “conformation sensitive gel electrophoresis”

“VLDL” means “very low density lipoprotein”

“EM” means “embryo medium”

“NBD cholesterol” means “nitrobenzoxadiazole cholesterol”

Definitions

As used herein, the term “agent” refers to any molecule, compound, or treatment that assists in the treatment of a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism.

As used herein, the term “screening” or “to screen” refers to a process in which a large number of potentially useful agents are processed in the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic of PED[0045] 6, a quenched PLA₂substrate. A. The dinitrophenol on the phospholipid headgroup effectively quenches any emission resulting from excitation at 505 nm of the BODIPY-labeled acyl chain. B. When the BODIPY-labeled acyl chain is liberated by PLA₂mediated cleavage, the quencher is separated from the fluorophore and emission (515 nm) is observed. C. NDB-labeled cholesterol in which the fluorophore replaces the terminal segment of cholesterol's alkyl tail.
FIG. 2. Intestinal differentiation in 84 hpf larvae. A. Cross-section of posterior intestine. Desmin immunoreactivity of intestinal smooth muscle (green) and enteric neurons as indicated by zn6 immunoreactivity (red) demonstrate development of the enteric neuromuscular system. B. Transmission Electromicrograph showing mature apical junctional complexes in enterocytes in the intestinal epithelium. Arrow points to desmosomes; A: Adherens junction. T: tight junction. C. Histochemical detection of enterocyte aminopeptidase activity (red); anterior intestinal cross section. D. Histological detection of goblet cell mucin (purple) in the posterior intestine. Also present at this developmental stage, immunoreactive pancreatic polypeptide in enteroendocrine cells. [0046]
FIG. 3. PED6: a fluorescent lipid reporter. A. Bright-field image of a 5 dpf larva soaked in PED6 (3 ug/ml, 2 hr). B. Corresponding fluorescent image, with intestinal (arrow) and gall bladder (arrowhead) labeling. C. Larva soaked in BODIPY-C5-PC (0.2 ug/ml). In contrast to B, unquenched fluorescent lipid labels the pharynx (arrowhead), confirming that lipid is swallowed before gall bladder labeling (arrow). [0047]
FIG. 4. Rate of fluorescence after PED6 labeling. Larvae (n=5) were placed in medium containing PED6 (0.17 mg/ml) and tricaine. Images were captured at various times and fluorescence intensity was determined in specific structures. Organ fluorescence intensity determined at specific times was normalized to the observed intensity at 45 min. Data are expressed as Mean±SEM. [0048]
FIG. 5. Lipid processing. A. and B. Atorvastatin (ATR) inhibits processing of PED6 (A) but not of BODIPY-FL-C5 (Molecular Probes). B. Larvae were bathed in fluorophore (0.6 uM) in the presence or absence of atorvastatin (Lipitor tablet suspension containing 1 mg/ml) (arrowhead, gall bladder). C. Mouse digestive organs. D. Gall bladder fluorescence after processing (t=30 min) of PED6 (1 ug), administered by gavage. Symbols: gb, ball bladder; d, common bile duct; lv, liver. Scale bars, 1.0 mm (C and D), 200 um (other images). [0049]
FIG. 6. BODIPY FR-PC reveals both the substrate and PLA[0050] ₂cleavage product. Upon integration into cells, excitation at 505 nm results in an emission at 568 nm (orange) due to fluorescence resonance energy transfer (FRET). After cleavage by PLA₂emission is observed only at 515 nm (green).
FIG. 7. Fluorescence emission spectrum of mixed micelles of 0.05 mol % BODIPY FR-PC in mixed-lipid vesicles. The fluorescence emission spectrum of BODIPY FR-PC (0.5 uM in ethanol; excitation, 505 nm) showed peaks at 514 nm and 568 nm with a ratio of 1.0 indicating FRET. An excitation scan (emission, 568 nm) showed peaks at 507 and 560 nm with a ratio (507/560) of 1.2. Solid line: before addition of PLA[0051] ₂. Dashed line: after addition of N. naja PLA₂. Vesicles were prepared by sonication of the dried lipids (from chloroform-methanol solution); 1.1 μM BODIPY FR-PC (0.05 mol %), 0.1 mM dimyristoylphosphatidycholine (46 mol %), 0.12 mM ditetradecylphospha-tidylmethanol (54 mol %), buffer (50 mM Tris, pH 8, 100 mM NaCl, 1 mM CaCl₂); excitation, 505 nm.
FIG. 8. BODIPY FR-PC is effective in paramecium. Paramecium incubated (1 hr) in BODIPY FR-PC (2.5 ug/ml) results in labeling of lipid droplets (orange). Digestion of the lipid results in green fluorescence. Excitation 505 nm, emission LP520. [0052]
FIG. 9. BODIPY FR-PC localizes to the intestinal epithelium. Fish were incubated (1-4 hr) in BODIPY FR-PC (2.5 ug/ml). BODIPY FR-PC metabolites are observed in gall bladder (green). Excitation 505 nm, emission LP520. A. Brightfield image. B. and C. Visualization of antigen presenting enterocytes in segment II of the larval zebrafish intestine after only 1 hr of labeling. Arrows mark segment II domain. D. 4 hr labeling with BODIPY FR-PC results in FRET effect throughout intestine. [0053]
FIG. 10. Lipid processing in intestinal mutants. A and C. Bright field images of 5 dpf mlt and pie larvae. B and D. Fluorescent images corresponding to A and C. Normal lipid processing of PED6 (0.3 ug/ml, 2 hr) in mlt larva (arrowhead marks gallbladder). D and E. Abnormal lipid processing in PED6 labeled pie and slj larvae; fluorescence is present in the intestinal lumen (white arrowhead) and reduced in the gallbladder (red arrowhead). [0054]
FIG. 11. Phospholipid processing and transport. A. Following uptake, PED6 is cleaved by PLA[0055] ₂liberating a labeled fatty acid. The fate and transport of this fatty acid remains unknown. B. TLC of fluorescent lipid standards. PED6 (black because it is quenched), D3803 (C5-BODIPY PC), and C5-fatty acid are easily resolved using a two solvent system silica gel plates (Whatman, LK5D). Solvent 1 (toluene, ether, ethanol, acetic acid; 25/15/2/0.2); Solvent 2 (chloroform, methanol, acetic acid, water; 25/15/4/2).
FIG. 12. Red BODIPY-PC given by gavage labels the gall bladder. Adult fish was anesthetized in tricaine, injected with 80 ug of D3806 (a BODIPY PC 582/593 nm), allowed to recover for 1 hr and dissected on ice. [0056]
FIG. 13. Larval zebrafish (5 dpf) labeled with NDB cholesterol—derivative labels gall bladder within 30 minutes of ingestion (3 ug/ml, solubilized with fish bile). [0057]
FIG. 14. fat free larvae lack gall bladder fluorescence. A. Bright-field images of wild-type (WT) and mutant 5 dpf larvae. B. Corresponding fluorescent image of the gall bladder after PED6 labeling (0.3 ug/ml) (arrowheads). C. Biliary secretion of phenol red in fat free larvae. Mutant larvae identified by PED6 labeling were injected with phenol red (arrowhead, gall bladder). D. Reduced lipid processing in fat free mutants. TLC of the pooled lipid fraction from eight larvae is shown after labeling with BODIPY C5-PC (12 hours, 0.16 ug/ml); red arrowhead denotes BODIPY-cholesteryl ester, black arrowhead indicates BODIPY C5-PC ([0058] lane 1, WT; lane 2, fat free; lane 3, endogenous fluorescent lipids present in unlabeled WT larvae). TLC: solvent 1 (chloroform:methanol:water 35/7/0.7); solvent 2 (ethyl ether:bensene:ethanol:acetic acid; 40/50/2/0.2). E. fat free larvae fail to accumulate 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol) (2 hrs, 3 ug/ml, solubilized with fish bile). Scale bars, 200 μm.

DETAILED DESCRIPTION

The present invention relates to a mutagenesis screen to identify genes that regulate lipid metabolism using fluorescently-tagged or quenched lipids such as cholesterol or lipids that are substrates for phospholipases such as PLA[0059] ₂and to a fish having a metabolic defect affecting phospholipid and cholesterol processing, which is identified using a mutagenesis screen. For example, cleavage of quenched phospholipid substrates by PLA₂results in an increase in fluorescence intensity or alters the spectral properties of fluorescent emission thus allowing lipid metabolism to be followed in vivo. In one embodiment of the instant invention, optically transparent zebrafish larvae exposed to the fluorescently-tagged or quenched lipids display intense gallbladder fluorescence, which reflects lipid cleavage by intestinal PLA₂and subsequent transport of the fluorescent cleavage products through the hepatobiliary system.
The instant invention presents evidence demonstrating that in the context of a mutagenesis screen, fluorescent PLA[0060] ₂substrates and fluorescently-tagged cholesterol or other lipids have the potential to identify genes that affect many aspects of lipid metabolism. Consequently, when used in the context of a genetic screen these reagents provide a high throughput readout of digestive physiology that cannot be assessed using standard screening strategies. Given current understanding of the pathway of lipid processing in zebrafish, specifically its shared features with lipid processing in mammals, the present invention has relevance for biomedical research related to, among other things, cancer, inflammatory and cardiovascular diseases, and congenital and acquired diseases of the intestine and liver.
Fluorescent Reagents to Assess Organ Physiology in vivo [0061]
The fluorescent lipids of the instant invention allow assaying of physiological processes. The reagents are fluorescent analogues of compounds that could serve as modifiable substrates in important metabolic and signaling pathways. The reagents of the instant invention were constructed by covalently linking fluorescent moieties to sites adjoining the cleavage site of phospholipids. Dye-dye or dye-quencher interactions modify fluorescence without impeding enzyme-substrate interaction (106). PLA[0062] ₂cleavage results in immediate unquenching and detectable fluorescence. Quenched phospholipids [N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-palmitoyl-2-BODIPY-FL-pentanoyl-sn-glycero-3-phosphoethanolamine (PED6)] allow subcellular visualization of PLA₂activity and reveal organ-specific activity (36). The evidence presented in the instant invention demonstrates that zebrafish larvae 5 days post-fertilization (dpf) bathed in PED6 show intense gall bladder fluorescence and, shortly thereafter, intestinal luminal fluorescence. Substrate modification allows localization of enzymatic activity by altering the emission spectrum of the fluorescent compounds. When used in the context of a genetic screen these fluorescent lipids provide a high throughput readout of organ function.
The reagents of the instant invention facilitated the development of genetic screens that are more sensitive than the whole-mount in situ and antibody based screening protocols now used to assay gene expression. The fluorescent reagents are simpler to use since they can be administered to and assayed in a wide range of organisms, including, but not limited to rodents and teleosts, and they offer the opportunity to screen for hypomorphic mutations that alter gene function but do not affect levels of gene expression. By providing a visual assay of metabolic processes, these reagents can be used to identify mutations that affect more than just the single gene responsible for substrate modification. Visualization of the fluorescent signal also is dependent upon the delivery and uptake of the substrate as well as the storage, metabolism or secretion of its modified metabolites. [0063]
Fluorescent PLA[0064] ₂Substrates
PLA[0065] ₂s were chosen as the “target” enzymes to assay because of the important role these enzymes play in the generation of lipid signaling molecules (32-34). PLA₂s are a large family of enzymes that can be categorized according to their cellular distribution, molecular weight, and calcium dependence (33). Some PLA₂s also exhibit a preference for phospholipids with arachadonyl sn2 acyl side chains (34, 37, 38). Given the wide range of processes the PLA₂family of enzymes is known or thought to regulate, the advantage of the fluorescent lipids as screening reagents is significant.
cPLA[0066] ₂. For example, one PLA₂gene family member, cytoplasmic PLA₂(cPLA₂), regulates eicosanoid production. Eicosanoids are ubiquitous signaling molecules generated by the biochemical modification of arachidonic acid, the principal fatty acid liberated from membrane phospholipids by cPLA₂s (42-44). cPLA₂s have a strong preference for arachidonyl phospholipids and cPLA₂cleavage of these phospholipids is known to be the rate-limiting step in eicosanoid synthesis (39, 41, 44-47). In comparison, secretory PLA₂s (sPLA₂s) exhibit little substrate preference and unlike cPLA₂, the arachidonic acid generated through sPLA₂activity is not directly coupled to eicosanoid production (37, 46).
The two major classes of eicosanoids produced by vertebrate cells are leukotrienes and prostaglandins (48, 54). Prostaglandins are a large family of signaling molecules that are synthesized through the action of cyclooxygenases (COX) and prostaglandin-isomerases on PLA[0067] ₂generated arachidonic acid (48). Prostaglandins are especially important in vertebrate physiology as they regulate a myriad of physiological processes, including hemostasis, cell proliferation, fertility, and inflammation (48-50). The importance of prostaglandin in humans is underscored by the widespread use of COX inhibitors (e.g., aspirin) as medicinal agents.
Given the focus of this invention, there is special interest in intestinal prostaglandins. Intestinal prostaglandins play an important role in regulating mucosal blood flow (51-54), and inhibition of prostaglandin synthesis by COX inhibitors, such as aspirin, is associated with the development of mucosal ulcerations (55). There is also strong evidence that intestinal prostaglandins regulate epithelial cell proliferation (56-58). COX inhibitors are known to have a chemopreventitive effect on several human digestive cancers, and COX-2 has been identified as a genetic modifier of APC, an important colon cancer gene. [0068]
Because arachidonic acid release is the rate-limiting step in eicosanoid production, characterization of the cell specific regulation of cPLA[0069] ₂s is important (33, 34). Although the genes in some of the signaling pathways that activate cPLA₂have been identified, this characterization is far from complete. A screen utilizing fluorescent lipid substrates will allow identification of mutations that perturb lipid processing and alter cPLA₂activity.
sPLA[0070] ₂. Another PLA₂gene family member, sPLA₂, plays a role in inflammation, host defenses, digestion, and cell proliferation (43, 70, 71). In comparison to cPLA₂s, sPLA₂s show little preference for arachidonic acid over other phospholipids sn2 acyl side chains (34).
There is a large body of experimental and clinical evidence supporting the role of sPLA[0071] ₂s in inflammation. First, serum levels of sPLA₂are increased in inflammatory conditions such as Rheumatoid Arthritis, Crohn's Disease, endotoxic shock, and atherosclerosis (32, 72-76). Second, sPLA₂cleavage of arachidonyl phospholipids has been shown to augment cPLA₂mediated eicosanoid production (46, 71, 77). Third, independent of its enzymatic activity, sPLA₂has been shown to bind to a specific cellular receptor that plays a role in endotoxic shock (78) and other aspects of the inflammatory response (43, 79, 80). Like cPLA₂, the regulation of inflammatory sPLA₂s (Groups II and V) is an important area of biomedical research.
Group IIA sPLA[0072] ₂recently has been shown to play a role in colon cancer tumorigenesis through its genetic interaction with APC in the min mouse, a colon cancer animal model (66-68). High levels of this gene are expressed in intestinal Paneth cells where it is also believed to function as an antimicrobial agent because of its ability to digest bacterial cell wall phospholipids (38, 76, 81). The precise mechanism of sPLA₂'s mitogenic effect is unclear. Since COX activity also has been shown to modify colon tumorigenesis in the min mouse, sPLA₂may be acting via prostaglandins (69). This hypothesis is counter-intuitive, however, because in the min mouse, loss of sPLA₂function is associated with a tumor promoting effect. An alternate hypothesis suggests that in the colon, sPLA₂also may function as a cell-signaling molecule, independent of its enzymatic activity, through its interaction with the sPLA₂receptor.
The role of sPLA[0073] ₂s in the digestion and absorption of dietary phospholipids has been studied in many vertebrate species, including teleosts (82). Pancreatic sPLA₂is the prototypical low molecular weight sPLA₂—its proenzyme is secreted into the intestinal lumen where it is activated and cleaves dietary phospholipids that have been modified by bile salts (83). PLA₂activity also is present in the intestinal brush border and undoubtedly contributes to the digestion of dietary phospholipids (84, 85). Intestinal PLA₂activity is present in the form of PLB, an enzyme that can function as a high molecular weight, calcium independent PLA₂as well as a lysophospholipase (86-89). Regulation of lipid absorption and transport is an important area of biomedical research that has implications for metabolic, inflammatory, and cardiovascular diseases.
Phospholipid Metabolism [0074]
In fish, like mammals, dietary phospholipid is cleaved by intestinal and pancreatic PLA[0075] ₂s to form free fatty acid, lysolipid, and phosphoglycerol (82). These molecules are absorbed by enterocytes, presumably by simple diffusion (lysolipid, phosphoglycerol) and receptor mediated processes (fatty acid) (90). Within the enterocyte, the free fatty acids are processed according to their size: long carbon chain fatty acids are re-esterified to form triglycerides and phospholipids, packaged into lipoprotein particles (chylomicrons or VLDL) and enter the general circulation via the lymphatics; shorter chain fatty acids presumably can enter the circulation directly via the portal vein (90-92). Lipoprotein bound phospholipids enter cells in the periphery by binding to specific receptors or via endocytosis (90, 91).
In comparison to the fatty acids, the fate of the phospholipid derived lysolipid and phosphoglycerol cleavage products are less certain. In mammals, they are largely re-esterified to form phospholipids, which then are incorporated into lipoproteins and absorbed via the lymphatics, like triglycerides (90-92). Vertebrate lipid absorption is further complicated by the existence of other intestinal phospholipases (PLD, PLA[0076] ₁) that cleave the polar head group or snl fatty acid from lysolipids and phospholipids. This raises the possibility that phospholipids undergo extensive re-arrangement within the intestinal epithelium prior to absorption. In fish, there is experimental evidence supporting this observation (82).
Materials, Methods, and Results [0077]
Zebrafish [0078]
Zebrafish, Danio reno, were housed in a separate facility consisting of approximately 500 tanks of varying sizes (1 liter, 3.75 liter, and 9 liter). Environmental conditions were carefully monitored for disease prevention and to maintain fish in perpetual breeding condition. Male and female fish were reared at a density of no more than 8 fish per liter at a constant temperature and light cycle (27-29° C. with the light/dark cycle kept at 14/10 hours) in pretreated water (heated, charcoal-filtered and UV-sterilized). Fish were fed twice daily with a variety of dried and live foods. [0079]
Zebrafish provide a relatively simple model system for more complex vertebrates, such as humans. They are small in size, easy to maintain and breed, and produce large numbers of progeny on a daily basis. Their embryos develop rapidly and are optically clear, permitting direct observation of the developing digestive system. Being vertebrates, zebrafish contain orthologues for almost all human genes. The species also is amenable to genetic methods so that one can screen for mutations that disrupt organ function or development. It is possible, therefore, to identify genes important for intestinal development and function by examining fish that carry random mutations. In addition, many techniques have been worked out for manipulating zebrafish, including in vitro fertilization, production of haploids and parthenogenic diploid embryos, mutagenesis, cell lineage and cell transplantation. [0080]
Generation of fluorescent PLA substrates to assay lipid processing [0081]
In the instant invention, a family of fluorescent phospholipids was generated with such phospholipids serving as substrates for PLA[0082] ₂. As previously noted, characterization of the genetic regulation of PLA₂is an active area of biomedical research (67, 68), and the reagents described were developed to function as in vivo biosensors of PLA₂activity that can be assayed using microscopy or simple biochemical techniques. When administered to zebrafish embryos and larvae, these reagents provide a rapid readout of a wide range of developmental and physiological processes that are amenable to high throughput genetic analyses. The fluorescent lipids described in this proposal are quenched fluorescent phosphatidylcholine analogues and NBD-labeled cholesterol (FIG. 1). The phospholipid reagents differ in both their fluorescent emission and their specificity for different PLA₂isoforms (93, 94). Cleavage of these phospholipids by PLA₂generates a fluorescent fatty acid or lysolipid that can be used to localize and quantify PLA₂activity in live fish (36, 94). It has been demonstrated that there is utility of these agents for revealing localized PLA₂activity in developing zebrafish embryos (94).
To determine whether these reagents could be used to study organ specific PLA[0083] ₂activity, the quenched fluorescent lipids were administered to zebrafish larvae at 5 dpf, a developmental stage when the major larval organ systems function (FIG. 2). 5 dpf larvae soaked in PED6 uniformly developed intense gallbladder fluorescence 15-20 minutes after ingesting the lipid (FIG. 3). Based upon established mechanisms of vertebrate lipid processing, it was theorized that the larval gallbladder fluorescence reflected ingestion of the lipid reagents followed by intestinal PLA₂cleavage, intestinal absorption, transport to the liver and biliary excretion of the fluorescent cleavage product.
This hypothesis was tested with three experiments. First, to establish that PED6 is swallowed by the larvae, an unquenched fluorescent lipid was administered to 5 dpf larvae, and the appearance of labeled lipid in the pharynx and intestinal lumen before the gallbladder was noted (FIGS. 3C & 4). Second, that gallbladder fluorescence reflects hepatobiliary transport of the fluorescent cleavage products was shown by demonstrating the absence of PLA[0084] ₂activity in dissected adult gallbladders with and without bile (4785±626 when full of bile vs. 7421±2043 when empty, arbitrary fluorescence units ±SEM, n=3). Dissected adult gall bladders were lysed in embryo medium (EM) (30 ul) containing BODIPY-FL-C₅-PC (0.1 ug) to release bile. PLA₂activity was determined as described (1). Measurements were compared with activity of bile-depleted gallbladders. Third, this finding was confirmed by demonstrating the early appearance of fluorescent cleavage products in the liver of larvae exposed to PED6 compared with the gallbladder (FIG. 4). Larvae were labeled with PED6 (0.3 ug/ml) in EM, anesthetized (tricaine, 170 ug/ml), and placed in depression slides. Fluorescent images were captured over 1 hr using a Zeiss Axiocam 2 mounted on a Leica MZFL-III. Because PLA₂activity was not detected in bile and fluorescent PED6 metabolites underwent rapid hepatobiliary transport, labeling the liver before the gall bladder, PED6 must be cleaved within the intestine.
Moreover, PED6 labeling of the gall bladder was completely blocked by atorvastatin (Lipitor, Parke-Davis) (FIG. 5A), a potent inhibitor of cholesterol synthesis in humans (108). Because the addition of exogenous bile reversed this effect [Bile was obtained from freshly killed tilapia ([0085] Orechromis mossambicus) and extracted with three volumes of methanol:chloroform (1:2). The aqueous fraction was recovered, reduced to one volume under nitrogen, and added to EM (20 ul/ml)], and atorvastatin failed to inhibit processing of a water-soluble short-chain fatty acid (BODIPY-FL-C5, Molecular Probes Inc.) (105) (FIG. 5B), the data demonstrate that atorvastatin blocks the synthesis of cholesterol-derived biliary emulsifiers needed for dietary lipid absorption (109). These results, coupled with the rapid appearance of biliary fluorescence in mice fed PED6 (FIG. 5D), demonstrate that in zebrafish larvae, lipids are processed in a similar, if not identical, manner as other vertebrates and that gallbladder fluorescence reflects intestinal absorption and hepatic excretion of the lipid reagents and its metabolites.
BODIPY FR-PC, a reporter of both substrate and cleavage product [0086]
To further demonstrate that PED6 and other fluorescent lipids are processed by intestinal lipases prior to transport to the liver and gallbladder (as compared to being absorbed from the intestinal lumen, transported to the liver, and first cleaved by hepatic PLA[0087] ₂activity prior to biliary excretion) a new fluorescent lipid was developed (FIG. 6). BODIPY FR-PC (FIG. 6) is a substrate that can be used to determine the site of PLA₂activity more precisely because it emits distinct fluorescent profiles before and after cleavage. This phospholipid contains two dyes that interact via fluorescence resonance energy transfer (FRET). Substrate and cleavage product possess unique spectral signatures, allowing their tissue distribution to be distinguished by fluorescence microscopy. Excitation (505 nm) of micelle-incorporated BODIPY FR-PC produces an orange emission (568 nm) (FIG. 7) from the sn2 fluorophore via the FRET effect (18). Cleavage of the substrate purified by PLA₂, however, releases the sn2 acyl chain and abolishes FRET (FIG. 7). Excitation of the resultant lysolipid produces a green emission (515 nm). The fluorescence emission spectrum of BODIPY FR-PC was determined using mixed micelles of 0.05 mol % BODIPY FR-PC in dimyristoyl phosphatidylcholine (46 mol %) and ditetradecylphosphatidylmethanol (54 mol %) prepared in buffer (50 mM Tris, pH 8, 100 mM NaCl, 1 mM CaCl₂) by sonication of the dried lipids.
Having established that BODIPY FR-PC behaves as anticipated in in vitro assays, its behavior in vivo was analyzed by exposing paramecia to the lipid for 1 hr, followed by excitation at 505 nm. As expected, FRET emission (orange) was observed from lipid droplets within individual paramecium soon after exposure, followed by the gradual appearance of BODIPY emission at 515 nm (green fluorescence) indicative of substrate cleavage (FIG. 8). [0088]
To further demonstrate the behavior of BODIPY FR-PC in vivo, 5 dpf zebrafish larvae were bathed in BODIPY FR-PC. Within 1 hr, larvae exposed to BODIPY FR-PC showed a bright green fluorescent gallbladder and orange fluorescence within the apical cytoplasm of posterior intestinal cells (FIG. 9C). These cells are known to absorb luminal macromolecules through pinocytosis (107). Orange fluorescence indicative of uncleaved substrate also was observed after 4 hr of incubation in the anterior intestine epithelium, the site of lipid absorption in fish (82, 95). FRET emission was never detected in the liver even after prolonged incubation (FIG. 9D), implying that gallbladder fluorescence following ingestion of labeled lipids is due to cleavage by intestinal PLA[0089] ₂s.
The evidence of the instant invention is consistent with the predicted pathway of lipid processing observed in mammals since uncleaved substrate (orange) was seen only in the intestinal epithelium. Over time, continuous exposure to BODIPY FR-PC either overwhelms intestinal lipase activity or leads to the integration of BODIPY FR-PC into the cellular phospholipid pool. Regardless, the data of the present invention provide further evidence that in zebrafish, as in mammals, lipids are absorbed and modified in the intestine prior to transport to the liver. [0090]
Lipid processing in zebrafish intestinal mutants [0091]
To determine the utility of the fluorescent lipids as reagents in a genetic screen, PED6 was administered to mutants known to perturb intestinal morphology (31), and the pattern and timing of gallbladder fluorescence was examined (FIG. 10). These mutations cause embryonic lethality and each of the affected genes regulate intestinal development in a region-specific manner. The mutations slim jim (slj) and piebald (pie) each cause degeneration of anterior intestinal epithelium and exocrine pancreas, whereas meltdown (mlt) results in cystic expansion of the posterior intestine. As shown in FIGS. 10B and 10C, mlt mutants retain PED6 processing in the anterior intestine and show normal levels of gall bladder fluorescence. In contrast, pie (FIGS. 10D & 10E) and slj (FIG. 10F) mutants display greatly reduced gall bladder labeling. Although slj and pie have similar histological phenotypes, gall bladder fluorescence was absent in pie larvae but visible in slj larvae, albeit at a reduced level compared with wild-type or mlt larvae (FIGS. 10B & 10C). Hence, fluorescent lipids can be used to identify mutants with abnormal digestive organ morphology. [0092]
Screening for mutations that perturb lipid processing using fluorescent [0093]
[0094] PLA 2 substrates
Lipid reporters also are effective tools for identifying mutations that perturb lipid metabolism without causing obvious morphological defects. In a pilot screen, larval progeny of individual F2 families derived from an ENU mutagenesis protocol were bathed in PED6 and screened for digestive organ morphology and phospholipid processing. PED6 fluorescent metabolites dramatically enhanced digestive organ structure, facilitating scoring of gallbladder development, intestinal folding, and bile duct morphology. From 190 genomes screened, two mutations were identified and confirmed in the subsequent generation. These mutations were recovered based on their pattern of gallbladder fluorescence, not morphological criteria, thereby supporting the use of the fluorescent lipids of the instant invention in large-scale mutagenesis screens. [0095]
Lipid processing was examined further in the identified mutants using a fluorescent cholesterol analog (FIG. 1). [0096]

EXAMPLE 1

Characterize the Biochemical Pathways Responsible for the Metabolism of the Fluorescent PLA[0097] ₂Substrates in Zebrafish
To characterize, biochemically, the identity of fluorescent compounds present in the bile of zebrafish following exposure to fluorescent PLA[0098] ₂substrates and other lipid molecules, zebrafish were exposed to a panel of fluorescent phospholipids and free fatty acids that differed in acyl chain length and fluorophore position. The experiments enabled the determination of the phenotypic significance of recovered mutants by identifying shared pathways of lipid metabolism in zebrafish and mammals.
Identity of the Fluorescent Lipid in the Bile of Fish Labeled with PED6 [0099]
To determine whether the fluorescence in the bile is due to a reacylated phospholipid (lacks the DNP quencher on the head group) or exists as a free BODIPY-labeled fatty acid, adult fish were labeled with PED6 by gavage. Adult fish were used because of the small amount of lipid in larvae bile. Under a fluorescence stereomicroscope, the gall bladders were easily identified by their intense fluorescence. The gall bladders were removed at various times post-gavage and placed in cold methanol, sonicated and extracted (97). [0100]
The organic fraction was then subjected to TLC analysis as described to determine whether the fluorescent sn2 acyl chain was re-esterified prior to biliary excretion (FIG. 11A & 11B) (36, 98). This procedure was performed on five fish using D3806 (a red BODIPY PC, 582/593) (FIG. 12). [0101]
Identity of the Fluorescent Lipids in the Bile of Fish Labeled with BODIPY- C5, C12, C16 Fatty Acids [0102]
To ascertain whether short chain fatty acids are transported via the portal system, adult zebrafish are injected with C16, C12 and C5 BODIPY-labeled fatty acids and then the fluorescent bile characterized biochemically using TLC. Based on work in other vertebrates, only short-chain fluorescent fatty acids can pass directly into the bile, consistent with transport via the portal vein, following injection of C16 BODIPY fatty acid (82, 99). The profile and kinetics of labeling are very different with the shorter chain analogues. [0103]
Labeling of 5 dpf Larvae with BODIPY Labeled Fatty Acids and Phospholipids [0104]
To extend the findings from the adult bile assays to younger fish, the rate of gallbladder fluorescence in zebrafish larvae exposegd to BODIPY-labeled fatty acids and phospholipids with different chain lengths is compared. Larvae are soaked in the identical BODIPY fatty acids injected into the adults, and the rate of gall bladder fluorescence is assayed. The labeling rate is determined by placing a fish in water containing the fluorescent substrate and capturing a digital image of a lateral view (Axiovision, Zeiss and ImageQuant, Molecular Dynamics Inc.). The gall bladder fluorescence is quantified at various time intervals to compute the rate of change over time. Delayed appearance of gallbladder fluorescence after exposure to long chain fatty acids and phospholipids lends further support to the hypothesis that acyl chain length plays a role in lipid transport in zebrafish. [0105]
PLA[0106] ₂Isoform Mediation of PED6 Processing
It is well established that cPLA[0107] ₂and some iPLA₂isoforms exhibit acyl chain specificity and favor phospholipids that contain arachidonic acid, the precursor of eicosanoids (41, 44). Despite the fact that the fluorescent phospholipids do not contain arachidonic acid, the placement of the BODIPY moiety on the sn2 position results in significant cleavage by cPLA₂(36, 94). The ability of the BODIPY moiety to disrupt the membrane and its hydrophobicity are both properties similar to arachidonic acid that enable cleavage by cPLA₂(100). Secreted PLA₂isoforms exhibit no acyl chain specificity (34). Fluorescent phospholipids that contain a saturated acyl chain on the sn2 position are fine sPLA₂substrates but poor cPLA₂ones.
To identify which PLA[0108] ₂isoforms mediate the appearance of fluorescence in the gall bladder, the rate of gall bladder fluorescence is compared in larvae exposed to two types of substrates. Larvae are labeled with two phospholipid substrates that differ in their specificity for cPLA₂: a PL with an sn1 BODIPY and an sn2 saturated acyl chain—a poor PLA₂substrate, or BODIPY FR-PC—a good cPLA₂substrate. If little difference is observed in the rates of gall bladder fluorescence using the two substrates, then the critical lipase important for the processing of these lipids is a secreted PLA₂isoform, a promiscuous enzyme that exhibits little acyl chain preference (96). BODIPY FR-PC is used because no other available fluorescent phospholipid contains sn2 arachidonic acid.
Taken together, the experiments clarify the relevant PLA[0109] ₂isoforms, the route(s) of phospholipid/fatty acid transport, and allow refinement of the screening reagents (e.g. by preparing cocktails of fluorophores to target specific transport processes).

EXAMPLE 2

A Mutagenesis Screen to Identify Genes that Regulate Lipid Processing in Zebrafish [0110]
In the instant invention a large-scale genetic screen for genes that regulate lipid processing in zebrafish using fluorescent PLA[0111] ₂substrates is disclosed. This screen leads to the recovery of mutations that regulate a wide range of developmental and physiological processes. As outlined, the screen identifies mutations that either resemble or are allelic to previously described intestinal mutations. Given the large number of steps required for processing of the fluorescent PLA₂substrates, however, this screen leads to the recovery of genes that could not be identified using standard screening strategies. These include, but are not limited to, genes that directly regulate PLA₂; genes responsible for the development of the liver, biliary system, and the intestinal vasculature and lymphatics; and genes that play a direct role in lipid metabolism and transport.
The mutagenesis and screening strategies of the instant invention incorporate several well established methodologies. First, adult male fish are mutagenized with ENU using established dosing schedules. There exist a number of established dosing schedules (e.g., 110, 111). Second, the F1 progeny of the mutagenized GO fish will be bred to homozygosity using a classical 3 generation protocol (F3 screen), as well as parthenogenetically, using the early-pressure (EP) technique (103). Progeny refers to any and all future generations derived or descending from a particular organism, whether the organism is heterozygous or homozygous for the mutation. Progeny of any successive generation are included herein, such that the progeny, the F1, F2, F3 generations, and so on indefinitely are included in this definition. Zebrafish larvae generated in this fashion are soaked in the BODIPY FR-PC, PED6, and /or NBD cholesterol (FIG. 13) lipid reagents at 5 dpf and then screened for defects in lipid processing based upon the timing and pattern of fluorescence in the intestine, liver and gallbladder. Parallel F3 and EP screens are employed because they offer the best opportunity to maximize the use of resources. Although EP screens are in many ways less efficient than classical F3 screens, they are suited to moderate sized fish facilities and require less manpower because of the reduced number of matings required. [0112]
ENU Mutagenesis [0113]
For the EP screen, adult male fish from the ‘*AB’ zebrafish line (this line of fish carries the fewest number of haploinsufficient or lethal mutations of common lab strains) are exposed to ENU (3.5 mM) in a secluded fume hood for 30 minutes on 3 consecutive days. After taking the necessary precautions to remove adherent ENU, the fish are returned to the main fish facility and outcrossed to WT fish from the Wik [0114] genetic background 3 weeks after mutagenesis, and the F1 progeny are raised to sexual maturity. To improve the likelihood that the *AB mutagenized males do not carry significant mutations in their genetic background, mutagenized males are outcrossed only if 5 of their female siblings produce large clutches of viable EP embryos that survive to 5 dpf. The efficacy of ENU mutagenesis is determined prior to outcrossing by calculating the specific locus rate for new mutations at the alb, spa, and bra loci.
One week after ENU exposure, the mutagenized GO males are mated to double-mutant “tester” female carriers of the alb, spa, and bra mutations and their 32 hpf progeny are analyzed for clones of alb/alb, spa/spa, and bra/bra mutant pigmented cells. Based upon published SLR's for these loci using our ENU dosing schedule there is a need to analyze approximately 1500-2000 progeny from each mutagenized GO fish to accurately determine the efficacy of mutagenesis. [0115]
Screening for Mutations that Perturb lipid Processing [0116]
For screening, 5 dpf larvae derived from pair-wise matings of F2 families or EP treatment of F1 females are soaked fluorescent lipids for 1-10 hrs and analyzed for perturbation of lipid processing using a fluorescent stereo-microscope (Leica MZ FLIII). The BODIPY FR-PC and PED6 reagents used in the screen are purified via TLC, resuspended in ethanol/DMSO and tested in vivo using WT 5 dpf larvae prior to screening. The F3 5 dpf larvae for screening are derived from pair-wise matings of fish from F2 families, while 5 dpf larvae produced parthenogenetically are produced using standard EP protocols: briefly, eggs are collected from anesthetized F1 females (tricaine) and exposed to UV irradiated sperm following standard in vitro fertilization (IVF) protocols. Immediately after IVF (1.4 min.), the fertilized eggs are exposed to 13,000 psi in a French Press for 4-6 min, then slowly returned to ambient atmospheric pressure and allowed to develop until 5 dpf (103). [0117]
Putative mutations that alter the pattern or rate of accumulation of lipid fluorescence in the digestive organs, as well as those that produce specific alterations in larval morphology, are recovered and outcrossed for future analysis. Mutations that result in a generalized delay in larval development are not analyzed given their high frequency of recovery in prior chemical mutagenesis screens. For the F3 screen, no less than 20, 5 dpf larvae are analyzed from ≧6 crosses per F2 family. Using this strategy, excluding lethal effects of haploinsufficiency in heavily mutagenized genomes, the theoretical chance of recovering a mutation present in the genome of an F2 family is ≧89%. For the EP screen, all of the viable progeny derived from each F1 female are analyzed. In contrast to the F3 screen, the statistical likelihood of recovering mutations in the genome of F1 females treated with EP is not predictable given the tendency to recover smaller viable clutches with this technique, and the potential for meiotic recombination to alter Mendelian inheritance patterns. Only those heritable defects that can be recovered in statistically significant Mendelian ratios when outcrossed, are considered mutations. [0118]
The data and analysis of known intestinal mutants suggest that lipid processing mutations are not rare mutations. A total of approximately 750-2000 mutanized genomes are screened. [0119]

Example 3

Determination of the Physiological Significance and Molecular Nature of Recovered Mutations [0120]
The instant invention discloses methods for characterizing, both phenotypically and molecularly, the generated mutations. Phenotypic analysis is approached first. Mutations that affect a wide range of developmental and physiological processes are recovered in the screen. Careful phenotypic analysis of these mutants is important for several reasons. First, it allows quantitation of the range and frequency of phenotypes actually recovered using the fluorescent lipid reagents. Second, it allows the performance of comprehensive complementation analyses so that hypomorphic alleles of interesting mutations are not overlooked. Third, it allow the selection of those mutations worthy of molecular analysis now versus mutations that may be of more immediate interest to other members of the zebrafish scientific community. The rationale for the molecular analysis of zebrafish mutations is well known to those of skill in the art. Recognition of the importance of phenotypic analyses of mutant phenotypes prior to molecular analyses is relevant given the large amount of work that is often required to identify the responsible gene. [0121]
Phenotypic Analyses [0122]
Mutant phenotypes recovered using the screen of the instant invention can be categorized into several broad categories. First, using morphological and histological criteria mutations that visibly perturb structural development of the pharynx, esophagus, intestine, liver and biliary tract are distinguished from those mutants that appear normal. The latter group is considered physiological mutants and is categorized based upon its handling of the panel of fluorescent lipids of the instant invention. This group encompasses, but is not limited to, mutations affecting the intestinal epithelium, liver, vasculature and lymphatics, enteric neuromusculature, and the tissue specific regulation of PLA[0123] ₂.
Embryological and transient expression assays also are important studies that can aid phenotypic analyses of zebrafish mutants. Unfortunately, mutations affecting development of the zebrafish digestive organs are, in general, less easily analyzed using these techniques than mutations affecting early development. The short half-life of injected RNA transcripts and DNA expression constructs coupled with the mosaic distribution of the micro-injected DNA limits the utility of transient expression assays for mutations that are not recognizable until 4-5 dpf. [0124]
In one embodiment of the instant invention, histological analyses are performed by fixing larvae in 4% paraformaldehyde, embedding the fixed larvae in glycolmethacrylate, and followed by sectioning. Sections are stained using toluene blue/azure II as described and analyzed using a Zeiss Axioplan compound microscope. When needed, selected immunocytochemical and molecular markers are employed to further categorize organ specific defects. If necessary ultrastructural studies are performed as well. For ‘physiological’ mutations, affected larvae are sequentially soaked in the fluorescent lipids of the instant invention, thereby allowing a more detailed categorization. [0125]
Molecular Analyses [0126]
The molecular characterization of zebrafish mutations can be performed using techniques widely known to those of skill in the art. These techniques include, but are not limited to, use of an ever expanding array of physical and genetic markers, several large insert genomic DNA libraries, outstanding bioinformatics, and a successful EST program. [0127]
Recent announcement of plans to physically map and sequence the zebrafish genome suggest that within 2-3 years molecular characterization of zebrafish mutants will be greatly simplified. One of the major advantages of working in zebrafish compared with other vertebrates is the potential to generate high-resolution maps of mutant loci. Since mutations can be confidently mapped to within 0.1 cM, the critical interval surrounding a mutant locus can be narrowed to include relatively few candidate genes. The recent identification of the genes responsible for several zebrafish mutants demonstrates this nicely. [0128]
In one embodiment of the instant invention, the molecular characterization of identified mutations is accomplished by introducing mutations into a polymorphic genetic background and assigning a chromosomal location using either bulk segregant analyses and polymorphic markers from the 25 zebrafish linkage groups or using half tetrads and polymorphic centromeric markers. Concomitantly, DNA from large numbers of mutant progeny from the ‘map cross’ are extracted and stored. Thereafter, the genetic map of the region surrounding the locus is refined using standard PCR based techniques and genetic markers from existing maps. [0129]
The closest known genetic markers flanking the mutant locus are then used to screen large insert genomic libraries to identify more closely linked markers. For BAC and PAC clones this involves direct sequencing, whereas for YAC clones this requires rescue and sequencing of the zebrafish genomic DNA adjacent to the YAC arms. Ultimately, flanking markers within 0.1 cM of the mutant locus are identified, and a BAC or PAC clone spanning the locus is analyzed for the responsible gene. The latter is accomplished by direct sequencing, using the genomic insert to probe an appropriately staged genomic library and/or exon trapping. Confirmation of mutation is accomplished via expression analyses and mutant rescue, using transient analyses, if possible, or via transgenesis. Identification of tightly linked flanking markers generally involves a BAC, PAC, or YAC chromosomal walk, using markers mapped genetically and physically (radiation hybrid panel). [0130]
In one embodiment of the instant invention, genetic mapping is performed using PCR based techniques. SSR polymorphisms are resolved using denaturing (SSR) polyacrylamide gel electrophoresis while single base-pair polymorphisms are resolved using Conformation Sensitive Gel Electrophoresis (CSGE), a non-denaturing technique related to SSCP. DNA detection is accomplished using a Molecular Dynamics Fluorescent Scanner; PCRs use Cy-5 end-labeled primers. PCR fragments generated using non-fluorescent primers are visualized on polyacrylamide gels after staining with the fluorescent intercalating dye Syto-61 (Molecular Probes). For bulk segregant analyses, separate pools of DNA from 25 mutant larvae and 25 sibling WT larvae are generated and the segregation of SSR or single base pair polymorphic markers derived from ESTs analyzed. [0131]
For physical mapping, the LN 540 (104) and Goodfellow Radiation Hybrid panels are employed. Markers are mapped using a PCR based strategy and computer assisted analysis of amplification patterns. Screening of genomic libraries, rescue of YAC ends, generation of YAC, BAC, and PAC clones is accomplished using the commercial suppliers or published protocols, which are known to those of skill in the art. All other molecular techniques outlined above employ published protocols, which also are known to those of ordinary skill in the art. [0132]

EXAMPLE 4

Fat Free Mutation [0133]
The instant invention discloses methods for screening for mutations that perturb lipid processing and characterizing those mutations, both phenotypically and molecularly. In one embodiment of the instant invention, a classic two-generation breeding scheme (110, 111) was employed in the screen. Adult male zebrafish were mutagenized with 3 mM ENU by placing them into an aqueous solution for three (3) 1 hr periods within [0134] 1 week. At >4 weeks after the ENU treatment, the G0 males were bred to homozygosity using a classical 3 generation protocol (F3 screen) (110, 111). Employing this approach, the segregation of recessive mutations was easily tested, and the F2 parents of a cross producing mutant F3 embryos were identified as heterozygous carriers for the mutation.
Larval progeny of individual F2 families were bathed in PED6 and screened for digestive and organ morphology and phospholipid processing. Of the 190 genomes screened, two mutations were identified and confirmed in the subsequent generation. The recessive lethal mutation canola produced a phenotype closely resembling that of slj and pie, with degeneration of the intestinal epithelium and reduced gall bladder and intestinal fluorescence. The fat free recessive lethal mutation causes a more pronounce reduction in fluorescence (FIG. 14B). However, in contrast to canola and other intestinal mutants, the fat free digestive tract has a normal appearance at 5 dpf (FIG. 14A). Therefore, fat free would have escaped detection in a screen based solely on morphological criteria. [0135]
The affect of the fat free mutation on larval digestive physiology was determined. In order to ascertain whether swallowing was normal in fat free mutants, larvae 6 dpf were placed in EM containing fluorescent latex microspheres (0.0025% Fluoresbrite plain YG 2.0 μm, Polysciences Inc.) for 1 hr, washed, and imaged over 1 hour using a [0136] Zeiss Axiocam 2 mounted on a Leica MZFL-III. Numbers of beads were 10±2 in the WT versus 14±3 beads in mutant larvae (mean±SEM, n=9, P>0.3). Next, to determine whether the fat free hepatobiliary system was functional, fat free larvae were injected (˜5 nl) with phenol red (2% in EM) dorsal to the anterior intestine and labeling of the gall bladder was monitored (FIG. 14C). TLC of metabolites produced by fat free larvae labeled with an unquenched BODIPY phosphatidylcholine analog (36) revealed that PLA₂processing was intact. Extracts prepared from mutants, however, contained significantly reduced levels of fluorescent cleavage products (FIG. 14D).
Lipid processing was examined further using a fluorescent cholesterol analog (112) whose absorption is dependent on biliary emulsification. Cholesterol was absorbed poorly by fat free larvae (FIG. 14E). Uptake of BODIPY FL-C5, a more soluble short-chain fatty acid, was reduced to a lesser extent than uptake of cholesterol, thus suggesting that fat free directly regulates bile synthesis or secretion rather than intestinal lipid absorption or transport. The fat free mutation provides in vivo evidence supporting the hypothesis, derived from mammalian cell culture experiments, that phospholipid turnover and cholesterol metabolism are coupled. [0137]
The fat free fish will be useful in assessing phospholipid and/or cholesterol processing and will aid in identifying treatments for diseases and disorders associated with phospholipid and/or cholesterol metabolism. [0138]
Although mutant zebrafish represent a preferred embodiment of the invention, other mutant organisms, including without limitation mutant amphibia, mutant teleosts, and mutant mammals (for example, rodents, pigs, cattle, and sheep) can be constructed by standard techniques are are included within the scope of this invention. [0139]

EXAMPLE 5

Drug Screening Assays [0140]
The invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by (or associated with) aberrant or abnormal lipid and/or cholesterol metabolism. These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent for the ability to modulate (e.g., stimulate or inhibit) lipid and/or cholesterol metabolism. Candidate/test compounds or agents that have one or more of these abilities can be used as drugs to treat disorders characterized by aberrant or abnormal lipid and/or cholesterol metabolism. Candidate/test compounds or agents include, for example, (1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see e.g., Lam, K. S. et al. ([0141] 1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; (2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see. e.g., Songyang, Z. et al. (1993) Cell 72:767-778); (3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and (4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).
In one embodiment, the invention provides a method for identifying a compound capable of use in the treatment of a disorder characterized by (or associated with) aberrant or abnormal lipid and/or cholesterol metabolism. This method typically includes the step of assaying the ability of the compound or agent to modulate lipid and/or cholesterol metabolism, thereby identifying a compound for treating a disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism. Disorders characterized by aberrant or abnormal lipid and/or cholesterol metabolism are described herein. The invention provides screening assays to identify candidate/test compounds or agents that modulate lipid and/or cholesterol metabolism. Typically, the assays include the steps of identifying at least one phenotypic perturbation of lipid and/or cholesterol metabolism in an organism, administering at least one quenched or fluorescently-tagged phospholipid, cholesterol or other lipid analogue to the organism having the phenotypic perturbation, administering a candidate/test compound or agent to the organism under conditions that allow for the uptake of the candidate/test compound or agent by the organism and wherein but for the presence of the candidate/test compound or agent the pattern of fluorescence would be unchanged, and detecting a change in the pattern of fluorescence by comparing the pattern of fluorescence prior to candidate/test compound or agent administration with that seen following administration of the candidate/test compound or agent. [0142]
In another embodiment, a screening procedure for identifying therapeutic compounds that affect phospholipid and/or cholesterol metabolism in vivo is disclosed. In general, the method involves screening any number of compounds for therapeutically active agents by employing a mutant organism, for example but not limited to fish having the fat free mutation, described herein. Based upon the above results, it will be readily understood that a compound that affects phospholipid and/or cholesterol metabolism in an organism (e.g., the mutant fish described herein) can provide an effective therapeutic agent in a mammal (e.g., a human patient). Since the screening procedures of the invention are performed in vivo, it can be ascertained whether candidate compounds will be highly toxic to a mammalian host organism (e.g., a human patient). In addition, the invention also makes available high throughput in vitro screening methods for screening of large quantities of candidate compounds using the cells and cell lines of the invention. [0143]
Accordingly, the methods, materials, and organisms of the instant invention simplify the evaluation, identification, and development of active agents, such as drugs, for the treatment of a variety of diseases associated with phospholipid and/or cholesterol metabolism. In general, the screening methods of the invention provide a facile means for selecting any number of compounds of interest from a large population that are further evaluated and condensed to a few active and selective materials. Constituents of this pool can then be evaluated in the methods of the invention to determine their activity. [0144]
Administration of the candidate compound to an organism of the invention can be by any known route, and at a range of concentrations. Following an appropriate period of time, the animal can be assessed for the effect of the compound compared to control animals. [0145]
Cells from the mutant organisms of the invention are also useful as a source of cells for cell culture. [0146]
Mutagenesis screens using fluorescent lipids exploit the advantages of combining genetic analyses with imagining of enzymatic function. The existence of related lipid processing mechanisms in mammals and teleosts, and the finding that therapeutic drugs used to modify lipid metabolism in humans are active in zebrafish, establish that genetic screens can be designed to probe the mechanistic basis of acquired and heritable human disorders. The evidence of the instant invention demonstrates that lipid metabolism in mammals and fish can be monitored with fluorescent lipids, and that such organisms metabolize ingested fluorescent lipids in an analogous manner. Consequently, the methods for using the fluorescent lipids of the instant invention to study lipid metabolism, identify diseases of lipid metabolism, and/or to identify agents to treat therapeutically or prophylatically diseases or disorders of lipid metabolism and genetic screening are applicable to all vertebrate model organisms, including, but not limited to, rodents, amphibia, and fish. Fluorescent reporters are predicted to identify genes, including but not limited to, genes involved in diseases of lipid metabolism, such as atherosclerosis; in disorders of biliary secretion, such as biliary atresia; and in cancer, a disease in which lipid signaling plays an important role. Identification of these genes has important implications for cancer research and research related to diseases of the liver, intestine and cardiovascular system. [0147]
While this invention has been described with a reference to specific embodiments, it will be obvious to those of ordinary skill in the art that variations in these methods and compositions may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims. [0148]

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Claims

We claim:

1. A mutant fish whose genome comprises a homozygous disruption in a metabolic pathway of cholesterol and/or lipid processing, wherein said disruption reduces cholesterol absorption in a gall bladder of said mutant fish and wherein said mutant fish are phenotypically normal.

2. A mutant fish of claim 1, wherein the homozygous disruption results in a reduction in cholesterol absorption in a gall bladder of said mutant fish as compared to wild-type fish.

3. A method of identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism, comprising:

(a) administering a fluorescent label to said mutant fish of claim 1 by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish;

(b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;

(c) administering said agent(s) to said mutant fish under conditions that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);

(d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and

(e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said mutant fish.

4. A method of identifying an agent(s) to reduce lipid and/or cholesterol metabolism, comprising:

5. A method of identifying an agent(s) to enhance lipid and/or cholesterol metabolism, comprising:

6. A method of identifying an agent(s) to reduce cholesterol absorption, comprising

(a) administering a fluorescent label to said mutant fish of claim 1 by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish such that fluorescence is observed in a gall bladder of said mutant fish;

(b) selecting at least one agent to reduce cholesterol absorption in said mutant fish;

(c) administering said agent(s) to said mutant fish under condition that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);

(d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to reduce cholesterol absorption; and

(e) identifying an agent(s) to reduce cholesterol absorption by a reduction in the pattern and/or rate of fluorescence in a gall bladder of said mutant fish.

7. A method of identifying candidate functional mediators of the homozygous disruption in the mutant fish of claim 1, comprising

(a) producing an F₁generation fish by crossing a first mutant fish whose genome comprises a heterozygous disruption in the metabolic pathway of cholesterol and/or lipid processing, wherein said disruption when homozygous reduces cholesterol absorption in a gall bladder of a fish having said homozygous disruption but produces no phenotypic change in a fish having said homozygous disruption, with a second mutant fish whose genome comprises at least one random mutation;

(b) selecting from the offspring of the cross of step (a) those fish whose genome comprises the heterozygous disruption of said first mutant fish;

(c) producing an F₂generation fish by intercrossing the offspring selected in step (b);

(d) selecting from the offspring of the cross of step (c) those fish whose genome comprises the heterozygous disruption of the mutant fish of claim 1;

(e) producing an F₃generation fish by intercrossing the offspring selected in step (d);

(f) administering a fluorescent label to the selected F₃offspring of step (d) by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said selected F₃offspring under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said selected F₃offspring;

(g) comparing the pattern and/or rate of fluorescence in said selected F₃offspring to the pattern and/or rate of fluorescence observed in a wild-type fish and/or mutant fish of claim 1, which wild-type fish and/or mutant fish of claim 1 was administered a fluorescent label by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said wild-type fish and/or mutant fish under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said wild-type fish and/or mutant fish of claim 1; and

(h) identifying candidate functional mediators of the homozygous disruption in the mutant fish of claim 1 by a change in the pattern and/or rate of fluorescence in said selected F₃offspring as compared to the pattern and/or rate of fluorescence in wild-type fish and/or mutant fish of claim 1.

8. A cell line derived from the mutant fish of claim 1.

9. The cell line of claim 8, wherein the cell line is derived from a liver of the mutant fish of claim 1.

10. The cell of claim 8, wherein the cell line is derived from a gall bladder of the mutant fish of claim 1.

11. A method of identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism, comprising:

(a) fluorescently labeling a cell isolated from at least one of the cell lines of claim 8, 9, or 10 by contacting said cell with at least one fluorescent lipid under conditions that allow for uptake of said fluorescent lipid by said cell;

(c) contacting said agent(s) with said cell under conditions that allow for uptake of said agent(s) by said cell prior to, after, or simultaneously with step (a);

(d) analyzing a change in the pattern and/or rate of fluorescence in said cell as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and

(e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said cell.

12. A method of identifying an agent(s) to reduce lipid and/or cholesterol metabolism, comprising:

13. A method of identifying an agent(s) to enhance lipid and/or cholesterol metabolism, comprising:

14. A method of identifying candidate functional mediators of the homozygous disruption in the mutant fish of claim 1, comprising

(a) providing a first fluorescent label with a cell isolated from at least one of the cell lines of claim 8, 9, or 10 by contacting at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol with said cell under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol to said cell;

(b) analyzing the pattern and/or rate of fluorescence of said first fluorescent label in said cell;

(c) randomly re-mutagenizing the DNA of said cell;

(d) providing a second fluorescent label to said cell by contacting at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol, wherein said second fluorescent label has a fluorescence emission profile that contrasts with the fluorescence emission profile of said first fluorescent label, with said cell under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol;

(e) comparing the pattern and/or rate of fluorescence of said second fluorescent label in said cell to the pattern and/or rate of fluorescence observed in said cell in step (b); and

(f) identifying candidate functional mediators of the homozygous disruption in the mutant fish of claim 1 by a change in the pattern and/or rate of fluorescence in said cell.