Deficiencies in C20 Polyunsaturated Fatty Acids Cause Behavioral and Developmental Defects in Caenorhabditis elegans fat-3 Mutants
a Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-63401p, http://www.100md.com
ABSTRACT1p, http://www.100md.com
Arachidonic acid and other long-chain polyunsaturated fatty acids (PUFAs) are important structural components of membranes and are implicated in diverse signaling pathways. The 6 desaturation of linoleic and linolenic acids is the rate-limiting step in the synthesis of these molecules. C. elegans fat-3 mutants lack 6 desaturase activity and fail to produce C20 PUFAs. We examined these mutants and found that development and behavior were affected as a consequence of C20 PUFA deficiency. While fat-3 mutants are viable, they grow slowly, display considerably less spontaneous movement, have an altered body shape, and produce fewer progeny than do wild type. In addition, the timing of an ultradian rhythm, the defecation cycle, is lengthened compared to wild type. Since all these defects can be ameliorated by supplementing the nematode diet with gamma-linolenic acid or C20 PUFAs of either the n6 or the n3 series, we can establish a causal link between fatty acid deficiency and phenotype. Similar epidermal tissue defects and slow growth are hallmarks of human fatty acid deficiency.
THE 6 fatty acid desaturase catalyzes the rate-limiting step in the conversion of the essential fatty acids, linoleic acid (18:2n6) and linolenic acid (18:3n3), into C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3). (Fatty acid nomenclature used here is the following: X:YnZ refers to a fatty acid chain of X carbon atoms and Y methylene-interrupted cis double bonds; Z indicates the position of the terminal double bond relative to the methyl end of the molecule.) PUFAs play critical roles in regulating membrane structure, dynamics, and permeability. In mammals, C20 PUFAs are substrates for oxygenases that produce powerful short-range eicosanoid effector molecules, including prostaglandins, leukotrienes, and thromboxanes (FUNK 2001 ). In response to mechanical, cytokine, or growth factor stimuli, phospholipase A2 cleaves PUFAs from cell membranes so they can be acted on by cyclooxygenase, lipoxygenase, and P450 monooxygenase enzymes. The eicosanoids mediate a variety of processes in many cell types, including pain, inflammation, and reproductive processes. In addition to these roles, liberated free fatty acid forms of C20 PUFAs, most notably 20:4n6, display considerable biological activity, including activation of nuclear hormone receptors, modulation of ion channels, and as second messengers in signal transduction (BRASH 2001 ). Studies examining the roles of specific fatty acids in these processes in animals are hampered by the difficulty of manipulating lipid composition in vivo.
Caenorhabditis elegans is an attractive animal model in which to investigate the physiological roles of specific fatty acids in growth, development, and the nervous system. Unlike mammals, C. elegans does not require essential fatty acids in its diet, but is capable of synthesizing 20:4n6 and 20:5n3 using only saturated and monounsaturated fatty acids from bacteria as precursors (HUTZELL and KRUSBERG 1982 ). This is possible because C. elegans expresses the full range of desaturase activities found in plants (12 and n3 desaturase) and animals (5 and 6 desaturase) as well as PUFA elongase activities found in animals (NAPIER and MICHAELSON 2001 ).@;, http://www.100md.com
To investigate the roles of various fatty acids in growth, development, and neurological function in an animal system, we recently isolated C. elegans mutants deficient in PUFA synthesis by direct analysis of fatty acid composition (WATTS and BROWSE 2002 ). These mutants revealed that C. elegans does not require n3 or 5 unsaturated PUFAs for normal development under laboratory conditions. The n3 and 5 desaturase mutants are deficient in certain classes of C20 PUFAs but accumulate higher levels of precursor C20 PUFAs as a consequence of these deficiencies. In contrast, the fat-3 mutants that lack 6 desaturase activity fail to produce any of the common C20 PUFAs and, as a result, their growth and behavior are compromised. Here we demonstrate that although the fat-3 mutants are viable and fertile, they exhibit neuromuscular defects, cuticle abnormalities, reduced brood size, and altered biological rhythms. These defects can be biochemically complemented by dietary supplementation of various 20-carbon PUFAs.
MATERIALS AND METHODS:, 百拇医药
Culture and measurement of nematodes::, 百拇医药
Nematodes were cultured and maintained according to standard methods (WOOD 1988 ). The strains were grown at 20° on nematode growth medium (NGM) plates unless otherwise indicated. The wild-type strain was N2 and the fat-3 strain used in these studies was fat-3(wa22). Body-length measurement of late L4 animals was obtained essentially as described in REINER et al. 1999 . For each genotype or fatty acid treatment, 10 animals were measured using an Alvin map wheel. Brood size and hatch rate was measured by placing 8–10 L4 animals onto individual plates. Worms were transferred to a new plate each day for 4 days, and the number of eggs and larvae on each were counted. Percentage viability was obtained by dividing the number of hatched L1 larvae by the total number of eggs laid for each worm. Cuticle disintegration was scored by directly observing gravid adult nematodes placed in 0.5 ml alkaline hypochlorite solution (1% sodium hypochlorite, 0.25 M NaOH) in 24-well culture plates and noting the time of the first major break in the cuticle. Plates were agitated gently every 30 sec during observation. Errors are SEM.
Behavioral assays:81, 百拇医药
The defecation cycles of first-day adult animals were scored by measuring the time from one posterior body contraction to the next. The presence or lack of an enteric muscle contraction at the end of each cycle was noted as well. For each strain a minimum of six animals were scored for 10 cycles each. Unless otherwise noted, defecation cycles and enteric muscle contractions were scored with the petri dishes closed. Movement assays were performed as described (MILLER et al. 1996 ) in M9 buffer. One "thrash" was defined as a change in direction of bending at the midbody. At least 10 animals for each genotype or fatty acid treatment were measured. Pharyngeal pumping was scored by direct observation of at least 10 animals for each genotype for 1 min. All behavioral assays were performed at room temperature (22°–23°).81, 百拇医药
Construction of the fat-3::GFP reporter gene:81, 百拇医药
The full-length translational fusion was constructed by fusion PCR of the amplified fat-3 promoter and coding sequences together with the green fluorescent protein (GFP) coding sequence amplified from pPD95.75 (HOBERT et al. 1999 ). The upstream regulatory region included 1086 bases upstream of the fat-3 ATG. This region included 97 bases of the last exon of the upstream fat-4 gene. The constructs were microinjected into wild-type or fat-3(wa22) worms together with the rol-6(su1006) dominant marker plasmid pRF4 (MELLO et al. 1991 ). Rolling transgenic worms were isolated and multiple independent lines that produced heritable rolling progeny were examined for GFP expression and analyzed for fatty acid composition.
Fatty acid supplementation and analysis:a$-&'[, http://www.100md.com
Fatty acid sodium salts were obtained from NuChek Prep (Elysian, MN) and stored at -20° in the dark. For each experiment, a fresh 0.1 M stock was prepared by dissolving fatty acids in sterile H2O. NGM agar was prepared with the addition of 0.1% tergitol (NP-40). Agar was cooled to 45°–50° and fatty acid stock was added slowly and stirred for 1 min. Plates were poured immediately and then covered to dry in the dark for 24 hr. Plates were then seeded with Escherichia coli and allowed to dry for 2 days in the dark at room temperature before the addition of embryos. Embryos were prepared by alkaline hypochlorite treatment of adult nematodes to obtain a semisynchronized population of early embryos. After phenotypic analysis of adult worms, nematodes were washed off the plates in H2O and centrifuged gently to pellet the worms. As much water as possible was removed and the worm pellets were frozen for determination of fatty acid composition as described in WATTS and BROWSE (2002).
RESULTS AND DISCUSSION?4[x\, http://www.100md.com
Fatty acid composition of 6 desaturase mutants and growth phenotypes:?4[x\, http://www.100md.com
The6 desaturase mutants were isolated without selection using gas chromatography analysis of fatty acids derived from mutagenized nematodes (WATTS and BROWSE 2002 ). The fatty acid composition of three independent fat-3 lines revealed elevated levels of the 6 desaturase precursors 18:2n6 and 18:3n3 and a deficiency in C20 fatty acids, most notably undetectable levels of dihomogamma-linolenic acid (20:3n6), arachidonic acid (20:4n6), and eicosapentaenoic acid (20:5n3). We found that the phenotypes described in this work were indistinguishable among the three fat-3 alleles (wa22, wa23, and wa25). Worms of all three genotypes display identical fatty acid compositions with undetectable 6 unsaturated PUFAs, implying that all three alleles represent loss of activity of the 6 desaturase. Detailed phenotypic characterization was carried out with fat-3(wa22).?4[x\, http://www.100md.com
The fat-3 homozygous worms are viable and fertile, indicating that C20 PUFAs are not essential for life in this organism. However, they grow at a slower rate than wild type, requiring one extra day of development at 20° before they become fertile adults (WATTS and BROWSE 2002 ). In this study, we examined brood size and hatch rate of fat-3 worms and embryos at three growth temperatures. We found that within a range of temperatures between 15° and 25° the fat-3 worms consistently produced smaller broods than wild type did, with the largest difference at 15° (1). In addition, at the relatively low growth temperature of 15°,(Jennifer L. Watts Eric Phillips Katharine R. Griffing and John Browse)
ABSTRACT1p, http://www.100md.com
Arachidonic acid and other long-chain polyunsaturated fatty acids (PUFAs) are important structural components of membranes and are implicated in diverse signaling pathways. The 6 desaturation of linoleic and linolenic acids is the rate-limiting step in the synthesis of these molecules. C. elegans fat-3 mutants lack 6 desaturase activity and fail to produce C20 PUFAs. We examined these mutants and found that development and behavior were affected as a consequence of C20 PUFA deficiency. While fat-3 mutants are viable, they grow slowly, display considerably less spontaneous movement, have an altered body shape, and produce fewer progeny than do wild type. In addition, the timing of an ultradian rhythm, the defecation cycle, is lengthened compared to wild type. Since all these defects can be ameliorated by supplementing the nematode diet with gamma-linolenic acid or C20 PUFAs of either the n6 or the n3 series, we can establish a causal link between fatty acid deficiency and phenotype. Similar epidermal tissue defects and slow growth are hallmarks of human fatty acid deficiency.
THE 6 fatty acid desaturase catalyzes the rate-limiting step in the conversion of the essential fatty acids, linoleic acid (18:2n6) and linolenic acid (18:3n3), into C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3). (Fatty acid nomenclature used here is the following: X:YnZ refers to a fatty acid chain of X carbon atoms and Y methylene-interrupted cis double bonds; Z indicates the position of the terminal double bond relative to the methyl end of the molecule.) PUFAs play critical roles in regulating membrane structure, dynamics, and permeability. In mammals, C20 PUFAs are substrates for oxygenases that produce powerful short-range eicosanoid effector molecules, including prostaglandins, leukotrienes, and thromboxanes (FUNK 2001 ). In response to mechanical, cytokine, or growth factor stimuli, phospholipase A2 cleaves PUFAs from cell membranes so they can be acted on by cyclooxygenase, lipoxygenase, and P450 monooxygenase enzymes. The eicosanoids mediate a variety of processes in many cell types, including pain, inflammation, and reproductive processes. In addition to these roles, liberated free fatty acid forms of C20 PUFAs, most notably 20:4n6, display considerable biological activity, including activation of nuclear hormone receptors, modulation of ion channels, and as second messengers in signal transduction (BRASH 2001 ). Studies examining the roles of specific fatty acids in these processes in animals are hampered by the difficulty of manipulating lipid composition in vivo.
Caenorhabditis elegans is an attractive animal model in which to investigate the physiological roles of specific fatty acids in growth, development, and the nervous system. Unlike mammals, C. elegans does not require essential fatty acids in its diet, but is capable of synthesizing 20:4n6 and 20:5n3 using only saturated and monounsaturated fatty acids from bacteria as precursors (HUTZELL and KRUSBERG 1982 ). This is possible because C. elegans expresses the full range of desaturase activities found in plants (12 and n3 desaturase) and animals (5 and 6 desaturase) as well as PUFA elongase activities found in animals (NAPIER and MICHAELSON 2001 ).@;, http://www.100md.com
To investigate the roles of various fatty acids in growth, development, and neurological function in an animal system, we recently isolated C. elegans mutants deficient in PUFA synthesis by direct analysis of fatty acid composition (WATTS and BROWSE 2002 ). These mutants revealed that C. elegans does not require n3 or 5 unsaturated PUFAs for normal development under laboratory conditions. The n3 and 5 desaturase mutants are deficient in certain classes of C20 PUFAs but accumulate higher levels of precursor C20 PUFAs as a consequence of these deficiencies. In contrast, the fat-3 mutants that lack 6 desaturase activity fail to produce any of the common C20 PUFAs and, as a result, their growth and behavior are compromised. Here we demonstrate that although the fat-3 mutants are viable and fertile, they exhibit neuromuscular defects, cuticle abnormalities, reduced brood size, and altered biological rhythms. These defects can be biochemically complemented by dietary supplementation of various 20-carbon PUFAs.
MATERIALS AND METHODS:, 百拇医药
Culture and measurement of nematodes::, 百拇医药
Nematodes were cultured and maintained according to standard methods (WOOD 1988 ). The strains were grown at 20° on nematode growth medium (NGM) plates unless otherwise indicated. The wild-type strain was N2 and the fat-3 strain used in these studies was fat-3(wa22). Body-length measurement of late L4 animals was obtained essentially as described in REINER et al. 1999 . For each genotype or fatty acid treatment, 10 animals were measured using an Alvin map wheel. Brood size and hatch rate was measured by placing 8–10 L4 animals onto individual plates. Worms were transferred to a new plate each day for 4 days, and the number of eggs and larvae on each were counted. Percentage viability was obtained by dividing the number of hatched L1 larvae by the total number of eggs laid for each worm. Cuticle disintegration was scored by directly observing gravid adult nematodes placed in 0.5 ml alkaline hypochlorite solution (1% sodium hypochlorite, 0.25 M NaOH) in 24-well culture plates and noting the time of the first major break in the cuticle. Plates were agitated gently every 30 sec during observation. Errors are SEM.
Behavioral assays:81, 百拇医药
The defecation cycles of first-day adult animals were scored by measuring the time from one posterior body contraction to the next. The presence or lack of an enteric muscle contraction at the end of each cycle was noted as well. For each strain a minimum of six animals were scored for 10 cycles each. Unless otherwise noted, defecation cycles and enteric muscle contractions were scored with the petri dishes closed. Movement assays were performed as described (MILLER et al. 1996 ) in M9 buffer. One "thrash" was defined as a change in direction of bending at the midbody. At least 10 animals for each genotype or fatty acid treatment were measured. Pharyngeal pumping was scored by direct observation of at least 10 animals for each genotype for 1 min. All behavioral assays were performed at room temperature (22°–23°).81, 百拇医药
Construction of the fat-3::GFP reporter gene:81, 百拇医药
The full-length translational fusion was constructed by fusion PCR of the amplified fat-3 promoter and coding sequences together with the green fluorescent protein (GFP) coding sequence amplified from pPD95.75 (HOBERT et al. 1999 ). The upstream regulatory region included 1086 bases upstream of the fat-3 ATG. This region included 97 bases of the last exon of the upstream fat-4 gene. The constructs were microinjected into wild-type or fat-3(wa22) worms together with the rol-6(su1006) dominant marker plasmid pRF4 (MELLO et al. 1991 ). Rolling transgenic worms were isolated and multiple independent lines that produced heritable rolling progeny were examined for GFP expression and analyzed for fatty acid composition.
Fatty acid supplementation and analysis:a$-&'[, http://www.100md.com
Fatty acid sodium salts were obtained from NuChek Prep (Elysian, MN) and stored at -20° in the dark. For each experiment, a fresh 0.1 M stock was prepared by dissolving fatty acids in sterile H2O. NGM agar was prepared with the addition of 0.1% tergitol (NP-40). Agar was cooled to 45°–50° and fatty acid stock was added slowly and stirred for 1 min. Plates were poured immediately and then covered to dry in the dark for 24 hr. Plates were then seeded with Escherichia coli and allowed to dry for 2 days in the dark at room temperature before the addition of embryos. Embryos were prepared by alkaline hypochlorite treatment of adult nematodes to obtain a semisynchronized population of early embryos. After phenotypic analysis of adult worms, nematodes were washed off the plates in H2O and centrifuged gently to pellet the worms. As much water as possible was removed and the worm pellets were frozen for determination of fatty acid composition as described in WATTS and BROWSE (2002).
RESULTS AND DISCUSSION?4[x\, http://www.100md.com
Fatty acid composition of 6 desaturase mutants and growth phenotypes:?4[x\, http://www.100md.com
The6 desaturase mutants were isolated without selection using gas chromatography analysis of fatty acids derived from mutagenized nematodes (WATTS and BROWSE 2002 ). The fatty acid composition of three independent fat-3 lines revealed elevated levels of the 6 desaturase precursors 18:2n6 and 18:3n3 and a deficiency in C20 fatty acids, most notably undetectable levels of dihomogamma-linolenic acid (20:3n6), arachidonic acid (20:4n6), and eicosapentaenoic acid (20:5n3). We found that the phenotypes described in this work were indistinguishable among the three fat-3 alleles (wa22, wa23, and wa25). Worms of all three genotypes display identical fatty acid compositions with undetectable 6 unsaturated PUFAs, implying that all three alleles represent loss of activity of the 6 desaturase. Detailed phenotypic characterization was carried out with fat-3(wa22).?4[x\, http://www.100md.com
The fat-3 homozygous worms are viable and fertile, indicating that C20 PUFAs are not essential for life in this organism. However, they grow at a slower rate than wild type, requiring one extra day of development at 20° before they become fertile adults (WATTS and BROWSE 2002 ). In this study, we examined brood size and hatch rate of fat-3 worms and embryos at three growth temperatures. We found that within a range of temperatures between 15° and 25° the fat-3 worms consistently produced smaller broods than wild type did, with the largest difference at 15° (1). In addition, at the relatively low growth temperature of 15°,(Jennifer L. Watts Eric Phillips Katharine R. Griffing and John Browse)