Genetic analysis of digestive physiology using fluorescent phospholipid reporters

Science

Volumn 292, Issue 5520, 2001, Pages 1385-1388

  Farber, Steven A ^a,b   Pack, Michael ^c   Ho, Shiu Ying ^b   Johnson, Iain D ^d   Wagner, Daniel S ^c   Dosch, Roland ^c   Mullins, Mary C ^c   Hendrickson, H Stewart ^e   Hendrickson, Elizabeth K ^e   Halpern, Marnie E ^a  

^a GEOPHYSICAL LABORATORY   (United States)

^b THOMAS JEFFERSON UNIVERSITY   (United States)

^c UNIVERSITY OF PENNSYLVANIA   (United States)

^d MOLECULAR PROBES INC   (United States)

^e UNIVERSITY OF WASHINGTON   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHOLESTEROL; DRUG DOSAGE; ENZYMES; FLUORESCENCE; GENES; GENETIC ENGINEERING; METABOLISM; MORPHOLOGY; MUTAGENS; PHOSPHOLIPIDS; PHYSIOLOGY;

VERTEBRATES;

DIGESTIVE SYSTEM;

PHOSPHOLIPID; TRIACYLGLYCEROL LIPASE;

ARTICLE; CHOLESTEROL SYNTHESIS; CONTROLLED STUDY; DIGESTIVE FUNCTION; ENZYME ACTIVITY; FLUORESCENCE; GENETIC ANALYSIS; GENETIC SCREENING; HEPATOBILIARY SYSTEM; INTESTINE; LARVA; LIPID METABOLISM; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; ZEBRA FISH;

ANIMALS; ANTICHOLESTEREMIC AGENTS; BILE ACIDS AND SALTS; BORON COMPOUNDS; CHOLESTEROL; DIGESTIVE PHYSIOLOGY; DIGESTIVE SYSTEM; FLUORESCENT DYES; GALLBLADDER; HEPTANOIC ACIDS; LARVA; LIPASE; MICE; MICROSCOPY, FLUORESCENCE; MICROSCOPY, VIDEO; MUTATION; PHOSPHOLIPIDS; PYRROLES; SIGNAL TRANSDUCTION; ZEBRAFISH;

DANIO RERIO; MAMMALIA; VERTEBRATA;

EID: 0035907008     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.1060418     Document Type: Article

References (30)

1. S. A. Farber, E. S. Olson, J. D. Clark, M. E. Halpern, J. Biol. Chem. 274, 19338 (1999).
2. H. S. Hendrickson, E. K. Hendrickson, I. D. Johnson, S. A. Farber, Anal. Biochem. 276, 27 (1999).
3. M. MacPhee et al., Cell 81, 957 (1995).
4. R. T. Cormier et al., Nature Genet. 17, 88 (1997).
5. E. A. Dennis, Trends Biochem. Sci. 22, 1 (1997).
6. D. Tocher, in Biochemistry and Molecular Biology of Fishes, P. Hochachka, T. Mommsen, Eds. (Elsevier, New York, 1995), vol. 4, pp. 119-157.
7. M. A. Sheridan, Comp. Biochem. Physiol. B 90, 679 (1988).
8. H. S. Hendrickson, Anal. Biochem. 219, 1 (1994).
9. Larvae were labeled in embryo medium (EM) [M. Westerfield, The Zebrafish Book (Univ. of Oregon, Eugene, OR, ed. 3, 1995)], anesthetized (tricaine, 170 μg/ml), and placed in depression slides. Fluorescent images were captured over 1 hour using a Zeiss Axiocam 2 mounted on a Leica MZFL-III.
10. 2 activity was determined as described (7). Measurements were compared with activity of bile-depleted gall bladders.
11. J. W. Nawrocki et al., Arterioscler. Thromb. Vasc. Biol. 15, 678 (1995).
12. Bile obtained from freshly killed tilapia (Orechromis mossambicus) was 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 μl/ml).
13. 27 bile alcohol (A. F. Hofmann and L. R. Hagey, personal communication).
14. T. Goto et al., Hepatology 26, 295A (1997).
15. M. Rubin, R. Pakula, T. Gilat, A. Tietz, Lipids 34, 571 (1999).
16. For methods, see Science Online (www.sciencemag. org/cgi/content/full/292/5520/1385/DC1).
17. P. R. Selvin, Nature Struct. Biol. 7, 730 (2000).
18. 2] by sonication of the dried lipids.
19. H. W. Stroband, H. van deer Meer, L. P. Timmermans, Histochemistry 64, 235 (1979).
20. R. E. Honkanen, M. W. Rigler, J. S. Patton, Am. J. Physiol. 249, 399 (1985).
21. M. Pack et al., Development 123, 321 (1996).
22. M. C. Mullins, M. Hammerschmidt, P. Haffter, C. Nusslein-Volhard, Curr. Biol. 4, 189 (1994).
23. To view time-lapse movies of gall bladder contraction and intestinal motility after PED6 labeling, see Science Online (www.sciencemag.org/cgi/content/full/ 292/5520/1385/DC1).
24. S. A. Farber, M. Pack, unpublished data.
25. Larvae (6 dpf) were placed in EM containing fluorescent latex microspheres (0.0025% Fluoresbrite plain YG 2.0 μm, Polysciences Inc.) for 1 hour, washed, and imaged as described (9). Numbers of beads were 10 ± 2 in the wild type versus 14 ± 3 beads in mutant larvae (mean ± SEM, n = 9, P > 0.3).
26. Phenol red (2% in EM) was injected (∼5 nl) into larvae dorsal to the anterior intestine, and labeling of the gall bladder was monitored.
27. For quantification of fluorescence data, see Science Online (www.sciencemag.org/cgi/content/full/292/ 5520/1385/DC1).
28. A. Frolov et al., J. Biol. Chem. 275, 12769 (2000).
29. R. Homan, M. K. Jain, in Intestinal Lipid Metabolism, C. M. Mansbach, P. Tso, A. Kuksis, Eds. (Kluwer Academic, New York, 2001), pp. 81-104.
30. Supported by a National Research Service Award (NRSA) and a Barbara McClintock Fellowship (Carnegie Institution) (SAF.), National Institute of Diabetes and Digestive and Kidney Diseases grant DK 54942 (M.P.), a NRSA (D.S.W.), the Deutscher Akademischer Austauschdienst (R.D.), the March of Dimes (M.C.M.), NSF grant MCB-9619859 (H.S.H.), and a Pew Scholar's Award (M.E.H.). We thank A. Dolan, M. Macurak, J. Thorpe, R. DeRose, T. Woodard, and D. Hooper for technical assistance and L. R. Hagey and A. F. Hofmann for mass spectrometry analysis of zebrafish bile.