In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.
Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.
Hama
Kotaro
K
Department of Embriology, Carnegie Institution, Baltimore, MD 21224, USA.
Provost
Elayne
E
Baranowski
Timothy C
TC
Rubinstein
Amy L
AL
Anderson
Jennifer L
JL
Leach
Steven D
SD
Farber
Steven A
SA
D000818:Animals; D002766:Cholecystokinin / Q000378:metabolism; D004063:Digestion / Q000187:drug effects / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D007422:Intestines / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D007814:Larva / Q000201:enzymology; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D046790:Pancreas, Exocrine / Q000196:embryology / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010743:Phospholipids; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D029961:Zebrafish Proteins; C062602:transcription factor PTF1; C513590:vps51 protein, zebrafish; D002766:Cholecystokinin; D010740:Phospholipases; D010447:Peptide Hydrolases