Imaging Liver Development/Remodeling in the See-Through Medaka Fish
© Hinton et al; licensee BioMed Central Ltd 2004
Published: 14 January 2004
One major function of the livers of fishes, essential for life, is the metabolism of xenobiotics, rendering lipophilic compounds water soluble and more easily excreted. This function is in turn linked to the formation and excretion of bile. Linked hepatocytic and biliary epithelial function in fishes involves hepatic tubules [1–3]. In these, hepatocytes are clustered about an axis of the biliary system. This pattern is analogous to that of an exocrine gland and is the adult phenotype for amphibians, birds, reptiles, and fishes .
Most fish species lack resident macrophages of the hepatic sinusoids (Kupffer cells) ; and, while mammalian studies – including recent findings reported at this meeting – clearly show important roles of these and other cells of the hepatic sinusoids in initiation and modulation of hepatoxic responses, studies in fishes lag far behind those of their mammalian counterparts. Our overall objective is to improve understanding of the biology of laboratory model fish so that future workers may have better tools with which to approach the complexity of environmental science including toxicology. Herein, we present, for the first time, results of in situ and vital studies on liver development, metamorphosis and formation of adult vascular elements in medaka. These studies were greatly facilitated by use of the see-through medaka , a vertebrate model with a transparent body throughout life.
Individual ST II (see-through) medaka (Oryzias latipes) were used. Production of these mutants is described by Wakamatsu et al. . From about 50 natural color mutants of medaka in the Laboratory of Freshwater Fish Stocks, Bioscience Center, Nagoya University (Nagoya, Japan) [5, 6], some were selected that showed deficiency in pigmentation. By crossing selected mutants, Wakamatsu, et al.  genetically removed pigments from the entire body, thereby generating a transparent fish. Breeding groups of ST II medaka fed a commercial ration (Otohime Beta, Nisshin Feed Co. Ltd., Tokyo) twice daily and supplemented with brine shrimp nauplii for four days each week were maintained under a 16:8 hr light/dark cycle at 26–C (spawning conditions). Fertilized eggs were collected daily and development recorded through the transparent chorion using a dissection microscope (Leica Wild M420; equipped with a Nikon 990 cool pix camera). To facilitate orientation and to improve imaging of certain embryonic stages, dechorionation was employed using medaka hatching gland enzyme as described [7, 8]. Embryonated eggs of other medaka were collected weekly for 12 wks. Iwamatsu  described and figured developmental stages of medaka and this was followed to select liver development from early organogenesis, through larval and juvenile stages into spawning females. Specimens for high-resolution light microscopy (HRLM) were directly placed in fixative (embryos) or were first killed by overdose in anesthetic (tricaine methane sulfonate; larvae and older) then immediately placed in fixative (10% neutral buffered formalin or 4% paraformaldehyde in phosphate buffer). After 72 hrs, fixed specimens were transferred to phosphate buffered saline containing 6% sucrose, stored in the cold until time of processing, embedded in glycol methacrylate, sectioned at 2–4 microns thickness, stained by Gill's hematoxylin and eosin and viewed using a Nikon Eclipse E600 binocular microscope with digital still camera system (DXM 1200).
Use of see-through medaka greatly facilitates correlation of liver development, metamorphosis and maturation. Pigment interference is not a problem in this model. Medaka are being used to investigate endocrine disruptors [10, 11], liver carcinogenesis [12, 13] and the efficiency of new waste water treatment and reuse systems (unpublished studies this laboratory). Their small size has led to their incorporation in studies in outer space as well. Their usefulness will be greatly enhanced following improved understanding of the roles of various liver cell types in health and disease.
This work was supported in part by NIEHS Superfund grant P42 ES 04699 and by 1 R01 RR18583 from the National Center for Research Resources of the U.S. National Institutes of Health.
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