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Imaging Liver Development/Remodeling in the See-Through Medaka Fish
Comparative Hepatology volume 3, Article number: S30 (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).
Imaging of the living ST II medaka at stage 37  is shown in Figure (1). The chorion (eggshell) was intact and had numerous small filaments on outer surface. Examination of the chorionated embryos was made complex due to the rounded confines of the embryonic space. When head was viewed from rostral end with embryo in anatomic position, the caudal peduncle curved and the more distal portions of the tail extended alongside the embryo and over the head with the tip of the tail reaching a point caudal to the otic vesicle (not shown in figure). At this time, the yolk sac is large and major veins pass in circuitous routes over the yolk sac and eventually converge at the sinus venosus. After dechorionation, intra-vital analysis of embryos near normal time of hatching (Stage 39), revealed topography and anatomical features of key abdominal organs (Fig. 2) as well as inner ear. Spleen was visible as a red disc lateral to the swim bladder (adjacent brown disc). The elongated liver was located within the left lateral portion of the abdominal cavity (Fig. 2). The intrahepatic circulation in stage 39 was visible and a single left hepatic vein conducted blood from the liver to the left duct of Cuvier that was visible as a wavy line on the yolk sac.
Remodeling of liver, major veins of yolk sac and abdomen was apparent when larvae of wk one after hatching were compared to those one week older (wk 2). When the liver was in the left lateral portion of the abdominal cavity, ducts of Cuvier and median yolk vein were apparent in ventral view of abdomen (Fig. 3A). One week later, a similar view revealed metamorphosis of liver. Position of liver was now transverse and location was in the rostral most portion of the abdominal cavity (Fig. 3B). Also, left and right ducts of Cuvier were not imaged and there was no indication of the median yolk vein. When medaka at this time of development were processed for HRLM, sagittal sections revealed incorporation of median yolk vein into liver (Fig. 4).
The adult pattern of hepatic tubular architecture was most apparent in actively spawning female medaka at 8 wks of age (Fig. 5). Hepatocytes were arranged as tubules with biliary lumens in center.
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.
JA Hampton, McCuskey PA, McCuskey RS, Hinton DE: Functional units in rainbow trout (Salmo gairdneri) liver. I. Arrangement and histochemical properties of hepatocytes. Anat Rec. 1985, 213: 166-175. 10.1002/ar.1092130208.
Hampton JA, Lantz RC, Hinton DE: Functional units in rainbow trout (Salmo gairdneri, Richardson) liver: III. Morphometric analysis of parenchyma, stroma, and component cell types. Am J Anat. 1989, 185: 58-73. 10.1002/aja.1001850107.
Hinton DE, Segner H, Braunbeck T: Chapter 4. Toxic responses of the liver. In: Target Organ Toxicity in Marine and Freshwater Teleosts. Volume 1 Organs. Edited by: Schlenk D, Benson WH. 2001, London, Taylor and Francis, 224-268.
Wakamatsu Y, Pristyazhnyuk S, Kinoshita M, Tanaka M, Ozato K: The see-through medaka: a fish model that is transparent throughout life. PNAS. 2001, 98 (18): 10046-10050. 10.1073/pnas.181204298.
Tomita H: Mutant genes in medaka. In: Medaka (Killifish): Biology and Strains. Edited by: Yamamoto T. 1975, Tokyo, Keigaku, 251-272.
Tomita H: The lists of the mutants and strains of the medaka, common gambusia, silver crucian carp, goldfish, and golden venus fish maintained in the laboratory of Freshwater Fish Stocks, Nagoya University. Fish Biol J Medaka. 1992, 4: 45-48.
Wakamatsu Y, Ozato K, Hashimoto H, Kinoshita M, Sakaguchi M, Iwamatsu T, Hyodo-Taguchi Y, Tomita H: Generation of germ-line chimeras in medaka (Oryzias latipes). Mol Marine Biol Biotech. 1993, 2 (6): 325-332.
Yasumasu S, Iuchi I, Yamagami K: CDNAs and the genes of HCE and LCE, two constituents of the Medaka hatching enzyme. Development Growth & Differentiation. 1994, 36 (3): 241-250. 10.1111/j.1440-169X.1994.00241.x.
Iwamatsu T: Stages of Normal Development in the Medaka (Oryzias latipes). Zoological Science. 1994, 11: 825-839.
Metcalfe CD, Metcalfe TL, Kiparissis Y, Koenig B, Khan C, Hughes RJ, Croley TR, March RE, Potter T: Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry. 2001, 20 (2): 297-308. 10.1897/1551-5028(2001)020<0297:EPOCDI>2.0.CO;2.
Nimrod A, Benson WH: Reproduction and development of Japanese medaka following an early life stage exposure to xenoestrogens. Aquatic Toxicology. 1998, 44 (1–2): 141-156. 10.1016/S0166-445X(98)00062-9.
Okihiro MS, Hinton DE: Progression of hepatic neoplasia in medaka (Oryzias latipes) exposed to diethylnitrosamine. Carcinogenesis. 1999, 20: 933-940. 10.1093/carcin/20.6.933.
Hawkins WE, Walker WW, Overstreet RM: Carcinogenicity Tests Using Aquarium Fish. Toxicology Methods. 1995, 5 (4): 225-263. 10.3109/15376519509084029.
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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Hinton, D.E., Wakamatsu, Y., Ozato, K. et al. Imaging Liver Development/Remodeling in the See-Through Medaka Fish. Comp Hepatol 3, S30 (2004). https://doi.org/10.1186/1476-5926-2-S1-S30
- Liver Development
- Hepatic Sinusoid
- Sinus Venosus
- Otic Vesicle
- Left Hepatic Vein