Onset of Apoptosis in the Cystic Duct During Metamorphosis of a Japanese Lamprey Lethenteron reissneri.код для вставкиСкачать
THE ANATOMICAL RECORD 293:1155–1166 (2010) Onset of Apoptosis in the Cystic Duct During Metamorphosis of a Japanese Lamprey, Lethenteron reissneri MAYAKO MORII,1 YOSHIHIRO MEZAKI,2 NORIKO YAMAGUCHI,2 KIWAMU YOSHIKAWA,2 MITSUTAKA MIURA,2 KATSUYUKI IMAI,2 HIROAKI YOSHINO,1 TAKU HEBIGUCHI,1 TATSUZO HEBIGUCHI,1 AND HARUKI SENOO2* 1 Department of Pediatric Surgery, Akita University Graduate School of Medicine, Akita, Japan 2 Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan ABSTRACT A nonparasitic lamprey in Japan, Lethenteron reissneri, stops feeding prior to the commencement of metamorphosis. Resumption of feeding cannot take place due to major alterations in the digestive system, including loss of the gall bladder (GB) and biliary tree in the liver. This degeneration of bile ducts is considered to depend on programmed cell death or apoptosis, but molecular evidence of apoptosis remains lacking. Using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunohistochemistry with an antibody against active caspase-3, we showed that epithelial cells of the cystic duct (CD) and GB became TUNELpositive by the early metamorphosing stage. Immunohistochemical staining of active caspase-3, a key mediator in the apoptotic cascade, showed that the apoptotic signal was initiated in the region around the CD in the late larval phase. In later stages, active caspase-3-positive epithelial cells were also observed in the large intrahepatic bile duct (IHBD) and peripheral small IHBDs. At the early metamorphosing stage, bile canaliculi between hepatocytes were dilated and displayed features resembling canaliculi in cholestasis. Onset of apoptosis around the CD, which is the pathway for the storage of bile juice, and progression of apoptosis towards the large IHBD, which is the pathway for the secretion of bile juice, may lead to temporary intrahepatic cholestasis. The present study represents the ﬁrst precise spatial and temporal analysis of apoptosis in epithelial cells of the biliary tract system during metamorphosis of any lamprey species. Anat Rec, 293:1155– C 2010 Wiley-Liss, Inc. 1166, 2010. V Key words: apoptosis; biliary atresia; cholestasis; lamprey; bile canaliculi; morphology; TUNEL; active caspase-3 Lampreys are extant representatives of vertebrates, the Agnatha. They can be divided into two categories, parasitic and nonparasitic, based on the adult-life history immediately following metamorphosis. Lethenteron reissneri is the most prevalent nonparasitic lamprey in Japan (Iwata et al., 1985; Yamazaki and Goto, 2000). The larvae of L. reissneri feed on detritus and some bacteria, and therefore carry out digestive absorption. However, they stop feeding before the commencement of metamorphosis, and then spawn and die without ever resuming feeding (Hardisty and Potter, 1971). The entire C 2010 WILEY-LISS, INC. V Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan [Grants-in-Aid for Young Scientists (B)]; Grant numbers: 20791296, 20790158. *Correspondence to: Haruki Senoo, Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Fax: þ81-18834-7808. E-mail: email@example.com Received 25 May 2009; Accepted 20 January 2010 DOI 10.1002/ar.21151 Published online 13 April 2010 in Wiley InterScience (www. interscience.wiley.com). 1156 MORII ET AL. bile-transport apparatus of larvae disappears completely during metamorphosis as normal ontogeny, but the cause of this biliary atresia is unknown. The only reason that we know that biliary atresia takes place is that part of the biliary duct system transforms into an endocrine pancreas (Youson and Elliott, 1989). In most vertebrates, bile juice is known to be harmful to the liver unless correctly discharged. However, despite the biliary atresia that occurs in lampreys, neither biliary cirrhosis nor liver dysfunction develop. The relevance of biliary atresia in lampreys to biomedical research was suggested from an analysis of a parasitic species of lamprey, Petromyzon marinus (Sterling et al., 1967, 1968), and was ﬁrst described in a nonparasitic species of lamprey, Lampetra planeri (De Vos et al., 1973). Detailed temporal descriptions of changes in the biliary duct system during metamorphosis have been presented (Youson and Sidon, 1978; Youson, 1993). The metamorphosis of P. marinus can be divided into seven stages (Youson and Potter, 1979), and seven stages are universally accepted for all species, including L. reissneri (Tsuneki and Ouji, 1983). In P. marinus, alterations of bile ducts are most dramatic by Stages 3 and 4, and a few subtle changes in liver morphology occur in the earliest phase (Stages 1 and 2); (Youson and Sidon, 1978; Sidon and Youson, 1983a,b). In some lamprey species, regression of bile ducts is asynchronous, with smaller peripheral ducts degenerating more rapidly than the large central ducts (Sidon and Youson, 1983a). Furthermore, the type of cell death occurring there is speculated to most likely represent apoptosis as deﬁned by Wyllie et al. (1981); (Youson and Ogilvie, 1990; Youson, 1993). However, molecular evidence of apoptosis as an event in biliary atresia has yet to be deﬁnitively observed in the metamorphic lamprey. Apoptosis plays an important role in a variety of physiological phenomena such as follicular atresia, epithelial cell renewal in the intestine, sculpture of the digits, and disappearance of the tail in the metamorphosing tadpole (Penaloza et al., 2006; Alberts et al., 2008). Apoptosis is characterized by a fragmentation of DNA that can be detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. The DNA fragmentation reﬂects complex biochemical events carried out by a family of cysteine proteases called caspases (Alnemri et al., 1996). Caspases are divided into initiator caspases with long prodomains (caspase-8, caspase-9, and caspase-10) and effector caspases with short prodomains (caspase-3, caspase-6, and caspase-7). Initiator caspases activate effector caspases. One of the effector caspases, caspase-3, has been implicated as a key mediator of apoptosis. In response to various death signals, the caspase3 proenzyme is activated by initiator caspases in the cytoplasm, and then enters the nucleus, leading to the fragmentation of DNA (Woo et al., 1998; Zheng et al., 1998; Kamada et al., 2005). By detecting DNA fragmentation with TUNEL staining and the upstream signaling with immunohistochemical identiﬁcation of active caspase-3-positive cells, we show in this report that the biliary atresia observed in the metamorphic lamprey is mediated by apoptosis. Furthermore, by precise time-course inspection of the liver and bile duct systems in metamorphosing lampreys, we demonstrate that this apoptosis is initiated in the region around the cystic duct (CD). The biological signiﬁcance of the onset of apoptosis in the CD during metamorphosis is discussed. MATERIALS AND METHODS Animals Larval, metamorphosing, and adult phases of nonparasitic lampreys, L. reissneri, were collected from several branches of the Koyosi river and Omono river in Akita prefecture once a month from April to October in 2008. Two species of the genus Lethenteron are known to live in Akita prefecture: L. japonicum and L. reissneri. These two species are easily discriminated by the color of ﬁns and number of trunk myomeres in larval and metamorphosing phases and by body size in the mature adult phase (Hubbs and Potter, 1971; Iwata et al., 1985). A total of 34 specimens of L. reissneri (14 larvae, 10 metamorphosing animals, three immature adults, and seven sexually maturing adults) were investigated in the present study. The life history of L. reissneri in Tottori prefecture is reportedly no less than 3.5 years (Tsuneki and Ouji, 1983). The immature adults were captured in October and the sexually maturing adults in April. All animals were transported to the laboratory in fresh water with aeration and used within a few days. With slight modiﬁcation of the criteria reported by Tsuneki and Ouji, individuals with hardly visible eyes (Stage 1) were treated as the late larval phase, and Stage 6 (macrophthalmia stage) and Stage 7 (adult without secondary sex characteristics) were classiﬁed as immature adults. Classiﬁcation of the metamorphosing stages into seven stages was too difﬁcult to achieve for this study, so we divided the animals into two stages, early and later. Three animals in the early stage were characterized by eyes distinctly visible as dark spots. Seven animals in the later stage were characterized by a white eye totally circumscribing the dark eye. The protocol for animal experimentation described in this article was approved by the Animal Research Committee of Akita University Graduate School of Medicine. All subsequent animal experiments adhered to the university’s guidelines for animal experimentation. Morphological Methods All 34 lampreys were prepared for morphological analysis. Following anesthetization in eugenol (4-allyl-2methoxyphenol), livers were surgically resected. Whole liver blocks were immersed in 4.0% formaldehyde at 4 C for 2 days. Parafﬁn-embedded sections were prepared for histological and immunohistochemical observations (Wake et al., 1987; Wold et al., 2004; Higashi et al., 2005). Light Microscopy Five-micrometer serial sections were made from livers of larval, adult, and metamorphosing lampreys. Alternate sections were stained with hematoxylin and eosin (HE) and examined under light microscopy (Axioskop; Carl Zeiss, Jena, Germany). Some slides were recorded digitally using NanoZoomer Digital Pathology (Hamamatsu Photonics, Hamamatsu, Japan). Morphometric and statistical analyses were performed using NDP.view software (Hamamatsu Photonics) and Excel software BILE SYSTEM APOPTOSIS DURING METAMORPHOSIS (Microsoft, Redmond, WA), respectively. The central part of the liver, which contains the large intrahepatic bile ducts (IHBDs), portal vein, and hepatic artery, was used for morphometric analyses. Randomly selected 0.1 mm2 regions were analyzed. The minor axes of all bile canaliculi within the selected region were measured and averaged, and this value was considered to be the diameter of the bile canaliculi. The statistical signiﬁcance of differences was evaluated using Student’s t-test. Values of P < 0.05 were considered to be statistically signiﬁcant. TUNEL Staining Sections of parafﬁn-embedded liver blocks were subjected to TUNEL assay using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI). In brief, deparafﬁnized sections were incubated with proteinase K for 10 min, washed in phosphate-buffered saline (PBS), and incubated with a terminal deoxynucleotidyl transferase enzyme and ﬂuorescein-12-dUTP as substrate. Nuclei were stained with TO-PRO-3 (Invitrogen, Carlsbad, CA). Labeled cells were observed under laser-scanning microscopy (LSM-510; Carl Zeiss). To deﬁne whether apoptosis takes place accompanying the degeneration of the biliary duct system, we stained several sections of various phases of the lamprey using TUNEL staining. In larval lampreys, the TUNEL labeling rate of the middle and small IHBDs, which do not show any sign of apoptosis accompanying biliary atresia, was 1.75% 1.43%. In the early metamorphosing stage, TUNEL labeling rate of the gall bladder (GB), which is largely degenerating, was 60.2% 21.1%. In humans, the TUNEL labeling rate of biliary epithelial cells under normal conditions is reported to be 3.6% 2.8%, compared to 48.9% 13.2% during human biliary atresia (Funaki et al., 1998). We deﬁned biliary epithelial cells as TUNEL-positive when more than 5% of the cell was stained, because apoptosis occurs even in normal physiological conditions such as that seen in cell turnover, as shown above. In larval and metamorphosing lampreys, TUNEL staining was performed on several sections containing hepatocytes, bile canaliculi, extrahepatic bile duct (EHBD), IHBDs, CD, and GB, to detect DNA fragmentation in these tissues. Small intestinal tissue from the C57BL/6 mouse was processed and treated as above, then used as a positive control for TUNEL staining. As a negative control for TUNEL staining, incubation without terminal deoxynucleotidyl transferase enzyme was performed. Active Caspase-3 Staining Cells undergoing apoptosis were also detected by immunohistochemical staining with an anti-active caspase3 antibody. In brief, deparafﬁnized sections were treated with 0.2% Triton X-100 in PBS for permeabilization, incubated with a blocking solution (1% bovine serum albumin), and then incubated with 1:50 rabbit anti-active caspase-3 antibody (Cell Signaling Technologies, Danvers, MA) at 4 C overnight. Following washes, sections underwent incubation with 1:100 biotin SP-conjugated AfﬁniPure goat anti-rabbit immunoglobulin (Ig)G (Jackson ImmunoResearch, West Grove, PA). Finally, sections were treated with 1:100 streptavidin-Alexa Fluor 488 1157 conjugate (Invitrogen). Nuclei were stained with TOPRO-3 (Invitrogen). Labeled cells were observed under laser-scanning microscopy (LSM-510; Carl Zeiss). For the negative control for antibody speciﬁcity, replacement of rabbit anti-active caspase-3 antibody with normal rabbit IgG (Millipore, Billerica, MA) was performed. Small intestinal tissue from C57BL/6 mouse was used as a positive control for immunohistochemical staining after processing and treatment as above. RESULTS Identiﬁcation of L. reissneri and Metamorphic Stages Figure 1 shows a larva (A), a metamorphosing animal in later stage (B and C), immature adults (D and E), and a sexually maturing male (F) and female (G) of L. reissneri. The ﬁns were transparent (Fig. 1A,B,F,G), trunk myomeres numbered between 57 and 65 (Fig. 1B,F,G), and the animals showed three pairs of lateral circumoral teeth and two supraoral plates (Fig. 1E). These morphological characteristics are typical for this species (Hubbs and Potter, 1971). In the late stage of metamorphosis, the white eye was recognized as totally circumscribing the dark eye, although this eye was very thin (Fig. 1C, arrow). Immature adults have a large silver iris (Fig. 1D) and teeth (Fig. 1E). Sexually maturing adults captured in the spawning season showed prominent secondary sex characteristics, such as a urinogenital papilla in males (Fig. 1F, arrow) and large eggs within the female abdomen (Fig. 1G, arrow). Microscopic Structure of the Liver and the Biliary Tree Observation of serial sections (data not shown) conﬁrmed that the liver in larvae is a lanceolate-shaped organ with a broad cranial end gradually tapering caudally (Youson, 1993). This is shown schematically in Fig. 2. The GB is located at the cranial end, and the CD connects the GB to a large IHBD that collects bile juice from IHBDs from the entire liver. The large IHBD ramiﬁes to the peripheral convoluted medium IHBDs and then small IHBDs and ﬁnally connects to the bile canaliculi (Fig. 2). The large IHBD comprised simple columnar epithelium surrounded by thick ﬁbrous connective tissue (Fig. 3A, large arrow). Medium IHBDs were also composed of simple columnar epithelia, but lacked surrounding connective tissues (Fig. 3A, arrowheads). The small IHBDs consisted of simple cuboidal epithelia, typical examples of which are shown in Fig. 3B (arrows). The alimentary canal was located at the dorsal surface of the liver (Figs. 2, 3A). The EHBD leaves the liver at approximately two-thirds of the length of the liver and enters the alimentary canal at the esophageal-intestinal junction. Microscopic Observation of the Liver in L. reissneri at Larval and Adult Phases A cross-section of the middle region of the liver in the larval phase was fan-shaped and single-lobed. Figure 3A shows a large IHBD in the central region of the fanshaped liver, along with a portal vein and hepatic artery (small arrow). The large IHBD was the only bile duct 1158 MORII ET AL. Fig. 1. Color photographs of lampreys, Lethenteron reissneri. A: Lateral view of a larva. B and C: Lateral views of a lamprey at the later metamorphosing stage, showing the thin white eye circumscribing the dark eye (arrow). D: Lateral view of the anterior region of the immature adult. E: Ventral view of the anterior region of the immature adult. Alignment of the teeth shows the characteristic feature of this species. F: Lateral view of the sexually maturing male, showing a urinogenital papilla (arrow). G: Lateral view of the sexually maturing female, showing large eggs within the abdomen (arrow and inset). surrounded by thick ﬁbrous connective tissue at this larval stage. Many medium IHBDs were also seen throughout the whole liver (Fig. 3A, arrowheads). Figure 3B shows small IHBDs (arrows) that drain bile acids produced by hepatocytes via bile canaliculi (arrowheads), which are tubular lumina surrounded by three to six hepatocytes. In adult lampreys, large, medium, and small IHBDs were absent, as were the GB and EHBD (Fig. 3C,D). Fibrous connective tissue was seen near the portal vein (Fig. 3C, asterisk). We could not identify any bile canaliculi between hepatocytes (Fig. 3D). metamorphosis. We therefore investigated more precisely when the degeneration of bile ducts occurs during the stages of metamorphosis. We also tried to observe the associated morphological changes of bile canaliculi. In the late larval phase, no morphological changes were observed in epithelial cells of the CD (Fig. 4A, arrow) or bile canaliculi (Fig. 4B, arrowheads). In the early metamorphosing stage, a few subtle morphological changes were observed in biliary epithelial cells of the CD (Fig. 4C, arrow), with reduced height of biliary epithelial cells and a thickened basal membrane. Signiﬁcant dilation of bile canaliculi around the portal vein of the liver was also observed in the early metamorphosing stage (Fig. 4D, arrowheads), from 2.34 0.76lm (N ¼ 131 over 2 animals) at the late larval phase to 4.69 1.85 lm (N ¼ 153 over 2 animals) at the early metamorphosing stage. In the later metamorphosing stage, the CD had disappeared and the height of biliary epithelial cells was Microscopic Observation of the Liver of L. reissneri During the Metamorphosing Period Observation of both larval and adult lampreys revealed that the bile duct system degenerated during BILE SYSTEM APOPTOSIS DURING METAMORPHOSIS Fig. 2. Schematic diagram of the bile duct system in the liver of lamprey. Bile juice produced by hepatocytes is secreted to bile canaliculi, and then passes through small, medium, and large intrahepatic bile ducts (IHBDs) and is temporarily stored in the gall bladder (GB) via the cystic duct (CD). Upon feeding, stored bile juice is excreted to the alimentary canal via the CD, large IHBD and extrahepatic bile duct (EHBD). reduced in the large IHBD (Fig. 4E, large arrow), as seen with the CD in the early metamorphosing stage, while small IHBDs (Fig. 4E, small arrows) remained intact. The lumina of bile canaliculi were occupied with debris (Fig. 4F, large arrowhead) and lateral interstitia between hepatocytes were dilated (Fig. 4F, small arrowheads). In the other individual of the later metamorphosing stage, which showed more advanced features, almost the entire bile duct system had disappeared and the lumen of the large IHBD was ﬁlled with debris (Fig. 4G, arrow), resembling that observed earlier in bile canaliculi (Fig. 4F). Many vacuoles were apparent in the cytoplasm of epithelial cells of the large IHBD in the late metamorphosing stage (Fig. 4G, arrow). In this stage, hepatocytes were arranged into cords like those of adult lampreys, and few bile canaliculi were observed (Fig. 4H). Detection of Apoptosis HE staining of metamorphosing lampreys showed that a morphological change in the bile duct system ﬁrst occurred in the early metamorphosing stage (Fig. 4C) and was most prominent in the CD. As a result, we then checked whether TUNEL-positive biliary epithelial cells were present within the CD at this stage. We found that epithelial cells of the CD (Fig. 5A–C) and the cranial half of the large IHBD were TUNEL-positive. Moreover, epithelial cells of the GB were also stained intensely with TUNEL staining (Fig. 5D–F,J,K). Middle and small IHBDs were TUNEL-negative at this stage (Fig. 5G–I). Incubation of the section without a terminal deoxynucleotidyl transferase enzyme resulted in no ﬂuorescent signal, indicating that the signal was speciﬁc and not derived from autoﬂuorescence (Fig. 5L,M). As a positive control, small intestine from C57BL/6 mice was sectioned and stained in the same manner (Fig. 5N), showing some TUNEL-positive signals that were comparable to signals from the sections of lampreys. 1159 To conﬁrm that these cells were undergoing apoptosis, cross-sections of lampreys from the early metamorphosing stage were stained with antibody against active caspase-3, a key regulator of the apoptotic cascade that is activated in an early phase of apoptosis before DNA fragmentation. Interestingly, epithelial cells of the CD in the early metamorphosing stage were negative for active caspase-3 staining (data not shown), despite being positive for TUNEL staining. Instead, epithelial cells of large (Fig. 6A, arrow and Fig. 6D), medium (Fig. 6A, large arrowheads and Fig. 6E), and small (Fig. 6A, small arrowhead and Fig. 6F) IHBDs showed positive results for active caspase-3 staining. Surprisingly, the stained region of large and medium IHBD cells was nuclear (Fig. 6D,E, arrowheads) and that of small IHBD cells was cytoplasmic (Fig. 6F, arrowheads). This indicates that apoptotic cascades in large and medium IHBDs precede those in small IHBDs, because caspase-3 is known to be ﬁrst activated in cytoplasm and then translocated to the nucleus (Kamada et al., 2005). Incubation of the lamprey section with normal rabbit IgG instead of rabbit anti-active caspase-3 antibody resulted in no ﬂuorescent signal (Fig. 6B). As a positive control, small intestine from C57BL/6 mouse was sectioned and stained in the same manner (Fig. 6C), showing active caspase-3 staining in epithelial cells. Despite the onset of apoptosis as demonstrated by TUNEL staining in the CD of early metamorphosing lampreys, cells in the CD were negative for active caspase-3 staining. We therefore undertook further analysis of cells in the biliary tract in late larval lampreys using TUNEL staining and immunostaining with antibody against active caspase-3 (Fig. 7). TUNEL staining in the late larva showed that epithelial cells of the CD (Fig. 7A) and large IHBD were TUNEL-negative, in contrast to the early metamorphosing stage (Fig. 5B). Instead, epithelial cells of the CD (Fig. 7B) and large IHBD were positive for active caspase-3 staining in nuclei, while cells in medium IHBDs were positive for active caspase3 staining in cytoplasm (Fig. 7C). These results suggest that apoptosis in the bile duct system had already begun in epithelial cells of the CD, large IHBD, and medium IHBDs in the late larval phase. The onset of apoptosis around the CD in the late larval phase, as evaluated by TUNEL and active caspase-3 staining, was rather earlier than that evaluated by HE staining (Fig. 4). DISCUSSION The results of the detection of apoptosis along with those obtained from HE staining are shown schematically in Fig. 8. In the late larval phase, active caspase-3 was detected in the nuclei of the CD and large IHBD, suggesting that apoptosis of the biliary system begins around these regions. At the same time, cytoplasmic active caspase-3 staining was observed in medium IHBDs, implying that apoptotic signaling may extend to the peripheral regions. Supporting this idea, active caspase-3 was seen in nuclei in medium IHBDs from the early metamorphosing stage, and cytoplasmic caspase-3 staining further extended to the small IHBDs. The CD, GB, and large IHBD were in the apoptotic stage of DNA fragmentation, as demonstrated by TUNEL staining. In the later metamorphosing stage, almost all parts of the bile duct system had degenerated, as shown by HE 1160 MORII ET AL. Fig. 3. Cross-sections of the livers of larval (A and B) and sexually maturing adult male (C and D) lampreys. A: There is no lobulation in the larval liver. Large IHBD (large arrow) surrounded by ﬁbrous connective tissue, portal vein (PV), and hepatic artery (small arrow) are located centrally. Several medium IHBDs (arrowheads) extend through the liver and conduct bile juice to the large IHBD. B: Biliary canaliculi (arrowheads) are tubular lumina surrounded by three to six hepatocytes. Arrows indicate small IHBDs. C: The bile duct system is absent in the liver of the sexually maturing lamprey, and ﬁbrous connective tissues (asterisk) are present in the region near the portal vein (PV). D: No biliary canaliculi are present in the liver of the sexually maturing adult lamprey. staining (Fig. 4). Notably, the CD was already absent at this stage. Considering that the CD is the region where the apoptotic signals were ﬁrst detected, we believe that the CD initiates the apoptotic signal within the bile duct system. During the later metamorphosing stage, almost all of the bile duct system had disappeared except the large IHBD and some of the medium IHBDs in the liver. In a detailed inspection of HE staining, epithelial cells of bile ducts seemed to disappear from peripheral small IHBDs to central medium IHBDs during the later metamorphosing stage. This observation is in accordance with a previous report stating that smaller peripheral ducts degenerate more rapidly than the large central ducts (Sidon and Youson, 1983a). Taken into account with the report by Sidon and Youson (1983a) and our new ﬁnding that apoptosis of the biliary duct system starts at the CD and progresses to the large IHBD, there is a possibil- ity that biliary leakage is due to the blockage of bile ﬂow along the bile acid storage and excretion pathways. The stage-by-stage feature of hepatocyte transformation with gradual loss of bile canaliculi (Sidon and Youson, 1983b) were described in detail from light and electron microscopic observations. A description and morphometric analysis of the gradual disappearance of tight junctions at the bile canaliculi and the increased area occupied by gap junctions has been reported (Youson et al., 1987a). As a consequence, relocalization of membrane enzymes such as alkaline phosphatase, adenosine triphosphatase, and 5’-nucleotidase occurs from apical to lateral membranes during lamprey biliary atresia (Sidon and Youson, 1984). Moreover, the degeneration of bile ducts is associated with the development of marked periductal ﬁbrosis due to ﬁbroblasts activated by leaked biliary materials (Yamamoto et al., 1986; Youson et al., 1987b). Our observation of the dilation of bile canaliculi Fig. 4. Degeneration of the bile duct system and morphological changes of bile canaliculi during metamorphosis. A and B: In the late larval phase, the GB and CD (arrow) are located near the portal vein (PV). Bile canaliculi (arrowheads) are not dilated. C and D: In the early metamorphosing stage, the CD (arrow) becomes degenerated, and bile canaliculi (arrowheads) are markedly dilated. E and F: In the later metamorphosing stage, the CD has disappeared and the large IHBD (large arrow) shows further degeneration. Small IHBDs (small arrow) remain intact. The lumina of bile canaliculi are occluded with debris (large arrowhead). Interstitia between hepatocytes are recognizable (small arrowheads). G and H: Late in the later metamorphosing stage, the large IHBD (arrow) is ﬁlled with debris and completely occluded. Few bile canaliculi are observed. 1162 MORII ET AL. Fig. 5. TUNEL staining of biliary epithelial cells of the early metamorphosing stage lamprey. A: Nuclei of epithelial cells in the CD are stained with TO-PRO-3 (blue). B: Most epithelial cells constituting the CD are TUNEL-positive (green). C: The adjacent section is stained with hematoxylin and eosin (HE) and the corresponding region is shown. D and J: Nuclei of epithelial cells of the GB are stained with TO-PRO-3 (blue). E and K: Continuous arrangement of TUNEL-positive cells (green) is detected in epithelial cells of the GB. F: The adjacent section is stained with HE and the corresponding region is shown. Arrowheads in A, B, D, and E indicate TUNEL-positive nuclei. G–I: Low magniﬁcation images of medium and small IHBDs in the early metamorphosing stage. Epithelial cells of the medium and small IHBDs are TUNEL-negative. L and M: Section adjacent to J and K, stained without terminal deoxynucleotidyl transferase enzyme. N: TUNEL staining of a section of small intestine from C57BL/6 mouse, showing some TUNEL-positive signals comparable to signals from the sections of lampreys. Fig. 6. Immunohistochemical staining of active caspase-3 in biliary epithelial cells of lampreys in the early metamorphosing stage. Livers of early metamorphosing stage lampreys were stained with anti-active caspase-3 antibody (green), followed by staining with TO-PRO-3 (blue) for nuclei. Merged images (D–F) and merged images overlaid on differential interference contrast (DIC) images (A–C) are shown. A: Active caspase-3 staining was detected in almost all epithelial cells of large (arrow), medium (large arrowheads), and small (small arrowhead) IHBDs. B: The section was stained with normal rabbit IgG instead of anti-active caspase-3 antibody, and showed no staining in medium IHBDs (arrowheads). C: Active caspase-3 staining of a section of small intestine from C57BL/6 mouse, showing some signals comparable to the signals from lamprey sections. D and E: Higher magniﬁcation images of the large (D) and medium (E) IHBDs, showing nuclear localization of active caspase-3 staining (arrowheads). F: Higher magniﬁcation image of the small IHBDs, showing cytoplasmic localization of active caspase-3 staining (arrowheads). 1164 MORII ET AL. Fig. 7. Detection of apoptosis of biliary epithelial cells of the late larval phase lamprey. A: Livers of late larval phase lampreys were stained with TUNEL staining (green), followed by staining with TO-PRO-3 (blue). TUNEL-positive cells were not detected in CD. B: Nuclear translocation of active caspase-3 (green) was seen in epithelial cells of the CD (arrowheads). C: Accumulation of active caspase-3 (green) in cytoplasm was seen in epithelial cells of the medium IHBD (arrowheads). and lateral interstitia between hepatocytes is consistent with these previous reports. Intrahepatic cholestasis may also regulate the remodeling of the hepatocytes, or more speciﬁcally the disappearance of tight junctions in hepatocytes surrounding the bile canaliculi. If this is the case, it would be interesting to know whether farnesoid X receptor, for which bile acids are known to be a ligand, is involved in the remodeling process of hepatocytes. We could not observe the EHBD in the advanced later metamorphosing stage animals. This observation is quite different from earlier observations that the EHBD transforms into a caudal endocrine pancreas in all Northern Hemisphere lampreys (Youson and Elliott, 1989; Youson and Cheung, 1990). We could actually observe pancreas-like tissues at the esophageal-intestinal junction throughout the metamorphosing period (data not shown), but whether these pancreas-like tissues were derived from the EHBD was not certain, as pancreatic cells are known to derive not only from the EHBD, but also from the diverticulum or the region around the esophageal-intestinal junction. Immunohistochemical analysis with anti-insulin and anti-somatostatin antibodies is needed to clarify the origin of the endocrine pancreas in L. reissneri, as previously demonstrated for other species (Youson and Elliott, 1989; Youson and Cheung, 1990). Biliary atresia is the most common and serious neonatal hepatobiliary disorder in humans. In human pathology, inﬂammatory obstruction of the EHBD is accompanied by a characteristic intrahepatic portal lesion. Our observation that, in the lamprey, obstruction of the CD and large IHBD precedes degeneration of BILE SYSTEM APOPTOSIS DURING METAMORPHOSIS 1165 small IHBDs and subsequent cholestasis indicates a similarity between human and lamprey biliary atresia, and suggests the possibility of using the lamprey as a model for various human biliary diseases such as biliary atresia, liver ﬁbrogenesis, and cholestasis, as previously suggested (Youson and Sidon, 1978; Yamamoto et al., 1986; Youson, 1993). Our TUNEL staining and active caspase-3 staining revealed that apoptosis was actually involved in the degeneration process of lamprey biliary atresia. Also in human biliary atresia, several reports have stated that biliary apoptosis may play an important role in obstructive cholangiopathy (Funaki et al., 1998; Erickson et al., 2008; Harada et al., 2008). Revealing the upstream factors that control the initiation of the apoptotic cascade may be useful for elucidating the etiology of not only lamprey, but also human biliary atresia. The lamprey loses the entire bile duct system during metamorphosis (Youson, 1993). However, no biliary cirrhosis is seen in the adult lamprey. In humans, the main method of cholesterol excretion is enterohepatic circulation, and obstruction of this circulation results in death (Ramm et al., 1998; Issa et al., 2001). Conversely, the adult lamprey can sulfonate bile acids in hepatocytes for solubility, and release them into the river environment via the gills (Venkatachalam, 2005). Recently, sulfation of bile acids has been reported to take place in the normal human liver, and sulfated bile acids are increased in several cholestatic diseases, including biliary atresia (Alnouti, 2009). Revealing the mechanisms that control the sulfation of bile acids in the lamprey liver during biliary atresia may contribute to the treatment of human obstructive cholangiopathy. ACKNOWLEDGMENTS The authors thank Hideki Sugiyama (Akita Prefectural Fisheries Promotion Center, Akita, Japan) and Masayuki Kumagai (Ziban Kankyo Consultant, Akita, Japan) for collecting and taking photographs of lampreys and also for useful discussions. LITERATURE CITED Fig. 8. Schematic presentation of apoptosis during metamorphosing period of a lamprey, L. reissneri. 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