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Onset of Apoptosis in the Cystic Duct During Metamorphosis of a Japanese Lamprey Lethenteron reissneri.

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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: senoo@ipc.akita-u.ac.jp
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.
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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).
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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.
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Fig. 8. Schematic presentation of apoptosis during metamorphosing period of a lamprey, L. reissneri. In the late larval phase, nuclear
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