First published online October 27, 2004
doi: 10.1242/10.1242/dev.01411
Development 131, 5753-5766 (2004)
Published by The Company of Biologists 2004
Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy
Kristin Lorent1,
Sang-Yeob Yeo2,
Takaya Oda3,*,
Settara Chandrasekharappa3,
Ajay Chitnis2,
Randolph P. Matthews4 and
Michael Pack1,5,
1 Department of Medicine, University of Pennsylvania School of Medicine, 421
Curie Boulevard, Philadelphia, PA 19104-6058, USA
2 Laboratory of Molecular Genetics, NICHD, NIH, 31 Center Drive, 9000 Rockville
Pike, Bethesda, MD 20892-2425, USA
3 Genome Technology Branch, NHGRI, NIH, 49 Convent Drive, 9000 Rockville Pike,
Bethesda, MD 20892-2152, USA
4 Division of Gastroenterology and Nutrition, Children's Hospital of
Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104,
USA
5 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6058, USA
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Fig. 1. Mammalian and teleost liver architecture. (A) Schematic representation of
the mammalian liver lobule. Portal tracts (white circles) surround bilayered
hepatocyte plates (h). Each portal tract contains a portal vein radicle (pv),
a hepatic artery radicle (ha), and 1 or 2 interlobular bile ducts (b). Apical
bicellular canaliculi (c) are located between adjacent hepatocytes.
Fenestrated sinusoidal endothelial cells that line the basal hepatocyte
membrane (depicted in blue) allow uptake and transport of proteins and other
macromolecules. Blood enters the liver lobule through pv and ha radicles, and
flows through sinusoidal channels lined by basal endothelial cells towards the
central vein (cv), the proximal branch of the hepatic venous system. (B)
Schematic representation of the teleost tubular liver. Portal vein radicles
(pv), hepatic artery radicles (ha) and bile ducts are not grouped together in
portal tracts. Note, portal venous and hepatic venous (cv) radicles are
indistinguishable, although they are depicted in different colors for this
schematic. Hepatocytes are arranged in tubules rather than in bilayered
plates, and are surrounded by fenestrated endothelia. Longitudinal, transverse
and oblique sections of hepatocyte tubules are present in histological
sections, but are often difficult to appreciate. Small bile ducts (ducts)
reside within hepatocyte tubules. In this schematic, a bile duct composed of a
single biliary epithelial cell (dc) anastomoses with three hepatocyte
canaliculi (c). Unicellular canaliculi of cyprinid fish are tubular
invaginations of the hepatocyte membrane that extend to a perinuclear
location. Note, biliary-arteriolar tracts (not shown) are described for some
fish. [Adapted from Hinton and Couch
(Hinton and Couch, 1998 ).]
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Fig. 2. Zebrafish adult liver. (A,B) Histological sections from an adult male
liver. In A, two intrahepatic veins are visible (arrows). Identification of
either vein as a portal or hepatic venous radicle is not possible. Sinusoidal
channels containing nucleated red blood cells are visible between hepatocytes,
most prominently in B (white arrowheads). Contiguous sinusoids linking
adjacent venous structures are not seen. Large bile ducts are also visible in
cross section (arrows in B), as is a smaller biliary radicle (arrowhead). Such
ducts are infrequently seen in the liver periphery. (C,D) Confocal projections
of adult liver samples processed for immunohistochemistry (IHC) using
anti-human cytokeratin 18 (C) and anti-human P-glycoprotein (D) antibodies.
Note branching anastomotic network of bile ducts, and tubular canaliculi
described in other teleosts. (E,F) Transmission electron micrographs of adult
liver. In each, ductular cells anastomose with hepatocyte canaliculi that have
prominent microvilli. Multiple canaliculi converging on a single bile duct are
evident (F). bd, bile duct; c, canaliculus; dc, ductular cell; h,
hepatocyte.
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Fig. 3. Zebrafish intrahepatic biliary development. (A-C) Tissue cross-sections of
60-hpf (A) and 70-hpf (B) embryos and a 80-hpf larva (C) processed for
cytokeratin IHC. Nascent ducts (arrows) within the developing liver are
evident at 60 hpf (A) and 70 hpf (B). By 80 hpf (C), a branching ductular
network (arrows) is evident. Arrowheads in A,B indicate the origin of the
extrahepatic duct. (D) Confocal projection of intrahepatic bile ducts in a
5-dpf larva processed for cytokeratin IHC. (E) Tissue section of a 70-hpf
larva processed for P-glycoprotein IHC. Developing canaliculi (arrowheads) are
evident between adjacent hepatocytes. (F) Confocal projection of hepatocyte
canaliculi within the liver of a 5-dpf larva – note the elongated,
tubular canalicular structure. (G,H) Confocal projection generated from
contiguous Z-sections of a 75-hpf larva (G) and a 5-dpf larva (H) processed
for cytokeratin IHC. Intrahepatic bile ducts emerge from the liver to form the
common hepatic duct (chd), and join the cystic duct (cd) and common bile duct
(cbd) that inserts into the intestine (i). (I) Transmission electron
micrograph of a 70-hpf larva, showing a developing canaliculus (c) near the
hepatocyte nucleus (hn). (J) Canaliculus of a 70-hpf larva anastomosing with a
bile duct composed of two ductular cells (dc). (K,L) Transmission electron
micrograph from a 5-dpf larva, showing a ductular cell within a hepatocyte
tubule anastomosing with several canaliculi from surrounding hepatocytes (K).
(L) Distal portion of a bile duct within the center of a hepatocyte tubule.
Electron-dense particles within bile are evident in the duct lumen. e,
esophagus; g, gall bladder; l, liver; pa, pancreas; p, pronephric duct.
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Fig. 4. Zebrafish intrahepatic biliary development occurs independently of liver
vasculature. (A,B) Histological cross-sections through the liver of wild-type
(A) and cloche mutant (B) 80-hpf embryos processed for cytokeratin
IHC. Developing bile ducts (arrows) are present in both wild-type and
cloche embryos. e, esophagus; l, liver; ph, pharynx; p, pronephric
duct. Asterisk indicates sinusoids.
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Fig. 5. Liver expression of zebrafish jagged and notch genes.
(A-C) Lateral view of wild-type 72-hpf larvae processed for RNA in situ
hybridization using jagged 1 (A), jagged 2 (B) and
jagged 3 (C) antisense probes. All three jagged genes are
expressed in the larval liver (arrow), but jagged 2 expression is
most pronounced. Expression in the branchial arches is also evident. (D-G)
Lateral view of wild-type 72-hpf larvae processed for RNA in situ
hybridization using notch 1a (D), notch 1b (E), notch
2 (F) and notch 5 (G) probes. All notch genes are
expressed in the developing larval liver and the branchial arches.
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Fig. 6. jagged gene knockdowns perturb craniofacial and cardiac
development. (A-E,G) Lateral views of (A) wild type, and (B) jagged
1, (C) jagged 2, (D) jagged 3 and (E,G) jagged
2/3 morphants. Note, forebrain and midbrain defects in
jagged 1 morphants (arrowheads) and mild craniofacial defects in the
jagged 3 (D) and jagged 2/3 (E,G) morphants. All
morphants, except jagged 2, have small ears (arrows). jagged
2/3 morphants have pericardial edema (open arrowhead). (F,H)
Alcian Blue staining of 5-dpf wild-type (F) and jagged 2/3
morphant (H) larvae, lateral view. These stainings show that the ethmoid (e),
palatoquadrate (pq) and ceratohyal (ch) cartilages of jagged
2/3 morphants are smaller than in wild-type siblings. Similar
findings are observed in jagged 3 morphants (not shown). cb,
ceratobranchial; hs, hyosymplectic; m, Meckel's cartilage; oa, occipital
arch.
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Fig. 7. jagged 2 and jagged 3 regulate development of the
zebrafish intrahepatic biliary system. (A-I) Confocal projections through the
liver of 5-dpf zebrafish larvae processed for cytokeratin IHC. Intrahepatic
biliary development is normal in jagged 1 (B), jagged 3 (E),
jagged 1/2 (F) and jagged 1/3 (not shown)
morphants, when compared to wild type (A). Biliary development is abnormal in
jagged 2 morphants. In low-dose jagged 2 morphants (C),
rosettes of liver cells with apical cytokeratin staining are noted (arrow).
High-dose jagged 2 morphants (D) have frequent rosettes (arrow) and a
small complement of normal bile ducts. Co-injection of the jagged 3
morpholino with either a low dose (G), or high doses (not shown), of the
jagged 2 morpholino severely disrupts intrahepatic bile duct
development. These larvae have few recognizable bile ducts. Instead,
immunoreactive cytokeratin is located apically, within hepatocyte rosettes
(arrow), or along vascular sinusoids (asterisk) that in teleosts normally
express low levels of cytokeratins. (H) Twenty percent of larvae co-injected
with full-length human Jagged 1 mRNA and jagged 2/3
morpholinos have only rare rosettes (arrow). (I) Low-power confocal projection
showing normal gallbladder and extrahepatic bile duct development in a
jagged 2/3 morphant larva. Arrow indicates liver cell
rosette; arrowhead indicates origin of common hepatic duct (chd) within the
liver. cbd, common bile duct; cd, cystic duct; chd, common hepatic duct; g,
gallbladder; i, intestine; l, liver; pa, pancreas; pd, pancreatic duct.
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Fig. 8. Jagged-mediated Notch signaling may regulate a binary cell-fate decision of
zebrafish hepatoblasts. (A-F,I) Histological sections through the liver of
5-dpf wild-type (A-C) and jagged 2/3 morphant (D-F,I) larvae
processed for cytokeratin and P-glycoprotein IHC. (A) A Branching network of
bile ducts is evident in this wild-type larva. (B) Section in A processed for
histology with superimposed pseudocolored cytokeratin pattern (magenta). (D)
Four hepatocyte rosettes (arrows) are shown in this section through the liver
of a jagged 2/3 morphant larva. Weak cytokeratin expression
is also present in endothelial cells lining sinusoids seen in cross-section
(arrowheads). (E) Section in D processed for histology with superimposed
pseudocolored cytokeratin pattern (magenta). These sections show cytokeratin
within the apical region of rosette cells (arrow indicates one of the four
rosettes identified in D) and in surrounding sinusoidal endothelial cells
(arrowhead). Dashed lines outline individual hybrid cells in two hepatocyte
rosettes. (C,F) Wild-type (C) and jagged 2/3 morphant (F)
larvae processed for P-glycoprotein IHC. Individual canaliculi are seen in the
liver of wild-type larvae. In morphants, the P-glycoprotein is clustered in
the central region of rosettes (arrow points to middle rosette). Compared with
wild type, there is much less P-glycoprotein staining in the morphant liver.
(I) Section shown in F stained for histological analysis. Red asterisks
identify the location of P-glycoprotein+ cells (F). (G,H) Electron micrographs
through the liver of 5-dpf jagged 2/3 morphant larvae.
Low-power view (G) shows rosette cells with apical canaliculi (c), best
appreciated in a high power view (H). Ultrastructurally, cells comprising the
rosettes (dashed line in G) resemble hepatocytes. However, cytokeratin, a
biliary marker, is also located apically in these cells (D).
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Fig. 9. Jagged-mediated Notch signaling regulates pancreatic duct development.
Confocal projections through the posterior pancreas of 5-dpf wild-type (A,C,E)
and jagged 2/3 morphant (B,D,F,I) larvae processed for
cytokeratin IHC (green; duct marker), and carboxypeptidase (red; acinar cell
marker) IHC. (A) Immunoreactive cytokeratin outlines ducts within the
wild-type pancreas (arrow), as well as a large ventral blood vessel
(*). (B) Only a few large ducts (arrowheads) are visible in
jagged 2/3 morphants. Most regions of the morphant pancreas
are devoid of ducts and instead contain enlarged acini (arrows) that
ectopically express cytokeratin. Acinar structure in morphant larvae was
confirmed ultrastructurally (not shown). (C) Immunoreactive carboxypeptidase A
(red) is localized in small acini in the wild-type pancreas. (D) Acini in
5-dpf jagged 2/3 morphants are enlarged (arrows) and may
have dilated lumens (lower arrow). Acinar cells express carboxypeptidase A
(red). (E,F) Superimposed confocal projections through wild-type and
jagged 2/3 morphant pancreas shown in A and C, and B and D,
respectively. The acinar cells within the enlarged morphant acini (arrows in
F) ectopically express the cytokeratin duct marker on the apical and lateral
cell surface of the acinar cells. (I) Thin optical section (10 µm) through
the lumen of the larger acinus depicted in B and F, showing apical cytokeratin
in cells lining the acinar lumen (arrow). (G,H) Low power, whole-mount image
of the 5-dpf wild-type (G) and jagged 2/3 morphant (H)
larval pancreas; larvae processed for carboxypeptidase A IHC (green). Note the
enlarged, dispersed acini in the jagged 2/3 larvae (H)
compared with wild-type sibling (G).
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Fig. 10. Multiple Notch receptors regulate zebrafish intrahepatic biliary
development. (A-I) Confocal projections through the liver of 5-dpf zebrafish
larvae processed for cytokeratin IHC. (A) Wild-type intrahepatic biliary
ducts. (B) Normal biliary development in notch 2 morphants. (C) Rare
hepatocyte rosettes are seen in notch 5 morphants (arrow). (D)
notch 2/5 morphants have rosettes (arrow) and reduced duct
density. (E,F) Severe biliary defects in jagged 2/notch 2
and jagged 2/notch 5 morphants. Note rosettes (arrows) and
also pronounced vascular cytokeratin staining in the jagged
2/notch 2 morphant. (G) jagged 3 knockdown does not
augment the mild notch 5 morphant biliary phenotype (arrow, rosette).
(H,I) 96-hpf hsp70:GAL4 and hsp70:GAL4; UAS:notch1aICD
transgenic larvae processed for cytokeratin IHC. Ectopic biliary ducts
(arrows) are only visible in the bigenic larvae following heat shock at 74 hpf
and 86 hpf. Asterisk indicates endothelial cytokeratin staining.
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