Mutations affecting development of zebrafish digestive organs

Abstract

The zebrafish gastrointestinal system matures in a manner akin to higher vertebrates. We describe nine mutations that perturb development of these organs. Normally, by the fourth day postfertilization the digestive organs are formed, the epithelial cells of the intestine are polarized and express digestive enzymes, the hepatocytes secrete bile, and the pancreatic islets and acini generate immunoreactive insulin and carboxypeptidase A, respectively. Seven mutations cause arrest of intestinal epithelial development after formation of the tube but before cell polarization is completed. These perturb different regions of the intestine. Six preferentially affect foregut, and one the hindgut. In one of the foregut mutations the esophagus does not form. Two mutations cause hepatic degeneration. The pancreas is affected in four mutants, all of which also perturb anterior intestine. The pancreatic exocrine cells are selectively affected in these four mutations. Exocrine precursor cells appear, as identified by GATA-5 expression, but do not differentiate and acini do not form. The pancreatic islets are spared, and endocrine cells mature and synthesize insulin. These gastrointestinal mutations may be informative with regard to patterning and crucial lineage decisions during organogenesis, and may be relevant to diabetes, congenital dysmorphogenesis and disorders of cell proliferation.

Full text

INTRODUCTION
The goal of this work is to identify genes that fashion the ver-
tebrate gut and its derivative organs. We hope to identify
signals that guide lineage assignment, promote region-specific
tubular growth and budding, and maintain the differentiated
state.
The principle cellular functions of the gut are the digestion
and absorption of nutrients, the former dependent upon
exocrine and the latter upon absorptive epithelial cells. These
cells differ in specialized function between the anterior and
posterior intestine. The hepatocytes of the liver are crucial for
metabolism of a broad array of endogenous and exogenous
substances and for excretion of bile. The pancreas is composed
both of acini of exocrine cells, which secrete digestive
enzymes such as lipases and peptidases, and the Islets of
Langerhans, which secrete insulin and other peptide hormones.
The gut in higher vertebrates originates from the endoder-
mal-lined anterior and posterior intestinal portals (AIP and
PIP), which will give rise to the fore- and hindgut, respectively.
Although the details of assembly vary among species, in
general a continuous tube is generated from these pouches by
endodermal growth and migration combined with ventral
folding of the embryo (Lamers et al., 1987). The foregut
becomes the esophagus, stomach, and duodenum. The hindgut
becomes the distal large intestine, and the region in between,
the midgut, becomes the intermediate zone of the small and
large intestines. Growth of the gut is accommodated by looping
and rotation as the absorptive surface is incresed by villus
formation. The pancreas and liver originate as endodermal
buds from the foregut.
During maturation of the intestine, the lining cells develop
from a simple cuboidal to a polarized columnar epithelium
composed of several distinctive cell types. Outside the
basement membrane there is growth of connective tissue,
blood vessels, lymphatics, and nerves. Epithelial buds from the
foregut extend into the adjacent mesenchyme and generate the
pancreas and liver.
The signals that guide these cell fate decisions and maintain
the differentiated state are unknown. Explant experiments
suggest that the competence of the AIP (Le Douarin et al.,
1968; Sumiya, 1976) differs from the PIP (Sumiya, 1976) in
that only the former can generate anterior gut derivatives, and
does so only in the presence of an appropriate inductive signal.
Precardiac mesoderm is necessary for the liver to develop from
foregut endoderm (Le Douarin, 1975).
When the experiments reported here were initiated it was not
clear how fruitful a genetic approach would be to a vertebrate
organ system such as the gastrointestinal tract, because it
develops relatively late and depends upon cascades of prior
embryological decision-making. However, targeted mutation
of known genes in mice has shown, for example, that absence
of IPF-1 blocks development of pancreas (Jonsson et al.,
1994), loss of neuronal nitric oxide synthase causes hypertro-
phy of the stomach (Huang et al., 1993), and mice homozy-
gous for a null mutation in Bcl-2 show degeneration of intesti-
321
Development 123, 321-328
Printed in Great Britain © The Company of Biologists Limited 1996
DEV3332
The zebrafish gastrointestinal system matures in a manner
akin to higher vertebrates. We describe nine mutations that
perturb development of these organs. Normally, by the
fourth day postfertilization the digestive organs are formed,
the epithelial cells of the intestine are polarized and express
digestive enzymes, the hepatocytes secrete bile, and the pan-
creatic islets and acini generate immunoreactive insulin and
carboxypeptidase A, respectively. Seven mutations cause
arrest of intestinal epithelial development after formation
of the tube but before cell polarization is completed. These
perturb different regions of the intestine. Six preferentially
affect foregut, and one the hindgut. In one of the foregut
mutations the esophagus does not form. Two mutations
cause hepatic degeneration. The pancreas is affected in four
mutants, all of which also perturb anterior intestine. The
pancreatic exocrine cells are selectively affected in these
four mutations. Exocrine precursor cells appear, as identi-
fied by GATA-5 expression, but do not differentiate and
acini do not form. The pancreatic islets are spared, and
endocrine cells mature and synthesize insulin. These gas-
trointestinal mutations may be informative with regard to
patterning and crucial lineage decisions during organogen-
esis, and may be relevant to diabetes, congenital dysmor-
phogenesis and disorders of cell proliferation.
Key words: gut, intestine, liver, pancreas, zebrafish
SUMMARY
Mutations affecting development of zebrafish digestive organs
Michael Pack, Lilliana Solnica-Krezel, Jarema Malicki, Stephan C. F. Neuhauss, Alexander F. Schier,
Derek L. Stemple, Wolfgang Driever and Mark C. Fishman*
Cardiovascular Research Center, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA and
Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
*Author for correspondence

322
nal villi (Kamada et al., 1995). These phenotypes illustrate the
potential power of genetics in dissecting the steps of organ-
otypic decision-making, despite the inherent complexities of
large multicellular tissue arrays.
MATERIALS AND METHODS
Fish, embryos and larvae
Fish were raised and handled as described by Westerfield (1993). Day
1 of development was considered to commence 24 hours after fertil-
ization. The ENU mutagenesis screen is described in the accompa-
nying paper (Driever et al., 1996). Progeny were screened for devel-
opmental defects involving the digestive organs on days 5 and 6 using
a Wild M10 dissecting microscope. Complementation analyses were
performed by pairwise matings between all members of groups
assigned locus names, with the exception of no relief
m264
, which was
not complemented to piebald
m497
. Three mutants (m73, m140, m750)
were lost as a result of illness during the course of this screen, and
are not available for further analysis.
Histology, immunocytochemistry and electron
microscopy
Embryos and larvae were fixed in 4% paraformaldehyde, embedded
in methacrylate (JB-4, Polysciences, Warrington PA) and 3-5
µm
serial sections were stained with hematoxylin and eosin, or
methylene blue-azure II. Labelling for glycogen was performed using
periodic acid-Schiff staining with and without diastase. For immuno-
cytochemistry, embryos and larvae were fixed in Bouin’s and
embedded in paraffin. Five µm serial sections were stained with a
guinea pig anti-porcine insulin antibody (Linco, Eureka MO), 1:100,
overnight at 4°C, or rabbit anti-bovine carboxypeptidase A antibody
(Rockland Inc., Gilbertsville PA), 1:1000, overnight at 4°C. Texas
red-conjugated goat anti-guinea pig and FITC-conjugated goat anti-
rabbit antibodies (Jackson Immuno Research, West Grove, PA) were
used as secondary antibodies, 1:100, for 2 hours at room tempera-
ture. For anti-cytokeratin staining, larvae and adult tissue were fixed
in 4% paraformaldehyde, embedded in paraffin, sectioned, and
treated with 0.2 mg/ml pronase XXV (Sigma) in PBS at 37°C for 30
minutes prior to application of the anti-cytokeratin antibody
(AE1/AE3; Boehringer Mannheim) 1:100, 4°C overnight, followed
by peroxidase-conjugated biotinylated goat anti-mouse secondary
antibody (ABC Elite Kit; VectorLabs). For laminin imunostaining,
rabbit anti-laminin antibody (Sigma) 1:200 was used at 4°C
overnight, followed by a biotinylated goat anti-rabbit antibody (ABC
Elite Kit, VectorLabs). For glucagon immunostaining, rabbit anti-
glucagon antibody (generously provided by Dr Julia Polak, Royal
Postgraduate Medical School, London, UK) was used 1:50, overnight
at 4°C, followed by a FITC-conjugated goat anti-rabbit secondary
antibody (Jackson Immuno Research, West Grove, PA.), 1:100 for 2
hours at room temperature.
Histochemical detection of leucine aminopeptidase, lipase and
ATPase was accomplished using standard protocols (Bancroft and
Stevens, 1990), as was alkaline phosphatase (Genius System,
Boehringer Mannheim).
For electron microscopy, embryos and larvae were fixed in 2% glu-
taraldehyde for 1 hour at room temperature and postfixed in 1%
osmium tetroxide, 1 hour, at 4°C. Specimens were dehydrated in
ethanol, infiltrated with epoxy resin (Polysciences, Warrington PA),
cut, mounted and stained following standard EM protocols and
viewed with a Philips CM10 electron microscope.
Whole mount in situ hydridization was performed as described
(Oxtoby and Jowett, 1993), using an anti-sense riboprobe generated
from a full-length zebrafish GATA-5 cDNA, a generous gift of
Leonard Zon (Boston, MA), and were viewed and photographed using
a Wild M10 dissecting microscope.
RESULTS
Zebrafish gastrointestinal tract development
The adult zebrafish digestive system is similar to that of other
vertebrates (Fig. 1). The intestinal epithelium (Fig. 1A)
includes most of the cell types observed in the small intestine
of other vertebrates (absorptive, endocrine, goblet) but lacks
crypts (Harder, 1975). Paneth cells have not been described in
the fish intestine and an intestinal stem cell has not been iden-
tified in the teleost intestine, although there are differentiated
proliferating cells in the base of the intestinal folds (Rombout
et al., 1984). In Cyprinids, fat is absorbed in the proximal
intestine and protein absorption occurs more distally (Rombout
and Taverne-Thiele, 1984). A short terminal segment of the
intestine is likely to be involved in ion transport and may be a
homologue of the colon. As in other vertebrates, peristalsis is
achieved by the contraction of an inner circular and outer
longitudinal layer of smooth muscle, and is regulated by
enteric nerves. The structure of the anterior end of the gut
varies among vertebrates. As in all Cyprinids, the zebrafish is
stomachless and the intestine is continuous with the pharynx
through a short esophagus composed of a stratified squamous
epithelium (Harder, 1975). The teleost liver (Fig. 1B)
M. Pack and others
Fig. 1. Histology of adult zebrafish digestive organs. (A) Cross
section of intestine. Arrow points to intestinal epithelium, arrowhead
to outer longitudinal smooth muscle; * indicates inner circular
smooth muscle. (B) Liver. Arrow points to intrahepatic bile duct.
(C) Pancreas. e, exocrine cells; h, hepatocytes; i, islet. Scale bars,
A, 25 µm; B and C, 15 µm.

323Zebrafish digestive organ mutations
resembles that of the mammal but lacks a lobular archi-
tecture with portal tracts (Harder, 1975). The hepato-
cytes are arranged as cords of cells bathed in sinsuoidal
portal blood. The intrahepatic bile ducts arise from bile
cannaliculi and are scattered throughout the liver
parenchyma. As in other vertebrates, a gall bladder is
present for the storage of bile. The adult zebrafish
pancreas (Fig. 1C) is a less coherent structure than that
of the mammal in that several prominent islets are
located near the proximal end of the pancreatic duct
and accessory islets and exocrine tissue are scattered
along the intestinal mesentery in close proximity to the
pancreatic ducts (M. Pack, personal observation).
Fate maps of the zebrafish blastula indicate that
endodermal precursors originate from marginal blas-
tomeres, which involute early in gastrulation (Warga
and Kimmel, 1990). As shown in Fig. 2A, at 36 hours
postfertilization (hpf) the developing zebrafish gut is
a cord of radially aligned cells. The anus and
esophagus have not yet developed. By 52 hpf, as
shown in Fig. 2B, a lumen is evident in the rostral gut,
although not yet connected to the pharynx. By 72 hpf
(Fig. 2C), differentiation is evident. Goblet cells have
appeared and proximal intestinal cells have begun to
polarize. The developing esophagus joins the
proximal intestine to the pharynx. By 4 days postfer-
tilization (dpf) the exocrine cells of the pancreas have
Fig. 2. Histology of the digestive organs in
the developing zebrafish. Cross sections of
proximal gut. (A) A small lumen is visible in
the developing intestine (arrow) of the 36
hour postfertilization (hpf) embryo. (B) The
intestine has enlarged (arrow) and the
pancreatic islet (i) and exocrine precursor
cells (arrowhead) are visible in the 52 hpf
embryo. (C) The intestinal epithelium
(arrow) has begun to polarize in the 72 hpf
larvae. Exocrine precursors surround the islet
(arrowhead). (D) The intestinal epithelium
(arrow) has fully polarized and the pancreatic
exocrine cells have differentiated
(arrowhead) at 4 dpf. The liver is rostral to
the plane of section in A and B. e, exocrine
pancreas; i, pancreatic islet; l, liver; n,
notochord; y, yolk; * labels pneumatic duct
of gas bladder. Scale bars, A and D, 25 µm;
B, 20 µm; C, 30 µm.
Fig. 3. Mutations affecting the developing zebrafish
intestine. (A,C,E,G,I) Lateral views of living 5 dpf larvae.
(B,D,F,H,J) Corresponding histological cross-sections
through the mid-intestine. Lumen size varies with position
along the anterior-posterior axis and with muscular
contractions. (A,B) Wild type. The intestinal epithelium
(arrow) is highly folded, bile is visible in the lumen and the
gas bladder is inflated. The intestinal epithelium is polarized
with basal nuclei and a microvillus brush border. (C,D) slj
m74
,
(E,F) m750, (G,H) sst
m311
, (I,J) pie
m497
. The intestinal wall
of all mutants is thin, lacks folds and the epithelium is not
fully polarized and shows signs of degeneration. Scale bar,
B, F and H, 20 µm; D, 15 µm; J, 25 µm.

324
differentiated, and the intestinal epithelium is polarized along
its entire length and has a prominent microvillus brush border
(Fig. 2D). Intestinal folds have developed in the rostral and
mid-gut and peristaltic contractions are obvious. At 5 dpf
proximal-distal regionalization is demonstrable by staining
for lipase in the proximal and aminopeptidase in the distal
epithelium (data not shown).
By Nomarski optics, liver cells and bile are visible by 3 dpf
and hepatic blood flow by 4 dpf. Hepatocytes are evident his-
tologically by 36 hpf, the extrahepatic biliary duct by 52 hpf,
and endothelial-lined sinusoids at 3 dpf. The pancreas is visible
using Nomarski optics at 4 dpf.
We have developed a series of molecular
markers that characterize specific cell types of the
zebrafish gut, liver, and pancreas and which have
been helpful for mutant analysis. These are
outlined in Table 1.
Mutations affecting digestive organs
The gastrointestinal tract is relatively obscured by
overlying tissues. Peristalsis is slow and irregular,
so cannot highlight defects as do contractions of
the heart for the cardiovasculature. In addition,
because the gut develops late relative to many
developmental decisions, it is secondarily
affected by mutations with effects elsewhere. We
selected for further studies those with relatively
gut-specific effects (Table 2).
Mutations that affect intestinal epithelium
Seven mutants affecting intestinal development
were isolated. In six (examples of four mutants
shown in Fig. 3), the intestinal tube develops
without evident abnormality until 4 dpf. At this
time the wild-type intestine (Fig. 3A,B) is folded
and contains bile, and the epithelial cells are
polarized and have a microvillus brush border. By
contrast, in slim jim (slj) (Fig. 3C,D), m750 (Fig.
3E,F), straight shot (sst) (Fig. 3G,H), piebald
(pie) (Fig. 3I,J) and no relief (not shown), the
intestinal epithelium is thin and lacks folds and
the cells have only few scattered microvilli and
lack evident polarization. In each of these mutants the intesti-
nal epithelium begins to degenerate by 4-5 dpf.
Limitation of the defect to specific regions is a feature of
several mutants. Anterior intestine is selectively perturbed in
slj, posterior intestine is selectively affected in meltdown (mlt)
and the esophagus in m140 embryos. In slj mutants (Fig.
3C,D), despite the apparent lack of polarization of the anterior
intestine, some cellular differentiation does occur. As shown
by electron microscopy at 4 dpf, adherens and tight junctions
are clearly visible between cells in the anterior intestinal
epithelium of wild type (Fig. 4A,C) and slj (Fig. 4B,D). In slj
M. Pack and others
Table 1. Cell-specific markers for the
digestive organs of zebrafish embryos and
larvae
Marker Cells
GATA-5 Hepatocytes, exocrine cells of
pancreas, intestinal epithelium
Cytokeratin Biliary cells of liver, ductular
cells of pancreas, intestinal
epithelium
Lipase Intestinal epithelium (proximal)
Leucine aminopeptidase Intestinal epithelium (mid-
distal)
Insulin Pancreatic islet
Glucagon Pancreatic islet
Carboxypeptidase A Exocrine cells of pancreas
Laminin Basement membrane of intestine
ATP-ase Intestinal epithelium, endothelial
cells of liver sinusoids
Alkaline phosphatase Intestinal epithelium
Fig. 4. Electron micrographs showing regional intestinal differentiation.
(A,C,E) wild type and (B,D,F) slj
m74
. (A) The wild-type anterior intestinal
epithelium is columnar and has a prominent microvillus brush border (arrow).
(B) slj
m74
anterior intestinal epithelial cells are cuboidal with few microvilli.
(C,D) Adherens (arrowhead) and tight junctions (arrow) are present in the anterior
intestine of both (C) wild type and (D) slj
m74
. (E,F) Posterior intestinal epithelium
appears normal in both (E) wild type and (F) slj
m74
. * labels the intestinal lumen.
Magnification: A, ×950; B, ×1100; C, ×22,000; D, ×28,500; E, ×2200; F, ×2950.

325Zebrafish digestive organ mutations
these cells are also immunoreactive for a cytokeratin marker,
the expression of which normally appears contemporaneously
with polarization, and they have apical alkaline phosphatase
activity (data not shown). The slj posterior intestinal epi-
thelium is normal, even as assessed by electron microscopy
(Fig. 4F; wild type, Fig. 4E). In mlt, however, the mid- and
distal intestinal epithelium is disorganized and the mes-
enchyme expanded while the anterior intestine, liver and
pancreas develop normally (Fig. 5). In m140, the pharynx,
proximal intestine and liver begin to develop normally, but
remain unconnected due to failure of esophageal development
(Fig. 6). Subsequently the pharyngeal and intestinal epithelium
degenerate.
In the four other intestinal mutants, epithelial degeneration
occurs throughout the intestine. In one of these examined by
electron microscopy, pie (Fig. 3I,J), the epithelial cells become
polarized and develop adherens and tight junctions before
degenerating (data not shown). In all mutations affecting the
intestine, pronephric and mesonephric epithelium develop
normally, indicating that the epithelial abnormality is limited
to the gastrointestinal system.
Mutations that affect the liver
Two liver mutants were identified
(Fig. 7). In m73 mutants (Fig. 7C,D),
reddish brown pigmentation accumu-
lates in the liver. This is evident by
Nomarski optics by 4 dpf, although
histologically it appears by 2 dpf.
Hepatocellular degeneration is evident
by histology (Fig. 7D), and the
abnormal liver pigmentation appears
to be due to blood pooling around the
degenerating hepatocytes. Develop-
ment of the intra- and extra-hepatic
bile ducts appears to be normal, as
indicated by the presence of bile in the
4 dpf m73 intestinal bulb and the
presence of anti-cytokeratin
immunoreactivity (data not shown).
m73 embryos also show brain degen-
eration. In humans, glycogen storage
diseases are known to affect liver and
brain. In the absence of feeding,
glycogen is depleted from wild-type
embryos by 6 dpf (Fig. 7G) but
persists in the liver of m73 embryos (Fig. 7H) at 6 dpf. This
is compatible with impaired glycogen utilization in the m73
liver. In the beefeater (bef) liver mutant (Fig. 7E,F) the
histology resembles m73, but there is no brain degeneration.
Mutations that affect the pancreas
The relationship of the endocrine and exocrine lineages of the
pancreas is uncertain. It has been proposed that they share a
common precursor (Alpert et al., 1988; Teitelman et al., 1987;
Gu et al., 1994; Guz et al., 1995), or that endocrine cells derive
from the neural crest (Alpert et al., 1988). Therefore, it is of
interest that four mutants develop islets in the essential absence
of pacreatic acini.
Normally, the exocrine pancreas is first evident by the end
of 3 dpf, a time which precedes overt intestinal differentiation,
and appears fully developed by 4 dpf (Fig. 8A). In slj (Fig. 8B)
and pie mutants, the islet is evident at 4 dpf, and expresses
immunoreactive insulin (Fig. 8D) as does the wild type (Fig.
8C). slj islets are essentially devoid of surrounding cells (Fig.
8B), and the few there are lack zymogen granules. The
exocrine cells of wild-type larvae at this stage express GATA-
Table 2. Mutations affecting the digestive organs
Genetic loci Alleles Phenotype Other phenotypes
Group I: Foregut
slim jim (slj) m74 Intestinal epithelial degeneration (anterior), exocrine pancreas fails to develop Reduced branchial arches (variable)
piebald (pie) m497 Intestinal epithelial degeneration, exocrine pancreas fails to develop
straight shot (sst) m311 Intestinal epithelial degeneration, exocrine pancreas fails to develop Reduced branchial arches
no relief (nor) m264 Intestinal epithelial degeneration, exocrine pancreas fails to develop Reduced branchial arches
- m750 Intestinal epithelial degeneration, small exocrine pancreas
- m140 Esophagus fails to develop, degeneration of intestinal and pharyngeal epithelium
Group II: Hindgut
meltdown (mlt) m498 Posterior intestine rendundant with expanded mesenchyme
Group III: Liver
beefeater (bef) m362 Liver degeneration
- m73 Liver degeneration Brain degeneration
Fig. 5. mlt
m498
perturbs posterior intestine. (A,C) Wild type, (B,D) mel. The posterior intestinal
epithelium is disorganized and the mesenchyme expanded. * labels the intestinal lumen;
arrowheads, posterior intestine; small arrows, pronephric duct. Scale bars, C and D, 30 µm.

326
5 and immunoreactive carboxypeptidase A (Fig. 8E,G). In slj,
carboxypeptidase A immunoreactivity is absent from the
pancreas (Fig. 8F). This failure of exocrine cell development
is also seen using a GATA-5 probe, a marker
of early gut development (Laverriere et al.,
1994). Normally, all exocrine cells of the
exocrine pancreas express GATA-5 at 4 dpf
(Fig. 8G). slj mutants lack GATA-5
expression in this location (Fig. 8H) but
retain it in other locations such as the heart,
liver and intestine. However, at 3 dpf there
is a normal pattern of GATA-5 expression in
the developing pancreas of all embryos of
heterozygous slj matings. This suggests that
in the slj pancreas there may be a transient
population of exocrine precursors which do
not survive. In pie, cellular exocrine differ-
entiation seems to proceed further. Even
though mature acini do not develop, a few
cells express GATA-5 and carboxypeptidase
A in a ring around the islet at 4-5 dpf, in a
pattern that resembles the 3 dpf islet. This
selective failure of pancreatic exocrine
development, with normal growth of islets,
is also seen in sst and nor mutants. In m750
embryos, the exocrine pancreas develops but
is smaller than the wild type.
DISCUSSION
Screening for gastrointestinal
mutations
We report here nine mutations that perturb
gastrointestinal development. This is from a
total of about 500 lines screened specifically
for digestive organ defects at 5-6 dpf. It does
not include mutations with generalized retar-
dation or markedly pleotropic effects. The
pancreas could not be identified reliably by
this type of visual screen independently of
the gut, but was involved in several foregut
defects. Additional screens undoubtedly
might enhance sensitivity by the use of
lineage- or organ-specific markers. For
example, cytokeratin and digestive enzyme
markers distinguish the early stages of
intestinal epithelial differentiation; the pan-
creatic exocrine cells express GATA-5 and
then carboxypeptidase; the pancreatic islets
synthesize insulin and glucagon. Peristalsis can be rendered
visible with dyes, and biliary excretion marked with phenol red
(M. Pack, personal observation).
M. Pack and others
Fig. 6. In m140 the esophagus fails to
develop normally. (A) wild type, saggital
section, 4 dpf. The intestine (arrow) is
joined to the pharynx by the esophagus (*).
(B) m140, 4 dpf. There is no connection
(large arrow) between the intestine (small
arrow) and pharynx, both of which have
begun to degenerate. p, pharynx; g,
glomerulus; y, yolk. Scale bars: A, 40 µm;
B, 30 µm.
Fig. 7. Liver mutants. (A,BG) Wild type. (C,D,H) m73. (E,F) bef
m362
. (A) Lateral view
of wild-type 5 dpf larva with bile (b) in intestinal lumen; arrow points to liver. (B) Cross
section of wild-type liver 5 dpf with erythrocytes in the sinusoids (*) and hepatocytes
(h). (C) The day-4 m73 liver (arrow) has a gray-brown hue. (D) m73 hepatocytes are
degenerating and erythrocytes (arrow) are pooling in the sinusoids. (E) bef
m362
liver
(arrow) has a red-brown hue. (F) bef
m362
hepatocytes also degenerate and compress the
sinusoids. (G) Wild-type liver 6 dpf does not stain with PAS. (H) m73 liver 6 dpf stains
for PAS. Scale bar, 15 µm.

327Zebrafish digestive organ mutations
Patterning and differentiation
Specialization along the length of the gut characterizes all ver-
tebrate digestive systems, and establishment of axial polarity
is assumed to be a key component of its organogenesis,
although it has been without molecular underpinning. Several
mutations disrupt the early stages of gut development in a
region-specific manner. m140 prevents formation of the
esophagus, of interest because poor development of this gut
region is responsible for a set of congenital human disorders
referred to as esophageal atresia, and because intestinal meta-
plasia in the esophagus, which is considered a forerunner of
esophageal cancer, may result from dis-
ordered cell renewal in this organ. slj has
a selective effect upon the anterior
intestine and mlt upon the posterior
intestine. These regions have their origins
in the endoderm of the foregut and
hindgut, respectively, and remain
different in function throughout life. The
molecular underpinning of this regional-
ization is unknown but several homeobox
(Yokouchi et al., 1995; Gaunt et al., 1990;
Geada et al., 1992) and forkhead
(Monagan et al., 1993; Sasaki and Hogan,
1993) genes are expressed in the devel-
oping gut in a region-specific manner and
have been proposed to play such pattern-
ing roles. Xlhbox8 (the Xenopus IPF-1
homologue; Wright et al., 1988), has
specifically been proposed in this regard
for anterior gut.
The signals that regulate the differen-
tiation, death, and renewal of gut epi-
thelial cells are largely unknown. Perhaps
because of the high rate of proliferation,
gut epithelium is an affected site in
mutations of N-myc (Stanton et al., 1992)
and Bcl-2 (Kamada et al., 1995), genes
which affect the balance of cell prolifer-
ation and cell death. In explant culture,
mesenchyme of developing gut induces
epithelial development (reviewed in
Haffen et al., 1987), and, in some cases,
can cause a heterotopic epithelium to
adopt a new fate concordant with that of
the region from which the mesenchyme
was derived (Haffen et al., 1987; Mizuno
and Yasugi, 1990).
In slj and most of the other gut mutants,
differentiation proceeds through the
original stages of organotypic assembly,
with enzyme synthesis and epithelial
polarization, and thereafter the intestine
begins to degenerate. One explanation
could be that a requirement for trophic
support is initiated after the onset of
differentiation, and that elements of this
process, which may result from epi-
thelial-mesenchymal interactions, are
perturbed by the mutations. Precursor and
differentiated cells certainly may have
different trophic dependencies. Along these lines, EGF
receptor mutant mice can generate an intestine, but villus
development is impaired and intestinal architecture becomes
disorganized and degenerates soon after birth (Threadgill et al.,
1995; Miettinen et al., 1995).
Lineage
The lineage relationship of pancreatic endocrine and exocrine
cells is of especial interest, partly because there is no signifi-
cant renewal of either cell type in the adult, which has
important therapeutic implications for diabetes and chronic
Fig. 8. Pancreas mutants. Pancreas development in (A,C,E,G) wild type and (B,D,F,H)
slj
m74
embryos. (A) Cross-section of the pancreas at 4 dpf showing islet (i) and exocrine (e)
cells. (B) In slj
m74
the islet appears normal but the exocrine cells have not developed.
(C) Immunoreactive insulin in the wild-type islet. (D) Immunoreactive insulin in the slj
m74
pancreas. (E) Immunoreactive carboxypeptidase A in the wild-type exocrine pancreas.
(F) There is little if any immunoreactive carboxypeptidase A in the slj pancreas at 4 dpf.
Arrrowheads mark the position of islet cells immunoreactive for insulin. Arrow, background
staining in intestine. (G) GATA-5 in situ hybridization in the wild type shows expression in
the exocrine pancreas (arrows). (H) slj
m74
does not express GATA-5 in the exocrine
pancreas. Scale bars, A,B,D and F, 15 µm; C,E, 20 µm.

328
pancreatitis. Whether there is a common precursor for the two
cell types is controversial. Most cells express exocrine or
endocrine markers alone, but recent reports suggest that there
may be a few cells expressing both (Guz et al., 1995; Gu et al.,
1994). The IPF-1 gene may be a marker of the cells in the
duodenum and pancreas with this bipotentiality (Guz et al.,
1995). Compatible with this suggestion is the observation that,
in the absence of the IPF-1 gene, neither cell type is generated
in the mouse. In slj mutants, islet cells can develop in the
absence of differentiated pancreatic exocrine cells. It is
possible that the GATA-5-expressing population, which tran-
siently surrounds the developing islet at 3 dpf in mutants and
wild type, represents an undifferentiated exocrine-endocrine
precursor, one which could give rise to islet precursors before
being lost by 4 dpf. Transplantation may help to determine
whether the defect affects the exocrine cells in a cell-
autonomous manner.
We thank Chris Simpson, Colleen Boggs and Margaret Boulos for
outstanding technical help, John Lydon and Dennis Brown for invalu-
able assistance with electron microscopy, and Daniel K. Podolsky for
advice and support. This work was supported by NIH RO1-HL49579
(M. C. F.), NIH RO1-HD29761 (W. D.) and a sponsored research
agreement with Bristol Myers-Squibb (M. C. F. and W. D.). Support
for M. P. came from in part from an NIH training grant to Dr Daniel
K. Podolsky, and NIH K11-DK02157 (M. P.). Support for D. L. S.
from the Helen Hay Whitney Foundation, for A. F. S. from Swiss
National Science Foundation and EMBO, and for J. M. from Walter
Winchell-Damon Runyon Cancer Research Fund.
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