Download Characterization of rat epimorphin/syntaxin 2 expression suggests a

Characterization of rat epimorphin/syntaxin 2 expression
suggests a role in crypt-villus morphogenesis
ALKA GOYAL,1 RENU SINGH,2 ELZBIETA A. SWIETLICKI,2
MARC S. LEVIN,2 AND DEBORAH C. RUBIN2
1Department of Pediatrics and 2Department of Medicine, Washington University School
of Medicine and Barnes-Jewish Hospital, St. Louis, Missouri 63110
intestine; mesenchyme; ontogeny; organogenesis
THE MAMMALIAN small intestine contains a continuously
proliferating and differentiating epithelium. The primary anatomic and functional component of this epithelium is the crypt-villus unit. Anchored stem cells are
located in the crypts of Lieberkühn and give rise to
proliferating daughter cells that differentiate into four
major intestinal epithelial cell types, including enterocytes, enteroendocrine and goblet cells, and Paneth
cells (8). Cellular differentiation occurs coincident with
migration from the stem cell/proliferative region onto
the villus or to the base of the crypts.
Crypt-villus axis morphogenesis begins in the rodent
small intestine during late gestation. Before that time,
the intestinal tube is composed of a single layer of
undifferentiated endoderm surrounded by mesenchymal cells. From gestational day 13 (G13) to G14, the
endoderm and mesenchyme proliferate and a stratified
epithelium is produced. In the next several days, a
central lumen forms, accompanied by the emergence of
small villi covered by a differentiating epithelium (26,
46). Proliferating cells, initially scattered randomly
G114
throughout the epithelium, become confined to the
intervillus epithelial region at birth (G22), as nascent
crypts are formed (19). From G18 through G22, the
villus-associated epithelium continues to differentiate,
preparing to digest and absorb nutrients immediately
after birth.
The molecular basis of gut morphogenesis and cryptvillus axis formation has been the subject of intensive
investigation (41). In the intestine and in other epithelial tissues such as skin and lung, direct cell-cell contact
between mesenchyme and endoderm is required for
epithelial morphogenesis and cellular differentiation
(4, 22, 25, 27). The presence of mesenchyme is essential
for generation of the normal crypt-villus axis containing all four differentiated cell types, as demonstrated
previously in elegant endodermal-mesenchymal coculture and implantation experiments (24, 25). Investigators have begun to identify the specific mesenchymal
molecules required for appropriate morphogenesis. For
example, hepatocyte growth factor is produced and
secreted by mesenchyme and interacts with the c-met
receptor on endodermal cells. Experiments in cell culture suggest the importance of hepatocyte growth
factor in lumen formation (47), and studies on human
embryos have revealed that MET immunostaining was
positive in the basal domain of the fetal intestinal
epithelia between 5 and 13 wk of gestation, which
coincides with the time of organ differentiation (28).
Another important mesenchymal gene is a member of
the forkhead family of winged transcription factors.
Deletion of the fkh 6 gene, normally expressed in
embryonic mesenchyme adjacent to endoderm, results
in enhanced proliferation in the crypts with distortion,
villus hyperplasia, and the formation of mucinous cysts
(23). Further identification of these critical factors is
essential for understanding the molecular basis of
crypt-villus axis formation.
Another potential candidate mesenchymal morphogenetic factor is epimorphin. Epimorphin is a membraneassociated protein that has been postulated to regulate
morphogenesis of the lungs and skin (22). Transfection
of the epimorphin cDNA into fibroblasts followed by
coculture with lung endoderm induced the formation of
a large lumen and well-developed air sacs. In addition,
incubation of skin or lung organ cultures with epimorphin antibody led to disorganization of hair follicle and
air sac structure, respectively (22). cDNA sequence
analysis revealed homology to the syntaxin family of
proteins, and cloning experiments designed to isolate
homologues of neuronal syntaxin led to the identification of a rat cDNA that shared 96% amino acid identity
with epimorphin and between 20% and 65% amino acid
0193-1857/98 $5.00 Copyright r 1998 the American Physiological Society
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
Goyal, Alka, Renu Singh, Elzbieta A. Swietlicki, Marc
S. Levin, and Deborah C. Rubin. Characterization of rat
epimorphin/syntaxin 2 expression suggests a role in cryptvillus morphogenesis. Am. J. Physiol. 275 (Gastrointest. Liver
Physiol. 38): G114–G124, 1998.—The rodent intestinal mucosa undergoes a remarkable morphogenesis as the cryptvillus axis is formed. Endoderm-mesenchymal interactions
play a critical role in this process. Epimorphin is a mesenchymal protein postulated to play a role in lung and skin
morphogenesis. The rat homologue, syntaxin 2, belongs to a
family of integral membrane proteins that function in vesicle
docking and fusion. To clarify its role in fetal gut morphogenesis, epimorphin expression was examined during ontogeny,
in an isograft model of ischemic injury and mucosal repair,
and during intestinal adaptation after small bowel resection.
Epimorphin/syntaxin 2 mRNA levels were increased in fetal
gut during lumen formation and villus morphogenesis. mRNA
levels remained elevated in the first 2 wk after birth and then
declined at weaning. In situ hybridization showed epimorphin/
syntaxin 2 mRNA in gestational day 14 (G14) and G15
intestinal mesenchymal cells and in the mucosal lamina
propria during villus formation. Epimorphin/syntaxin 2
mRNA expression increased during villus repair in the isograft. In contrast, in the early stages of intestinal adaptation
after small bowel resection, epimorphin/syntaxin 2 mRNA
expression was suppressed in the adapting gut. We conclude
the cell-specific and temporal patterns of epimorphin expression in the models used in this study suggest a role in the
morphogenesis of the crypt-villus axis.
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
MATERIALS AND METHODS
Animals. Sprague-Dawley rats were housed under a strict
light-dark cycle and fed standard laboratory chow. Pregnant
female Sprague-Dawley rats (Sasco) were precisely timed for
gestation based on the presence of a vaginal plug (defined as
day 0 of gestation) and killed on G14–G22 (birth). Pups were
killed at postnatal week 1 (P1), P2, P3, P4, P6, and P8.
Suckling pups remained with their dams until they were
killed. Male Sprague-Dawley rats (220–260 g) were used for
small intestinal resection experiments. Fetuses from pregnant female Fisher rats killed on G19 were used as donors for
fetal intestinal isografts, and 4- to 6-wk-old male Fisher rats
were used as hosts.
Tissues and RNA isolation. Total RNA was isolated from
intestinal tissues using the single-step method of Chomczynski and Sacchi (9). On G14–G17, RNA was isolated from the
whole intestine, without further subdivision due to its small
size. From G18 through P8, proximal intestine (jejunum) or
ileum was used for analysis. For fetal intestinal RNA isolation, three to six pregnant females were killed for each time
point, each containing 6–10 fetuses. For RNA isolation on P1,
P2, and P3, two to three litters of 8–10 pups were killed for
each date. For P4, P6, and P8, at least four rats were killed for
each time point.
Cloning of the rat intestinal epimorphin/syntaxin 2 cDNA.
The reverse transcriptase-polymerase chain reaction (RTPCR) method was used to isolate a partial cDNA encoding rat
intestinal epimorphin/syntaxin 2. Nucleotide primers were
designed based on the mouse epimorphin sequence, from
nucleotides 600 to 620 (sense primer) and nucleotides 924 to
946 (antisense primer). The first strand was synthesized from
E17 rat intestinal RNA using reverse transcriptase, and PCR
(40 cycles) was performed using Taq polymerase. As predicted, a 350-bp fragment was isolated. Complete sequence
analysis of this partial cDNA revealed 96% nucleic acid
identity with mouse epimorphin and 100% identity with rat
syntaxin 2 (3).
RNase protection assays. We determined by Northern blot
analysis that epimorphin/syntaxin 2 mRNA levels were detectable but rare in the adult gut. Therefore RNase protection
assays were utilized to examine the ontogenic and spatial
expression of epimorphin/syntaxin 2 and its regulation during gut adaptation. The 350-bp PCR fragment was subcloned
into the plasmid pGEM-2, and a 32P-labeled epimorphin/
syntaxin 2 antisense RNA probe was prepared with high
specific activity [32P]CTP and T7 polymerase. The probe was
purified by 6% polyacrylamide gel electrophoresis and hybridized overnight at 42–45°C to 10 µg of total RNA from each
intestinal segment and developmental time point. After RNase
digestion, samples were run on a 6% polyacrylamide gel, and
autoradiography was performed. A single band was noted and
quantitated using NIH Image 1.55 analysis of digitized
images (W. Rasband, National Institute of Mental Health)
obtained with a UMAX PS-2400X scanner, using UMAX
Magicscan v. 2.1 (UMA Technologies, Fremont, CA). To verify
the results, these experiments were repeated three to six
times. A low specific activity 18S ribosomal probe (Ambion,
Austin, TX) was used to normalize for differences in RNA
concentration or loading. Hybridization of this probe to .0.5
µg total RNA produces two closely migrating bands, as
indicated by the manufacturer (Ambion) and shown in Fig. 1.
The band at the top was used to normalize the RNase
protection experiments.
Northern blot hybridization. Northern blot hybridization
was performed to quantitate epimorphin mRNA expression in
Fig. 1. Epimorphin/syntaxin 2 mRNA levels are regulated during fetal gut ontogeny. Top: representative
RNase protection assay used to quantitate epimorphin/
syntaxin 2 expression during gut ontogeny. Total RNA
from intestinal segments (10 µg per sample) was hybridized to an epimorphin/syntaxin 2 antisense cRNA probe,
digested by RNase, and run on a 6% polyacrylamide gel,
as described in MATERIALS AND METHODS. Bottom: fetal
ontogeny. Lane 1, gestational day 13 (G13) whole rat
fetal RNA. Lanes 2–5, whole intestine obtained on G14,
G15, G16, and G17, respectively. Lane 6, G18 jejunum.
Lane 7, G19 jejunum. Arrow shows 350-bp fragment
protected by epimorphin/syntaxin 2 probe. Arrowhead
indicates 80-bp 18S fragment used for normalization
(Ambion).
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
identity with other members of the syntaxin family (3).
These integral membrane proteins act as target membrane receptors for vesicle docking and fusion in a
variety of tissues, including neurons and nonneuronal
secretory tissues such as pancreas, where they play a
role in calcium-regulated exocytosis (13). Yet, in several
instances, syntaxins appear to play a different role in
development, distinct from their function in mature
tissues (6, 10, 15, 32).
The role of epimorphin/syntaxin 2 in organ morphogenesis has not been clarified. To address the hypothesis that epimorphin/syntaxin 2 is important in lumen
formation and villus morphogenesis in the developing
fetal gut, the cell-specific and temporal regulation of
epimorphin expression was examined during ontogeny.
Also, epimorphin regulation was studied in an isograft
model system of fetal villus ischemic injury and regeneration. To begin to understand the role of epimorphin
in the adult intestine, expression patterns were analyzed in the mature intestine and during intestinal
adaptation, when epithelial cell proliferation, migration, and differentiation are altered in the remnant gut.
The results suggest a role for epimorphin/syntaxin 2 in
gut morphogenesis, particularly in lumen and cryptvillus axis formation.
G115
G116
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
Fig. 2. Postnatal intestinal expression of epimorphin.
RNase protection assay was performed as described in
Fig. 1 legend. Lanes 1–4, jejuna from postnatal weeks 1
through 4 (P1–P4), respectively. Lane 5, P6 jejunum.
Top: 350-bp protected fragment. Bottom: 18S bands as
per Fig. 1.
Intestinal isograft implantation analyses. Rat intestines
were harvested from fetuses of pregnant female Fisher rats
on G18–G19. Jejunal segments were implanted into the
dorsal subcutaneous space of young adult male Fisher rat
hosts as previously described (16, 37, 39). Briefly, intestinal
segments were isolated from fetal pups and placed into RPMI
medium on ice. Hosts were anesthetized, and a midline
incision along the dorsal paravertebral surface was made.
The subcutaneous fascia was dissected by forceps, and isografts were placed into this space and sutured using 7-O silk.
Isografts were harvested 24–96 h after implantation and
analyzed for cellular epimorphin/syntaxin 2 expression by
Northern blot analysis. The developmental age of the 96-h
isograft was considered equal to G22, calculated as previously
described (16, 37, 38). Briefly, 24 h were allowed for vascularization of the isograft. The age of the graft was then expressed
as the fetal age plus the total number of days of implantation
minus 1 (for vascularization).
70% Small bowel resection surgical protocol. Male SpragueDawley rats (220–260 g) were randomly assigned to receive
either 70% small intestinal resection or control, sham intestinal resection surgery, as previously described (11, 39). All
animals were fasted overnight after being acclimated to our
animal facility. For 70% small bowel resection surgery, the
intestine was cut 5 cm distal to the ligament of Treitz and 15
cm proximal to the ileocecal valve. The resected part of the
intestine was removed, with construction of an end-to-end
anastomosis. In sham-resected animals, bowel bisection and
reanastomosis were performed 5 cm distal to the ligament of
Treitz. After surgery, animals received buprenorphine (0.4
mg/kg sc) for analgesia and were caged individually with
access to 5% dextrose in water containing oxytetracycline (4.5
g/l) for up to 2 days. Animals were killed at 2, 4, 8, 16, 48, and
168 h after surgery (n 5 3 per group). Portions of the middle
part of the remnant ileum and the equivalent fragment in the
control animals were rapidly frozen and saved for total RNA
isolation.
RESULTS
Isolation of a partial cDNA encoding rat intestinal
epimorphin/syntaxin 2. To isolate the rat cDNA encoding intestinal epimorphin/syntaxin 2, we used RT-PCR.
PCR primers were based on the mouse epimorphin
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
isograft epithelium, as previously described (36). Total RNA
was prepared from individual isografts, and 20 µg RNA were
electrophoresed on 1.2% agarose and 2.2 M formaldehyde
gels. Gels were stained with ethidium bromide and photographed to verify mRNA integrity. RNA was transferred onto
nitrocellulose membranes, prehybridized at 42°C for 2–3 h in
50% formamide, 63 SSPE (13 SSPE is 0.15 M NaCl, 0.01 M
Na2HPO4, and 0.001 M EDTA, pH 7.4), 53 Denhardt’s
solution, 0.5% SDS, and 50 mg/l denatured salmon sperm
DNA, and probed with an [a-32P]dCTP radiolabeled epimorphin cDNA probe. Probe was labeled using the random oligo
priming method (Amersham Life Sciences, Arlington Heights,
IL), hybridized at 42°C overnight, and washed at 55°C in
0.13 SSC (13 SSC is 0.15 M NaCl and 0.015 M sodium
citrate, pH 7.0). Blots were placed on film at 270°C and
developed after 1 wk. Quantitation of hybridization bands
was performed by scanning laser densitometry. Blots were
normalized to the ethidium bromide signal of the 18S ribosomal band, which was quantified with the Eagle Eye image
analysis system (Stratagene, La Jolla, CA) to control for
differences in gel loading. Relative mRNA levels were reported as the ratio of specific transcripts to 18S RNA.
In situ hybridization analyses. In situ hybridization analysis to detect the cell-specific expression of epimorphin/
syntaxin 2 in fetal and postnatal intestine was performed as
previously described (16, 35). Briefly, tissues were fixed in 4%
paraformaldehyde in PBS overnight, incubated in 30% sucrose in PBS until saturated, and then frozen in optimal
cutting temperature solution. Cryostat sections (8 µm) were
prehybridized by proteinase K digestion (1 µg/ml for 10 min
at 37°C) and acetylated. A radiolabeled antisense epimorphin/
syntaxin 2 RNA probe or sense (control) probe was synthesized using high specific activity 35S-labeled UTP. Sections
were hybridized with 5 3 106 disintegrations per minute
(dpm) of probe per milliliter of hybridization solution at 55°C
overnight. Standard washing conditions were performed as
described previously (16, 35) and included a stringent wash in
0.13 SSC at 55°C. Slides were dipped in Kodak NTB-2
emulsion, exposed for 10–16 days at 4°C, developed, and
counterstained with hematoxylin and eosin. Photographs
were taken using a Nikon Microphot camera under dark-field
and light microscopy. For each time point, tissues were used
from at least four to six animals, and at least four sections for
each animal were analyzed.
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
sequence isolated from a C3H10T1/2 expression library
(22). A 350-bp fragment of the epimorphin/syntaxin 2
cDNA was cloned. Alignment of this partial cDNA
sequence with the mouse epimorphin cDNA revealed
96% nucleotide identity. Alignment with the rat syntaxin 2 cDNA, the rat homologue of mouse epimorphin
cloned from several cDNA libraries, including liver,
brain/spinal cord, macrophages, pancreas, and pituitary gland (3), indicated 100% identity in this region.
Thus this part of the coding sequence of rat intestinal
epimorphin/syntaxin 2 is identical to syntaxin 2 from
other rat tissues.
Epimorphin/syntaxin 2 mRNA is maximally expressed in the developing fetal intestine. Crypt-villus
axis morphogenesis begins on approximately G13–G14
and continues until several days after birth. To test the
hypothesis that epimorphin/syntaxin 2 might play a
role in regulating part of this morphogenetic process,
we examined the developmental regulation of its expression in the intestine using RNase protection assays
(Figs. 1–4). Fetal and postnatal rat intestine were harvested, and total RNA was prepared from each segment. An antisense epimorphin/syntaxin 2 cRNA probe
was hybridized to 10 µg of total RNA from each sample.
Typical RNase protection assays are shown in Figs. 1
and 2. A single protected band of ,350 bp represents
epimorphin/syntaxin 2 mRNA. The results of several
independent RNase protection assays are quantitated
in Fig. 3A (fetal ontogeny) and Fig. 3B (postnatal
ontogeny). Epimorphin/syntaxin 2 mRNA was most
abundantly expressed in intestine on fetal days 14–17,
coincident with the commencement of crypt-villus morphogenesis (Fig. 3A). By G18, mRNA levels decreased
in proximal gut but remained elevated in distal gut
(Fig. 4). Subsequently, on G19 through birth (G22),
mRNA levels declined in all segments but were still
readily detectable. A second peak of epimorphin/
syntaxin 2 mRNA expression was found during the
suckling period (P1 and P2; Figs. 2 and 3B), but mRNA
levels decreased to basal levels by P3, the onset of
weaning. mRNA levels remained low but were still
detectable in adult jejunum (P6 and P8).
Epimorphin/syntaxin 2 mRNA is expressed in fetal
mesenchymal cells and in the lamina propria of the
nascent villus, coincident with villus formation. To
begin to determine the function of epimorphin/syntaxin 2
in gut morphogenesis, we examined its cell-specific expression, using in situ hybridization techniques (Figs. 5 and 6).
On fetal day 15, the endoderm is a stratified, multicellular layer of undifferentiated cells surrounded by mesenchyme. There is a central lumen, and villus structures
have not yet formed. Expression of epimorphin/syntaxin 2 mRNA was most prominent in the mesenchyme
that surrounds endoderm (Fig. 5, A and B). Small
thumblike villi appeared by G16, and mesenchymal
expression persisted (Fig. 5, C and D). At this stage,
epimorphin/syntaxin 2 mRNA was not detectable in
endoderm (Fig. 5, C and D; thin arrows). A sense
epimorphin/syntaxin 2 control RNA probe hybridized to
fetal day 15 intestine demonstrated the specificity of
the probe; no specific signal is seen (Fig. 5, E and F).
Fig. 4. Ontogeny of epimorphin expression along the horizontal axis of the intestine. RNase protection assay was
performed as described in legend to Fig. 1. RNA samples were prepared from whole intestine from G14 through G17
or were divided into proximal (p) and distal (d) segments for G18, G19, and G20. Intestine harvested at birth (G22)
(B) was further subdivided into proximal, mid- (m), and distal segments because of its larger size. 1wp, 1 wk
proximal segment; 6wp, 6 wk proximal segment.
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 3. Quantitative analysis of ontogeny of epimorphin mRNA
expression. Data are expressed as mean relative mRNA level 6 SE.
Fetal intestinal values (A) were derived from n 5 3–6 independent
RNase protection assays, and postnatal values (B) were from n 5 3
independent RNase protection assays. Fetal RNA samples were
prepared from whole intestine from fetal days 14, 15, 16, and 17, and
from jejunum only for fetal days 18, 19, 20, and 22. All postnatal RNA
was prepared from jejunum.
G117
G118
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
By P1, well-defined villi were present and epimorphin expression was found in the villus mucosal lamina
propria and muscularis layers (Fig. 6, A and B, black
arrows). There was no expression in the villusassociated epithelium (white arrows) or in the
crypt epithelium. Probe specificity was again demonstrated by using a sense RNA probe hybridized to P1
intestine (Figs. 6, C and D). White grains indicating
the presence of epimorphin mRNA were not found
in the lamina propria (thick black arrows) or on the
villus (thin black arrows). The large white cells noted at
the base of the villi had no specific grains overlying
them. As we have shown previously, these intestinal
cells are intensely refractile under dark-field microscopy, leading to the white color (11), but inspection of
multiple sections revealed no specific grains on these
cells.
Epimorphin/syntaxin 2 mRNA levels increase after
isograft implantation. Intestinal isograft implantation
techniques have been used by our laboratory and
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 5. Epimorphin/syntaxin 2 mRNA is expressed in mesenchymal cells of fetal intestine. In situ hybridization
analyses were performed to detect cell-specific epimorphin/syntaxin 2 mRNA expression, using a 350-bp antisense
epimorphin/syntaxin 2 cRNA probe (A-D) or a sense control RNA probe (E, F). Sections were counterstained with
hematoxylin and eosin. For each time point, at least 4 fetal pups and 4 sections from each pup were analyzed.
Representative sections for each time point are shown. A: G15 whole intestine viewed under dark-field microscopy.
Yellow-white grains represent epimorphin/syntaxin 2 mRNA expression. Thin arrows indicate endoderm; small
thick arrows show mesenchyme. B: same section as in A, viewed by light microscopy. C: G16 intestine, viewed by
dark-field microscopy. Small thumblike villi have begun to form. Thin arrows indicate endoderm, relatively devoid
of grains. D: same section as in C, viewed by light microscopy. E: G15 intestine before villus formation, hybridized to
a sense control cRNA epimorphin probe. Note absence of specific white grains overlying the mucosa. F: same section
as in E, viewed by light microscopy. Magnification: 3100 (A and B); 3250 (E and F); and 3320 (C and D).
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
G119
others to examine the influence of luminal contents on
spatial differentiation in the intestine, from duodenum
to colon and from crypt to villus tip (16, 37, 38). When
fetal intestine is removed from its normal environment
and isografted into the subcutaneous tissue of a host
rat, it develops into a well-differentiated intestine that,
despite the absence of luminal substances, expresses
enterocytic and enteroendocrine gene products in an
appropriate region-specific manner.
The isograft can also be used as a model for examining neonatal villus repopulation and repair after isch-
emic injury. G19 intestine contains nascent villi. When
implanted, this isograft epithelium is rapidly sloughed
due to ischemia, resulting in intraluminal hemorrhage
(29). From 48 h to several days after implantation,
surviving cells proliferate and the epithelium is repaired, accompanied by the formation of new villi.
We have used this isograft model to begin to determine whether or not epimorphin/syntaxin 2 may play a
role in villus repopulation after ischemic injury to
neonatal bowel after nascent villi have formed. G19
jejunum was implanted in the subcutaneous space of
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 6. Epimorphin/syntaxin 2 mRNA is expressed in lamina propria of postnatal intestine. Cell-specific expression
of epimorphin was assessed in P1 (A-D) and P6 jejunum (E-H). Antisense probe was hybridized to P1 (A, B) and P6
sections (E, F). Sense control probe was hybridized to P1 jejunum (C, D) or P6 jejunum (G, H). In situ hybridization
analyses were performed as described for Fig. 5. A: P1 jejunum, viewed by dark-field microscopy. Black arrows show
signal in villus core and mucosal lamina propria. Thin white arrows indicate epithelium that does not express
epimorphin. B: same section as in A, viewed by light microscopy. C: P1 jejunum viewed under dark-field microscopy,
hybridized to sense control probe. No specific lamina propria signal (thick black arrows) or epithelial signal (thin
black arrows) is seen. Cells at base of villi, which appear white under dark-field microscopy, do not exhibit white
grains specific for epimorphin mRNA. D: same section as in C, viewed under light microscopy. E: section of P6
jejunum, hybridized to antisense epimorphin cRNA probe, viewed under dark-field microscopy. Thick white arrows
show large mesenchymal lamina propria cells that express epimorphin. Thin white arrows indicate negative
epithelium. F: high-power view of P6 jejunum hybridized to antisense epimorphin cRNA probe, viewed under
dark-field microscopy. Thick white arrows point to large cells positive for epimorphin mRNA; thin white arrows
point to negative epithelium. G: P6 jejunum hybridized to sense control probe (dark-field). No specific signal is seen
in epithelium (thin white arrow) or in lamina propria (thick white arrows). H: same section as in G, under light
microscopy. Magnification: 3320 (A and B); 3250 (C and D); 3100 (E); 3200 (F-H).
G120
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
Fig. 7. Intestinal isografts express epimorphin/syntaxin 2 during
villus regeneration after implantation. G19 intestine was implanted
as an isograft in normal Fisher rat hosts as described in MATERIALS
AND METHODS. Isografts were harvested at 48, 72, and 96 h after
implantation, and total RNA was prepared from each graft separately. Loss of nascent G19 villi at 24–48 h after implantation,
followed by regeneration after implantation (at 72–96 h), was
verified by routine hematoxylin and eosin staining of grafts (data not
shown). A: Northern blot hybridization of total RNA prepared from
individual isografts harvested at various times after implantation.
Total RNA (20 µg/lane) was electrophoresed on an agarose formaldehyde gel and transferred to nylon membranes. Blots were probed
with [32P]dCTP-radiolabeled 350-bp epimorphin cDNA, and autoradiography was performed. Each lane represents total RNA isolated
from an individual isograft. B: quantitation of relative epimorphin
mRNA levels in isografts. Autoradiographic bands were quantitated
by scanning laser densitometry and were normalized to the ethidium
bromide signal of the 18S ribosomal band. Data are expressed as
mean relative densitometric units for each time point (n 5 3–4 grafts
per time point). C: Northern blot hybridization of total RNA prepared
from intact jejunum harvested at birth or from a fetal day 19 isograft
harvested at 96 h after implantation. A sevenfold increase in epimorphin mRNA expression was seen in the isograft compared with
normal intestine.
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
young adult male rats. At 24 h after implantation, we
confirmed that isografts demonstrate intraluminal hemorrhage with villus loss and denuding of the intestinal
epithelium. However, surviving cells proliferate and
nascent villi reappeared by 48–72 h after implantation
(data not shown).
The expression of epimorphin/syntaxin 2 was examined in the repairing bowel 48–96 h after implantation.
Epimorphin/syntaxin 2 mRNA was readily detectable
even by Northern blot hybridization as early as 48 h
after implantation (Fig. 7A). Furthermore, mRNA levels increased by 96 h after implantation, coincident
with the formation of many new villi (Fig. 7B). To
compare epimorphin/syntaxin 2 expression in isografts
with the equivalently aged normal intestine, total RNA
was isolated from a G22 pup (birth) and from a fetal day
19 isograft harvested 96 h after implantation (thus
equivalent to G22 intact intestine; see MATERIALS AND
METHODS ), and a Northern blot was prepared. Epimorphin/syntaxin 2 mRNA levels were sevenfold higher in
the isografted intestine than in the equivalent segment
of intact intestine harvested from a pup at birth (Fig. 7C).
Due to the relatively small size of the grafts, direct
histological correlation of crypt-villus formation in the
same grafts from which RNA was prepared was not
possible. However, hematoxylin and eosin staining was
performed on other grafts harvested at the same time
points, and a marked increase in the size and number of
villi was noted by 72–96 h (data not shown).
Epimorphin/syntaxin 2 mRNA is expressed in the
lamina propria of the adult gut, and mRNA levels
decrease in the early stages of intestinal adaptation
after small bowel resection. Epimorphin mRNA levels
declined to low but detectable levels in the adult
intestine, as shown in Fig. 2. To begin to determine the
function of epimorphin in mature intestine, we determined cell-specific expression in adult gut. As shown in
Fig. 6, E and F, a subpopulation of large lamina propria
cells expresses epimorphin in the adult gut. The diffuse
expression in the mucosal and submucosal lamina
propria noted in fetal and early postnatal life is no
longer apparent. When stained with hematoxylin and
eosin, these cells resemble macrophages. Expression in
this cell type is consistent with previous data indicating
that macrophages produce syntaxin 2, which functions
in phagocytic pathways (17). Comparison of these
sections with those in Fig. 6, G and H, hybridized to the
sense control probe, shows the specificity of the signal.
Although large white cells are seen, there are no
specific white grains overlying them.
After loss of functional small bowel surface area, the
gut epithelium undergoes marked morphological and
functional changes in a remarkable process of adaptation. In the early stage of the adaptive response (i.e.,
the first 48 h after resection), there is a rapid increase
in the crypt cell production rate (12, 18, 48). In addition,
we have shown that the enterocytic expression of
several genes involved in lipid uptake and processing
increases rapidly after resection (39). In the second
stage of adaptation, which peaks 1–2 wk after resection, enhanced crypt cell production rates persist, result-
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
ing in increased crypt depth and villus height, villus
hyperplasia, and upregulation of absorptive capacity.
To assess the role of epimorphin/syntaxin 2 as a
potential regulator of proliferation and/or differentiation in the adult gut, we examined its regulation in
control and adaptive small bowel after massive intestinal resection (Fig. 8). Rats were subjected to 70% small
intestinal resection. Epimorphin/syntaxin 2 mRNA expression was quantitated in the adaptive or control
ileum at 2, 4, 8, 16, 48, and 168 h (1 wk) after resection,
by RNase protection assay (Fig. 8). During the initial
stages of adaptation, from 4 to 16 h, we found that
epimorphin/syntaxin 2 mRNA levels were suppressed
in adaptive ileum compared with control, shamresected intestine. However, by 48 h postoperatively,
epimorphin/syntaxin 2 mRNA expression was unchanged in resected compared with sham-resected animals.
DISCUSSION
We have shown that epimorphin/syntaxin 2 mRNA
expression is developmentally regulated in the rodent
small intestine. mRNA levels increase in the fetal gut
coincident with lumen and villus formation. A second
peak of expression occurs in the suckling period during
P1 and P2, followed by a decline at weaning. Abundant
expression can be detected in fetal mesenchymal cells
that underlie the nascent villus epithelium. Gut endoderm and newly differentiating enterocytes do not
appear to express epimorphin/syntaxin 2 mRNA. We
also found that epimorphin/syntaxin 2 mRNA expression is increased in the isografted intestine during
villus repopulation after ischemic injury to its nascent
epithelium. Yet in the adult intestine, during intestinal
adaptation early after small bowel resection, epimor-
phin/syntaxin 2 mRNA levels are suppressed in adaptive ileum compared with sham-resected controls.
This study is the first to examine in detail the
cell-specific and temporal patterns of expression of
epimorphin during ontogeny. Our in situ hybridization
analyses are particularly important in establishing the
cell-specific expression of epimorphin in fetal intestine,
since there are conflicting data from studies of other
tissues. For example, epimorphin mRNA has been
shown to be present in ameloblasts, which are epithelium-derived cells (31). Also, immunohistochemical
analyses have also yielded variable results, since the
epimorphin antibody cross-reacts with syntaxin 1 (7).
Our data agree with those reported originally by Hirai
et al. (22), describing epimorphin as a mesenchymal
skin and lung protein in fetal tissues.
The patterns of epimorphin/syntaxin 2 expression
during gut development and in the isograft model of
villus repair suggest that its critical function in the
intestine is during ontogeny, specifically in regulating
lumen formation and villus morphogenesis. In particular, the persistently increased epimorphin mRNA levels
in fetal day 18 distal gut compared with fetal day 18
proximal gut also support a role in morphogenesis,
since the formation of the villus proceeds in a wavelike
fashion from proximal to distal gut during fetal life.
This presumed function is consistent with previous
studies demonstrating that epimorphin expression can
be upregulated by retinoic acid, a potent morphogen
(34), and with experiments in other epithelia suggesting that epimorphin may be an important mesenchymal regulator of morphogenesis. For example, transfection of epimorphin into NIH/3T3 fibroblasts followed by
coculture with fragments of lung endoderm induces the
formation of air sacs that appear normal, with a large
lumen (22). In comparison, native fibroblast-endoderm
cocultures resulted in small tubular structures without
a dominant lumen. Also, in fetal organ cultures, air sac
formation from fetal lung and hair follicle formation
from fetal lip skin were blocked by incubation with
epimorphin antibody. The formation of a central lumen
is important in gut as well as lung morphogenesis, and
the morphology of the hair follicle (in which there is a
proliferative compartment that gives rise to welldifferentiated cells) mimics the organization of the
crypt-villus axis. Thus the existence of general morphogenetic processes in most epithelial organs suggests
that common molecules may play similar roles in
different tissues. It is likely that epimorphin/syntaxin 2
functions in the same manner in the intestine. Studies
in which intestinal endoderm or cultured intestinal
cells are grown with epimorphin-transfected fibroblasts could provide further support for this hypothesis.
The mechanism by which epimorphin/syntaxin 2
influences tissue morphogenesis is not well understood.
Sequence analysis of the encoded epimorphin protein
revealed homology to the syntaxin family (3, 44). These
are integral membrane proteins that are thought to act
as target membrane receptors during secretory vesicle
docking and fusion. There are multiple isoforms ex-
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 8. Epimorphin/syntaxin 2 mRNA levels are suppressed in
adaptive compared with sham-resected control ileum. Rats were
subjected to 70% small intestinal resection as described in MATERIALS
AND METHODS. Epimorphin/syntaxin 2 mRNA levels were detected in
adaptive ileum or control sham-resected ileum by RNase protection
assay and normalized by simultaneous hybridization to an 18S probe,
as described in Figs. 1 and 2. Digitized images were quantitated as
described in MATERIALS AND METHODS. Data are expressed as the ratio
of resected to sham-resected intestine, in arbitrary units (resected
units/sham-resected units).
G121
G122
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
neuronal tissues. Its localization changes during rat
neuronal development (2, 15, 32). In fact, it is expressed
before synapse formation, again suggesting a role for
this protein in ontogeny that is independent of regulating synaptic transmission, its presumptive function in
the adult brain (15, 32). The very different cell-specific
patterns of expression found in developing vs. mature
intestine suggest that epimorphin/syntaxin 2 plays
distinct roles depending on the ontogenic stage.
Epimorphin/syntaxin 2 mRNA levels declined in
adapting compared with sham-resected control intestine 4–16 h after resection and then returned to basal
adult levels. Decreased expression does not appear to
be due to a general suppressive effect of surgery, since
mRNA levels decreased in comparison with shamoperated controls that underwent identical anesthesia
as well as transection and reanastomosis. We have also
shown that expression of a variety of other intestinal
gene products increases early in adaptation (11, 38).
Little is known about the molecules that may regulate
epimorphin expression, so it is difficult to postulate a
specific humoral, neuroendocrine, or other intestinal
product that may mediate the decrease in expression. It
has been suggested that epimorphin has an antiproliferative effect, since proliferation of HUVECs, an endothelial cell line, is inhibited when these cells are
cultured on epimorphin-coated wells (33). If this is
indeed true, suppression of epimorphin expression
might be required to permit enhanced cell proliferation
during adaptation, particularly in the crypt and submucosal compartments. However, these data are difficult
to interpret in light of other findings regarding the role
of epimorphin/syntaxin 2 as a vesicle docking and/or
fusion protein in mature cells and our discovery that its
expression in adult gut is confined to lamina propria
cells, although some positive cells do lie in close proximity to the crypts.
A major gap in the understanding of mesenchymalepithelial interactions during intestinal epithelial morphogenesis is that critical mesenchymal regulatory
genes are still largely unknown. Although its precise
function in intestinal ontogeny is unclear, the unique
cellular and ontogenic patterns of expression of epimorphin make it a valuable model gene for identifying
mesenchymal trans-acting factors that regulate its
expression. Once identified, the role of these factors in
the gut and in general epithelial tissue morphogenesis
can be explored, similar to experiments in which the
function of critical intestinal endodermal-epithelial
trans-acting factors (43, 45) or mesenchymal factors in
other epithelial tissues [e.g., isl 1 in pancreas (1)] has
been clarified. In addition, future experiments that
elucidate the role of epimorphin/syntaxin 2 in developing and mature mammalian tissues may provide novel
insights into how a specific ontogenic stage influences
the function of individual proteins.
We thank Kathleen Grapperhaus for excellent technical assistance.
This work was supported in part by a Basic Research Grant from
the March of Dimes Birth Defects Foundation and by National
Institutes of Health Grant DK-46122. A. Goyal was supported by an
American Digestive Health Foundation Advanced Research Training
Award.
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
pressed in different tissues. This family was defined by
low-stringency screening of rat cDNA libraries, using
syntaxins 1A and 1B (neuronal syntaxins) as probes (3).
Syntaxin 2 is the rat homologue of epimorphin, sharing
,60% amino acid homology with syntaxins 1A and 1B.
Most studies have shown that syntaxin 2 is located on
the plasma membrane, although there are multiple
isoforms that differ in their carboxy terminals, and one
in particular has a truncated domain of insufficient
length to span the membrane (3). When transfected
into Madin-Darby canine kidney cells, syntaxin 2 targets both apical and basolateral membranes (30), although in pancreatic acinar cells it was found in the
apical plasma membrane and in the cytoplasm (14). It
is postulated to be important in targeting zymogen
granules to the apical membrane during regulated
secretion (14) and appears to play a role as a calciumsensitive regulator of exocytosis in pancreatic acinar
cells (13). These proteins have generally been found to
have an intracellular orientation (3). However, when
syntaxin 1 was expressed in Xenopus oocytes, it exhibited glutamate receptor properties, with an extracellular orientation (5, 42). Also, Hirai (20) obtained data
supporting an extracellular domain for epimorphin.
Oka and Hirai (33) have also suggested a different role
for epimorphin, indirectly regulating cell proliferation
and mesenchymal cell migration. A specific NH2terminally modified fragment of the epimorphin molecule inhibited proliferation and induced morphogenetic changes in human umbilical vein endothelial cells
(HUVECs) cultured in collagen, resulting in their rearrangement into circular venule-like formations. In addition, HUVECs adhered to dishes coated with this same
modified fragment. The effects of this polypeptide on
the morphology of cultured intestinal epithelial cells
have not yet been examined.
Thus the precise localization and function of epimorphin, particularly during ontogeny, have not been clarified. How can these disparate data, suggesting very
different roles for epimorphin/syntaxin 2 (i.e., regulation of vesicle docking and fusion vs. epithelial morphogen) be reconciled? One unifying hypothesis is that
epimorphin may play a role in morphogenesis as a
vesicle docking protein, by regulating the secretion of
diffusible morphogens such as hepatocyte growth factor
(44). Recent studies by Hirai et al. (21) indicate that
growth factors such as hepatocyte growth factor and
epidermal growth factor require the presence of epimorphin to induce growth and branching morphogenesis of
mouse mammary epithelial cells. A second possibility is
that the role of this gene product is different in developing compared with adult intestine. The function of
syntaxins in other in vivo systems provides support for
this premise. For example, the Drosophila homologue
of syntaxin 1, syx, is expressed at the earliest stages of
Drosophila development and is found on newly forming
membranes (40). Mutations of syx prevented normal
cellularization of the Drosophila embryo, which is
dependent on massive membrane addition and the
migration and segregation of cytoskeletal proteins (6).
In addition, syntaxin 1 is developmentally regulated in
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
Address for reprint requests: D. C. Rubin, Washington Univ.
School of Medicine, Division of Gastroenterology, 660 South Euclid
Ave., Box 8124, St. Louis, MO 63110.
Received 12 November 1997; accepted in final form 27 March 1998.
REFERENCES
20. Hirai, Y. Sodium-dodecyl sulfate resistant complex formation of
epimorphin monomers, and interaction of the 150-kDa complex
with the cell surface. Eur. J. Biochem. 25: 1133–1139, 1994.
21. Hirai, Y., A. Lochter, S. Galosy, S. Koshida, S. Niwa, and
M. J. Bissell. Epimorphin functions as a key morphoregulator
for mammary epithelial cells. J. Cell Biol. 140: 159–169, 1998.
22. Hirai, Y., K. Takebe, M. Takashina, S. Kobayashi, and M.
Takeichi. Epimorphin: a mesenchymal protein essential for
epithelial morphogenesis. Cell 69: 471–481, 1992.
23. Kaestner, K. H., D. G. Silberg, P. G. Traber, and G. Schutz.
The mesenchymal winged helix transcription factor Fkh6 is
required for the control of gastrointestinal proliferation and
differentiation. Genes Dev. 11: 1583–1595, 1997.
24. Kedinger, M., P. Simon-Assmann, E. Alexandre, and K.
Haffen. Importance of a fibroblastic support for in vitro differentiation of intestinal endodermal cells and for their response to
glucocorticoids. Cell Differ. 20: 171–182, 1987.
25. Kedinger, M., P. M. Simon-Assman, B. Lacroix, A. Marxer,
H. P. Hauri, and K. Haffen. Fetal gut mesenchyme induces
differentiation of cultured intestinal endoderm and crypt cells.
Dev. Biol. 113: 474–483, 1986.
26. Klein, R. M. Small intestinal cell proliferation during development. In: Human Gastrointestinal Development, edited by E.
Lebenthal. New York: Raven, 1989, p. 367–392.
27. Klein, R. M. Cell proliferative regulation in developing small
intestine. In: Human Gastrointestinal Development, edited by E.
Lebenthal. New York: Raven, 1989, p. 393–436.
28. Kolatsi, J. M., R. Moore, P. J. D. Winyard, and A. S. Woolf.
Expression of hepatocyte growth factor/scatter factor and its
receptor MET, suggests roles in human embryonic organogenesis. Pediatr. Res. 41: 657–665, 1997.
29. Leapman, S. B., A. A. Deutsch, R. J. Grand, and J. Folkman.
Transplantation of fetal intestine: survival and function in a
subcutaneous location in adult animals. Ann. Surg. 179: 109–
114, 1974.
30. Low, S. H., S. J. Chapin, T. Weimbs, L. G. Komures, M. K.
Bennett, and K. E. Mostov. Differential localization of syntaxin
isoforms in polarized Madin-Darby canine kidney cells. Mol.
Biol. Cell 7: 2007–2018, 1996.
31. Matsuki, Y., N. Amizuka, H. Warshawsky, D. Goltzman, and
Y. Yamada. Gene expression of epimorphin in rat incisor ameloblasts. Arch. Oral Biol. 40: 161–164, 1995.
32. Miya, F., A. Yamamoto, K. Akagawa, K. Kawamoto, and Y.
Tashiro. Localization of HPC-1/syntaxin 1 in developing rat
cerebellar cortex. Cell Struct. Funct. 21: 525–532, 1996.
33. Oka, Y., and Y. Hirai. Inductive influences of epimorphin/
syntaxin 2 on endothelial cells in vitro. Exp. Cell Res. 222:
189–198, 1996.
34. Plateroti, M., D. C. Rubin, I. Duluc, R. Singh, C. FoltzerJourdainne, J.-N. Freund, and M. Kedinger. Subepithelial
fibroblast cell lines from different levels of the gut axis display
regional characteristics. Am. J. Physiol. 274 (Gastrointest. Liver
Physiol. 37): G945–G954, 1998.
35. Rubin, D. C. Spatial analysis of transcriptional activation in
fetal rat jejunal and ileal gut epithelium. Am. J. Physiol. 263
(Gastrointest. Liver Physiol. 26): G853–G863, 1992.
36. Rubin, D. C., D. E. Ong, and J. I. Gordon. Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic
patterns of gene expression. Proc. Natl. Acad. Sci. USA 86:
1278–1282, 1989.
37. Rubin, D. C., K. A. Roth, E. H. Birkenmeier, and J. I.
Gordon. Epithelial cell differentiation in normal and transgenic
mouse intestinal isografts. J. Cell Biol. 113: 1183–1192, 1991.
38. Rubin, D. C., E. Swietlicki, K. A. Roth, and J. I. Gordon. Use
of fetal intestinal isografts from normal and transgenic mice to
study the programming of positional information along the
duodenal-to-colonic axis. J. Biol. Chem. 267: 15122–15133, 1992.
39. Rubin, D. C., E. A. Swietlicki, J. L. Wang, B. D. Dodson, and
M. S. Levin. Enterocytic gene expression in intestinal adaptation: evidence for a specific cellular response. Am. J. Physiol. 270
(Gastrointest. Liver Physiol. 33): G143–G152, 1996.
40. Schulze, K. L., and H. J. Bellen. Drosophila syntaxin is
required for cell viability and may function in membrane formation and stabilization. Genetics 144: 1713–1724, 1996.
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017
1. Ahlgren, U., S. L. Pfaff, T. M. Jessell, T. Edlund, and H.
Edlund. Independent requirement for ISL1 information of pancreatic mesenchyme and islet cells. Nature 385: 257–260, 1997.
2. Bennett, M. K., N. Calakos, and R. H. Scheller. Syntaxin: a
synaptic protein implicated in docking of synaptic vesicles at
presynaptic active zones. Science 257: 255–259, 1992.
3. Bennett, M. K., J. E. Garcia-Arraras, L. A. Elferink, K.
Peterson, A. M. Fleming, C. D. Hazuka, and R. H. Scheller.
The syntaxin family of vesicular transport receptors. Cell 74:
863–873, 1993.
4. Birchmeier, C., and W. Birchmeier. Molecular aspects of
mesenchymal-epithelial interaction. Annu. Rev. Cell Biol. 9:
511–540, 1993.
5. Brose, N. Membrane fusion takes an excitatory turn: syntaxin,
vesicle docking protein, or glutamate receptor? Cell 75: 1043–
1044, 1993.
6. Burgess, R. W., D. L. Dietcher, and T. L. Schwarz. The
synaptic protein syntaxin 1 is required for cellularization of
Drosophila embryos. J. Cell Biol. 138: 861–875, 1997.
7. Butt, K. I., M. Manabe, H. Yaguchi, R. Tsuboi, and H.
Ogawa. Immunolocalization of epimorphin in skin. J. Dermatol.
Sci. 13: 193–201, 1996.
8. Cheng, H., and C. P. Leblond. Origin, differentiation and
renewal of the four main epithelial cell types in the mouse small
intestine: unitarian theory of the origin of the four epithelial cell
types. Am. J. Anat. 141: 537–562, 1979.
9. Chomczynski, P., and N. Sacchi. Single step method of RNA
isolation by acid guanidinium thiocyanate, phenol-chloroform
extraction. Anal. Biochem. 162: 156–159, 1987.
10. Dhingra, N. K., Y. Ramamohan, and T. R. Raju. Developmental expression of synaptophysin, synapsin I and syntaxin in the
rat retina. Dev. Brain Res. 102: 267–273, 1997.
11. Dodson, B. D., J. L. Wang, E. A. Swietlicki, D. C. Rubin, and
M. S. Levin. Analysis of cloned cDNAs differentially expressed
in adapting remnant small intestine after partial resection.
Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G347–G356,
1996.
12. Dowling, R. H. Cellular and molecular basis of intestinal and
pancreatic adaptation. Scand. J. Gastroenterol. 27, Suppl. 193:
64–67, 1992.
13. Edwardson, J. M., and S. An. The secretory granule protein
syncollin binds to syntaxin in a calcium sensitive manner. Cell
90: 325–333, 1997.
14. Gaisano, H. Y., M. Ghai, P. Malkus, L. Sheu, A. Bouquillon,
M. K. Bennett, and W. S. Trimble. Distinct cellular locations of
the syntaxin family of proteins in rat pancreatic acinar cells. Mol.
Biol. Cell 7: 2019–2027, 1996.
15. Grant, V. P., and H. C. Pant. Expression of p67 (Munc-18),
Cdk5, P-NFH and syntaxin during development of the rat
cerebellum. Dev. Neurosci. 19: 172–183, 1997.
16. Gutierrez, E. D., K. J. Grapperhaus, and D. C. Rubin.
Ontogenic regulation of spatial differentiation in the crypt-villus
axis of normal and isografted small intestine. Am. J. Physiol. 269
(Gastrointest. Liver Physiol. 32): G500–G511, 1995.
17. Hackam, D. J., O. D. Rotstein, M. K. Bennett, A. Klip, S.
Grinstein, and M. F. Manolson. Characterization and subcellular localization of target membrane soluble NSF attachment
protein receptors (t-SNAREs) in macrophages. J. Immunol. 156:
4377–4383, 1996.
18. Hanson, W. R., J. W. Osborne, and J. G. Sharp. Compensation
by the residual intestine after intestinal resection in the rat. I.
Influence of amount of tissue removed. Gastroenterology 72:
692–700, 1977.
19. Hermos, J. A., M. Mathan, and J. S. Trier. DNA synthesis and
proliferation by villous epithelial cells in fetal rats. J. Cell Biol.
50: 255–258, 1971.
G123
G124
EPIMORPHIN/SYNTAXIN 2 EXPRESSION IN GUT MORPHOGENESIS
41. Simon, T. C., and J. I. Gordon. Intestinal epithelial cell
differentiation: new insights from mice, flies and nematodes.
Curr. Opin. Genet. Dev. 5: 577–586, 1995.
42. Smirnova, T., J. Stinnakre, and J. Mallet. Characterization of a presynaptic glutamate receptor. Science 262: 430–433,
1993.
43. Soudais, C., M. Bielinska, M. Heikinheimo, C. MacArthur,
N. Narita, J. E. Saffitz, M. C. Simon, J. M. Leiden, and
D. B. Wilson. Targeted mutagenesis of the transcription factor
GATA-4 gene in mouse embryonic stem cells disrupts visceral
endoderm differentiation in vitro. Development 121: 3877–3888,
1995.
44. Spring, J., M. Kato, and M. Bernfield. Epimorphin is related
to a new class of neuronal and yeast vesicle targeting proteins.
Trends Biochem. Sci. 18: 124–125, 1993.
45. Suh, E., L. Chen, J. Taylor, and P. G. Traber. A homeodomain
protein related to caudal regulates intestine-specific gene transcription. Mol. Cell. Biol. 14: 7340–7351, 1994.
46. Trier, J. S., and P. C. Moxey. Morphogenesis of the small
intestine during fetal development. In: Development of Mammalian Absorptive Processes, edited by K. Elliot and J. Whelan. New
York: Excerpta Medica, 1979, p. 3–29. [CIBA Foundation Symposium]
47. Tsarfaty, I., J. H. Resau, S. Rulong, I. Keydar, K. L. Faletto,
and G. F. Vande Woude. The met proto-oncogene receptor and
lumen formation. Science 257: 1258–1261, 1992.
48. Wang, J. L., D. A. Swartz-Basile, D. C. Rubin, and M. S.
Levin. Retinoic acid induces early cellular proliferation in the
adapting remnant small intestine after partial resection. J. Nutr.
127: 1297–1303, 1997.
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.1 on June 18, 2017